New The Biocompatibility Of The Chitosan Derivatives Multifunctional Drug Delivery Carrier

Chitosan is a natural aminopolysaccharide possessing useful characteristics such as biocompatibility and biodegradation, which can be further chemically modified to develop chitosan derivatives as promising drug carriers due to high density of reactive amino and hydroxyl groups. Considering their potential application, it highlights the need to perform the biocompatibility evaluation to insure their safety in use, since foreign materials may cause toxic effects, inflammation or immune reaction.In the present study, linoleic acid and poly(/?-malic acid) (PMLA) double grafted chitosan (LMC), trimethyl chitosan (TMC), and poly(ethylene glycol)-graft-trimethyl chitosan block copolymers (PEG-TMC) were investigated in the biocompatibility.1 Biocompatibility of LMC nanoparticlesBiocompatibility of LMC nanoparticles (NPs) was explored at molecular, cellular, tissue and animal levels in terms of blood compatibility, cytotoxicity, genotoxicity and acute toxicity (i.v. and i.p.). Residual solvents were analyzed by gas chromatography-mass spectrography (GC-MS) analysis to insure the exactness of biocompatibility evaluation.As an amphiphilic chitosan derivative, LMC can form NPs in aqueous environment, which exhibited lower contact angle than that of chitosan due to the grafting of hydrophilic PMLA chains. Results of hemolysis test, as well as prolonged plasma recalcification time and improved anticoagulation potential suggested that LMC NPs displayed good blood compatibility. A battery of cytotoxicity assays, including "FDA/PI" staining, LDH, neutral red, protein content and MTT assays, were performed on Chinese hamster ovary cells (CHO) and human hepatocarcinoma cell line (SMMC-7721), suggesting LMC NPs (0.05-2 mg/mL) induced minimal loss of cell membrane integrity, cell viability and metabolic activities. The rise in cellular malondialdehyde content and depletion of glutathione showed slight oxidative stress insult. However, flow cytometry assay, comet assay in vitro and in vivo and the bone marrow micronucleus assay in mice indicated that LMC NPs did not cause cell apoptosis, DNA damages or chromosome damage. In the acute toxicity study, no toxic signs were observed in mice following i.v. administration of LMC NPs at 500 mg/kg or i.p. administration of LMC NPs at 1250 mg/kg. The lysozyme-catalyzed degradation of LMC NPs was slightly lower than that of chitosan after 84-d incubation and the degradation products were no cytotoxicity. Contents of benzene, chloroform and toluene detected were 0.284,0.069 and 0.091 ppm, respectively, which were below the acceptable levels of residual solvents in pharmaceutics.2. Biocompatibility of TMC and PEG-TMCTwo partially quaternized derivatives of chitosan were synthesized, named TMC1 and TMC2, respectively. PEG-TMC was obtained by grafting 2 kDa and 5 kDa activated methoxypoly(ethylene glycol) (mPEG) onto TMC2 and was nominated as PEG2K-TMC and PEG5K-TMC.The elemental analysis results indicated that the N-substitution degree of TMC 1 and TMC2 were 23.7%and 49.5%, respectively. Results of XRD and DSC indicated that the structure of chitosan chains has been changed due to the introduction of N-trimethylaminoethylmethacrylate chloride and mPEG Contact angle assay demonstrated that the PEG moieties presented on the backbone of TMC could increase the hydrophilicity of the copolymer. TMC1 and TMC2 at 5 mg/mL exhibited the hemolysis ratio higher than 5%and the erythrocyte aggregation. No damage to the erythrocytes was induced by PEG-TMC. The longer PEG chains were attributed to the anticoagulation of the copolymers. MTT assay suggested a dose-dependent cytotoxic response in CHO and SMMC-7721 cells. The PEGylation can decrease the cytotoxicity of TMC2. The LDH leakage assay indicated that TMC could cause serious damage to the endothelial cell in intestinum angustum and the LDH leakage of PEG5K-TMC treatment group was similar to the negative control (saline), suggesting minimal damage to endothelial cells. The grafting of quaternary ammonium salt increased bovine serum albumin (BSA) adsorption of chitosan while PEGylation dramatically decreased BSA adsorption of TMC2. TMC and PEG-TMC exhibited a concentration-dependent manner in antioxidative ability. Higher degree of ^/-substitution or molecular weight of PEG could improve hydroxyl radicals scavenging activity. PEGylation would decrease the reducing power and lipid peroxidation inhibition in rat liver hemogenate of TMC2. The degradation products had no cytotoxicity following 84-d lysozyme degradation.