ORIGINAL PAPER
Albumin-based nanoparticles as magnetic resonance contrastagents: II. Physicochemical characterisation of purifiedand standardised nanoparticles
A. A. Abdelmoez • G. C. Thurner • E. A. Wallnofer •
N. Klammsteiner • C. Kremser • H. Talasz •
M. Mrakovcic • E. Frohlich • W. Jaschke • P. Debbage
Accepted: 30 June 2010 / Published online: 14 July 2010
� Springer-Verlag 2010
Abstract We are developing a nanoparticulate histo-
chemical reagent designed for histochemistry in living
animals (molecular imaging), which should finally be useful
in clinical imaging applications. The iterative development
procedure employed involves conceptual design of the
reagent, synthesis and testing of the reagent, then redesign
based on data from the testing; each cycle of testing and
development generates a new generation of nanoparticles,
and this report describes the synthesis and testing of the
third generation. The nanoparticles are based on human
serum albumin and the imaging modality selected is mag-
netic resonance imaging (MRI). Testing the second particle
generation with newly introduced techniques revealed the
presence of impurities in the final product, therefore we
replaced dialysis with diafiltration. We introduced further
testing methods including thin layer chromatography,
arsenazo III as chromogenic assay for gadolinium, and sev-
eral versions of polyacrylamide gel electrophoresis, for
physicochemical characterisation of the nanoparticles
and intermediate synthesis compounds. The high grade of
chemical purity achieved by combined application of these
methodologies allowed standardised particle sizes to be
achieved (low dispersities), and accurate measurement of
critical physicochemical parameters influencing particle size
and imaging properties. Regression plots confirmed the high
purity and standardisation. The good degree of quantitative
physicochemical characterisation aided our understanding
of the nanoparticles and allowed a conceptual model of them
to be prepared. Toxicological screening demonstrated the
extremely low toxicity of the particles. The high magnetic
resonance relaxivities and enhanced mechanical stability of
the particles make them an excellent platform for the further
development of MRI molecular imaging.
Keywords Albumin nanoparticles � MRI � Gadolinium �Targeting � PEGylation � Molecular imaging
Introduction
Histochemistry in living animals has become known as
molecular imaging. Molecular imaging by magnetic reso-
nance imaging (MRI) (MRMI) promises early detection of
malignancies (Harisinghani et al. 2003; Jaffer and Weiss-
leder 2005) and of vulnerable atherosclerotic plaques
(Winter et al. 2003), in addition to monitoring of drug
distribution (Saito et al. 2004; Griffiths and Glickson 2000)
A. A. Abdelmoez and G. C. Thurner contributed equally to this work.
A. A. Abdelmoez � G. C. Thurner � E. A. Wallnofer �C. Kremser � W. Jaschke
Department of Radiology, Innsbruck Medical University,
Anichstrasse 35, 6020 Innsbruck, Austria
N. Klammsteiner � P. Debbage (&)
Department of Anatomy, Histology and Embryology, Innsbruck
Medical University, Mullerstrasse 59, 6020 Innsbruck, Austria
e-mail: [email protected]
H. Talasz
Biozentrum of the Medical University Innsbruck,
Section for Clinical Biochemistry, Fritz-Pregl-Straße 3,
6020 Innsbruck, Austria
M. Mrakovcic � E. Frohlich
Center for Medical Research, Stiftingtalstrasse 24,
8010 Graz, Austria
Present Address:A. A. Abdelmoez
Department of Pharmaceutical Organic Chemistry,
Faculty of Pharmacy, Assiut University, Assiut, Egypt
123
Histochem Cell Biol (2010) 134:171–196
DOI 10.1007/s00418-010-0726-6
and application as surrogates for clinical testing (Rehman
and Jayson 2005). MRMI is therefore under development
by numerous groups around the world (Flacke et al. 2001;
Hengerer and Grimm 2006; Mulder et al. 2006; Utsumi
et al. 2006). MRMI requires use of nanoparticles as
amplifiers for specific signals (Debbage and Jaschke 2008).
Since nanoparticles are novel agents with largely unknown
toxicological and immunological potential (Debbage 2009;
Stollenwerk et al. 2010; Robbens et al. 2010; Vega-Villa
et al. 2008; Suh et al. 2009), adequate development and
testing in iterative developmental cycles are necessary to
develop safe and effective agents for clinical use. This
paper is the second of a series describing our design,
synthesis and optimisation of successive generations of
albumin-based nanoparticles for eventual application in
clinical imaging and therapy. We recently reported syn-
thesis and characterisation of two generations of nanopar-
ticles formed from HSA emulsified with polylactic acid,
the nanoparticles of approximately 30 nm diameter, and
each bearing several hundred gadolinium-DTPA-chelates
(Stollenwerk et al. 2010). The major advance achieved at
this stage was upscaling the synthesis to produce 3–5 g of
nanoparticles as product. However, the variability of the
nanoparticles was too high to allow precise interbatch
comparisons, and much too high to consider use of the
particles in any clinical application (Stollenwerk et al.
2010). In this paper, we address the question of standar-
dising the nanoparticles, aiming to reduce the intra- and
interbatch variability and to increase the purity of the
batches. We considered it likely that the variability had two
possible causes, the first being the variability in the starting
raw materials, and the other being due to the presence of
educts (starting materials) remaining as contaminating
impurities in the final nanoparticle preparations. To detect
chemical impurities, we extended the range of analytical
techniques applied at different stages of the synthesis
procedure. A further major concern was the stability of the
nanoparticles in the physiological environments within
living animals. We noted earlier that complex structures
such as nanoparticles can show different types of insta-
bility. We focus here on one particular type of stability,
namely mechanical stability. We aimed to increase the
internal cohesion of the nanoparticles, so that they would
retain their integrity as individual particles and not break
up into fragments during passage through the bloodstream
or during intercellular processing. We describe the cross-
linked nanoparticles, and also procedures for testing the
degree of nanoparticle stability. Finally, for protein-based
nanoparticles, it is essential to ensure that the protein
molecules retain their native configuration, in order to
preserve their specific functions [for HSA, this is the
binding and transport of small molecules (Fehske et al.
1981; Frokjaer and Otzen 2005; Goldwasser and Feldman
1997; Gonzalez and Kannewurf 1998; Griffel and Kaufman
1992; Kragh-Hansen 1981; Montero et al. 2007; Peters
1985; Putnam 1984; Rainey and Read 1994)], and to avoid
the potentially dangerous effects of denaturation, such as
triggering amyloid formation (Schnabel 2010; Goldschmidt
et al. 2010; Balbirnie et al. 2001; Nelson et al. 2005).
In the first paper of this series, we described the syn-
thesis and characterisation of ‘‘naked’’ nanoparticles,
which were derivatized in only one way, by incorporation
of gadolinium to allow detection of the particles by MRI.
The ultimate aim of this work is to develop specific
histochemical staining in living animals, which requires
targeting of the nanoparticles to specified sites within
living tissues. In this paper, we explore the result of
adding targeting groups to the particles, aiming to exploit
well-standardised, mechanically stable nanoparticles as a
platform for targeting applications. Adjunct to this, we
check the nanoparticle properties after attachment of
PEG chains (Veronese 2001; Zalipsky 1995a), which are
frequently used to confer ‘‘stealth’’ properties on nano-
particles, reducing nanoparticle uptake into the reticulo-
endothelial system (Abuchowski et al. 1977; Zalipsky
1995b).
This paper describes four further steps in our develop-
ment of nanoparticles for use as intravital histochemical
stains. As conceptual background, the potential application
of the nanoparticles for clinical use is always present, and a
major criterion of success is the possibility of transferring
the synthesis protocols to industrial production. Assess-
ment of this possibility is an integral part of this paper.
Materials and methods
Materials
In addition to materials listed in our previous paper (Stol-
lenwerk et al. 2010), charcoal, sodium chloride, caprylic
acid, gadolinium chloride (GdCl3), 2,7-bis(o-arseno-
phenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acid
(arsenazo III), sodium cyano-borohydride (NaCNBH3),
iodine and ninhydrine were purchased from Sigma–Aldrich
(Munich, Germany). Rotilabo� injection filter (PP-Geha-
use, sterile, PVDF 0.2 lm), Rotilabo� paper filter (type
600P, ø 185 mm) were purchased from Carl Roth GmbH &
Co (Karlsruhe, Germany). Materials used for polyacryl-
amide gel electrophoresis (PAGE) (SDS, Native, pI),
namely the gels (Ready Gel� precast gels, 10 well comb,
30 ll load volume), the standards, the running and the
sample buffers were purchased from BioRad (Vienna,
Austria). The sheets for thin layer chromatography (TLC)
and the membranes for diafiltration were purchased from
VWR (Vienna, Austria). Adjustments of pH values were
172 Histochem Cell Biol (2010) 134:171–196
123
done with a pH 211 Microprocessor pH meter, Hanna
instruments (Kehl am Rhein, Germany); the electrode
cleaning solution HI 7073, the electrode storage solution
HI 70300 and the pH meter calibration buffers (pH 4, pH 7,
pH 10) were purchased from Carl Roth GmbH & Co
(Karlsruhe, Germany). Throughout this work, the water
used was purified Millipore water (Millipore, Billerica,
MA, USA).
Purification and extraction procedures
To purify the reaction products diafiltration was intro-
duced, replacing simple dialysis (Stollenwerk et al. 2010).
Diafiltration was carried out after each derivatization step
to separate the reaction product (conjugate, nanoparticles)
from contaminating component molecules (HSA-DTPA-
Gd and also small molecular weight species), by use of a
Minimate TFF System, and Minimate TFF membrane
cassettes with membranes of Omega� type (Pall Life Sci-
ences, MI, USA), having cutoffs of 10, 100 and 500 kDa.
To ensure good purification distilled water was added to an
amount at least seven times larger than that used as starting
volume of the nanoparticle solution, additionally pressure
was applied in the range 10–20 psi and the sample solution
was stirred. The quality and end point of diafiltration were
checked by TLC and purification was stopped as soon as no
traces of contaminants remained. If not stated differently
the procedure of diafiltration always was carried out
according to this protocol.
Preparation of the HSA-DTPA-Gd conjugates
Cleaning and stabilisation of HSA
Albumin was prepared by the method of Chen (1967) and
then stabilised by addition of octanoate according to
Shrake et al. (2005): a solution containing 50 mg/ml of
human serum albumin in distilled water was prepared at
room temperature. Then charcoal powder was added under
continuous stirring (HSA:charcoal = 2:1 w/w) and the pH
was adjusted to 3 with 1 M HCl. The suspension was
placed in the fridge at 4�C and stirred magnetically for 1 h.
Afterwards the suspension was filtered through a folded-
filter paper and centrifuged for 10 min at 5,000g. The
supernatant was further filtered through a 0.2 lm filter. The
pH of the solution containing the cleaned HSA molecules
was raised to 4 with 0.1 M NaOH. NaCl, predissolved in
distilled water, was added to a final concentration of
150 mM and the HSA concentration was adjusted to
30 mg/ml. Then Na-Caprylate was added (30 mM Na-
Caprylate per ml nanoparticle sample) and the pH was
slowly increased to 10 by addition of 3 M NaOH. After
stirring magnetically for 1 h at room temperature the pH
was decreased to 7 by addition of 1 M HCl and stirred
overnight at room temperature.
Conjugation of DTPABA to HSA
The procedure described earlier (Stollenwerk et al. 2010)
was slightly modified, omitting the suspension of DTPABA
in DMSO. The following procedure was used instead: the
pH of the protein solution (30 mg/ml as measured by UV
spectrometry) was adjusted to 8.5 by means of 3 M NaOH
and a 50-fold molar excess of solid DTPABA was added
portionwise. The pH of 8.5 was maintained throughout the
chelate addition by adequate provision of 3 M NaOH. After
the reaction mixture was stirred for 2 h at RT, diafiltration
was performed using a membrane of 100 kDa cutoff. The
resulting HSA-DTPA conjugates were now ready for che-
lation of a metal, in this investigation gadolinium (Gd).
Preparation of trisodium bis(nitrilotriacetate) gadolinate
(Na3Gd(NTA)2)
In our previous work (Stollenwerk et al. 2010), gadolinium
oxide was used as starting material. In the work reported
here, this was replaced with gadolinium chloride, as fol-
lows: 4.6 g of gadolinium chloride hexahydrate
(1.24e-02M) were dissolved in 60 ml water and 6.38 g
Na3NTA (2.48e-02M) were added under continuous stir-
ring. After complete dissolution of Na3NTA, the pH was
lowered from 7.5 to 6.0 using 1 M HCl, the volume was
then made up to 100 ml with water and the solution was
stored at 4�C.
GdCl3 þ 2Na3NTA! Gd NTAð Þ2Na3 þ 3NaCl
The product was assayed for the presence of non-
chelated gadolinium using arsenazo III.
Gadolinium complexation to HSA-DTPA
The principle of this chelation is shown in our earlier paper
(Stollenwerk et al. 2010); during the work reported here the
chelation of the conjugate was performed as follows: A 20-
or 50-fold molar excess of Na3[Gd(NTA)2] solution
(116 mM, pH 6.0) was added to HSA-DTPA in 0.1 M
citrate buffer. The sample was then stirred for 24 h at 4�C
followed by diafiltration using a membrane of 100 kDa
cutoff. A subsequent arsenazo III assay was carried out to
check there was no remaining free gadolinium.
Additionally, the following assays were carried out to
examine any uptake of gadolinium by HSA which was
independent of the chelator:
1. Gd-NTA was added to albumin bearing no DTPA, and
the resulting mixture was passed through 14 cycles
of diafiltration. Afterwards the Gd:HSA ratio was
Histochem Cell Biol (2010) 134:171–196 173
123
quantified by atomic absorption spectrometry (AAS)
and Pierce assays.
2. Gd-NTA was added to albumin after prior incubation of
the albumin with 1 M citrate buffer (HSA:cit-
rate = 10:1 v/v). The resulting mixture was passed
through 14 cycles of diafiltration. Afterwards the
Gd:HSA ratio was quantified by AAS and Pierce assays.
Preparation of the nanoparticles
Emulsification
A mixture of PLA plus HSA-DTPA-Gd, based on 1:10 M
ratio PLA:HSA, was emulsified, using the protocol
described in detail for series II by Stollenwerk et al. (2010).
The resulting turbid suspension was filtered through paper
filters to clear the solution prior to diafiltration using a
membrane of 500 kDa cutoff.
Crosslinking
The nanoparticles were crosslinked by addition of 1%
glutaraldehyde solution (glutaraldehyde:nanoparticle ratio
1:10 v/v) and stirred overnight at room temperature followed
by diafiltration using a membrane with 500 kDa cutoff.
Additionally, the following variant procedures were
carried out to examine the nanoparticle aggregation
behaviour and stability:
1. Different concentrations of glutaraldehyde (2–5%)
were added (glutaraldehyde:nanoparticle ratio 1:10 v/
v) and stirred at RT for different durations (1–12 h).
(a) The surplus glutaraldehyde in the crosslinked
nanoparticle samples was quenched with 1% of
aqueous NH4Cl solution (NH4Cl:nanoparticle
ratio was 1:5 v/v). After further stirring for 1 h
the nanoparticle solutions were diafiltered using a
membrane with 500 kDa cutoff.
(b) After crosslinking NaCNBH3 was added to stabi-
lise the Schiff bases formed by glutaraldehyde,
reducing them to covalency according to the
methods described by Means and Feeney (1995).
Briefly, for 10 mM glutaraldehyde 12.5 mM NaC-
NBH3 were added and stirred for 2 h at room
temperature. Then the sample was diafiltered using
a membrane with 500 kDa cutoff.
Physicochemical and immunohistochemical
characterisation: analytical techniques
Nanoparticles were characterised by use of several techniques
described in detail in Stollenwerk et al. (2010), as follows:
1. The molecular weights of HSA and of HSA-DTPA-Gd
conjugate (referred to in the following simply as
‘‘conjugate’’) were estimated by SDS–PAGE.
2. The Gd content of conjugates and nanoparticles was
determined by AAS.
3. The protein content of conjugates and nanoparticles
was determined by the Pierce reaction, and by UV
spectrometry.
4. MR relaxivity properties of conjugates and nanopar-
ticles were measured by use of an inversion recovery
sequence for T1, and by use of a CPMG-type multi
echo spin-echo sequence for T2.
5. Electrical charge properties of the nanoparticles were
determined by measuring their electrophoretic mobi-
lity using a PSS NICOMPTM 380 DLS/ZLS.
6. The presence of albumin in its natural configuration
was tested by checking the presence of epitopes using
immunohistochemistry to detect HSA, carried out on
formvar-coated grids.
7. Size measurements of the nanoparticles were made
from negatively contrasted preparations on formvar-
coated grids by transmission electron microscopy
(TEM), and analysed by use of the Metamorph
programme (Zeiss, Germany). Ultrastructural analyses
were also carried out on pelleted nanoparticles fixed in
glutaraldehyde, postfixed in osmium tetroxide, embed-
ded in Epon and ultrathin-sectioned.
8. Size measurements were also carried out by photon
correlation spectroscopy (PCS); the PCS data was
collated with the TEM data.
9. Nanoparticle masses were calculated by use of the
following formulae, in which diameter d (nm) of the
particles; conjugate density of HSA-DTPA-Gd =
1.4 g/cm3; packing density d of albumin molecules in
the nanoparticles *0.40. The results were obtained
first as nanoparticle mass Mg measured in grams, then
converted to nanoparticle mass MDa expressed as
Daltons:
Mg ¼ 4=3p d=2ð Þ3�10�21 � 1:4� dh i
g;
MDa ¼ Mg=1:66054e�24� �
Da:
10. Nanoparticle structural stability was tested by
exposing them to ultrasound, to detergents, to heat,
and to various combinations of these agents.
11. Nanoparticle cytotoxicity was examined by assess-
ing the effect of incubating the endothelial cell line
EAhy926 with nanoparticles, using the activity of
intracellular dehydrogenases, intracellular ATP con-
tent, and lactate dehydrogenase release as indicators
for cellular damage; these tests are in conformity
with the ‘‘Biological Evaluation of Medical Devices-
part 5: tests for in vitro cytotoxicity guidelines (ISO
174 Histochem Cell Biol (2010) 134:171–196
123
10993-5:1999)’’. Blood smears were also evaluated
to identify changes in platelets due to treatment with
nanoparticles. Whole blood sampled in citrate tubes
was incubated, within 10 min after venipuncture, for
10 min at room temperature with an equal volume of
particles diluted in phosphate-buffered saline (PBS).
Whole blood incubated with ADP (20 lM), for
2 min at room temperature, served as positive
control. After incubation, blood smears were pre-
pared and fixed for 5 min in 100% methanol.
Subsequently, smears were stained, firstly for
3 min with May–Grunwald (Gatt-Koller, Austria)
staining solution, secondly for 1–2 min with May–
Grunwald solution diluted 1:2 in PBS and thirdly for
40 min with the Giemsa staining reagent diluted
1:10 in distilled water. After rinses in distilled water
the slides were coverslipped and viewed under an
Olympus IX51 microscope.
12. Nanoparticle hemocompatibility was examined by
assessing the effect of incubating human erythrocyte
suspensions with the nanoparticles and determining
the degree of hemolysis. Plasma from human volun-
teers was also incubated with the nanoparticles, and
levels of complement C3a and of prothrombin and D-
dimer measured as indicators of activation of the
complement system and of coagulation; these tests
are in conformity with the ‘‘Biological Evaluation of
Medical Devices-part 4: selection of tests for inter-
action with blood (ISO 10993-4:2002)’’, and were
approved by the local Ethics Committee.
13. The yields of nanoparticles were determined for each
batch, to allow estimation of interbatch variability
and of overall synthesis efficiency.
The above analytical procedures were carried out
exactly as described in our previous paper (Stollenwerk
et al. 2010), to allow precise comparison with the nano-
particles of series I and series II described in that report.
The nanoparticles described in the present paper can
therefore be considered as series III in our ongoing trans-
lational research in iterative design, synthesis and optimi-
sation of successive generations of albumin-based
nanoparticles. In order to gain closer control over our
synthetic procedures, however, we introduced a suite of
further analytical techniques aimed at real-time analysis of
intermediate-product and product quality during the syn-
thetic procedures. In this way it was possible to vary sen-
sitive parameters rapidly in a directed fashion and thus to
fine-tune batch optimisation. These newly introduced
assays are described in detail below; furthermore, the
assays described above were also sometimes applied in
modified form, which will be noted as appropriate in the
subsequent text. The newly introduced assays were as
follows:
14. Preparation of lectin-targeted nanoparticles Lyco-
persicon esculentum agglutinin (LEA) was extracted
and purified from tomato fruits as described in
our earlier paper (Paschkunova-Martic et al. 2005).
A nanoparticle colloidal solution in PBS (pH
8.0, 0.15 M) was used. N-(3-dimethylaminopropyl)-
N0-ethyl-carbodiimide hydrochloride was added
(0.63 mg carbodiimide per mg nanoparticle) in order
to activate the carboxylic groups on the albumin
molecules of the nanoparticle surface. The mixture
was stirred for 2 h at room temperature and, in some
batches, excess carbodiimide reagent was removed,
then LEA presuspended in distilled water was added
dropwise to the activated nanoparticles (LEA:nano-
particle ratio 1:20 w/w). The pH was adjusted to 6
with 1 M HCl and the sample was stirred over night
at room temperature, thus coupling the LEA to the
nanoparticles. Aliquots of the resulting nanoparticle
colloidal solutions were analysed by PCS, TEM, and
by SDS gel electrophoresis.
15. Preparation of PEGylated nanoparticles a-Methoxy-
x-carboxylic acid succinimidyl ester polyethylene
glycols having molecular weights of 5, 10 and
20 kDa, respectively, were used (Iris Biotech GmbH
Marktredwitz, Germany). The activated PEGs were
added in solid form to the nanoparticle samples
(HSA:PEG molar ratio 1:1) and stirred for 30 min at
room temperature. Afterwards the nanoparticles were
crosslinked with 1% glutaraldehyde (1:10 v/v) and
stirred over night at room temperature.
16. TLC was used to determine the purity of the starting
materials, conjugates, and nanoparticles. TLC was
carried out on Silica Gel 60 UV254 sheets (Polygram�
Sil G/UV254, 0.2 mm; Macherey–Nagel, Germany).
The solvent system employed was citric acid and
sodium citrate in a 1:2 v/v ratio. The samples were
spotted onto the sheets using micropipettes. After the
spots were completely dry the sheets were developed
in the citrate solvent system. Afterwards the sheets
were dried and detection of the samples was
performed using UV light at 254 and 366 nm as well
as iodine vapour or ninhydrine spraying-reagent.
Some sheets were prepared for detection of free
gadolinium, by brief immersion in the Arsenazo III
solution (see below).
17. Colorimetric testing with Arsenazo III (10 lmol
solution in 0.1 M acetate buffer, pH 5) was per-
formed according to a protocol from Nagaraja et al.
(2006). Briefly, 200 ll of arsenazo III were spiked
with 50 ll of conjugate or nanoparticle samples; a
Histochem Cell Biol (2010) 134:171–196 175
123
change in the colour of arsenazo III then indicated the
chemical state of gadolinium (chelated metal gave a
purple colour, and non-chelated metal gave a green
colour).
18. Pyknometry was employed to measure the density of
conjugates and of nanoparticles. Conjugates as well
as nanoparticles were measured as liquids and their
densities were calculated based on their concentra-
tions. Concentrations of HSA, HSA-DTPA-Gd and of
nanoparticles were measured by UV spectrometry at
280 nm. Density measurements were carried out
using a pyknometer containing a thermometer (Brand
GmbH und Co KG, 97861 Wertheim/Main, Ger-
many; nominal volume 10.0 cm3, certified measured
volume of 9.8162 cm3). The density was calculated
as mass/volume at 20�C.
19. Polyacrylamide gel electrophoresis (PAGE) for all
PAGE methods described here the Mini-PROTEAN�
Tetra Cell from BioRad (Vienna, Austria) as well as
their gels, standards, running and sample buffers were
used. Only the fixative, staining and destaining
solutions were prepared manually but according to
the protocols given by BioRad. Three different
conditions of PAGE were performed, also following
the protocols by BioRad:
– SDS PAGE this was slightly modified from the
protocol described in our previous paper (Stol-
lenwerk et al. 2010). Briefly, 7.5% Tris–HCl gels
containing no SDS were used. The amount of
SDS necessary to denature the proteins was
present only in the sample and running buffers.
The sample concentrations were adjusted to 0.5 or
1 lg/ll and after addition of the sample buffer
they were heated to 95�C for 5 min. Immediately
afterwards they were loaded onto the gel together
with a molecular weight standard. The loading
volumes were 10 ll for the standard and 20 ll for
each sample, the gel running conditions were set
to 50 V for 5 min and 150 V for 60 min. After
the electrophoresis was complete the gel was
removed, stained with Coomassie blue for 1 h and
destained until the desired background was
reached.
– Native PAGE also here 7.5% Tris–HCl gels
containing no SDS were used. Under native
conditions SDS is neither present in the sample
nor in the running buffers. The samples were not
heated but loaded directly onto the gel after
addition of the sample buffer. All other conditions
were the same as for SDS PAGE.
– Isoelectric focussing (IEF) PAGE IEF gels were
used having a pH range from 4 to 8.5. The sample
concentrations were adjusted to 0.5 or 1 lg/ll.
After addition of the sample buffer they were
loaded directly, without being heated, onto the gel
together with an IEF standard. The loading
volumes were 10 ll for the standard and 20 ll
for each sample. The gel running conditions were
set to 100 V for 60 min, 250 V for 60 min and
500 V for 30 min. After the electrophoresis was
complete the gel was removed, stained with IEF
stain for 1 h and destained until the desired
background was reached.
20. Modelling the reliability of TEM measurements of
nanoparticle size a model ensemble of particles
(glass marbles of various sizes) was photographed to
provide well-defined images allowing particles of
many sizes to be counted and measured. Particle
collectives were counted at different numbers
(n = 1 - 1,000), then recounted with the removal
of a single small particle, a large-sized particle, or
one of each. For each collective, the average size
and the polydispersity index (PDI) were calculated
as described earlier in detail (Stollenwerk et al.
2010).
21. Numbers of nanoparticles in 1 g the concentration of
a nanoparticle colloidal solution was determined by
UV spectrometry at 280 nm. The solution was
adjusted to a concentration of 1 lg/ml (10-6 g/ml)
by adding water. A dilution series was prepared in
which each member was tenfold more dilute, the final
concentration being 10-12 g/ml. From each solution
in the dilution series, a 1 ll drop was allowed to dry
onto a formvar-coated slot copper grid. Each grid was
dipped for 10 s into a 1% solution of uranyl acetate in
30% methanol, then dried under a lamp. Grids were
viewed in a Philips CM 120 electron microscope, and
a grid selected for counting which bore easily
countable populations of nanoparticles. The dilution
factor associated with this grid was noted, and was
employed in the subsequent calculations, which were
used to calculate the number of nanoparticles per
milliliter at that concentration of the nanoparticle
solution.
Efficiency calculations
Synthesis yields were calculated on the basis of the amount
of HSA protein used as starting material. UV spectrometric
measurements made immediately after diafiltration of the
purified protein were used to calculate the efficiency of this
step, and similar measurements made immediately after
diafiltration of the HSA-DTPA conjugates or, in some
176 Histochem Cell Biol (2010) 134:171–196
123
cases, the HSA-DTPA-Gd conjugate-chelates, were used to
calculate the efficiency of the synthesis prior to emulsifi-
cation with PLA. The calculations were carried out for
each batch individually and for the conjugation/chelation
step incorporated the assumptions that a single gadolinium
ion was chelated by a DTPA moiety and that there were no
unoccupied DTPA moieties; the absence of non-chelated
gadolinium ions had been previously checked by arsenazo
III analyses.
Storage of the nanoparticles
Nanoparticle preparations were stored in water in the
presence of 0.1% sodium azide, at 4�C.
Results
Monitoring and improving purity
Dialysis was used for early work on batch 1 of series III. Its
efficacy was tested, using cutoffs (100, 500 kDa) well
above the molecular weight of HSA (MW 66,400). Pierce
and AAS (Fig. 1a) and MRI (Fig. 1b) analyses showed that
dialysis of HSA proceeded extremely slowly in water and
that it failed entirely in saline and PBS; use of the higher
cutoff (500 kDa) did not improve the results. In conse-
quence, diafiltration was introduced in late stages of batch
1 and used in all later batches for purification of interme-
diate stage products and of nanoparticles. At the same time,
a suite of analytical techniques was assembled to allow
rapid detection of contaminants and thus monitor progress
of the purification. TLC was the first of these techniques. It
was used to check both conjugates and nanoparticles. It
confirmed earlier Pierce, AAS and MRI results in series II
and batch 1 of series III, which in some batches indicated
the presence of unexplained excesses of gadolinium and
implied the presence of varying amounts of educts con-
taminating both the conjugates and the nanoparticles. TLC
also aided analysis of intermediate steps during the course
of the reactions which conjugated DTPA to HSA, and then
chelated Gd to the HSA-DTPA conjugates (Fig. 2); it
revealed the presence of major contaminant materials, in
this case non-bound DTPA and non-chelated gadolinium.
Development of the TLC sheets in arsenazo III solution
(see below) showed non-chelated gadolinium present in
the [Gd(NTA)2]Na3 (NTA-Gd) stock solution (Fig. 2a).
Figure 2b demonstrates that the (diafiltered) conjugates were
not contaminated with non-chelated gadolinium (lane 5 is a
positive control). This method is a novel means of visualis-
ing non-chelated gadolinium, and Fig. 2a, b illustrate its
utility. TLC developed by exposure to iodine vapour
revealed impurities in preparations of both conjugates and
nanoparticles. Stabilised HSA was free of contaminants,
whereas HSA-DTPA-Gd conjugates were contaminated by
NTA and/or DTPA prior to diafiltration (Fig. 2c). The large
total amounts of educt present in filtrates (Fig. 2c, d, lanes
4, 5, respectively) show the effectiveness of diafiltration in
purifying the conjugates and the nanoparticles. Diafiltra-
tion, however, altered the conformation of the albumin
(compare lanes 1, 4 in Fig. 2c, and 1, 3 in d). For nano-
particles, Fig. 2e shows a particularly favourable result, in
which the nanoparticles exhibited ideal purity and also
good stabilisation. Figure 2f, in contrast, shows two
nanoparticle batches in which glutaraldehyde fixation
appears to have displaced some DTPA from the conjugates.
Arsenazo III chromogenic analysis provided a second
rapid procedure for monitoring the progress of purification.
Arsenazo III solutions distinguished between chelated and
non-chelated gadolinium: the presence of chelated gado-
linium was indicated by a change of arsenazo III colour
from bright pink to lighter or darker purple, depending on
the concentration of chelated gadolinium present, whereas
a change to green indicated the presence of non-chelated
gadolinium (Fig. 3a). In diafiltered preparations of
Fig. 1 Retention of albumin and albumin-DTPA-Gd conjugates in
dialysis tubing throughout periods 15 min to 50 h. a Dialysis using
both 100 and 500 kDa cutoffs, results analysed by AAS. b Dialysis
using 100 kDa cutoff, results analysed by MRI
Histochem Cell Biol (2010) 134:171–196 177
123
conjugates or nanoparticles, this method showed that non-
chelated gadolinium was not present (Fig. 3b). The arse-
nazo III method allowed the course of gadolinium binding
to HSA, followed by the removal of the bound gadolinium,
to be visualised effectively: Fig. 3c shows an experiment
to assess whether gadolinium binds to HSA which bears no
DTPA moieties. Tubes 2 and 7 in the Figure showed
that the gadolinium did indeed bind to the pure HSA, and
this was confirmed by AAS/Pierce analysis, which mea-
sured *1.7 gadolinium ions per HSA molecule (we con-
sider it likely that these gadoliniums were bound to the two
metal-binding sites of HSA). Further testing with arsenazo
Fig. 2 Analysis of intermediate products by thin layer chromatog-
raphy (TLC). a TLC in the citric acid/citrate system, developed by
immersion in arsenazo III. Lane 1 NTA, lane 2 NTA:Gd stock
solution, lane 3 GdCl3, lane 4 DTPA, lane 5 Magnevist = DTPA-Gd.
The green line shows non-chelated gadolinium. Note that Magnevist
shows no non-chelated gadolinium. b TLC in the citric acid/citrate
system, developed by immersion in arsenazo III. The arsenazo has
stained the TLC sheet lightly pink, except where the presence of large
molecules or particles has hindered its access to the sheet material.
Lane 1 HSA-DTPA-Gd of batch 15, before diafiltration, lane 2 HSA-
DTPA-Gd of batch 15, after diafiltration: retentate (i.e.: containing the
purified conjugate), lane 3 HSA-DTPA-Gd of batch 15, after
diafiltration: filtrate (i.e.: containing the impurities which have been
removed by diafiltration), lane 4 nanoparticles of batch 15, after
glutaraldehyde fixation, lane 5 GdCl3 as positive control. Note that
only lane 5 shows the presence of non-chelated gadolinium, in the
form of a green line at the forward edge (asterisk). c TLC in the citric
acid/citrate system, developed by exposure to iodine vapour, showing
preparations from batch III/11. The iodine has stained the TLC sheet
material yellow, and all amines brown. Lane 1 stabilised HSA, lane 2HSA-DTPA, before diafiltration: note the presence of a dark heart-
shaped spot indicating the presence of DTPA not attached to the HSA,
lane 3 HSA-DTPA-Gd before diafiltration: here also, a prominent
spot indicates the presence of DTPA not attached to the HSA, lane 4HSA-DTPA-Gd, after diafiltration: retentate: we suggest that the faint
spot (beneath the DTPA spot in lane 3) represents DTPA covalently
attached to the HSA. Lane 5 HSA-DTPA-Gd after diafiltration:
filtrate; note that the material in lane 5 was a small sample (*2 ll)
from a large volume (600 ml) of diluted filtrate: in this strongly
diluted sample the moderately intense spot represents a much larger
total of DTPA in the total filtrate. d TLC in the citric acid/citrate
system, developed by exposure to iodine vapour, showing prepara-
tions from batch III/13. Lane 1 stabilised HSA, lane 2 HSA-DTPA-
Gd before diafiltration: note the intense heart-shaped band indicating
the presence of DTPA not bound to the HSA. Note also an extended
faint spot (in blue oval) which we consider to represent NTA, lane 3HSA-DTPA-Gd, after diafiltration: retentate, lane 4 HSA-DTPA-Gd,
after diafiltration: filtrate, in this strongly diluted sample the
moderately intense spot represents a much larger total of DTPA in
the total filtrate. e TLC in the citric acid/citrate system, developed by
exposure to iodine vapour, showing preparations from batch III/3.
Lane 1 HSA-DTPA-Gd, after diafiltration: retentate, lane 2 stabilised
nanoparticles after diafiltration: retentate, lane 3 stabilised nanopar-
ticles after diafiltration: filtrate, lane 4 stabilised nanoparticles not
diafiltered (and the result of an experiment in which HSA:PLA was
emulsified in the ratio 1:1). Note that in lanes 2, 4, the nanoparticles
occupy a round spot at the origin, indicating the absence of
conformational isoforms, i.e.: an ideal result—these nanoparticles
are shown again in Fig. 4a, lanes 3–4. f TLC in the citric acid/citrate
system, developed by exposure to iodine vapour, showing prepara-
tions from batches III/14 (lanes 1, 2) and III/15 (lanes 3, 4). Lane 1batch 14 nanoparticles before glutaraldehyde fixation, Lane 2 batch
14 nanoparticles after glutaraldehyde fixation: note the presence of
non-bound DTPA which is not evident in lane 1, lane 3 batch 15
nanoparticles before glutaraldehyde fixation, Lane 4 batch 15
nanoparticles after glutaraldehyde fixation: note again the presence
of non-bound DTPA which is not evident in lane 3
c
178 Histochem Cell Biol (2010) 134:171–196
123
III then showed that it was possible to remove all the bound
gadolinium by adequate diafiltration (tubes 3–5 in the
Figure), and that this was more effective in the presence of
citrate ions (tubes 6–10 in the Figure); this was confirmed
by AAS. A further example of the use of arsenazo III
resulted from our query whether our NTA stock solution
contained non-chelated gadolinium. Figure 3d shows that
the stock NTA-Gd solution did contain non-chelated gad-
olinium (tube 2 in the Figure), and that increasing the
proportion of NTA only gradually chelated more of the
gadolinium (tubes 3–7 in the Figure), and all the gadolinium
was chelated only when high molar ratios (NTA:Gd [ 5:1)
were used (tubes 8, 9 in the Figure).
Polyacrylamide gel electrophoresis (PAGE)
PAGE was used to assess mechanical stability of the nano-
particles under a range of various preparative procedures,
and was also used to assess the charge:mass properties of
both nanoparticles and conjugates. To assess the mechanical
stability of individual nanoparticles, nanoparticle colloidal
solutions were placed on SDS–PAGE gels. The particles are
larger than the pores within the gel, so that cohesive (stable)
particles should not enter the gel: they remained at the gel
origin. Macromolecules such as HSA and HSA-DTPA-Gd,
being much smaller, entered the pores within the gel and
migrated through the gel according to their mass and charge
properties (Fig. 4a, lanes 2, 6, 7). The presence of SDS in the
gel aided the disintegration of unstable particles, reducing
them to their macromolecular constituents (Fig. 4a, lanes 3,
4, 8, 9). In certain nanoparticle batches, some mechanically
stable particles were present, and these did not enter the gel
(Fig. 4a, lanes 3, 4); less stable nanoparticles entered the gel
and disintegrated there [note that the batch of nanoparticles
shown in lanes 3, 4 also exhibited favourable properties in
the TLC assay (Fig. 2e, lanes 2, 4)]. In other batches of
nanoparticles, none of the particles exhibited adequate
mechanical cohesion to resist entering the gel (Fig. 4a, lanes
8, 9). Batches of nanoparticles which were not only glutar-
aldehyde fixed (generating Schiff bases between the protein
Fig. 3 Analysis of gadolinium chelation by arsenazo III staining.
a The three colours of arsenazo III, photographed from a 12-well
plastic dish. At top, the pink colour characterises the arsenazo III
solution in which no gadolinium is present. At centre, the greencolour shows that gadolinium is present in non-chelated (‘‘free’’)
form. At bottom, the purple colour shows that gadolinium is present,
but is fully chelated: no free gadolinium ions are present. Since
arsenazo colour depends on pH of the solution, it should be noted that
the colours shown here were recorded at pH 4.0. Note that b, c have
been lightly retouched to remove background details. b Conjugates
and nanoparticles. Tube 1 stock NTA-Gd, Tube 2 conjugate 5 min
after addition of stock NTA-Gd, Tube 3 conjugate after 24 h at 4�C
before diafiltration, Tube 4 conjugate after diafiltration: retentate,
Tube 5 pure arsenazo III solution, Tube 6 glutaraldehyde-fixed
nanoparticles before diafiltration, Tube 7 glutaraldehyde-fixed nano-
particles after diafiltration: retentate, Tube 8 glutaraldehyde-fixed
nanoparticles after diafiltration: filtrate. c Experiment testing potential
binding of gadolinium ions to HSA in pure form (without DTPA
attached). The five tubes (1–5) at left show (1) arsenazo III in pure
solution; (2) HSA plus gadolinium prior to diafiltration; (3) HSA plus
gadolinium diafiltered with 150 ml water; (4) HSA plus Gd diafiltered
with 300 ml water; (5) HSA plus gadolinium diafiltered with 450 ml
water. Tubes 6–10 show the same experiment as tubes 1–5, except
that citrate ions are present at a concentration of 0.1 M. d Nine
Eppendorff tubes containing arsenazo III solution (pH 4.0) to which
different combinations of NTA-Gd have been added. At left (1) is
arsenazo III solution without NTA-Gd; the following eight tubes (2–
9), from left to right, contain NTA-Gd in different proportions. Tube 2
contains NTA:Gd at 2:1 ratio, each subsequent tube is a further
increase in this ratio, until the tube at right (number 9) is reached,
which contains 8 NTAs per 1 Gd. Note the development of the colour
from green (tube 2) through blue (tubes 5, 6) to purple (tubes 8, 9),
indicating that an excess of NTA in the ratio of 7:1 or 8:1 is necessary
to ensure that all the gadolinium ions in the solution are chelated
Histochem Cell Biol (2010) 134:171–196 179
123
Fig. 4 Analysis of intermediate products by polyacrylamide gel
electrophoresis (PAGE). a SDS–PAGE of stabilised HSA (lane 6), of
conjugates after diafiltration (lanes 2, 7) and of glutaraldehyde-fixed
nanoparticles after diafiltration (lanes 3, 4, 8, 9). (Note that the particles
shown here in lanes 3, 4 are shown again in Fig. 2e, lanes 2, 4). The
nanoparticles in lanes 8 and 9 break up completely under the denaturing
effect of SDS, only a small proportion of the nanoparticles in lanes 3 and4 (red arrows) remain at the origin. Lane 1 shows the molecular weights
(kDa) obtained by running standards in the same gel. No differences
between conjugates and nanoparticles are visible. b SDS–PAGE of
nanoparticles fixed with different concentrations of glutaraldehyde
(lanes 1–5 1–5% v/v) and quenched with 1% (v/v) NH4Cl, and also
nanoparticles fixed with the same concentrations of glutaraldehyde
(lanes 6–10 1–5%) but reduced with NaCNBH3. A better stability is
reached when the nanoparticles are treated with NH4Cl but they are also
more likely to form visible aggregates (data not shown). (No molecular
weight standard was applied to this gel but interpretation of the masses
is according to the standard shown in a.) c Native-PAGE of stabilised
HSA (lanes 1, 6), of HSA/DTPA after diafiltration (lane 2), of conjugate
after diafiltration (lane 3), of non-glutaraldehyde-fixed nanoparticles
before (lane 4) and after (lane 5) diafiltration and of glutaraldehyde-
fixed nanoparticles after diafiltration (lanes 7, 8). Under native
conditions the molecules migrate through the gel according to their
mass–charge ratio; also here no difference between conjugates and
nanoparticles is visible. (No molecular weight standard is available
for native gel conditions.) d shows SDS–PAGE of stabilised HSA
(lane 6), of conjugate after diafiltration (lane 7), of glutaraldehyde-fixed
nanoparticles (lanes 2, 8), of glutaraldehyde-fixed nanoparticles
PEGylated with PEG 5 kDa (lane 3), with PEG 10 kDa (lane 4) and
with PEG 20 kDa (lane 5), and of glutaraldehyde-fixed nanoparticles
targeted with LEA lectin (lane 9). Lane 1 shows the molecular weight
(kDa) obtained by running standards in the same gel. A clear difference
is visible between PEGylated and non-PEGylated nanoparticles.
PEGylated nanoparticles do not break up to the same extent as non-
PEGylated nanoparticles, and the degree of breakup decreases as the
length of the PEG chains increases (lanes 3–5). e shows IEF-PAGE of
non-stabilised (lane 2) and of stabilised HSA (lane 3), of series IIIconjugate after diafiltration (lane 4), of series III glutaraldehyde-fixed
nanoparticles after diafiltration (lanes 5–7) and of series II lyophilised
nanoparticles (lanes 8–10). HSA clearly shows its natural isoelectric
point (pI) of about 4.7 but no difference in the pI is visible between
conjugates and nanoparticles. Lane 1 shows the pI values obtained from
running standards in the same gel, these bands are blocked out where
they overlapped with lane 2
180 Histochem Cell Biol (2010) 134:171–196
123
constituents) but were also ‘‘quenched’’ with ammonium
chloride, or reduced to covalency by reaction with sodium
cyanoborohydride, contained significant proportions of
nanoparticles with mechanical stability sufficient to resist
entering the gel (Fig. 4b). The ‘‘quenched’’ particles con-
tained large majorities of stable nanoparticles, and the
minorities of particles that disintegrated and entered the gel
produced only weak bands representing macromolecular
constituents (i.e.: disintegration products) migrating through
the gel (Fig. 4b, lanes 1–5). In batches which were reduced
to covalency, the proportions of mechanically cohesive
particles were much smaller and the bands representing
particle disintegration were much more prominent (Fig. 4b,
lanes 6–10). Another form of PAGE, ‘‘native PAGE’’, in
which no SDS detergent is present and sample migration
depends on charge:mass relationships, showed that no dif-
ference could be observed between the HSA-DTPA-Gd
conjugates and nanoparticle samples (Fig. 4c), thus dem-
onstrating that the major component of the nanoparticles is
indeed HSA-DTPA-Gd. Further PAGE (SDS–PAGE)
analyses showed that ammonium chloride quenching and
covalent reduction were not the only methods that protect
nanoparticles against mechanical disintegration: PEGyla-
tion of glutaraldehyde-fixed nanoparticles provided an
additional stabilisation which was clearly seen in SDS–
PAGE (Fig. 4d), and which increased as the mass of the PEG
chains increased (compare lanes 3, 4, 5 in Fig. 4d). On the
other hand, LEA-derivatized nanoparticles showed little
mechanical stability and disintegrated in SDS–PAGE
(Fig. 4d, lane 9). A further form of PAGE, isoelectric
focussing (IEF-PAGE), provided information confirming
the variability of the HSA-DTPA conjugates: a clear pI of
4.7 was seen for HSA, with well-defined, sharp bands
(Fig. 4e, lanes 2, 3). In contrast, conjugates and nanoparti-
cles produced smears in the range pH 5.5–7.5, indicating the
presence of numerous charged variants within the molecular
and particle populations (lanes 4–10, Fig. 4e); we attributed
these to the varying numbers of DTPA-Gd chelates attached
to the HSA.
Size and size standardisation
HSA-DTPA-Gd conjugates
After removal of all ligands from fraction V HSA, and
their replacement by caprylate as single major ligand, the
HSA molecules assumed a standard and highly repro-
ducible set of properties, most easily demonstrated by
PCS as a narrow distribution of particle size close to
10 nm hydrodynamic diameter (Fig. 5a). The size
increased to 14 nm hydrodynamic diameter after attach-
ment of the DTPA groups (Fig. 5b) and after chela-
tion of the gadolinium ions, but remained uniform
(Fig. 5c); both the size and the variability of the protein
remained similar throughout these different stages of the
synthesis. Diafiltration of the conjugates, and emulsifi-
cation of conjugates with PLA, led to some aggregation
(Fig. 5d).
Reliability and validation of single particle counts
The number of particles that should be counted in order to
obtain reliable valid PDI values was investigated by use of
a simple glass marble model (Fig. 6a, b). The sensitivity to
omission of a single particle from the ensemble was high
for small ensembles, for example omitting a single rela-
tively very small particle from an ensemble of size n = 29
(Fig. 6a) altered PDI from 6.168 to 5.96 (-3.37%);
omission of a single large particle changed PDI from 6.168
to 6.595 (?6.92%). Removing a single particle from larger
ensembles altered the PDI values obtained by smaller
amounts, the exact value depending on the relative size of
the particle removed. Removal of a very small particle
resulted in a divergence of the PDI value by \ 1% if 100
particles were counted; removal of a very large particle
resulted in a divergence of \ 1% only after [ 500 particles
had been counted (Fig. 6b). The sample size n = 500 was
therefore selected as the minimum to obtain reliable PDI
values; a sample of nanoparticles of this population size is
shown in Fig. 7a.
Characterisation of the nanoparticles’ size
Starting with homogenous HSA-DTPA-Gd conjugates,
emulsification with PLA produced a turbid solution
containing aggregates of various sizes. Removal of these
aggregates by filtration through paper filters resulted in
populations of nanoparticles which had PDI values in the
range 1.2–1.6, and generally *1.4–1.5. Aggregation also
occurred after later steps in the synthesis, including
diafiltration of fixed or unfixed nanoparticles (Fig. 5e, f).
The almost uniform size of the filtered nanoparticles was
evident in TEM images (Fig. 7a). In most batches the
nanoparticles had average diameters close to 18 nm
(Fig. 7b). The uniform nanoparticle size was reflected in
PDI values in the range 1.2–1.6 (Fig. 7c). In both their
nanoparticle size and the size variability characteristics,
these batches represented a clear improvement on the
previous series II of nanoparticles (Stollenwerk et al.
2010, Fig. 6).
Numbers of nanoparticles per gram
For batch 15, *500 nanoparticles were counted in a single
electron microscopic field of view at 88,0009 (Fig. 7a),
the field of view covering an area *1.7 lm2. The 0.2 ll
Histochem Cell Biol (2010) 134:171–196 181
123
drop of nanoparticle solution applied to the grid had dried
down to cover an area *1 mm2 = 106 lm2) so it had
contained approximately [500 9 106]/1.7 = 2.94 9 108
nanoparticles. Noting our definition of 6.022 9 1023
nanoparticles as representing 1 mol of nanoparticles, we
find that the 0.2 ll drop had contained [2.94 9 108/6.022 9
1023] = 4.88 9 10-16 mol of nanoparticles, which we
rounded to 5 9 10-16 mol.
Numbers of conjugates per nanoparticle
The measured concentration of HSA in the nanoparticle
solution of batch 15, used to prepare Fig. 7a, was
1.5 9 10-5 mol/ml, and that solution was diluted by a factor
106 prior to application to the grid, so the 0.2 ll drop of this
solution had contained 3.01 9 10-15 mol of HSA. The
solution contained*3 9 10-15 mol of HSA arranged in the
Fig. 5 Follow-up of our nanoparticle synthesis shown by volume-
weighted PCS read-outs, the numbers given at each peak represent its
mean size, its standard deviation and the percentage of the total
particle population of that peak. a Cleaned and stabilised HSA,
b shows HSA/DTPA (not diafiltered), c shows HSA-DTPA-Gd before
diafiltration, d shows HSA-DTPA-Gd after diafiltration, note that
diafiltration causes mechanical stress which disturbs the protein
conformation and leads to minor aggregation, e shows nanoparticles
after emulsification with PLA, not yet fixed with glutaraldehyde and
not diafiltered, note that emulsification leads to strong formation of
aggregates, f shows nanoparticles, fixed with glutaraldehyde and
diafiltered
182 Histochem Cell Biol (2010) 134:171–196
123
form of *5 9 10-16 nanoparticles, indicating that each
mole of nanoparticles contained [3 9 10-15/5 9 10-16] =
6 mol of HSA. Thus each 18 nm nanoparticle comprised six
molecules of HSA-DTPA-Gd (plus a small amount of PLA).
Further calculations become possible when the packing
density of the nanoparticles is known (see below).
Packing density of the nanoparticles
Nanoparticles in batch 15 were (on average) 18 nm in
diameter, and we calculate the packing density for a
nanoparticle of this size and containing six conjugate
molecules. The nanoparticle volume was [(4/
3)p(9)3] nm3 = 3,054 nm3. Taking the volume of HSA to
be 90 nm3 (Stollenwerk et al. 2010; Sugio et al. 1999), a
nanoparticle containing six HSA molecules (total vol-
ume = 540 nm3) has a packing density (540/
3,054) 9 100% = 17.7%. This calculation ignores, how-
ever, the DTPA-Gd chelates attached to the nanoparticles,
which increase their volume; this will be considered more
closely in the ‘‘Discussion’’. Taking the packing density
value based simply on HSA, a nanoparticle of 20 nm
diameter, with a volume of 4,188 nm3, of which 17.7%
(741 nm3) consists of protein material, would contain (741/
90) = 8.2 conjugate molecules. In our series III batches,
nanoparticle sizes are between 18 and 22 nm diameter, so
our evidence indicates that the nanoparticles each consist
of 6–10 HSA-DTPA-Gd conjugate molecules, together
with a small amount of PLA (see below).
Electrical charge on the conjugates and nanoparticles
Surface charge measured as zeta potential
HSA-DTPA-Gd conjugates carried a small positive charge,
between 1 and 4 mV. Nanoparticles formed from these
conjugates carried a larger and negative charge, -30 to
-40 mV, which, however, disappeared after the nanoparticle
Fig. 6 Glass marble model experiment to determine the minimum
counts necessary to generate reliable size and PDI data from TEM
images of the nanoparticles. a Glass marble collectives to demonstrate
the concept underlying the PDI. At left, 29 marbles form a population
of various sizes, the variability amongst the marbles being summa-
rised by the large value for PDI: 6.16. At right, 17 marbles form a
population of marbles of closely similar size, the uniformity of the
sizes being summarised by the small value for PDI: 1.15. For marbles
of identical size, PDI = 1.0. A small marble (red asterisk) or a large
marble (blue asterisk) were removed from the population at left
during counting experiments, see b. b Data from repeated counts of
the marble population shown in a. During one of the many counting
sessions, one large marble, one small marble, or one large and one
small marble, were removed, thus altering the PDI. After repeated
counting sessions the alteration (carried out once only) became
insignificant in its effect on PDI, and the calculated PDI therefore
converges on the original value. The convergence is seen as the
‘‘error’’ curves approach the zero line. For each curve, counts of
increasing size were made, the largest being 579 counts. Pink line:
removal of a single large marble; green line: removal of one small and
one large marble; blue line: removal of a single small marble. The red
number placed against each line indicates the number of counts
necessary to achieve an ‘‘error rate’’ less than 1% for this type of
‘‘variation’’
Histochem Cell Biol (2010) 134:171–196 183
123
solutions were lyophilised. Nanoparticles targeted by
attachment of the LEA lectin also carried significant neg-
ative charge (-20 to -30 mV).
Density of the conjugates and nanoparticles
Conjugates and nanoparticles were assayed by pyknometry
in concentrations ranging from 5 to 25 mg/ml, the upper
limit being set by the maximum nanoparticle concen-
tra-tion available after diafiltration; HSA was assayed up to
30 mg/ml. All results obtained from solutions at concen-
trations below 10 mg/ml were below the detection limit of
our pyknometry. The data obtained from solutions above
20 mg/ml showed a trend: based on the remaining small
number of measurements, the density of stabilised HSA
was 1.86 g/cm3, that of the HSA-DTPA conjugate 1.58
g/cm3, that of the HSA-DTPA-Gd conjugate 1.75 g/cm3
and that of the nanoparticles 1.38 g/cm3.
Structural stability of the nanoparticles
As noted above, treatment of the nanoparticles with glu-
taraldehyde, or with glutaraldehyde followed by exposure
to ammonium ions, or to glutaraldehyde followed by
reduction to covalency, all enhanced the mechanical sta-
bility of the nanoparticles. PEGylation with 20 kDa PEG
chains also protected the nanoparticles from breaking up in
polyacrylamide gels.
Fig. 7 Size analysis of the nanoparticles. a A negative contrast
transmission electron microscopical image of nanoparticles of batch
III/15, following glutaraldehyde fixation and diafiltration. Original
magnification 988,000. Calibration bar 200 nm. b A dot diagram of
the nanoparticle sizes in series III batches. Each dot represents a
series III batch and is denoted by the batch number. The scale is the
same as that used in our previous paper (Stollenwerk et al. 2010). c A
dot diagram of the PDI values in series III batches. Each dotrepresents a series III batch and is denoted by the batch number. The
scale is the same as that used in our previous paper (Stollenwerk et al.
2010)
184 Histochem Cell Biol (2010) 134:171–196
123
Conformation of protein components
in the nanoparticles
After glutaraldehyde fixation and ammonium chloride
quenching, the nanoparticles were unaltered in size
(20–30 nm diameter), and retained the antigenic epitopes
characteristic for human serum albumin (Fig. 8).
Number of gadolinium ions in the nanoparticles
Characterisation of the HSA-DTPA-Gd conjugates
The conjugates were checked for purity, in particular for the
absence of contaminating educts such as non-chelated
gadolinium, as described above. Then the Gd:HSA ratios
were determined by Pierce/AAS assays. As shown in Fig. 9,
early batches in this series showed relatively low Gd:HSA
ratios, but later batches had higher ratios and the final bat-
ches tended to cluster in the Gd:HSA = 15–20 range. An
experiment carried out to assess how many gadolinium ions
could bind to each molecule of pure HSA (not bearing
DTPA) gave the result 1.7 (there are two metal-binding sites
in each HSA molecule); adequate diafiltration removed
these gadolinium ions from the HSA molecules (Fig. 3),
and since adequate diafiltration was routinely applied, this
type of non-specific binding was ignored during the pre-
paration of all later batches. A nanoparticle with six conju-
gate molecules has packing density *17%, mass 461 kDa
and relaxivity r1 = 0.83 9 106/Ms. It carries 114 Gd ions
each with relaxivity *7,280/Ms.
Relaxivities of the conjugates and nanoparticles
The conjugates in this series III exhibited higher relaxivi-
ties than in the previous series II nanoparticles. After an
initial learning period, comprising the first five batches of
series III, we obtained conjugates with r1 values routinely
exceeding 3 ml/mg s, and clustered between 3 and 4 ml/
mg s; their r2 values all exceeded 4 ml/mg s, clustering
between 4.5 and 5.5 ml/mg s (Fig. 10a). Similarly, the
nanoparticles, after the first five batches, exhibited r1
Fig. 9 Dot diagram of Gd:HSA ratios for nanoparticle batches in
series III. Each dot represents a single batch, denoted by its series IIIbatch number. Values for LEA-targeted nanoparticles are shown in
red. Batch 5 was derivatized with too small an amount of gadolinium.
Its low Gd:HSA ratio resulted in a low relaxivity (compare Fig. 10);
this batch is shown here because it helps exemplify the relation
between Gd:HSA ratio and relaxivity (Fig. 11). a Data for the HSA-
DTPA-Gd conjugates, b data for the nanoparticles
Fig. 8 Series III batch six nanoparticles after glutaraldehyde fixation
and ‘‘quenching’’ with ammonium chloride. Original magnification
940,000. The sample was acquired from a PAGE application well
after the gel had been run, and essentially no nanoparticles had
entered the gel. The sample on the grid was incubated for anti-HSA
immunohistochemistry, with the second antibody bearing gold
particles
Histochem Cell Biol (2010) 134:171–196 185
123
values exceeding 1.5 ml/mg s, clustering between 1.5 and
2.0 ml/mg s; their r2 values all exceeded 2.5 ml/mg s,
clustering close to 2.6 ml/mg s (Fig. 10b).
The analytical data presented above showed that the
nanoparticles were of similar sizes and chemistry in bat-
ches 2–15 of series III. The coefficient of determination,
R2 *81%, seen in the regression plot (Fig. 11) between
Gd:HSA and relaxivity, was therefore an indication that
nanoparticle relaxivity depended directly on the number of
gadolinium ions present on the nanoparticles. The average
number of gadolinium attached to the HSA conjugate
molecules of the series III nanoparticles was 19 (Fig. 9a),
so the conjugate molecules had average molecular weight
([393.35 ? 157.25 = ] 550.6 9 19) ? 66,400 = 76,861
Da = *76,9 kDa. Consisting of six conjugate molecules,
a nanoparticle with diameter 18 nm therefore weighed
(6 9 76.9) = 461 kDa = 0.461 MDa. A typical value for
relaxivity r1 was 1.8 ml/mg s (Fig. 10), so the relaxivity
for the series III nanoparticles was close to r1 =
(0.461 9 1.8) = 0.830 9 106 /Ms. A similar calculation,
taking typical R2 values for series III as 2.6, indicates that
the nanoparticles had r2 = 1.199 9 106 /Ms.
Toxicological assessment of the nanoparticles
Cell viability remained above 90% at nanoparticle doses of
2 mg/ml, as measured by ATP content, and no membrane
damage was seen, as assessed by release of the cytoplasmic
enzyme lactate dehydrogenase; some loss in viability was
seen at 5 mg/ml (Fig. 12a); LDH release data showed a
similar pattern (Fig. 12b). Three of the four batches tested
did not cause hemolysis even at 5 mg/ml nanoparticle
concentration, and the fourth batch caused a maximum of
5% hemolysis at 5 mg/ml nanoparticle concentration
(Fig. 12c). Plasmatic coagulation was assessed by quanti-
fication of prothrombin F1 ? 2 fragments, which arise
during activation of the cascade. The level of the fragments
increased 12 times upon treatment with the positive control
(5 mg/ml kaolin) but was around 80% for the particles;
platelets showed aggregation upon treatment with the
positive control 20 lM ADP. In the negative control and in
the nanoparticle samples up to 5 mg/ml, pseudopod for-
mation of thrombocytes was seen occasionally, but no
aggregation of platelets occurred (Fig. 13).
Fig. 10 Dot diagram of HSA-DTPA-Gd conjugate and nanoparticle
relaxivities in series III. a Conjugate relaxivities are shown in the
form ‘‘ml/mg s’’; the red entries show the relaxivity r1, the blueentries show relaxivity r2. Due to improvements in our synthesis
technique, this diagram extends further to the right than the equivalent
diagram in our previous paper (Stollenwerk et al. 2010). The value for
batch 11 (off-scale in this diagram) was 6.11 ml/mg s. b Nanoparticle
relaxivities are shown in the form ‘‘ml/mg s’’; the red entries show
the relaxivity r1, the blue entries show relaxivity r2. Here also the
scale extends further to the right than in our previous paper
Fig. 11 This plot relates the Gd:HSA ratios of most series III batches
to their r1 relaxivities (batches 9, 10 were not derivatized with
gadolinium, and batch 1 was not fully purified). About 81% of the
variation in the response variable (r1 relaxivity) can be explained by
the explanatory variable (Gd:HSA ratio)
186 Histochem Cell Biol (2010) 134:171–196
123
Attaching targeting groups to the nanoparticles
Four of the fifteen batches (numbers 1, 2, 4, 11) were
derivatized by attaching the LEA lectin to the nanoparti-
cles. Serial dilution experiments allowed the minimum
concentration of LEA-bearing nanoparticles to be identi-
fied at which the particles agglutinated fresh human blood;
the minimum necessary LEA-nanoparticle concentration
was then compared with the minimum necessary LEA
concentration (Fig. 14). The results, shown in Table 1,
show that LEA-bearing nanoparticles were between 0.669
and 20.29 as effective as pure LEA in hemagglutination of
fresh human blood, with most of the ratios lying in the
range 69–129.
PEGylation of the nanoparticles
As shown by PAGE analysis, glutaraldehyde-fixed nano-
particles (Fig. 4d, lane 2) showed some resistance to
mechanical disruption. PEGylated nanoparticles showed
slightly more resistance if the PEG chains were of 5 kDa
mass (Fig. 4d, lane 3), significantly more resistance with
PEG chains of 10 kDa mass (lane 4), and strong resistance
to disruption with PEG chains of 20 kDa mass (lane 5).
Yield efficiencies of nanoparticle syntheses
Yields were calculated on the basis of HSA content, and
expressed as per cent fraction of the amount of HSA pro-
tein used as starting material for any particular batch.
Figure 15 plots two of the major synthetic steps in the
synthesis protocol, namely the cleaning and stabilisation of
the HSA protein, and the conjugation of DTPA/chelation of
Fig. 12 Toxicological data for series III batches 9 and 11. In batch 9,
the nanoparticles were PEGylated, and control nanoparticles were left
non-PEGylated. In batch 11, the nanoparticles were targeted with
LEA, and control particles were left untargeted. The plots show
a viability data, b LDH release data and c hemolysis data; in each
case no toxicity is evident at concentrations of any nanoparticles up to
2 mg/ml. At 5 mg/ml toxic effects begin to become visible
Fig. 13 Platelet aggregation test. a The HSA-based nanoparticles
cause no aggregation of the platelets; rarely a slight activation
(pseudopod formation) of a few platelets is seen (arrow). Calibrationbar 20 lm. b In the positive control, platelet aggregation is common
(arrows). Calibration bar 20 lm
Histochem Cell Biol (2010) 134:171–196 187
123
gadolinium to the HSA. The Figure shows that cleaning of
the protein was associated with relatively high loss rates,
typically close to 30%. The approximately 65% efficiency
of this step was the major determinant of overall efficiency:
the further *10% loss during conjugation/chelation, with
their associated purification procedures, did not involve a
critical reduction in overall synthesis efficiency. A learning
effect is evident in Fig. 15: only after the first five batches
did the yields become predictable; this is evidence for the
complex nature of the conjugation/chelation step, with its
numerous—and sometimes critical—variable parameters.
Discussion
The work described in this paper was a further step towards
optimising the HSA-based nanoparticles we described
previously (Stollenwerk et al. 2010); the improvements in
nanoparticle standardisation, purity and mechanical sta-
bility will be considered here. The question which chelator
to use for attachment of gadolinium will be left to a later
paper: for this work, the linear chelator DTPA was used as
previously. Suspecting that the intra- and interbatch vari-
ation seen in series II was partly due to contamination of
the HSA-DTPA-Gd conjugates by educts, we used protein
analyses and AAS to measure Gd concentration, PAGE to
demonstrate stability, mass and charge:mass properties of
the HSA protein and the HSA conjugates, TLC to visualise
several types of impurities, MRI to measure nanoparticle
relaxivity, PCS and TEM to determine nanoparticle size
and zeta measurements to determine surface charge. These
analyses showed that dialysis, as used in series II, had
indeed failed to remove most educts, leaving for example
single HSA-DTPA-Gd molecules amongst the nanoparticle
products; the presence of remaining educts skewed
size distributions, because singlets, duplexes and higher
aggregates of HSA and HSA-DTPA-Gd conjugates
remained in the samples and were measured together with
Fig. 14 Hemagglutination data from batches 2, 4, comparing LEA
with LEA-bearing nanoparticles. The figure has been lightly
retouched to remove background details. a A positive control series
in which LEA in pure form was applied, b, c two experimental series
in which the LEA was attached to nanoparticles. a 200 ll fresh
human blood mixed with 200 ll LEA solution, as a dilution series.
LEA concentration in tube 1 1 mg/ml, 2 500 lg/ml, 3 250 lg/ml, 4125 lg/ml, 5 62.5 lg/ml, 6 31.25 lg/ml, 7 15.63 lg/ml, 8 7.82 lg/
ml, 9 negative control, PBS only. Red asterisk at tube 6 shows the
minimal LEA concentration causing hemagglutination. b 200 ll fresh
human blood mixed with 200 ll LEA-bearing nanoparticles in
colloidal solution, as a dilution series. Nanoparticle-LEA concentra-
tion in tube 1 1 mg/ml, 2 500 lg/ml, 3 250 lg/ml, 4 125 lg/ml, 562.5 lg/ml, 6 31.25 lg/ml, 7 15.63 lg/ml, 8 7.82 lg/ml. Red asteriskat tube 6 shows the minimal LEA concentration causing hemagglu-
tination. c 200 ll fresh human blood mixed with 200 ll LEA-bearing
nanoparticles in colloidal solution, as a dilution series. Nanoparticle-
LEA concentration in tube 1 200 lg/ml, 2 100 lg/ml, 3 50 lg/ml, 425 lg/ml, 5 12.5 lg/ml, 6 6.25 lg/ml, 7 3.13 lg/ml, 8 1.56 lg/ml.
Red asterisk at tube 3 shows the minimal LEA concentration causing
hemagglutination
Table 1 The results of hemagglutination assays to determine the efficacy of LEA-bearing nanoparticles in agglutination of fresh human blood
Nanoparticle
batch details
Minimal nanoparticle concentration
causing hemagglutination (M)
Minimal LEA concentration
causing hemagglutination (M)
LEA:Nanoparticle ratio, to compare molar
efficacy in agglutinating human blood
Batch series III, #1 0.8–1.9 9 10-11 4.5–9.0 9 10-11 2.4–11.25
Batch series III, #2 2.1 9 10-11 1.8 9 10-10 8.6
Batch series III, #4 8.9 9 10-12 1.8 9 10-10 20.2
Batch series III, #4 2.86 9 10-11 1.8 9 10-10 6.3
Batch series III, #4 1.43 9 10-11 1.8 9 10-10 12.6
Batch series III, #11 5.4 9 10-10 3.57 9 10-10 0.66
188 Histochem Cell Biol (2010) 134:171–196
123
the nanoparticles. This partial failure of purification
explained the strong variability of the nanoparticles syn-
thesized in series II. Whereas dialysis failed to remove
these contaminants, diafiltration did so efficiently. By
repeated cycles of diafiltration it was possible to obtain
nanoparticles in which all the Gd was present as DTPA
chelates conjugated to the nanoparticles. The first major
alteration to our previous synthesis protocol was therefore
to replace dialysis with diafiltration.
The second major alteration to our previous synthesis
protocol was to de-fat and stabilise the HSA prior to pre-
paring HSA-DTPA conjugates. This was motivated by our
suspicion that variability in fraction V HSA played a major
role in causing variability in intra- and interbatch physi-
cochemical properties. The high interbatch variability of
HSA in fraction V (Cohn) was documented 40 years ago
(Chen 1967; Foster et al. 1965). We suspected that HSA
variability affected its properties as surfactant for PLA,
causing unpredictable variations in nanoparticle size. Pre-
viously we had used HSA as obtained from the commercial
supplier to prepare the nanoparticles (series I, series II),
finding variable composition (Stollenwerk et al. 2010). We
now followed Chen’s protocol to de-fat and purify fraction
V HSA: this reduced yield significantly, causing loss of
about one-third of the starting HSA. However, the resulting
HSA and HSA-DTPA were homogenous in size. These
HSA molecules were so reproducible that we could use
them as an informal means of calibrating PCS data. The
nanoparticles made from them were also reliably homo-
genous in size. In order to obtain nanoparticles consisting
of correctly conformed HSA molecules, we added octa-
noate (‘‘caprylate’’), because albumin must bind at least
one of its major ligands in order to be stable in its correct
configuration (Carter and Ho 1994; Spector 1975; Stewart
et al. 2003; Sugio et al. 1999). The resulting albumin was
highly homogenous, chemically well defined, pure, and
consisted of HSA molecules in their correct native con-
formation, as shown by immunohistochemistry.
To overcome further challenges associated with nano-
particle purity and size, we established an on-site suite of
methods providing rapid and sensitive assessment of
whether a synthesis was ‘‘on course’’. TLC, PCS, TEM and
arsenazo III were the techniques used most often on a
routine basis. This series of evaluations gave a much more
comprehensive picture of the composition of the compo-
nents than could a single method. We observed the
importance of comparing different measures of the same
parameter. For example, TEM visualised the nanoparticles
but ignored the aggregates, whereas PCS highlighted the
presence of aggregates but provided only indirect, mod-
elled indications of nanoparticle size (for a full discussion,
see Stollenwerk et al. 2010). Diafiltration caused mechani-
cal stress and led to aggregation; glutaraldehyde fixation
sometimes led to displacement of DTPA moieties from the
nanoparticles. A further example was the assessment of
conjugate and nanoparticle concentrations. UV spectro-
metry was rapid, but its validity for measuring protein
concentrations is doubtful; it is less accurate than Pierce
measurements, which, however, are much slower to per-
form. We noted that Pierce data routinely showed results
about 10% lower than those produced by UV spectrometry.
Both these methods were compared with the results of
drying down conjugate and nanoparticle solutions, and this
underlined the difference between Pierce data and the true
weights: Pierce measures the HSA concentrations and does
not include the DTPA and gadolinium components, and
therefore needs correction by a factor of *1.29. When this
correction is included, the Pierce data are good indicators
of true conjugate concentration. They are good indicators
of true nanoparticle concentration only if the nanoparticles
have been diafiltered, which removes contaminant educts
having a mass approximately equal to that of the nano-
particles themselves. In sum, we repeatedly found that no
single assay could be relied upon to provide a true picture
in the physicochemical characterisation of nanoparticles
and their intermediate synthesis products. Combined
application of the analytical tests at selected stages of
synthesis allowed production of highly purified nanoparti-
cles and facilitated standardisation of the nanoparticle
properties.
TLC was simple to perform, cheap and fast, and could
be used during a synthesis procedure to check for the
Fig. 15 Dot diagram showing efficiency values for the first two steps
in the nanoparticle synthesis. The efficiency of the HSA cleaning and
stabilisation procedure is shown in blue dots, that of the HSA-DTPA-
Gd conjugation and chelation procedure is shown in red dots. Eachdot represents an individual batch synthesis, as shown by the number
associated with the dot
Histochem Cell Biol (2010) 134:171–196 189
123
presence of impurities. Also, TLC sheets were easily
developed by use of a range of different staining methods,
each highlighting one particular aspect of the nanoparticle
chemistry. We used iodine to show all types of amino
groups, ninhydrin to show primary amino groups, fluores-
cence to show conjugated electron systems, and arsenazo
III to show non-chelated gadolinium.
Arsenazo III is a naphthalene derivative with metallo-
chromic indicator properties (Alimarin and Savvin 1966;
Basargin et al. 2000; Rowatt and Williams 1989). It has
been routinely used for detection and assay of lanthanides
in industry, and has also been applied in biomedicine and
nanotechnology (Nagaraja et al. 2006; Soenen et al. 2007;
Magnotti 2008). For example, Nagaraja et al. (2006)
reported the possibility of quantitative assay of Gd(III)
concentration in HSA-DTPA-Gd conjugate samples.
Another protocol (Magnotti 2008) describes the quantita-
tive assay of Gd-chelates in biological samples using
arsenazo III at a lower pH, where Gd binds more strongly
to the arsenazo III than to the chelating agent (DTPA),
resulting in a colour change that can be detected spectro-
photometrically. We found arsenazo III staining a good
complement to TLC analyses, because its striking colour
changes distinguished between free and chelated Gd
(Fig. 3a). We noted thereby that arsenazo III colours are
pH-dependent (Rohwer and Hosten 1997) and therefore
worked always with pH = 4.0; furthermore we were aware
that different gadolinium concentrations result in colour
shifts. The arsenazo analysis of NTA-Gd chelates showed
that they varied significantly in their content of free Gd
(compare Fig. 3d), and that chelation was only complete
for high molar ratios ([5) of NTA to Gd. The incomplete
degree of chelation by NTA suggests that use of this che-
lator might prevent accurate control of conjugation con-
ditions. Using this assay, all nanoparticle batches in series
III were found to be free from non-chelated gadolinium,
though like the conjugates, they varied considerably in
their content of gadolinium chelates. As a first approxi-
mation, the low toxicity of the nanoparticles derives from
this absence of free gadolinium ions.
Gel electrophoresis provided sensitive, stringent tests for
several properties of both conjugates and nanoparticles. It
provided sensitive detection of nanoparticle mechanical
cohesion, with contrasty indication of nanoparticle break-
down. It also visualised variability in chemical properties,
for example variation of DTPA ratios in populations of
conjugates or of nanoparticles were visualised as extended
smears, compared with narrow bands defining homogenous
populations. Electrophoresis also provided evidence for
stabilisation and protection of the nanoparticles in the
presence of PEGylation, showing that 20 kDa PEG chains
were more effective for this than were 5 or 10 kDa chains.
Electrophoresis also indicated that nanoparticles bearing
LEA lectin targeting groups exhibited less mechanical
stability than non-targeted nanoparticles.
Analytical results
Gadolinium content of HSA-DTPA-Gd conjugates
The molar ratios of Gd on the nanoparticles (Gd:HSA)
ranged from 6 to 32, so that series III overall did not differ
from the ratios observed in the previous generation (series
II) of the nanoparticles (Stollenwerk et al. 2010). However,
learning effects in our team are evident in this data (Fig. 9),
see for example that the later batches in series III cluster in
a small range (16–21). In later discussion of these nano-
particles we will use the Gd:NP ratio = 19 as representa-
tive value. Although the Gd:HSA ratio became more
predictable as our skill improved, we consider that the
standardisation of this feature remains one of the more
challenging problems to solve prior to industrial upscaling
of the particles. During synthesis of series III nanoparticles
we applied the gadolinium ions to the HSA-DTPA conju-
gates in the form of NTA-Gd chelates, in order to avoid
any binding of gadolinium ions to pure HSA (i.e.: bearing
no DTPA). However, the arsenazo analysis showed that the
stock solution of NTA-Gd chelate that we used did itself
contain free gadolinium ions, and that these could only be
chelated by adding a large excess (69) of NTA (Fig. 3).
We therefore investigated the results of adding gadolinium
ions in the form of gadolinium chloride, and found that this
resulted in binding of, on average, 1.7 gadolinium ions per
albumin molecule (these albumin molecules bearing no
DTPA). This result is readily explained by the presence of
two metal-binding sites in each HSA molecule, and which
in physiological conditions bind zinc (Stewart et al. 2003;
Andre and Guillaume 2004; Rowe and Bobilya 2000). This
gadolinium binding would be of potential toxicological
significance, because these gadolinium ions could escape
into the tissues and exert significant toxic effects. However,
we found that they could be removed by adequate diafil-
tration, and since in our protocol diafiltration is a routine
purification procedure for HSA-DTPA-Gd conjugates,
these ions would be removed routinely from our nanopar-
ticle preparations. In future work, therefore, we will add
the gadolinium ions to the HSA-DTPA conjugates in the
form of gadolinium chloride, and not as NTA-Gd chelates.
Nanoparticle sizes and size variability
Nanoparticle size and its variability were improved in
series III, when compared with series II. The nanoparticles
in series III were slightly smaller, being generally close to
18 nm diameter rather than 25–30 nm diameter as in series
II (Stollenwerk et al. 2010). The obvious clustering of the
190 Histochem Cell Biol (2010) 134:171–196
123
PDI values to the left of the scale in the dot diagrams for
series III (Fig. 7b, c) indicates a clear improvement over
the diffuse distributions of these values in series II. The
reasons for this improvement lie in the better standardisa-
tion of the HSA-DTPA-Gd conjugates, especially in view
of the indications (see below) that the DTPA-Gd chelate
groups play a significant role in defining the nanoparticle
size. Nonetheless, the series III nanoparticles are not an
entirely uniform particle population. This is illustrated by
the following consideration, which ignores the fact that the
nanoparticles possess not only an outer surface but also a
variety of internal surfaces. The smallest particles, with
diameters 16 nm, have a much larger outer surface area
(1 gm containing 2 9 1018 of these particles has a surface
area of 1,575 m2), whereas the largest particles with
diameters 25 nm have a smaller outer surface area (1 gm
containing 5 9 1017 of these particles has a surface area of
1,026 m2). This difference can be set in perspective by the
fact that the surface area of an adult human being is close
to 1.5 m2, so that the difference in surface area between 1 g
of each of these two sizes of nanoparticles is equal to the
total surface area of *360 adult human beings (weighing a
total of more than 25 tons). Thus, since volume/area ratios
alter rapidly with size in the nanoscale, even the low vari-
ability of these nanoparticles involves dramatic scaling
effects (Gao et al. 2003; Wang et al. 2007). For these
reasons, nanoparticles designed for pharmaceutical appli-
cations should have extremely low size dispersities.
Nanoparticle density
Our exploratory measurements of density defined the
concentrations useful for assays in future work; when data
outside this range were eliminated, there remained a small
number of measurements allowing us to determine the
densities of the stabilised HSA, the intermediate synthesis
products, and various versions of the final nanoparticles. A
trend was evident in these densities, and although the small
data set prohibits definitive assessment, we can readily
explain the trend that we observed. Stabilised HSA was the
most dense part of the conjugate and of the final nano-
particle, with density 1.85 g/cm3 in approximate agreement
with results reported by other workers (Chick and Martin
1912). The addition of DTPA moieties reduces the density
by defining a hydrodynamic (rotator) volume or shell, in
which approximately 20 Gd-DTPA molecules protrude
from the HSA: the major part of this volume is occupied by
water and only a small fraction by DTPA. DTPA-Gd has a
density given as 1.01 g/cm3 (ch.oddb.org) or as 1.02 g/cm3
(Bayer product sheet). The larger apparent volume of the
HSA-DTPA is due mainly to water, thus reducing the
mass/volume (density). The resulting density (1.58 g/cm3)
is later raised to 1.75 g/cm3 by the addition of a dense
metal (gadolinium) in the ratio one metal ion per DTPA
moiety. The nanoparticle is constructed by packing 6–10
HSA-DTPA-Gd molecules into a volume of about
3,000 nm3, The packing density of 0.17 accounts for
essentially the entire volume of the nanoparticle, but not all
the water in the nanoparticle is bound in the HSA-DTPA-
Gd shells: as discussed in our previous paper (Stollenwerk
et al. 2010), a spheroidal volume cannot be packed 100%
with spheres, the maximum packing density being about
85%. Thus, a further *15% non-bound water is included
in the nanoparticle, in addition to the water included in the
HSA-DTPA-Gd conjugates; this reduces the nanoparticle
density yet further, to 1.36 g/cm3.
Nanoparticle integrity
The total dissolution of the particles in 1% SDS gels,
containing anionic detergents, showed that the Schiff bases
formed by glutaraldehyde crosslinking may in some cir-
cumstances be inadequate to guarantee particle cohesion.
‘‘Quenching’’ of glutaraldehyde-crosslinked nanoparticles
by application of ammonium salts resulted in nanoparticles
with a high degree of internal cohesion, and which there-
fore did not enter the gels. Ammonium salts evidently
mediate a type of strong crosslinking. The nature of this
crosslinking is unclear, but it requires the presence of both
glutaraldehyde (or of glutaraldehyde-induced Schiff bases)
and the ammonium ion. We consider it likely that nitrogen
atoms become involved in covalent bonds at the sites of the
glutaraldehyde-induced Schiff bases. The ammonium-
mediated crosslinking did not destroy HSA antigenic epi-
topes, indicating that the proteins were present in the
nanoparticles in their native conformations. Reducing the
glutaraldehyde-formed Schiff bases in the nanoparticles to
covalency by treatment with sodium cyanoborohydride
resulted in nanoparticles that were more stably crosslinked
than those treated with glutaraldehyde alone, but less stably
crosslinked than those treated with glutaraldehyde then
subsequently with ammonium ions (Fig. 4). We do not yet
know which degree of crosslinking will be appropriate
within animal tissues, so simply note that varying strengths
of crosslinking can be obtained by simple methods.
Nanoparticle relaxivity
In purified and standardised nanoparticle preparations, all
the contrast enhancement (relaxivity) is due to gadolinium
chelated to DTPA which is covalently bound to the HSA.
In series III nanoparticles, we found a relationship between
the number of gadolinium atoms on the nanoparticles and
their relaxivity, only *19% of the variability in the
relaxivity remaining unexplained (Fig. 11). In the multi-
step path leading to this data, numerous types of error can
Histochem Cell Biol (2010) 134:171–196 191
123
occur, including inaccuracies in synthesis, partial failure of
purification procedures, and measurement errors in
assessment of the concentrations of HSA, Gd, and of r1
relaxivity. In view of these numerous sources of potential
error, we consider 81% explanation of the relaxivity to
show the data set to be of good quality. It furthermore
suggests a valid comparability between the several batches
shown, indicating a good purity and standardisation level
had been achieved. Figure 11 also charts our learning
progress as we adjusted the molar concentrations of the
reagents and optimised the reaction conditions. The prin-
ciple example of this was the improvement in the Gd
loading ratio from 8 to 25 after recognising the importance
of using fresh dry DTPABA. Towards the end of this
learning process, we could reliably produce HSA-DTPA
conjugate molecules each bearing typically 19 DTPA
chelators, and responsible for [81% of the observed
relaxivity.
Description of the nanoparticles
At the nanoscale familiar scaling relationships, incorpo-
rated in our intuitive assessments of phenomena and pro-
cesses, are no longer valid (Gao et al. 2003; Wang et al.
2007). The connections between electrical charge, stability,
viscosity and many other properties are different to those
valid at the meter scale. It is therefore useful to attempt to
form an image of the nanoparticles, to facilitate under-
standing of their internal arrangement and cohesion, and of
their external interactions (Rocke 2010). The quantitative
physicochemical data obtained for this series of nanopar-
ticles was of high quality, as shown by comparable data
obtained from entirely different approaches (see for
example, Fig. 11), and was adequate to allow conceptu-
alisation of the ‘‘average’’ nanoparticle; the relatively good
standardisation of the nanoparticle properties implied that
most nanoparticles would be similar in properties to the
‘‘average’’ nanoparticle. The nanoparticle size (average
18 nm) and packing density (18–49%, depending on
whether the calculation is based on HSA only, or on HSA-
DTPA-Gd conjugates) allow the average nanoparticle to be
reconstructed as follows. The nanoparticle occupies a total
volume of 3,054 nm3, and our counts show that this vol-
ume contains 6 HSA molecules. Since one HSA molecule
occupies *90 nm3 (Stollenwerk et al. 2010; Sugio et al.
1999), the nanoparticle has packing density = 0.17.
However, this calculation ignores the presence of DTPA-
Gd chelates on the conjugate molecule, which therefore
occupies more volume than the HSA molecule alone. As
noted above, the stabilised HSA molecules have average
sizes (hydrodynamic diameters, measured in PCS) close to
10 nm, whereas the HSA-DTPA-Gd conjugate molecules
have average sizes close to 15 nm. These hydrodynamic
diameters represent freely moving (rotating, tumbling)
molecules and thus give the appearance of a solid surface.
For immobilised molecules, as found in the interiors of the
nanoparticles, the (typically) 19 DTPA-Gd chains do not
fill the surrounding volume to create a spheroidal structure.
Instead, they protrude from the HSA molecules as hairs or
filaments, and do not fill the space entirely. The DTPA
chain folds around the metal (gadolinium) ion during
chelation, resulting in a structure which is approximately
1–2 nm in diameter (Choppin and Schaab 1996; Maceke
et al. 1989). We consider here that DTPA length is
approximately 2 nm. In this case, the axes of the conju-
gate molecule are (8 ? 4) 9 (3 ? 4) 9 (3 ? 4) = 12 9
7 9 7 nm in length, so that the molecule defines a spatial
volume of *588 nm3, though its substantial mass fills only
90 nm3. On this basis, the packing density could be cal-
culated as [(6 9 588) / 3,054] 9 100% = 115%, but the
large increase in nominal packing density, from *18 to
*115%, is accounted for mainly by water. In summary,
the protein conjugate molecules occupy essentially the
entire volume of the nanoparticle, though substantially
filling only about 18% of this volume. Most of the material
in the volume defined by the presence of the DTPA-Gd
chelates is water; but by limiting the close packing of the
molecules, these chelates might define the spatial ordering
of the conjugate molecules in the interior of the nanopar-
ticle, and therefore determine the size of the nanoparticle.
Knowledge of the packing density allows us to calculate
the total weight of the average 18 nm diameter nano-
particle, as follows. Since the HSA-DTPA-Gd conju-
gate contains (on average) 19 Gd-DTPA chelates (each
with molecular weight [393.35 ? 157.25 = ] 550.6 Da),
it weighs (66,400 ? [19 9 550.6]) = 76.9 kDa. The
total weight of the average nanoparticle is there-
fore (6 9 76.9 kDa =) 0.461 MDa. We note that 1 Da
weighs 1.66 9 10-24 g so, with rounded figures, a nano-
particle of 18 nm diameter weighs 0.46 MDa, which is
7.7 9 10-19 g. It follows that the number of nanoparticles
in 1 g is 1.3 9 1018, and taking the Avagadro number of
nanoparticles to represent ‘‘1 mol’’ of nanoparticles, then
1 mol of nanoparticles weighs 461 kg, and 1 g of nano-
particles is 2.17 9 10-6 mol; a 1 g/ml solution of nano-
particles is 2.17 lM.
The PLA in the nanoparticles has been ignored in the
preceding calculations, but its concentration is so small that
it does not alter those results significantly. In the series II
particles, for which this part of the synthesis protocol was
essentially identical to series III, PLA accounted for 1–5%
of the particle mass (Stollenwerk et al. 2010). These small
amounts could not be determined with great accuracy using
the method we employed. We consider the range 1–3% in
the following. In a nanoparticle weighing 0.46 MDa, 1%
PLA would weigh *4.6 kDa, 2% PLA would weigh
192 Histochem Cell Biol (2010) 134:171–196
123
9.2 kDa and 3% PLA would weigh 13.8 kDa. One lactide
subunit (monomer) of PLA weighs 90 Da, so a PLA
polymer weighing 4.6 kDa contains 51 lactide monomers,
that weighing 9.2 kDa contains 102 lactide monomers,
and that weighing 13.8 kDa contains 153 lactide mono-
mers. The lactide monomer has length 0.5 nm, so the
three PLA chains comprising, respectively, 1, 2 or 3% of
the 18 nm diameter nanoparticles would be 25.5, 51 and
76.5 nm long. Evidently they must lie coiled within
the nanoparticle interior, because each is longer than the
nanoparticle diameter. We note that in preparing the
nanoparticles we used PLA chains nominally of 90 kDa
length, and they are much longer (1,000 monomers,
*500 nm chain length). PLA is hydrophobic, and there-
fore enters into physical and geometric configurations
which minimise its exposure to aqueous environments. In
the interior of a nanoparticle it must either associate with
HSA, or must form coils and knots to generate hydro-
phobic pockets. The six HSA conjugate molecules in our
18 nm nanoparticles would be large enough to ‘‘coat’’
about 50 nm of a PLA chain, such as the 51 nm chain
which would represent 2% of the mass in our 18 nm
nanoparticles. They would not suffice to coat the 76.5 nm
chain which would represent 3% of the nanoparticle mass,
so that a PLA chain 76.5 nm long must form coils or
knots along its length, or fold back to associate itself
repeatedly with the HSA molecules. The 6 conjugate
molecules in one nanoparticle would coat less than 10%
of the 500 nm long PLA chain (90 kDa) which we
applied when emulsifying the HSA and PLA to form the
nanoparticles. However, such long chains are not present
in our nanoparticles: we know this because, if they were
present, their mass would represent (90 kDa/461 kDa =)
19.5% of the entire nanoparticle mass. This large fraction
lies well within the detection limits of the PLA assay we
used and we would have measured it repeatedly; in fact
we always obtained PLA measurements close to 3% of
the nanoparticle mass. We therefore consider our nano-
particles to contain short PLA chains, even although our
starting material was a long PLA chain. In sum, our
evidence indicates that the nanoparticles contain chains
short enough to be fully coated by HSA, so that PLA does
not form knots in the nanoparticles. We do not know how
the short PLA chains arose, though we suspect that the
long and extremely energetic mechanical stresses of the
emulsification procedure (20,000 rpm for 15 min) might
have shredded the original long chains, by aiding hydro-
lysis of the PLA (Shih 1995; Karst and Yang 2006).
The nanoparticles contain a high proportion of water.
Since 18 g of water (= 18 cm3 at 15�C) contain 1 mol of
water, one nanoparticle of 18 nm diameter contains (3,054/
18 9 1021 =) 1.70 9 10-19 mol of water. The nanoparticle
volume thus contains (1.70 9 10-19 9 6.022 9 1023 =
102,374) *100,000 molecules of water. This calculation
ignores the *18% of the nanoparticle volume occupied by
the HSA molecules, but it should be noted that the HSA
molecule contains a significant amount of water internally.
The DTPA chelates define a volume close to the entire
volume of the nanoparticle, and we consider the water
within the ‘‘shell’’ defined by the DTPA chelates to be
bound water. Spheroidal structures cannot be packed with
packing densities higher than *0.85 (see detailed discus-
sion in Stollenwerk et al. 2010), and the HSA-DTPA-Gd
conjugates, though rather oblate, also cannot be packed
with packing densities much greater than 0.85. The water
between them, outside the shells formed by the DTPA
chelates, is not bound. This is important for fast exchange
between the unbound and bound water pools, which is a
requirement for achieving high MR relaxivity. Two fea-
tures of the nanoparticles are highlighted by considering
the water contained in the particle. First, each of the
approximately 100–200 gadolinium ions in the nanoparti-
cle is surrounded by an adequate number of water mole-
cules and this water has full access to the water
surrounding the particle, thus enabling fast exchange and
hence an increase of the relaxation rate. Secondly, the
nanoparticle should not be considered a rigid structure but
rather a gelatinous one; this consideration becomes
important in designing nanoparticle mechanical integrity.
To aid understanding and discussion, a model of the
nanoparticles as they are prepared in series III is shown in
Fig. 16.
Fig. 16 The internal structure of a ‘‘typical’’ nanoparticle, showing a
section through the centre of the nanoparticle. PLA polymer is
coloured purple, pale blue shows ‘‘internal’’ bound water of the
nanoparticle, dark grey-blue shows internal non-bound water, greyshows the HSA protein—note the heart-shaped HSA molecule at the
right side of the nanoparticle—and dark blue shows the water
surrounding the nanoparticle. A single DTPA-gadolinium chelate is
shown enlarged protruding from the right side of the nanoparticle: the
gadolinium ion is shown gold. Similar chelates protrude from the
surfaces of all HSA protein molecules in the nanoparticle, defining
shells around the HSA molecules which are filled with (bound) water
Histochem Cell Biol (2010) 134:171–196 193
123
Toxicological assays
Gd is highly toxic in non-chelated form, and recent FDA
alerts concerning its application in persons with kidney
illnesses have been well publicised in the literature (Bro-
ome et al. 2007; Kuo et al. 2007; Rinck 2008; Rofsky et al.
2008); the presence of non-chelated gadolinium on the
nanoparticles must be prevented. Arsenazo testing showed
that our nanoparticles did not contain any non-chelated
gadolinium. The results of the toxicological cell-screening
studies agreed with those carried out on the previous series
II nanoparticles (Stollenwerk et al. 2010), showing that at a
nanoparticle concentration of 2 mg/ml, no loss of viability
and no membrane damage was detected for any of the
nanoparticle batches. The particles were fully hemocom-
patible, they did not induce damage of erythrocytes
(hemolysis), activate plasmatic coagulation, nor induce
aggregation of platelets. The slightly decreased levels of
prothrombin fragments (F1 ? 2 levels) may be due to the
binding of proteins to the particle surface, a phenomenon
called formation of a ‘protein corona’ (Lundqvist et al.
2008). Only at local concentrations of 5 mg/ml could the
first evidence of toxic effects be discerned. Our investi-
gations in animal models show that concentrations
exceeding 2 mg/ml will not be necessary for imaging
studies (manuscripts in preparation).
Nanoparticles bearing PEG chains
This initial exploration of PEGylation showed that the
HSA nanoparticles were readily PEGylated with PEG
chains of sizes 5–20 kDa. PAGE analysis showed that
these chains stabilise the nanoparticles, and that this sta-
bilisation is greater for longer PEG chains (Fig. 4d). Since
stabilisation of the nanoparticles is likely to be important
for their successful targeting within living tissues, and
since variable degrees of stabilisation may be desirable,
this stabilisation by PEG chains will be an important topic
for study in subsequent work.
Nanoparticles bearing LEA targeting molecules
This initial exploration of lectin targeting showed that LEA
is readily covalently bound to HSA-based nanoparticles, by
an appropriate modification of the two-stage carbodiimide
method (Irache et al. 1994). This is generally used as a
carboxyl activating agent for the coupling of primary
amines to yield amide bonds by reaction with free amino
groups of the ligand polypeptide chains (Olde Damink
et al. 1996); in albumin, these carboxylic groups can be
found on aspartic and glutamic acid residues. We did not
determine directly the number of LEA molecules attached
to each nanoparticle, though hemagglutination studies
provided an indirect assessment of this. Hemagglutination
was used as an in vitro surrogate to test specific binding
capacity of the LEA-bearing nanoparticles to their ligands
(oligolactosamines), which in living animal tissues are
present on the surface of erythrocytes and, indeed, of all
epithelial cells (Davidson et al. 1988; Debbage 1996;
Konska et al. 2003). Hemagglutination results from the
divalent binding of LEA molecules to the erythrocytes so
that, in a blood sample containing LEA, a single LEA
molecule can bind to two different erythrocytes and cause
them to adhere to one another; many LEA molecules will
bind twice to a single cell, so the hemagglutination assay is
only semiquantitative. In contrast, in a blood sample con-
taining LEA-bearing nanoparticles, each LEA molecule
attached to one particular nanoparticle needs to bind to
only one cell to cause agglutination, because other LEA
molecules attached to the nanoparticle will bind to one or
several other cells and thus cause agglutination. Since the
orientation of the individual LEA molecules on the nano-
particles was not optimised in our work, many will be
wrongly oriented for binding their ligand: these LEA
molecules will have no agglutination effect so that, for this
reason also, the hemagglutination assay is only semi-
quantitative. The results obtained in our four nanoparticle
batches, in experiments carried out during 2 years by dif-
ferent operators, all showed that the LEA-bearing nano-
particles were comparable to LEA in their efficacy to cause
hemagglutination, and the consensus result (compare
Table 1) was that, on a molar basis, the nanoparticles were
69–129 more effective than was LEA in causing hem-
agglutination. A result like this would be obtained if each
of the 6–10 HSA-DTPA-Gd molecules packed into each
individual nanoparticle (see below) were to bear a single
LEA molecule, in correct orientation for binding its ligand
on the erythrocytes. We consider this match between data
obtained in very different fashions (hemagglutination,
TEM measurements of nanoparticles) as an indication that
our experiments reveal the physicochemical nature of the
nanoparticles accurately. Closer consideration of Fig. 4d
(lane 9), however, suggests that the LEA-bearing nano-
particles, as prepared in this work, may not have sufficient
mechanical stability to function as single units when
applied in the turbulent environment of the blood flow,
rather than in the non-turbulent environment of the test-
tube.
Upscaling of the nanoparticles
For several reasons, these nanoparticles are not yet suitable
for upscaling into industrial production. First, their sizes are
not adequately standardised; with PDI *1.2–1.6, they are
much more uniform than earlier series of the same nano-
particles (Stollenwerk et al. 2010), but industrial production
194 Histochem Cell Biol (2010) 134:171–196
123
and pharmaceutical applications would require PDI \1.1.
Some of the variability remaining in our nanoparticle bat-
ches may be due to the varying, and not fully quantified,
lengths of the PLA chains within the particles. Secondly,
the emulsification step generates nanoparticles with a cer-
tain rate of aggregation, and purification by diafiltration also
causes some aggregation, and this varies between batches.
Thirdly, the numbers of DTPA-Gd chelates attached to the
nanoparticles varies from batch to batch, and as a result the
relaxivities of the nanoparticles are not sufficiently stand-
ardised for pharmaceutical applications. Glutaraldehyde
crosslinking appears sometimes to remove a (variable)
number of DTPAs from the nanoparticles. In spite of these
blemishes, this generation of nanoparticles has several
qualities which make it interesting for application in animal
models, for example nanoparticle production in gram
amounts, and the final nanoparticles characterised by high
uniformity and high relaxivity and by enhanced stability.
The synthesis efficiencies are adequate for laboratory
preparations. Although it is not sufficiently advanced for
translational research, this generation of HSA-based nano-
particles provides excellent reagents for exploratory work
in Molecular Imaging in animal models; our next manu-
scripts in this series will explore MR imaging, biodistri-
bution and pharmacokinetics of these particles in animal
models (manuscripts in preparation). It also provides a good
starting platform for studies of drug loading.
Acknowledgments The Austrian Nano-Initiative, the Austrian
Science Foundation (FWF) (Project N201-NAN) and the Austrian
National Bank Jubilee Programme supported this work (Projects
9273, 10844, 11574 and 13096).
References
Abuchowski A, van Es T, Palczuk NC, Davis FF (1977) Alteration of
immunological properties of bovine serum albumin by covalent
attachment of polyethylene glycol. J Biol Chem 11:3578–3581
Alimarin IP, Savvin SB (1966) Application of arsenazo III and other
azo-compounds in the photometric determination of certain
elements. Pure Appl Chem 13:445–456
Andre C, Guillaume YC (2004) Zinc–human serum albumin associ-
ation: testimony of two binding sites. Talanta 63:503–508
Balbirnie M, Grothe R, Eisenberg DS (2001) An amyloid-forming
peptide from the yeast prion Sup35 reveals a dehydrated b-sheet
structure for amyloid. Proc Natl Acad Sci 98:2375–2380
Basargin NN, Ivanov VM, Kuznetsov VV, Mikhaliova AV (2000)
40 years since the discovery of the arsenazo III reagent. J Anal
Chem 55:204–210
Broome DR, Girguis MS, Baron PW, Cottrell AC, Kjellin I, Kirk GA
(2007) Gadodiamide-associated nephrogenic systemic fibrosis:
why radiologists should be concerned. Am J Roentgenol
188:586–592
Carter DC, Ho JX (1994) Structure of serum albumin. Adv Protein
Chem 45:153–203
Chen RF (1967) Removal of fatty acids from serum albumin by
charcoal treatment. J Biol Chem 242:173–181
Chick H, Martin CJ (1912) The density and solution volume of some
proteins. Chem Ind Kolloide Zeitsch 11:102–107
Choppin GR, Schaab KM (1996) Lanthanide(III) complexation with
ligands as possible contrast enhancing agents for MRI. Inorga-
nica Chim Acta 252:299–310
Davidson SE, McKenzie JL, Beard MEJ, Hart DNJ (1988) The tissue
distribution of the 3a-fucosyl-N-acetyl lactosamine determinant
recognized by the Cd15 monoclonal antibodies Cmrf-7 and 27.
Pathology 20:24–31
Debbage PL (1996) A systematic histochemical investigation in
mammals of the dense glycocalyx glycosylations common to all
cells bordering the interstitial fluid compartment of the brain.
Acta Histochem 98:9–28
Debbage P (2009) Targeted drugs and nanomedicine: present and
future. Curr Pharm Des 15:153–172
Debbage P, Jaschke W (2008) Molecular imaging with nanoparticles:
giant roles for dwarf actors. Histochem Cell Biol 130:845–875
Fehske KJ, Muller WE, Wollert U (1981) The location of drug
binding sites in human serum albumin. Biochem Pharmacol
30:687–692
Flacke S, Fischer S, Scott MJ, Fuhrhop RJ, Allen JS, McLean M,
Winter P, Sicard GA, Gaffney PJ, Wickline SA, Lanza GM
(2001) Novel MRI contrast agent for molecular imaging of
fibrin: implications for detecting vulnerable plaques. Circulation
104:1280–1285
Foster JF, Sogami M, Peterson HA, Leonard WJ (1965) The
microheterogeneity of plasma albumins. II. Preparation and
solubility properties of subfractions. J Biol Chem 240:
2503–2507
Frokjaer S, Otzen DE (2005) Protein drug stability: a formulation
challenge. Nat Rev Drug Discov 4:298–306
Gao H, Baohua J, Jager IL, Arzt E, Fratzl P (2003) Materials become
insensitive to flaws at nanoscale: lessons from nature. Proc Nat
Acad Sci USA 100:5597–5600
Goldschmidt L, Teng PK, Riek R, Eisenberg D (2010) Identifying the
amylome, proteins capable of forming amyloid-like fibrils. Proc
Natl Acad Sci 107:3487–3492
Goldwasser P, Feldman J (1997) Association of serum albumin and
mortality risk. J Clin Epidemiol 50:693–703
Gonzalez ER, Kannewurf BS (1998) Clinical review of appropriate
uses for albumin. US Pharm 23:HS15–HS26
Griffel MI, Kaufman BS (1992) Pharmacology of colloids and
crystalloids. Crit Care Clin 8:235–253
Griffiths JR, Glickson JD (2000) Monitoring pharmacokinetics of
anticancer drugs: non-invasive investigation using magnetic
resonance spectroscopy. Adv Drug Deliv Rev 41:75–89
Harisinghani MG, Barentsz J, Hahn PF, Deserno WM, Tabatabaei S,
van de Kaa CH, de la Rosette J, Weissleder R (2003)
Noninvasive detection of clinically occult lymph-node metasta-
ses in prostate cancer. N Engl J Med 348:2491–2499
Hengerer A, Grimm J (2006) Molecular magnetic resonance imaging.
Biomed Imaging Interv J 2:e8. doi:10.2349/biij.2.2.e8.
http://www.biij.org/2006/2/e8
Irache JM, Durrer C, Duchene D, Ponchel G (1994) In vitro study of
lectin–latex conjugates for specific bioadhesion. J Control
Release 31:181–188
Jaffer FA, Weissleder R (2005) Molecular imaging in the clinical
arena. JAMA 293:855–862
Karst D, Yang Y (2006) Molecular modeling study of the resistance
of PLA to hydrolysis based on the blending of PLLA and PDLA.
Polymer 47:4845–4850
Konska G, Zamorska L, Pituch-Noworolska A, Szmaciarz M, Guillot
J (2003) Application of fluorescein-labelled lectins with different
glycan-binding specificities to the studies of cellular glycocon-
jugates in human full-term placenta. Folia Histochem Cytobiol
41:155–160
Histochem Cell Biol (2010) 134:171–196 195
123
Kragh-Hansen U (1981) Molecular aspects of ligand binding to serum
albumin. Pharmacol Rev 33:17–53
Kuo PH, Kanal E, Abu-Alfa AK, Cowper SE (2007) Gadolinium-
based MR contrast agents and nephrogenic systemic fibrosis.
Radiology 242:647–649
Lundqvist M, Stigler J, Elia G, Lynch I, Cedervall T, Dawson KA
(2008) Nanoparticle size and surface properties determine the
protein corona with possible implications for biological impacts.
Proc Natl Acad Sci 105:14265–14270
Maceke HR, Riesen A, Ritter W (1989) The molecular structure of
indium-DTPA. J Nucl Med 30:1235–1239
Magnotti R (2008) Detection of gadolinium chelates. World Patent
WO 2008/045767 A2
Means GE, Feeney RE (1995) Reductive alkylation of proteins. Anal
Biochem 224:1–16
Montero EI, Benedetti BT, Mangrum JB, Oehlsen MJ, Qu Y, Farrell
NP (2007) Pre-association of polynuclear platinum anticancer
agents on a protein, human serum albumin. Implications for drug
design. Dalton Trans 43:4938–4942
Mulder WJM, Strijkers GJ, van Tilborg GAF, Griffioen AW, Nicolay
K (2006) Review article: lipid-based nanoparticles for contrast-
enhanced MRI and molecular imaging. NMR Biomed
19:142–164
Nagaraja TN, Croxen RL, Panda S, Knight RA, Keenan KA, Brown
SL, Fenstermacher JD, Ewing JR (2006) Application of arsenazo
III in the preparation and characterization of an albumin-linked,
gadolinium-based macromolecular magnetic resonance contrast
agent. J Neurosci Meth 157:238–245
Nelson R, Sawaya MR, Balbirnie M, Madsen AØ, Riekel C, Grothe
R, Eisenberg D (2005) Structure of the cross-ß spine of amyloid-
like fibrils. Nature 435:773–778
Olde Damink LH, Dijkstra PJ, van Luyn MJ, van Wachem PB,
Nieuwenhuis P, Feijen J (1996) In vitro degradation of dermal
sheep collagen cross-linked using a water-soluble carbodiimide.
Biomaterials 17:679–684
Paschkunova-Martic I, Kremser C, Mistlberger K, Shcherbakova N,
Dietrich H, Talasz H, Zou Y, Hugl B, Galanski M, Solder E,
Pfaller K, Holiner I, Buchberger W, Keppler B, Debbage P
(2005) Design, synthesis, physical and chemical characteriza-
tion, and biological interactions of lectin-targeted latex nano-
particles bearing Gd-DTPA chelates: an exploration of magnetic
resonance molecular imaging (MRMI). Histochem Cell Biol
123:283–301
Peters T Jr (1985) Serum albumin. Adv Protein Chem 37:161–245
Putnam FW (1984) The Plasma Proteins, vol 4, 2nd edn. Academic
Press, London
Rainey TG, Read CA (1994) The pharmacological approach to the
critically ill patient, 3rd edn. Williams & Wilkins, Baltimore,
pp 272–290
Rehman S, Jayson GC (2005) Molecular imaging of antiangiogenic
agents. Oncol Cancer Imaging 10:92–103
Rinck PA (2008) Radiologists meet with heavy collateral damage.
Diagnostic Imaging Europe, November 2008, pp 19–22
Robbens J, Vanparys C, Nobels I, Blust R, van Hoecke K, Janssen C,
de Schamphelaere K, Roland K, Blanchard G, Silvestre F,
Gillardin V, Kestemont P, Anthonissen R, Toussaint O, Vank-
oningsloo S, Saout C, Alfaro-Moreno E, Hoet P, Gonzalez L,
Dubruel P, Troisfontaines P (2010) Eco-, geno- and human
toxicology of bio-active nanoparticles for biomedical applica-
tions. Toxicology 296:170–181
Rocke AJ (2010) Image and reality: Kekule, Kopp, and the scientific
imagination. University of Chicago Press, USA, p 416
Rofsky NM, Sherry AD, Lenkinski RE (2008) Nephrogenic systemic
fibrosis: a chemical perspective. Radiology 247:608–612
Rohwer H, Hosten E (1997) pH dependence of the reactions of
arsenazo III with the lanthanides. Anal Chim Acta 339:271–277
Rowatt E, Williams RJP (1989) The interaction of cations with the
dye arsenazo III. Biochem J 259:295–298
Rowe JD, Bobilya DJ (2000) Albumin facilitates zinc acquisition by
endothelial cells. Proc Soc Exp Biol Med 224:178–186
Saito R, Bringas JR, McKnight TR, Wendland MF, Mamot Ch,
Drummond DC, Kirpotin DB, Park JW, Berger MS, Bankiewicz
KS (2004) Distribution of liposomes into brain and rat brain
tumor models by convection-enhanced delivery monitored with
magnetic resonance imaging. Cancer Res 64:2572–2579
Schnabel J (2010) The dark side of proteins. Nature 464:828–829
Shih C (1995) Chain-end scission in acid catalyzed hydrolysis of
poly(D, Llactide) in solution. J Control Release 34:9–15
Shrake A, Frazier D, Schwarz FP (2005) Thermal stabilization of
human albumin by medium- and short-chain n-alkyl fatty acid
anions. Biopolymers 81:235–248
Soenen SJH, Desender L, De Cuyper M (2007) Complexation of
gadolinium(III) ions on top of nanometre-sized magnetolipo-
somes. Int J Environ Anal Chem 87:783–796
Spector AA (1975) Fatty acid binding to plasma albumin. J Lipid Res
16:165–179
Stewart AJ, Blindauer CA, Berezenko S, Sleep D, Sadler PJ (2003)
Interdomain zinc site on human albumin. Proc Natl Acad Sci
100:3701–3706
Stollenwerk MM, Pashkunova-Martic I, Kremser C, Talasz H,
Thurner GC, Abdelmoez AA, Wallnofer EA, Helbok A,
Neuhauser E, Klammsteiner N, Klimaschewski L, von Guggen-
berg E, Frohlich E, Keppler B, Jaschke W, Debbage P (2010)
Albumin-based nanoparticles as Magnetic Resonance contrast
agents: I. Concept, first syntheses and characterisation. Histo-
chem Cell Biol 133:375–404. doi:10.1007/s00418-010-0676-z
Sugio S, Kashima A, Mochizuki S, Noda M, Kobayashi K (1999)
Crystal structure of human serum albumin at 2.5 A resolution.
Protein Eng 12:439–446
Suh WH, Suslick KS, Stucky GD, Suh YH (2009) Nanotechnology,
nanotoxicology, and neuroscience. Prog Neurobiol 87:133–170
Utsumi H, Yamada K, Ichikawa K, Sakai K, Kinoshita Y, Matsumoto S,
Nagai M (2006) Simultaneous molecular imaging of redox reac-
tions monitored by overhauser-enhanced MRI with 14N- and 15N-
labeled nitroxyl radicals. Proc Natl Acad Sci 103:1463–1468
Vega-Villa KR, Takemoto JK, Yanez JA, Remsberg CM, Laird
Forrest M, Davies NM (2008) Clinical toxicities of nanocarrier
systems. Adv Drug Deliv Rev 60:929–938
Veronese FM (2001) Peptide and protein PEGylation: a review of
problems and solutions. Biomaterials 22:405–417
Wang J, Karihaloo BL, Duan HL (2007) Nano-mechanics or how to
extend continuum mechanics to nano-scale. Bull Polish Acad Sci
55:133–140
Winter PM, Morawski AM, Caruthers SD, Fuhrhop RW, Zhang H,
Williams TA, Allen JS, Lacy EK, Robertson JD, Lanza GM,
Wickline SA (2003) Molecular imaging of angiogenesis in early-
stage atherosclerosis with avß3-integrin-targeted nanoparticles.
Circulation 108:2270–2274
Zalipsky S (1995a) Chemistry of polyethylene glycol conjugates with
biologically active molecules. Adv Drug Deliv Rev 16:157–182
Zalipsky S (1995b) Functionalized poly(ethy1ene glycol) for prepa-
ration of biologically relevant conjugates. Bioconjug Chem Rev
6:150–165
196 Histochem Cell Biol (2010) 134:171–196
123
Top Related