J. Chitin Chitosan 16(1), 7-14 (2011)

7

Http://www.chitosan.or.kr

Biomedical and Environmental Applications of Chitosan-based Nanomaterials

Lam Dai Tran1,*, Hoang Vinh Tran

2, Trang Thu Mai

1, Thu Phuong Ha

1, Binh Hai Nguyen

1, Hoang Thai

3,

Hoang Dinh Vu2, Dien Gia Pham

4, Phuc Xuan Nguyen

1, and Jae Kweon Park

5,**

1Institute of Materials Science, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Hanoi, Viet Nam

2Faculty of Chemical Technology, Hanoi University of Technology, 1 Dai Co Viet, Hanoi, Vietnam

3Institute of Tropical Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Hanoi, Viet Nam

4Institute of Chemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Hanoi, Viet Nam

5Department of Pharmaceutical Science, Gachon University of Medicine and Science, Yeonsu-dong, Yeonsu-gu, InCheon, Korea

ABSTRACT

Being naturally abundant resources and having many interesting physicochemical and biological properties, chitin/chitosan

have been found useful in many fields. This paper describes the strategy to design a multifunctional, chitosan based

nanomaterials and their biomedical and environmental applications. Different physicochemical methods including FESEM/

TEM, PPMS were used to characterize the obtained nanomaterials. For each application, a series of specific characterizing

methods were used for evaluating the applicability/capacity of materials.

Keywords: biomedical, environment, chitosan, nanmaterials

INTRODUCTION

Chitin, a natural polysaccharide and first identified in 1884,

is the most abundant polymer after cellulose. The most

important derivative of chitin is chitosan (CS), obtained by

deacetylation of chitin in the solid state under alkaline

conditions, resulting in a heterogeneous distribution of acetyl

groups along its chains, or by enzymatic hydrolysis in the

presence of chitin deacetylase (Fig. 1). A wide variety of

biomedical applications in tissue engineering (1, 2), wound

healing (3), drug and gene delivery (1, 4-6) was based on

biocompatible, biodegradable and non-toxic CS. This paper

demonstrates synthetic strategies, characterizations and the

most important biomedical and environmental applications of

chitosan-based nanoparticles and nanocomposites.

MATERIALS AND METHODS

Preparation of glucosamine sulphate

All the chemicals were of reagent grade used without

further purification. Ferric chloride hexa-hydrate (FeCl36H2O),

ferrous chloride tetra-hydrate (FeCl24H2O) and sodium

hydroxide (NaOH) were purchased from Aldrich. Aluminium

sulfate octadecahydrate (Al2(SO4)3.18H2O), copper(II) sulfate

pentahydrate (CuSO4.5H2O), cobalt(II) chloride hexahydrate

(CoCl2.6H2O), cadmium chloride hemipentahydrate (CdCl2.

2.5H2O), lead(II) nitrate (Pb(NO3)2), Nickel(II) sulfate

hexahydrate (NiSO4.6H2O),4-(2-Pyridylazo)resorcinol (PAR)

were purchased from Acros.

NaOH, NH4OH (26% of ammonia), oleic acid (C17H33

COOH) were purchased from Aldrich. Chitin, chitosan was

purchased from Nha Trang Aquatic Institute (Vietnam),

OMSCS was from Aldrich. Glucosamine hydrochloride was

obtained by acidic hydrolysis of chitin (with concentrated

HCl) at 60~70oC. Glucosamine hydrochloride could convert

into glucosamine sulphate with the help of Na2SO4 under

room temperature (4).

Biocompatibility of the materials

In vitro biocompatibility of the materials was tested in

simulated body fluid (SBF) at 37oC, for period of 0 to 7 days.

SBF solutions have ionic concentrations similar to those of

human blood plasma. SBF solutions were prepared according

to the detailed protocol given elsewhere and denoted as 1x

SBF and 5x SBF, corresponding to 1-fold and 5- fold

concentrations respectively. pH of solution were buffered at

the value of 7.4 using 0.01 M of tris-(hydroxymethyl)-

aminomethane (CH2OH)3CNH2). Curcumin (1,7-bis(4-hydroxy-

3-methoxyphenyl)-1,6-heptadiene-3,5-dione) was from Institute

of Chemistry (Vietnam). Cells were cultured in RPMI 1640

(Roswell Park Memorial Institute) (Gibco) medium. This

medium was supplemented with 10% Fetal Bovine Serum

(Invitrogen), 100 IU/ml penicillin-streptomycine (Invitrogen),

2 mM – Glutamine (Invitrogen). Cells were grown in a

humidified chamber in the presence of 5% CO2, at 37oC.

*To whom all correspondence should be addressed

Tel: +84-4-3756-4129; Fax: +84-4-3836-0705; E-mail: lamtd@ims.vast.ac.vn

**Co-corresponding author. Tel. +82-32-820-4781; E-mail : jamyeong@yahoo.co.kr

8 Biomedical and Environmental Applications of Chitosan-based Nanomaterials

Evaluation of macrophage-colony stimulating factor

Human buffycoat was obtained from National Institute

of Hematology and Transfusion (Vietnam). Mononuclear

cells were isolated by density gradient centrifugation using

1.077 g/ml Ficoll. Cells were cultured in RPMI 1640 medium

with 1 µg/ml HGM- CSF (human granulocyte macrophage-

colony stimulating factor) (MP Biomedicals). 7 to 12 week

old Swiss mice were obtained from National Institute of

Hygiene and Epidemiology (Vietnam). Human monocytes or

mouse primary peritoneal macrophages were grown for 24 h

on glass coverslips. 106 cells were incubated with 0.05 mg

MNPs for 2-15 h, then treated with either anti-human CD14

antibody (Bio Legend) or actins antibody (Invitrogen) for

taking LSCM images.

FT-IR Spectrometer analysis

Infra red (IR) spectra were recorded with Nicolet 6700 FT-

IR Spectrometer, using KBr pellets, in the region of 400-

4000 cm-1, with resolution of 4 cm

-1. Field Emission Scanning

Electron Microscope (FE-SEM) and Transmission Electron

Microscope (TEM) images was analyzed by Hitachi S-4800

and JEM-1200EX (Voltage:100kV, magnification X200,000),

respectively. Dynamic light scattering (DLS) was analyzed

with Zetasizer 2000 instrument (Malvern, UK).

Confocal microscope analysis

Laser Scanning Confocal Microscope (LSCM) images with

excitation light of 488nm were collected with use of a ZEISS

510 LSCM with a 20x or 40x or 63x oil immersion objectives.

The magnetic properties were measured using Physical Properties

Measurement System (PPMS) from Quantum Design at fields

ranging from 20 to 20 kOe at 25oC, with accuracy of 10

-5emu.

RESULTS AND DISCUSSION

Synthesis of glucosamine sulfate sodium chloride

Glucosamine, an amino monosaccharide derived from

chitin, has been demonstrated to be useful and effective in

osteoarthritic therapy for nearly 40 years (7-10). Among its

different derivatives including glucosamine hydrochloride,

and N-acetyl-glucosamine, glucosamine sulfate (11), glucosamine

sulfate is considered to be one of the most effective one (12).

The synthesis of Glucosamine sulfate sodium chloride (Glu-

) from chitin was performed (Fig. 2). FTIR, 1H–NMR,

13C–NMR and HPLC spectra (shown in Fig. 3) confirmed that

synthesized (Glu- ) matched well with the standard

sample. Its purity, quality and other characteristics with

respect to United State Pharmacopoeia 26 were also fulfilled.

Synthesis of chitosan/hydroxyapatite for tissue engineering

The biomaterial Hydroxyapatite (HAp) has attracted many

scientists thanks to its excellent physicochemical (structure,

composition) and biological properties (bioactivity, biocompatibility,

biodegradability). However, its mechanical (micro hardness, tensile)

properties do not meet those of nature scaffolds. To overcome this

limitation, HAp was combined with CS to improve mechanical

properties of the resulting composites (13, 14). The HAp powder,

first synthesized by coprecipitation of Ca2+

and HPO4

2-, then

was immersed in chitosan solution at the weight ratio of 1 : 1,

and finally dried to obtain the composite. To investigate its

bioactivities, HAp/CS composites were submerged in simulated

body fluid (SBF) at different testing periods. XRD spectra in Fig.

4 show that the crystality of HAp/CS, manifested in its typical

peak of 211, increased gradually. FESEM images in Fig. 5 also

confirmed that the HAp/CS has needle shapes, uniform

dimensions and homogeneous dispersion after 5 and 7 days,

whereas it still agglomerated at the very few days.

SO4

2 –

SO4

2 –

Fig. 1. Structure of chitin and chitosan.

Lam Dai Tran et al. 9

Antibacterial and proliferative activities of silver/

chitosan nanoparticles

Silver nanoparticles (AgNPs), including ionic silver (Ag+)

or metallic silver (Ago), were shown to have good antibacterial

and proliferative activities. Numerous methods have been

proposed and reported in literature (15-19). In our study,

AgNPs were prepared by reducing silver from silver salt

solutions using chitosan as both reducing and stabilizing

agent. It can be observed clearly that core-shell structure of

AgNPs/CS was successfully obtained (composite diameter

was about 12 nm, whereas the structural coating shell of

Chitosan was about 2 nm). The investigation of AgNPs/CS

into antibacterial activities was conducted with six bacterial

lines, including Lactobacillus feremetum, Bacillius subtillis,

Staphylococcus aureus, Samonella enterica, Escherichia coli,

Pseudomonas aeruginosa, and one fungal line named Candida

albicans (Table 1) using the multimicrodilution assay. Figure

6 and Table 2 show the bactericidal effects of AgNPs/CS

samples (SK-W, SO-W and SA-W, corresponding to 60, 85,

900C of AgNPs synthesis respectively) against Bacillius

subtillis and Pseudomonas aeruginosa along with the testing

conditions (Table 2). It is obvious that the AgNPs have

significant effect on controlling the growth of Bacillius

subtillis and Pseudomonas aeruginosa. It can also be observed

in Table 1 that the half maximal inhibitory concentration

(IC50), the minimum inhibitory concentration (MIC) and the

minimum bactericidal concentration (MBC) values of SO-W,

SA-W and SK-W samples against bacteria and fungi lines

Fig. 2. Chemical synthesis of glucosamine-Cl- and glucosamine-SO4

2- from chitin.

Fig. 3. HPLC curves of synthesized (solid line) and standard

(dot line) glucosamine-SO4

2- .

Fig. 4. XRD patterns of CS/Hap in simulated body fluid after: 0,

1, 3, 5, 7 days.

10 Biomedical and Environmental Applications of Chitosan-based Nanomaterials

were achieved as promising ones. In our study, AgNPs/CS

exerted a bactericidal effect against all bacterial strains of

negative gram (S. enterica, E. coli and P. aeruginosa), positive

gram (L. fermentum, S. aureus and B. subtilis) and yeast (C.

albicans). To our best understanding so far, we have achieved

the surprisingly low number (1.5-6 g/ml, except for S.

enterica) with respect to IC50, in comparison to the best result,

recently reported in literature (20), which studied on 25 nm

AgNPs using Maltose as reducing agent.

Drug delivery chitosan-based system

Unique physicochemical and biological characteristics of

CS make it suitable for drug delivery systems. The strategy to

design a multifunctional, nanosized magnetofluorescent water-

dispersible Fe3O4-curcumin conjugates (those diameters was less

than 500 nm) and its multiple ability to label, target and treat

the tumor cells was developed. The conjugate possesses

magnetic nano Fe3O4 core, CS as outer shell and entrapped

curcumin (Cur, an anti-oxidant, anti-inflammatory and anti-

tumor substance), serving dual function of naturally

autofluorescent dye as well as antitumor model drug, delivered

to the cells with the help of macrophage whose role is to

phagocytise (engulf and then digest) cellular debris and

pathogens either as stationary or as mobile cells, and to

stimulate lymphocytes and other immune cells to respond to

the pathogen. Primary peritoneal macrophages isolation was

described in details elsewhere (21). Hence, it can be used as potential

vehicles for transport of MNPs into the core of tumor cells.

Fig. 5. FE-SEM images of CS/HAp in simulated body fluid after: 0, 1, 5, 7 days.

Fig. 6. Microplates (96-wells) image of testing samples against Bacilius Subtilis (a) and Psudomonas Aeugunosa (b).

Lam Dai Tran et al. 11

Fe3O4-Cur conjugate could be visualized dually by

Fluorescence Microscope, Laser Scanning Confocal Microscope

(LSCM) as well as magnetization measurement (Physical

Properties Measurement Systems, PPMS). LCSM images,

taken at 1, 2, 4 and 6 h of incubation, showed that the number

of Fe3O4-Cur that took into macrophage cytoplasm increased

clearly with incubation time (Fig. 7). The green fluorescence

was observed clearly, started at about 0.5-1h and then reached

its maximal at 6 h, confirming that the Fe3O4-Cur particles

were efficiently loaded. Alternatively, PPMS magnetization

curves of macrophage samples at 1, 2, 4 and 6 h of incubation

also confirmed that the magnetization of macrophage increases

(magnetization of the remaining supernatants decreases). This

magnetization result is, therefore, in good accordance with

that done by the above demonstrated in-situ observation by

LSCM fluorescence.

Magnetic inductive heating capacity of the ferrofluids

Hyperthermia is a promising approach to cancer therapy. It

is well known that MNPs exhibit a so-called magnetic heating

(MH) effect - a very specific property that has attracted much

research interest recently because of its potential applications

in cancer hyperthermia (2-4,8,9), drug release (10) and also

desorption of toxic substances (11). However, to be applicable

in such applications Fe3O4 have to be encapsulated to avoid

the agglomeration or to make them monodisperse in suspension.

Table 1. Antibacterial activities testing on bacterial and fungal lines

Bacterial and Fungal lines ConcentrationTesting samples

SA-W SO-W SK-W

Gram (+)

Lactobacillus Fermentum

IC50 0.248 4.245 1,54

MIC 0.421 6.75 0.421

MBC 1.687 >27 1.687

Bacilius Subtilis

IC50 1.084 4.469 4.484

MIC 1.687 6.75 6.75

MBC >27 27 >27

Staphylococcus Aureus

IC50 4.496 1.141 5.230

MIC >27 27 27

MBC >27 27 27

Gram (-)

Salmonella Enterica

IC50 15.058 13.74 >27

MIC >27 27 >27

MBC >27 27 >27

Escherichiala Coli

IC50 5.021 1.188 >27

MIC >27 27 >27

MBC >27 27 >27

Psudomonas Aeugunosa

IC50 1.087 1.105 4.630

MIC 1.687 1.687 27

MBC 1.687 1.687 27

Yeast Candida Albican

IC50 4.324 5.178 2.641

MIC 6.75 27 >27

MBC >27 27 >27

Table 2. Experimental conditions of 3 testing samples: SA-W;

SO-W and SK-W

Testing SampleTemperature

(oC)

Reaction time(h)

CS/Ag+ mole

ratio

SA-W 95 48 10:1

SO-W 80 48 10:1

SK-W 60 48 10:1

Fig. 7. LCSM image of Fe3O4-Cur taken into macrophage with various incubation times (1-6h).

12 Biomedical and Environmental Applications of Chitosan-based Nanomaterials

Therefore, it is essential to modify the surface of these particles

to increase the stability by surfactants, among which

biocompatible polymers like CS and its derivatives (eg, O-

carboxymethylchitosan, OCMCS) are the most potential

candidates owing to its biocompatibility and biodegradability.

Figure 8 presents FE-SEM images of CS/Fe3O4 and

OCMCS/Fe3O4 nanoparticles. Accordingly, for each samples,

the heating curve measurements was carried out under the

same AC field conditions (frequency of 236 kHz and

amplitude of 80Oe, Fig. 9). OCMCS has the backbone

structure similar to CS but hydroxyl group (-OH) is substituted

by carboxyl group (-COOH). This substitution helps increasing

the ability to disperse in neutral and alkaline solution of

OCMCS, and thus broadening their biomedical applications

(22, 23). The so called saturation temperature for suspensions

of the magnetite NPs modified by CS and OCMCS are

gathered in Table 3. Comparing heating curves for the Fe3O4-

CS and Fe3O4-OCMCS magnetic fluids with the same

concentration (10 mg/ml), it can be seen that the saturation

heating temperatures are different. In the concentration range

from 0.5 to 1 mg/ml, the heating capacity of Fe3O4/CS is

better than that of Fe3O4/OCMCS, when the concentration is

below 0.5 mg/ml the saturation temperature of Fe3O4-

OCMCS is higher. This might be explained as because of the

fact that CS is not dissolvable in aqueous solution. So when

reducing the concentration, CS will precipitate, the stability of

magnetic fluid decrease and this also lessen the saturation heating

temperature. And therefore, the applications of CS in biomedicine

fields are limited despite of its higher hyperthermia ability.

Heavy metal ions removal

Apart from the above mentioned applications, CS and its

derivatives can be used for Heavy metal ions removal. CS has

excellent properties for the adsorption of metal ions, principally

due to the presence of amino groups (–NH2) in the polymer

matrix, which can interact with metal ions in solution by ion

exchange and complexation reactions (11). The high content of

amino groups also makes possible many chemical modifications

in polymer with the purpose of improving selectivity and

adsorption capacity.

In our group, chitosan/magnetite nanocomposite beads

Fig. 8. FESEM images of unmodified (a), CS (b) and OCMCS (c) modified Fe3O4 particles.

Fig. 9. Magnetic heating curves measured for suspensions of OCMCS-modified (a) and CS-modified (b) Fe3O4 NPs.

Table 3. Magnetic heating parameters

NoConcentration

(mg/ml)

Saturation temperature (Ts, oC )

CS – Fe3O4 OCMCS – Fe3O4

1 1 > 100 98

2 0.7 > 98 90

3 0.5 90 78.5

4 0.3 59 72

5 0.2 53.3 60.7

Lam Dai Tran et al. 13

were prepared, characterized and used for removal of toxic

metal ions such as Pb(II), Ni(II) in the pH range from 4 to 6.

Langmuir isotherms were used to analyze the equilibrium

data at different pH. These nanocomposite beads can be

removed easily from water with the help of an external

magnet thanks to their exceptional magnetic properties (24).

Further, to increase the adsorption capacity, Fe3O4/Al(OH)3/

CS was designed by co-precipitation of Fe2+

, Fe3+

and Al3+

in

CS solution, using concentrated alkaline solution as a

precipitation agent. Al(OH)3 is expected to form intercapillary

structure and thus to synergize the adsorbability of Fe3O4/

Al(OH)3/CS composite. Figure 10 shows the FESEM images

of Fe3O4/Al(OH)3/CS nanocomposites before and after

exposure to heavy metal ion (Ni2+

, Pb2+

, Cd2+

and Cu2+

)

solutions, clearly indicating that the morphology of materials

changed from smoothness and fold into roughness and

swelling. This change brings an evidence that that heavy

metal ions were efficiently adsorbed (as in metal complexes

with Chitosan) onto the surface of Fe3O4/Al(OH)3/CS composite.

It should be noted that saturation magnetization of nanocom-

posites decreases slightly, even with or without Al(OH)3, so that

they can be manipulated by external magnet to retrieve and

Fig. 10. FESEM of CS/MNPs/Al(OH)3 surface before (a-c) and after adsorption metals ions: Co2+

(d-f); Cu2+

(g-i); Pb2+

(j-l) and Cd2+

(m-o) with different magnification.

14 Biomedical and Environmental Applications of Chitosan-based Nanomaterials

reuse afterwards. In conclusion, some CS based nanomaterials

have been successfully synthesized and used in biomedical

and environmental applications.

ACKNOWLEDGEMENTS

The authors are grateful for the financial support for this

work by Korean-Vietnamese joint research project (2010-

2011, code 59/2615/2010/HD-NDT).

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Received December 1, 2010

Revised December 25, 2010

Accepted December 30, 2010