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Selective The Cholesteric Chitosan Derivatives Nano Drug Carriers

Posted on:2012-02-08Degree:DoctorType:Dissertation
Country:ChinaCandidate:M M ChenFull Text:PDF
GTID:1114330335482169Subject:Biomedical engineering
Abstract/Summary:PDF Full Text Request
Chitosan is a cationic polysaccharide with outstanding biological properties such as biocompatibility, biodegradability, non-toxicity and antimicrobial activity. In recent years, much attention has been paid to hydrophobically modified chitosan as carriers for drug delivery, because they can spontaneously form nanoparticles with an inner hydrophobic core and an outer shell of hydrophilic groups in aqueous solution. In this study, 6-0-cholesterol modified chitosan (0-CHCS) conjugates with succinyl linkages were synthesized and its self-aggregated nanoparticles were prepared. This novel nanoparticle system may be used as a carrier for hydrophobic drug or DNA. The main contents of this work are as follows:6-O-cholesterol modified chitosan (O-CHCS) conjugates with succinyl linkages were synthesized through a protection-graft-deprotection method with phthaloylchitosan as an intermediate. O-CHCS conjugates were characterized by fourier transform infrared spectroscopy (FT-IR), proton nuclear magnetic resonance (1H NMR) and differential scanning calorimetry(DSC), and the degrees of substitution (DS) of the cholesterol moiety determined by elemental analysis were 1.7%,4.0% or 5.9%. O-CHCS self-aggregated nanoparticles prepared by the dialysis method displayed the classic "core-shell" structures and their sizes were in the range of 101.9-238.5 nm. The self-aggregation property of O-CHCS and its critical micelle concentration (CMC) were determined using fluorescence spectroscopy with pyrene as a fluorescent probe. The CMC values of O-CHCS in aqueous solution were in the range of 0.0072-0.0175 mg/mL. The size and the CMC values decreased with DS of cholesterol moiety increasing.All-trans retinoic acid (ATRA) and doxorubicin (DOX), as the model drugs, were physically entrapped inside O-CHCS self-aggregated nanoparticles by the dialysis method. With increasing initial levels of the drug, the drug loading content increased, but the encapsulation efficiency decreased. The release profiles in vitro demonstrated that ATRA or DOX showed slow sustained released over 72 h, which indicated that O-CHCS nanoparticles had the potential to be used as a carrier for hydrophobic drugs. The cellular cytotoxicity and cellular uptake were accessed by using human cervical carcinoma (HeLa) cells. The blank O-CHCS nanoparticles did not show any cytotoxic effect on HeLa cells even at the concentration of 200μg/mL. However, The DOX-loaded O-CHCS nanoparticles showed anti-tumor activity. Flow cytometry and confocal image analysis revealed that free DOX appeared much easier cell endocytic than DOX-loaded O-CHCS nanoparticles, and free DOX was mainly distributed in the nucleus, while the DOX-loaded nanoparticles were in the cytoplasm.The interaction between O-CHCS nanoparticles and bovine serum albumin (BSA) was investigated by spectroscopy and calorimetric methods. The morphology of the complex between O-CHCS nanoparticles and BSA observed by transmission electron microscope (TEM) was almost spherical shape. The size and the zeta potential of the complex increased with the concentration of O-CHCS nanoparticles increasing. The release profiles in vitro showed a minor change after interacted with BSA at a low concentration, and released slower with BSA at a high concentration. The results of fluorescence and UV spectroscopy, CD and ITC indicated that the conformation of BSA was not significantly changed in the interaction process, and the binding force between O-CHCS nanoparticles and BSA was mainly driven by hydrogen bonding, hydrophobic and electrostatic interactions. Therefore, O-CHCS nanoparticles would have the potential applications as a novel carrier for drugs due to non-toxic effect on proteins in vivo.pCMV-hBD2 plasmid was introduced to mouse fibroblast cells (L929) by O-CHCS nanoparticles as a gene vector for humanβ-defensin-2 (hBD2). The expression of hBD2 was verified by RT-PCR and western blot, respectively. The antimicrobial activity of recombinant hBD2 was investigated using K-B disc agar diffusion test. The results indicated that hBD2 was effectively expressed both in the transcriptional and protein level. The antimicrobial activity results showed that the supernatant of transcription cells had obvious antimicrobial effects toward Staphylococcus aureus. It indicated that O-CHCS nanoparticles could be a gene vector for hBD2, which provided an economic and convenient way for gene transcription.In conclusion, O-CHCS, a novel polymeric amphiphile, could form nanoparticles by self-assembly manner in aqueous solution. O-CHCS nanoparticle system would have the potential to be used as a novel carrier of hydrophobic drugs or DNA due to biocompatibility.
Keywords/Search Tags:Chitosan, Cholesterol, Phthaloylchitosan, Self-aggregated nanoparticles, Drug delivery system
PDF Full Text Request
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