Preparation Of A Hepatic Targeting Galactosylated Chitosan Nanoparticle Loaded Lactosyl-norcantharidin.

Objective Asialoglycoproteins receptor (ASGP-R), expressed plentifully on the surface of hepatoma cells, can recognize terminalβ-D-galactose or N-acetylgalactosamineresidues. A hepatic targeting nanopartical was prepared using GC and Lac-NCTD to obtain active liver-targeting in addition to passive liver-targeting. Metabolism in rat liver microsomes of Lac-NCTD, which had not been researched extensively as a new derivative of norcantharidin, was investigated initially .Methods (1)Lac-NCTD GC NPs was obtained by ionic cross-linkage process and orthogonal design was used to optimize the formulation and preparation procedure with the particle size, PDI , entrapment efficiency and drug-loaded amount. (2) FT-IR, X-ray diffraction, DSC, TEM, etc. were used to evaluate the characteristics of Lac-NCTD GC NPs ; in vitro drug release of nanoparticles investigated by dialysis membrane. (3) In vivo anti-tumor activity of Lac-NCTD GC-NPs was evaluated in mice bearing H22 hepatoblastoma tumor with neoplasm volume, inhibition rate on tumor weight and Pathology characteristic photose. (4) IC50 of Lac-NCTD to SMMC-7721 and HepG 2 was studied by MTT Assay in order to provide basisi of uptake dosage. (5) HPLC was used to research the uptake of Lac-NCTD and nanoparticles in SMMC 7721 and HepG 2. (6) The rat liver microsomes was prepared by differential centrifugation technique and the enzyme kinetics parameters were calculated by Lineweave-Brurk. (7) Experiments incubated with inhibitors including Ketoconazole(CYP3A), Quinidine(CYP2D6), Phenacetin(CYP1A2) and 4-methylpyridine(CYP2E1) were carried out to investigate which subfamily of cytochrome P450 play important roles in Lac-NCTD metabolism.Results (1) The optimal preparation condition was as follows: GC concentrations was 2.5 mg·mL^-1, Lac-NCTD:TPP was 1:4(m/m) and the temperature was 30℃. The average particle size of the nanoparticles were (142.22±5.20)%, PDI was (0.121±0.049), entrapment rate was (73.82±0.51)%, drug-loading rate was (13.40±0.08)%. (2) The nanoparticles appear uniform and round under TEM and drug release followed Higuchi equation. (3) Among the treatment groups, both Lac-NCTD and Lac-NCTD-GC NPs were more powerful against the tumours than NCTD, and Lac-NCTD-GC NPs were the most powerful of all [28]. It was indicated that the Lac-NCTD entrapped in galactosylated nanoparticles administered in vivo may be able to display active and passive liver-targeting characteristics. (4) The uptake of Lac-NCTD-CS NPs indicated that adsorptive endocytosis existed as a nonspecific interaction of the cytomembrane compared with that of Lac-NCTD. The cellular uptake of Lac-NCTD-GC NPs showed that receptor-mediated endocytosis was generated by ligand binding to galactose receptors on hepatocytes. (5) Lac-NCTD-GC NPs had lower Vmax , Km and CLint than Lac-NCTD, indicating that NPs could step down the motebolism of Lac-NCTD in rat liver microsomes. It was suggested NPs could improve bioavailability of Lac-NCTD due to delaying the accretion rate. (6) In presence of Ketconazole the metabolism was inhibited significantly while other inhibitors had no inhibitory action.Conclusions The Lac-NCTD GC NPs appear uniform and round under TEM and drug release followed Higuchi equation. Compared with Lac-NCTD-CS NPs and Lac-NCTD , Lac-NCTD-GC NPs displayed active liver-targeting characteristics in addition to passive liver-targeting, more satisfactory compatibility with hepatoma cells and significant antitumor effect in hepatoma-bearing mice. Lac-NCTD-GC NPs could improve bioavailability of Lac-NCTD due to delaying the accretion rate, and Lac-NCTD is likely to be metabolism by CYP3A of rat liver microsomes, but further studies also should be taken on metabolism.