Randomly 50% N-acetylated low molecular weight chitosan as a novel renal targeting carrier

Selective targeting of drugs to kidneys may improve renal effectiveness and reduce extrarenal toxicity. Using fluorescence imaging, we found for the first time that randomly 50% N-acetylated low molecular weight chitosan (LMWC) selectively accumulated in the kidneys, especially in the renal tubules after i.v. injection in mice. To develop and evaluate the novel renal drug carrier, prednisolone, used as a model drug, was covalently coupled with various molecular weight LMWCs via a succinic acid spacer. The mean residence time (MRT) in plasma of prednisolone conjugates increased as the molecular weight increased. The conjugate with molecular weight 19 kD (conjugate-19k) displayed the highest accumulation rate in the kidneys, which was 14.06 ± 2.81% of the administered dose 15 min after i.v. injection. The total amount of the conjugate-19k in the kidneys was 13-fold higher than that of controlled prednisolone group. Both conjugate-19k and conjugate-31k had higher retention and about 10% of injected dose was still retained in the kidneys after 120 min. Additionally, MTT assay showed LMWCs were noncytotoxic towards L929 and NRK-52E cells. Conclusion can be drawn that the coupling of prednisolone to the proper molecular weight LMWC results in increased prednisolone concentration in the kidneys. Therefore, LMWC with a proper molecular weight can be applied as a promising carrier for renal targeting.     

Randomly 50% N-acetylated low molecular weight chitosan as a novel renal targeting carrier

Selective targeting of drugs to kidneys may improve renal effectiveness and reduce extrarenal toxicity. Using fluorescence imaging, we found for the first time that randomly 50% N-acetylated low molecular weight chitosan (LMWC) selectively accumulated in the kidneys, especially in the renal tubules after i.v. injection in mice. To develop and evaluate the novel renal drug carrier, prednisolone, used as a model drug, was covalently coupled with various molecular weight LMWCs via a succinic acid spacer. The mean residence time (MRT) in plasma of prednisolone conjugates increased as the molecular weight increased. The conjugate with molecular weight 19 kD (conjugate-19k) displayed the highest accumulation rate in the kidneys, which was 14.06+/-2.81% of the administered dose 15 min after i.v. injection. The total amount of the conjugate-19k in the kidneys was 13-fold higher than that of controlled prednisolone group. Both conjugate-19k and conjugate-31k had higher retention and about 10% of injected dose was still retained in the kidneys after 120 min. Additionally, MTT assay showed LMWCs were noncytotoxic towards L929 and NRK-52E cells. Conclusion can be drawn that the coupling of prednisolone to the proper molecular weight LMWC results in increased prednisolone concentration in the kidneys. Therefore, LMWC with a proper molecular weight can be applied as a promising carrier for renal targeting.     

Galactosylated liposomes as oligodeoxynucleotides carrier for hepatocyte-selective targeting

18mer oligodeoxynucleotides (ODNs) which can inhibit survivin gene expression were selected as a model gene drug. The glycolipid (5-cholestan-3beta-yl)-1-[2-(lactobionyl amido) ethylamido] formate (CHE-LA) which specific target to the cells expressing galactose receptors was synthesized through the reaction of lactone of lactobiono-1,5-lactone (LA) and the amino-group of 2-(cholesteryloxycarbonylamino) ethylamine (CHE). The galactosylated liposome incorporated with CHE-LA containing oligodeoxynucleotides was prepared with SPC, cholesterol, CHE-LA and oligodeoxynucleotides by the thin-film hydration method. 1,1'-Dioctadecyl-3,3,3',3'tetramethylindocarbocyanine perchlorate (Dil) was used as a marker for all the liposome preparations. Compared with conventional liposomes (CL), the galactosylated liposomes (GL) exhibited a drastically increased distribution to the liver in vivo and the galactosylated liposomes containing oligodeoxynucleotides (GLO) can also more efficiently induced an apoptosis of HepG2 cells in vitro than the conventional liposome containing oligodeoxynucleotides (CLO). In addition, the GLO represented an improving of the ODNs entrapment efficiency.     

一种低分子壳聚糖硫酸酯铝的制备

本文采用正交试验设计方法对壳聚糖进行氧化降解,获得了一种低相对分子质量的水溶性壳聚糖,并在此基础上经硫酸化和盐交换制得了一种低相对分子质量壳聚糖硫酸酯式铝盐,壳聚糖氧化降解实验结果表明降解的最佳条件是温度为80度,过氧化氢浓度为5％,降解时间为1h,在此条件一产品收率可达52．68％,其粘均相对分子质量为3990,对制是的低相对分子质量壳聚糖硫酸酯铝进行了红外光谱分析和部分理化性质测定,经测定定样品中有机硫（S）含量为9．10％,铝（Al)含量为20．58％,样品制酸力为188．59mL.g^-1。     

Effect of chitosan salts and molecular weight on a nanoparticulate carrier for therapeutic protein

The objective of this study was to investigate the potential of chitosan salts as a carrier in the preparation of protein-loaded nanoparticles. Glutamic and aspartic acids were used to prepare chitosan salts of 35, 100, and 800 KDa. Nanoparticles of chitosan base, chitosan glutamate, and chitosan aspartate were produced by ionotropic gelation with sodium tripolyphosphate (TPP). Bovine serum albumin (BSA) was applied as a model protein at loading concentrations ranging from 0.2 to 2 mg/mL. The size of the nanoparticles, as measured by photon correlation spectroscopy, was in the range of 195 to 3450 nm, depending on type and molecular weight of chitosan. Nanoparticles prepared with higher molecular weight chitosan showed larger sizes. The encapsulation was controlled by the competition of BSA in forming ionic cross-linking with chitosan and by the entrapment of BSA during the gelation process. Higher BSA encapsulation efficiency (EE) was obtained for nanoparticles prepared with chitosan salts compared to those prepared with the base. The higher EE was a result of a higher degree of ionization, causing more active sites to interact with BSA. In addition, a higher and faster release of BSA from the nanoparticles into pH 7.4 buffer medium was observed for nanoparticles of the chitosan salts than was observed for nanoparticles of the chitosan base. The higher and faster release was attributed to higher EE and lower entrapment of BSA within the matrix of the nanoparticle during the gelation process. The influence of molecular weight on the property of nanoparticles exhibited different effects. The difference was a result of different organic acids used to prepare nanoparticles leading to the difference in polymer conformation and viscosity of organic acid solution. Therefore, this study showed that the characteristics of chitosan nanoparticles loaded with a protein drug could be readily modulated by changing the salt form or the molecular weight of the chitosan carrier.     

Galactosylated chitosan nanoparticles for hepatocyte-targeted delivery of oridonin

In this study, oridonin-loaded nanoparticles coated with galactosylated chitosan (ORI-GC-NP) were prepared for tumor targeting and their characteristics were evaluated for the morphologies, particle size and zeta potential. Oridonin-loaded nanoparticles (ORI-NP) without galactosylated chitosan were prepared as a control. The entrapment efficiency of ORI-GC-NP and ORI-NP were 72.15% and 85.31%, respectively. The in vitro drug release behavior from nanoparticles displayed biphasic drug release pattern with initial burst release and consequently sustained release. Next, the pharmacokinetics and tissue distribution of ORI-GC-NP, ORI-NP and ORI solution were carried out. Pharmacokinetic analysis showed that ORI-GC-NP and ORI-NP could prolong the drug plasma levels compared with ORI solution. Meanwhile, the distribution of ORI-GC-NP to liver was higher than that of ORI-NP and free drug. In conclusion, ORI-GC-NP, as a promising intravenous drug delivery system for ORI, could be developed as an alternative to the conventional ORI preparations.     

A method for direct preparation of chitosan with low molecular weight from fungi.

By modifying the common method for the preparation of chitosan from fungi, low molecular weight chitosan with an average MW of 4.5 x 10(4) g/mol and a numerical MW of 1.7 x 10(4) g/mol can be directly extracted from the raw material without the need of thermal or chemical depolymerization. Based on the solubility of low molecular chitosan up to alkaline pH ranges, reprecipitation and washing with ethanol is required to keep the low molecular fraction within the preparation. The use of water for washing between the preparation steps would cause solving and discarding of the low molecular chitosan. The chitosan was analyzed by laser light scattering and 1H-NMR spectroscopy.     

Interaction of indomethacin with low molecular weight chitosan,and improvements of some pharmaceutical properties of indomethacin by low molecular weight chitosans

The interaction of four kinds of low molecular weight chitosans, which were different in molecular weight and degree of deacetylation, were studied in solution and the solid state using an anti-inflammatory drug, indomethacin (IM), as an acidic model molecule. The solubility of IM enhanced with increasing concentration of low molecular weight chitosan, especially C-I which had the lowest molecular weight and least degree of deacetylation. The C-I crystal complex was obtained in a molar ratio of 16:1 (IM: C-I) from aqueous solution. The data suggested that the acetyl group and amino group of chitosan played an important role in the complexation. The IM dissolution rates from kneaded mixtures with low molecular weight chitosans were enhanced in the order of C-I >C-II >C-III >C-IV (molecular weight: C-IV >C-III >C-II >C-I). The oral absorption rate of IM from C-I kneaded mixture was improved compared with IM alone.     

Antidiabetic action of low molecular weight chitosan in genetically obese diabetic KK-Ay mice

Recently, we reported that low molecular weight (LMW) chitosan (chitosan lactate, average MW: 20,000) prevents the progression of low dose (100 mg/kg, i.p.) streptozotocin-induced slowly progressive diabetes mellitus in male ICR mice. The present study was designed to clarify the effects of LMW chitosan on hyperglycemia, hyperinsulinemia and hypertriglyceridemia in genetically obese diabetic male KK-Ay mice. LMW chitosan (0.05%, 0.2% or 0.8% water solution) was given daily as drinking water to male KK-Ay mice for 11 weeks, from 5 weeks of age. The non-fasting serum glucose levels of control mice continued to increase slowly throughout the experimental period. LMW chitosan lowered the serum glucose levels in a dose-dependent manner. In these diabetic mice, hyperinsulinemia and hypertriglyceridemia were observed, and LMW chitosan was dose-dependently effective in improving both serum biochemical parameters. LMW chitosan at three doses improved overdrinking and polyuria observed in these diabetic mice. It is concluded from these results that LMW chitosan may be useful for the treatment of obesity-related type 2 diabetes mellitus.     

Doxorubicin-loaded galactose-conjugated poly(d,l-lactide-co-glycolide) nanoparticles as hepatocyte-targeting drug carrier

The objective of this work is to produce doxorubicin-loaded galactose-conjugated poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) to be specifically recognised by human hepatoma cellular carcinoma (Hep G2) cells and assess NPs cytotoxicity. Doxorubicin-unloaded and doxorubicin-loaded galactose-conjugated PLGA NPs were prepared using an emulsion method and characterised for morphology, size, drug release behaviour, Hep G2 recognition and cell cytotoxicity. The produced doxorubicin-loaded PLGA-galactose-conjugate nanoparticles (PLGA-GAL NPs) are spherical in shape with a size of 365?±?74?nm, a drug encapsulation efficiency of 69% and released in a biphasic pattern with higher release rates at pH 5. In vitro cell studies confirmed the specific interaction between the receptors of Hep G2 and the PLGA-GAL NPs. Cell cytotoxicity tests showed that unloaded NPs are non-toxic and that doxorubicin-loaded NPs caused a cellular viability decrease of around 80%, therefore representing a promising approach to improve liver-specific drug delivery.     

Phosphorylatable short peptide conjugated low molecular weight chitosan for efficient siRNA delivery and target gene silencing

Small interfering RNA (siRNA) has been widely investigated as a potential therapeutic approach for diseases with genetic defects. However, its application was greatly hampered by the rapid degradation and poor cellular uptake. Recently, chitosan (CS) and its derivant have been considered as a promising siRNA transporter with the advantages of low toxicity, good biodegradability and biocompatibility. Chitosan of different molecular weight (Mw) and degrees of deacetylation (DD) showed significantly varied target gene silencing efficacy, and it is still not well clarified how these characteristics influence CS mediated siRNA transfection. To compare the aspects of cell permeability and intracellular unpacking of CS/siRNA complex on the effect of CS/siRNA transfection. A radiolabeled siRNA, targeting firefly luciferase gene, was loaded by chitosan of different molecular weight (varying from 2000 to 800,000 Da) and subjected to the transfection against MDA-MB-231/Luc human breast cancer cell line which stably expressed knocked in firefly Luciferase reporter gene. Following transfection intracellular radioactivity was measured to represent cell entrance ability of the CS/siRNA, while, luciferase activity in the cell lysate was also determined to reflect target gene silencing effect. The results revealed that although low molecular weight chitosan (LMWC) condensed siRNA has the highest cell permeability of almost two folds of medium molecular weight chitosan and lipofactamine, its target gene silencing effect is really low of almost eight times less than lipofectamine. This conspicuous contradiction gave us the hypothesis that LMWC generated more condensed CS/siRNA complex to facilitate cell entrance but the tight electrostatic interaction probably limited intracellular siRNA unpacking as well and unfavorably hindered target gene silencing as the final consequence. To approve this hypothesis a phosphorylatable short peptide conjugated LMWC was adopt to promote intracellular siRNA unpacking. Which was demonstrated of perfect target gene knock down ability to the extent of being even superior to lipofactamine 2000. In a conclusion, low molecular weight chitosan has the great potential to be an ideal siRNA vehicle if the issue of siRNA unpacking could be properly resolved.     

Galactosylated chitosan (GC)-<Emphasis Type="Italic">graft</Emphasis>-poly(vinyl pyrrolidone) (PVP) as hepatocyte-targeting DNA carrier:<Emphasis Type="Italic">in vitro</Emphasis> transfection

Galactosylated chitosan-graft-poly(vinyl pyrrolidone) (GCPVP) was synthesized and characterized for hepatocyte-targeting gene carrier. GCPVP itself as well as GCPVP/DNA complex had negligible cytotoxicity regardless of the concentration of GCPVP and the charge ratio, but GCPVP/DNA complex had slightly cytotoxic effect on HepG2 cells only in the case of the higher charge ratio and 20 mM of Ca2+ concentration used. Through the confocal laser scanning microscopy, it is shown that the endocytosis by interaction between galactose ligands of GCPVP and ASGPR of the hepatocytes was the major route of transfection of GCPVP/F-plasmid complexes.     

Low molecular mass chitosan as carrier for a hydrodynamically balanced system for sustained delivery of ciprofloxacin hydrochloride

Chitosan has become a focus of major interest in recent years due to its excellent biocompatibility, biodegradability and non-toxicity. Although this material has already been extensively investigated in the design of different types of drug delivery systems, it is still little explored for stomach specific drug delivery systems. The objective of the present investigation was to explore the potential of low molecular mass chitosan (LMCH) as carrier for a hydrodynamically balanced system (HBS) for sustained delivery of water soluble drug ciprofloxacin hydrochloride (CP). Various formulations were prepared by physical blending of drug and polymer(s) in varying ratios followed by encapsulation into hard gelatin capsules. All the formulations remained buoyant in 0.1 mol L(-1) HCl (pH 1.2) throughout the experiment. Effect of addition of xanthan gum (XG) or ethyl cellulose (EC) on drug release was also investigated. Zero order drug release was obtained from the formulations containing LMCH alone or in combination with XG, and in one instance also with EC. Our results suggest that LMCH alone or in combination with XG is an excellent material for stomach specific sustained delivery of CP from hydrodynamically balanced single unit capsules.     

Eco-dyeing with biocolorant from spent coffee ground on low molecular weight chitosan crosslinked cotton

Growing concern about the environmental effects of synthetic dyes is encouraging the use of natural dyes. Spent coffee ground (SCG), a potential renewable source of natural dye, was investigated as a biocolorant for dyeing of cotton. SCG contains the brown pigment melanoidin. Low molecular weight chitosan (LMWCTS) was used to introduce amino groups, with citric acid (CA) as a crosslinker. LMWCTS was applied to cotton at various concentrations using a simple pad-dry-cure technique, followed by dyeing with SCG extract using an exhaustion process. Fourier transform infrared spectrometry analysis confirmed the formation of ester linkages between the citric acid and cellulose or chitosan. The dyed fabrics were also evaluated for color properties using CIELAB (L*, a* and b*) and K/S values, color fastness, and whiteness index, for physical properties (wrinkle recovery angle, drape coefficient, and tensile strength), and for surface morphology. The combination of chitosan and citric acid provided fabrics with a rich natural brown shade. The samples showed the increase in color strength and wrinkle resistance compared with untreated and alum-mordanted samples. Fabrics treated with CA/LMWCTS had the ability to retain drape and exhibited good wrinkle resistance through 20 wash cycles. Color fastness to washing and rubbing was good.     

Low molecular weight chitosan nanoparticles as new carriers for nasal vaccine delivery in mice.

High molecular weight (Mw) chitosan (CS) solutions have already been proposed as vehicles for nasal immunization. The aim of the present work was to investigate the potential utility of low Mw CS in the form of nanoparticles as new long-term nasal vaccine delivery vehicles. For this purpose, CS of low Mws (23 and 38 kDa) was obtained previously by a depolymerization process of the commercially available CS (70 kDa). Tetanus toxoid (TT), used as a model antigen, was entrapped within CS nanoparticles by an ionic cross-linking technique. TT-loaded nanoparticles were first characterized for their size, electrical charge, loading efficiency and in vitro release of antigenically active toxoid. The nanoparticles were then administered intranasally to conscious mice in order to study their feasibility as vaccine carriers. CS nanoparticles were also labeled with FITC-BSA and their interaction with the rat nasal mucosa examined by confocal laser scanning microcopy (CLSM). Irrespective of the CS Mw, the nanoparticles were in the 350 nm size range, and exhibited a positive electrical charge (+40 mV) and associated TT quite efficiently (loading efficiency: 50-60%). In vitro release studies showed an initial burst followed by an extended release of antigenically active toxoid. Following intranasal administration, TT-loaded nanoparticles elicited an increasing and long-lasting humoral immune response (IgG concentrations) as compared to the fluid vaccine. Similarly, the mucosal response (IgA levels) at 6 months post-administration of TT-loaded CS nanoparticles was significantly higher than that obtained for the fluid vaccine. The CLSM images indicated that CS nanoparticles can cross the nasal epithelia and, hence, transport the associated antigen. Interestingly, the ability of these nanoparticles to provide improved access to the associated antigen to the immune system was not significantly affected by the CS Mw. Indeed, high and long-lasting responses could be obtained using low Mw CS molecules. Furthermore, the response was not influenced by the CS dose (70-200 microg), achieving a significant response for a very low CS dose. In conclusion, nanoparticles made of low Mw CS are promising carriers for nasal vaccine delivery.     

Liposome coated with low molecular weight chitosan and its potential use in ocular drug delivery

In this study liposome coated with low molecular weight chitosan (LCH) was proposed and investigated its in vitro and in vivo properties, and its potential use in ocular drug delivery was evaluated. LCH with a molecular weight of 8 kDa was prepared and coated on liposome loaded with diclofenac sodium. The LCH coating changed the liposome surface charge and slightly increased its particle size, while the drug encapsulation was not affected. After coating, the liposome displayed a prolonged in vitro drug release profile. LCH coated liposome also demonstrated an improved physicochemical stability at 25 °C in a 30-day storage period. The ocular bioadhesion property was evaluated by rabbit in vivo precorneal retention, and LCH coated liposome achieved a significantly prolonged retention compared with non-coated liposome or drug solution. The LCH coating also displayed a potential penetration enhancing effect for transcorneal delivery of the drug. In the ocular tolerance study, no irritation or toxicity was caused by continual administration of LCH coated liposome in a total period of 7 days. In conclusion, the LCH coating significantly modified the properties of liposome and brought a series of notable advantages for ocular drug delivery.     

低分子量壳聚糖衍生物促胰岛细胞增殖作用的研究

目的探讨低分子量壳聚糖衍生物对胰岛β细胞的促增殖作用。方法在细胞水平上,通过细胞形态学观察和MTT法,研究3种低分子量壳聚糖衍生物对胰岛β细胞系RIN-m5f的促增殖作用。结果低分子量50%D.D.壳聚糖(LM-50%D.D.CTS)、低分子量羧甲基甲壳素(LM-CMC)、低分子量磺酸化壳聚糖(LM-SCTS)对于胰岛β细胞具有明显促增殖作用,其最适作用浓度分别为500,250,500mg.L-1。结论3种低分子量壳聚糖衍生物对于胰岛β细胞系体外增殖具有明显的促进作用。     

Characteristics of vitamin C encapsulated tripolyphosphate-chitosan microspheres as affected by chitosan molecular weight

In this paper, the effect of chitosan molecular weight on the characteristics (size, encapsulation efficiency, zeta potential, surface morphology and release rate) of vitamin C encapsulated tripolyphosphate cross-linked chitosan (TPP-chitosan) microspheres. The molecular weight of chitosan had a noticeable influence on the size, encapsulation efficiency, zeta potential, surface morphology and controlled release behaviour of the vitamin C encapsulated TPP-chitosan microspheres. The mean particle size and encapsulation efficiencies of TPP-chitosan microspheres were 3.1, 4.9 and 6.7 microm and 67.25, 60.43 and 52.74% for the microspheres prepared using low, medium and high molecular weight chitosan, respectively. All the TPP-chitosan microspheres (low, medium and high molecular weight) had positive charge on their surface. The zeta potential of the TPP-chitosan microspheres prepared using low, medium and high molecular weight chitosan was 41.25, 40.84 and 39.13 mV, respectively. The particle sizes of TPP-chitosan microspheres increased with increases in chitosan molecular weight. Molecular weight of chitosan did not affect significantly the % yield of TPP-chitosan microspheres prepared by spray-drying. The influence of chitosan molecular weight on the surface morphology of vitamin C encapsulated TPP-chitosan microspheres was examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). It was observed that, as the molecular weight of chitosan increases, TPP-chitosan microspheres with uniform spherical shape could be obtained. The physical state of vitamin C (amorphous or crystalline) in TPP-chitosan matrix was studied by X-ray diffraction (XRD) and it was found that vitamin C is dispersed at the molecular level (amorphous) in the TPP-chitosan matrix. Release rate of the vitamin C from TPP-chitosan microspheres was significantly affected by the chitosan molecular weight. The release rate decreased with increase in the chitosan molecular weight. The release of vitamin C from TPP-chitosan microspheres followed Fick's law of diffusion.     

Neurotrophin-like low molecular weight compounds

A large number of compounds have been reported to prevent ischemia-induced neuronal death, whereas there are few described to enhance recovery of brain functions. Since neurotrophins do not only prevent neuronal death but also protect neuronal circuits, they may be potential candidates. However, their poor penetration of the blood-brain-barrier hampers their development as therapeutic agents. In this context, low-molecular-weight compounds that possess neurite outgrowth- and neuronal survival-promoting activities may be alternative candidates. Neurite outgrowth-promoting prostaglandins, which were recently-synthesized based on the chemical structure of anti-tumor cyclopentenone prostaglandin derivatives, have been characterized by their neurotrophic effects on neurons in the central nervous system. In this paper, we present a review of these compounds as therapeutic agents against several neurodegenerative diseases.     

Characteristics of vitamin C encapsulated tripolyphosphate-chitosan microspheres as affected by chitosan molecular weight

In this paper, the effect of chitosan molecular weight on the characteristics (size, encapsulation efficiency, zeta potential, surface morphology and release rate) of vitamin C encapsulated tripolyphosphate cross-linked chitosan (TPP-chitosan) microspheres. The molecular weight of chitosan had a noticeable influence on the size, encapsulation efficiency, zeta potential, surface morphology and controlled release behaviour of the vitamin C encapsulated TPP-chitosan microspheres. The mean particle size and encapsulation efficiencies of TPP-chitosan microspheres were 3.1, 4.9 and 6.7?µm and 67.25, 60.43 and 52.74% for the microspheres prepared using low, medium and high molecular weight chitosan, respectively. All the TPP-chitosan microspheres (low, medium and high molecular weight) had positive charge on their surface. The zeta potential of the TPP-chitosan microspheres prepared using low, medium and high molecular weight chitosan was 41.25, 40.84 and 39.13?mV, respectively. The particle sizes of TPP-chitosan microspheres increased with increases in chitosan molecular weight. Molecular weight of chitosan did not affect significantly the % yield of TPP-chitosan microspheres prepared by spray-drying. The influence of chitosan molecular weight on the surface morphology of vitamin C encapsulated TPP-chitosan microspheres was examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). It was observed that, as the molecular weight of chitosan increases, TPP-chitosan microspheres with uniform spherical shape could be obtained. The physical state of vitamin C (amorphous or crystalline) in TPP-chitosan matrix was studied by X-ray diffraction (XRD) and it was found that vitamin C is dispersed at the molecular level (amorphous) in the TPP-chitosan matrix. Release rate of the vitamin C from TPP-chitosan microspheres was significantly affected by the chitosan molecular weight. The release rate decreased with increase in the chitosan molecular weight. The release of vitamin C from TPP-chitosan microspheres followed Fick's law of diffusion.