Intracellular Kinetics And Anticancer Effect Of Hydroxycamptothecin Submicron Emulsions

Hydrocamptothecin (HCPT) submicroemulsions were prepared by microfluid technology. The physicochemical characteristics, intracellular kinetics, subcellular biodistribution, mechanism of cellular uptake and antitumor effect of HCPT submicroemulsions (HCPT-SE) were studied. The relation between phase distribution and in vitro release of HCPT submicroemulsions, and overcoming multidrug resistance were also investigated. In the present work we provide some academic and experimental data for submicroemulsions as carrier of anticacer agents.The method of high performance liquid chromatography (HPLC) was set up to determine the concentration of HCPT. The apparent solubility of HCPT in distilled water and octanol was 2.98μg/mL and 47.06μg/mL, respectively. The apparent partition coefficient of HCPT in octanol/distilled water was 12.62. Lipophilicity of HCPT is more than hydrophilicity. The pKa was calculated from the process of carboxylate form of HCPT into lactones form in diferent pH solution. The solubility of HCPT in different oil was also determined.HCPT-SE containing HCPT, soybean oil, lecithin, stearylamine cholesterol and vitamine E as oily phase and Poloxamer188 and (or) as lecithin emulsifier were prepared by microfluid technology. As a result, HCPT-SE of different charge, cationic submicroemulsion (HCPT-SAE) and anionic charged submicroemulsion (HCPT-LipoE) were prepared. The HCPT-SE were characterized in terms of physical appearance, droplet size, polydispersity and zeta potential. The result showed that the characterizations of HCPT-SE were not significantly affected by autoclaving. HCPT-SE accorded with the quality standards of injections, although the pH value and content of HCPT were slight decrease. The HCPT in submicroemulsions mainly distributed in oil/water interface (～80%). Distribution of HCPT in oil phase (～14%) was more than that in water phase. In vitro, the release of HCPT from HCPT-SAE and HCPT-LipoE were similar. The release pattern of HCPT was slow and related to distribution of HCPT in submicroemulsions. HCPT-SE significantly decreased the conversion of lactone to carboxylate and enhanced proportion of the serum HCPT with the lactone fragment.To study the intracellular kinetics behavior and distribution of HCPT-SE, reversed-phase HPLC mothod was established to determine intracellular HCPT concentration. The cellular uptake kinetics study showed that HCPT-SE could significantly increase intracellular accumulation and nuclear delivery of HCPT compared with HCPT-I at same dose.The intracellular elimination kinetics study in SGC7901 cells showed that the AUC of HCPT-SAE and HCPT-LipoE were 9.88 and 7.69 folds higher than that of HCPT-I, when half-life was enhanced by 1.67 and 3.1 folds, respectively. The AUC and halt-life of HCPT-SE in nuclei and cytoplasm were significantly higher than that of HCPT-I (P < 0.01), whereas the AUC of HCPT-SAE extended the AUC of HCPT-LipoE by 1.76 times. The AUC of HCPE-SE in nuclei was lower than that in cytoplasm.In SGC7901/VCR cells, the AUC of HCPT-SAE and HCPT-LipoE were 29.16 and 8.54 folds higher than that of HCPT-I, when half-life was enhanced by 5.37 and 6.23 folds, respectively. The AUC of HCPT-SE in nuclei were significantly higher than that in cytoplasm (P< 0.01), whereas the half-life of HCPT-LipoE in nuclei was longer than that in cytoplasm. The AUC of HCPT-SAE in nuclei and cytoplasm was significantly higher than that of HCPT-LipoE (P < 0.01). The half-life of HCPT-SE in nuclei was no difference although the half-life of HCPT-SAE in cytoplasm was significently longer than that of HCPT-LipoE (P < 0.01).The AUC of HCPT-SAE and HCPT-LipoE were 35.26 and 15.44 folds higher than that of HCPT-I in HeLa cells, when half-life was enhanced by 1.67 and 1.75 folds, respectively. The AUC of HCPT-SE in nuclei were significantly higher than that in cytoplasm (P<0.01), whereas the half-life of HCPT-LipoE in nuclei was longer than that in cytoplasm. The AUC of HCPT-SAE in nuclei and cytoplasm was significantly higher than that of HCPT-LipoE (P < 0.01).The cellular uptake of HCPT-SE was in proportion to the concentrtion, and the pathway was saturable in high concentrtion (1.0μg/mL). The uptake of HCPT-SE was energy-dependent and was influenced by temperature. Endocytosis inhibitors (sodium azide, mannitol and chloroquine) decreased significantly accumulation of HCPT (p<0.01). The result revealed that HCPT-SE was uptaken through the endocytosis of energy-dependent.HCPT-SE showed higher antiproliferative activity than HCPT-Injection (HCPT-I), and the antiproliferative activity of HCPT-SE was in proportion to the incubation time and HCPT concentration. The proliferation of SGC7901cells, SGC7901/VCR cells and HeLa cells were signifcantly inhibited by HCPT-SAE (p<0.01). Whereas HCPE-LipoE only inhibited proliferation of SGC7901cells and HeLa cells and did not show significantly cytotoxicity in SGC7901/VCR (multidrug resistance cells). HCPT-SAE was suggested to overcome multidrug resistance in SGC7901/VCR cells by many approaches, such as fluorescence microscopy, flow cytometric ananlysis and HPLC, etc.After IV administration of HCPT-SE and HCPT-I to rat, pharmacokinetics parameters were calculated. It turned out that HCPT-SE significantly prolonged half-life of HCPT, and ehanced bioavailability. HCPT-SAE and HCPT-LipoE could extend the half-life of HCPT-I by 5.75 and 3.72 times, when AUC was enhanced by 4.03 and 4.01folds, respectively.The biodistribution test was carried out on S-180 tumor bearing mice. HCPT-SAE enhanced HCPT content of tumor, and had significantly tumor targeting effects. The AUC and C_max of HCPT-SAE in tumor were 11.57 and 1.91 folds higher than that of HCPT-I, respectively.Pharmacological test showed that HCPT-I at 4 mg/kg induced a 31.03% tumor inhibition rate. Compared with HCPT-I, HCPT-SAE and HCPT-LipoE showed tumor inhibiton rate of 90.75% and 70.21%, respectively. HCPT-SAE had tumor inhibiton rate of 64.31% at 2 mg/kg, which was 2 folds higher than that of HCPT-I at 4 mg/kg. Pathologic slice of tumor tissue also showed the effect of antitumor was higher than that of HCPT-LipoE and HCPT-I.