Studies On The Liver Targeted Solid Lipid Nanoparticles Lording With Cucurbitacin B Mediated By N-Hexadecyl Lactobionamide

Receptor-mediated cell targeting can be obtained by properly selection of a ligand that can be specifically recognized by the receptor over-expressed on the surface of target cells. Mammalian hepatocytes (parenchymal cells) exclusively possessing a large number of asialoglycoprotein receptors (ASGPr) can recognize terminalβ-D-galactose or,N-acetylgalactosamine residues. Recently, a great deal of concern was focused on ASGPr-mediated endocytosis to fulfill nanoparticles targeting to liver. In this paper, a galactosylated lipid, N-hexadecyl lactobionamide (N-HLBA) was synthesized. As a "homing molecule", N-HLBA was used to modify cucurbitacin B (Cue B) loaded solid lipid nanoparticles (SLN) and form galactosylated SLN (GalSLN). The freeze drying preparation of Cue B-SLN was prepared by lyophillization to enhance the storage stability. The pharmacokinetics and tissue distribution in rats was studied to reveal the fate in vivo of Cue B-SLN. The anti-tumor efficiency of Cue B-SLN in vitro and in vivo was investigated.The inhibitory effect of Cue B in vitro was investigated using the Bel-7402, Hela and SGC-7901 cells with MTT method. Cue B greatly inhibited the growth of Bel-7402 and Hela cells, and moderately inhibited the growth of SGC-7901 cells, both demonstrating the clear dose-dependent manner. The results of IC50 showed that the inhibitory effect of Cue B on the Bel-7402 was better than that of 5-Fu.Lactobionic acid (bearing a galactosyl group) and hexadecylamine were chemically attached via the amidation reactions to produce N-hexadecyl lactobionamide (N-HLBA) with a mono-galactoside moiety. The chemical structure was characterized by IR, NMR and ESI-MS. The synthesis process of N-HLBA is simple and easy-controlled.Cue B-SLN was prepared by high pressure homogenization, which Compritol 888 ATO was lipid material and lecithin and poloxamer 188 as emulsifier. The single factor experiments were investigated the effects of technology factors (adding order, homogenization pressure and cycles, ultrasound power and time, et al) and formulation factors (the kinds and content of lipid phase, the kind and ratio of emulsifier and the content of Cue B) on particle size and distribution of Cue B-SLN.The orthogonal design was carried to optimize the formulation further. Cue B-SLN was prepared into freeze drying preparation by lyophillization after the process and formulation parameters were optimized. At last, the optimal formulation with 7.5% of trehalose and 5.0% of mannitol presented good appearance and reconstitution.The pharmaceutical properties of Cue B-SLN including the shape, particle size, drug status in SLN, encapsulation efficiency and release in vitro were studied. The micrographs of transmission electron microscope (TEM) of CSLN and GalSLN showed that the particles had quasi-spherical physical shapes and uniform size. The particle size was measured by laser diffraction, and the mean diameter before and after lyophilization was 123 nm and 204 nm for CSLN,135 nm and 218 nm for GalSLN respectively.ξpotential of Cue B-SLN was determined by electrophoretic method, which was negative.ξpotential of the two types of SLNs was about-30 mV. DSC analysis showed that Cue B was amorphously dispersed within the SLN. The drug entrapment efficiency (EE) was measured by HPLC after ultracentrifugation. The EE was 93.1% for CSLN and 90.7% for GalSLN. The in vitro drug release behavior revealed that drug release was fitted well by Higuchi equation.HPLC method was developed for the determination of Cue B in biological specimen. The rat intravenous pharmacokinetic behaviors of three formulations (solution, CSLN, GalSLN) were investigated. The results showed that t the two types of SLNs had higher AUC and MRT than drug solution. Cue B-SLN showed the most obvious effect in prolonging the drug circulation time. The rat tissue distribution of the three formulations were examined after intravenous. The drug concentrations in the plasma, heart, liver, spleen, lung, kidney were determined at different time. The results suggested that compared with drug solution, the two types of SLNs changed the tissue distribution character obviously, The (Te) liver values suggested that the liver targetability of GalSLN was 2.5 times greater than that of CSLN. The biodistribution confirmed that the GalSLN could produce more efficient delivery into the target organ-liver. The cytotoxicity of drug solution, CSLN and GalSLN was investigated in the HepG2 cell line with MTT method. The three formulations all displayed obvious cytotoxicity, and had time-dependent and dose-dependent manner. Besides, SLNs had more potent cytotoxicity compared with drug solution and GalSLN had the most potent cytotoxicity.The efficiency of tumor inhibition after intravenous administration of different Cuc B-SLN were evaluated in KM mice bearing subcutaneous engrafted H22 murine hepatoma or S180 murine sarcopma. The result shows that GalSLN (0.11,0.055 mg/kg) and CSLN (0.11,0.055 mg/kg) exhibited siginificant anti-tumor effect in vivo against H22 murine hepatoma or S180 murine sarcopma. The effect of antitumor growth of GalSLN (0.11 mg/kg) was better compared with the CSLN (0.11 mg/kg).