친수성기와 소수성기로 치환된 저분자량 수용성 키토산의 합성

손 소 희, 장 미 경, 나 재 운*

순천대학교 공과대학 고분자공학과

jwnah@sunchon.ac.kr

The Synthesis of Low Molecular and Water Soluble Chitosan Modified with Hydrophobic and Hydrophilic group

So-hee Son, Mi-Kyeong Jang, Jae-Woon Nah*

Depart. of Polym. Sci. & Eng., Sunchon National University, Suncheon,

Jeonnam 540-742, Korea

jwnah@sunchon.ac.kr

Abstract

The low molecular, water-soluble chitosan nanoparticle (LMWSC-NP) was prepared as modify with hydrophilic and hydrophobic moieties to evaluate the potential for pharmaceutics application. The synthesis of LMWSC-NP was identified by FT-IR and 1H-NMR spectrum. Also, we measured the photon correlation spectroscopy (PCS), transmission electron microscope (TEM) and atomic force microscope (AFM) to investigate the characteristics and morphology of the LMWSC-NPs. At the PCS measurement, the more increase the number of substitutive group, the more decrease the positive charge of LMWSC-NP surface. From the results of TEM and AFM, spherical morphologies were observed, and its size was 30∼100 nm. Resultantly, LMWSC-NP prepared in this experiment will be expected a suitable device for the drug targeting system.

Introduction

Generally, nanoparticles are useful device for carrying drugs or bioactive agents in the human body due to their small particle size. However, conventional nanoparticles still have several problems such as surfactant removal in process, undesirable side effects, rapid clearance due to the reticuloendothelial system (RES), thermal instability, short blood circulation, and lower loading efficiency[1-2]. In a previous study[3], we first synthesized nanoparticles without surfactant by the dialysis method, then solved the problem of toxicity by surfactant in the body. Until now, the materials used to prepare the nanoparticles were PLGA and diblock copolymers composed of poly(aspartic acid) or poly(-benzyl-L-aspartate) as the hydrophobic component and poly(ethylene glycol) as the hydrophilic component[4-5]. Unlike these synthetic polymers, we used low molecular, water soluble chitosan (LMWSC) having a structure similar to that of cellulose, natural polysaccharide, with a free amine group synthesized in the our laboratory[6].

In this study, using the low molecular, water soluble chitosan (LMWSC) developed in our lab, we prepared LMWSC-NP with a hydrophobic core using novel method which included modification with cholesterol as the PEG. The nanoparticles were formed as introduce hydrophilic group, PEG, and hydrophobic group, cholesterol, at chitosan chains. PEG chains can prevent cell adhesion by entropically driven steric repulsion and increasing hydrophilicity of carrier surfaces. The introduction of the cholesterol can enhance the association behavior of chitosan and, stability and activity of the hydrophobic drug can be enhanced by formation of hydrophobic core of the chitosan-cholesterol derivatives. We anticipate that hydrophobically modified nanoparticles will be found effective in serving as drug carriers for anticancer agents.

Experimental

An LMWSC with Mw of 19 K Da and DAc of 93% was supplied from KITTOLIFE Co. KOREA. Methoxy poly(ethylene glycol) p-nitrophenyl carbonate (MPEG-pNP), the hydrophilic moiety, was purchased from Sigma Co. and cholesteryl chloroformate, the hydrophobic group, was purchased from Aldrich Co. The synthetic route of LMWSC-NP was carried out in two steps. First, LMWSC was dissolved in the phosphate buffered saline. MPEG-pNP solution dissolved into PBS slowly dropped in LMWSC solution and reactant was stirred at the room temperature for two hours. After reacting, the solution was dialyzed using a molecular cut-off 12 000 g/mol dialysis tube against 1. 0L × 3 of cold distilled water for 3 h and then distilled water was exchanged at intervals of 34 h for a 24 h. The solution was freeze-dried. For the second reaction, the product was dissolved in PBS . A Cholesteryl chloroformate solution dissolved in DMF was dropped into the resulting solution. After two hours, reactant was created using the above procedure.   The characterization of LMWSC-NP was determined by FT-IR(Shimadzu, FT-IR 8700) and 1H-NMR spectrometer (Bruker, DRX-500MHz). The morphologies observation of LMWSC-NPs were examined with a JEOL JEM-2000 FX-II transmission electron microscope (TEM) and PARKs Science, Autoprobe CP Atomic Force Microscope (AFM). Also, the size of LMWSC-NP was measured with a Zetasizer 3000 (Malvern instruments, England).

Results and Discussion

In this study, we used LMWSC prepared by a novel method. LMWSC has a free-amine group at the C-2 position of the glucose amine unit. With this in mind, we chose to modify the free-amine group, using MPEG-pNP as the hydrophilic group and cholesteryl chloroformate as the hydrophobic group. At the FT-IR spectrum, amide I and amine peak were identified at 1635 and 1539 cm-1, respectively. However, it was discovered that the amide I band at 1650 cm-1 increased and the amine peak at 1539 cm-1 decreased due to the formation of amide bonds between LMWSC and PEG. Also, the salient peak of PEG by aliphatic -CH appears at 2880 cm-1. From 1H-NMR data, the character peak by 14-H at cholesteryl chloroformate was observed at 5.4 ppm. The proton by aceteamide group of chitosan and the proton by methoxy group of MPEG-pNP peaked at 2.0 and 3.3 ppm, respectively. The evidence of polymeric micelle formation of LMWSC-NP was obtained with 1H-NMR in CDCl3 and D2O. At the 1H-NMR spectrum of CDCl3 where micelle formation is not expected. All the peaks correspondence to LMWSC, MPEG as hydrophilic part and cholesterol as the hydrophobic part were detected in CDCl3. But, in D2O, no signals corresponding to cholesterol were shown. These results indicated restricted motions of these protons within the micellar core and rigid structure of cholesterol core of the LMWSC-NP. The shape of the LMWSC-NPs with the ratio of 10:2:2, 10:2:4, 10:2:6, the ratio for the units of glucosamine : MPEG-pNP : cholesteryl chloroformate, is spherical and the diameters range from about 30 nm to100 nm. When the MPEG ratio to glucosamine unit is constant, the more increase the ratio of cholesterol, the smaller the size of LMWSC-NP due to the increase of hydrophobicity.     From the AFM observation, LMWSC-NPs were spherical and the sizes were ranged about 30 nm∼100 nm in diameter. From these results, it was identified that the morphology and size of LMWSC-NPs is agree with the TEM results. Table 1 shows the zeta potential of LMWSC-NPs according to the number of the substituted group at C-2 position of chitosan and the size of LMWSC-NPs by TEM, AFM measurements. As the results, the more increase the number of substitutive group, the more decrease the positive charge of nanoparticle surface. This means that the positive charge having amine group of chitosan was neutralized by substitution with other group.

Conclusions

This study shows the synthesis of LMWSC-NP with a hydrophobic core for delivery of hydrophobic drugs such as Taxol. The LMWSC was modified with MPEG-pNP and cholesteryl chloroformate. These polymers are known to be biocompatible, biodegradable and nontoxic. The resulting synthesis was identified by FT-IR and 1H-NMR spectrum analysis and the morphology was also revealed through AFM and TEM. It was shown that LMWSC-NP prepared successfully in this manner can yield the necessary modified chitosan nanoparticles suitable for drug delivery systems. Therefore, it is expected that LMWSC-NP modified with hydrophobic groups will increase the solubility of hydrophobic drugs.

Table I. Particle size and zetapotential of LMWSC-NPs

LMWSC-NPs	The number of substituted group per [1]0 units of glucosamine	Zeta potential (mV)	Particle size (nm)
LMWSC-NP1	4	11.5	70-100
LMWSC-NP2	6	3.2	50-80
LMWSC-NP3	8	1.1	30-50

Reference

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