Studies On Conventional, Long-circulating And Temperature-sensitive Liposomes Containing Topotecan Hydrochloride

As a water-soluble derivate of camptothecin, topotecan (TPT) has been studied extensively recently due to its better anti-cancer activity and lower toxicity in comparison with other camptothecins. It has been approved for therapy of ovarian cancer and small cell lung cancer by FDA. But as other camptothecins, its lactone structure also easily hydrolyzes to be inactive carboxylate form at physiology condition. Based on the characteristics of Hposome - a new drug carrier, conventional Hposome and PEGlyated Hposome containing TPT were prepared by ammonium sulfate gradient method - an active loading procedure, and they were studied systematically. Moreover, the new temperature-sensitive polymers were synthesized, and the temperature-sensitive liposome modified with one polymer was prepared and its therm-sensitive characteristics in vitro were studied. The temperature-sensitive liposome containing TPT was also prepared and the preliminary studies of its therm-sensitive characteristics were preformed. The following parts are included in this paper:1. Preparation of liposomes containing TPT. To study the best prepare method of liposomal TPT, liposomes of TPT were prepared by the traditional methods, for example thin-film dispersion, ethanol injection, reverse-phase evaporation, and active remote loading-ammonium sulfate gradient. And the effect of different methods on encapsulation efficiency was studied, and the results showed that a higher encapsulation efficiency and drug-to-lipid ratio were got by ammonium gradient method. The effects of different influencing factors in preparation of liposomal TPT by ammonium sulfate gradient were also studied. The lipids type and compositions, the content of ammonium sulfate, the ratio of lipid to drug and vesicle size of blank liposome were the main factors influencing the encapsulation efficiency of TPT liposomes by ammonium sulfate gradient method.2. Study on the stability of TPT liposomes in vitro. The effect of cholesterol content, saturation extent of phosphlipid and PEG modification on the in vitro release of TPT liposomes were studied. The TPT leakage from liposome was decreased by increasing the content of cholesterol. Compared with SPC liposome, the TPT liposome composed with saturated HSPC having higher phase transition temperature was more stable. PEG modification could increase the liposome stability in serum or plasma. The results on determination of ring opening ratio of lactone showed that liposomal encapsulation improved the stability of TPT and its hydrolysis rate decreased. Moreover, in comparison with SPC liposomes, HSPC liposome improved the lactone stability of TPT.3. The preliminary stability study of TPT liposomes. Based on the study on preparation and stability in vitro, several TPT liposomes with different compositions were prepared. Their encapsulation efficiencies were all about 90ï¼…and the average vesicle size were about 140nm. The results of preliminary stability study demonstrated that the stability of TPT liposomes were acceptable, and there were no significant change on the vesicle size, encapsulation efficiency and content of TPT after 1, 3 (or 6) months at 4℃.4. Study on distribution of TPT liposomes in vivo. The biodistribution studies were preformed by bolus i.v. injection different samples into the lateral tail vein of S₁₈₀ tumor-bearing mice, and the drug content in plasma and other tissues were determined at each time point. The results showed that liposomal encapsulation changed the biodistribution behavior of TPT, and the stability of TPT in vivo was improved. Compared with free TPT, liposomal encapsulation decreased the clearance ratio of TPT and increased its distribution in plasma. Moreover, the ratio of lactone TPT increased significantly. The membrane fluidity of liposome has an important effect on biodistribution pattern of TPT liposome. In comparison with S-Lip, the distribution of TPT in plasma and other tissues increased for H-Lip. The tumor target activity of H-Lip improved, and the relative tumor uptake ratio (compared with free drug) was 2.7-fold of S-Lip. Compared with unmodified liposome, PEG modification improved the stability of TPT liposome and H-PEG showed 3.7-fold increase in AUC_{0→∝} based on lactone of TPT compared with H-Lip. Moreover, H-PEG dramatically decreased the uptake of liposome by liver and spleen and increased the accumulation of TPT in tumor, and the relative tumor uptake ratios (compared with free drug) improved from 4.1 to 5.2.5. Study on cytotoxicity of TPT liposomes in vitro. The cytotoxic effect of TPT liposomes against human ovarian carcinoma (A2780) and human colon carcinoma (HCT-8) cells were studied by MTT assay. The results showed that liposomal encapsulation enhanced antiproliferation ability of TPT against A2780 and HCT-8 in comparison with free drug. Moreover, PEG modified liposomes increased the cytotoxieity of TPT, and the values of IC50 for H-PEG against A2780 and HCT-8 decreased 5- and 9- fold compared with that of H-Lip, respectively.6. Study on anti-tumor effect of TPT liposomes in vivo. To investigate the therapeutic advantage of TPT liposomes, the effect of different groups in the H₂₂ tumor-bearing mice model were studied. The results showed that liposomal encapsulation improved the anti-tumor effect of TPT, and the tumor inhibition ratio of S-Lip and H-Lip were respectively compared with free drug. Compared with S-Lip, the anti-tumor effect of H-Lip enhanced, but the body weight of tumor-bearing mice decreased significantly. In comparison with conventional liposomes, the anti-tumor effect of PEG-modified liposomes containing TPT improved and the change ratio of mice weight decreased.7. Synthesis of temperature-sensitive polymers. The PNIPAm and its copolymers were synthesized and exosyndromed, and their temperature-sensitive characteristics were also studied. The results showed that the LCST of PNIPAm was 32～33℃and its LCST changed in different mediums. In comparison with PNIPAm, the LCST of Poly(NIPAm-co-MA) and Poly(NIPAm-co-EA) were all decreased and the LCST of Poly(NIPAm-co-AM) increased.β-CD was reshaped and Poly(NIPAm-co-MAH-β-CD) was synthesized. The LCST of Poly(NIPAm-co-MAH-β-CD) increased with the increase of MAH-β-CD content, and its LCST value was higher than that of PNIPAm when the molar ratio of MAH-β-CD increased to 2ï¼….8. Study on temperature-sensitive liposomes containing calcein. Poly(NIPAM-co-AM-co-ODA) was synthesized and its LCST was about 38℃which slightly lower than that of Poly(NIPAm-co-AM). The temperature-sensitive liposome modified with this polymer containing ca1lcein was prepared and its therm-sensitive characteristic in vitro was studied. The results showed that calcein released from liposome modified with polymer increased above 37"C compared with unmodified liposome. With the increase of ratio of polymer to lipid, the release of calcein from liposome increased and the release did not significantly increase at the ratio of 0.1. Moreover, the release of calcein from HSPC liposome modified with polymer was higher than that of SPC liposome.9. Preliminary study on temperature-sensitive liposome containing TPT. The temperature-sensitive liposome modified with Poly(NIPAM-co-AM-co-ODA) containing TPT was prepared and preliminary studies of its therm-sensitive characteristics in vitro was preformed. The liposome showed the temperature-dependent release characteristics. TPT almost not release from liposome at 37℃, but the release increased quickly at 40℃. The release ratio of TPT from liposome modified with polymer was about 20ï¼…within 15 min at 40℃.