Study On PH-sensitive Liposomes Loaded With Lactosyl-norcantharitin Phospholipids Complex

Objective: To prepare liposomes loaded with lactosyl-norcantharitin phospholipid complexes (LPC), in which soybean phosphatidylcholine was used to improve the liposolubility of lactosyl-norcantharitin (Lac-NCTD). The pH-sensitive LPC liposomes (pH-LPC-lips) were obtained by electrostatic adsorption of the carboxymethyl chitosan (CMCT) onto the surface of the liposomes, thus leading to the active release of Lac-NCTD in the tumor tissue which had the lower pH than the normal tissue. As a result, pH-LPC-lips can be regarded as liver-targeting agents, which combined targeting and active releasing.Methods: (1) LPC was prepared using solvent - evaporation and the complexed Lac-NCTD yield was monitored; orthogonal design was used to optimize the preparation technique of LPC; the formation of LPC was confirmed by FT-IR, DSC and 1H-NMR; the characteristics of LPC were determined by HPLC. (2) To prepare Lac-NCTD-liposomes (Lac-lips) and LPC-lips using reverse phase film-evaporation and reverse phase evaporation method respectively; uniform design was used to optimize the preparation technique of these liposomes; the particle size, entrapment efficiency (EE), loading capacity (LC) and zeta potential of the liposomes were monitored. (3) CMCT was adsorbed on the surface of LPC-lips by the electrostatic adsorption,then pH- LPC -lips were obtained; in vitro drug release of pH-LPC-lips and LPC-lips were investigated by means of a dialysis method in various dissolution media, then the influence of CMCT on drug release was discussed. (4) MTT Assay was used to study the cytotoxic effects of Lac-NCTD and its lipsomes on HepG2. (5) Lac-NCTD accumulated in HepG2 cells was determined by high performance liquid chromatogram (HPLC). (6) In vivo anti-tumor activity of Lac-NCTD, Lac-lips and pH-LPC-lips were evaluated in mice bearing H22 liver tumors. (7) The hepatocyte specificity of near-infrared fluorescence dye (Cy7)-labeled pH-LPC-lips in H22 tumor-bearing mice was monitored through NIR fluorescence real-time tumor imaging.Results: (1) The yield of the complexed Lac-NCTD was 97.2±2.01%, the LPC significantly increased the lipophilicity of Lac-NCTD for the P of LPC was about 135-fold more than that of Lac-NCTD. The results of FT-IR,DSC and 1H-NMR demonstrated that the LPC was a complex but not the physical complex; the result of HPLC showed that LPC had the same retention time as Lac-NCTD, thus LPC was not a new compound. (2) The pH- LPC -lips were obtained with average particle size of 47.18±4.16nm,entrapment efficiency of 70.00±1.30%,loading capacity of 2.24±0.43% and zeta potential of -26.0±2.97 mV, most of the liposomes appear uniform and round under TEM; while the Lac-lips were obtained with average particle size of 83.14±9.64nm,entrapment efficiency of 38.12±1.12 %,loading capacity of 1.22±0.21 % and zeta potential of -11.0±2.42mV. (3) In vitro release of Lac-lips and pH-Lac-lips in diverse media followed the Higuchi and Weibull equation, respectively. Furthermore, the drug release of pH-Lac-lips exhibited obvious pH-dependence and explosive release effect. (4) The pH-LPC-lips demonstrated strong cytotoxicity against the cells and easily permeated the cell membrane, compared with Lac-NCTD and Lac-lips. (5) The results of in vivo antitumor activity showed that pH-LPC-lips displayed best tumor inhibition effect and was effective in protecting organs and enhancing immunity. (6) The optical imaging results indicated that Cy7-labeled pH-LPC-lips showed excellent hepatocyte specificity in H22 tumor-bearing mice, which could reduced the side effect, and increased antitumor activity.Conclusions: Optimized preparation technique of LPC was satisfied with the simple process, low cost, and high yield. The hydrophilic drug, such as Lac-NCTD, was able to be formulated into liposomes with high entrapment efficiency due to the lipophilic enhancement of LPC. The liposomes modified with CMCT showed pH sensitivity and could release the drug actively when the liposomes reached the tumor tissue to maximize the therapeutic effect. The antitumor activity and hepatocyte-targeting of pH-LPC-lips was improved both in vitro and in vivo. As a result, pH-LPC-lips can be regarded as liver-targeting agents, which combined targeting and active releasing.