Study On Sulfated Chitosan Derivative Hybrid Material And Nano - Drug Carrier

Chitosan is a cationic polysaccharide with outstanding biological properties such as biocompatibility, biodegradability, non-toxicity and antimicrobial activity, low immunogenicity, stability. In recent years, much attention has been paid to hydrophllically modified chitosan as carriers for drug delivery, because they can spontaneously for nanoparticles with an inner hydrophobic core and an outer shell of hydrophilic groups in aqueous solution. Expecially, the nanoparticles can be used to build hybrid nanoparticles and also can be used as a carrier of anticancer drugs attracted the interest of researchers.In this study, PEG and PEI modified chitosan(CS-PEG-PEI) were synthesized and its self-aggregated nanoparticles were prepared. This novel nanoparticle system may be used as a carrier for hydrophobic drug and may be also used to build hybrid nanoparticles with low toxicity and biocompatibility. The main contents of this work are as follows:1. The chitosan derivatives were designed and synthesized with different substitution degree of PEG and PEI. First, double carboxy-terminated PEG was synthesized by esterification of poly(ethylene) glycol (PEG) and succinic acid anhydride (SA). Second, partly thioled polyethylenimine was prepared by branched polyethylenimine (PEI) and methyl thioglycolate. The degrees of substitution of the thiol moiety were determined by Ellman’s method. A modified version of the NHS/EDC coupling approach was used to synthesize PEG2000-g-chitosan conjugate and PEI-PEG-g-chitosan conjugate. The structures of PEG2000-g-chitosan and PEI-PEG-g-chitosan were characterized by1H NMR and FT-IR spectra. The degrees of substitution of the PEG or PEI moiety were influenced by the molar ratio.2. CS-PEG-PEI self-aggregated nanoparticles were prepared by the dialysis method and displayed the classic "core-shell" structure. The nanoparticles were characterized by transmission electron microscope (TEM), scanning electron microscope (SEM) and dynamic laser particle size analyzer (DLLS) morphology. The results exhibited that the structure of nanoparticles were into spherical and diameters of nanoparticles were in the range of157.9-233.1nm. The chitosan derivatives with PEG and substitution degree was24%and3.4%were choose for further studies due to the physical and chemical properties of nanoparticles.3. GNRs were synthesized through seed mediated growth procedure capped by CTAB bilayeron the surface, and characterized by UV-vis spectra and TEM. The chitosan-modified-GNRs nanoparticles (CS-GNRs) were prepared by two methods based on Au-S bond as follow:(1) The mixture of GNRs and chitosan derivatives was treated by ultrasonic probe, and CS-GNRs with uniform spherical structure exhibitted well dispersion. GNRs were embedded on the surface of nanoparticles.(2) CS-GNRs were also obtained only by stirred the mixture of GNRs and chitosan derivatives was stirred24h, and the structure of nanoparticles were similar as above nanoparticles. However, the number of GNRs of each nanoparticle was less than above nanoparticles. In a word, the first method was chosen to prepare CS-GNRs due to the stability, size and combination property. In in vitro studies, crude GNRs affected the metabolic activity of cells even at low concentration. CS-GNRs exhibited low cytotoxicity. These results suggest that CS derivatives can improve GNRs biocompatibility.4. Doxorubicin (DOX), as the model drugs, was physically entrapped inside CS-PEG-PEI self-aggregated nanopartieles by the dialysis method. The properties and in vitro drug release behavior of DOX-loaded CS-PEG-PEI nanoparticles (DOX-CS) were studied. And the results showed that, with the initial levels of the drug increasing, the drug loading content of DOX-CS spherical nanoparticles inereased, but the encapsulation effiency decreased. The release profiles in vitro demonstrated that DOX showed slow sustained released over72h, which indicated that CS-PEG-PEI nanopartieles had the potential to be used as a carrier for hydrophobic drugs. The cellular cytotoxicity and cellular uptake of CS-PEG-PEI nanopartieles and DOX-CS were studied and the results shown that CS-PEG-PEI nanoparticles exhibited very low cytotoxicity on HeLa cells even at the concentration of200μg/mL. However, The DOX-CS nanoparticles showed anti-tumor activity. Flow cytometry revealed that DOX-CS appeared much easier cell endocytic than free DOX, and confocal image analysis obtained that free DOX was mainly distributed in the nucleus, while DOX-CS were in the cytoplasm.