Tailoring the (bio)activity of polymeric and
metal oxide nano- and microparticles in
biotic and abiotic environments
Sathish Ponnurangam
Submitted in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
in the Graduate School of Arts and Science
Columbia University
2012
ABSTRACT
Tailoring the (bio)activity of polymeric and metal oxide nano- and microparticles in biotic
and abiotic environments
Sathish Ponnurangam
Polymeric and metal oxide micro- and nanoparticles are being increasingly introduced into
biomedical applications such as tissue engineering as well as in consumer products, which has boosted
extensive research towards developing predictive paradigms of their (bio-)activity. The core hypotheses
which are tested in the four interrelated studies of this work is that the (bio-)activity of the particles is
defined not only by their intrinsic properties such as the composition/structure, functional groups, surface
charge, and size/morphology, but also on the concentration of particles which in turn is determined by
specific applications. In addition, the (bio)activity of the particles can be controlled by the application-
specific biomolecules or surfactants. These hypotheses are tested on polymeric and metal oxide particles
from the perspective of their application in tissue engineering of articular cartilage and consumer products
(antioxidant additives and dyes), respectively. The modeling of the transport properties of biomaterials, as
well as of the adsorption properties of metal oxide nanoparticles can help to determine or interpret the
observed relationships.
To test this hypothesis, the first study addresses the effect of the concentration of the polysebacic
anhydride microparticles on the cellular viability of chondrocytes (cartilage cells) and on the properties of
in vitro grown tissue. The polysebacic anhydride based microparticles, which are one most common
biocompatible and biodegradable polymer, is used in this study for short term delivery of bioactive
molecules such as growth factors and Vitamin C in articular cartilage tissue engineering. An in situ short
term delivery of nutrients and growth factors is hypothesized to overcome the problem of the decrease in
the diffusion caused by the progressive elaboration of extracellular matrix in 3D agarose scaffolds with
time. I found that, contrary to the reported safety of polyanhydrides, the polysebacic anhydride -based
microparticles are cytotoxic to chondrocytes (cartilage cells) at concentrations relevant to delivery in
articular cartilage engineering. The cytotoxicity is explained by the lipotoxicity from the polysebacic
anhydride degradation products. The available data allow suggesting that the bioactivity of polysebacic
anhydride polymer is collectively and uniquely determined by its degradation rate and hydrophobicity
which requires further verification. It is found that the cytotoxicity of polysebacic anhydride can be
mitigated significantly by administering of bovine serum albumin. The bovine serum albumin-based
mitigation strategy makes polysebacic anhydride microparticles effective in the short-term delivery of
growth factors in articular cartilage engineering. With this bovine serum albumin-based mitigation
strategy, the loading concentrations of fast degrading polyanhydride-based particles can be increased to
levels relevant to in situ delivery requirements for articular cartilage tissue engineering. This opens up
possibility of obtaining tissues with spatially homogenous properties.
Another critical factor that arises due to the presence of the polymeric particles is the modification
of transport properties of agarose hydrogel scaffold. To address this issue, a model is developed to
extract the pore/void size as a function of agarose concentration from the turbidity measurements.
The second study verifies the importance of functional groups of polymer particles in the model
system of polyacrylate-based nanoparticles and chondrocytes in a 3D agarose hydrogel scaffold. From
this study, the charge on the functional groups of the polymers is found to have a significant effect on
their bioactivity. Specifically, the polyacrylate-based nanoparticles are shown to be biocompatible (at
0.2% w/v) to chondrocytes in terms of cellular viability. However, they exhibit bioactivity which is
detrimental to the synthesis of extracellular matrix even at such low concentrations. An inverse correlation
is established between mechanical strength and the negative surface charge of these nanoparticles. The
cause for poor matrix synthesis is suggested to be the disruption of the inter-cellular signaling process by
higher anionicity. The main impact of this study is that the utility of anionically charged polymeric particles
in articular cartilage engineering should be scrutinized carefully, since it is shown that for a clinically
relevant scaffold system such as agarose hydrogels, these particles may act detrimentally.
Study 3 focuses on the (bio-)activity of redox-active metal oxide nanoparticles toward
chondrocytes. For the first time, nanoceria is shown to greatly improve the biochemical as well as
mechanical properties of the cartilage tissue grown in vitro. The effect is explained by the unique redox
properties of the nanoparticles. Specifically, nanoceria is biocompatible toward chondrocytes at
concentrations at least up to 1000 μg/mL, while the constructs cultured with embedded nanoceria at 1000
μg/mL have mechanical and biochemical properties significantly better than those of the control.
Interestingly, the bioactivity of nanoceria in combating inflammatory chemical such as interleukin-1α
depends on the mode of its administration. In particular, when nanoceria is suspended in the growth
medium, it offered significant chondroprotectivity against interleukin-1α and partial prevention of the
matrix degradation. However, when nanoceria is directly embedded into the constructs, it does not offer
any protection against interleukin-1α. These results show the great potential of nanoceria in improving
tissue properties and combating the arthritic inflammation. If the mode of nanoceria administration and
cellular uptake are further optimized, the long term protection of cartilage can be achieved.
In contrast to nanoceria, ferric (hydr)oxide nanoparticles, which are also redox active,
detrimentally affect the growth of cartilage tissue. The adverse effect increases with increasing
nanoparticle size and confirms the initial hypothesis of the size dependence of bioactivity of ferric
(hydr)oxide nanoparticles, which is based on earlier observation from our group of the catalytic activity of
these nanoparticles. Specifically, the mechanical properties of the tissue constructs embedded with
hematite and ferrihydrite nanoparticles degrade with increasing nanoparticle size. On the other hand, the
cellular viability remains similar among all these sample sets. These two results together imply that the
bioactivity is directly related to the redox potential and the catalytic redox mechanism (including either the
deactivation or generation of reactive oxygen species) of the metal oxide nanoparticles toward redox
active constituents at the biointerface.
Study 4 addresses the effects of nanoparticle size and morphology on the adsorption and
dispersion of the ferric (hydr)oxide nanoparticles in the presence of surfactants, which is relevant to the
dispersion of the nanoparticles in their application in image and contrast agents, cancer therapy,
ferrofluids, paints, and cosmetics. The first experimental evidence is obtained for the dependence of the
surface density, speciation, and packing order of adsorbed fatty acids on the nanoparticle size and
morphology. The conditions under which fatty acids form self-assembled monolayers and bilayers on
such nanoparticles in water are distinguished and the electric polarization of the nanoparticles is
demonstrated to be a powerful tool for manipulating the interfacial properties of the nanoparticles.
Specifically, an increase in nanosize improves the adsorption capacity and affinity of hematite
nanoparticles, in agreement with the nanosize-induced changes in the structural and electronic properties
of the nanoparticles. Consequently, an increase in nanosize of hematite nanoparticles improves the
packing order of laurate and hence hydrophobicity of the nanoparticles provided a similar hexagonal habit
of these nanoparticles. It was also observed that, independent of morphology, the dispersibility of the
nanoparticles by fatty acids in water degrades as nanoparticle size decreases. This effect is attributed to
the decrease in the adsorption capacity which ends up in the formation of a loosely packed bilayer. This
implies that the surface electronic and structural properties, which can be controlled by size for metal
oxide nanoparticles, offer important ways to manipulate adsorption properties.
Morphology of the nanoparticles is critical for the self-assembly if (i) the majority of the molecules
are chemisorbed and (ii) the morphology-determined surface arrangement of the adsorption sites is
geometrically incompatible with a surfactant ordering. The surface ratio of the physisorbed/chemisorbed
surfactant depends on the surface charge of nanoparticles. Negative polarization suppresses
chemisorption. This effect can be used to remove the geometrical constraints for self-assembly. The
dominance of the chemisorbed (inner-sphere) complexes results in the isotropic surface filling by fatty
acids, while the prevalence of outer-sphere complexes favors the cluster-like filling pattern.
A new approach to density functional theory modeling of the in situ FTIR spectra of adsorbed
species is developed to solve the technically challenging problem of determining the adsorbed structures
especially at the magnetic oxide-water interfaces. This new approach constitutes searching of the
adsorbed structure that provides the best correspondence of the trends of the theoretical and
experimental spectra upon variations of the solution pH, ionic strength and protonation-deprotonation of
the carboxylate group, instead of conventional approach of searching for the structure that provides the
best one-to-one correspondence between the theoretical and experimental spectra. The new density
functional theory approach is used to interpret the vibrational spectroscopic data in obtaining information
on adsorption forms and packing of fatty acids on ferric (hydr)oxide nanoparticles. These molecular
information is used to interpret the dispersion properties of ferric (hydr)oxide nanoparticles. The
developed approach advances the utility of density functional theory modeling of adsorption phenomena
by incorporating physical variables such as pH, surface charge and ionic charge. This new approach, if
applied, can potentially solve many of controversies in literature in interpreting the adsorption forms
obtained from spectroscopic and density functional theory data.
Although there is still no general paradigm predicting the (bio-)activity for both the polymeric and
metal oxide micro- and nano-particles, the experimental, methodological and theoretical results presented
in these studies are an important step and base towards developing such a paradigm. In this context, the
following factors determine the bioactivity of the polymeric particles considered (polyanhydrides and
polyacrylates): degradation rate, hydrophobicity, functional group and surface charge. In addition to
above parameters, concentration of these particles with respect to cell density is critical factor determining
their (bio-)activity. Among the numerous physico-chemical variable that can affect the (bio-)activity of
metal oxide nanoparticles, the catalytic redox properties and the redox mechanism are general predictors
for ceria and ferric (hydr)oxide nanoparticles. These properties can be varied by varying nanosize of the
nanoparticles. Both nanosize and morphology of nanoparticles can also significantly affect their
adsorption properties and, through them, their bioactivity and bioavailability. In addition, the density
functional theory-based methodology to interpret the adsorption phenomena developed in this work can
further augment the possibility of formulating such a general paradigm for (bio-)activity.
i
Table of Contents
Table of Contents ........................................................................................................................................... i
List of abbreviations ...................................................................................................................................... v
List of Figures ............................................................................................................................................... vii
List of Tables ................................................................................................................................................ xii
List of Schemes ........................................................................................................................................... xiii
Acknowledgements ..................................................................................................................................... xiv
Chapter 1 Introduction, Literature review, and Goals ................................................................................... 1
1.1 Literature review and gaps in the current knowledge ......................................................................... 1
1.1.1 Bio-activity of polysebacic anhydride microparticles used for in situ delivery in articular cartilage
engineering............................................................................................................................................ 1
(a). Challenges in articular cartilage engineering.............................................................................. 2
(b). Rationale for using polymeric microparticles for in situ delivery of bioactive molecules ............ 5
(c). Comparison of biomedical properties of natural and synthetic polymeric biomaterials .............. 6
(d). Sebacic acid based polyanhydride polymer for delivery device in articular cartilage
engineering: Advantages and challenges ......................................................................................... 8
1.1.2. Bioactivity of polyacrylate nanoparticles in articular cartilage tissue engineering: Effects of
functional groups ................................................................................................................................. 11
1.1.3 (Bio-)chemical activity of metal (hydr)oxide nanoparticles ......................................................... 13
Current paradigm of the cytotoxicity and biocompatibility of metal oxide nanoparticles................. 14
1.1.4 (Bio-)activity of engineered ceria oxide nanoparticles: Anti-inflammatory and reactive-oxygen
species scavenging properties ............................................................................................................ 16
(a). Structural, electronic, and abiotic redox properties of CeO2 nanoparticles .............................. 16
(b). Rationale for selection of nanoceria to combat inflammation in articular cartilage .................. 18
1.1.5 (Bio-)activity of ferric (hydr)oxide nanoparticles: adsorption, dispersion and biocompatible
properties ................................................................................................................................................ 20
(a) Structural, electronic, and abiotic redox properties of hematite nanoparticles .......................... 20
(b). Size, shape and morphology dependence of (bio-)activity of ferric (hydr)oxide nanoparticles 23
1.1.6. Challenges in modeling the adsorption of fatty acids at the metal oxide nanoparticle-water
interface ............................................................................................................................................... 28
1.2. Goals and objectives ........................................................................................................................ 30
Chapter 2 Materials and Methods ............................................................................................................... 33
2.1 Materials ............................................................................................................................................ 33
2.2 Tissue culture studies ....................................................................................................................... 33
2.3 Characterization and physico-chemical experimental methods ....................................................... 35
2.4 Density Functional Theory (DFT) Methodology ................................................................................ 38
ii
2.5 Error Analysis of data ........................................................................................................................ 39
Chapter 3 Synthesis and characterization of polymers and polymeric micro- and nano-particles ............. 40
3.1 Synthesis of polysebacic anhydride polymer using melt condensation method ............................... 40
3.2. Functionalization of polysebacic anhydride with fluorescein label and vitamin C ........................... 42
3.2.1 Functionalization with Fluorescein ............................................................................................. 42
3.2.2 Functionalization with vitamin C (L-ascorbic acid) ..................................................................... 42
3.3 Characterization of polysebacic anhydride and its derivatives using NMR and Raman
spectroscopies ........................................................................................................................................ 44
3.4 Fabrication of polysebacic anhydride microparticles and encapsulation of bioactive molecules using
double emulsion method ......................................................................................................................... 46
3.5 Characterization and release kinetics of polysebacic anhydride microparticles using optical,
electron, and confocal fluorescence microscopies, as well as UV-Vis absorption spectroscopy. .......... 48
3.6. Synthesis and characterization of acrylate nanoparticles ................................................................ 50
3.6.1. Ternary phase diagrams ........................................................................................................... 50
3.6.2.Synthesis of poly(acrylic acid) nanoparticles and its functional derivatives ............................... 51
3.6.3.Size determination using SEM and dynamic light scattering ..................................................... 53
3.6.4 Atomic composition using X-Ray photoelectron spectroscopy .................................................. 54
3.6.5 Surface charge using electrophoretic mobility (Zeta potential) measurements ......................... 54
3.7 Pore-size determination of agarose-based hydrogel as a function of agarose concentration and
presence of polysebacic anhydride microparticles ................................................................................. 55
Chapter 4 Synthesis and characterization of ferric (hydr)oxide nanoparticles ........................................... 62
This chapter presents the synthesis procedures used to obtained hematite nanoparticles of different
sizes, as well as the characterization of these nanoparticles using TEM, HRTEM, XRD, Raman, XPS,
acid-base titration, and zeta potential methods. ..................................................................................... 62
4.1 Synthesis of hematite nanoparticles ................................................................................................. 62
4.2 Characterization of ferric (hydr)oxide nanoparticles ......................................................................... 62
Chapter 5 Bioactivity of polymeric particles in articular cartilage engineering: In situ delivery using
poly(sebacic anhydride) microparticles and functional group effects of poly(acrylate) based nanoparticles
.................................................................................................................................................................... 68
5.1 Effects of in situ delivery of bioactive molecules on the chondrocytes cultured in 3D scaffold ........ 68
5.1.2. Dose dependent cellular viability in the presence of poly(sebacic anhydride) ......................... 69
5.1.3 Mechanisms of the cytotoxicity of poly(sebacic anhydride) microparticles ................................ 69
(a). Are cytotoxic effects of PSA-microparticles on chondrocytes due to local pH changes? Testing
the hypothesis through increased buffer capacity........................................................................... 71
(b). Are cytotoxic effects due to cellular uptake PSA-microparticles or its degradation products and
the resulting lipotoxicity? Confocal fluorescence studies ................................................................ 72
5.1.4 Mitigation of the cytotoxicity of PSA on chondrocytes: positive impact of bovine serum albumin
(BSA) ................................................................................................................................................... 73
iii
5.1.5 Delivery of TGF-β3 using PSA-microparticles in BSA-based system ........................................ 74
5.2 The effects of functionalization of polyacrylate nanoparticles on the cellular viability and metabolism
................................................................................................................................................................ 76
5.2.1 Sterilization tests: ....................................................................................................................... 76
5.2.2 Biocompatibility tests: ................................................................................................................. 76
5.2.3 Three-dimensional tissue culture studies with acrylate nanoparticles ....................................... 77
5.3 Conclusions: ...................................................................................................................................... 80
Chapter 6 Anti-inflammatory and anti-oxidant bioactivity of ceria nanoparticles in articular cartilage
engineering ................................................................................................................................................. 82
6.1 Experimental design .......................................................................................................................... 82
6.2 Effects of nanoceria embedded into the 3D-agarose scaffold on tissue growth as well as combating
inflammation due to interleukin-1α .......................................................................................................... 84
6.3 Effects of nanoceria suspended in the growth medium on tissue growth as well as combating
inflammation due to interleukin-1α .......................................................................................................... 87
6.4 Conclusions ....................................................................................................................................... 88
Chapter 7 Dependence of the (bio)activity of hematite NPs on their size, morphology, and surface charge
.................................................................................................................................................................... 89
7.1 Size, shape and surface-charge dependent bioactivity of hematite NPs on chondrocytes .............. 89
7.2 Size, shape and surface-charge dependent interaction of hematite NPs with sodium laurate
(dodecanoate) ......................................................................................................................................... 93
7.2.1 Dispersion and adsorption properties of hematite NPs in the presence of sodium laurate. ...... 93
7.2.2 Speciation of carboxylic groups in the first monolayer. .............................................................. 97
a. Inner-sphere monodentate mononuclear complex (ISMM): ....................................................... 99
b. Outer-sphere hydration shared complex (OS): ........................................................................... 99
c. Protonated outer-sphere hydration shared complex (OS-H): ................................................... 100
7.2.3 Packing order of hydrocarbon chains. ..................................................................................... 101
7.2.4 Controls of NP dispersion by fatty acids. Formation of a bilayer. ............................................ 102
7.2.5 Effect of negative polarization on the structure of the first monolayer. .................................... 104
7.2.6 Speciation of adsorbed fatty acid. Effects of the oxide acidity. ................................................ 106
7.2.7 Conditions for SAM formation. Effects of the NP size, morphology, and surface charge. ...... 107
7.3 Conclusions ..................................................................................................................................... 109
Chapter 8 A new approach to DFT modeling of the adsorbed species at the metal (hydr)oxide NPs-water
interfaces ................................................................................................................................................... 111
8.1 A combined DFT-FTIR approach to resolve the adsorption forms at the oxide-water interface .... 111
8.2 A combined DFT-FTIR approach to resolve the adsorption forms at the oxide-water interface .... 113
8.3 Identification of adsorption forms of fatty acid on ferric (hydr)oxide NPs ....................................... 116
8.2.1 FTIR results .............................................................................................................................. 117
iv
8.2.2 DFT modeling of carboxylate binding to hematite surface........................................................... 119
8.4 Conclusions ..................................................................................................................................... 123
Chapter 9 Summary .................................................................................................................................. 124
Chapter 10 Suggested Future Studies ...................................................................................................... 131
REFERENCES .......................................................................................................................................... 133
APPENDIX ................................................................................................................................................ 150
A. Estimation of buffer capacity needed to offset the drop in pH due to polsebacic anhydride
degradation ........................................................................................................................................... 150
B. Determination of wavelength exponent ............................................................................................ 151
v
List of abbreviations
AFM Atomic force microscopy
ANOVA Analysis of variance
AOT Aersol-OT
ATR Attenuated total reflection
BET Brunauer-Emmett-Teller
BSA Bovine serum albumin
CM Culture medium
CMC Critical micellar concentration
COSMO Continuum solvation model
DFT Density functional theory
DNP Double numerical polarization
ECM Extracellular matrix
FH Ferrihydrite
fPSA Fluoresceinated-polysebacic anhydride
FTIR Fourier transform infrared spectroscopy
GAG(s) Glycosaminoglycan(s)
GRGD Glycine-arginine-glycine-aspartic acid
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
HLB Hydrophilic-lipophilic balance
IEP Isoelectric point
IL-1α Interleuklin-1alfa
ISBB Innersphere bidentate binuclear
ISMM Innersphere monodentate mononuclear
IRE Internal reflection element
ML Monolayer
MSC Mesenchymal stem cells
NMR Nuclear magnetic resonance
NP(s) Nanoparticle(s)
OS Outersphere
PAA Polyacrylic acid
PAM Polyacrylamide
PAAP Polyacrylic acid-phosphonate
P(CPP-SA) Poly(carboxy phenoxy propane-co-sebacic anhydride)
vi
PEG Poly(ethylene glycol)
PEGF Poly(ethylene glycol-co-fumarate)
PLGA Poly(lactic-co-glycolic acid)
PSA, P(SA) Polysebacic anhydride
PSA-M Polysebacic anhydride-microparticles
PZC Point of zero charge
RGD Arginine-glycine-aspartic acid
ROS Reactive oxygen species
RNS Reactive nitrogen species
SA Sebacic acid
SAED Small area electron diffraction
SAM Self-assembled monolayer
SEM Scanning electron microscope
SL Sodium laurate
SOD Superoxide dismutase
TDW Triple distilled water
TEM Transmission electron microscope
TGF-β3 Transformer growth factor beta 3
THF Tetrahydrofuran
TMAO Trimethyl-amine oxide
XPS X-ray photoelectron spectroscopy
XRD X-Ray Diffraction
vii
List of Figures
Figure 1.1 A typical cross section of articular cartilage showing four zones4 .............................................. 2
Figure 1.2 The fcc cell of CeO2 with the fluorite structure. Ce is white, O is red. ...................................... 16
Figure 1.3 A model of the reaction mechanism for the oxidation of hydrogen peroxide by nanoceria and
the regeneration via reduction by superoxide. An oxygen vacancy site on the nanoceria surface (1)
presents a 2Ce4+
binding site for H2O2 (2), after the release of protons and two-electron transfer to the two
cerium ions (3) oxygen is released from the now fully reduced oxygen vacancy site (4). Subsequently
superoxide can bind to this site (5) and after the transfer of a single electron from one Ce3+
, and uptake of
two protons from the solution, H2O2 is formed (6) and can be released. After repeating this reaction with a
second superoxide molecule (7) the oxygen vacancy site returns to the initial 2Ce4+
state (1). It is also
possible that the third Ce3+
indicated, which gives rise to the oxygen vacancy, could participate directly in
the reaction mechanism. The square Ce–O matrix is shown here only to illustrate the model and does not
correspond to the actual spatial arrangement of the atoms in the crystal structure. Reproduced from
Ref.70
........................................................................................................................................................... 18
Figure 1.4 Crystal structure of α-Fe2O3. Red atoms are O; Blue atoms are Fe. ........................................ 21
Figure 1.5 Conceptual scheme of electrochemical mechanism of redox reaction Ox2 + Red1 Ox1 +
Red2 in the dark.116
FL = Fermi level, VB = valence band, CB = conduction band, △s = gradient of
surface potential. ......................................................................................................................................... 22
Figure 3.1 NMR spectra of polymers on their precursors; (a) Sebacic acid, (b) Poly(sebacic anhydride)
(inset: expanded and curve fitted region from 2.18-2.48 ppm), (c) Ascorbic acid (d) Poly(Sebacic
anhydride)- Ascorbic acid. ........................................................................................................................... 45
Figure 3.2 Raman spectra of polymers on their precursors; Poly(sebacic anhydride) and Sebacic acid.. 46
Figure 3.3 SEM images of PSA-microparticles encapsulated with casein-FITC after 24 hours in aqueous
suspension. All the images are from the same batch of microparticles but at different magnifications. .... 48
Figure 3.4 Particle size distribution of PSA-microparticles with casein-FITC after 24 hours in aqueous
suspension. The size distribution was obtained from SEM image analysis performed using ImageJ
program. ...................................................................................................................................................... 49
Figure 3.5 L-ascorbic acid loaded polyanhydride microparticles embedded in agarose scaffold. Green
color indicates the fluorescein-functionalized PSA polymeric shell. Black core is the encapsulated L-
ascorbic acid. Both the images are from the same microparticle sample .................................................. 49
Figure 3.6 Release profile of amitriptyline drug from poly(sebacic anhydride)-based microparticles........ 50
Figure 3.7 Phase diagrams of (a) hexane/water/AOT (b) hexane /(water+acrylic acid)/AOT systems at
room temperature. Only inverse emulsion region is indicated by colors .................................................... 52
viii
Figure 3.8 Phase diagram of hexane /(water+acrylic acid)/AOT systems at 50 C. Only inverse emulsion
region is indicated by the enclosed region. ................................................................................................. 52
Figure 3.9 Synthesis of functionalized polyacrylate nanoparticles, (a) polyacrylic acid (b) polyacrylamide
(c) poly(acrylic acid-phosphonate) (d) poly(acrylic acid-sulfonate); Bisacrylamide is the crosslinker and
ascorbic acid is the initiator ......................................................................................................................... 53
Figure 3.10 SEM micrographs of poly(acrylic acid) particles synthesized using concentrations at (a)
phasepoint-1 (b) phasepoint-2 .................................................................................................................... 53
Figure 3.11 Size of nanoparticles using dynamic light scattering technique, (a) poly(acrylic acid), 130-
340-nm (b) poly(acrylic acid-acrylamide(10%)), 100-320-nm (c) poly(acrylic acid-phosphonate(10%)),
140-260-nm (d) poly(acrylamide) 70-90-nm nanoparticles ......................................................................... 54
Figure 3.12 X-ray photoelectron spectroscopy of poly(acrylic acid) nanoparticle. Carbon 1S spectral
region is shown. .......................................................................................................................................... 55
Figure 3.13 Zeta potential of poly(acrylamide), poly(acrylic acid-phosphonate), poly(acrylic acid)
nanoparticles ............................................................................................................................................... 55
Figure 3.14 Theoretical calculated wavelength exponent as a function of pore size for wavelength range
of 700-800 nm. ............................................................................................................................................ 59
Figure 3.15 Pore size of agarose hydrogel as function of cooling time. The temperature at time t=0 is 95 0C................................................................................................................................................................. 61
Figure 3.16 Effect of agarose concentration (C, % w/v) on the pore size (ξ, nm) of agarose hydrogel..... 61
Figure 4.1 TEM images of hematite and FH NPs. NPs are labeled according to the average size as
measured by the analysis of TEM images. ................................................................................................. 64
Figure 4.2 Typical (a) HR-TEM image and (b) SAED pattern of 38-nm hematite (H38) ........................... 65
Figure 4.3 (a) XRD and (b) Raman spectra of dry hematite and ferrihydrite NPs. “T” marks the peak due
to tetrahedral defects, LO Eu is the peak activated by the lattice disorder.222
............................................ 65
Figure 4.4 Effect of size of hematite nanoparticles on their point of zero charge and isoelectric point ..... 66
Figure 5.1 Effect of polymer concentration on chondrocytes viability on day-1 in 3D agarose scaffolds.
PSA concentrations are (a) 0.2% (b) 2% and (c) 10% w/v. Green color indicates cytoplasm of the live
cells and red color indicates dead cells and probably microparticles ......................................................... 70
Figure 5.2 Equilibrium Young’s modulus of hydrogel constructs with different concentrations of
poly(sebacic anhydride) microparticles. The y-axis values are normalized by modulus of the
corresponding control constructs (without microparticles). The concentration of polymeric microparticles in
x-axis is represented as w/v% of hydrogel constructs ................................................................................ 70
Figure 5.3 Effect of increased in buffer capacity (5mM HEPES) of growth medium on chondrocytes
viability on day-23 in 3D agarose scaffolds using Live/Dead cytotoxicity kit. The agarose constructs
contain (a) no PSA (control) (b) 2 % PSA microparticles. The microparticles are encapsulated with
chondroitinase-ABC. Green color indicates cytoplasm of the and red color indicates dead cells and
probably microparticles ............................................................................................................................... 72
ix
Figure 5.4 Cellular uptake of fluoresceinated-PSA (F-PSA) by chondrocytes. F-PSA is added nearby a
chondrocytes aggregate at time t=0 and images were obtained at time, t= a) 5 min b) 20 min. c) 60 min.
Gradual increase in the fluorescence intensity is due to intracellular accumulation of F-PSA ................... 73
Figure 5.5 Mechanical properties of control without BSA (BSA-), control with BSA (BSA+) and TGFβ3
microparticles-containing (ugel-TGF-β3-BSA+) constructs. (a) Equilibrium Young’s modulus, and dynamic
modulus at (b) 0.1 Hz. Using two way ANOVA analysis, there was no statistically significant difference
was found between Control BSA+ vs. ugel-TGF-β3-BSA+ for p< 0.05 ..................................................... 75
Figure 5.6 Mechanical properties of control, polyacrylamide containing (PAM) and polyacrylic acid-
Phosphonate (PAA-P) nanoparticles-containing constructs. (a) Equilibrium Young’s modulus, and (b)
dynamic modulus at 0.1 Hz. ........................................................................................................................ 78
Figure 5.7 Total GAG & DNA content (in mg) of control, polyacrylamide containing (PAM) and polyacrylic
acid-Phosphonate (PAA-P) containing scaffolds ........................................................................................ 79
Figure 5.8 Live/Dead assay of scaffolds using fluorescence microscopy, for control, polyacrylamide
containing (PAM) and polyacrylic acid-Phosphonate (PAA-P) containing scaffolds on day0 and day33... 79
Figure 6.1 Mechanical properties of control and nanoceria-containing constructs on day 16 and day 30.
Interleukin-1α was added from day 16 to 30. Nanoceria concentration in constructs are 10, 100 and 1000
μg/mL. Control constructs contain no nanoceria (a) Equilibrium Young’s modulus, (b) dynamic modulus at
0.1 Hz. The significance are *p<0.001 and **p<0.001 ................................................................................ 85
Figure 6.2 Biochemical composition of control and nanoceria-containing constructs on day 16 and day 30
as a fraction of wet weight. Interleukin-1α was added from day 16 to 30. Nanoceria concentration in
constructs are 10, 100 and 1000 μg/mL. (a) Glycasoaminoglycan (GAG) (b) Collagen (c) DNA .............. 85
Figure 6.3 Live/dead analysis of control and nanoceria-containing constructs on day 30 using cytotoxicity
kit and confocal fluorescence microscopy. Green color indicates live and red color indicates dead cells.
Top row contains images of control, nanoceria-containing constructs at 10, 100, 1000 μg/mL
concentration without interleukin-1α insult. Bottom row has the same sets with interleukin-1α insult from
day 16 to day 30. ......................................................................................................................................... 86
Figure 6.4 Mechanical and biochemical properties of constructs at day 34 as a function of nanoceria
concentration in the growth medium. (a) compressive Young’s modulus, (b) dynamic modulus at 0.1 Hz,
(c) glycosaminoglycan. Interleukin-1α is added from day 21 to 34 and its concentrations are given on the
top of the bars (0, 0.5, 10 ng/mL). For IL-1α 10ng/mL and 20000 μg/mL vs. 0 μg/mL *p<<0.00001 ......... 88
Figure 7.1 Mechanical properties of control and feriic (hydr)oxide NPs-containing constructs on day
30.The concentration of the NPs is plotted as total surface area. (a) Equilibrium Young’s modulus,
dynamic modulus at (b) 1 Hz (c) 0.5 Hz (d) 0.1 Hz. .................................................................................... 92
Figure 7.2 Live/dead analysis of control and ferric (hydr)oxide NPs-containing constructs on day 72 using
cytotoxicity kit and confocal fluorescence microscopy. Green color indicates live cells, red color indicates
dead cells. Top row contains images of control, FH, and H9-containing constructs at 25 μg/mL
concentration and bottom row that of H38 and H150 containing-constructs at two different NP
concentrations (25, 50 μg/mL) .................................................................................................................... 93
Figure 7.3 Suspensions of FH and hematite NPs at initial SL concentrations of 3 mM and ionic strength
of 5 mM NaCl. ............................................................................................................................................. 94
x
Figure 7.4 Adsorption isotherms of sodium laurate on FH (wine) and H150 (blue) NPs at pH ~ 7.2. ....... 95
Figure 7.5 Contact angles of SL-coated H150 nanoparticulate film. Formal monolayer coverage of 4.3
molecules/nm2 was estimated from the adsorption isotherm. Hydrophobicity of 150-nm hematite
increases as the surface coverage of laurate increases up to 1 ML. At this point, the particulate film is
superhydrophobic (water droplets are repelled from the surface). With a further increase of surface
coverage, hydrophobicity gradually decreased, indicating that laurate is adsorbed with the head group
pointing out into the solution. ...................................................................................................................... 96
Figure 7.6 Effect of the initial concentration of laurate (written at the bottom as well as on the vials) on the
dispersion of H150 particles at pH 7.2 ........................................................................................................ 96
Figure 7.7 (a) Adsorption isotherms of sodium laurate on FH (wine), H9 (orange), H38 (red) and H150
(blue) NPs at pH ~ 7.2. The last data point corresponds to 1 mM initial laurate concentration (CMC of
laurate) (b) Zeta potential of H150 and FH as a function initial laurate concentration in 5mM NaCl
aqueous solution. Both electrophoretic (red and blue curves) and electroacoustic (green curve) results
are shown. Connecting lines are drawn for reading ease. .......................................................................... 97
Figure 7.8 in situ FTIR-HATR spectra of FH and hematite NPs in contact with a 10–3
M solution of sodium
laurate at pH 10.00 ± 0.05 with no added electrolyte in spectral region of (a) COO and (b) CH
vibrational modes: (olive) H150, (red) H38, (blue) H9. Conditioning time is 2530 min. ........................... 98
Figure 7.9 in situ FTIR-HATR spectra of FH and hematite NPs in contact with a 10–3
M solution of sodium
laurate at pH 7.15 ± 0.05 with no added electrolyte in spectral region of (a) COO and (b) CH
vibrational modes: (olive) H150, (red) H38, (blue) H9, (magenta) FH. Conditioning time is 25 min.
Intensity of the spectra in the 12001800-cm–1
region is normalized by the intensity of the sCH2 band
(see text for more detail). ............................................................................................................................ 99
Figure 7.10 Correlation between frequency of sCH2 band of ISMM laurate in the spectra shown in
Figure 7.9a and PZC of FH and hematite NPs measured in open air (Figure 7.7b) ................................ 100
Figure 7.11 Effect of surface coverage on the position of the sCH2 band of laurate adsorbed on (a)
H150, (b) H38, (c) H9 hematite, and (d) FH from 1 mM solution at pH 10.0 and 7.15. Surface coverage,
evaluated using the sCH2 peak intensity, increases from (red) ~ 0.030.1 to (magenta) 1.0 formal ML
which is assumed to be formed at the maximum interaction time. It is increased with increasing the
conditioning time which is indicated in the graph. The band intensity is normalized to the maximal value in
the series. Color codes for H150, H38, and H9 are the same. FH was conditioned only at pH 7.15
because it does not adsorb 1 mM laurate at pH 10. ................................................................................. 103
Figure 7.12 Arrangement of surface cations on (001) basal, (101) hexagonal as well as on (012) and
(104) rhombohedral hematite surfaces. .................................................................................................... 104
Figure 7.13 Effect of the substrate on in situ FTIR-HATR spectra of H38 hematite NPs in contact with a
10–3
M solution of sodium laurate at pH 7.15 ± 0.05 with no added electrolyte in spectral region of (a)
COO- and (b) CH vibrational modes: NPs were deposited on a (green) Ge and (red) ZnSe IRE.
Conditioning time is 25 min. ...................................................................................................................... 105
Figure 7.14 Comparison of in situ FTIR-HATR spectra in the CH region of (red) H38 deposited on Ge
and (violet) H150 deposited on ZnSe. The NPs were conditioned similarly with a 10–3
M solution of
sodium laurate at pH 7.15 ± 0.05 with no added electrolyte for 2530 min. ............................................. 106
xi
Figure 8.1 A general predictive paradigm for identifying adsorption forms on metal (hydr)oxides using
density functional theory simulations and FTIR spectroscopy. ................................................................. 115
Figure 8.2 Ferric trioctahedral cluster extracted form hematite (104) surface. The cluster contains both
edge-shared and corner-shared oxygen ions. The surface terminal OH/H2O groups that are involved in
coordination with carboxylate group are shown in yellow spheres. (Iron – blue spheres; oxygen – large
red spheres; carbon – large gray sphere; hydrogen – small green spheres) ........................................... 116
Figure 8.3 in situ FTIR-HATR spectra in region of (a) CO2 and (b) scisCH2
vibrational modes of (1)
hematite reacted for 25 min with a 10–3
M solution of sodium laurate at pH 7.15 ± 0.05 with no added
electrolyte. For comparison, shown are spectra of (2) aqueous 10–2
M sodium laurate at pH 10.2
measured with a Ge IRE, (3) solid sodium laurate solid, and (4) solid lauric acid. Baseline in the
16001660-cm-1
region of the adsorbed laurate was corrected by adding spectrum of water (water is
removed from the interface upon the adsorption). This figure is taken from Ref. 190
. Spectral regions of the
C=O and scisCH2 modes of the surfactant are shadowed. Band assignment is taken from Ref.253
....... 118
Figure 8.4 in situ FTIR-HATR spectra of 150-nm hematite reacted successively with SL at (a) pH 7.15
and SL concentration of (black) 0.1 mM, (blue) 0.5 mM and (green) 1.0 mM and (b) pH 10.0 and SL
concentration of 1 mM. Time of the interaction is shown in the graphs. It was measured from the moment
of the introduction of a SL solution into the FTIR-ATR cell till the beginning of the spectrum scanning
(which takes 5 min). This figure is taken from Ref. 190
. The background spectrum was that of the
deposited particles pre-equilibrated with water of pH 7.15 for 1 hour. Spectra are shifted along the Y axis
for clarity. ................................................................................................................................................... 119
Figure 8.5 Geometry optimized DFT models of a butanoate ion attached as ISMM and OS hydration-
shared complexes to a ferric trioctahedral cluster with charge q = 0 which represents the (104) hematite
surface. Numbers are the CO and CC and H bond lengths in Å Dashed lines show the surface
coordinating H bonds. ............................................................................................................................... 120
Figure 8.6 The predictive methodology applied in this work for identifying adsorption forms of sodium
laurate on 150-nm hematite NPs using density functional theory simulations and FTIR spectroscopy ... 122
xii
List of Tables
Table 1.1 Characteristics of human articular cartilage using zonal representation4 .................................... 3
Table 4.1 Morphological characteristics of hematite NPs .......................................................................... 67
Table 6.1 Two way ANOVA for all the mechanical properties (equilibrium Young’s modulus, dynamic
modulus at 0.1, 0.5, and 1 Hz) on day 30 of control and nanoceria-containing constructs those were not
insulted with interleukin-1α. ......................................................................................................................... 86
Table 8.1 Calculated vibrational frequencies of IS and OS complexes of butanoate .............................. 121
xiii
List of Schemes
Scheme 1.1 In situ delivery of bioactive molecules using encapsulated polymeric microparticles
embedded in a hydrogel matrix. The surface eroding polymeric microparticles allows release the bioactive
molecules in a uniform and sustained manner. ............................................................................................ 5
Scheme 1.2 Hydrolysis of acetyl-endcapped poylanhydride polymer into polyacid-anhydride oligmers and
acetic acid. The red ellipsoid is a lipophilic backbone chain with or without anhydride interlinks ................ 9
Scheme 1.3 Cluster models of different laurate adsorption complexes. ISBB-Inner sphere bidentate
bridging; ISMM – Innersphere monodentate mononuclear; OS – Outersphere; Blue octahedra – Fe3+
; red
spheres – oxygen; gray spheres – carbon; green spheres – hydrogen. .................................................... 29
Scheme 3.1 Synthesis of polyanhydrides using melt condensation method ............................................. 41
Scheme 3.2 Synthesis of L-ascorbic acid functionalized PSA polymer ..................................................... 43
Scheme 3.3 Synthesis of microparticles using double emulsion and solvent evaporation method ........... 47
Scheme 6.1 Two different ways of introducing nanoceria in a 3D-agarose based culture of chondrocytes.
(a) nanoceria is embedded in 3D agarose constructs during casting on day-0 (b) nanoceria was added in
to the growth medium along with interleukin from day-21 .......................................................................... 83
Scheme 7.1 Cluster models of surface binding of fatty acid: (a) inner sphere monodentate mononuclear
(ISMM) carboxylate complex with H-bonded second oxygen (b) outer sphere (OS) surface-hydration
shared ion pair, and (c) OS fatty acid. Inner-sphere monodentate mononuclear complex: specific
adsorption of one of the oxygen of laurate molecule with one of surface ferric ion, whereas the second
oxygen H-bonds with other surface hydroxyl or water. Outer-sphere surface-hydration complex: non-
specific adsorption of laurate (or lauric acid) on surface ferric ions mediated by a hydration layer. .......... 98
Scheme 7.2 Stretching of the sCO2– dipole of adsorbed carboxylate by positive charge of the NP
surface. ...................................................................................................................................................... 100
Scheme 7.3 Deprotonation of lauric acid by basic surface sites upon adsorption. ................................. 107
Scheme 8.1 Cluster models of different laurate adsorption complexes. Blue octahedra – Fe3+; red
spheres – oxygen; gray spheres – carbon; green spheres – hydrogen. ................................................. 117
xiv
Acknowledgements
I would like to express my most sincere thanks and intense gratitude to my advisor Prof. Ponisseril
Somasundaran for his guidance, motivation and support. His extraordinary ability to motivate, energize
and train me to work hard helped me in solving different problems. His ‘Saturday sermons’ are very
inspiring and are valuable as advice and benefits from his decades of experience. There is never a dull
moment when Prof. Som is around and is always pleasant to work with. However careful I was, I did
become part of large repertoire of his jokes, which of course, I enjoyed thoroughly. He propelled and
inspired me to attack and tackle problems far from my expertise and continuously encouraged me
through the difficult times in the course of this work. I am highly grateful to Prof. Somasundaran, for
teaching and training in various concepts and skills, in particular where I was found wanting. I convey my
deepest thanks to him for giving me great freedom to pursue my ideas.
I would like to take this opportunity to convey my profound gratitude to Prof. Clark T. Hung for
agreeing to collaborate with me in the tissue engineering part of this work, guiding and motivating me on
this part. In particular, this work would not have been possible without his encouragement and
commitment. I would also like to thank him greatly for taking time to teach me and for access to his
laboratory facilities. I would also like to specially show my indebtedness to Dr. Grace O’Connell for her
constant guidance in tissue engineering fundamentals and experimental methods. I enjoyed greatly
working and communicating with her.
I express my deepest appreciation for Dr. Irina Chernyshova for her collaborating with me during
my graduate study and teaching me and educating me not only in the spectroscopic methods, but also in
accurate and reproducible experimentation, as well as writing scientific papers and proposals. She is a
constant source of inspiration and criticism, and I am greatly indebted to her for her crucial help in
achieving and contextualizing the spectroscopic interpretations presented in the work.
I would like to extend my special thanks to Dr. Raymond Farinato for his teaching, advice and
guidance which helped me immensely in choosing my research topics and overcoming the problems in
xv
particle dispersions and polymeric particle synthesis. I would also like to thank Dr. Sun-yi Huang whose
advice solved problems that I was facing in microemulsion systems. I express my sincere thanks to Dr.
Nagaraj for his constant guidance in critical thinking and for further providing me with avenues where I
could contribute. I would like to thank Prof. Paul Duby, Prof. Nick Turro, Drs. Pradip, Venkat Runkana and
K. Ananthapadmanabhan for their advice, discussions, and motivation throughout this work.
I would also like to take this opportunity to acknowledge the financial support provided by NSF
Center for Particulate and Surfactant Systems, Columbia University and Tata Research Design and
Development Center.
Not least of all, I wish to acknowledge and thank all my current and previous lab members for
constant willingness to help me. In particular, I appreciate contributions, experimental help, and very
useful comments of Drs. P Purhoit, X. Fang, B. Li, S. Lu, and J. Wu. I also would express my genuine
appreciation for all my friends, in particular Jonathan Levine and Karthik for their constant interest in my
progress, support, encouragement, and great time spent together. I deeply appreciate the administrative
personnel from Earth and Environmental Engineering for the sustained support, and Katherine Hickey for
proof reading.
Finally, I would like to convey my love and gratitude towards my parents, Ponnurangam
Muniswamy and Banumathi Renu, for without their love, support and encouragement none of my
endeavors would have been possible.
1
Chapter 1 Introduction, Literature review, and
Goals
Polymeric nano- and micro-particles have attracted attention as promising candidates for
biomedical applications, such as encapsulation and drug-delivery. It has been demonstrated that these
particles can be biocompatible, biodegradable, and, moreover, obtained from renewable resources. They
have already found applications in medical imaging and diagnostic devices, tissue engineering, and cell
therapeutics. Another important relevant area, with no general or simple solution, is the safe, uniform and
controlled delivery of drugs, nutrients and growth factors using polymeric particles. Metal (hydr)oxide NPs
are one of the main nanomaterial in the current and researched nanotechnologies.1 The societal concern
about the safety of these NPs for human health and the environment has recently triggered an additional
interest to the nano-biointerfaces.2
In general, structural, compositional, dimensional, and morphological properties of nano- and
microparticles determine their technological and biomedical performance, as well as cytotoxicity.
However, each particular application and each particular type of material require a specific inquiry.
This work is focused on the structure-(bio)activity relationship for four types of particlestwo
types of polymeric nano- and microparticles, and two types of metal (hydr)oxide nanoparticles. Here, the
bioactivity of polymeric particles is defined as their biocompatibility and cell-stimulating activity in articular
cartilage engineering, whereas for metal(hydr)oxide nanoparticles, it is defined as their antioxidant
properties and cytotoxcity, as well as the adsorption and dispersion properties.
1.1 Literature review and gaps in the current knowledge
1.1.1 Bio-activity of polysebacic anhydride microparticles used for in situ delivery in articular
cartilage engineering
This section outlines
2
(a) Challenges in articular cartilage engineering,
(b) Rationale for using polymeric microparticles for in situ delivery of bioactive molecules that address
these challenges,
(c) Different classes of polymeric biomaterials that are used for encapsulation and
(d) Rationale for the selection of polyanhydrides, physico-chemical and biocompatible and biodegradable
properties of polyanhydrides,
(a). Challenges in articular cartilage engineering
Articular cartilage is a connective tissue between the articular surfaces of joint bones which helps
reduce friction between the joints, absorbs shock, and enables the sliding movement of joints. The major
component of native human articular cartilage is water (~72% by weight). Other significant components
include collagens and glycosaminoglycans (GAGs) weighing ~66% and 18% of dry mass, respectively.3
There are four zones of cartilage (superficial, middle, deep, and calcified) and they differ biochemically as
well structurally from each other (See Figure 1.1 and Table 1.1).4 For example, collagen content is
observed to be maximum in the superficial zone and decreases as we go deeper, whereas aggrecan
content reaches a maximum in the deep zone and calcified zone has mineralized cartilage tissue.(Table
1.1) It is obvious that a multiphasic scaffold that can guide the chondrocytes to elaborate a extracellular
matrix mimicking the native cartilage zones is necessary.
Figure 1.1 A typical cross section of articular cartilage showing four zones4
3
Table 1.1 Characteristics of human articular cartilage using zonal representation4
The regeneration of damaged or diseased sites of articular cartilage either through medical
treatment or implantation of engineered tissue constructs seeded with chondrocytes has been particularly
challenging due to the avascular nature and poor self-repairing ability of the cartilage, stringent
requirements of biomechanical properties (compressive, tensile, and shear resistances) and associated
problems of integration with subchondral bone.5, 6
Because of this poor self-repair capacity of articular cartilage, any damage due to injury or
disease can lead to an inflammatory arthritic condition which results in chronic pain and dysfunction in the
joints of afflicted patients. One treatment method introduced to relieve the pain and swelling is the
implantation of autologous chondrocytes around the lesions.7 However, this method is temporary and has
several drawbacks including lack of full load-bearing capacity, generation of inferior quality fibrocartilage
compared to articular cartilage, and the need for repeated surgeries have been reported.8, 9
Zones Structure Composition Functions
Superficial Zone
Chondrocytes are flat and parallel to the surface
Highly polarized and self-assembled parallel collagen network
Smallest collagen fibrils diameter (20 nm)
Collagen: Type II
Proteoglycan: decorin (leucine-rich), biglycan
Lowest aggrecans
Surface zone of the cartilage in contact with synovial fluid
Secretes lubricin: essential for lubrication
Highest tensile properties
Middle zone
Cell density lower
Rounded cells
Collagen fibrils larger in diameter and arranged randomly
Proteoglycan: aggrecans in medium concentration
Extensive ECM
Compressive modulus higher than superficial zone
Deep zone Cell density lowest
Collagen fibrils largest in diameter (120 nm)
Proteoglycan: Maximum aggrecans concentration
Collagen content lowest
Highest compressive modulus
Calcified zone
Hypertrophic phenotype Collagen: Type X
Intermediate mechanical properties between uncalicified deep zone and subchondral bone
4
Several novel ideas and growth factor schemes are being researched and tested to address the
shortcoming of this autologous chondrocyte implantation method.6, 10
Namely, 1) the seeding of
chondrocytes into a resorbable scaffold and culture ex vivo so as to partially elaborate the tissue before
implanting the constructs in vivo, 2) use of autologous stem cells or allogenic sources of chondrocytes 3)
growth factors and their schemes of addition 4) bio-functional scaffolds with cell-adhesive and chondro-
inductive properties 4) mechano-induction of chondrocytes through external dynamic load schemes 5)
bioreactors design 6) in situ delivery of nutrients and growth factors.
Several hundred biomaterials, both natural and synthetic varieties, have been tested as scaffolds
for seeding cells. Of these materials, the class of materials that form hydrogel matrix possess distinct
advantages due to their highly interconnected porous structure (better suited for sustained transport of
nutrients and growth factors) and large volume fraction of water (>98) which provides space for
accumulation of the extra-cellular matrix elaborated by chondrocytes.
Of the natural materials, agarose, a polysaccharide that forms hydrogel is a very popular scaffold
material, as it is found to maintain the phenotype of chondrocytes as well as to possess beneficial
mechanical properties.11-14
The advantage of using agarose scaffolds has also been shown by the
engineering of the cartilage tissue that has the Young’s modulus similar to that of native tissues11, 15
and
has already been developed into a practical technology named Cartipatch, autologous chondrocyte
implantation therapy.16
Some of the challenges in the regeneration of articular cartilage include,
1) Mimicking native cartilage tissues in structure and functionality. In particular, Type II
collagen content and the dynamic modulus of regenerated tissue is lower than the
native tissue values
2) Overcoming the detrimental spatial gradient in the properties of engineered tissue due
to poor transport & diffusion of nutrients and growth factors
3) Minimizing the inflammatory response under both in vivo and in vitro conditions
4) Achieving smooth integration of regenerated cartilage with original cartilage as well as
subchondral bone
5
5) Minimizing cell and extracellular matrix leakages during culture period
6) Cuing in the chondrogenic differentiation of stem cells
7) Effect of the composition and functional groups of the scaffold on tissue generation is
only beginning to be understood
(b). Rationale for using polymeric microparticles for in situ delivery of bioactive molecules
One of the critical challenges listed above is to overcome the decrease in the nutrients diffusivity
over a period of time in a homogenous scaffold. Diffusivity decreases due to the barriers created by the
progressive matrix elaboration. The resulting scaffold has spatially-inhomogeneous mechanical
properties.17
In particular, the internal regions of the scaffold exhibit poorer mechanical properties. The
creation of macrochannels has been suggested as a solution to this diffusion limitation.17
An alternative
solution is to embed the nutrient-encapsulated polymeric micro- or nano-particles which can release the
nutrients in a desired timeframe through controlled degradation rates (Scheme 1.1).
Scheme 1.1 In situ delivery of bioactive molecules using encapsulated polymeric microparticles
embedded in a hydrogel matrix. The surface eroding polymeric microparticles allows release the bioactive
molecules in a uniform and sustained manner.
Encapsulated polymeric nano- and micro-spheres have been extensively researched for
controlled release drugs and bio-active molecules. These polymeric carriers have several advantages
including the ability to vary their molecular weight and thereby control the release rate, degradation rate,
6
and hydrophilicity. Additionally it is known that supplementation of growth factors such as TGF-β3, has to
be transient (only for initial 2 weeks) at least for in vitro culture of juvenile bovine chondrocytes.18
This
necessitates that the delivery device and the selected polymer should have such erosion and degradation
rate such that release of most of the bioactive molecules should happen within this time frame.
(c). Comparison of biomedical properties of natural and synthetic polymeric biomaterials
The biocompatible and functionalized polymeric materials are being investigated for several
applications as drug, genes or vaccine delivery agents, scaffolds as well as guides for tissue growth in
regenerative medicine, as temporary prosthesis to name few. 19
20
In particular, use of polymeric
biomaterials (both natural and synthetic varieties) in the regeneration of damaged or diseased tissue is
fast emerging as an attractive alternative over artificial prosthetic implants due to the long-term
biocompatibility and prospects of permanent cure without repeated surgeries. However, there are several
challenging requirements for a biomaterial before it can be applied in vivo,21
1) Biocompatibility: Not only the initial material but also its degradation products should be
biocompatible. The long-term biocompatibility of the polymer and degradation products needs to
be confirmed before applying in vivo. The biocompatibility is determined not only by its cellular
toxicity but also by the elicited inflammatory response, genotoxicity, modification of cellular
morphology and biochemical processes, as well as impact on the secretion of extracellular
matrices.
2) Biodegradability: Unlike artificial prosthetic implants, the biodegradable polymers offer the scope
for restoring the regenerated tissue to its native state through the degradation and resorption or
elimination of the polymers in the body. However, their rate of degradation should be matched
with that of growing tissue such that overall biofunctionality (eg. mechanical properties) of the
implant and the tissue is not lost during the course of healing. Moreover, both the initial and
degraded components of the biopolymers should be either metabolized or eliminated quickly in
the body.
7
3) The other properties of polymers that are important include, porosity, gellability, crystallinity, glass
transition temperature, hydrophilicity/hydrophobicity, bulk or surface erosion properties, pH –
response, surfactancy, simpler synthesis, purification and sterilization processes, ease of
functioanlization and shelf life. The choice of these properties that are important is dictated by the
particular needs of a given biomedical application.
The biocompatible and biodegradable polymers can be classified based on the source as natural or
synthetic polymers. Some examples of polymers derived from natural materials include collagen, gelatin,
agarose, alginate polysaccharides and derivatives thereof and examples of synthetic polymers include
anhydrides, (ortho-)esters, urethanes, amides, carbonates and co-polymers in various combinations. The
(dis-) advantages of both natural and synthetic polymers in biomedical applications are shown in Table
1.2.
Table 1.2 A comparison of properties of natural and synthetic polymers relevant to biomedical
applications
Property Natural polymers Synthetic polymers
Biocompatiblity Typically good Needs extensive testing
Biomimetic activity and presence of receptor ligands
Good Typically not biomimetic and requires conjugation of biological motifs for targeted bio-functionality
Degradation Proteolytic degradation and typically elicits benign cellular response
Hydrolytic and requires careful studies to understand cellular response
Immunogenicity & disease transmission
High risk and difficult to control Low risk
Batch-batch variations High low
Functionalization with biological motifs
Difficult Relatively easy
Tunable properties Molecular weight Bad Good Hydrophobicity Bad Good Gellability Good Good Porosity Good Bad Degradation rate Bad Good Ease of processing Bad Good
8
(d). Sebacic acid based polyanhydride polymer for delivery device in articular cartilage
engineering: Advantages and challenges
The important criteria in selecting the encapsulating polymer for tissue engineering applications
are the faster degradation rate and surface erosion characteristics. Specifically, in articular cartilage
engineering, the in situ release of bioactive molecules from the microparticles is preferred to be within 2-3
weeks for maximum benefit. Thus polymer degradation rate should be necessarily in the faster range.
The synthetic polymers were selected for this study because of the ability to tailor the degradation rate
and further scope for surface modification with biological motifs. A survey of literature of different classes
of synthetic polymer indicates the following typical decreasing order of hydrolytic degradable bonds
anhydrides> orthoesters > esters > amides. The polyanhydrides are a class of biodegradable and
biocompatible polymers that used for a uniform, sustained and short-term delivery of bioactive agents.
They typically have a hydrophobic backbone chains linked to each other by easily cleavable anhydride
bonds. The polyanhydrides were first synthesized in the beginning of 20th century and at present, several
dozens of new polyanhydrides have been synthesized and researched for various biomedical
applications. Apart from the claim that they are non-mutagenic and non-cytotoxic, they have several
advantages, including surface erosion properties and tunable degradation rates suitable for short-term
release. The high hydrophobicity of the polyanhydride-based microparticles excludes the contact
bioactive encapsulation from water or water-based oxidants thus protecting their bioactivity until release
through surface erosion. Thus, polyanhydrides have found applications in delivery of vaccines, adjuvants,
and proteins where the immunogenicity of these actives is preserved. In particular, polyanhydride-based
wafers have already obtained FDA clearance for delivery of drugs in the treatment of brain tumor.22
One of the most common component of anhydride (co-) polymer used for these purposes sebacic acid
with either acetyl or methacryl end groups. Several parameters control the degradation rate of
polyanhydrides, namely, molecular weight, hydrophobicity, pH, crystallinity, porosity, and surface area of
the polymeric device whereas, the thickness of the polymer layer determines the total time for
degradation.23-29
In particular, hydrophobicity of polyanhydrides has significant influence on the
degradation rate.25
By of incorporation of aromatic group (poly(carboxyphenoxy propane-co-sebacic
9
anhydride), P(CPP-SA)) in the anhydride copolymer, degradation rate was observed to decrease by
several orders of magnitude when compared simple poly(sebacic anhydride) (P(SA or PSA).
Interestingly, the in vivo degradation of polyanhydrides is found to be slower than the in vitro rate
due to decrease in water content in the former.30, 31
The polyanhydrides and their degradation products
are considered to be safe, non-cytotoxic,32-39
non-mutagenic40, 41
and non-carcinogenic39
under both in
vitro and in vivo conditions. However, MRI imaging of P(SA) & P(CPP-SA) tablets implanted in the neck of
Wister rats, showed muscle edema at around Day 7 but was resolved eventually.30, 31
Unlike aromatic
polyanhydrides, aliphatic ones, such as P(SA) are shown to be extensively metabolized by rat brain cells
using radio-labeling technique. In particular, 80% of the SA was metabolized and eliminated as CO2.42, 43
It was further speculated that SA-based degradation product can participate in β-oxidation metabolic
pathway producing acetyl-CoA, which is an important intermediate molecule.44
Other cells such as aorta
epithelial osteoblast-like, and smooth-muscle cells also did not show cytotoxic effects due to
polyanhydrides.39, 45
However, in the case of SA dominated copolymer, lack of cell adhesion was
observed at latter times, probably due to faster degradation rate this component.
Scheme 1.2 Hydrolysis of acetyl-endcapped poylanhydride polymer into polyacid-anhydride oligmers and
acetic acid. The red ellipsoid is a lipophilic backbone chain with or without anhydride interlinks
H2
O
10
In contrast, other studies,46
show that maximum tolerable concentration of polyanhydride (P(CPP-
SA)) is as low as 2.8 mg/mL, above which cytotoxic effects are observed. However, no mechanism for the
cytotoxic effects at higher concentration was elucidated. Moreover, there are no reported mitigation
routes for the cytotoxicity of this polymer.
Generally, several pathways can be hypothesized for the inflammatory, cytotoxic, genotoxic or altered
biochemical response of cells due to the presence and uptake of biomaterials above certain limits. These
include variation in local pH and osmolality at biomaterial-chondrocyte interfaces, cytoplasmic membrane
disruption, cellular uptake of the biomaterials and corresponding changes in the intracellular pH, Ca2+
levels, lipotoxicity, and induction of reactive oxygen or nitrogen species.47-49
It is also known that a
decrease or increase in pH from physiological value of 7.2 significantly decreases matrix synthesis. The
hydrolytic degradation of polyanhydrides in to fatty acids and corresponding uptake by cells can decrease
the intracellular pH. It has been noted that an increase in extracellular pH and corresponding increase in
the intracellular pH can cause increase Ca2+
, which in turn can initiate the signaling cascades for cellular
apoptosis. Similarly, it has been shown that changes in osmolality away from native values of 350-400
mOsm/kg can significantly alter the morphology of chondrocytes and hence their metabolism. The
potential cytotoxicity due to polyanhydride can also be caused by their intracellular lipotoxicity due to their
possible cellular uptake.49
In summary, the polymeric microparticles can be used for in situ delivery of growth factors and
nutrients to overcome the transport limitations of elaborated matrix. There are no studies that address the
decrease in nutrient transport using short-term in situ delivery in articular cartilage engineering and show
an improvement the tissue properties.
The polysebacic anhydride with surface eroding property and highly tunable degradation rate as
well as hydrophobicity was selected for this work to encapsulate bioactive materials such as TGF-β3 and
vitamin-C. The poly(sebacic anhydride), undergoes hydrolysis to form poly(sebacic acid) and both the
polymer and its degradation products are reported to be biocompatible. There are few investigations
using of copolymeric anhydrides containing smaller quantities of sebacic acid in tissue engineering, 50, 51
.
However, none of them have studied encapsulation and delivery of bioactives such as TGF-β3, vitamin C
11
or chondroitinase-ABC in cartilage regeneration within a time frame of delivery of 2-3 weeks. The
unaddressed challenge in delivering appropriate quantities of these bioactives in situ is the weight
fraction of polymeric microparticles needed for this purpose calls their established biocompatibility in to
question. A rough estimate for a sufficient in situ release of bioactive molecules such as TGF-β3 or
vitamin C for initial 2-3 weeks of culture indicates a 50-200 mg/mL of PSA-M in the casting melt.
However, there is no data in the literature that points towards biocompatibility of such high doses of PSA-
M with chondrocytes. As a consequence there are no studies exploring the potential mechanisms of
cytotoxicity of polyanhydrides. Furthermore, mitigation strategies for such toxicity are non-existent.
Without answering these questions, the utility of polyanhydrides (in particular polysebacic anhydride) as
delivery device in articular cartilage tissue engineering will be very limited.
1.1.2. Bioactivity of polyacrylate nanoparticles in articular cartilage tissue engineering:
Effects of functional groups
Many recent advances in the design of multi-functional biomaterials for tissue engineering are
geared towards the objective of manipulating the cell-bioscaffold interactions to control and direct the
tissue morphogenesis.52-58
In this section, functionalization of polymeric biomaterials with bioactive motifs,
their biomedical applications, and their relevance in articular cartilage engineering are reviewed.
Two important design features that can enable cell-scaffold interactions are, (i) functionalization
of scaffolds and (ii) engineering of nanoscale roughness and structures of the scaffolds. Polymeric
nanoparticles if fabricated with these design features have high potential to be used as a nanocomposite
material in bio-scaffolds. There are several examples in literature where functionalization of the substrate
has been shown to have markedly influenced the extracellular expression of cells as well as the
phenotypic expression of stem cells. For example, types I and X collagens, as well as aggrecan, are
found to be constitutively expressed by human MSCs on polystyrene dishes,59
whereas type X collagen
expression is inhibited by the presence of Nylon-6 and polypropylene.59
Polyethylene glycol (PEG)
12
tethered with decorin results in 10 times more Collagen type II and 2.5 times more glycosaminoglycan
(GAG) content. An increased collagen type II and GAG content is desired for better mechanical
properties. The KLER sequence, a binding site from decorin protein, is known to bind strongly to collagen
type II and is responsible for matrix organization, while RGD promotes the general survival of cells.60
A
poly(lactic-co-glycolic acid) (PLGA) scaffold, grafted with TGF-β and loaded with dexamethasone, has
showed enhanced type II collagen production.61
Crosslinked poly(ethylene) glycol-fumarate (PEGF)
copolymer has been functionalized with Gly-Arg-Gly-Asp (GRGD) peptide to enhance attachments of
cells.62
The interesting effects of functional groups of the substrate on the phenotypic expression of
human mesenchymal stem cells is observed in a study where, PEG functionalized with carboxylic,
phosphate and t-butyl groups yield cartilage-, bone-, and adipose-like tissues, respectively.63
This study
shows the importance of the functional groups in maintaining the phenotype of stem cells and also the
chemical constituents of their extracellular expression.
However, most studies have not addressed the effects of functional groups on their
biocompatibility with cells such as chondrocytes. The effects that arise due to the presence of the
functional groups, such as changes in the local pH and changes in the overall charge state of the
extracellular space and their impact on biocompatibility are not well studied. In particular, cartilage tissue
is already highly anionic due to the presence of sulfate-containing proteoglycans in extracellular matrix.
The additional presence of anionically functionalized polymer can alter not only the hydration state of the
scaffolds but also the local pH and thereby affecting their overall functions. The scaffold which is part of
the extracellular space during the tissue elaboration can also alter the cellular behavior by influencing the
inter-cellular signaling processes.64
In particular, 3D scaffold in conjunction with secreted matrix provides
not only a substrate for cellular attachment but can also potentially act as a repository for morphogenetic
protein storage and transport. It is known that extracellular matrix components such as proteoglycan and
collagen possess motifs that can bind to the morphogenetic proteins leading to their accumulation in the
interstitial space. This allows a crucial mechanism by which the cells communicate with each other on the
status of extracellular environment thereby eliciting appropriate responses. Only recently there have been
some attempts to engineer these functionalities onto the biomaterials that are used as scaffolds. 64
13
To summarize, functional groups present in scaffold are known to play an important role in
directing the cells into synthesizing matrix with different biochemical and biophysical characteristics. In
particular, the cell-scaffold interaction is considered crucial for tissue morphogenesis. To prevent articular
cartilage implant failure or rejection, it is important that the engineered tissue mimics the biochemical and
structural variations seen in the different zones of the native cartilage (Figure 1.1). There are several
studies directed towards this objective. However, most of these studies focus on the functionalized
scaffold-cell interaction, whereas, there are no studies which instead uses functionalized polymeric
nanoparticles embedded in an inert hydrogel scaffold to achieve similar results. This preserves the
advantages the inert scaffold (present in larger fraction) and functionalized polymeric nanoparticles
(present in smaller fraction) and allows a greater flexibility in the design of multiphasic scaffolds. Though
acrylate-based scaffolds have been well studied, the effects of various functional groups, such as amides,
phosphonates and sulfonates on the cellular viability as well as cellular metabolism have not been
explored.
The functional groups and the chemical composition of these scaffolds or nanoparticulate
systems themselves can cause (1) changes in the local pH (2) changes overall charge state of the
extracellular space (3) interfere in the intercellular signaling. The presence of additional anionically
functionalized polymer can create disequilibrium in the ionic state of extracellular matrix and thereby
affect the overall functions of chondrocytes. Thus, it is possible that presence of nanoparticles with high
anionic charge can potentially mimic biofunctions of sulfated proteoglycans and thus can either be
beneficial or harmful when present in the interstitial space. There are no studies which consider the effect
of surface charge of functionalized polymeric nanoparticles in clinically relevant hydrogel culture of
chondrocytes.
1.1.3 (Bio-)chemical activity of metal (hydr)oxide nanoparticles
Development of the nanomedical, bioanalytical and theranostic applications of metal oxide NPs
calls for a deeper understanding of the chemical, redox and dispersion properties of such NPs, as well as
their interactions with live cells. This knowledge is also required to assess the safety of the growing
amount of nanomaterials exposed to the human beings and environment, as well as to develop predictive
14
models of the fate and transport of the released nanomaterials in the environment. Finally, this knowledge
is needed to find out ways to mitigate the adverse effects induced by the NPs.
Cerium dioxide (CeO2) and ferric (hydr)oxide NPs are of special interest in this context. Ceria
nanoparticles (nanoceria) are an important technological nanomaterial which has found applications in
catalysis, sensing, and solid oxide fuel cells (SOFCs). In particular, nanoceria is a well-known component
of the modern three-way exhaust-gas catalyst, where it acts as an oxygen reservoir, releasing oxygen
under fuel-rich conditions, and absorbing oxygen under deficient conditions. In the nano-biomedicine,
CeO2 NPs has been demonstrated as a regenerating anti-inflammatory agent, scavenger of reactive-
oxygen & reactive nitrogen species and as anti-oxidants. This allows nanoceria to act as in situ redox
buffers of the oxidative stresses exerted on live cells by unfavorable conditions.
The interest in the adsorption and dispersion properties, as well as to the cytotoxicity of ferric
(hydr)oxide NPs stems from their application as pigments and MRI contrast agents, as well as from their
abundance in the environment, which significantly increases the frequency of the interaction of these NPs
with living organisms.
The following literature review presents the current paradigms of the cytotoxicity and biocompatibility of
metal oxide nanoparticles
Current paradigm of the cytotoxicity and biocompatibility of metal oxide nanoparticles
It is known that the presence of (nano)particles can exert oxidative stress on live cells. A way to
mitigate this stress is by intervention with antioxidants such as vitamins C and E or -carotene. These
substances, when present in low concentrations compared with those of an oxidizable substrate, can
significantly delay or prevent the substrate oxidation. The term ‘oxidizable substrate’ includes every type
of molecule found in vivo.65, 66
Reactive oxygen species (ROS) is a common name for oxygen-based radicals and
hydrogen peroxide (H2O2). Main ROS are superoxide anion (O2), hydroxyl radical (OH),
singlet oxygen (1O2), and hydroperoxyl HO2. Hydrogen peroxide, even though not a free radical,
is classified as a ROS because it acts as a conduit to transmit free radicals induced damage
15
across cell compartments and between cells.67 All the ROS are formed by partial reduction of
dioxygen O2. Due to the unpaired electrons, ROS are strong and nonspecific oxidants. Human
cells produce ROS as byproducts of their aerobic metabolic activity, in the course of normal
growth factor signaling, and specific enzymatic reactions.67, 68 Specifically, O2 is the final
electron acceptor for cytochrome-c oxidase, the terminal enzymatic component of a
mitochondrial enzymatic complex that catalyzes the four-electron reduction of O2 to H2O.
Superoxide anion and hydrogen peroxide are formed by one- and two-electron reductions of O2
during these (and other) electron transfer reactions, respectively. Although hydroxyl radical
formation can occur in several ways, by far the most important mechanism in vivo is likely to be
the transition metal catalyzed decomposition of superoxide and hydrogen peroxide.69
In the normal physiological state, cells protect themselves from the elevated concentrations of
ROS using antioxidant enzymes such as superoxide dismutase (which reduces O2
to H2O2), catalase,
and glutathione peroxidase (which reduces H2O2 to H2O) (Figure 1.2). However, due to endogenous or
environmental factors, the ROS–antioxidant balance can be broken in favor of the former, imparting
oxidative stress on the cells and eventually leading to the cell damage. In fact, ROS can react rapidly with
most biological molecules and damage lipids, protein, and DNA (Figure 1.3).68
In general, nanoparticles can exert oxidative stress on live cells by several mechanisms, including
catalytic redox activity, solubility, cellular uptake, damage to cell wall, hydrophilicity, etc. 2 In the case of
metal oxide nanoparticles, the main reasons have recently been identified as the catalytic redox activity
and solubility leading to the damage of biomolecules, disruption of the signaling pathways, and leaching
out of toxic cations.2, 70
In particular, more catalytic active Mn2O3, CoO, Cr2O3 are more cytotoxic than
less catalytically active TiO2 and Fe2O3. At the same time, the catalytic properties of some metal oxide
NPs such as CeO2 and Y2O3 make them biocompatible and even effective in scavenging oxygen free
radicals.71-73
Though there is an emerging speculative paradigm70
addressing the causes for such varied
biocompatibility among catalytically active metal oxide, it is still not well understood.
16
1.1.4 (Bio-)activity of engineered ceria oxide nanoparticles: Anti-inflammatory and reactive-
oxygen species scavenging properties
The goal of this study is to explore the potential of nanoceria as an anti-inflammatory agent for
treating damaged or diseased articular cartilage. Accordingly, this section is organized as follows: (a) the
structural, electronic and abiotic redox properties of CeO2 nanoparticles surveyed. (b) the rationale for
selection of nanoceria to combat inflammation in articular cartilage
(a). Structural, electronic, and abiotic redox properties of CeO2 nanoparticles
Cerium is the most abundant of the lanthanides, and forms compounds with two oxidation states,
+3 and +4, and hence is redox active. Cerium dioxide, CeO2, crystalizes in a lattice with the fluorite-like
(CaF2) structure (space group Fm3m). This structure (Figure 1.2) can be viewed as a face-centered cubic
(fcc) array of Ce (white) ions with O (red) ions residing in the tetrahedral holes.
Figure 1.2 The fcc cell of CeO2 with the fluorite structure. Ce is white, O is red.
In the reduced form, cerium oxide lattice has oxygen vacancies or defects which are
associated with the loss of oxygen in the reaction:
CeO2 CeO2-δ + δ/2O2. (1)
17
The deviation from stoichiometry can be as large as 0.5.74
Each released O atom leaves behind an
oxygen vacancy and creates two Ce3+
(Ce 4f1) cations by transferring electrons to two Ce
4+ cations (Ce
4f0). This property determines the oxygen storage capacity of ceria. The nonstoichiometric oxide CeO2-δ is
readily reoxidised back to CeO2 absorbing oxygen. In nanoceria, the reducibility, oxygen storage capacity,
and high ionic conductivity are remarkably enhanced. The redox properties of ceria arise from the
reversibility of the Ce3+
/Ce4+
redox couple with consequent formation/annihilation of surface defects
(oxygen vacancies).
An important property of ceria is its ionic conductivity: The defective fluorite structure allowa
oxygen conductivity of ceria through the hopping of oxygen ions to the vacant sites. The pure CeO2 is
paramagnetic, but acquires ferromagnetism upon partial reduction of Ce ions from 4+ to 3+ state. The
effect is likely to have close relationship with oxygen vacancies.75, 76
The nature of bonding in CeO2 and
Ce2O3 may be described as polarized ionic.77
The origin of the nanosize effect on the chemical and physical properties of ceria is under debate.
The effect can be due to the size-induced partial removal of oxygen atoms in the surface layer which is
paralleled by an increase of Ce3+
defects (bigger radius compared to Ce4+
) and leads to the lattice
expansion.78
Given that annealing at high temperature suppresses significantly the oxygen storage
property and catalytic activity of ceria, it is suggested that the interstitial oxygen ions associated with Ce3+
are the "active" species that provide necessary oxygen mobility crucial for the functioning of ceria as a
catalyst.74
In contrast, it has been shown79
that under ambient conditions, the oxidation state of ceria NPs
is always close to 4+, and there is no stabilization of Ce3+
in smaller ceria nanoparticles but rather
stronger structural disorder in nanoceria compared to bulk CeO2.79
As stated above, the redox catalytic activity of nanoceria that arises from the presence of both
Ce4+
and Ce3+
state. This redox activity forms the basis for their superoxide dismutase-like (SOD) and
catalase-like enzyme-mimetic scavenging ability (Figure 1.3). 72, 80
It has been proposed that SOD- and
catalase-mimetic activities arise due to oxidation of Ce3+
and reduction of Ce4+
states by radicals
18
respectively.72
Though the mechanism of auto-regeneration of nanoceria oxidation state is not clear, a
simple suspension in water81
or a PEG-coating82
were observed to restore the Ce3+
oxidation state.
Figure 1.3 A model of the reaction mechanism for the oxidation of hydrogen peroxide by nanoceria and
the regeneration via reduction by superoxide. An oxygen vacancy site on the nanoceria surface (1)
presents a 2Ce4+
binding site for H2O2 (2), after the release of protons and two-electron transfer to the two
cerium ions (3) oxygen is released from the now fully reduced oxygen vacancy site (4). Subsequently
superoxide can bind to this site (5) and after the transfer of a single electron from one Ce3+
, and uptake of
two protons from the solution, H2O2 is formed (6) and can be released. After repeating this reaction with a
second superoxide molecule (7) the oxygen vacancy site returns to the initial 2Ce4+
state (1). It is also
possible that the third Ce3+
indicated, which gives rise to the oxygen vacancy, could participate directly in
the reaction mechanism. The square Ce–O matrix is shown here only to illustrate the model and does not
correspond to the actual spatial arrangement of the atoms in the crystal structure. Reproduced from
Ref.72
(b). Rationale for selection of nanoceria to combat inflammation in articular cartilage
Some of the major problems encountered in cell-based tissue engineering repair strategies for
injured articular cartilage are due to the adverse inflammatory response to the grafts leading to its
19
rejection or reduced functionality and poor integration with host tissues.10, 83-85
Several strategies have
been tested to overcome these problems. For instance, it was shown that the constructs grown in vitro to
a partially or fully matured state and then implanted in vivo can withstand harsh catabolic environment of
the diseased region better than a freshly seeded or under-developed constructs.86, 87
Other strategies
include application of molecules combating inflammation such as dexamethasone and exogenous
crosslinkers.87-89
Alternatively, an in situ approach for combating inflammation that is clinically attractive through
incorporation of auto-regenerative biocompatible substances in to the implants capable of scavenging
reactive species. Such an in vivo approach has the benefits of speedy recovery and reduced in-patient
time since it allows in vivo maturation of grafts.
The cerium oxide nanoparticles has the potential for such an in vivo role since it has been shown
in vitro and in vivo to scavenge reactive oxygen and nitrogen species, combat inflammation, down-
regulate cytokine levels and confer cell-protection. The inhibition of nitrated proteins is particularly
significant since they are found to be one of the important causes for the rejection of grafts.90, 91
The
nanoceria has already been shown to protect several mammalian cell types including neural,92
retinal,93
hepatic,94
epithelial,73
cardiac,92
breast,95
and epidermal cells.96
Interestingly, viability and invasive
capacity of tumor cells are shown to decrease in the presence of ceria nanoparticles at concentrations
that are non-toxic to normal cells.97-100
It is suggested that due to the Warburg effect, tumor cells have
acidic pH which results in inhibition of catalase-like activity of nanoceria and accumulation of H2O2. This
accumulation selectively kills the tumor cells but not normal cells because at physiological pH both SOD-
and catalase-like activities of nanoceria are preserved. The effects of nanoceria in tissue engineering
have been reported recently101
where it was shown that nanoceria had improved both the osteoblastic
differentiation of h-MSC and collagen production.
In summary, the presence of nanoceria during the in vitro culture of chondrocytes can not only
enhance the collagen production but can also protect the elaborated extracellular matrix from degradation
20
due to exogenously added inflammatory chemical, interleukin-1α (IL-1α). However, no study has looked
at exploring the potential of nanoceria in articular cartilage engineering or in treatment of arthritic-like
conditions.
1.1.5 (Bio-)activity of ferric (hydr)oxide nanoparticles: adsorption, dispersion and
biocompatible properties
The goal of this study is to understand the size, morphology and surface dependence of
adsorption and dispersion properties of ferric (hydr)oxide NPs as well as their biocompatibility.
Accordingly, this section discusses the following, (a) the structural, electronic and abiotic redox properties
of hematite nanoparticles (b) the size, shape and morphology dependence of (bio-)activity of ferric
(hydr)oxide NPs
(a) Structural, electronic, and abiotic redox properties of hematite nanoparticles
Hematite (-Fe2O3) NPs have been used since ancient times as pigments and more recently
have found application as (photo)catalysts, sorbents, and sensors due to their special physicochemical
and electronic properties, low processing cost, high resistance to corrosion, and the environmental
compatibility.102-106
Hematite-like clusters are found in living organisms (as a core of ferritin) and, along
with other iron (hydr)oxides, are common in the environment (oceans, ground and surface waters, dust,
soils), where they control transport of elements and present a nutrient source for bacteria and
phytoplankton. 106
Hematite (-Fe2O3) adopts the corundum-type structure which has hexagonal symmetry with R ̅c
space group (Figure 1.4). This structure can be viewed as a hexagonal close-packed lattice of oxygen
atoms with two-thirds of the interstitial sites occupied by Fe atoms (Figure 1.4). The oxygen atoms occur
in layers along the z axis, three atoms per layer, within the hexagonal cell. Between each of these layers
there are two Fe atoms in a noncoplanar arrangement.
21
Figure 1.4 Crystal structure of α-Fe2O3. Red atoms are O; Blue atoms are Fe.
Hematite is a charge-transfer insulator (energy gap of ~2 eV), which exhibits interesting magnetic
properties. Below the Neel temperature TN = 953K, the stable phase is antiferromagnetic (space group
R3), with alternating (001) layers of spin-up and spin-down iron atoms in the hexagonal cell (Figure 1.4).
However, due to a slight canting of the spins, hematite is weakly ferromagnetic at room temperature.106
Hematite is the most stable ferric (hydr)oxide polymorph at bulk sizes and oxidizing conditions.
However, on the nanoscale, it becomes thermodynamically unstable relatively to several less stable
polymorphs,52,53
in common with corundum, rutile, zirconia, and other oxides. As the end-member of the
nanosize-induced phase transformation under hydrous conditions, goethite (-FeOOH) has been
suggested based on the XPS spectra.54
In contrast, vibrational spectroscopy data suggest that the end-
member is ferrihydrite (Fe10O14(OH)2+~H2O in the more ordered form107, 108
).52
The nanosize-induced structural changes of hematite are associated with changes in the
electronic properties such as an increase in ionicity and a decrease in electron affinity (opening of the
band gap).109
These electronic changes have been shown to be the driving force for the degradation of
the oxidative properties, as well as for the enhancement of the reductive dissolution of this mineral with
decreasing NP size.110, 111
The changes in the reactivity can be related to a decrease of the toxic effects
towards bacteria with decreasing NP size.112
Accordingly, 50-nm hematite NPs provoke an inflammatory
response in endothelial cells, 113
while smaller and less catalytically active114
30-nm NPs do not.115
22
The redox properties of hematite NPs in aqueous media are described by the electrochemical
mechanism.116
Figure 1.5 Conceptual scheme of electrochemical mechanism of redox reaction Ox2 + Red1 Ox1 +
Red2 in the dark.116
FL = Fermi level, VB = valence band, CB = conduction band, △s = gradient of
surface potential.
The term “electrochemical” means that the redox reaction consists of two spatially separated half-
reactionsanodic and cathodic.93-100
For the oxidation of a reductant (Red1) by O2
Red1 + 1/2O2 + 2H+ + 2e = Ox1 + H2O, (1)
117-124117-124the anodic reaction is the oxidation of Red1 by either ejected hole or by injection of electron
Red1 = Ox1 + 2e. (2)
The cathodic counterpart is the oxygen reduction reaction (ORR) by ejected electron
O2 + 4H+ + 4e
− 2H2O. (3)
However, at relatively low cathodic overpotentials, the “direct” four-electron reaction (3) has a
much higher kinetic barrier than a less thermodynamically favorable production of ROS which can be
schematized as
O2 + 2H+ + 2e
− H2O2. (4)
Thus, hematite NPs can oxidize biomacromolecules such as proteins, DNA and carbohydrates
and reduce the dissolved O2 molecules to generate ROS which will further affect the cells.
redox reaction in the dark
Anodic
reaction
Cathodic
reaction
23
(b). Size, shape and morphology dependence of (bio-)activity of ferric (hydr)oxide nanoparticles
Both the biotic and abiotic activity of ferric (hydr)oxide NPs is a complex function of their, size, shape,
crystallinity, solubility, porosity, hydrophilicity, catalytic activity, surface charge and aggregation state. In
the biotic case, the phenomenon becomes even more complicated by the profound role of the dynamics
of nano-bio interface including protein corona formation, favorable pathways for cellular uptake,
interference in cellular biochemical processes to name a few.2 Surfactants
125 and proteins
2, 126-129 are
known as modifiers of the (bio)chemical reactivity, stability, and availability of ferric (hydr)oxide NPs.
Specifically, these (bio)molecules can change the wettability, surface charge, and aggregation state of
NPs, as well as can dissolve-re-precipitate them.
Sorption and adsorption of fatty acids and size, shape and surface charge-dependent dispersion
phenomena
It is well recognized that the adsorption of organic (bio)molecules provides a way to manipulate
the nanotechnologically critical interfacial properties of nanoparticles (NPs) such as wettability, surface
selectivity towards specific moieties, surface dipole, surface charge, magnetization, and population of
surface states.130-132
Specifically, wettability determines the stability and foamability of nanoparticulate
dispersions, which need to be controlled in the engineering of nanocomposite materials (e.g., hybrid
materials, polymers, composite electrochemical coatings, ceramics, and paints), biomedical applications
of NPs, and processing of ultrafine minerals. Adjustment of surface dipole and charge, passivation of
surface states, and physical protection of the NPs (e.g., by making their surfaces hydrophobic or
impermeable for gases) are necessary design elements in the NP-based (opto-)electronics,133
while
magnetic properties are critical for the theragnostic applications.134
Although the NP uptake by a cell is a
rather complicated phenomenon,2, 135
it is affected by the surface charge and wettability of the NPs.136
In general, it is assumed that smaller or more porous nanoparticles are more reactive because of
a relatively higher fraction of surface and under-coordinated atoms as compared to the larger or less
porous counterparts, respectively.137-139
However, hematite seems to exhibit the opposite trend. Namely,
the cytotoxicity,112, 140
redox activity,116, 141
and bacterial activity142
decrease with decreasing NP size. This
24
size dependence is consistent with the size-driven variations of the electronic properties of the NPs.109
However, the effects of size can be masked by the effects of the morphology and synthesis history of the
NPs.143
There is also a general understanding that the adsorption properties of particles are determined
by their composition and stoichiometry as well as structural and electronic properties, which in turn can be
manipulated by nanoscaling the solid or varying its morphology (habit/texture, phase distribution, surface
porosity).1, 137, 143-146
At the same time, no study has systematically addressed and delineated the
contribution of NP size and morphology to the interaction of metal (hydr)oxide NPs with carboxylic groups
of organic molecules in aqueous solution. Distinguishing the effect of NP size from the effects of the NP
morphology and synthesis history is challenging problem. The main reason is the practical limitations to
independently vary the nanosize and the morphology of the NPs without employing capping agents
during the synthesis and annealing. Moreover, no data is available on the effect of electric polarization on
the adsorption properties of metal (hydr)oxide NPs in water, although this effect is established for metal
films interfaced with solid electrolytes (known as electrochemical promotion of catalysis)147
and for
semiconducting sulfides in water.120, 148, 149
Semiconducting (nano)particles can electrically be polarized by varying the redox potential of the
suspensions, through contact with other (semi)conducting particles with different work functions (the so-
called galvanic interactions)148, 150
or, as shown in our recent paper,151
upon aggregation or deposition on
different substrates. Hence, being implemented by one of these approaches, electric polarization
presents an important extrinsic control of the adsorption properties of NPs, which has escaped attention
of previous researchers.
Why ferric (hydr)oxides and fatty acids?
The choice of this model system is dictated by substantial interest in commercialization of green and cost-
effective materials and reagents. Ferric (hydr)oxide NPs are industrially used as sorbents,
(photo)catalysts, abrasives, polishing agents, pigments, and components of optoelectronic and
optomagnetic metamaterials,104-106
and are tested in alternative energy152, 153
(“using rust to create
fuels!”154
), sensorics,102, 103
magnetic resonance imaging (MRI)155
and magnetic hyperthermia treatment,
25
as well as in drug delivery.104, 156
These NPs are a natural component of the environment and the living
organisms.106
Long, straight chain monocarboxylic, or fatty acids and their salts (soaps) have long been used
for laundry and personal care since the ancient Egyptian period. They represent by far the highest volume
of all surfactants.157
These reagents are green in the sense that: They have low toxicity and high
biodegradability and can be obtained from renewable resources by the hydrolysis of the plant or animal
fat.158
In modern technology, fatty acids and their derivatives are used to modify surface and dispersion
properties of iron (hydr)oxides in the personal care, biomedical, micro- and opto-electronics, abrasive,
paint and plastics formulations as well as in the mineral processing, corrosion protection, lubrication, and
tar-sands extraction operations. The interaction of ferric (hydr)oxides with fatty acids is also related to
many processes in nature. In particular, being a component of the natural organic matter (NOM), aliphatic
carboxylic acids regulate the fate and transport of the soil components, contaminants, and different types
of bacteria and pathogens in the environment.159, 160
Sodium laurate (dodecanoate), CH3(CH2)10COONa, which is used in this study is classified as a
weak electrolyte.161
Its state in water is controlled by hydrolysis to lauric acid (pKa = 5.0), precipitation (3
mM at pH 7.1), and micellization.161, 162
Critical micellar concentration (CMC) for sodium laurate (SL)
depends on pH, being 1 and 20 mM at pH 7.1 and 10.0, respectively.161
A range from 25 to 42C has
been reported for the Krafft point.161
According to Refs.,163-165
fatty acids can form acid-soap dimers or
low-molecular weight sub-micellar aggregates, although others have argued against their existence.166-168
The formation of self-assembled monolayers (SAM) and bilayers of fatty acids on ferric
(hydr)oxide NPs directly relates to the NP applications in the areas listed above, as well as to
the corrosion protection of iron and steel surfaces given the nanoparticulate structure of rust.
One of the main difficulties in distinguishing between the size and morphology effects of hematite
NPs on their interaction with fatty acids is the controversy in the current knowledge on the adsorption
forms of fatty acids. In earlier macroscopic studies, it is suggested that the chemisorption and
physisorption dominate at pH > PZC and pH < PZC, respectively (based on argument that electrostatic
26
repulsion between the anionic surfactant and negatively-charged surfaces is the determining factor).169, 170
This assumption has been recently questioned using spectroscopic results171-173
and is shown that the
electrostatic repulsion can be compensated by the favorable formation of surfactant hemimicelles and
structured interfacial water (influenced by surfactants).
As discussed in Section 1.1.6 below, to resolve the structures of the adsorbed fatty acids,
spectroscopic data should be complemented by the theoretical modeling. However, to the best
knowledge of the author, density functional theory (DFT) modeling has not been performed for this
system. Also, the effects of NP size and morphology on the interaction of metal oxide NPs in general and
ferric (hydr)oxide in particular have not been addressed, despite their practical importance.
Taking the previous results of the size-dependence of electronic properties of ferric (hydr)oxide
NPs in to account,109
it is hypothesized that the adsorption capacity/affinity of fatty acids should decrease
with decreasing NP size, although effects due to morphology is unknown and may affect this trend. As a
consequence, the stability of the NP suspensions in solutions of fatty acids should also vary. A knowledge
of the size-dependent variation in the dispersion properties of NPs is critical for many applications
including electrophoretic deposition, dewatering of mineral tailings,174
polymer composites, water
purification, bioavailability, and the mitigation of the toxicity of NPs.
Ferric (hydr)oxide nanoparticle-Cell interaction
The interest to the cytotoxicity of ferric (hydr)oxide NPs stems from a high probability of their
contact with living beings. Such NPs are found in oceans, ground and surface waters, dust, and soils.
They are added as pigments to paints, plastics, rubber, building materials, food and pharmaceutical
products, and are among the most widely used catalysts. 106
Superparamagnetic Fe2O3 NPs have been
used for biomedical applications for over two decades as MRI contrast agents for the detection of liver
tumors, and also have been commercialized for the diagnosis and therapy of other specific diseases. 156,
175 Ferric (hydr)oxide NPs are present in the most living organisms, where iron is a biologically important
element as a core of ferritin (an intracellular protein that serves as an iron depot in a body). Since the
released ferric iron can participate in the normal iron metabolism, ferric (hydr)oxide NPs were initially
considered to be non-cytotoxic. At the same time, as a transition metal, iron is able to catalyze the
27
production of hydroxyl radicals (by a Fenton or Haber–Weiss reaction) and thus induce oxidative stress,
which is considered as the principal injury mechanism in cells 70
.
So far, a series of studies have been performed mostly for superparamagnetic - Fe2O3
(maghemite) NPs to address the concerns about the safety of ferric (hydr)oxide NPs. The results, which
are summarized in several reviews,135, 175-177
demonstrate that such NPs can exert drastic effects on the
cell’s wellbeing, and their impact depends on many parameters, such as the exposure concentration and
time, NP size, fraction of Fe2+
(the redox state of iron), surface functionalization, cell line. 176
However,
the great variety in types of the NPs, cells and incubation protocols used have rendered it impossible to
make any conclusions about the safety of magnetic iron oxide NPs.176
Moreover, all the previous works
consider the NP-cell interaction from the nanotoxicological and nanomedical perspectives, being focused
on the assessment of the level/manifestation of inflammatory response, if any, as well as on the fate and
transport of the NPs in the culture media. The results are mostly interpreted in terms of the amount of
leachable iron. At the same time, to our knowledge, no attempt has been made to relate the cytological
effects to the emerging physico-chemical paradigm,70
according to which, along with the acute solubility,
the redox activity is the determining factor of the bioactivity of metal oxide NPs. Moreover, the nanosize
effect on the bioactivity of uncoated ferric (hydr)oxide NPs has not been addressed systematically using a
multidisciplinary approach, in which the cell-NP interaction is considered from the combined perspective
of colloid chemistry, catalysis, cytology, and nanotoxicology.
The current knowledge on - Fe2O3 is in a similar situation, except that the number of
nanotoxicology studies on these NPs is much lower112, 113, 115, 140, 178-181
as compared to maghemite.
Specifically, unclear is the dependence of the cytotoxicity of hematite NPs on nanosize: 50-nm hematite
is genotoxic for human bronchial fibroblasts and epithelial cells, and its adverse effects are more
pronounced as compared to microsized hematite at the same weight doses.178
The 50-nm hematite also
indices functional neurotoxicity of brain cells.180
In contrast, low toxicity was observed for 12-nm hematite
towards human bronchial epithelial cells and rat alveolarmacrophage cells,70
as well as for 30-nm
hematite towards human lung epithelial cells. 115
.
28
Given that the abiotic activity of hematite NPs decreases with decreasing NP size,110
it is
hypothesized, based on the emerging paradigm,70
that the cytotoxicity of hematite normalized to the
surface area of the NPs should also increase with increasing size of the NPs
1.1.6. Challenges in modeling the adsorption of fatty acids at the metal oxide nanoparticle-
water interface
Macroscopic/thermodynamic adsorption measurements can provide a wealth of information on the
interaction of the carboxylate groups with metal oxide NPs. Specifically, using macroscopic methods, the
adsorption of fatty acids on iron (hydr)oxides and corresponding hydrophobicity were related to the
surface tension minimum of the surfactant.163, 182-185
The formation of highly surface active premicellar
species such as acid-soap dimers were suggested as the reason for this relation. However, the
“premicellar” model is inapplicable for hematite and C6-C18 homologues of saturated fatty acids.169
Moreover, this model does not account for the chemisorption of the fatty acid adsorption,182
and neglects
the variation of the oxide surface chemistry with size of the particles.184
A better mechanistic understanding and hence modelling of the adsorption process requires
knowledge of the surface speciation of fatty acids on ferric (hydr)oxides which is currently debated. Based
on the adsorption measurements, the fatty acids are suggested to preferentially physisorb on hematite at
pH < IEP but chemisorb at pH > IEP (IEP-isoelectric point).169, 170
Moreover, the chemisorption was
inferred based on electrostatic repulsion arguments for pH > IEP. However, there are several recent
studies that question this model by suggesting a thermodynamically favourable formation of hemimicelles
and strong hydrophobic interactions can overcome the electrostatic repulsion.173,183, 186
On the other hand,
oleate adsorbed on iron oxides were interpreted as chemisorption based on the conventional spectral
correlations of the splitting between the asCO2– and sCO2
– modes = asCO2
– sCO2
– in the FTIR
spectra and the speciation of the adsorbed carboxylate.171, 172
If so, it is unclear why the hydrophobicity
maximum of oleate-coated hematite is coincident with the highest solution activity of the premicellar
associates of fatty acids.
29
At the same time, resolving the surface speciation of fatty acids at the metal-oxide water interface
is challenging problem, given the several possible adsorption modes (Scheme 1.3) and inefficiency of X-
ray adsorption spectroscopy to study such systems.
Scheme 1.3 Cluster models of different laurate adsorption complexes. ISBB-Inner sphere bidentate
bridging; ISMM – Innersphere monodentate mononuclear; OS – Outersphere; Blue octahedra – Fe3+
; red
spheres – oxygen; gray spheres – carbon; green spheres – hydrogen.
The only remaining practical microscopic alternative is FTIR spectroscopy. However, in common
with all vibrational spectroscopies, FTIR spectroscopy is not directly sensitive to the atomic positions, but
rather to the symmetry and bond strengths. Additionally, the traditional correlation of to the binding type
of the adsorbed carboxylate group187-189
can be misleading because these correlations are established
for bulk coordination compounds and species formed at the solid-gas interface. They neglect strong
effects of the polarization of the anion in the double layer, the surface charge of the NP, H-bonding with
coadsorbed water, and the coupling of the carbonate modes with modes of coadsorbed water, to mention
a few (See Ref.190
for more details).
Thus, there is a need to further develop the DFT method to resolve the bonding type (outer vs.
inner sphere) and configuration of carboxylate groups at the metal oxide-water interface based on their in
situ FTIR spectra.
b) ISMM a) ISBB c) Chelating d) OS surface hydration
-shared
e) OS surface
hydration-separated
30
1.2. Goals and objectives
Based on the many critical unresolved issues formulated in the literature review sections, the following
goals and objectives are determined, and the hypotheses are proposed
Goal:1. Tailoring of the bioactivity of (poly)sebacic anhydride-based microparticle-delivery
systems for articular cartilage tissue engineering
Objective: To overcome the problem of the decrease in the diffusion of nutrients and growth factors due
to progressive elaboration of extracellular matrix in 3D agarose scaffolds with time.
Hypotheses: The problem can be solved using nutrient-encapsulated microparticles based on
(poly)sebacic anhydride polymer which can release the bioactive molecules in situ in a desired timeframe
controlled by the polymer degradation rate. This also has the advantage of a uniform, localized and in situ
delivery of the nutrients or growth factors.
On this basis, the following working hypotheses are formulated,
a. Due to the short term release faster degradation rates required for this objective, there may be an
upper limit for the amount of polyanhydrides-based microparticles in vitro above which cytotoxic
issues may arise (due to the accumulation of its degradation products). This is particularly important
since most of the reported studies that attest to the biocompatibility of polyanhydrides either have
longer degradation profile or have been for delivering adjuvants or cancer therapy.
b. It is hypothesized that serum-albumin, which naturally regulates the intracellular lipid content, can
be applied to mitigate the potential lipotoxicity of polyanhydrides by supplying an abundant quantity
bovine serum albumin in the culture medium.
Goal 2. Understanding the effects of functional groups of polyacrylate nanoparticles on their
bioactivity in articular cartilage tissue engineering
31
Objective: Synthesize functionalized acrylate-based polymeric nanoparticles and study their effects on
biochemical composition and mechanical properties of cartilage
Hypothesis: The surface charge of the polymer alone may play an important role in the chondrocyte-
scaffold interaction.
To test this hypothesis, the effects of acrylate-based nanoparticles with different charge characteristics
when introduced into agarose 3D scaffolds seeded with chondrocytes is studied.
Goal 3. Anti-inflammatory and scavenging (bio-)activity of engineered ceria oxide nanoparticles
Objective: The bioactivity of cerium oxide NP will be explored for their potential benefit in scavenging
inflammatory and oxidative chemicals that are relevant to in articular cartilage regenerative treatments.
Hypothesis: It is hypothesized that presence of nanoceria during the in vitro culture of chondrocytes will
not only enhance the collagen production but also protect the elaborated extracellular matrix from
degradation due to exogenously added interleukin-1α (IL-1α). I have selected IL-1α isoform for our study
since it was already shown that under both in vivo and in vitro condition they not only inhibit the
maturation but also damage the tissues.87, 89, 191
Goal 4. Size, shape and morphology dependence of (bio-)activity of ferric (hydr)oxide
nanoparticles-Interactions with chondrocytes and surfactants
Objectives:
1) To answer the still open questions about whether there are any effects of size, morphology,
and surface charge of the ferric (hydr)oxide NPs in their interaction with cells and surfactants? and if so
why?
2) To rationally control the dispersion and other interfacial properties of ferric (hydr)oxide NPs
using fatty acids
32
3) To understand the effect of size, morphology and surface charge of the ferric (hydr)oxide NPs
on the cellular viability and metabolism of chondrocytes in 3D culture
Hypothesis: The (bio-)activity of ferric (hydr)oxide NPs increases with increasing particle size
and depends on the semiconducting properties of hematite NPs. Thus, it is further hypothesized that the
adsorption density of fatty acids decreases with decreasing NP size, although variations in morphology
can affect this trend. As a consequence, the stability of the NP in fatty acid containing suspensions can
also vary. Additionally, it is also surmised that increase of the particle size of ferric (hydr)oxide NPs might
have larger impact on chondrocytes
Goal 5: To develop a predictive methodology for identification of adsorption forms using density
functional theory (DFT) simulations and FTIR spectroscopy
Objective: The overall objective is to develop a comprehensive methodology to predict the
adsorption forms of fatty acids using density functional theory (DFT) simulations by comparing
theoretically calculated vibration spectra to the experimental spectroscopic data.
Hypothesis: The inclusion of effects of ionic strength, pH and co-adsorption on the DFT
calculated spectra
33
Chapter 2 Materials and Methods
2.1 Materials
Chemicals required for synthesis of polymeric and hematite nano- and micro-particles were
obtained commercially and are of analysis or higher grade. The aqueous solutions were always made
from triple distilled water or nanopure water having less than 18 MΩ/m. Polymeric nanoparticles made up
of the acrylate backbone and different functional groups are synthesized by inverse microemulsion
method. The size of the particles varied between 70-300 nm. Poly(sebacic anhydride) microparticle
particles are synthesized using double-emulsions and solvent-evaporation method. The size of the
microparticles particles ranged between 2-20 microns. Hematite nanoparticles of average sizes 7, 9, 30,
38, and 120-nm are synthesized by forced hydrolysis. Hematite particles of size 150-nm and ferrihydrite
particles of 4-nm size are obtained commercially.
2.2 Tissue culture studies
Agarose-based hydrogel are used in this study as scaffold for culturing chondrocytes. The advantages of
using agarose are outlined in Section 1.1.1. In the first set of studies, the fabricated polymeric nano- and
micro-particles are embedded within the agarose matrix during the casting of the hydrogel scaffolds.
Aqueous melt of agarose of 4% by weight was prepared at 40 0C and mixed with chondrocytes and
polymeric particle suspensions such that final concentration of agarose is 2%. The polymeric particles are
incorporated at varying weight percentages. In the second set of studies, inorganic ceria nanoparticles
and hematite nanoparticles are incorporated at different concentrations. Control sets in both of these
studies are the agarose scaffolds that have neither nano- nor micro-particles. Chondrocytes harvested
from cartilage of bovine calves are passaged up to 4 generations and are added to the agarose melt
diluted with growth media such that the final concentration of chondrocytes in the suspensions is ~30
million cells/ml. These chondrocyte loaded suspensions which are at 40 0C are cast into rectangular mold
and cooled to 37 0C and maintained at this temperature overnight. After which the scaffolds were
punched into cylindrical constructs of ~ 4mm diameter and ~ 2.4 mm length. These constructs were
cultured in Dulbecco's modified essential medium supplemented with amino acids (0.5x minimal essential
34
amino acids, 1x nonessential amino acids), buffering agents [10 mM HEPES, 10 mM sodium bicarbonate,
10 mM TES, and 10 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid], and antibiotics (100 U/ml
penicillin, 100 mg/ml streptomycin). The growth media was regularly replenished and 3-7 constructs from
each experimental and control groups were used for mechanical testing, biochemical and histological
analysis at specified time intervals during entire culture period. The mechanical properties studied include
equilibrium Young’s modulus and dynamic modulus in under confined compression, and biochemical
content studied include proteoglycan represented by glycasoaminoglycans (GAG), collagen and DNA
analyses.
Mechanical properties
A computer- controlled custom made mechanical tester capable of measuring equilibrium
Young’s modulus (EY) and dynamic moduli, G*, (at 0.1, 0.5 & 1 Hz) of constructs in unconfined
compression was used. The EY is defined as the stress generated by the construct under 10% strain and
no external load conditions. The 10% compressive strain is achieved by gradual application of external
load (strain rate of 0.05 %/s). Once the 10% strain is achieved the external load is removed and the
construct is allowed to equilibrate for 30 minutes and this equilibrium compressive stress is used for
calculating the Young’s modulus. The dynamic modulus is measured by imposing 2% sinusoidal strain at
specified frequencies on the above equilibrated constructs.
Cellular viability
The viability of chondrocytes were tested using live/dead cytotoxicity kit from Molecular Probes and
confocal laser scanning microscope from Olympus (Fluoview-100). The cell permeant calcein-AM
fluoresces green only in the cytoplasm of live cells, whereas the cell-impermeant ethidium bromide
fluoresces red when it complexes with the nucleus of compromised cells (dead cells).
Biochemical content
The constructs were dried by lyophilization for 48 hours and digested using Proteinase K for 16 hours at
56 oC.
192 The agarose is centrifuged and the supernatant collected for further analysis of DNA, GAG &
collagen. The concentration of GAG in digested samples is measured spectrophotometrically using 1,9-
35
dimethylmethylene blue dye-binding assay and by comparing with chondroitin-6-sulfate standards193, 194
The collagen present in the digested sample is calculated by measuring ortho-hydroxyproline (OHP)
content through a colorimetric procedure.195
The cellular viability is additionally inferred from the amount
of DNA present in the samples measured using PicoGreen assay. The biochemical contents (GAG,
collagen, and DNA) of constructs are normalized with either wet or dry weight of the constructs.
2.3 Characterization and physico-chemical experimental methods
The primary particle size and morphology are characterized using scanning (SEM) and
transmission (TEM) electron microscopies. The SEM was used for characterizing polymeric nano- micro-
particles used in this study. The SEM is best suited for polymeric particles because particles are larger in
size and can be coated with gold to enhance conductivity as well of contrast. The polymeric particles
were deposited on a carbon substrate and were used with or without a coating of Au-Pd layer. The SEM
images were obtained using a 4700-Hitachi SEM microscope with voltage set at 0.8 kV and current at 20
mA. The ceria and ferric (hydr)oxide NPs were imaged using a Jeol JEM 100CX Transmission Electron
Microscope (TEM) that is operated in the bright field mode at 100 keV. HRTEM images were obtained by
Evans Analytical Group (EAG) with a FEI Tecnai TF-20 FEG/TEM operated at 200kV in bright-field. The
use of TEM is justified since these nanoparticles are small and have poor conductivity. The sample was
prepared by dispersion of nanoparticles on to a lacey copper grid. The SAED aperture of the microscope
used for the TEM imaging was 150 nm, which did not allow selecting a single particle.
The X-ray diffraction (XRD) was performed to measure the overall crystallinity of the ferric
(hydr)oxide NPs. The XRD analysis was conducted using a Scintag Model X2 X-ray powder
diffractometer. A CuK( = 0.154 nm) radiation source operated at 45 kV and 35 mA is used. The scan step
size was 0.05 deg.
The impact of NPs size, morphology or surface charge on a given phenomenon can be plotted
based on either unit mass or unit surface area. The estimates of the surface area are made with a single-
point BET N2 adsorption isotherm (Monosorb, Quantachrome surface area analyzer). The samples are
36
degassed with N2 at 50 oC for 30 minutes before measurements are taken. Two or three replicate
measurements are performed, which gave results that agreed within less than 5%.
The measure of surface charge of NPs, zeta potential ξ, was characterized using electrokinetics
and salt titration method. These two methods provide information on the macroscopic basicity of the NPs
and characterize the point of zero charge and iso-electric point, respectively. Electrokinetic
measurements are carried out using a Zeta Sizer instrument, Nano-ZS from Malvern. Zeta potential (ξ)
measurements are performed within a pH range of 4–10 and at a NPs loading of 0.005–0.01 by vol%.
The concentration of the background electrolyte, NaNO3, is 0.01 M and the pH of each subsample is
adjusted using NaOH and/or HNO3. The pH-adjusted hematite NPs suspensions are allowed to
equilibrate for 24 h and pH is re-adjusted to initial values two hours before the actual measurement.
Since electrokinetic measurements performed did not exclude adsorption natural CO2, salt
titration technique was employed for determining true PZC of NPs.17, 18
Experiments are conducted in a
glove box continuously flushed with N2 gas at room temperature. Freshly decarbonated triple-distilled
water (TDW) is always used and the decarbonation is realized by boiling the TDW for several hours under
continuous N2 bubbling. The NPs suspensions in 0.0015M NaNO3 are decarbonated by N2 bubbling for a
day. For pH measurements, an Accumet combination double-junction Ag/AgCl reference pH electrode is
used under slow stirring condition of the suspensions. pH adjustments are carried out using 0.01M
solutions of either HNO3 (prepared in decarbonated TDW) or NaOH (prepared from a Dilut-It analytical
NaOH, Baker). The total surface area of NPs in the suspension is kept constant at 200 m2. These
suspensions are divided into 5 portions 40 ml each and pH is adjusted to selected values. After
stabilization of pH (0.5–1 h), a known quantity of NaNO3 is added to increase the ionic strength up to
0.1M of suspensions. Another 10-30 min is allowed for pH stabilization to a new pH value. The resulting
change in pH, ΔpH, is determined from the difference (inclusive of sign) between the final pH and the
initial pH. Point of zero charge is the pH at which pH = 0.
To delineate the adsorption modes, whether chemisorption or physisorption, x-ray photoelectron
spectroscopic (XPS) measurements were performed. The XPS spectra are collected with a Perkin-Elmer
PHI 5500 instrument using monochromatic AlK X-rays with pass energies of 17.6 eV at resolution of 0.9
37
eV at take-off angle of 45 at pressures of less than 1 10–8
Torr, calibrated using the Ag 3d peak. Scans of
X-ray induced Auger peaks are performed at 0.1 eV steps. All samples were prepared by spreading a thin
layer of an aqueous suspension of NP on a UHV metallic holder followed by air-drying.
The adsorption isotherms of laurate on ferric (hydr)oxide NPs were measured to understand the
size-dependent surface coverage and packing. Adsorption isotherms of sodium laurate were measured
using depletion method and total organic carbon analysis (TOC-5000A, Shimadzu). 0.15 g (0.2 g in the
duplicate experiments) of particles was suspended in 20 ml of aqueous solution with a 0.005 M NaCl ionic
strength and different concentration of dissolved sodium laurate surfactant at natural pH of 7.5-9.0.
Afterwards, pH of the suspensions was adjusted to 7 using HCl solution and the adsorption is allowed to
proceed for 2 hours under vigorous stirring condition. The suspensions were then centrifuged, the clear
supernatants are pipetted out for the total carbon analysis, while the fraction of the wet particles was used
for the contact angle measurements.
The molecular mechanisms of adsorption of laurate on ferric (hydr)oxide NPs was followed using
vibrational spectroscopy. The Fourier transform infrared spectroscopy-Attenuated total reflection (FTIR-
ATR) spectra were measured using a Perkin-Elmer Spectrum 100 FTIR spectrometer equipped with an
MCT detector. A horizontal ATR accessory consisting of a ZnSe internal reflection element (IRE) (10
internal reflections, angle of incidence 45°) and a cap on IRE to prevent evaporation of water was
employed. A particulate film of hematite NPs was deposited onto the ZnSe IRE. This film was air dried
and then rinsed several times with water to remove any detached particles. The plate with the NPs-
coated IRE was mounted on to the ATR accessory, then water at a preset pH was added to 3 ml IRE cell
and the system was equilibrated for at least 1 h with water being replaced manually every 10 minutes. In
the case of laurate, protein and sulfate adsorption experiments, the background water or electrolyte
solution was replaced with 3 ml of adsorbate solution and the sample spectrum was measured after a
selected equilibration time, both without and with refreshing the SL solution in the FTIR cell. The spectra
were collected for 300-400 scans at a resolution of 4 cm–1
and represented in the absorbance scale. As a
background, either the ATR spectrum of the clean ZnSe IRE covered by water of a selected pH or the
ZnSe IRE covered by the NP layer and the water was used. All the FTIR spectra were measured in
38
duplicate on the NPs synthesized in different batches and were reproducible in terms of the NP size-
induced regularities.
FTIR spectra of polymer samples were measured both as dry sample and as aqueous solution
using ATR setup. Background spectra of air and water were suitably subtracted.
Raman spectra were obtained using Horiba JobinYvon’s Aramis confocal Raman microscope.
Adsorbed protein on ferric (hydr)oxide nanoparticles and polymeric nanoparticles were measured using
785 nm and 532 cm lasers respectively.
2.4 Density Functional Theory (DFT) Methodology
DFT calculations are performed to develop a combined methodology with FTIR spectroscopy to
elucidate adsorption forms of laurate on a hematite surface. The exchange-correlation functional used is
Perdew–Burke–Ernzerhof (PBE) generalized gradient approximation (GGA)19
as implemented in DMol
code and available in Material Studio package (Accelrys Inc., San Diego, CA, USA). The numerical basis
set, namely, double numerical polarization (DNP), which is capable of accurate description of hydrogen
bonding, is employed. Full geometry optimization is performed without spatial or symmetry constraints.
Spin-polarized orbitals are utilized to account for the anti-ferromagnetic properties of hematite. A real
space cut off in the range of 3 – 4.3 Å is chosen for orbitals. Vibrational frequencies are estimated
through a two-point finite difference scheme of analytical forces. The computed frequencies did not
exhibit imaginary frequencies. They are not corrected for anharmonicity and are not scaled. The
calculations are performed both in the gas-phase approximation (at dielectric constant r of 1) and within
the continuum solvation model, COSMO,20, 21
at r of 12, 45, and 78 corresponding to the bulk hematite,
water-hematite interface22
and bulk water, respectively. The problem of modeling surfaces of metal oxide
NPs is generally complicated by a lack of knowledge of their surface structures, which are different from
the structures of their bulk counterparts. In particular, under oxidizing hydrous conditions, hematite NPs’
surface region undergoes partial transformation to the FH phase.23
However, the structure of FH is still
under debate24-26
which poses an essential obstacle for the interpretation and modeling of spectroscopic
and microscopic adsorption data on both hematite NPs and FH. Therefore, in the first approximation, to
elucidate the effect of co-adsorption as such, we model the hematite surface as a cluster consisting of
39
both corner-shared and edge shared multiple ferric octahedra extracted from the (012) hematite surface.
The surface charge of hematite, which depends on the pH of the system, is modeled using different
hydration states of the ferric octahedra.
2.5 Error Analysis of data
The standard error is calculated for all the reported data here using the below formula. The
standard error is an estimate of standard deviation of the mean based on sample population.
√
Where, σ is the standard deviation of the sample and n is the population size
The analysis of variance (ANOVA) was performed to test for the statistically significant difference
between different experimental sets. The ANOVA uses three or more classes of data for these sets. For
eg., To determine if any statistically significant difference exist between control and experimental
constructs, the data from four different mechanical tests - Young’s modulus and three dynamic moduli
(0.1, 0.5 and 1 Hz)) of the constructs were used. In particular, a two way ANOVA with replication was
performed and the statistical significant difference is reported as p-value. If p < 0.05, it implies that there
is a 95% chance that the compared data sets are different.
40
Chapter 3 Synthesis and characterization of
polymers and polymeric micro- and nano-
particles
In the first part of this chapter (Section 3.1-3.5), the synthesis and characterization results of
polymers and polymeric microparticles are presented. The details provided include, (1) synthesis of
polysebacic anhydride polymer using melt condensation method (2) functionalization of this polymer with
fluorescein label and vitamin C (3) characterization of polysebacic anhydride and its derivatives using
NMR and Raman spectroscopies (4) fabrication of polysebacic anhydride microparticles and
encapsulation of bioactive molecules using double emulsion method (5) characterization and release
kinetics of polysebacic anhydride microparticles using optical, electron, and confocal fluorescence
microscopies, as well as UV-Vis absorption spectroscopy.
The second part (Section 3.6) is devoted to the synthesis of acrylate-based functionalized nanoparticles
using inverse microemulsion and their characterization using light scattering, XPS, and SEM methods.
In the final part (Section 3.7), the pore size of agarose-hydrogel scaffold was modeled and determined as
function of agarose alone as well as in the presence polymeric microparticles.
3.1 Synthesis of polysebacic anhydride polymer using melt condensation method
Several methods have been reported for synthesizing polyanhydrides and polyanhydride-esters
including, 196, 197
a) Bulk melt condensation
(b) Ring opening polymerization
(c) Acyl chloride acid-base reaction
41
(d) Interfacial polymerization
Of these methods, melt condensation is the simplest and most common method for synthesis of both
aliphatic and aromatic polyanhydrides. Thus, the melt condensation method was applied for synthesis of
poly(sebacic anhydride) (PSA),with either acetyl and methacryl endcapped. Additionally, to functionalize
the polyanhydrides with L-ascorbic acid (Vitamin C), acyl chloride acid-base reaction was adapted.198, 199
Synthesis of polyanhydrides using melt condensation method involves two-steps (Scheme 3.1): (1)
de-hydration and acetylation of the diacid groups of monomer with acetic anhydride. (2) polymerization of
this precursor anhydride molecule in vacuum and at high temperature to increase both the molecular
weight and the hydrophobicity of polymer.
Scheme 3.1 Synthesis of polyanhydrides using melt condensation method
The poly(sebacic anhydride) (P(SA)) was synthesized using sebacic acid (SA) as a monomer.
(Scheme 3.1) The sebacic acid is a di-carboxylic acid connected by 8 methylene groups. A typical
synthesis procedure adopted is described below: 5 mg of recrystallized SA was mixed with 50 mL of
acetic anhydride and stirred for 20 minutes at 40 0C until a clear solution was obtained. The excess of
acetic anhydride was removed by vacuum evaporation at 70-120 0C. The temperature was further
(CH2)8 - Sebacic acid
(CH2)8
(CH2)8 (CH2)8 (CH2)8 (CH2)8
Acetic anhydride
42
increased and maintained at 150 0C under vacuum and constant stirring for 2 hours. The final product
was dissolved in a minimum amount of methylene chloride and recrystallized using a large excess of
ethyl ether and petroleum ether. The resulting suspension was centrifuged, dried at room temperature
under vacuum, and stored at 20 0C in tightly-capped vials. A typical yield of ~20% was observed after the
purification step and this lower value is due to the losses during the purification step.
3.2. Functionalization of polysebacic anhydride with fluorescein label and vitamin C
3.2.1 Functionalization with Fluorescein
The conjugation of PSA with a cell-permeant dye such as fluorescein will allow studies of cellular
monitoring and uptake of the polymer. For attaching fluorescein label to the PSA, methacrylic anhydride
was used instead of acetic anhydride during the first step in the melt condensation method. The
methacrylic endgroups in PSA was used to link the acrylate double bond in the fluorescein o-acrylate, a
fluorescein derivative. A radical initiated linking between methacrylic group of P(SA) and acrylic group of
fluorescein o-acrylate was carried out in an organic solvent (methylene chloride) at 40-50 0C. The
fluorescein o-acrylate of 1 wt% and two radical initiators 2, 2’ Azobis (2-methyl propionitrile) and 2, 2’
Azobis(2-(2-imidazolin-2-yl) propane] dihydrochloride were used. The reaction was carried out for ~30
minutes under stirring. The resulting polymer was purified by repeated dissolution in acetone and
recrystallization using hexane. The polymer was washed with water several times to remove any residual
unreacted fluorescein o-acrylate.
3.2.2 Functionalization with vitamin C (L-ascorbic acid)
The L-ascorbic acid (Vitamin C) is an essential nutrient for articular cartilage engineering. A local in situ
delivery of vitamin C can also be achieved by degradation of PSA chemically linked to vitaminC. To
functionalize the PSA with L-ascorbic acid (Vitamin-C), esterification procedure based on acid-base
reaction method was adapted. Typically, this method involves reaction between an acyl chloride (eg.,
sebacoyl chloride) and diacids (eg., sebacic acid) or hydroxy acids (eg., salicylic acid) in an appropriate
solvent such as tetrahydrofuran (THF). Pyridine is added not only to activate acyl chloride by formation
43
the acyl pyridinium ion but also to deprotonate the hydroxy- acid-groups. Thus formed precursor diacid
molecule can be dehydrated using acetic anhydride and further polymerized using melt condensation
under vacuum.
The L-ascorbic acid (Scheme 3.2) has several hydroxy groups that can form ester bonds with sebacoyl
groups. This process involves two steps (Scheme 3.2). In the first step, an equimolar concentration of
sebacic acid and sebacoyl chloride were reacted to form a diacid precursor molecule: A 15 mmol of
sebacoyl chloride was dissolved in 10 mL of tetra hydrofuran (THF) and was added drop wise into 40 mL
of THF containing 15 mmol of sebacic acid and 3.5 mL of pyridine. The mixture was stirred at room
temperature for 2 hours and poured over water at pH 2 (adjusted using HCl) to form an off-white
precipitate of oligomeric poly(sebacic acid). The precipitate was filtered and washed with water and dried
overnight under vacuum. In the second step, 1.5 grams of the above product, 375 mg of L-ascorbic acid,
and 365 mg of pyridine were dissolved in 100 mL of THF.550 mg of sebacoyl chloride dissolved in 1.5 mL
was added drop wise in the pyridine mixture. After two of stirring at room temperature, the mixture was
added to 500 mL of HCl solution at pH 2 resulting in an off white precipitate which was filtered and
washed with water and dried at room temperature under vacuum.
Scheme 3.2 Synthesis of L-ascorbic acid functionalized PSA polymer
44
3.3 Characterization of polysebacic anhydride and its derivatives using NMR and Raman
spectroscopies
The chemical identity of the products and the degree of polymerization were measured by NMR
and vibrational spectroscopy.
The NMR spectrum of sebacic acid is shown in Figure 3.1a, where the ratio of the area of
different proton peaks match with its stoichiometry. The protons and their chemical shifts are, a(8H)-1.25
ppm; b(4H)-1.48 ppm; c(4H)-2.18 ppm and e(2H)-11.94 ppm. The corresponding ratio of area are
8H:4H:4H:2H = 1:0.49:0.49:0.22.
If polymerization of sebacic acid into PSA is successful, several changes in peak positions and
area are expected. Namely, the peak at 11.94 ppm that corresponds to the protons of carboxylic acid
group should disappear, the methylene protons conjugated to the anhydride groups should have peaks at
around 2.45 and 2.55 ppm,200
and an additional peak due to methyl protons in the acetic anhydride end
groups should be observed at ~ 2.2 ppm. In fact, all these three requirements are confirmed by the NMR
spectra of PSA (Figure 3.1b). However, the peak at 2.2 ppm is convolution of methylene (conjugated to
anhydride groups) and methyl (from acetyl group) protons. The first peak at 2.17 ppm is due to the methyl
protons and the other at 2.16 ppm is due to the methylene protons. Such a fast degradation of PSA is
expected even in organic solvents.201
The areal ratio of methylene:methyl protons was calculated by a
curve fitting procedure and was found to be 0.7:0.3. Furthermore, the peak at 2.45 ppm is also a
combination of these methylene protons and the residual protons from the solvent d-DMSO.
Decomposition of this band into its components yields areal ratio of methylene:DMSO = 0.78:0.22. In
summary, the protons and their chemical shifts of PSA are, a(8H)-1.32 ppm; b(4H)-1.6 ppm; c(4H)-2.16,
2.45 and 2.55 ppm and d(6H)-2.17 ppm.
To obtain information on the degree of polymerization of the PSA achieved, the area of the
individual proton groups (Figure 4.1b) and their ratio are calculated. Assuming, n, is the number of
repeating units of sebacate monomer in PSA,
Protons in the polymer back bone:
45
Area of the 4 inner most methylene groups (‘a’) = nX8H = 1
Area of the 2 penultimate methylene groups (‘b’) = nX4H = 0.49
Area of the 2 outermost methylene groups (‘c’) = nX4H = 0.7*0.07 + 0.78X0.48 +0.09 = 0.51
Methyl protons in the end groups of PSA chain:
Area of methyl protons in the acetyl end groups (‘d’) = 6H = 0.3*0.07 = 0.021
To calculate n, a:d= nX8H:6H = 1/0.021; n/6 = 47.6; n ≈ 36
Therefore, number averaged molecular weight of the polymer, Mn, Mn ≈ 36X184+74 Da; Mn ≈ 6700 Da
Figure 3.1 NMR spectra of polymers on their precursors; (a) Sebacic acid, (b) Poly(sebacic anhydride)
(inset: expanded and curve fitted region from 2.18-2.48 ppm), (c) Ascorbic acid (d) Poly(Sebacic
anhydride)- Ascorbic acid.
The diagnostic carbonyl region in the Raman spectra of the PSA and their precursors is shown in
the Figure 3.2. In particular, there is only one carbonyl stretching band (νC=O) at 1642 cm-1
for carboxylic
acids. On the other hand, anhydrides have two carbonyl groups that are connected by an oxygen atom,
and hence exhibit two carbonyl bands. One band represents the symmetric stretching (νsC=O) of
46
carbonyls at 1808 cm-1
and the other is asymmetric stretching (νasC=O) at 1742 cm-1
. The symmetric
stretching band is always at higher frequency and is separated from the asymmetric by 50-80 cm-1
,
independent of their actual position.202
Thus the formation of poly(sebacic anhydride) was confirmed by
the disappearance of carboxylic band and appearance of these two diagnostic bands of anhydrides
1H NMR spectra of the PSA functionalized with L-ascorbate along with precursors were
measured (Figure 3.1c,d) to verify the reaction. The proton shifts at 4.98 and 4.02 ppm in the spectrum of
the functionalized polymer (Figure 4.1d) are not present for the original polymer implying successful
binding of ascorbic acid molecule.
Figure 3.2 Raman spectra of polymers on their precursors; Poly(sebacic anhydride) and Sebacic acid
3.4 Fabrication of polysebacic anhydride microparticles and encapsulation of bioactive
molecules using double emulsion method
Microparticles based on acetylated or methacrylated sebacic anhydride polymers (PSA) were
synthesized using a double emulsion method (Scheme 3.4). A double emulsion consisting of three
phases, internal water/oil/external water phases, and poly(vinyl) alcohol (PVA) as a emulsifier at the
interface between the external water and oil phases were employed. Dichloromethane (DCM) was used
as oil phase and an evaporative process was used for elimination of DCM leaving behind the polymer and
encapsulation of the bioactive cargo loaded in the internal water phase. Alternatively, bioactive solids
were directly encapsulated by using them as internal phase (internal solid/oil/external water) instead of
47
water. This overcomes the solubility imposed by water. Even though PVA is a biocompatible surfactant,
the DCM is a toxic organic solvent. Hence, even traces of DCM left behind in the microparticles can be
quite harmful. However, the use of DCM and other microparticle fabrication conditions adopted have been
shown to be harmful neither to cells nor to the bioactivity of encapsulated proteins.203, 204
Scheme 3.3 Synthesis of microparticles using double emulsion and solvent evaporation method
The microparticle fabrication procedure adopted in this study is given below. A 100 mg of PSA
was dissolved in the 1 mL of DCM and the bioactive internal phase either as aqueous solution or solid
phase was emulsified by applying ultra-sonication and homogenization for 60 seconds. The wt. % of the
internal phase is typical set at 50% by weight of PSA (50 mg). The emulsion was then added drop wise to
2mL of the external phase consisting of aqueous solution of an 1% emulsifier (PVA) under continuous
sonication and homogenization for 120 seconds more. The obtained double emulsion was then diluted
with 20 mL aqueous solution of 0.5 % PVA and stirred vigorously for 4 hours to evaporate the oil-phase
(DCM). A dense layer of polymer was left behind encapsulating the internal bioactive-phase thus forming
microparticles. The encapsulated microparticle suspensions were then centrifuged and washed with
48
water for three times. The microparticles were freeze dried and stored at 20 0C until their use. However,
these microparticles were utilized within 24 hours of their fabrication to minimize the effects of their
degradation products. Several variations of the polymers and bioactive internal phase have been utilized
for microparticle fabrication: the variations of the polymers include acetylated-PSA, methacrylated-PSA,
or fluorescein-functionalized PSA; the variations of the bioactive internal phases include aqueous solution
of L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate or their solid form, solutions of TGFβ-1,
TGF-β3, chondroitinase-ABC, amitriptyline, casein-FITC or glucose. The microparticles intended for the
delivery in the tissue constructs were fabricated under sterile conditions. The water was replaced with
DMEM solution at all stages of fabrication procedure.
3.5 Characterization and release kinetics of polysebacic anhydride microparticles using
optical, electron, and confocal fluorescence microscopies, as well as UV-Vis absorption
spectroscopy.
SEM image of microparticles fabricated using above procedure and encapsulated with casein-
FITC is shown in the Figure 3.3. The size distribution of microparticles extracted using image analysis
program ImageJ is shown in Figure 3.4. The average size of the particles was found to be ~1.5 μm. The
particles smaller than 1 μm were excluded from the analysis to avoid counting the image artifacts. The
dispersed state of the internal phase (aqueous L-ascorbic acid) in polymer was found using fluorescein-
functionalized PSA and confocal microscopy (Figure 3.5). The extent of dispersion of the internal phase
was clearly not uniform: some of the microparticles have one big droplet, whereas, others have several
well dispersed smaller droplets.
Figure 3.3 SEM images of PSA-microparticles encapsulated with casein-FITC after 24 hours in aqueous
suspension. All the images are from the same batch of microparticles but at different magnifications.
49
Figure 3.4 Particle size distribution of PSA-microparticles with casein-FITC after 24 hours in aqueous
suspension. The size distribution was obtained from SEM image analysis performed using ImageJ
program.
Figure 3.5 L-ascorbic acid loaded polyanhydride microparticles embedded in agarose scaffold. Green
color indicates the fluorescein-functionalized PSA polymeric shell. Black core is the encapsulated L-
ascorbic acid. Both the images are from the same microparticle sample
50
Release kinetics of PSA microparticles
Release of amitriptyline molecules from encapsulated-microparticles was studied to verify the
linearity of the release profile. Absorption of UV-light at the wavelength 239 nm by amitriptyline was used
to monitor its release. The concentration of the released amitriptyline molecules in microparticle
suspension was measured for every few days by centrifuging the suspension and extracting 5 mL of the
supernatant and was stirred to resuspend the microparticle particles. The encapsulation efficiency was
found to be ~50%. Within 2 weeks, ~60 % of encapsulated amitriptyline was released from the PSA
microparticles and a linear profile was observed (Figure 3.6). This shows that PSA-based polymeric
microparticles can be utilized to provide sustained linear delivery of enzymes or drugs with no initial burst
effect.
Figure 3.6 Release profile of amitriptyline drug from poly(sebacic anhydride)-based microparticles
3.6. Synthesis and characterization of acrylate nanoparticles
3.6.1. Ternary phase diagrams
Cross-linked polymeric nanoparticles were synthesized using the microemulsion technique. Nano-sized
water droplets stabilized in oil continuous phase by low hydrophilic-lipophilic balance (HLB) surfactant
51
such as Aerosol-OT (AOT) were used as a nano-container for synthesis of acrylate nanoparticles through
radical polymerization and cross-linking reactions. The inverse-microemulsion system of
water/AOT/hexane was used due to higher stability in the temperature range (40-60 0C) required for the
initiation of radical polymerization. The phase behavior was measured and phase diagrams were
constructed for water/AOT/hexane and (water+acrylic acid)/AOT/hexane systems at room temperature
(Figure 3.7a, b, respectively). Addition of acrylic acid monomer had in fact enlarged the stable region of
the inverse microemulsion phase. The phase diagram of (water+acrylic acid)/AOT/hexane system at 50
oC were constructed (Figure 3.8) and was found to be very similar to room temperature phase diagrams.
Two concentration points were selected for nanoparticle synthesis experiments (Figure 3.8), one of the
point is closer to the phase boundary of inverse microemulsion (phasepoint 1) and another point is well
inside this phase region (phasepoint 2).
3.6.2.Synthesis of poly(acrylic acid) nanoparticles and its functional derivatives
The poly(acrylic acid) nanoparticles were synthesized as shown in Figure 3.9a using bisacrylamide as a
crosslinker. The acrylic acid monomer was added to the water phase (~40 % by weight of water) along
with crosslinker (1% by weight monomer) and initiators (ammonium per sulfate and ascorbic acid, ~ 1%
each by weight monomer). The oil phase containing hexane was prepared by dissolving Aerosol-OT
surfactant (~50% by weight of oil phase). The water phase was added drop wise into hexane in a three-
necked flask under constant stirring condition. A clear inverse emulsion solution was noted which was
then flashed with ultra-pure N2 gas for 30 min. A reflux condenser was fitted and temperature was ramped
up to 50 – 700 C. The radical polymerization and crosslinking reactions were initiated due to the
increasing temperature and when the crosslinked nanoparticle particles were formed, the clear
microemulsion turned into a dull white emulsion system. The polymerization in the turbid emulsion was
then terminated (after ~ 30 minutes of maintaining at 50 – 700 C), cooled down to room temperature, and
the resulting polymeric nanoparticles were precipitated by addition of acetone.
52
Figure 3.7 Phase diagrams of (a) hexane/water/AOT (b) hexane /(water+acrylic acid)/AOT systems at
room temperature. Only inverse emulsion region is indicated by colors
The precipitate was filtered and washed repeatedly with hexane and acetone to remove the Aerosol-OT
surfactant and unreacted monomers. These polyacrylic acid nanoparticles were then freeze-dried for 2
days and stored at -20 0C. Polyacrylamide nanoparticles were synthesized using the same procedure and
chemicals except for the monomer which was changed from acrylic acid to acrylamide (Figure 3.9b). A
similar procedure was followed for polyacrylic acid nanoparticles functionalized with sulfonates, and
phosphonates, and amides by replacing only 10 % of the acrylic acid monomer with vinyl sulfonates, and
vinyl phosphonates, respectively (Figure 3.9c, d).
Figure 3.8 Phase diagram of hexane /(water+acrylic acid)/AOT systems at 50 C. Only inverse emulsion
region is indicated by the enclosed region.
(a) (b)
53
Figure 3.9 Synthesis of functionalized polyacrylate nanoparticles, (a) polyacrylic acid (b) polyacrylamide
(c) poly(acrylic acid-phosphonate) (d) poly(acrylic acid-sulfonate); Bisacrylamide is the crosslinker and
ascorbic acid is the initiator
3.6.3.Size determination using SEM and dynamic light scattering
The size of nanoparticles synthesized at the two phasepoints from Figure 3.8, were observed to
be widely different (Figure 3.10). In the case of phasepoint-2 (interior of inverse emulsion phase region),
the nanoparticles of smaller size were noted (70-nm to 300-nm) as seen in Figure 3.10b. However for the
phasepoint-1 (closer to the phase boundary), the size of nanoparticles increased by an order of
magnitude to become microparticles (Figure 3.10a). The size range of nanoparticles prepared using
phasepoint-2 were independently confirmed by dynamic light scattering measurements (Figure 3.11).
Figure 3.10 SEM micrographs of poly(acrylic acid) particles synthesized using concentrations at (a)
phasepoint-1 (b) phasepoint-2
54
Figure 3.11 Size of nanoparticles using dynamic light scattering technique, (a) poly(acrylic acid), 130-
340-nm (b) poly(acrylic acid-acrylamide(10%)), 100-320-nm (c) poly(acrylic acid-phosphonate(10%)),
140-260-nm (d) poly(acrylamide) 70-90-nm nanoparticles
3.6.4 Atomic composition using X-Ray photoelectron spectroscopy
X-ray photoelectron (XPS) spectroscopy were utilized to confirm the successful completion of
chain polymerization and crosslinking reactions. A sample XPS spectra of poly(acrylic acid) nanoparticles
was shown in Figure 3.12, where the CH2, C=O, and COO- peaks were identified to be the nanoparticle
peaks. A very small peak of C=C at 285 eV indicates that small amounts of monomers were left in the
solid unreacted.
3.6.5 Surface charge using electrophoretic mobility (Zeta potential) measurements
Zeta potentials of the nanoparticles were measured, and the isoelectric point was found to be <
pH 2 for all types of nanoparticle particles considered in this study (Figure 3.13). However, there is a clear
difference in pH dependence of zeta potential between poly -acrylamide, -phosphonate and -acrylic acid
nanoparticles. As a result, at pH ~7 (physiological pH ) acrylamide had lowest negative charge and
phosphonate nanoparticles have highest negative charge.
(a) (b)
(c) (d)
55
Figure 3.12 X-ray photoelectron spectroscopy of poly(acrylic acid) nanoparticle. Carbon 1S spectral
region is shown.
Figure 3.13 Zeta potential of poly(acrylamide), poly(acrylic acid-phosphonate), poly(acrylic acid)
nanoparticles
3.7 Pore-size determination of agarose-based hydrogel as a function of agarose
concentration and presence of polysebacic anhydride microparticles
It is known that pore-size of the agarose hydrogel varies with agarose concentration. To select
the optimum concentration of agarose in the agarose hydrogel scaffold, it can be suggested that the pore
size should be large enough for the easy transport of growth factors and nutrients (> 30 nm), but smaller
than 300 nm so as to retain secreted tissue components such as collagen fibrils. However, there is no
56
direct method to evaluate this quantity. This problem is very technically challenging, especially in the
presence of polymeric particles.
Turbidimetric measurements offer one of the fast and elegant method for measuring average
pore size of hydrogels formed by biopolymers.205, 206
The turbidity of an aqueous suspension of particles that are monodisperse and optically isotropic
was given by Doty and Steiner207
Turbidity, ( ) ( ) ( ) (3.1)
Where,
H(λ) is optical constant; Q(λ) is intraparticle dissipation factor
S(λ) is interparticle correction factor; M is molar mass of the particle
c is concentration of particles; λ is wavelength of incident light
The optical constant, H, depends on the refractive index of solvent (n0) and particle (n) as well as
wavelength of incident light (λ). The wavelength dependence of refractive indices of particles and medium
can be ignored in the first approximation. Then, the optical constant, H, can be approximated by the
following expression,208
( )
(
)
(3.2)
Where,
n0 and n are solvent and particles refractive indices, respectively; Na is Avogadro number
The intraparticle correction factor, Q(λ), represents integration of reduction in scattering intensity
due particle shape over all the viewing angles above acceptance angle, α. The value of Q varies from 0 to
57
1. For particles much smaller than wavelength of the incidents (D<<λ), there is no reduction in scattered
light intensity due to shape of particles. The particles are said to be in Raleigh regime and Q≈1.205
For
particle sizes equal to or greater than 5% of incident wavelength, Q deviates significantly from 1.205
The Q is defined for unpolarized light as follows,
( )
∫ ( )( )
(3.3)
Where, P(θ) is form factor and θ is scattering angle.
The form factor, P, can be either obtained by experiments through measuring scattered light at
various angles or calculated theoretically from shape-dependent expressions derived from pair-pair
correlations.209-211
The agarose chains in the hydrogel forms bundles of radii 0.9-10 nm212
allowing formation of
large pores of water.213
Thus, agarose can be approximated as Gaussian coils which allows defining the
form factor P as
( ) (
) ( ) (3.4)
Where, q = 4πn0 sin(θ/2)/λ;
ξ is correlation length ( ≈ average pore size)
N is apparent number of scattering units.
In the case of agarose fibers in water, N can be neglected since it is proportional to ξ/r0, the
monomer radii and the r0 << ξ as well as P0(q) can be assumed to be 1.208
Then equation 3.4 reduces to
( )
(3.5)
The final correction factor in the turbidity equation (3.1), the interparticle correction factor (S), can
be assumed to be 1 under the dilute particle concentration used.7
58
To obtain any structural information such as particle size or pore size using equation (3.1), it is
necessary to measure the absolute values of turbidity, which is non-trivial. The absorbance values
reported by most of the spectrophotometers are in arbitrary units. Thus
An alternative method that obviates the above problem is to measure the turbidity as a function of
wavelength. Thus equation (3.1) can be rewritten as,
( ) ( ) ( ) ( ) (3.6)
Differentiating with respect of log(λ),
( )
( )
( )
(3.7)
Using Eq. (3.2) into (3.7)
( )
(
)
(3.8)
The second and third term on the right hand side of the eq. (3.8) is evaluated to be 0.0248 and
0.0922 for wavelengths between 700 and 800 nm.214, 215
Thus the equation (3.7) can be written as,
( )
( )
(3.9)
Melik and Fogler216
has suggested that for extracting approximate particle sizes, the left hand
side of equation (3.9) (slope of turbidity and wavelength plotted in log-log scale) can be
considered as wavelength exponent (WLE).
( )
(3.10)
59
Using the procedure adopted by Aymard et al.,208
the dependence of wavelength exponent (WLE)
on pore size (ξ) for agarose-like material can be theoretically calculated using equations (3.2),
(3.4) and (3.10). The calculated WLE as a function of pore size is shown in Figure 3.14 for
different acceptance angles
Figure 3.14 Theoretical calculated wavelength exponent as a function of pore size for wavelength range
of 700-800 nm.
The experimental WLE from 700 to 800-nm is extracted from turbidity measurements as follows,
Turbidity, ( ) ( )
(3.11)
Where, l is the path length = 1 cm.
The WLE is obtained by fitting a slope of log-log plot of τ and λ from 700-800 nm
( ( )
)
(3.12)
60
Thus, agarose solutions were melted by increasing temperature to 95 0C. The absorbance of the
melt from 700-800 nm was measured immediately by placing them in UV-Vis spectrophotometer. The
absorbance was measured every 5 minutes for next 2 hours as the melt cools down to room temperature
and forms hydrogel network. The wavelength exponent (WLE) is extracted from the absorbance values
as mentioned before and pore size values were obtained using the established relationship (Figure 3.14).
The details of extracting WLE from the absorbance data is given in Appendix 2. The average pore size as
a function of cooling time is shown in Figure 3.15. The average pore size of agarose hydrogel at room
temperature as a function of agarose concentration is shown in Figure 3.16. It is noted that as the
agarose concentration was decreased from 4% to 1%, pore-size increased from 40-nm to 520–nm
(Figure 3.15).
Using this method, the variation of pore size with respect to agarose concentration was
empirically derived to be,
ξ = 521.95 C-1.833
where ξ is the pore size and C is the agarose concentration in w/v %.
When the polysebacic anhydride microparticles are introduced at 0.3% w/v to the agarose melt,
there was small an increase in pore size from 150-nm to 190-nm. This method failed to determine the
pore size when the microparticles concentration was greater than 0.3 %. This is due to the fact the
increased fraction of large microparticles themselves act as strong scatterers of the radiation apart from
the agarose fibers.
61
Figure 3.15 Pore size of agarose hydrogel as function of cooling time. The temperature at time t=0 is 95
0C
Figure 3.16 Effect of agarose concentration (C, % w/v) on the pore size (ξ, nm) of agarose hydrogel
ξ = 521.95 C-1.833
R² = 0.9998
0
100
200
300
400
500
600
0 2 4 6
Po
re s
ize,
ξ, n
m
Concentration of agarose, w/v %
62
Chapter 4 Synthesis and characterization of ferric
(hydr)oxide nanoparticles
This chapter presents the synthesis procedures used to obtained hematite nanoparticles of
different sizes, as well as the characterization of these nanoparticles using TEM, HRTEM, XRD,
Raman, XPS, acid-base titration, and zeta potential methods.
4.1 Synthesis of hematite nanoparticles
Two samples of hematite nanoparticles NPs (H7 and H9) are synthesized according to Ref.217
by slowly
dripping 60 mL of 1 M ferric nitrate (Sigma-Aldrich, 99.99% purity) solution into 750 mL of boiling TDW.
After the drip solution is consumed, the NP suspension is removed from the heat source. Difference
between H7 and H9 is that in the first case heating of the solution is stopped immediately after dripping is
started. H38 is synthesized according to Ref.217
by heating 8.08 g of ferric nitrate in 1 L of 0.002 HCl at
98C. This suspension is aged in the oven at 98°C for 7 d. In all cases, the precipitation by hydrolysis is
followed by cooling the suspensions overnight and dialysis for 2 weeks using singly distilled water until
the conductivity of the dialyzed water reaches that of the pure water. Sample H150 synthesized by the
annealing of ferric sulfate is purchased from Fisher. 2-line ferrihydrite (FH) is purchased from Alfa Aesar
as Iron(III) Oxide, 99.95% (metal basis), 3nm APS powder, surface area 250 m2/g. Star-shaped H400 are
synthesized by adapting the protocol of Ref.218
4.2 Characterization of ferric (hydr)oxide nanoparticles
Morphology and size of the NPs are characterized using TEM (Figure 4.1). The samples are labeled
according to mean NPs size as measured by averaging sizes of 10–15 NPs. FH NPs are strongly
agglomerated. Shape and size of the primary FH NPs cannot be resolved by the instrument used.
Reported crystal size of 2-line FH is about 2 nm,219, 220
while the extension range of local structural order
63
is as low as 4 Å.219
H7 and H9 hematite NPs exhibit average diameter of 7.0 0.5 and 9 1 nm,
respectively and a rounded irregular hexagonal shape as viewed along the optical axis of the microscope.
H30 have agglomerate-like irregular shape and average diameter of ~30 nm. These globules are porous,
which is confirmed by the fact that their geometrical surface area (GSA) is smaller than the BET surface
area value (Table 4.1). H38 NPs have well developed rhombohedral shape. Examination of the TEM
image does not reveal pores on the surface of H38 NPs and gives their average diameter of 38 5 nm.
The GSA value for H38 matches the BET surface area within the experimental error, confirming that H38
NPs are non-porous. H120 are rhombohedral, while H150 NPs are rounded polyhedral. Both H120 and
H150 are highly monodisperse and non-porous.
Additionally, the morphology of H38 was studied using HRTEM and selected area electron
diffraction (SAED) (Figure 4.2). In particular, it can also be noted that the faces of the H38 rhombohedra
are essentially flat, although the corners are slightly truncated. Furthermore, H38 NPs are enclosed by
rhombohedral facets of the {104} and {012} families. The morphology of the hematite NPs with the same
shape and HR-TEM pattern was characterized in detail in Ref.221
It was demonstrated that this pattern
corresponds to the (104) and (0-14) facets of rhombohedral hematite with zone axis of [-441]. At the
same time, the diffraction spots in the SAED pattern are attributed to the (012) and (014 ) planes of
rhombohedral hematite with the [100] zone axis. Aspect ratio of the NPs used in this study is similar
(ranging from 1 to 2).
The phase identity of hematite NPs and FH used in this study is verified using XRD (Figure 4.3a).
The diffractogram of FH is dominated by two broad lines characteristic of 2-line FH. There are also minor
peaks (eg. at 2θ=33) which point to the presence of a small admixture of hematite phase in FH. Except
for H7, all the hematite NPs show only hematite diffraction patterns, with no lines due to other impurity
phases. H7 too has hematite peaks but also has some admixture of 6-line ferrihydrite. For all hematite
NPs, the (110) and (300) lines are sharper than lines (104), (018), (202), and (024). It follows that
hematite NPs have either plate-like shape with an excess of the planes (near) perpendicular to the c axis
or better developed basal facets. The XRD lines gradually sharpen as NP size increases, which is a
signature of an increase in the average crystallinity size. The latter was quantified using the Scherrer
64
formula with a shape factor of 1 after removing the instrumental broadening by simple subtraction. The
instrumental broadening was taken as the line widths in the XRD pattern of micron-size natural hematite.
All the samples are tested for the presence of surface impurities using XPS. In addition to adventitious
carbon, only ferric iron and oxygen are detected on hematite NPs, while FH is found to contain Si at the
Si/Fe atomic ratio of 0.025. (See Ref.109
for more details on XPS and XRD analysis)
Comparative analysis of crystallinity of the NPs was performed using relative intensities of the
crystallinity-sensitive bands at 660 and 690 cm1 in the Raman spectra.
222 Intensity of both these bands
and hence crystallinity of the NPs increases with increasing NP size (FH < H9 < H38 < H150) (Figure
4.3b). UV-Vis spectroscopy revealed that the band-gap of the NP decreases in the same order.109
As
seen from Figure 4.4, PZC and the iso-electric point (IEP) of H9 and H38 are within the 8.5–9.5 range
typical for hematite NPs synthesized by the hydrolysis of a ferric nitrate/chloride solution and not
subjected to drying,106, 223, 224
although IEP of 9.2 of H38 is at the high limit of the range. H150 is more
acidic (PZC = 6.2 and IEP 7.0), which can be attributed to either a trace amount of acidic surface
impurities or the high-temperature route of the H150 synthesis that eliminates the most basic single-
cation coordinated hydroxyls. FH is characterized by IEP of 7.00.1, comparable with reported values of
7.0 (Ref. 225
), 7.2 (Ref. 226
), and 7.5 (Ref. 227
). H38 and H150 are the most basic and acidic among the
NPs, respectively. The macroscopic acid-base properties of H9 are intermediate, while FH is more acidic
than H9. Both PZC and IEP do not change monotonically with increasing the size of the NPs.
Figure 4.1 TEM images of hematite and FH NPs. NPs are labeled according to the average size as
measured by the analysis of TEM images.
65
Figure 4.2 Typical (a) HR-TEM image and (b) SAED pattern of 38-nm hematite (H38)
Figure 4.3 (a) XRD and (b) Raman spectra of dry hematite and ferrihydrite NPs. “T” marks the peak due
to tetrahedral defects, LO Eu is the peak activated by the lattice disorder.222
115
65
(0 ̅4)
5 nm
220 mm b
66
Figure 4.4 Effect of size of hematite nanoparticles on their point of zero charge and isoelectric point
As seen from Figure 4.17, values of the point of zero charge (PZC) and the iso-electric point (IEP) of
hematite NPs vary non-monotonically, with H38 and H150 are the most basic and acidic, respectively. FH
is characterized by IEP of 7.00.1, comparable with reported values of 7.0 (Ref. 225
), 7.2 (Ref. 226
), and
7.5 (Ref. 227
). The macroscopic acid-base properties of H9 are intermediate, while FH is more acidic than
H9.
67
Table 4.1 Morphological characteristics of hematite NPs
Sample
Average
Particle
Size
(TEM),
nm
Particle Habit
(TEM)
BET,
m2/g
GSA, m2/g
Approximation of shape,
heighta)
(h) in calculating
GSA
FH 2 b)
250 c)
–– ––
H9 9 Rounded
platelets 128.3 440/165
d)
Hexagonal platelets h = 1.5
nm/spheres
H38 38 Hexagons and
rhombohedra 37.0 40 Rhombohedra h = 26 nm
H150 150 Rounded
polyhedra 9.5 8.8
Rectangular prisms h = 80
nm
a) Sizes for hexagons, rhombohedra, and rectangles were taken based on the TEM images. Values
of h for H7 and H9 were approximated by the values provided by AFM for similarly synthesized
NPs,228
for H30–H60 h is taken as a mean coherence length in the [104] direction, while for H120
and H150 h is taken the average shortest dimension as measured by TEM. Size dependence of
the density of hematite NPs was obtained through linear interpolation between values for FH (3.96
g/cm3) for 2 nm NPs and a bulk value of 5.23 g/cm
3 for 120 nm NPs.
b) Cannot be resolved.
c)
Provided by manufacturer. e)
Second value is obtained approximating the NP shape by a sphere
with diameter taken as mean coherence length in the [110] direction;
68
Chapter 5 Bioactivity of polymeric particles in
articular cartilage engineering: In situ delivery
using poly(sebacic anhydride) microparticles and
functional group effects of poly(acrylate) based
nanoparticles
This chapter is divided into two parts: In the first part, the in situ delivery of bioactive molecules
using polysebacic anhydride microparticles (PSA-microparticles) is discussed. A cytotoxic limit for PSA-
microparticles is determined and the cytotoxicity is found to be due to the PSA and its degradation
products. The mechanism of cytotoxicity is deduced to be due to lipotoxicity of the PSA degradation
products. Several mitigation strategies for the PSA were tested and serum albumin treatment is found to
be quite effective. In the second part, the effects of functionalization of polyacrylate nanoparticles on the
cellular viability and metabolism are detailed. Finally, a summary of the results is presented.
5.1 Effects of in situ delivery of bioactive molecules on the chondrocytes cultured in 3D
scaffold
The objective of the first study is to overcome the problem of the decrease in the diffusion of
nutrients and growth factors due to progressive elaboration of extracellular matrix in 3D agarose scaffolds
with time. The polysebacic anhydride has high potential for fabricating microparticles with and
encapsulating critical nutrients (Vitamin C) or growth factors (TGF-β3) as well as for short-term delivery
(2—3 weeks) of the nutrients in tissue engineering applications. Thus, the goal of this study is to tailor of
the bioactivity of polysebacic anhydride microparticle-delivery systems that are cast together with
chondrocytes in a 3D hydrogel scaffold (Scheme 1.1).
69
5.1.2. Dose dependent cellular viability in the presence of poly(sebacic anhydride)
The dose-dependence of the biocompatibility of PSA-microparticles towards chondrocytes was
determined using a 3D agarose-scaffold.
Briefly, the samples were prepared as follows: the PSA-microparticles was encapsulated with
stabilized-vitamin C (Section 3.5) and cast along with chondrocytes in an agarose hydrogel slab. The final
concentrations of the components of slab were agarose-2%, chondrocytes-30 million/mL and PSA-
microparticles at 0.2%, 2% and 10% of w/v of the hydrogel scaffold. More details were provided in
Section 2.2. Each of these concentrations was cast and studied in separate batches and thus had its own
control set.
At 0.2 w/v % of PSA-microparticles, there is no difference in cellular viability as compared to
control (Figure 5.1). However, the viability decreases significantly for both 2% and 10% concentrations: At
the 10% polymer concentration, almost all the cells were dead, whereas 2% shows very little viable cells.
From visually inspecting the culture medium of the 2% and 10% concentrations, no metabolic activity was
observed throughout the culture period. Metabolic activity can be inferred from a decrease in pH due to
secreted acidic metabolic products (ex. lactic acid) which change the color of the phenol red indicator in
the culture medium from red to yellow. A comparison of the equilibrium Young’s modulus (EY) of these
constructs confirmed these findings (Figure 5.2). The y-axis of Figure 5.2 represents a normalized EY
value of microparticle-containing constructs with that of control constructs without microparticles. The x-
axis represents the concentration of polymeric microparticles (w/v %). These results together (figure 5.1
and 5.2) indicated that the presence of PSA polymeric microparticles even as low as 2% (20 mg/mL) was
detrimental to the cellular viability and their metabolism. This is a highly surprising result considering the
fact that majority of the literature considers the PSA to be highly biocompatible.
5.1.3 Mechanisms of the cytotoxicity of poly(sebacic anhydride) microparticles
Following the conclusion from the previous section that PSA has detrimental bioactivity above a
certain concentration, it is necessary to identify the mechanism of cytotoxicity for devising mitigation
strategies. As outlined in the Section 1.1.1(d), several reasons can account for the cytotoxicity of
70
Figure 5.1 Effect of polymer concentration on chondrocytes viability on day-1 in 3D agarose scaffolds.
PSA concentrations are (a) 0.2% (b) 2% and (c) 10% w/v. Green color indicates cytoplasm of the live
cells and red color indicates dead cells and probably microparticles
Figure 5.2 Equilibrium Young’s modulus of hydrogel constructs with different concentrations of
poly(sebacic anhydride) microparticles. The y-axis values are normalized by modulus of the
corresponding control constructs (without microparticles). The concentration of polymeric microparticles in
x-axis is represented as w/v% of hydrogel constructs
0
0.2
0.4
0.6
0.8
1
1.2
PSA-0.2% (day-15) PSA-2% (day-52) PSA-10%No
rma
lize
d e
qu
ilib
riu
m
Yo
un
g's
mo
du
lus
,
Poly(sebacic anhydride) microparticles, w/v %
Equilibrium Young's modulus vs. polymeric microparicles
increasing polymer concentration
71
poly(sebacic anhydride) microparticles. These include (a) shock to chondrocytes due to changes in local
pH; (b) cellular uptake or the damage to the cell wall by the polymer or its degradation products and the
generation of ROS due to the lipotoxicity of the degradation products. To test the hypotheses, (a) the
buffer capacity of the growth medium was increased to offset any local pH changes and (b) cellular
uptake was monitored using fluorescein-functionalized PSA-microparticles and confocal fluorescence
microscopy.
(a). Are cytotoxic effects of PSA-microparticles on chondrocytes due to local pH changes?
Testing the hypothesis through increased buffer capacity
A preliminary acellular experiment showed a sudden drop in pH of growth (CM) media from 7.5 to
6.8 when PSA microparticles were added at a concentration of 30 mg/mL. The pH decreased further to ~
6.3 within 5 hours. This implies a partial hydrolysis of anhydride bonds of polymeric shell of the
microparticles that produces carboxylic acid groups during the fabrication, freeze drying and storage
steps.
To verify if the pH drop due to PSA is the cause for observed cytotoxicity, the chondrocytes were cast
using CM growth media with increased buffer capacity by adding HEPES at 5mM concentration (for
justification of the selected HEPES concentration, see Appendix-A where the amount of buffer capacity
needed for offsetting the pH drop due to polymer degradation is estimated). Additionally, the carboxylic
groups of PSA were neutralized by pre-suspending the microparticles in 5 mL of growth medium (CM)
containing sodium bicarbonate. The particles were centrifuged and the medium was replenished. The
process was repeated until no visual drop in pH was observed. The microparticles were then
resuspended in a fresh growth media (with 5mM HEPES buffer) in appropriate quantity before mixing into
agarose melt. The microparticles were added at a concentration of 2% w/v. In this particular study,
microparticles were encapsulated with chondroitinase-ABC enzyme. The chondroitinase-ABC selectively
digests the GAG molecules and thus makes space for collagen accumulation. Even though the buffer
capacity was increased, as seen from Figure-5.3, cellular viability did not improve. In fact, right from the
beginning of the culture period, no change in the media color and hence no metabolic activity was
72
observed for the constructs with embedded microparticles (as opposed to the control). These results
prove that the local pH changes are not likely the cause of lack of metabolic activity.
(b). Are cytotoxic effects due to cellular uptake PSA-microparticles or its degradation products
and the resulting lipotoxicity? Confocal fluorescence studies
The main degradation product of polysebacic anhydride is polysebacic acid which mimics alkyl
fatty acids due to the presence of carboxylic acid groups and long hydrophobic chains. It is known from
the literature that excessive intracellular concentrations of alkyl fatty acids can cause abnormal levels of
ROS and can result in cellular death.49, 229
If the chondrocytes ingest the PSA-microparticles or its
degradation products, then a priori, the alkyl fatty acid-like concentrations inside cells can be expected to
increase thus causing lipotoxicity. To test if the cytotoxicity arises due to cellular uptake of PSA polymer,
a fluorescein label was attached the PSA as detailed in Section 3.2. This fluorescein-containing PSA
(fPSA) was then added to a 2D culture of chondrocytes aggregate and observed under confocal
fluorescence microscope for cellular uptake of fPSA (Figure 5.4). It can be seen that within 60 minutes
there was significant accumulation of fluorescein-labeled PSA in cells (Figure 5.4).
Figure 5.3 Effect of increased in buffer capacity (5mM HEPES) of growth medium on chondrocytes
viability on day-23 in 3D agarose scaffolds using Live/Dead cytotoxicity kit. The agarose constructs
contain (a) no PSA (control) (b) 2 % PSA microparticles. The microparticles are encapsulated with
chondroitinase-ABC. Green color indicates cytoplasm of the and red color indicates dead cells and
probably microparticles
73
Figure 5.4 Cellular uptake of fluoresceinated-PSA (F-PSA) by chondrocytes. F-PSA is added nearby a
chondrocytes aggregate at time t=0 and images were obtained at time, t= a) 5 min b) 20 min. c) 60 min.
Gradual increase in the fluorescence intensity is due to intracellular accumulation of F-PSA
5.1.4 Mitigation of the cytotoxicity of PSA on chondrocytes: positive impact of bovine serum
albumin (BSA)
Several strategies were applied to mitigate or reverse the cytotoxicity due to the PSA. The mitigation
strategies were tested on a 3D agarose systems seeded with chondrocytes and compared to control
scaffolds without the PSA microparticles. A summary of these studies and rationale for the selection of a
particular strategy are given below.
Initially, pH changes due to PSA uptake were thought to be the reason for cytotoxicity and hence the
buffer capacity of the medium was increased by increasing concentration of NaHCO3, Tris-HCl or HEPES
buffers. In the next set of experiments, osmolality increase due to PSA uptake was hypothesized to be
cause of cytotoxicity and hence trimethyl-amine oxide (TMAO), which is a known osmolyte, was used.
Additional tests were performed by decreasing the osmolality of growth media to offset any increase due
to PSA. None of these methods had any positive impact in mitigating the cytotoxicity due to PSA. Thus,
an alternative strategy was adopted to mitigate the toxicity, that is, to bind the degradation products of
PSA polymer in situ using proteins such as gelatin, which can additionally act as scaffold. Furthermore,
gelatin with amino acid functional group can act as buffer for pH shocks. For this purpose, a mixture of
agarose and gelatin scaffold was prepared and seeded with chondrocytes and PSA microparticles.
However, this strategy too did not help to reverse the toxic effects of PSA.
74
In fact, the natural pathway by which cells cope with lipotoxicity is through intracellular regulation of lipids
by serum albumin proteins.230, 231
That is, by maintaining a ratio of serum albumin/lipids of 0.7 - 2, cells to
combat adverse effects due to increase in fatty acid concentration.231
Thus, it can be hypothesized that
the presence of serum albumin can also combat the lipotoxicity, if any, of PSA degradation products. This
was tested by adding bovine-serum albumin both in the 3D constructs during casting and in culture
medium at 1 mg/mL concentration. It was found that BSA significantly reverses the cytotoxicity of these
anhydride polymers.
5.1.5 Delivery of TGF-β3 using PSA-microparticles in BSA-based system
The effect of uniform and sustained delivery of TGF-β3 using PSA-microparticles was tested
utilizing the above found mitigation strategy using BSA. For this purpose, the PSA microparticles were
fabricated with encapsulated TGF-β3. For a more consistent comparison with the control, only a fraction
of total TGF-β3 (one-fourth) needed for the first two weeks of culture period was delivered in situ using
encapsulated microparticles. The rest of TGF-β3 (three-fourth) was supplied externally through the
media. Given the encapsulation efficiency of ~50%, 1% w/v of microparticles is needed for delivering ¼ of
the TGF-β3 in situ of culture period of two weeks. The BSA protein was added both during casting
process and also in the growth medium throughout the culture period at a concentration of 1 mg/mL. Two
type of control scaffolds were cast, one without BSA and the other with BSA. The equilibrium Young’s
modulus and dynamic modulus at 0.1 are shown in Figure 5.5. It can be seen that the presence of BSA
has significantly mitigated the cytotoxicity of the PSA polymer and makes the PSA-microparticles effective
for short-term delivery. However, it is clear from presented results that biomedical applications using
polyanhydrides as a delivery device should not only incorporate BSA protein but also minimize
polyanhydride content by other means such as mixing polyanhydrides with slow degrading hydrophobic
polymers or reduce the polymer-to-bioactive molecules ratio.
75
Figure 5.5 Mechanical properties of control without BSA (BSA-), control with BSA (BSA+) and TGFβ3
microparticles-containing (ugel-TGF-β3-BSA+) constructs. (a) Equilibrium Young’s modulus, and dynamic
modulus at (b) 0.1 Hz. Using two way ANOVA analysis, there was no statistically significant difference
was found between Control BSA+ vs. ugel-TGF-β3-BSA+ for p< 0.05
To summarize the results,
(a) The PSA microparticles were found to be cytotoxic at a concentration of 20 mg/mL, but benign at
2 mg/mL.
(b) The mechanism of cytotoxicity is determined to be due to intracellular accumulation of PSA or its
degradation products and the resulting lipotoxicity. Changes in local pH due to polymer hydrolysis
was ruled out to have major impact on cellular viability
(c) Of the several mitigation strategies tested, bovine serum albumin treatment was found to offer the
best protection from adverse effects due to the polymer. Since it is known, in general, that serum
albumin is a natural regulator for intracellular lipids and prevents lipid overdose, the
a
b
76
chondroprotection of BSA against PSA polymer provides additional proof for its lipotoxicity apart
from its structural similarity to lipids
(d) The BSA-based mitigation strategy makes PSA microparticles effective in the short-term delivery
of TGF-β3 in articular cartilage engineering.
5.2 The effects of functionalization of polyacrylate nanoparticles on the cellular viability
and metabolism
In this section, the results of the effects of functionalized polyacrylate-based nanoparticulate system
(polyacrylic acid, polyacrylamide and polyphosphonate nanoparticles) embedded in a 3D hydrogel matrix
on the biochemical and biomechanical properties are presented. The results suggest that though these
nanoparticles are biocompatible, the negative surface charge on these nanoparticles correlates inversely
to the extracellular matrix synthesis and mechanical strength.
5.2.1 Sterilization tests:
Three nanoparticles, (1) polyacrylic acid (PAA), (2) polyacrylamide (PAM), polyacrylic acid-
phosphonate (PAAP), were kept under UV light for 20 minutes. These irradiated nanoparticles were
tested for sterility by suspending them in two separate media (water and tissue culture media with growth
factors). These were incubated at 37 oC in 5% CO2 environment for 4 days. The media remained clear
which indicated the absence of bacteria or other invasive microorganisms and success of sterilization
procedure. The next step was to culture chondrocytes in the presence of nanoparticles to find out its
biocompatibility.
5.2.2 Biocompatibility tests:
Three nanoparticles (PAA, PAM, PAAP) and a control (without nanoparticle) were used for these tests.
Chondrocytes obtained from calf-bovine source were plated on tissue culture flasks with or without the
nanoparticles at 10 μg/mL. The cell-to-nanoparticles concentration was initially maintained at 10,000
cells/μg. The cells were cultured for a week under these conditions viable cell density was counted. The
cell density for all the samples (control, PAA, PAM, PAAP) was found to be very similar (~ 10 million
77
cells). Thus it is concluded that nanoparticles are biocompatible with chondrocytes in a two dimensional
substrate at this concentration (10, 000 cells/μg). Following the biocompatibility studies, in vitro studies
using three-dimensional constructs of agarose hydrogels with embedded nanoparticles were conducted.
5.2.3 Three-dimensional tissue culture studies with acrylate nanoparticles
Agarose constructs embedded with three acrylate nanoparticles (PAA, PAM and PAAP) and a control
(without NPs) were used in this study. The juvenile bovine chondrocytes obtained from1st passage were
cast at 30 million cells/mL concentration. A total of four scaffolds, one control, and three nanoparticles-
containing (PAA, PAM, and PAAP) slabs were cast. The final w/v % of agarose and acrylate
nanoparticles were 2 and 0.2, respectively (see Section 2.2 for more details). Mechanical strength
(Equilibrium Young’s compressive modulus and dynamic modulus (at 0.1 Hz), cell viability (confocal
fluorescence microscopy), and biochemical analysis (glycosaminoglycan & DNA) were conducted on
constructs from day-0, day-14, day-28 and day-60 and are shown in Figure 5.6, 5.7 and 5.8. On day-3,
one of the experimental set (PAA) was lost due to infection and hence excluded from the study.
On the day-0, all the samples had the equilibrium compressive and dynamic moduli very similar to each
other (Figure 5.6). However, as time progressed, nanoparticles-containing scaffolds showed lower values
of the mechanical strength than the control. In the case of PAA-P nanoparticles, there was a very little
improvement in the mechanical strength up to day14 after which properties started degrading back to the
initial values. Though these moduli of the constructs containing PAM-nanoparticles were observed to
increase monotonically with time, the values were still lower than that of the control scaffolds.
The biochemical analysis provided interesting insights into the possible reason for lower values.
The glycosaminoglycan content of control on day-60 (Figure 5.7) was higher than both PAM and PAA-P-
containing constructs (similar to mechanical strength), whereas the DNA content per dry weight basis was
higher for PAM and PAA-P containing constructs when compared to control. This implies that, despite
having enough viable cells, the synthesis of extracellular matrix (GAG) was limited in the presence of the
acrylate nanoparticles. The confocal images (Figure 5.8) offer additional evidence for no difference in the
cellular viability between the control and nanoparticle scaffolds.
78
Figure 5.6 Mechanical properties of control, polyacrylamide containing (PAM) and polyacrylic acid-
Phosphonate (PAA-P) nanoparticles-containing constructs. (a) Equilibrium Young’s modulus, and (b)
dynamic modulus at 0.1 Hz.
These results indicate that the reason for the poor mechanical strength of the polymer
nanoparticles-containing scaffolds cannot be either cellular viability or pH shock. Even though the
nanoparticles are biocompatible, they may possess bioactivity detrimental to the synthesis of extracellular
matrix. It can be noted from the zeta potential of these nanoparticles at physiological pH ~7 (Figure 3.13)
that the higher the anionic charge of the polymer introduced externally into extracellular space the poorer
the matrix synthesis. In particular, anionic charge of the PAAP nanoparticles was greater than -45 mV
(Figure 3.13) and thus can disrupt the inter-cellular signaling process more effectively. It clear from these
results that chondrocytes has low tolerance for anionically charged polymers.
a
b
79
Figure 5.7 Total GAG & DNA content (in mg) of control, polyacrylamide containing (PAM) and polyacrylic
acid-Phosphonate (PAA-P) containing scaffolds
Figure 5.8 Live/Dead assay of scaffolds using fluorescence microscopy, for control, polyacrylamide
containing (PAM) and polyacrylic acid-Phosphonate (PAA-P) containing scaffolds on day0 and day33
0 10 20 30 40 50 60
0
10
20
30
40
50
60
70
80
90
GA
G/d
ry w
eig
ht
(w/w
)
Days
Control PAM PAAP
0 10 20 30 40 50 600.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
DN
A/d
ry w
eig
ht
w/w
Days
Control PAM PAAP
80
5.3 Conclusions:
In summary,
1) The PSA microparticles were found to be cytotoxic at a concentration of 20 mg/mL, but
benign at 2 mg/mL.
2) The mechanism of cytotoxicity is determined to be due to intracellular accumulation of PSA or
its degradation products and the resulting lipotoxicity. Changes in local pH due to polymer
hydrolysis was ruled out to have a major impact on cellular viability
3) Of the several mitigation strategies tested, bovine serum albumin treatment, was found to
offer the best protection from adverse effects due to the polymer. Since it is known, in
general, that serum albumin is a natural regulator for intracellular lipids lipids and prevents
lipid overdose, the chondroprotection of BSA against PSA provides additional proof for its
lipotoxicity
4) The BSA-based mitigation strategy makes PSA microparticles effective in the short-term
delivery of TGF-β3 in articular cartilage engineering.
5) The effect of functional groups present in poly-acrylate nanoparticles on cellular viability and
metabolism was studied. The polyacrylate nanoparticles are found to be biocompatibile .
6) Even though the nanoparticles are biocompatible, they possess a bioactivity which is
detrimental to the synthesis of extracellular matrix. This is confirmed by the poor mechanical
strength of the polymer nanoparticles-containing constructs when compared to control
constructs.
7) There is an inverse correlation between mechanical strength and the negative surface charge
of these nanoparticles. In particular, phosphonate nanoparticles containing constructs
exhibited the worst mechanical properties and is correlated to their high anionic charge (> -45
mV)
8) Disruption of the inter-cellular signaling process due to higher anionicity is proposed to be the
cause for poor matrix synthesis.
81
9) It clear from these results that chondrocytes have low tolerance for the extraneous anionically
charged polymers. Thus, further studies are needed to understand if the anionicity affects the
chondrogenic potential of stem cells too!
82
Chapter 6 Anti-inflammatory and anti-oxidant
bioactivity of ceria nanoparticles in articular
cartilage engineering
The interleukin-1α is one of the cytokines that cause inflammation at the damaged, diseased, or
implanted cartilage site, leading to tissue damage and cellular death. Here, the objective of this study is to
explore the chondroprotective potential of nanoceria as well as the applicability of nanoceria as an anti-
inflammatory agent against interleukin-1α.
This chapter is organized as follows: first section briefly details the experimental design and
parameters. The following two sections detail the effects of two different ways of adding nanoceria to a
3D-agarose based culture. The final section lists the main conclusion of this study.
6.1 Experimental design
The chondrocytes from articular cartilage were harvested from the knee joints of juvenile bovine.
The chondrocytes from the 1st passage were trypsinized and cast into a 2% wv agarose slab at 60M
cells/mL concentration with or without nanoceria at different concentrations.
Nanoceria were added into the system in two different modes:
In the first, nanoceria along with chondrocytes were seeded into the 3D hydrogel slab on day-0
but not added to the growth medium (Scheme 1a). The nanoceria was embedded at different
concentrations (10, 100, and 1000 μg/mL) during casting (day 0) whereas, the insult by IL-1α began on
day 16. The IL-1α was added to the growth medium at 10 ng/mL and replenished whenever the medium
was changed.
83
In the second, nanoceria was added only to the growth medium but not cast into the hydrogel
(Scheme 1b). The constructs were cultured for 21 days to obtain constructs with mechanical strength
suitable for IL-1α insults. On day 21, constructs were divided into 12 combinations of control and
experimental sets: combination of three IL-1α concentrations (0, 0.5 ng/mL, and 10ng/mL) and four
nanoceria concentration (0, 100, 1000, and 20000 μg/mL). In the experimental sets both nanoceria and
IL-1α were added simulataneously. One of the samples (nanoceria:1000 μg/mL and IL-1α: 10 ng/mL) was
lost due to infection.
Scheme 6.1 Two different ways of introducing nanoceria in a 3D-agarose based culture of chondrocytes.
(a) nanoceria is embedded in 3D agarose constructs during casting on day-0 (b) nanoceria was added in
to the growth medium along with interleukin from day-21
a
b
84
6.2 Effects of nanoceria embedded into the 3D-agarose scaffold on tissue growth as well
as combating inflammation due to interleukin-1α
On days 16 and 31, the equilibrium compressive Young’s modulus and dynamic moduli (at
0.1, 0.5, 1 Hz), as well as glycosaminoglycans (GAGs), collagen, and DNA content were measured.
Additionally, cellular viability was monitored using the Live/Dead cytotoxicity kit. The Young’s and
dynamic modulus at 0.1 Hzare reported in the Figure 6.1.
As can be seen from Figure 6.1, nanoceria at loadings of 100 and 1000 μg/mL improved
significantly the mechanical properties of the constructs in the absence of IL-1α. The direct contribution of
nanoceria to the mechanical strength can be easily ruled out because the increase in strength of
nanoceria-containing construts with time in relation to control was not constant.The nanoceria at 100 and
1000 μg/mL showed statistically significant difference in mechanical properties (Table 6.1). The presence
of nanoceria at 10ug/mL had minimal effect upon their properties and was not better than the control. The
biochemical analyses of these samples reveal an interesting pattern (Figure 6.2). Though DNA content
remianed the same across the sample sets, the GAG content and collagen content was higher for 1000
μg/mL nanoceria samples when compared to the rest. Thus, it can be concluded that nanoceria increases
the extracellular matrix production.
However, when IL-1α is present at a concentration of 10 ng/mL, nanoceria did not prevent
tissue degradation (Figure 6.1). As a result, the loss of mechanical strength was comparable to that of
control+IL-1α. The biochemical analysis revealed that both GAG and collagen content was reduced due
to the action of IL-1α across all the sample sets.
It should be noted, however, that the concentration of IL-1α (10 ng/mL) used in this study is very
high compared to its in vivo concentration (~500 times smaller than 10 ng/mL). As noted earlier, this
concentration was selected because it was found in previous studies to be appropriate for in vitro
experiments. Furthermore, the probablity of IL-1α coming into contact with cells may be higher than the
nanoceria getting to be internalized by the cells. The cellular viabilitiy on day 30 determined by Live/Dead
analysis remained the same across all the sets of samples (Figure 6.3). This fact provides additional
85
evidence (apart from DNA content (Figure6.2c)) that IL-1α was more active in degrading the matrix
degradation rather than being cytotoxic to chondrocytes .
Figure 6.1 Mechanical properties of control and nanoceria-containing constructs on day 16 and day 30.
Interleukin-1α was added from day 16 to 30. Nanoceria concentration in constructs are 10, 100 and 1000
μg/mL. Control constructs contain no nanoceria (a) Equilibrium Young’s modulus, (b) dynamic modulus at
0.1 Hz. The significance are *p<0.001 and **p<0.001
Figure 6.2 Biochemical composition of control and nanoceria-containing constructs on day 16 and day 30
as a fraction of wet weight. Interleukin-1α was added from day 16 to 30. Nanoceria concentrations in
constructs are 10, 100 and 1000 μg/mL. (a) Glycasoaminoglycan (GAG) (b) Collagen (c) DNA
86
Table 6.1 Two way ANOVA for all the mechanical properties (equilibrium Young’s modulus, dynamic
modulus at 0.1, 0.5, and 1 Hz) on day 30 of control and nanoceria-containing constructs without
interleukin-1α insults.
Figure 6.3 Live/dead analysis of control and nanoceria-containing constructs on day 30 using cytotoxicity
kit and confocal fluorescence microscopy. Green color indicates live and red color indicates dead cells.
Top row contains images of control, nanoceria-containing constructs at 10, 100, 1000 μg/mL
concentration without interleukin-1α insult. Bottom row: with interleukin-1α insult from day 16 to 30.
Samples ( only no IL-1α sets) P-valueⱡ
Control vs. nanoceria 10 μg/mL 0.5276
Control vs. nanoceria 100 μg/mL 0.0482
Control vs. nanoceria 1000 μg/mL 0.0004
nanoceria 10 μg/mL vs. nanoceria 100 μg/mL 0.0312
nanoceria 10 μg/mL vs. nanoceria 1000 μg/mL 0.0009
nanoceria 100 μg/mL vs. nanoceria 1000 μg/mL 0.1163
ⱡStatistically significant values (P<0.05) are shown in bold letters
87
6.3 Effects of nanoceria suspended in the growth medium on tissue growth as well as
combating inflammation due to interleukin-1α
In this study, instead of embedding nanoceria in the 3D-agarose scaffold, they were suspended
only in the growth medium. The rationale for the nanoceria addition to the growth medium was to
deactivate the inflammation by acting directly on IL-1α in the suspension medium itself. Several
conclusions can be drawn from the mechanical strength measured on day 34 (Figure 6.4). The anti-
inflammatory activity of nanoceria was observed at the highest concentration of both IL-1α and nanoceria
(Figure 6.4, nanoceria: 2000 μg/mL and IL-1α: 10 ng/mL). The Young’s modulus and dynamic moduli
were nearly twice higher than the control. The lower concentration of IL-1α (0.5 ng/mL) had negligible
effect on the constructs in vitro. In the absence of effects of IL-1α (0 ng/mL), there was a maximum
concentration (1000 μg/mL) of nanoceria at which mechanical properties were also maximum (see Figure
6.4). This again proves that nanoceria exerts postive effects on the mechnical and probably on
biochemical properties as well. The anti-inflammatory and chondroprotective actions of nanoceria were
also confirmed by GAG content measured on day 34 (Figure 6.4c). The GAG content of samples insulted
by 10 ng/mL of IL-1α in the presence of 20000 μg/mL nanoceria was higher than the control and other
concentrations. The maximum concentration of nanoceria for such positive effects can be between 1000
μg/mL and 20000 μg/mL. This maximum limit above which properties degrade can be explained as
follows.The reducing capacity of nanoceria, which is important for its ROS scavenging activity (Section
1.1.3), might become detrimental at high very particle concentrations. That is, a decrease in the oxygen
partial pressure of the growth medium due to high nanoceria concentration (20000 μg/mL) can be the
reason for drop in the mechanical strength in the absence of effects of IL-1α.
88
Figure 6.4 Mechanical and biochemical properties of constructs at day 34 as a function of nanoceria
concentration in the growth medium. (a) compressive Young’s modulus, (b) dynamic modulus at 0.1 Hz,
(c) glycosaminoglycan. Interleukin-1α is added from day 21 to 34 and its concentrations are given on the
top of the bars (0, 0.5, 10 ng/mL). For IL-1α 10ng/mL and 20000 μg/mL vs. 0 μg/mL *p<<0.00001
6.4 Conclusions
Nanoceria is found to combat inflammation due to interleukin-1α as well as enhances the mechanical
properties of cartilage through increased GAG and collagen production.
1) At concentrations up to 1000 μg/mL, nanoceria was biocompatible with chondrocytes.
2) The nanoceria has a beneficial impact on the biochemical as well as mechanical properties of cartilage
tissue grown in vitro.
3) An insult of the sample sets with interleukin-1α at 10 ng/mL for 12 days significantly degraded the
mechanical properties, GAG and collagen content.
4) The presence of nanoceria at 20000 μg/mL in the growth media was chondroprotective against the
inflammatory action of interleukin-1α and partially prevents matrix degradation.
89
Chapter 7 Dependence of the (bio)activity of
hematite NPs on their size, morphology, and
surface charge
The interactions of ferric (hydr)oxide nanoparticles (NPs) with chondrocytes and fatty acids are
reported. The impact of nanoparticle size and shape of ferric (hydr)oxide NPs on chondrocyte’s viability
and metabolism is reported in the first section. It was found that the mechanical strength of the NP-
containing engineered cartilage constructs increases with decreasing NP size.
In the second section, the interaction of these NPs with sodium dodecanoate (sodium laurate)
and the effects of size, morphology, and surface charge on this interaction is studied using results from
adsorption isotherms, zeta potential, contact angle, and FTIR measurements. Both the studies include
ferrihydrite (low crystalline hydrous ferric (hydr)oxide NPs of 2-5 nm size) for comparison as the end-
member the nanosize phase transformation of hematite. Contrary to the common correlation, the
adsorption capacity and affinity of hematite NPs decreases with decreasing particle size. The pattern with
which fatty acids fill the NP surface depends on the NP morphology. These results are rationalized taking
into account the nanosize induced changes in the catalytic and electron-accepting properties of hematite,
as well as the geometrical compatibility of the surface site arrangement and the attachment of carboxylate
groups.
7.1 Size, shape and surface-charge dependent bioactivity of hematite NPs on
chondrocytes
The goal of this study is to verify the hypothesis that the bioactivity of hematite NPs increases
with an increase in the NP size. This hypothesis is based on the previous results on the abiotic catalytic
activity of hematite NPs from our group.116
90
To test this hypothesis, three different sizes of hematite NPs (H9, H38 and H150) and 2-nm FH
were incorporated into the 3D-agarose hydrogel along with juvenile bovine chondrocytes at 30 million
cells/mL and cultured for 30 days. The NPs were added to the hydrogel in the weight range of 25-50
g/mL. A control hydrogel slab was cast at the same cell density but without NPs. The mechanical
properties and cellular viability were determined at the end of day-30 and day-71, respectively.
For comparison, the equilibrium Young’s modulus and the dynamic moduli are plotted (Figure
7.1) as a function of the loaded total surface area of the NPs, SAloaded. This was calculated as SAloaded =
SA m, where SA is the surface area per unit mass obtained using single point BET, m is the loaded
weight of the NPs per unit volume of the scaffold. As seen from Figure 7.1, the detrimental effect of the
hematite NPs on the mechanical properties of the grown tissue decreases with decreasing NP size, in
agreement with the initial hypothesis. The maximum and minimum effects on chondrocytes are for 150-
nm hematite and 2-nm FH, respectively. The following power law relationship between the decrease in
equilibrium Young’s modulus of the tissue constructs due to the presence of ferric (hydr)oxide
nanoparticles compared to control and the size of the nanoparticles is obtained (See Appendix C).
Normalized decrease in Young’s modulus = 0.048. (nanoparticle size)0.63
At the same time, no difference due to size in the cellular viability is observed among the NPs. In
fact, there is no difference in the viability between the control and the NP-exposed sets (Figure 7.2). This
implies that the NPs affect mostly the chondrocyte’s function of extracellular matrix production rather than
trigger cascades of cellular reactions leading to apoptosis. This finding matches with the previously
reported low or no toxicity (no DNA damage and no reactive oxygen species (ROS) production) of
hematite NPs toward cardiac, microvascular, endothelial and lung/bronchial epithelial cells.70, 115, 179, 181
Significant genotoxic effects have previously been observed for hematite only at loadings higher than 50
g/mL,178
that is, higher than the amount used in the present study.
To interpret this finding, our previous result of the nanosize effect on the oxidative catalytic
activity of hematite NPs may be recalled.116
Using batch kinetic measurements coupled with X-ray
photoelectron spectroscopy (XPS) as well as a new method that combines in situ FTIR spectroscopy and
ex situ XPS, the oxidative catalytic performance of hematite NPs was found to degrade with a decrease in
91
their size. It was demonstrated that these results can be accounted for by the nanosize-induced changes
in the electronic properties of ferric (hydr)oxides, along with the thermodynamic properties of the system,
given that the catalytic reaction proceeds through the electrochemical pathway. Briefly, as NP size
decreases, hematite NPs experience phase transformation to FH. These structural changes are
accompanied by an increase in the ionicity of the FeO bonds, as well as by opening of the
semiconductor band gap. The latter effect decreases the electron affinity of the NPs, and hence their
capacity to abstract electron from the reductant and to conduit it to a cathodic spot where O2 is reduced.
Therefore, the enhanced catalytic activity of larger NPs can lead to the oxidation of extracellular
biomacromolecules such as GAG and collagen, resulting in the lower strengths of the produced matrix.
A further non-invasive Raman spectroscopic study in future of the molecular composition and
structure of the extracellular matrix, as well as TEM studies of the NP localization in the scaffolds may
provide more detail on the effects observed.
92
Figure 7.1 Mechanical properties of control and feriic (hydr)oxide NPs-containing constructs on day
30.The concentration of the NPs is plotted as total surface area. (a) Equilibrium Young’s modulus,
dynamic modulus at (b) 1 Hz (c) 0.5 Hz (d) 0.1 Hz.
a
b
c
d
Control
Control
Control
93
Figure 7.2 Live/dead analysis of control and ferric (hydr)oxide NPs-containing constructs on day 72 using
cytotoxicity kit and confocal fluorescence microscopy. Green color indicates live cells, red color indicates
dead cells. Top row contains images of control, FH, and H9-containing constructs at 25 μg/mL
concentration and bottom row that of H38 and H150 containing-constructs at two different NP
concentrations (25, 50 μg/mL)
7.2 Size, shape and surface-charge dependent interaction of hematite NPs with sodium
laurate (dodecanoate)
This section is organized as follows: First, variation in the dispersion and adsorption properties of
laurate-coated ferric (hydr)oxide NPs is discussed including hydrophobicity and surface charge. Next,
spectroscopic data on the adsorption configuration of the fatty acid, as well as on the adsorption
mechanism and the chain packing order are presented.
7.2.1 Dispersion and adsorption properties of hematite NPs in the presence of sodium
laurate.
I found that at pH 7.2 and concentrations of sodium laurate (SL) lower than CMC (~1mM at pH
7.2),161
all the sizes ferric (hydr)oxide NPs flocculate and settle particles in 5mM NaCl aqueous solution.
In contrast, at SL concentrations higher than the precipitation limit (~ 2.5 mM at pH 7.2), H150 and H38
particles are well dispersed for at least two weeks, FH aggregates and settles, and H9 is partially
94
H150 FH H9 H38
dispersed (Figure 7.3). As compared to FH, hematite NPs are better dispersed: An increase in size of the
ferric (hydr)oxide NPs is accompanied by a regular improvement in their dispersion properties.
Figure 7.3 Suspensions of FH and hematite NPs at initial SL concentrations of 3 mM and ionic strength
of 5 mM NaCl.
To understand this difference in the NP dispersibility, the adsorption isotherms were measured for
the largest and smallest NPs (H150 and FH, respectively) which represent the well crystalline hematite
and FH end-members of the phase transformation sequence of hematite NPs in water.222
As depicted in
Figure 7.4, at concentrations lower than ~3.5 mM, 150-nm hematite has a higher sorption capacity.
However, at higher concentrations, FH abstracts the surfactant much more efficiently. The corresponding
part of the FH isotherm presents a vertical branch shifted to a lower equilibrium (residual) concentration.
Although the mechanism for such abstraction is not clear, it is hypothesized that FH coagulates with
surfactant precipitates, thereby occluding surfactant above its precipitation limit. Additionally, the effect
can be related to a higher solubility of FH as compared to coarser-sized hematite NPs,106
resulting in
precipitation of ferric laurate. Importantly, no such phenomenon is observed for H150 at the initial laurate
concentration up to ~7 mM. Instead, the adsorption isotherm consists of two steps and two plateaus. The
first plateau, which starts at ~0.5 mM, signifies the formation of the first monolayer. This monolayer has a
relatively high density of 4.3 0.1 molecules/nm2
and imparts superhydrophobicity to H150 (Figure 7.5).
This surface density is slightly below 4.8 molecules/nm2 typical for the tilted condensed phase of fatty
acids at the water-air interface.232
Hence, these particles are almost fully wrapped by the SAMs of laurate.
As the surfactant concentration is further increased, the second monolayer is assembled with the
95
surfactant head groups pointing out into the solution, as follows from a decrease in the contact angle
(Figure 7.5). The second plateau is caused by the pinning of the chemical potential of the surface by the
surfactant precipitation at 2.5 mM. In this concentration range, a second monolayer is already assembled,
as evidenced by the doubling of surface density and the evident onset of strong dispersion (Figure 7.6).
On this basis, it is concluded that the propensity to be more fully wrapped by a bilayer brings about a
better dispersibility of the oxide NPs.
To study the effects of NP size and morphology on the adsorption properties, the
adsorption isotherms were measured on all the four sizes at pH 7.2 and an initial SL concentration up to 1
mM (CMC). The last data point of the all the isotherms in Figure 7.7a corresponds to 1mM initial SL
concentration. As seen from Figure 7.7a, both the adsorption capacity and affinity of hematite NPs
decrease with decrease in size. The laurate surface densities at CMC are 4.2, 3.3, 2.6, and 2.1
molecules/nm2 on H150, H38, H9, and FH, respectively. A power law fit is obtained between the
monolayer coverage and the size of particles. Monolayer coverage values are normalized with maximum
coverage value of H150 (4.2 molecules/nm2), see appendix C for details: Normalized monolayer coverage
= 0.4 . (size)0.19
Figure 7.4 Adsorption isotherms of sodium laurate on FH (wine) and H150 (blue) NPs at pH ~ 7.2.
96
Figure 7.5 Contact angles of SL-coated H150 nanoparticulate film. Formal monolayer coverage of 4.3
molecules/nm2 was estimated from the adsorption isotherm. Hydrophobicity of 150-nm hematite
increases as the surface coverage of laurate increases up to 1 ML. At this point, the particulate film is
superhydrophobic (water droplets are repelled from the surface). With a further increase of surface
coverage, hydrophobicity gradually decreased, indicating that laurate is adsorbed with the head group
pointing out into the solution.
Figure 7.6 Effect of the initial concentration of laurate (written at the bottom as well as on the vials) on the
dispersion of H150 particles at pH 7.2
The electrophoretic data (Figure 7.7b) indicates that at SL concentration of at least up to 1mM
H150 has a higher negative zeta-potential than the other extreme in size, FH. Unfortunately, the visual
particle-tracking method in the electrophoretic technique failed for the H150 suspensions with initial
laurate concentrations above 1 mM, where the particles become dispersed and strongly scatter light.
Nevertheless, the available data confirm that the SAM on H150 has higher density, with the electrostatic
0mM 0.25 mM 0.75 mM 1 mM 3 mM 5 mM
0.25 mM
97
mechanism of stabilization of the NP suspensions by laurate. In contrast, a lower (by a factor of 2)
adsorption capacity of FH gives rise to their less negative charge which may have caused their
aggregation.
Figure 7.7 (a) Adsorption isotherms of sodium laurate on FH (wine), H9 (orange), H38 (red) and H150
(blue) NPs at pH ~ 7.2. The last data point corresponds to 1 mM initial laurate concentration (CMC of
laurate) (b) Zeta potential of H150 and FH as a function initial laurate concentration in 5mM NaCl
aqueous solution. Both electrophoretic (red and blue curves) and electroacoustic (green curve) results
are shown. Connecting lines are drawn for reading ease.
7.2.2 Speciation of carboxylic groups in the first monolayer.
To elucidate the effects of the size, morphology, and surface charge of the ferric (hydr)oxide NPs on the
speciation of the carboxylic groups and the packing order of adsorbed fatty acids, in situ FTIR-ATR
spectra were measured on the NPs interacted with a 1 mM solution of SL at pH 10.0 (Figure 7.8) and
7.15 (Figure 7.9) for 25 min. It was observed that at pH 10, FH does not adsorb laurate at initial SL
concentration up to 1 mM, while H9up to 0.5 mM. Three main adsorption forms are identified from the
fingerprint region of asymmetric (asCO2) and symmetric (sCO2
) stretching modes of the carboxylate
group (Scheme 7.1 and Figure 7.8 & 7.9). These are (1) Inner-sphere monodentate mononuclear
complex (ISMM): specific adsorption of one of the oxygen of laurate molecule with one of surface ferric
ion, whereas the second oxygen H-bonds with other surface hydroxyl or water. (2) Outer-sphere surface-
hydration complex (OS): non-specific adsorption of laurate on surface ferric ions mediated by a hydration
layer (3) Protonated outer-sphere surface-hydration complex (OS-H): non-specific adsorption of lauric
a b
98
acid on surface ferric ions mediated by a hydration layer. The details of such identifications using
combination DFT and spectroscopic data are provided in Chapter 9.
Figure 7.8 in situ FTIR-HATR spectra of FH and hematite NPs in contact with a 10–3
M solution of sodium
laurate at pH 10.00 ± 0.05 with no added electrolyte in spectral region of (a) COO and (b) CH
vibrational modes: (olive) H150, (red) H38, (blue) H9. Conditioning time is 2530 min.
Scheme 7.1 Cluster models of surface binding of fatty acid: (a) inner sphere monodentate mononuclear
(ISMM) carboxylate complex with H-bonded second oxygen (b) outer sphere (OS) surface-hydration
shared ion pair, and (c) OS fatty acid. Inner-sphere monodentate mononuclear complex: specific
adsorption of one of the oxygen of laurate molecule with one of surface ferric ion, whereas the second
oxygen H-bonds with other surface hydroxyl or water. Outer-sphere surface-hydration complex: non-
specific adsorption of laurate (or lauric acid) on surface ferric ions mediated by a hydration layer.
ISMM OS surface
hydration-shared
b a c
OS surface hydration-
shared protonated
99
Figure 7.9 in situ FTIR-HATR spectra of FH and hematite NPs in contact with a 10–3
M solution of sodium
laurate at pH 7.15 ± 0.05 with no added electrolyte in spectral region of (a) COO and (b) CH
vibrational modes: (olive) H150, (red) H38, (blue) H9, (magenta) FH. Conditioning time is 25 min.
Intensity of the spectra in the 12001800-cm–1
region is normalized by the intensity of the sCH2 band
a. Inner-sphere monodentate mononuclear complex (ISMM):
The main bands which dominate at ~1540 and ~1410 cm–1
are assignable, respectively, to the
asymmetric (asCO2) and symmetric (sCO2
) stretching modes of the carboxylate group of an ISMM
complex (Scheme 7.1).233
b. Outer-sphere hydration shared complex (OS):
The second asCO2/sCO2
pair at 1530/1425 cm–1
is attributed to an OS hydration-shared
complex.233
The positions and relative intensities of these two pairs of bands vary among the NPs.
Specifically, H38 and H150 are characterized by the lowest and highest positions of the sCO2 bands. It
is known from previous studies234, 23518,59
when there is an increase in the positive charge of the adsorbent
100
surface, the sCO2–
band of chemisorbed carboxylates decreases (Scheme 7.2). Indeed, as seen from
Figure 7.10, the red shift of the sCO2–
frequency can be correlated with an increase in PZC and hence
the surface charge of the NPs.
c. Protonated outer-sphere hydration shared complex (OS-H):
Another feature of the FTIR spectra measured at pH 7.15 (Figure 7.9a), which is missing at pH
10.0 (Figure 7.8a), is the C=O band at 1707 cm–1
of the OS complexes of lauric acid.233
For comparison
purposes, the spectra are normalized by peak intensity of the sCH2 band at ~2850 cm
-1. As seen from
Figure 7.7a, the C=O band is hardly distinguishable for FH and is maximal for H150. For H9 and H38, its
intensity is similar and intermediate (H150 > H38 ≈ H9 >> FH).
The Figure 7.9a also demonstrates a variation in the OS/ISMM intensity ratio for the sCO2
band
among the NPs: This ratio decreases in the following order FH H150 > H9 H38. As follows from the
effect of negative polarization on the intensity ratio (vide infra), it can be argued that the OS/ISMM ratio is
controlled by the adsorption capacity of the NPs towards the ISMM complexes (i.e., the concentration of
suitable strong adsorption sites).
Scheme 7.2 Stretching of sCO2– dipole of adsorbed carboxylate by positive charge of the NP surface.
Figure 7.10 Correlation between frequency of sCH2 band of ISMM laurate in the spectra shown in
Figure 7.9a and PZC of FH and hematite NPs measured in open air (Figure 7.7b)
6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
1402
1404
1406
1408
1410
1412
H38
H9
FH
sC
OO
-
PZC, air
H150
101
7.2.3 Packing order of hydrocarbon chains.
The conformational order of the aliphatic chains is an critical parameter to be studied, as it is
directly related to the wetting properties of the SAM. The symmetric stretching vibrations of methylene
groups sCH2 at ~2850 cm1 is used for
assessing this information: This band shifts to lower
wavenumbers and narrows with increasing packing order of an ensemble of long-chain molecules.149
Then, as seen from Figures 7.8b and 7.9b, the conformational order of the aliphatic chains in the adlayers
formed at both pH 7.15 and pH 10.0, respectively, increases in the following order H38 < FH < H9 <
H150. This order correlates neither with the adsorption density of the surfactant (Figure 7.7), nor with the
composition of the ML (the fractions of the OS complexes and the lauric acid) (Figure 7.9a).
To get more details, the patterns of the monolayer filling with time were analyzed. As seen from
Figure 7.11a,c,d, except for the initial stage (the formation of ~0.030.1 ML), the sCH2 band does not
change its position when the surface coverage of laurate increases up to 1 ML on FH, H9, and H150,
suggesting the cluster-like pattern of the monolayer growth. In contrast, the sCH2 band continuously
shifts downwards in the case of H38 (Figure 7.11b), which is typical of the isotropic filling. Hence, H38
demonstrates distinct monolayer filling, which is reasonable to ascribe to the distinct rhombohedral
morphology of these NPs (Figure 4.1 and 4.2), as well as to their high capacity to form chemical bonds
(ISMM complexes) with carboxylic groups (Figure 7.9a). Due to the predominance of the ISMM
adsorption, it can be suggested that the headgroup-surface interaction is possibly stronger than the
lateral hydrophobic, dispersion, and H-bonding interaction between the assembled surfactant molecules.
The morphological homogeneity of H38 (which is enclosed by rhombohedral facets of similar surface
energy106
) is also expected to contribute to this phenomenon.
Analysis of the main crystallographic surfaces of hematite and typical chain-chain packing
distance of laurate confirms this interpretation:
In adsorbed monolayers, the chains can pack in four different subcells: orthorhombic, triclinic,
monoclinic or disordered pseudohexagonal subcells.236,149
In all these packing motifs listed, the chains
are arranged such that the chain-chain distance of 4.75.5 Å.237, 238
Given that only tilt angles between 0
and 30 have experimentally been observed for adsorbed monolayers,149, 237
the surface projected
distances of well packed chains are 4.76.3 Å.
102
As seen from Figure 7.12c,d, ferric cations are arranged on the (104) and (012) rhombohedral
hematite planes in a parallelogramic fashion, with the FeFe distances of 3.71 in the rows of ferric
octahedra and 5.04 Å between the rows. Because the distance of 3.71 Å is much shorter than the low
limit of ~4.7 Å of the FeFe range which supports ordering, the adsorption sites in the rows can be
occupied only partially, with the minimum FeFe distances of 7.42 Å. This distance of 7.42 Å is
incompatible with a conformational ordering of the fatty acids if they are chemisorbed on these sites.
Thus, the geometrical mismatch and the dominance of the chemisorbed complexes on H38 preclude a
dense packing of the SAM.
In contrast, cations are (pseudo)hexagonally distributed on the {101} and iron-depleted {001}
facets (Figure 7.12a,b) which enclose hematite platelets.239
Moreover the FeFe distances of 5.44 and
5.04 Å on these surfaces are within the region of 4.76.3 Å suitable for the chain ordering.
7.2.4 Controls of NP dispersion by fatty acids. Formation of a bilayer.
Although the stabilization of NPs in suspensions by fatty acid bilayers is a known phenomenon,240
the current study is the first to observe both the formation of such bilayers on metal (hydr)oxide NPs in
water and the dependence of this phenomenon on NP size. Namely, the fatty acids are found to better
disperse larger ferric (hydr)oxide NPs, independent of their morphology. The effect is related to a higher
adsorption capacity of the larger NPs: A denser formal monolayer attaches more molecules to the second
layer, because the bilayer formation by a surfactant at the solid-water interface is governed by the
hydrophobic and the London-van der Waals dispersion interactions.125
The formation of a bilayer imparts
the highest hydrophilicity and negative charge to the particles, which corresponds to the best conditions
of the electrostatic stabilization of the suspension.
Interestingly, the smallest particles, FH, have displayed strong ability to occlude laurate above its
precipitation limit. The mechanism of this effect is still unclear: It can tentatively be related to the
enhanced tendency of FH to coagulate with the surfactant precipitates due to the partial coating of FH by
a bilayer. Another reason can be a higher solubility of FH as compared to hematite.
103
Figure 7.11 Effect of surface coverage on the position of the sCH2 band of laurate adsorbed on (a)
H150, (b) H38, (c) H9 hematite, and (d) FH from 1 mM solution at pH 10.0 and 7.15. Surface coverage,
evaluated using the sCH2 peak intensity, increases from (red) ~ 0.030.1 to (magenta) 1.0 formal ML
which is assumed to be formed at the maximum interaction time. It is increased with increasing the
conditioning time which is indicated in the graph. The band intensity is normalized to the maximal value in
the series. Color codes for H150, H38, and H9 are the same. FH was conditioned only at pH 7.15
because it does not adsorb 1 mM laurate at pH 10.
2880 2870 2860 2850 2840 2830 28202880 2870 2860 2850 2840 2830 2820
pH10 5'
pH10 47'
pH10 65'
pH7.15 5'
pH 7.15 30'
pH7.15 50'
2880 2870 2860 2850 2840 2830 2820
2880 2870 2860 2850 2840 2830 2820
2'
8'
25'
35'
130'
2880 2870 2860 2850 2840 2830 2820
2'
8'
25'
35'
130'
2880 2870 2860 2850 2840 2830 2820
pH10 5'
pH10 47'
pH10 65'
pH7.15 5'
pH 7.15 30'
pH7.15 50'
H9
a
b
c
H150
H38 FH
d
Wavenumber, cm-1
Wavenumber, cm-1
Wavenumber, cm-1
Wavenumber, cm-1
Ab
so
rba
nce
, A
U
Ab
so
rba
nce
, A
U
Ab
so
rba
nce
, A
U
Ab
so
rba
nce
, A
U
surf
ace c
overa
ge
incre
ases up t
o 1
ML
104
Figure 7.12 Arrangement of surface cations on (001) basal, (101) hexagonal as well as on (012) and
(104) rhombohedral hematite surfaces.
7.2.5 Effect of negative polarization on the structure of the first monolayer.
To verify if the morphology-driven geometrical incompatibility and the high propensity to
chemisorb carboxylates are the main obstacles in the chain self-assembly, the effect of negative
polarization on the speciation and packing of the first monolayer of laurate on these NPs was studied. As
105
shown in a recent study,151
the surface charge of the metal oxide NPs can be changed by depositing
them on substrates with different work functions. Specifically, deposition on Ge renders negatively charge
to hematite NPs. As seen from Figure 7.13a, the negative polarization of H38 shifts the adsorbate
equilibrium from the ISMM to OS complexes and from laurate to lauric acid within the OS complexes. This
conclusion is based on an increase in the relative intensity of the OS sCO2– band at ~1420 cm1
and the
C=O band at 1707 cm–1
, respectively. The blue shift of both the OS and ISMM sCO2– bands reflects the
negative polarization of the NPs on Ge. Importantly, the change in the speciation of the carboxylate
groups is accompanied by an increase in the chain packing in the monolayer which is manifested by a
pronounced red shift and narrowing of the sCH2 band (Figure 7.13b). As a result, the chains become
even more ordered than in the SAM formed on H150 when the latter is deposited on ZnSe (Figure 7.14).
Hence, partial suppression of chemisorption can bring about a better chain ordering and higher
hydrophobicity of the fatty-acid capped NPs.
Figure 7.13 Effect of the substrate on in situ FTIR-HATR spectra of H38 hematite NPs in contact with a
10–3
M solution of sodium laurate at pH 7.15 ± 0.05 with no added electrolyte in spectral region of (a)
COO- and (b) CH vibrational modes: NPs were deposited on a (green) Ge and (red) ZnSe IRE.
Conditioning time is 25 min.
106
Figure 7.14 Comparison of in situ FTIR-HATR spectra in the CH region of (red) H38 deposited on Ge
and (violet) H150 deposited on ZnSe. The NPs were conditioned similarly with a 10–3
M solution of
sodium laurate at pH 7.15 ± 0.05 with no added electrolyte for 2530 min.
7.2.6 Speciation of adsorbed fatty acid. Effects of the oxide acidity.
Although the surface density and packing order of laurate is not affected by the intrinsic proton
surface charge of NPs, the speciation of laurate is affected. Namely, a higher OS/ISMM surface ratio of
laurate on H150 and FH which have lower proton surface charge (Figure 7.9a) is observed. Although a
further theoretical and thermodynamic study is required to understand this effect, it is suggested that the
chemisorbed complex is destabilized by negative proton surface charge due to the hindered ligand
exchange. In fact, a ligand exchange with surface hydroxyls has a higher energy penalty than with more
weakly chemisorbed water.241
The same effect is observed when the NPs are negatively polarized by Ge
(Figure 7.13 and 7.14). Therefore, both proton and electron charges control the OS/ISMM ratio.
Interestingly, in contrast to the OS/ISMM surface ratio, the fraction of the protonated OS
carboxylates does not depend on the macroscopic surface charge but on its size: It decreases as NP size
decreases (H150 > H38 H9 > FH). To rationalize this finding, It should be recalled that at pH 7.2 and 1
mM concentration, the surfactant exists in the bulk solution as acid-containing associates (either
aggregates165
or precipitates162
). The negligible amount of the OS lauric acid on FH suggests that the acid
-0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
Ab
sorb
an
ce
2800 2850 2900 2950 3000
Wavenumbers (cm-1)
107
molecules are deprotonated upon physisorption on these NPs (Scheme 3), suggesting a higher basicity
of the negatively-charged oxygen sites on FH as compared to the larger NPs. On the other hand, the
surface fraction of the physisorbed acid increases with increasing surface coverage.233
Therefore, the net
result is a sum of the contribution of the intrinsic acid-base properties and their adsorption capacity of the
NPs.
Scheme 7.3 Deprotonation of lauric acid by basic surface sites upon adsorption.
7.2.7 Conditions for SAM formation. Effects of the NP size, morphology, and surface charge.
It is observed that only the largest (150 nm), well-crystalline particles have surface energy high
enough to form self-assembled monolayers (SAMs) upon interaction with fatty acids in water. Within the
set of the NPs used in this study, a decrease in nanosize causes a decrease in both the adsorption
affinity and capacity of the NPs (H150 > H38 > H9 > FH), suggesting that the NP morphology (H38) and
porosity (H9) are secondary for this particular system.
At a first glance, this finding is at odds with the conventional belief that smaller sizes having more
defects and cus cations should have larger adsorption affinity and capacity. At the same time, this size-
dependence agrees with the observed decrease in the surface energy of hematite with a decrease in NP
size242
which can be related to the nanosize-driven phase transformation of hematite into metastable
FH.222
The phase transformation of hematite NPs is accompanied by the lattice expansion50,51
and a
decrease in the average electron affinity.109
As a result, the surface area per adsorption site increases,
while their average affinity towards the HOMO electron of the carboxylate group decreases in the row
H150 > H38 > H9 > FH. The positions of H9 and FH in the adsorption trend are consistent with the
nanosize-induced phase transformation of hematite NPs into FH and the presence of FH-like defects on
108
the hematite NPs smaller than ~40nm.1 In line with this finding is the observed degradation of the
oxidative catalytic properties of the NPs in the same order.110
The monotonic size-dependence of the adsorption capacity towards the surfactant suggests that
this property does not depend on the macroscopic intrinsic proton surface charge (originated from the NP
acid-base properties and measured using a macroscopic method): In fact, FH and H150 are the least
positively charged NPs at pH ~7.2 (Figure 7.7b), but display the lowest and highest adsorption capacity
for fatty acids, respectively. This is contrary to the adsorption of small ions,243
short-chain
monocarboxylates,244
and aminoacids,245
the surface density of which strictly follows the variation in the
surface charge of the NPs. The lack of the effect in the case of fatty acids can be explained by the major
role of non-electrostatic (hydrophobic and H-bonding) interactions in determining the adsorption.233
At the
same time, surface charge affects the binding mode of the carboxylic group (see below).
However, the above size dependency does not hold for the packing order of the hydrocarbon
chains in the formal monolayer: The packing order decreases as H150 > H9 > FH > H38. The worst
packing order on H38 is somewhat surprising given the relatively high adsorption density on these NPs.
This effect is explained by the dominance of chemisorbed fatty acid on H38 (Figure 7.9a) and the
geometric incompatibility of the rhombohedral (104) facets of these NPs with self-assembly. Fortunately,
the morphological obstacle can be removed by negatively charging the NPs: The resulting upward shift of
the Fermi level of the NPs decreases their capacity to accept electrons151
and decreases the surface
population of the ISMM laurate. The increased spatial gap between remaining chemisorbed fatty acids is
now filled by OS complexes which have more freedom to rearrange laterally and promote better packing
and the formation of SAMs, in agreement with the general trend observed for alkanoate SAMs on
extended alumina surfaces.246-248
In the case of weak adsorption affinity (FH), although both the ISMM
and OS complexes are formed, their low adsorption density precludes their ordering. It is then expected
that the positive polarization of FH can SAMs can be formed on such NPs upon.
109
7.3 Conclusions
The interaction of ferric (hydr)oxide NPs with chondrocyte can be summarized as follows,,
1) The mechanical properties of the constructs embedded with these NPs degrade with increasing
NP size as follows: Control > FH (2-nm) > H9 (9-nm) > H38 (38-nm) >> H150 (150-nm).
2) The cellular viability remains similar between all these sample sets.
3) It is suggested that the size effect is driven by the catalytic activity of NPs which is responsible for
the reactive oxygen species (ROS) generation.
4) These results together with beneficial effects due to nanoceria imply that the bioactivity is directly
related to the redox potential and the catalytic redox mechanism (including either the deactivation
or generation of reactive oxygen species) of the metal oxide NPs toward redox active constituents
at the biointerface.
In the study of ferric (hydr)oxide NPs-laurate interactions, the first experimental evidence is obtained for
the dependence of the surface density, speciation, and packing order of adsorbed fatty acids on the
morphology, size (and, hence, crystallinity109
), and surface charge/electric polarization of metal
(hydr)oxide NPs. The conditions under which fatty acids form SAMs and bilayers on such NPs in water
are distinguished and the electric polarization of the NPs is demonstrated to be a powerful tool for
manipulating the interfacial properties of the NPs.
Specifically,
1) Laurate is adsorbed on hematite mainly as a mixture of chemisorbed (ISMM) and physisorbed (OS
hydration-shared) complexes, both at pH > PZC and pH < PZC;
2) The physisorbed laurate is partially protonated by a coadsorbed hydronium cation as either surface
coverage increases or solution pH decreases;
3) Independent of morphology, an increase in nanosize improves the adsorption capacity and affinity of
hematite NPs, as well as increases the fraction of the protonated physisorbed laurate, in agreement with
the nanosize-induced changes in the structural and electronic properties of the NPs.
4) In the case of hematite NPs with a similar hexagonal habit, an increase in nanosize improves the
packing order of laurate.
110
5) Independent of morphology, the dispersibility of the NPs by fatty acids in water degrades as NP size
decreases. This effect is attributed to the decrease in the adsorption capacity which ends up in the
formation of a loosely packed bilayer.
6) Morphology of the NPs is critical for the self-assembly if (i) the majority of the molecules are
chemisorbed (ISMM) and (ii) the morphology-determined surface arrangement of the adsorption sites is
geometrically incompatible with a surfactant ordering.
7) The surface ratio of the physisorbed/chemisorbed surfactant depends on the surface charge of NPs.
8) Negative polarization suppresses chemisorption. This effect can be used to remove the geometrical
constraints for self-assembly.
9) The dominance of the chemisorbed complexes results in the isotropic surface filling by fatty acids,
while an increased fraction of OS complexes favors the cluster-like pattern.
111
Chapter 8 A new approach to DFT modeling of the
adsorbed species at the metal (hydr)oxide NPs-
water interfaces
The first section of this chapter provides details of the computational quantum chemical method-
Density Functional Theory (DFT) used in this study. The second section details the new approach to
modeling the adsorption properties of metal oxides in aqueous media using DFT simulations and FTIR
spectroscopy. The third section presents the results obtained by applying this approach for adsorption of
fatty acids on hematite NPs and final section summarizes the results.
8.1 A combined DFT-FTIR approach to resolve the adsorption forms at the oxide-water
interface
The density functional theory (DFT) has become one of the most applied computational
techniques among the several available quantum chemical simulation methods for understanding
molecular processes in solids, fluids and interfaces. This is mainly due to its relatively low computational
cost, ease of implementation and conceptual simplicity which allows further innovations.
In quantum mechanics, all the properties of a system can be, in principle, obtained a-priori, if the
wavefunction (ψ) of the system is known. The ψ can be calculated using Schrodinger equation, under
time-independent and non-relativistic conditions,
[∑ (
( ))
∑ ( )] (8.1)
Where, m and N are mass and number of electrons, is reduced Planck’s constant, ν and U are
potential functions and E is energy of the system.
112
The first term of the left hand side represents kinetic energy term, second and third terms are
interaction energies between nuclei-electron and electron-electron, respectively. Several approximate
methods such as Born-Oppenheimer, Hatree-Fock were developed. All these electron wavefunction-
based methods have the major limitation of being computationally intensive and their application in
surface and interfacial problems are still relatively impractical.
At the same time, a useful theorem derived by Hohenberg and Kohn249
stated that all the ground
state properties of a system are a function of charge density functional, ρ. This implies that Schrodinger’s
equation can be solved for charge density (ρ) instead of multiple electronic wavefunctions, Ψel. The
Schrodinger equation (8.1) can be written in terms of ρ as,
( ) ( ) ( ) ( ) (8.2)
The electron-electron interaction energy, U(ρ), can be further divided into single particle
electrostatic term, UH, and many-body exchange-correlation energy (Exc). Furthermore, the electron
kinetic energy term can also be divided into non-interacting single particle term, Tint (ρ) and a correlated
interacting term which can be added to Exc term above.
Thus, the Schrodinger equation is simplified as follows,
( ) ( ) ( ) ( ) ( ) (8.3)
The non-interacting kinetic energy Tnon-int(φ) is a function of molecular orbital functions, φ. Using
Thomas-Fermi approximation, Tnon-int(φ) can be expressed as,
( )
∑ ∫
( ) ( ) (8.4)
The single electrostatic term between electrons is given
( )
∫ ∫ ( ) ( )
| | (8.5)
113
There are no exact functional forms for exchange-correlation term, Exc. Several approximate
functionals have been developed either based on local density approximation (LDA) or more generalized
gradient approximation (GGA).250
The LDA approach assumes a uniform Exc in the smallest volume
considered in the DFT scheme. This approach is inadequate where there are strong electron correlations
(for eq. transition metal ions). Thus, the GGA approach is superior since it includes the rate of spatial
variation of charge density (ρ) in the Exc functionals.250
[ ] ∫ ( ( )) ( )) (8.6)
However, the equation (8.3) still cannot be minimized for energy directly with respect to charge
density, ρ, alone because the kinetic energy term, Tint, is dependent on the molecular orbital functions (φ).
However, the molecular orbitals themselves are a function of charge density functional, φ(ρ). To
circumvent this problem, Kohn and Sham251
suggested solving the Schrodinger equation for an auxiliary
non-interacting system to obtain molecular orbitals (φ). These orbitals were further used to calculate
charge density function (ρ) and solve equation (8.3) using a self-consistent field procedure.250
The DFT
simulations, using above procedure, were performed using DMol code available in Material Studio
program from Accelrys Inc.
8.2 A combined DFT-FTIR approach to resolve the adsorption forms at the oxide-water
interface
As mentioned in Section 1.6.1, to extract the structures of adsorbed carboxylate groups from their
in situ FTIR spectra, the DFT approach should be further developed to circumvent the inherent limitations
of the direct one-to-one correspondence between the theoretical and experimental spectra.
114
One way to solve this problem is to search for the adsorption structure that provides the best
correspondence in the trends of the theoretical and experimental spectra upon variations of the solution
pH and protonation-deprotonation of the carboxylate group.241
Specifically, the following algorithm of the spectrum interpretation can be suggested for fatty acids
adsorption.
1) The number of adsorbed species is determined from the fingerprint region of the FTIR
spectrum by the correlation analysis of the spectrum dependence on the surfactant concentration,
adsorption time, pH, ionic strength, and the electric polarization of the metal oxide.
2) The adsorbed species can be divided into weakly adsorbed or strongly adsorbed through the
effects of ionic strength variation and polarization of the substrate on the spectra.
3) The protonation state of these species can be delineated by observing spectral change with
change in pH, and the absence/presence of the typical band of carboxylic groups at 1700-1730
cm-1
.
4) The impact of H/D exchange on the spectra allows to further classify these species into outer-
sphere (OS) & monodentate or bidentate due to different solvating properties of light and
heavy water.
In parallel to these experimental methods, DFT simulations can be used to verify independently
any of the proposed structures and its dependence of the above mentioned parameters.
a. Firstly, using DFT simulations several adsorption forms can be a priori postulated and
tested to ascertain if their formation is energetically favorable.
b. The impact of ionic strength can be modeled in DFT simulations by introducing counter
cation near the double layer and verify if the calculated spectra of the proposed structure
show similar dependencies.
c. Similarly, pH can be modeled in DFT by providing a charge to the surface through
changing the ratio of surface hydroxyls to the water (attached to the surface cations)
115
d. The impact of H/D exchange on the FTIR spectra can also be compared by replacing the
hydrogen atoms at the interface with deuterium atoms for the hypothesized adsorption
forms in DFT simulation.
e. Finally the site specificity data from the etching and TEM/HRTEM experiments can be
understood by simple geometric modeling cation-cation distances of the specific facets of
the substrates and the corresponding parking distance for adsorbates
Figure 8.1 A general predictive paradigm for identifying adsorption forms on metal (hydr)oxides using
density functional theory simulations and FTIR spectroscopy.
116
8.3 Identification of adsorption forms of fatty acid on ferric (hydr)oxide NPs
As a model for ferric (hydr)oxide NPs, hematite NP of 150-nm size (H150) was selected and for
fatty acid, sodium laurate (SL), CH3(CH2)10COONa, a commonly used anionic surfactant. In DFT
simulations, the hematite surface was modeled using a ferric trioctahedral cluster obtained from the (104)
hematite surface containing two corner-shared and one edge-shared ferric sites (Figure 8.2). The (104)
surface is one of the naturally available surfaces of hematite.106
A butanoate ion (C3H7COO) was used to
model laurate to reduce the calculation time, because there is no significant impact of the chain length on
the electrostatic or steric structure of the carboxyl head group.252
Spin-polarized orbitals were utilized to
account for the anti-ferromagnetic properties of hematite. A continuum solvation model, COSMO, with
dielectric constant, εr, of 12 was employed to model the interfacial species.241
A two point finite difference
scheme of analytical forces was employed to calculate vibrational frequencies of the model systems. The
vibrational modes were assigned through a visual inspection of the atomic displacement trajectories
created during the frequency calculations.
Figure 8.2 Ferric trioctahedral cluster extracted form hematite (104) surface. The cluster contains both
edge-shared and corner-shared oxygen ions. The surface terminal OH/H2O groups that are involved in
coordination with carboxylate group are shown in yellow spheres. (Iron – blue spheres; oxygen – large
red spheres; carbon – large gray sphere; hydrogen – small green spheres)
Iron
117
Scheme 8.1 Cluster models of different laurate adsorption complexes. Blue octahedra – Fe3+; red
spheres – oxygen; gray spheres – carbon; green spheres – hydrogen.
8.2.1 FTIR results
The FTIR spectrum of the monolayer of laurate (Figure 8.3) is characterized by three adsorption species
identified using the spectral asymmetric asCO2 and symmetric sCO2
stretching bands of the
carboxylate group and C=O mode of carboxylic group (–COOH). The asCO2 and symmetric sCO2
stretching bands of the two adsorbed carboxylate groups are at 1540 and 1410 cm–1
, and 1530 and 1425
cm–1
respectively. Whereas, the C=O mode of carboxylic group (–COOH) is present at 1707 cm–1
.
Based on the valence saturation argument, lauric acid can exist at the NP-water interface only as an OS
complex.
An interesting observation is the absence lauric acid at low surface coverage of the surfactant at
both pH 7.15 and 10.0 (Figure 8.4). It appears at pH 10.0 at coverage higher than 0.25 ML, which is
reached after ~1 h of the conditioning with 1 mM SL. Given laurate is present in the 1 mM solution at pH
10 exclusively as anion,161, 162
its protonation on the hematite surface can be attributed to the adsorption-
induced decrease in the local interfacial pH. This conclusion is supported by the DFT results described
below.
b) ISMM a) ISBB c) Chelating d) OS surface hydration
-shared
e) OS surface
hydration-separated
118
Figure 8.3 in situ FTIR-HATR spectra in region of (a) CO2 and (b) scisCH2
vibrational modes of (1)
hematite reacted for 25 min with a 10–3
M solution of sodium laurate at pH 7.15 ± 0.05 with no added
electrolyte. For comparison, shown are spectra of (2) aqueous 10–2
M sodium laurate at pH 10.2
measured with a Ge IRE, (3) solid sodium laurate solid, and (4) solid lauric acid. Baseline in the
16001660-cm-1
region of the adsorbed laurate was corrected by adding spectrum of water (water is
removed from the interface upon the adsorption). This figure is taken from Ref. 190
. Spectral regions of the
C=O and scisCH2 modes of the surfactant are shadowed. Band assignment is taken from Ref.253
119
Figure 8.4 in situ FTIR-HATR spectra of 150-nm hematite reacted successively with SL at (a) pH 7.15
and SL concentration of (black) 0.1 mM, (blue) 0.5 mM and (green) 1.0 mM and (b) pH 10.0 and SL
concentration of 1 mM. Time of the interaction is shown in the graphs. It was measured from the moment
of the introduction of a SL solution into the FTIR-ATR cell till the beginning of the spectrum scanning
(which takes 5 min). This figure is taken from Ref. 190
. The background spectrum was that of the
deposited particles pre-equilibrated with water of pH 7.15 for 1 hour. Spectra are shifted along the Y axis
for clarity.
Based on H/D exchange and pH response of FTIR spectra of adsorbed laurate,190
the following
adsorption forms can be tentatively assigned (1) the strongly adsorbed carboxylate species is assigned to
inner-sphere monodentate mononuclear complex (ISMM), (2) the weakly adsorbed carboxylate to outer-
sphere complex (OS) and (3) the adsorbed lauric acid to OS species.
8.2.2 DFT modeling of carboxylate binding to hematite surface
DFT calculations were performed to verify the foregoing interpretation of the vibrational
spectra as well as to determine the structure of the OS complex. In an earlier study of the
120
carbonate adsorption, I suggested a methodology which was based on analysis of stability of
different surface complexes and comparison of the theoretical and experimental frequencies of
these configurations with those of the dissolved species.241 However, the uncertainty in the
coordination of dissolved laurate (Section 1.1.6) hinders the direct application of this
methodology. Instead, a comparison of the stability of the OS hydration-separated and
hydration-shared complexes (Scheme 1) as well as the relationship between calculated
frequencies of these and ISMM complexes was performed.
The geometry optimization of the OS hydration-separated, OS hydration-shared, and
ISMM complexes were conducted at the ferric cluster charges q = 0 and q = +1. As opposed to
the OS hydration-shared complex, which was stable (Figure 8.5), the OS hydration-separated
counterpart was found to be unstable at both the cluster charges, moving away from the cluster.
Figure 8.5 Geometry optimized DFT models of a butanoate ion attached as ISMM and OS hydration-
shared complexes to a ferric trioctahedral cluster with charge q = 0 which represents the (104) hematite
surface. Numbers are the CO and CC and H bond lengths in Å Dashed lines show the surface
coordinating H bonds.
This finding can be attributed to the expected fact that two hydrating water layers better screen
the electrostatic attraction of the carboxylate group by the positively-charged surface as
ISMM
1.271 1.289
1.527 1.729
1.739
OS hydration-shared
1.517 1.275
1.286
1.85
1.685 1.862 1.788
Iron
121
compared to one layer. Moreover, similar to what was experimentally observed for the weakly
bound laurate, the sCO2 and asCO2
modes of the hydration-shared complexes are higher and
lower than that the ISMM complexes (Table 8.1), respectively. It follows that the OS complex
more likely adopts the hydration shared configuration.
Additionally, it was observed that at a charge of the ferric cluster q > +2 the OS
hydration-shared complex of the alkanoate was protonated by the water molecule that
coordinates the complex to a ferric cation. At the same time, protonation by the co-adsorbed
hydronium cation took place at a charge as low as q = 1. Therefore, the interfacial protonation
of the carboxylate group was more feasible by the co-adsorbed hydronium cation rather than by
the hematite surface. This result concurs with the relationship between the pKa values of the
involved functionalities. Namely, pKa = 5.0 of laurate is lower than IEP of hematite of 7.1 and
hence pKa the adsorbed water (see also Ref.254), but higher than pKa = 1.74 of hydronium. The
protonation by the co-adsorbed proton was further supported by the absence of protonated
laurate in the layer adsorbed from a 0.1 mM solution at IEP of hematite (Figure 5a).
The presented DFT results are consistent with co-adsorption of the OS hydration-shared and
ISMM complexes. They show that protonation of the hydration-shared OS complexes is more likely
caused by a co-adsorbed hydronium ion.
Table 8.1 Calculated vibrational frequencies of IS and OS complexes of butanoate
Configuration of
adsorbed butanoate
Charge on
ferric cluster
Frequencies (cm-1
)
sCO2 asCO2
ISMM 0 1383 1520
ISMM 1 1391 1521
OS hydration-shared 0 1391
1419
1499
OS hydration-shared 1 1382
1405
1480
122
The application of this paradigm for the prediction of laurate is summarized in Figure 8.6
Figure 8.6 The predictive methodology applied in this work for identifying adsorption forms of sodium
laurate on 150-nm hematite NPs using density functional theory simulations and FTIR spectroscopy
123
8.4 Conclusions
It is shown that the ambiguity of the conventional approach to employ DFT modeling for unraveling the
complex vibrational spectra of species at the oxide-water interface can be overcome for adsorbed fatty
acids by searching for the structure that provides the best correspondence of the trends of the theoretical
and experimental spectra upon variations of the solution pH and protonation-deprotonation of the
carboxylate group, instead of searching for the structure that provides the best one-to-one
correspondence between the theoretical and experimental spectra.
124
Chapter 9 Summary
Study 1.Polymeric nano- and micro-particles are being increasingly considered for delivery in
tissue engineering, though they have long attracted attention in other biomedical applications such as
cancer therapy or delivery of vaccines and adjuvants. Two of the important requirements for delivery in
tissue engineering of articular cartilage are the short-term and sustained release of bioactive molecules
such as vitamin C or growth factors.
To satisfy these criteria, polymeric microparticles were fabricated using polysebacic anhydride
(PSA) which has faster degradation rate and possess surface erosion profile. The PSA polymer was
synthesized using the polycondensation method. Its composition and structure were characterized and
confirmed using NMR and Raman spectroscopies. The PSA microparticles were fabricated and
encapsulated using the solvent evaporation based double emulsion method. The average size of the
microparticles was ~1.5 μm and had wide size distribution. A release of the drug (amitriptyline) from the
encapsulated microparticles was linear and sustained.
Although the PSA-based polymers are reported in literature to be highly biocompatible, their
biocompatibility and bioactivity are poorly understood at concentrations higher than 10mg/mL, which are
relevant for delivery in tissue engineering. Moreover, the transport properties of agarose hydrogel matrix
need to addressed and modelled so as to enhance the retention of secreted biomacromolecules. With
these main goals in mind, I (a) modeled and established a correlation between the pore size variation of
agarose hydrogel and agarose concentration; (b) found the optimum concentration of agarose using the
established model and evaluated the range of the applicability of the model when polymeric particles are
incorporated. (c) explored the biocompatibility of PSA microparticles with chondrocytes in a 3D hydrogel
scaffold.
To select the optimum concentration of agarose in the agarose hydrogel scaffold, one needs to
know the pore/void size. The pore size should be large enough for the easy transport of growth factors (>
30 nm), but smaller than 300 nm so as to retain tissue components such as collagen fibrils. However,
there is no direct method to evaluate this quantity. This problem is very technically challenging, especially
125
in the presence of polymeric particles. Through modeling the effect of the agarose concentration on the
turbidity, I established a correlation between the pore size and concentration of the agarose. In particular,
the correlation length between agarose fibers was obtained using the wavelength exponent method.
( ( )
)
( )
Using this method, the variation of pore size with respect to agarose concentration was
empirically derived as
ξ = 522 C-1.83
,
where ξ is the pore size, and C is the agarose concentration in w/v %. The optimal concentration of
agarose was found to be 2 % w/v. When the PSA microparticles are introduced at 0.3% w/v to the
agarose hydrogel, there was increase in pore size from 100-nm to 150-nm. The hydrogel pore size
determination using wavelength exponent method was found to be inadequate when the microparticles
concentration exceeded 0.3% w/v. This is due to the strong contribution to the scattering of radiation by
larger-sized microparticles or their aggregates, apart from the agarose fibers.
In the next step, the PSA microparticles were tested for their biocompatibility to chondrocytes and
their efficacy for delivery in articular cartilage. The PSA microparticles were found to be cytotoxic at a
concentration of 20 mg/mL, but benign at 2 mg/mL. I explained the cytotoxicity by the lipotoxicity resulting
from the intracellular accumulation of PSA or its degradation products, rather than by the changes in local
pH due to the polymer hydrolysis. It follows that the variables that collectively and uniquely determine the
bioactivity of PSA polymer are its degradation rate, functional groups and hydrophobicity but requires
further verification.
Among several strategies applied to mitigate the cytotoxicity of PSA, the administering of bovine
serum albumin (BSA) was found to be the most efficient. The chondro-protective action systemof BSA is
proportional to the ratio of BSA to the polymer. The BSA-based mitigation strategy makes PSA
microparticles effective in the short-term delivery of TGF-β3 in articular cartilage engineering.
Study 2. Functional groups of polymeric materials are known to be important in tissue
engineering, for example, for directing tissue morphogenesis. I addressed for the first time the effects of
126
functional groups on the bioactivity of polymeric nanoparticles in the tissue engineering of cartilage. For
this purpose, I synthesized polyacrylate-based nanoparticles and studied the effect of the
functionalization on the chondrocyte viability and metabolism in an agarose hydrogel. Since the published
protocols for synthesis of the polyacrylate nanoparticles using inverse microemulsion failed, the new
synthesis conditions were determined. For this purpose, a ternary phase diagram was constructed for the
water-in-hexane inverse microemulsion system stabilized by Aerosol-OT surfactant at room temperature.
It was found that the addition of a monomer such as acrylic acid increased the stable inverse emulsion
region. A similar phase diagram was constructed at 50 C, where the stable region remains more or less
similar to that at room temperature. On the basis of these diagrams, the functionalized polyacrylate
nanoparticles were obtained through radical polymerization of monomers (acrylic acid or its functional
derivatives) with crosslinkers. The size of nanoparticles was in the range of 70-300 nm, and surface
charge was characterized using the electrokinetic method.
These polyacrylate-based nanoparticles when introduced to chondrocytes in agarose hydrogel
are found to be biocompatible (2 mg/mL). However, they exhibit bioactivity that is detrimental to the
synthesis of extracellular matrix. There is an inverse correlation between mechanical strength and the
negative surface charge of these nanoparticles. The cause for the poor matrix synthesis is suggested to
be the disruption of the inter-cellular signaling process by higher anionicity. Thus, it is concluded that the
charge on the functional groups of the polymers has a significant effect the polymer bioactivity and can be
used to manipulate the cellular metabolism.
Study 3. Another control of the tissue growth can be the bioactivity of metal oxide nanoparticles. I
discovered, for the first time, a beneficial impact of nanoceria on the biochemical and mechanical
properties of articular cartilage tissue grown in vitro. Specifically, it was demonstrated that nanoceria is
biocompatible toward chondrocytes at concentrations at least up to 1000 μg/mL, while the constructs
cultured with embedded nanoceria at 100 and 1000 μg/mL have mechanical properties and collagen
content significantly better than the control. The mode of administering of nanoceria is also found to be
important in their efficacy to suppress the cartilage tissue damage caused by inflammatory agents such
as interleukin-1α (IL-1α). In particular, if nanoceria is just embedded into the constructs, it does not offer
127
any protection against IL-1α. However, significant chondroprotectivity against IL-1α and partial prevention
of the matrix degradation are observed when the nanoceria is suspended in the growth media at 20000
μg/mL. The found effects have potential in treating the inflammation of articular cartilage due to disease
or damage.
In contrast to nanoceria, ferric (hydr)oxide nanoparticles detrimentally affect the growth of
cartilage tissue. The cytotoxicity of these nanoparticles is unexpected given the literature data. It is shown
for the first time that the adverse effect increases with increasing nanoparticle size, following the trend
earlier observed for the catalytic activity of the nanoparticles. Specifically, the mechanical properties of
the tissue constructs embedded with hematite and ferrihydrite (FH) nanoparticles degrade with increasing
nanoparticle size as follows: Control > FH (2-nm) > H9 (9-nm) > H38 (38-nm) >> H150 (150-nm). On the
other hand, the cellular viability surprisingly remains similar among all these sample sets.
Correlating this result with the earlier observed trend in the catalytic activity116
and the decrease
in the adsorption activity (measured as normalized monolayer coverage of sodium laurate) found in Study
4 (see below), it can be concluded that both the catalytic redox and adsorption activities of metal oxide
nanoparticles contribute to their bioactivity. Moreover, given the beneficial effect of nanoceria, these
results imply that not only the catalytic redox activity per se, but also the catalytic redox mechanism
(including either the deactivation or generation of reactive oxygen species) controls the bioactivity of the
metal oxide at the biointerface. Thus, the emerging paradigm based on the overlap of the conduction
band energy with the redox potential range of redox active cellular components should be further
expanded with incorporation of the adsorption properties and the catalytic redox mechanism.
Study 4. This study was focused on the adsorption and dispersion properties of the metal
(hydr)oxide nanoparticles in the presence of fatty acids in aqueous media, as these properties can also
play an important role in the biochemical and environmental activity and bioavailability of the
nanoparticles. Specifically, I explored the effects of the size and morphology of ferric (hydr)oxide
nanoparticles in these phenomena. Using adsorption isotherm, electrokinetic, FTIR measurements as
well as DFT simulations, the first experimental evidence is obtained for the dependence of the surface
density, speciation, and packing order of adsorbed fatty acids on these properties. The conditions under
128
which fatty acids form self-assembled monolayers (SAMs) and bilayers on such nanoparticles in water
are distinguished. For the first time, the electric polarization of the nanoparticles is demonstrated to be a
powerful tool for manipulating the interfacial properties of the nanoparticles.
Specifically, it is shown that sodium laurate is adsorbed on hematite mainly as a mixture of
chemisorbed (inner sphere monodentate, ISMM) and physisorbed (outer sphere hydration-shared, OS)
complexes, both at pH > PZC and pH < PZC. The OS complex is partially protonated by a coadsorbed
hydronium cation as either surface coverage increases or solution pH decreases. Independent of
morphology, an increase in nanosize improves the adsorption capacity and affinity of hematite
nanoparticles, as well as increases the fraction of the protonated physisorbed (OS) alkanoate, in
agreement with the nanosize-induced changes in the structural and electronic properties of the
nanoparticles. This implies that the surface electronic and structural properties, which can be controlled
by size for metal oxide nanoparticles, offer important ways to manipulate adsorption properties.
Consequently, an increase in nanosize of hematite nanoparticles improves the packing order of laurate
and hence hydrophobicity of the nanoparticles provided a similar hexagonal habit of these nanoparticles.
It was also observed that, independent of morphology, the dispersibility of the nanoparticles by fatty acids
in water degrades as nanoparticle size decreases. This effect is attributed to the decrease in the
adsorption capacity which ends up in the formation of a loosely packed bilayer.
Morphology of the nanoparticles was found to be critical for the self-assembly if (i) the majority of
the molecules are chemisorbed (ISMM) and (ii) the morphology-determined surface arrangement of the
adsorption sites is geometrically incompatible with a surfactant ordering. The surface ratio of the
physisorbed/chemisorbed surfactant depends on the surface charge of nanoparticles. Negative
polarization suppresses chemisorption. This effect can be used to remove the geometrical constraints for
self-assembly. The dominance of the chemisorbed complexes results in the isotropic surface filling by
fatty acids, while the prevalence of OS complexes favors the cluster-like filling pattern.
A new approach to DFT modeling of the in situ FTIR spectra of adsorbed species was developed
to circumvent the intrinsic ambiguities of the conventional DFT modeling in interpreting vibrational
spectra, especially at the oxide-water interfaces. This new approach constitutes searching of the
129
adsorbed structure that provides the best correspondence of the trends of the theoretical and
experimental spectra upon variations of the solution pH, ionic strength and protonation-deprotonation of
the carboxylate group, instead of conventional approach of searching for the structure that provides the
best one-to-one correspondence between the theoretical and experimental spectra. This approach was
utilized to interpret the FTIR data in obtaining information on the adsorption forms and packing of fatty
acids on ferric (hydr)oxide nanoparticles.
Thus, the experimental, methodological and theoretical results obtained in these studies are an
important step toward developing a general paradigm predicting the (bio-)activity for both the polymeric
and metal oxide micro- and nano-particles. For the polymeric particles considered (polyanhydrides and
polyacrylates), the following factors are found to determine their bioactivity: degradation rate,
hydrophobicity, functional group and surface charge. In addition to above parameters, concentration of
these particles with respect to cell density is critical factor determining their (bio-)activity. In the case of
metal oxide nanoparticles, the catalytic redox properties, the redox mechanism, and the adsorption
properties have been suggested as the dominating factor controlling their cyto-protective and cytotoxic
properties, provided other physical parameters such as dissolution and aspect ratio are not critical.
Additionally, the DFT-based methodology to interpret the adsorption phenomena developed in this work
can further augment the possibility of formulating such a general paradigm for (bio-)activity.
130
Summary of main results
Effect of Material Significant physicochemical properties (Bio-)activity
Effect of polymers
a. Polyanhydrides
b. Polyacrylates
Degradation rate; hydrophobicity; carboxylic groups
Surface anionic charge
Lipotoxic effects
Decreased tissue synthesis
Effect of cerium oxide
nanoparticles
Mode of particles addition; redox properties Improved protein synthesis;
Protection against inflammation
Effect of hematite
nanoparticles
Nanoparticle size, morphology, redox and catalytic
properties.
Cytotoxicity due to size-
dependent catalytic activity
Size and morphology
dependent adsorption and
dispersion properties
131
Chapter 10 Suggested Future Studies
The following future studies are suggested based on the results of the present work.
1) To decrease the impact of lipotoxicity of polysebacic anhydride (PSA) due to the polymer
degradation, fabricate encapsulated microparticles using a physical composite of a slow
degrading polymer such as poly-caprolactone (in higher fractions) and the fast degrading PSA (in
lower fractions). This can create a bulk and surface erosion profile more effective for short-term
delivery.
2) To decrease the concentration of degrading PSA polymer, perform a study of the encapsulation
efficiency and release profile as a function of the thickness of the hydrophobic shell of the PSA
microparticles.
3) To enhance the cell-polymer interaction, functionalize the PSA polymer or polyacrylate
nanoparticles with either transforming growth factor (TFG-β3) or cell-adhering peptide sequence
Arginine-Glycine-Aspartic acid (RGD).
4) To improve the biocompatibility of polyacrylate nanoparticles (NPs) for chondrocytes, adapt the
stem cells to tolerate these NPs before their chondrogenic differentiation.
5) To increase the anti-inflammatory effect of nanoceria on chondrocytes, enhance nanoceria
uptake during the expansion stage of chondrocytes using mechanical motion or through
functionalization of nanoceria with cell-adhering RGD peptide sequence.
6) To protect tissue damage from oxidants, embed electron-rich nanomaterials such as zinc or
selenium nanoparticles in the tissue constructs and study its impact.
132
7) To rationally control the biocompatibility of ferric (hydr)oxide NPs, determine the effect of NP size
on their cellular uptake using electron microscopy and ICP atomic emission spectroscopy and
measure the impact of these NPs on the degradation of extracellular matrix components such as
GAG and collagen. Establish a direct correlation, if any, between degradation of matrix and
physicochemical properties of NPs
8) Explore the generality of the found nanosize effects on the adsorption and dispersion properties
of metal oxide NPs by studying other technologically important oxides that are thermodynamically
instable at the nanoscale such as corundum (-Al2O3) and rutile (TiO2)
9) To explain the size-dependence of the dispersion properties of (hydr)oxide NPs in the presence
of fatty acids in water, use DLVO or extended DLVO and flocculation models to quantify the
dispersibility.
10) To understand the (bio-)activity of ferric (hydr)oxide NPs, study the effect of size and morphology
on adsorption properties of proteins on ferric (hydr)oxide NPs. Establish any similarity to the (bio-
)activity ferric (hydr)oxide vis-a-vis chondrocytes and sodium laurate. This may enable to suggest
a general paradigm of the (bio-)activity for ferric (hydr)oxide NPs in particular or metal (hydr)oxide
in general
133
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150
APPENDIX
A. Estimation of buffer capacity needed to offset the drop in pH due to polsebacic
anhydride degradation
It was observed from experiments that when polysebacic anhydride microparticles were added to
culture medium there is pH drop from 7.2 to ~6.3. Thus additional buffer capacity, other than bicarbonate
that is present in the growth medium, is needed to offset this drop.
The buffer capacity is defined as infinitesimal quantity of base needed to change the pH by
infinitesimal amount.255
( )
( ) (
[ ]
( [ ]) [ ] [ ]) - (1)
Where,
β is buffer capacity, B is moles of strong base added
A is moles of strong base added, C is the total concentration of the buffer
Ka is acid dissociation constant for buffer
Assuming the worst case of 50% prior degradation of polysebacic anhydride molecules into basic sebacic
acid molecules and subsequent release of two protons from each sebacic acid molecules when they are
suspended in growth medium. Molecular weight of sebacic acid is 202 g/mol
A 50 mg of polymer in 1mL of growth medium will then release = 25/202 ≈ 0.125 mmoles of sebacic acid.
Thus 0.25 mmoles/mL protons will be released. This corresponds to 0.25 M addition of strong acid.
Thus d(A) = 0.25M and let the change of pH be set 0.2 (from 7.2 to 7.0)
Therefore –d(A)/d(pH) = 1.25.
If the Tris-HCl buffer is added to maintain the pH then the pKa = 8.07
Solving the equation (1) for the needed total Tris concentration at pH 7.2,
C= (0.54 – [H+] –[OH
-])/0.10631 = 5.4 mM of Tris buffer
Thus ~ 5mM of Tris or buffers with similar pKa and pH range such as HEPES is even in the worst case of
pH changes.
151
B. Determination of wavelength exponent
The wavelength exponent experimental WLE from 700 to 800-nm is extracted from turbidity
measurements as follows,
Turbidity, ( ) ( )
Where, l is the path length = 1 cm.
The wavelength exponent (WLE) is obtained by fitting a slope of log-log plot of τ and λ from 700-800 nm
( ( )
)
For 4% agarose hydrogel
For 3% agarose hydrogel
152
For 2% agarose hydrogel
For 1% agarose hydrogel
For 2% agarose hydrogel+ 0.3% polysebacic anhydride microparticles
153
Table Pore size and wavelength exponent as function of agarose
concentration and cooling time (from 95 C at 0 mins to room temperature
by 2 hours)
4 % Agarose 3 % Agarose
Time (min)
Wavelength exponent
Correlation Length (nm)
Time (min)
Wavelength exponent
Correlation Length (nm)
22 -5.4 0 34 -4.32 0
26 -4.19 0 41 -3.67 34
34 -3.71 29 46 -3.5 41
40 -3.65 31.5 54 -3.35 53
46 -3.61 33.5 61 -3.3 59
52 -3.58 35 66 -3.27 62
59 -3.56 36 71 -3.25 63.5
63 -3.55 36.5 76 -3.24 64
71 -3.54 37 98 -3.21 68
77 -3.52 37.5 106 -3.2 69
89 -3.51 39 112 -3.2 69
102 -3.51 39 118 -3.2 69
108 -3.5 41 124 -3.18 66.6
117 -3.5 41
121 -3.5 41
154
2 % Agarose 1 % Agarose
Time (min)
Wavelength exponent
Correlation Length (nm)
Time (min)
Wavelength exponent
Correlation Length (nm)
48 -4.05 8 25 -5.63 0
53 -3.68 30 37 -4.96 0
58 -3.41 47 44 -4.09 0
66 -3.15 74.5 51 -3.52 39
73 -3.05 96 57 -3.21 63
78 -3 110 64 -2.98 104
83 -2.96 114 71 -2.84 156
88 -2.93 121 77 -2.72 225
94 -2.9 128.5 83 -2.65 347
101 -2.88 135.7 102 -2.54 400
106 -2.87 139.3 108 -2.51 503
111 -2.86 142.8 113 -2.5 505
120 -2.85 146.3 118 -2.49 517
124 -2.84 149.8 124 -2.47 540
155
2 % Agarose with 0.3 % polysebacic anhydride microparticles
Time (min)
Wavelength exponent
Correlation Length (nm)
21 -3.2 65
28 -2.97 100
33 -2.89 132
38 -2.85 151
43 -2.83 164
50 -2.81 175
56 -2.8 179
65 -2.79 184
73 -2.79 184
78 -2.78 190
156
y = 530.5e-37.47x R² = 1
y = 530.5e-195.3x R² = 1
y = 530e-416.3x R² = 0.3982
y = 530.5e-1735x R² = 0.9656
0
100
200
300
400
500
600
0 0.001 0.002 0.003 0.004 0.005 0.006 0.007
Eq
uilib
riu
m Y
ou
ng
's m
od
ulu
s,
EY,
kP
a
Surface areaof nanoparticles in suspension, m2/mL
FH
H9
H38
H150
C. Curve fitting of mechanical properties of engineered cartilage tissues and monolayer
coverage of sodium laurate as a function of size of ferric (hydr)oxide nanoparticles.
Mechanical properties of tissues
The dependence of Young’s modulus of cartilage tissues Figure 7.1a on the total surface area of
nanoparticles in the scaffold is modeled using exponential functions (Figure C1). Except for the H38, the
fitting error was negligible. The mechanical strength of tissue constructs at nanoparticle concentration of
0.002 m2/mL were obtained from the exponential function. The concentration was chosen arbitrarily. For
H38 particles, the experimentally measured value of Young’s modulus at 0.002 m2/mL was used. The
values are tabulated in Table C1. The detrimental catalytic activity of ferric (hydr)oxide is reported as
decrease in Young’s modulus from the control value (scaffolds without nanoparticles = 530.5 kPa). This
decrease is normalized with the decrease for H150 for comparison purposes.
Figure C1 Decrease of the Young’s modulus of tissue constructs as a function of surface area per of the
ferric (hydr)oxide nanoparticles per unit volume in the constructs. FH-4nm; H9-9nm; H38-38nm and
H150-150nm.
157
y = 0.048x0.63 R² = 0.8557
0
0.2
0.4
0.6
0.8
1
1.2
0 50 100 150 200
No
rmal
ize
d d
ecr
eas
e in
Yo
un
g's
mo
du
lus
of
tiss
ue
co
nst
ruct
s
Size of ferric (hydr)oxide nanoparticles, nm
Table C1 Decrease in Young’s modulus of tissue constructs due to detrimental catalytic activity of ferric
(hydr)oxide nanoparticle: effect of nanosize
Ferric (hydroxide) nanoparticles size, nm
Young's modulus values obtained at 0.002 m
2/mL
from exponential curves fitted on Figure 7.1a
Decrease in Young's modulus due to nanoparticles from control value (530.5 kPa)
Decrease in Young's modulus due to nanoparticles normalized with the maximum value (H150: 514 kPa)
4 492 39 0.08
9 359 172 0.33
38 300 231 0.45
150 16.5 514 1
A power law fit of the size of ferric (hydr)oxide nanoparticles and their bioactivity (represented by the
normalized decrease in Young’s modulus is shown in Figure C2.)
Catalytic bioactivity (normalized decrease in Young’s modulus) = 0.048. (size)0.63
Figure C2 Normalized decrease in Young’s modulus of tissue constructs as a function of size of the ferric
(hydr)oxide nanparticles present in the tissue constructs at concentration of 0.002 mg/mL
158
y = 0.42x0.19 R² = 0.9922
0
0.2
0.4
0.6
0.8
1
1.2
0 50 100 150 200
No
rmal
ize
d m
on
ola
yer
cove
rage
o
f so
diu
m la
ura
te
Size of ferric (hydr)oxide nanoparticles, nm
Size-dependent adsorption activity
The monolayer coverage of sodium laurate on ferric (hydr)oxide nanoparticles with respect size is given
in the Table C.2.
Table C.2 Normalized monolayer coverage of sodium laurate on ferric (hydr)oxide nanoparticles
Size of ferric (hydr)oxide nanoparticles, nm
Monolayer coverage of sodium laurate at pH 7.1, molecules/nm
2
Normalized monolayer coverage with the maximum value (H150: 4.2 molecules/nm
2)
4 2.1 0.5 9 2.6 0.619048 38 3.3 0.785714 150 4.2 1
A power law fit was obtained between size of the nanoparticles and the normalized values of monolayer
coverage (with that of maximum coverage value of 150 nm particles: 4.2 molecules/nm2).
Normalized monolayer coverage of sodium laurate, y = 0.4 . (size)0.19
Figure C3 Normalized monolayer coverage of laurate as a function of size of the ferric (hydr)oxide
nanoparticles
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