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Study of the Uptake of Chitosan Oligosaccharide Nanoparticle Delivery

Author: WanLiQing
Tutor: HuFuQiang
School: Zhejiang University
Course: Microbial and Biochemical Pharmacy
Keywords: Chitooligosaccharides Nanoparticles Stearic acid Polymer micelles A549 cells Cytotoxicity Cellular uptake Endocytosis Mitomycin C
CLC: R96
Type: Master's thesis
Year: 2004
Downloads: 584
Quote: 1
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Abstract

Chitosan is a cationic polymer, by the composition of the glucose has good biocompatibility, low toxicity and biodegradability. Study found that chitosan and its derivatives such polymers has a protective effect the carrier prepared peptides, proteins, nucleic acid oral formulations of the drug has become the research focus of this class of drugs, biological macromolecular drugs in recent years and began to be used to carry the plasmid DNA in gene therapy research. The chitosan pKa value of approximately 6.5, the large molecular weight of chitosan in the physiological pH conditions (7.2 to 7.4) that does not dissolve; digestive tract also has no direct effect on the chitosan stars: the β glycosidic Degradation substructure chitosan enzyme, resulting in a large molecular weight chitosan as a drug carrier is difficult absorbed by the human digestive tract. Therefore, the study of low molecular weight chitosan (chitooligosaccharides, chitosan oligosaccharide, CSO) as a pharmaceutical carrier material has a very important significance. This topic obtained by hydrolysis and ultrafiltration separation techniques chitooligosaccharides amount of stars I was measured by gel permeation chromatography (gelpermeation chromatogram, GPC); solvent diffusion method Preparation of chitooligosaccharides nanoparticles by (CSO-NPs); with hard fatty acid (stearic acid, SA) modified chitooligosaccharides copolymer self-aggregation in water to form polymeric micelles of a few tens of nanometers in size (CSO-SA); determination of particle size and surface potential analyzer nanoparticles and polymer glue the particle size of the group, the surface potential (Zeta potential); mitomycin C (mitomycin C) as a model drug, the preparation of drug-loaded nanoparticles and micelles, the establishment of the the drug with Yuk determination, determination of drug encapsulation efficiency. The type Ⅱ A549 human lung epithelial cells as a model cell evaluation to nanocarrier contained cytotoxic drug nanoparticles and micelles. The CSO a PS and CSO-SA micelles labeled with the isothiocyanato fluorescein (fluorescein isothiocyanate, Thesis: of nanocarrier Oligochitosan cells uptake mechanism research FITC), used to study the mechanism of cellular uptake; inverted fluorescence microscope, fluorescence spectrophotometer, flow cytometry and laser scanning confocal microscopy and other methods of qualitative and quantitative study of A549 cells chitooligosaccharides nanoparticles, the uptake of polymer micelles, exploring nanoparticles, micelles cells The mechanism of uptake. Chitosan oligosaccharide obtained by hydrolysis and ultrafiltration separation techniques, gel permeation chromatography measured weight average molecular weight M, of 18.7K seven La, the molecular weight of the shell oligosaccharides can be dissolved in a PBS solution of physiological pH values. Chitooligosaccharides nanoparticles prepared by solvent diffusion method, the analysis of the particle size and surface potential measured by the number-average and volume average particle diameter were 133.3nm and 368.2nln and surface potential (Zeta potential) 53.9 Soil 3.1mV. To carbodiimides (l a-ethyl-3-(3-dimet field the lamin rapping ropyl) earbodiimide hydrochloride, EDC) as a crosslinking coupling agent in the main chain of the chitooligosaccharides grafted stearate formed chitooligosaccharides with hard The fatty acid of the copolymer. Determination of the degree of substitution, the proportion of different feeding chitooligosaccharides stearic acid grafted polymer, the actual is stearic acid substituted amino percentage were 13.08%, 7.48% and 4.20%, respectively. The polymer was a comb-shaped structure, with the amphiphilic hydrophobic chains as the core, and the hydrophilic chain is a particle diameter of the casing is several tens of nanometers (CSO SA) of polymer micelle formed spontaneously in the aqueous medium. Determination of the particle size and surface potential analyzer, to 1,10:1 and 20:1 ratio grafted stearate formed after CSO SA micelle number average particle size decreases with the grafting ratio of increase, respectively, 75.67nln, 53.glun 50.snln, surface potential were 49.2 Guests 1 .6 mV, 58.5 Guests disabilities l.omV 2.3 mV and 54.1. A549 lung epithelial cells as a model cell evaluation chitooligosaccharides nanoparticles (CSO PS), CSO-SA micelles cytotoxicity. The experimental results showed that the toxicity of the chitooligosaccharides nanoparticles cells were very low, Ie50 were 944.36 Lin g · mL '643.16 Lin g · mL'; grafting stearic eso sA micelles cells the toxicity increased slightly, 5:1, 10:1 and 20:1 ratio grafting stearic acid to form the xe50 eso a sA micelles value were 41 1.09 g · mL disabilities 43.29 Association, and 402.26 ± 22.05 09 · mL-'and 223.02 Guests 34.20 Association g · mL one. Is obtained by hydrolysis of the low molecular weight chitosan used in this study, the cell toxicity was significantly less than reported in the literature of high molecular weight chitosan; after nanoparticles formed by Oligochitosan and stearate modified chitooligosaccharides polymer micelle of Cell toxicity is also lower than other commonly used polycation (such as poly-L-lysine, poly-cationic carrier ICS. only 50 ± 10 Lin g · mL). Therefore, chitooligosaccharides the nanocarrier Thesis: the nanocarrier Oligochitosan cellular uptake mechanism of Hope safe biological macromolecular drug delivery system. Chitooligosaccharides preparation of drug-loaded nanoparticles and CSO-SA micelles as a model drug mitomycin C (mitomycinC, MMc) investigated the rate of drug-loaded nanocarrier. Drug-loaded nanoparticles drug encapsulation efficiency was 9832 Guests 2.15%; the stearic chitooligosaccharides grafting ratio of 5:1,10:1,20: l, the encapsulation efficiency of its corresponding drug-loaded micelles were 56.5,43.81 and 53.46%, respectively. A549 lung epithelial cells as a model cell, the evaluation containing the chitooligosaccharides nanoparticles mitomycin C and CSO-SA micelles cytotoxic. MTT assay determination of the MMc solution ICS. Value of 1.743 Tuo · mL one; the chitooligosaccharides nanoparticles wrapped the MMc cytotoxic decreases, the ICS. Value of 5.654 the female · mL A '; these three different proportion of stearic acid grafted close to the CSO a SA micelles wrapped MMC cytotoxicity with MMC solution, IC50 of 1 .543,1 .655 and 1.453, respectively. Lin g · mL l. Fluorescent labeling technology can be simple and rapid cellular uptake of qualitative and quantitative research. Using the fluorescent substance of the physical parcels method, there is a fluorescent substance is lost in the transfer process, resulting in a difficult to study the results of quantitative. Chemical coupling method of this experiment, the direct fluorescence of the carrier material, part of the free amino group on the main chain in the chitooligosaccharides (CSO) and the stearic acid modified chitooligosaccharides (CSO SA), and with FITC-isobutyl thiocyano addition reaction occurs, forming a CSO, and FITC cSO a SA and conjugate FITC, further preparation of the FITC-labeled the CSO a PS and CSO SA micelles, used to study the mechanism of cellular uptake of the two nano-carrier . An inverted microscope with fluorescent the chitooligosaccharides molecules, nanoparticles and CSO-SA micelles cellular uptake. Available from fluorescence photo the chitooligosaccharides nanoparticles and the CSO SA micelles in A549 cells?

Study of the Uptake of Chitosan Oligosaccharide Nanoparticle Delivery

Abstract

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