The Effects Of OCT-poloxamer 407 Thermosensitive Gel On Proliferation And Apoptosis Of Hca-F Cell And Study On Its Residence Time Within The Solid Tumor In Mice

Objective: At present the role of Octreotide(OCT) on treatment of hepatocellular carcinoma was clear, and it has little toxic and side-effects, but its half-life is short. We prepared OCT-poloxamer 407 thermosensitive gel ( OCT-P407)as a novel drug delivery system for intratumoral injection to prolong the residence time of OCT inside tumor. In this paper, we investigated the effects of OCT-P407 on the proliferation and apoptosis of Ascitic Hepatocellular Carcinoma Cell Strain with High Metastatic Potential in Lymphatic System of Mice (be called Hca-F cell) in vitro, meanwhile observed the holding period of OCT-P407 in the solid tumor cavity of mice in vivo after intratumoral injection. It was aimed to seek a new OCT sustained release method for intratumoral injection applied to patients with advanced liver cancer.Methods1. OCT-P407 was prepared with OCT and P407 by stirring method.2. After Hca-F cells were being intervened with OCT-P407 containing OCT with concentrations of 0.1μg/ml, 1μg/ml, 10μg/ml or 100μg/ml for 24 hours, the cell proliferation of Hca-F cell line was assessed by tetrazolium salt reduction assay(or called MTT method). And also the effects of OCT-P407 containing 20μg/ml OCT and OCT solution with the same concentration on the Hca-F cell line after 12 hours, 24 hours and 48 hours of intervention were observed.3. AnnexinV-FITC/PI double staining combined with flow cytometry (Flow cytometry, FCM) was used to detect the apoptosis of the Hca-F cell line which was intervened with OCT-P470 containing 20μg/ml OCT and the same concentration of OCT solution at the same times mentioned above.4. The cell morphological change was observed by inverted microscope.5. After establishment of transplantation tumor model of mice with Hca-F cell, (1) methylene blue and P407 or methylene blue and normal saline were mixed and injected respectively into solid tumor cavity under the guidance of colorultrasound ,we observed the coloration of local skin in both groups at 3min and 24 hours after injecting and the coloration in tumor cavity after 24 hours. (2) OCT-P470 or OCT solution was injected into solid tumor cavity, and then the contents of the OCT in cavities of both groups were detected after 12, 24 and 48 hours using high performance liquid chromatography for comparion of the residence times of OCT in solid tumor cavity between two groups.Results1. Inhibition of proliferation in vitro: OCT-P470 could significantly inhibit the Hca-F cell, moreover, with the increased concentration of OCT, the inhibiting role of OCT-P470 on Hca-F cells enhanced after interventation for 24 hours(P<0.05). The inhibition roles of OCT-P470 containing 20μg/ml OCT and OCT solution with the same concentration on Hca-F cells increased as time prolonging (P<0.05), however, the former showed more obviously (P <0.05).2. Induction of apoptosis in vitro: OCT-P470 and OCT solution could induce Hca-F cells apoptosis, and both effects were enhanced as time going.The former had a stronger effect(P <0.05).3. Histomorphological observation: After 48 hours of intervention by OCT-P407, parts of Hca-F cells changed their shapes from round to irregular, cells conglomerated, the refractivity of cells became weaker, and chromatin margination was showed.4. Detection of residence time of drugs in solid tumor cavity:(1) compared with the group stained by the methylene blue solution, the coloration of local skin was significantly lightened in the methylene blue-P407 group, and the coloration in tumor cavity was still evident after 24 hours. (2) the content of OCT in cavity after injection of 12 hours could be detected more in the OCT-P470 group than OCT solution group by HPLC (P <0.05). The OCT contents of two groups gradually reduced following the time prolonging. However, the content could not be determined in OCT solution group at 24 hours. We could still detect it at 48 hours in OCT-P470 group.Conclusions1. OCT-P470 may inhibit the proliferation of Hca-F cells, and its inhibition is more powerful with the increasing OCT concentration contained in OCT-P470, and also stonger following the prolonging contact time with Hca-F cells, which is greater than OCT solution with the same concentration.2. OCT-P470 may induce the apoptosis of Hca-F cell, with the extending of contact time with Hca-F cells, the effect of OCT-P470 on inducing Hca-F cells apoptosis is strengthened. The role of it on inducing apoptosis is stronger when compared to that of OCT solution.3. Compared with ordinary OCT solution, OCT-P470 has a long residence time in solid tumor cavity, and takes a good sustained action.