Research Of Loading-Norcantharidin Low Molecular Weight Chitosan Nanoparticles

Objective: To prepare nanoparticles with low particle size but high entrapment, using low molecular weight chitosan as carriers and Norcantharidin (NCTD) as model medicine against primary carcinoma of liver, based on the targeting distribution characteristic of nanoparticles with certain particle size. That would contribute to increase the liver target of NCTD and reduce its nephrotoxicity.Methods: (1) the NCTD detection method in vitro was established by High Performance Liquid Chromatogram (HPLC), and physicochemical properties such as solubility, hydrolytic substance, oil/water partition coefficient, etc. were determined. (2) The quality evaluation of nanoparticles was carried out, and after the preparation comparison, orthogonal design (OD) combining with multi-variable regression and artificial neural networks was applied to synchronously optimize two indexes (particle size and entrapment). (3) The surface characteristic of chitosan nanoparticles was determined by the modified alkalimetry, and Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), X-ray diffraction, etc. were applied to evaluate nanoparticle apatial representable structure; the release in vitro was investigated by dialysis membrane. (4) Chitosan nanoparticles loading-norcantharidin for oral administration was investigated according to the experiment of drug absorption via small intestine in vivo. (5) Pharmacokinetic characteristic of nanoparticles for intravenous administration was estimated through injection via mouse caudal vein.Results: (1) Norcantharidin was determined by HPLC as follows: twenty microliter sample was injected into a chromatograph (Shimadzu LC-10AT, Japan) equipped with a UV detector (Shimadzu SPD-M10A) and reversed phase column (Hypersil ODS2, 4.6×250mm, Elite, Dalian, China). The mobile phase was a mixture of acetonitrile: H2O=10:90 (adjusted to pH 3.1 by adding phosphoric acid), and the flow rate was 0.8ml/min with the wavelength of 210nm at 25℃. The solubility of 84.27mg/ml and oil/water partition coefficient of 0.087 were determined by HPLC, and it was proved that norcantharidin would convert into norcantharidate acid in water. (2) Particle size, entrapment and loading capacity were taken as indexes, and the optimal condition was calculated by multi-variable regression and ANNs: LCS and TPP concentrations (5:2 v/v) are 2mg/ml and 1.1mg/ml, and the temperature is 40℃, when the weights of temperature, particle size and entrapment were 0.1, 0.4 and 0.5. On this condition, the particle size of 131±7nm and entrapment of 45.12% were performed. (3) The contents of free amino groups on the surface of chitosan nanoparticles was 1.08×10-2mol/g, determined by the modified alkalimetry. In FT-IR spectrogram, the peak of chitosan at 3437cm-1 ofν(OH) shifted to 3420cm-1, and the peak at 1654 cm-1 disappeared. The peak at 1636cm-1 arose, and the peak at 1610cm-1ν(-NH2+) shifted to 1534 cm-1. In DSC,X-ray diffraction graph, the diagnostic absorption peak of LCS and TPP vanished or shifted. TEM graph shows that nanoparticles were sphere and distinct. The curve of the release in vitro was suitable for Weibull equation, and attain equilibrium in 70min. (4) On the experiment of drug absorption via small intestine in vivo, it was found that the K a and PA of nanoparticles was larger than NCTD aqueous solution. (5) The concentration-time curve equation of mice belonged to two-compartment model, and K12 of nanoparticles was larger than NCTD aqueous solution but K21 smaller.Conclusions: (1) As a matter of fact, it was norcantharidate acid that was analyzed by HPLC method, and mobile phase pH was adjusted to 3.1 so as to inhibit the ionization of norcantharidate acid. (2) After combining orthogonal design with analysis of multi-variable regression and ANNs, the method could dig up the data meaning. The optimal formulation was desired because of the synchronous optimization. (3) It was necessary to determine the contents of free amino on the surface of chitosan nanoparticles, because ionic inducement made -NH2+ on nanoparticles reduce, which could be linked by some targetable groups. (4) In the intestine circulation in vivo, it was proved that nanoparticles contributed to intestine absorption, not depending on dose. (5) Pharmacokinetics of nanoparticles showed that they are more targetable than NCTD aqueous solution.