Preparation And Characteristic Of Norcantharidin-associated Galactosylated Chitosan Nanoparticles

Objective: The aim in this study was to synthesize a novel chitosan derivative, galactosylated chitosan (GC), and prepare norcantharidin-associated galactosylated chitosan nanoparticles (NCTD-GC-NPs) with GC as hepatocyte-targeting carriers and norcantharidin (NCTD) as model drug against primary liver carcinoma. It was expected that NCTD-GC-NPs could be specifically recognized by asialoglycoprotein receptor (ASGP-R) of hepatocyte, and a dual hepatocyte-targeted delivery for NCTD, combine passitive targeting with active targeting, would be developed.Methods: (1) The deacetylation degree (D.D.) of chitosan was increased by intermittent hydrolysis method and GC was synthesized by carbodiimide condensation reaction; the chemical structure of GC was confirmed through FT-IR and 1H-NMR spectroscopy; the degree of substitution of galactose residues in GC was estimated by 1H-NMR and characteristic viscosity of GC was determined by using an Ubbelohde viscometer and a stopwatch; the characteristics of chitosan nanoparticles were determined by X-ray diffraction and DSC. (2) NCTD-GC-NPs were achieved by ionic cross-linkage process with GC as carrier; four factors (deacetylation degree of chitosan, pH, temperature, weight proportion of NCTD to GC) influencing the particle size, entrapment efficiency (E.E.), drug-loaded amount (D.L.) of NCTD-GC-NPs were constantly monitored; orthogonal design was used to optimize the preparation technique of NCTD-GC-NPs. (3) The characteristics of NCTD-GC-NPs were determined by FT-IR, X-ray diffraction, DSC, etc.; in vitro drug release of nanoparticles colloid and lyophilized powder were also investigated. (4) The various nanoparticles were fluorescence labeled with FITC so that the cellular uptake action could be detected by ?ow cytometry; in vitro anti-tumor activity of NCTD-GC-NPs was studied as cellular uptake and cytotoxicity on human hepatoma cell lines SMMC-7721 and HepG2 by flow cytometry and MTT Assay respectively. (5) In vivo anti-tumor activity of NCTD-GC-NPs was studied as tumor inhibitory activity in mice bearing H22 hepatoblastoma tumor, including neoplasm volume, inhibition on body weight, inhibition rate on tumor weight, immune organ coefficient; Pathology characteristic of stripped tumor was observed by biological microscope.Results: (1) D.D. of chitosan was increased to (93.06±2.38)%; the degree of substitution of galactose residues in GC estimated by 1H-NMR was about 8.92%; the characteristic viscosity of GC was 0.7809, and that of CS was 0.9558 contrastively, the declines of characteristic viscosity stemmed mainly from degradation of main chain on chitosan in the reaction; X-ray diffraction and DSC showed respectively that GC had satisfactory hydrophilicity and lower thermal stability. (2) It was thus evident in one-factor analysis that the chitosan D.D value and pH had valuable impacts on EE of nanoparticles, and no effect to the particle size, however, it was hard to raise EE of NCTD-CS-NPs further because of the pH contradiction between GC and NCTD; temperature had remarkable influence on particle size; weight proportion of NCTD to GC had an important effect on DL of NCTD-GC-NPs, and no conspicuous impact on particle size. Novel NCTD-GC-NPs were obtained by optimized preparation technique with 118.68±3.37nm of average particle size, 57.92±0.40% of entrapment efficiency and 10.38±0.06% of drug-loaded amount; most of the nanoparticles appear uniform and round under TEM, and the particle size was similar to the results of zeta-sizer nano particle analyzer. (3) In vitro NCTD release of nanoparticles had pH sensitivity characteristic: lower pH was favorable for drug release, contrastively release was more retardant at pH values near neutrality, and drug release followed Higuchi equation. (4) The excessive uptake of NCTD-CS-NPs by hepatoma cells compared with the control NCTD-GC-NPs could hold promise for the specific receptor-mediated cellular endocytosis of nanoparticles. Through galactosylated modification, novel nanoparticles were obtained with active liver-targeting characteristic and more satisfactory compatibility with hepatoma cells. Comparison with NCTD, the results of NCTD-GC-NPs displayed the highest inhibition level, next is NCTD-CS-NPs. The dosage form of galactosylated chitosan nanoparticles improved in vitro cytotoxicity activity of NCTD to hepatoma cells. (5) NCTD-GC-NPs improved in vivo tumor inhibitory activity of NCTD, reduced the side effect, and increased therapeutical ratio of NCTD in mice bearing H22 hepatoblastoma tumor.Conclusions: Optimized preparation technique of NCTD-GC-NPs was satisfied with the simple process, low cost, and stable reproducibility. The pH sensitivity and sustained drug release characteristic of NCTD-GC-NPs were showed in vitro, hepatocyte-targeting and anti-tumor activity of NCTD was improved both in vitro and in vivo. As dual hepatocyte-tageting carrier, novel galactosylated chitosan nanoparticles had wide perspective and practicability on the clinical application of norcantharidin.