Novel amphiphilic ternary polysaccharide derivates chitosan-g-PCL-b-MPEG: synthesis, characterization, and aggregation in aqueous solution

Chitosan-g-PCL-b-MPEG copolymers of various compositions were successful synthesized via a protection-graft-deprotection procedure, by the esterification of phthaloyl-protected chitosan (PHCS) with MPEG-b-PCL-COOH, which was synthesized from MPEG and epsilon-caprolactone and carboxylated by maleic anhydride. The chemical structure of the chitosan-g-PCL-b-MPEG was characterized by Fourier transform infrared and NMR spectroscopy. The chitosan-g-PCL-b-MPEG was obtained as amphoteric hybrid with amino polysaccharide backbone and amphiphilic MPEG-b-PCL side chain. Their crystallinity and aggregation behavior in aqueous solution were also investigated.     

Synthesis and property of chitosan graft copolymer by RAFT polymerization with tosylic acid-chitosan complex

pH- and thermo-sensitive (1→4)-2-amino-2-deoxy-β-d-glucan (i.e. chitosan) graft copolymer was prepared by reversible addition fragmentation chain transfer polymerizations of N-isopropylacrylamide with 4-methylbenzenesulfonic acid (i.e. tosylic acid)-chitosan complex. The polymerization was controlled well, and the amino group of chitosan could be deprotected easily and mildly with 15% Tris solution. The model aldehyde vanillin was conjugated with amino group of chitosan-g-PNIPAM via Schiff base bond (Loading efficiency, LE=77.6 mg/g), and the drug release could be controlled with temperature and pH. This property may promote the chitosan graft copolymer to be used in the field of "smart" drug delivery.     

Superoxide anion scavenging activity of graft chitosan derivatives

Two kinds of graft chitosan derivatives (CMCTS-g-MAS and HPCTS-g-MAS) were prepared by the graft copolymerization of maleic acid sodium onto etherified chitosans-carboxymethyl chitosan (CMCTS) and hydroxypropyl chitosan (HPCTS), respectively. Superoxide anion scavenging activity of the derivatives was evaluated in a luminal-enhanced autoxidaton of pyrogallol by chemiluminescence techniques. Compared with chitosan, the graft chitosan derivatives have much improved scavenging ability against superoxide anion. They have similar 50% inhibition concentrations (IC₅₀s) as ascorbic acid and superoxide dismutase (SOD). Graft chitosan derivatives with hydroxypropyl groups have relatively higher superoxide anion scavenging ability owing to the incorporation of hydroxyl groups. The graft chitosan derivatives (HPCTS-g-MAS 1, 2, and 3) with different grafting percentages exhibit IC₅₀s values ranging from 243 to 308 μg/mL, which could be related to the contents of active hydroxyl and amino groups in the polymer chains.     

Preparation, characterization, and in vitro drug release behavior of biodegradable chitosan-graft-poly(1, 4-dioxan-2-one) copolymer

A novel copolymer of chitosan-g-poly(p-dioxanone) (CGP) was synthesized in bulk by ring-opening polymerization of p-dioxanone (PDO) initiated by the hydroxyl group or amino group of chitosan using SnOct₂ as catalyst. The chemical structure was determined by ¹H NMR. It was found that the feed ratio of chitosan to PDO had a great effect on the degree of polymerization (DP) and the substitution (DS) of PDO. The thermal stability and crystallization behavior of graft copolymer CGP were closely related to the values of DP and DS. When the resulting copolymer was used as Ibuprofen carrier, the release rate of Ibuprofen decreased compared with that of pure chitosan carrier. The drug release behavior was also influenced by the structure of graft copolymers.     

低聚壳聚糖与α-丙氨酸/天冬酰胺的美拉德反应及其衍生物的抗氧化性能研究

研究低聚壳聚糖(COS)与α-丙氨酸/天冬酰胺的美拉德反应,考察了两个体系(低聚壳聚糖的羰基与氨基的物质量比均为1∶1)的pH、吸光度和荧光值的变化。醇沉法提取低聚壳聚糖衍生物CA和CN。对两种衍生物进行红外表征和分子量测定,并研究其对超氧阳离子O2-.、DPPH自由基的清除能力以及还原能力。结果显示:抗氧化能力强弱次序为CA>CN>COS,即美拉德反应后低聚壳聚糖衍生物抗氧化能力得到显著提高,且CA的抗氧化活性优于CN,表明与小分子氨基酸进行美拉德反应制得的壳聚糖衍生物具有更好的抗氧化性。     

Synthesis and bioactivities of poly(ethylene glycol)–chitosan hybrids

Poly(ethylene glycol)–chitosan hybrids of various molecular weights having different degree of substitution were synthesized, by reductive N-alkylation of chitosan with poly(ethylene glycol) aldehyde, to study their bioactivities. The influence of these chitosan derivatives on the reactive oxygen species generation from canine polymorphonuclear leukocyte cells was investigated in vitro by chemiluminescence response. Reactive oxygen species generation by the influence of poly(ethylene glycol)–chitosan hybrids was decreased with the increase of degree of substitution. The reduction of interaction of poly(ethylene glycol)–chitosan hybrids with polymorphonuclear leukocyte cells might be caused by the decrease of amino group in chitosan main chain and increase of the steric hindrance by poly(ethylene glycol) chain. The influence of the poly(ethylene glycol)–chitosan hybrids on complement component C3 activation was investigated by single radial immunodiffusion method. Influence on complement component C3 activation by poly(ethylene glycol)–chitosan hybrids was almost same as chitosan.     

Synthesis and self-assembly of chitosan-based copolymer with a pair of hydrophobic/hydrophilic grafts of polycaprolactone and poly(ethylene glycol)

Chitosan-based copolymers with binary grafts of hydrophobic polycaprolactone and hydrophilic poly(ethylene glycol) (CS-g-PCL&PEG) were prepared by a homogeneous coupling reaction of phthaloyl-protected chitosan with functional PCL-COOH and PEG-COOH, following deprotection to regenerate free amino groups back to chitosan backbone. They were characterized by ¹H NMR, Fourier transform infrared and X-ray diffraction analysis. These CS-g-PCL&PEG copolymers could form nano-size self-aggregates in acidic aqueous solution without a specific processing technique, which were investigated using dynamic light scattering and transmission electron microscopy. The formed self-aggregates become smaller with weakened stability upon pH increasing. Moreover, the aggregates of copolymer with higher content of PEG and PCL grafts could remain stable for over 30 days in both acid and neutral condition. A possible mechanism was proposed for the formation of self-aggregates from CS-g-PCL&PEG and their structural changes as pH. It is warranted to find promising application of these self-aggregates based on chitosan as drug carriers.     

Chitosan-graft poly(p-dioxanone) copolymers: preparation, characterization, and properties

A new biodegradable copolymer of chitosan and poly(p-dioxanone) (PPDO) was prepared through a protection-graft-deprotection procedure using N-phthaloyl-chitosan as an intermediate. PPDO terminated with the isocyanate group was allowed to react with hydroxyl groups of the N-phthaloyl-protected chitosan, and then the phthaloyl group was cleaved to give the free amino groups. The length of PPDO graft chains can be controlled easily by using the prepolymers of PPDO with different molecular weights. The resulting products were thoroughly characterized with FT-IR, ¹H NMR, TG, DSC, SEM, and WAXD. The copolymers were used as drug carriers for sinomenine (7,8-didehydro-4-hydroxy-3,7-dimethoxy-17-methyl-9α,13α,14α-morphinan-6-one) and these exhibited a significant controlled drug-releasing behavior whether in artificial gastric juice or in neutral phosphate buffer solution.     

Thiolation of chitosan. Attachment of proteins via thioether formation

Chitosan has a variety of biological functions through conjugating of other compounds to their amino and hydroxyl groups. To further expand applicability of chitosan, we have modified the amino group of chitosan with 2-iminothiolane to bestow thiol groups and obtained about 20% yield, which is equivalent to 913 microequiv SH/g chitosan or 457 nequiv SH/nmol chitosan. Bovine serum albumin (BSA) was reacted with N-(epsilon-maleimidocaproyloxy)sulfosuccinimide ester (sulfo-EMCS), and maleimide-modified BSA (MalN-BSA) was obtained. The yield of sulfo-EMCS addition was 12.8-36.8 mol MalN/mol BSA. When the chitosan-SH was reacted with MalN-BSA via thioether, 97.8% of the maleimide group was reacted, and 37.2% of the SH group was consumed. The remaining SH group was quenched by bromoacetamide. This is the first report of covalent conjugation of a protein to chitosan. Our method should find many applications in developing new chitosan-based biomedical materials containing other components such as growth factors and cell adhesion molecules, known to be crucial to cells. Our thiolated chitosan will facilitate conjugation of such biomedical components to provide new types of materials for tissue engineering.     

Synthesis of methylated chitosan containing aromatic moieties: Chemoselectivity and effect on molecular weight

N-Arylated chitosans were synthesized via Schiff bases formed by the reaction between the primary amino group of chitosan with aromatic aldehydes followed by reduction of the Schiff base intermediates with sodium cyanoborohydride. Treatment of chitosan containing N,N-dimethylaminobenzyl and N-pyridylmethyl substituents with iodomethane under basic conditions led to quaternized N-(4-N,N-dimethylaminobenzyl) chitosan and quaternized N-(4-pyridylmethyl) chitosan. Methylation occurred at either N,N-dimethylaminobenzyl and N-pyridylmethyl groups before the residual primary amino groups of chitosan GlcN units were substituted. The total degree of quaternization of each chitosan varied depending on the extent of N-substitution (ES) and the sodium hydroxide concentration used in methylation. Increasing ES increased the total degree of quaternization but reduced attack at the GlcN units. N,N-dimethylation and N-methylation at the primary amino group of chitosan decreased at higher ES’s. Higher total degrees of quaternization and degrees of O-methylation resulted when higher concentrations of sodium hydroxide were used. The molecular weight of chitosan before and after methylation was determined by gel permeation chromatography under mild acidic condition. The methylation of the N,N-dimethylaminobenzyl derivative with iodomethane was accompanied by numerous backbone cleavages and a concomitant reduction in the molecular weight of the methylated product was observed. The antibacterial activity of water-soluble methylated chitosan derivatives was determined using Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria; minimum inhibitory concentrations (MIC) of these derivatives ranged from 32 to 128 μg/mL. The presence of the N,N-dimethylaminobenzyl and N-pyridylmethyl substituents on chitosan backbone after methylation did not enhance the antibacterial activity against S. aureus. However, N-(4-N,N-dimethylaminobenzyl) chitosan with degree of quaternization at the aromatic substituent and the primary amino group of chitosan of 17% and 16–30%, respectively, exhibited a slightly increased antibacterial activity against E. coli.     

Antioxidant activity of water-soluble chitosan derivatives.

Water-soluble chitosan derivatives were prepared by graft copolymerization of maleic acid sodium onto hydroxypropyl chitosan and carboxymethyl chitosan sodium. Their scavenging activities against hydroxyl radical *OH were investigated by chemiluminescence technique. They exhibit IC(50) values ranging from 246 to 498 microg/mL, which should be attributed to their different contents of hydroxyl and amino groups and different substituting groups.     

Facile preparation of biodegradable chitosan derivative having poly(butylene glycol adipate) side chains

Various modes are being explored for the construction of functional materials from nanoparticles. Despite these efforts, the assembly of nanoparticles remains challenging with respect to the requirement of multiple component organization on varying dimensions and length scales. The graft copolymers of chitosan with poly(butylene glycol adipate) (PBGA) were prepared due to the esterification reaction between PBGA and 6-O-succinate-N-phthaloyl-chitosan (PHCSSA) in the presence of toluene as a swelling agent. The graft copolymers are nanoparticles with the size of few hundred nanometers as observed from TEM. It is a potential method to combine chitosan with the hydrophobic synthetic polymers. The grafting reactions were conducted with various PBGA/PHCSSA feed ratios to obtain chitosan-g-PBGA copolymers with various PBGA contents. FT-IR, NMR, XRD, spectrofluorophotometer, and TEM were detected to characterize the copolymers.     

Synthesis of chitosan–polycaprolactone blend for control delivery of ofloxacin drug

In the present research work chitosan has been blended with different amounts of polycaprolactone (PCL) (80:20, 75:25, 60:40 and 50:50) for using them for control delivery of ofloxacin. The blends were characterized by Fourier transmission infra red spectroscopy (FTIR), UV–visible spectroscopy (UV), scanning electron microscopy (SEM), X-ray diffraction (XRD) analysis. From the FTIR spectra the various groups present in chitosan and PCL blend were monitored. The homogeneity, morphology and crystallinity of the blends were ascertained from SEM and XRD data, respectively. The swelling studies have been measured at different drug loading. The kinetics of the drug delivery system has been systematically studied. Drug release kinetics was analyzed by plotting the cumulative release data vs. time by fitting to an exponential equation which indicated the non-Fickian type of kinetics. The drug release was investigated at different pH medium and it was found that the drug release depends upon the pH medium as well as the nature of matrix.     

Galactosylated derivatives of low-molecular-weight chitosan: production and properties

Chitosan, a binary heteropolysaccharide consisting of 2-acetamide-2-deoxy-beta-D-glucopyranose and 2-amino-2-deoxy-beta-D-glucopyranose residues linked in different proportions via beta-glycosidic bonds. The presence of a primary amino group in the chitosan structure allows for the synthesis of various derivatives. The procedure of obtaining activated N-hydroxysuccinimide esters with the use of lactobionic acid was applied to obtain galactosylated derivatives of low-molecular-weight chitosan with a substitution degree varying from 8 to 23%. The properties of these derivatives (viscosity, solubility, and biodegradability) were studied. These derivatives are well soluble at pH values greater than the acidity constant of amino groups of chitosan (6.5). Broadening the pH range towards increase and the presence of galactose residues allows these derivatives to be used in working with biological objects.     

Synthesis and characterization of sugar-bearing chitosan derivatives: aqueous solubility and biodegradability

The extended use of chitosan in biomedical fields has been limited by its insoluble nature in a biological solution. To endow the water solubility in a broad range of pH, chitosan derivatives were prepared by the covalent attachment of a hydrophilic sugar moiety, gluconic acid, through the formation of an amide bond. These sugar-bearing chitosans (SBCs) were further modified by the N-acetylation in an alcoholic aqueous solution. Thereafter, the effect of the gluconyl group and the degree of N-acetylation (DA) on the water solubility at different pHs and on the biodegradability of chitosan were investigated. The SBCs showed the water solubility in a broader range of pH than chitosan, whereas they were still insoluble at neutral and alkali pH. The N-acetylation of SBCs significantly affected the water solubility, for example, the SBCs with the DA, ranging from 29% to 63%, were soluble in the whole range of pH. This might result from the improved hydrophilicity by the gluconyl group, accompanied by the role of the N-acetyl group that disturbed the hydrogen bonding between amino groups of chitosan. From the biodegradation tests, determined by the decrease in the viscosity of a polymer solution exposed to lysozyme, it was evident that the gluconyl group attached to chitosan improved the biodegradability. Thus, it was possible to control the biodegradability of chitosan by adjusting the amounts of gluconyl and N-acetyl groups in the chitosan backbone. The N-acetylated SBCs, soluble in the broad range of pH, might be useful for various biomedical applications.     

Visualization of the Penetrative and Mucoadhesive Properties of Chitosan and Chitosan-Coated Liposomes Through the Rat Intestine

The objectives of this study were to observe the penetrative and mucoadhesive behavior of polymer-coated liposomes into the intestinal mucosa of rats. Chitosan (CS) and negatively charged liposomes were chosen as model polymer-coated liposomes. In order to observe their behavior, chitosan was labeled with Fluorescence Isothiocyanate (FITC) via chemical reaction at the isothiocyanate group of FITC and the primary amino group of chitosan; the liposomes (Lips) were marked by incorporation of DiI into the liposomal formulation. FITC-labeled chitosan (FITC-CS), Non-Lips, and FITC-labeled CS-coated Liposomes (FITC-CS-Lips) were intragastrically administered into male Wistar rats, and the behavior of the molecules was subsequently visualized by CLSM (Confocal Laser Scanning Microscopy). The results demonstrated that the chitosan molecules themselves, as well as the liposomes, could penetrate across the intestinal mucosa. Moreover, the CLSM images demonstrated a lack of separation of the chitosan molecules from the surface of the liposomes after the administration of chitosan-coated liposomes.     

Visualization of the penetrative and mucoadhesive properties of chitosan and chitosan-coated liposomes through the rat intestine

The objectives of this study were to observe the penetrative and mucoadhesive behavior of polymer-coated liposomes into the intestinal mucosa of rats. Chitosan (CS) and negatively charged liposomes were chosen as model polymer-coated liposomes. In order to observe their behavior, chitosan was labeled with Fluorescence Isothiocyanate (FITC) via chemical reaction at the isothiocyanate group of FITC and the primary amino group of chitosan; the liposomes (Lips) were marked by incorporation of DiI into the liposomal formulation. FITC-labeled chitosan (FITC-CS), Non-Lips, and FITC-labeled CS-coated Liposomes (FITC-CS-Lips) were intragastrically administered into male Wistar rats, and the behavior of the molecules was subsequently visualized by CLSM (Confocal Laser Scanning Microscopy). The results demonstrated that the chitosan molecules themselves, as well as the liposomes, could penetrate across the intestinal mucosa. Moreover, the CLSM images demonstrated a lack of separation of the chitosan molecules from the surface of the liposomes after the administration of chitosan-coated liposomes.     

Synthesis and characterization of the biodegradable polycaprolactone-graft-chitosan amphiphilic copolymers

A novel synthetic approach to biodegradable amphiphilic copolymers based on poly (epsilon-caprolactone) (PCL) and chitosan was presented, and the prepared copolymers were used to prepare nanoparticles successfully. The PCL-graft-chitosan copolymers were synthesized by coupling the hydroxyl end-groups on preformed PCL chains and the amino groups present on 6-O-triphenylmethyl chitosan and by removing the protective 6-O-triphenylmethyl groups in acidic aqueous solution. The PCL content in the copolymers can be controlled in the range of 10-90 wt %. The graft copolymers were thoroughly characterized by 1H NMR, 13C NMR, FT-IR and DSC. The nanoparticles made from the graft copolymers were investigated by 1H NMR, DLS, AFM and SEM measurements. It was found that the copolymers could form spherical or elliptic nanoparticles in water. The amount of available primary amines on the surface of the prepared nanoparticles was evaluated by ninhydrin assay, and it can be controlled by the grafting degree of PCL.     

Synthesis and properties of temperature-responsive chitosan by controlled free radical polymerization with chitosan-RAFT agent

The temperature-responsive chitosan was synthesized by free radical polymerization of N-isopropylacrylamide (NIPAM) at 60 degrees C in the presence of RAFT-chitosan agent. The chitosan was subsequently modified with phthalic anhydride and with S-1-dodecyl-S'-(alpha,alpha'-dimethyl-alpha'-acetic acid) trithiocarbonate to serve as reversible addition fragmentation chain transfer (RAFT) agent. The polymerization results show that the graft polymerization proceeded via RAFT process, while the "well-defined" graft polymers were successfully synthesized. The temperature played an important role on the self-assembly in H2O dispersion and the morphologies of chitosan-g-PNIPAMs. To our knowledge, this is the first thermosensitive chitosan prepared from controlled graft modification of chitosan by RAFT polymerization.     

壳聚糖中胺基对其抑菌性能的影响及与DNA的作用

采用抑菌圈法研究了壳聚糖对大肠杆菌(E.coli)和金黄色葡萄球菌(St.aureus)的抑菌活性。利用壳聚糖的席夫碱反应,对壳聚糖的胺基进行保护后,研究了壳聚糖中胺基对其抑菌性能的影响。同时,运用紫外吸收光谱和电化学的方法,研究了壳聚糖与DNA的相互作用,提出了壳聚糖对E.coli和St.aureus的抑菌机理。研究结果表明,壳聚糖对E.coli和St.aureus具有很好的抑制作用,且抑菌活性与其胺基有关;壳聚糖能与细胞内带负电的核酸结合,使细胞正常DNA复制生理功能受到影响,抑制细菌的繁殖,从而达到抑菌的目的。