Preparation Of A Novel Liver-targeting Nanoparticles Of Norcantharidin Derivative

Objective: Norcantharidin (NCTD) against primary carcinoma of liver was lactosylated to obtain lactosyl-norcantharidin(Lac-NCTD)as initiative derivative, based on the asialoglycoprotein receptor (ASGP-R), situated on the hepatocyte membrane, can recognize the galactose residue of drug, then the safety of Lac-NCTD was preliminarily evaluated. Lac-NCTD-NPs were prepared and their anti-tumor activity in vivo and in vitro were evaluated. As a result, the safe and effective nanoparticles loaded norcantharidin derivative, which combine both active and passive liver-targeting were achieved.Methods: (1) Lac-NCTD was synthesized using ethanediamine as a truss arm and its physicochemical properties such as solubility, oil/water partition coefficient, stability, etc. were determined. (2) MTD for Lac-NCTD was calculated by acu-toxicity test by intraperitoneal injection. (3) Lac-NCTD-NPs were achieved by ionic cross-linkage process and orthogonal experiment design was applied to optimize the formulation and preparation procedure with particle distribution, entrapment rate and drug-loading rate as integrated indexes. (4) Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), X-ray diffraction, transmission electron microscopy (TEM), etc. were applied to evaluate nanoparticle apatial representable structure; the release in vitro was investigated by dialysis membrane. (5) MTT was used to study the cytotoxic effects of Lac-NCTD and Lac-NCTD-NPs on HepG2, SMMC-7721 and SGC-7901 and the inhibition effects of Gal-FBS. (6) Lac-NCTD accumulated in SMMC-7721 cells was established by high performance liquid chromatogram (HPLC). (7) The H22 hepatoblastoma cell line bearing mice was establishmented to evaluate the in vivo anti-tumor activity of Lac-NCTD and Lac-NCTD-NPs.Results: (1) Lac-NCTD was a kind of polyhydroxy compound and the yield of it was 68.35%. (2) Single and multiple dosing of MTD for compound Lac-NCTD exceeded 20 g?kg-1 and 30 g?kg-1 respectively,which exceed the amount of human clinical usage of NCTD fairly far. (3) The optimal preparation condition was as follows: CS concentrations was 2.0 mg?mL-1, drug concentrations was 2.0 mg?mL-1 and CS:TPP was 3:1(m/m). The average particle size of the prepared nanoparticles were (149.46±1.79) nm, entrapment rate was (80.29±0.56)%, drug-loading rate was (9.58±0.09)%. (4) In FT-IR,DSC and X-ray diffraction graph, the diagnostic absorption peak of LCS and TPP vanished or shifted. TEM graph showed that nanoparticles were sphere and distinct. The curve of the release in vitro was suitable for Higuch equation. (5) The in vitro studies showed that the cytotoxic effects of Lac-NCTD-NPs against HepG2, SMMC-7721 cells were the most powerful, then Lac-NCTD and they were inhibited remarkably by Gal-FBS; as for SGC-7901, the cytotoxic effects of Lac-NCTD-NPs and Lac-NCTD were not stronger than NCTD and Gal-FBS had no influence on them at all. (6) The amount of Lac-NCTD accumulated in SMMC-7721 cells was 3.89μg?106cell-1 after treatment for 12 h. (7) The results of the anti-tumor activity in vivo suggested that the tumor-growth of H22 were inhibited effectively by Lac-NCTD-NPs.Conclusions: (1) The synthesis procedure of Lac-NCTD was simple and the yield was high. It should be protected against the tide when stored because of its strong moisture absorption. (2) Acute toxicity test demonstrated the compound Lac-NCTD was safe. (3) The large scale hydroxy group on the new chemical, Lac-NCTD, could combine with the positive charge of chitosan, shaped in the diluted acid, through intermolecular hydrogen bond to form nanoparticles by the means of adsorbing and encapsulation. As a result, the entrapment rate of Lac-NCTD-NPs was significantly higher than the norcantharidin nanoparticles(NCTD-NPs)prepared in the same condition. (4) The results of FT-IR,DSC,X-ray diffraction and TEM graph showed that the nanoparticles were shaped by the linkage of tripolyphosphate acid radical and the -NH2+ on the surface of chitosan. Effect of sustained drug release of Lac-NCTD-NPs was evident. (5) The ASGP-R on the surface of HepG2 and SMMC-7721 can recognize the galactose residue of Lac-NCTD, therefore, the cytotoxic effects of Lac-NCTD and Lac-NCTD-NPs on these two cell lines were stronger than NCTD and they were inhibited significantly by Gal-FBS. (5) The results of Lac-NCTD accumulated in SMMC-7721 cells further indicated that Lac-NCTD can partly permeate the cell membrance with the prime type through the ASGP-R pathway. (6) Lac-NCTD-NPs combined both the active targeting of Lac-NCTD and passive targeting of nanoparticles itself and were recognized specially by ASGP-R on the hepatocyte surface to achive liver-targeting and show powerful anti-tumor activity in vivo.