Anti-tumor Activity Of Folate Targeted Biodegradable Polymer–paclitaxel Conjugate Micelles On EMT-6 Breast Cancer Cell Line And EMT-6 Breast Cancer Model

Introduction: Breast cancer is the most common solid malignant tumor for women in China, in the United States and in other countries Paclitaxel(PTX) is a first line chemotherapy drug for breast cancer. However, because of its low water solubility and strong adverse reactions such as allergies, low blood pressure, etc., its clinical use has been greatly limited. Great efforts have been made to develop new delivery systems of PTX,including liposomes, polymeric micelles, protein–PTX conjugates, etc. Recently, our group synthesized a series of functionalized poly(ethylene glycol)-b-poly(L-lactide-co-carbonate)and their conjugates with PTX and targeting moieties. In this paper, therefore, we chose FA as the targeting moiety, and attached it to a carrier polymer as its side-chain groups rather than a terminal group. PTX, a real anti-tumor drug, is used instead of a model drug. These micelles(PTX micelles, M(PTX) and folic acid-bearing polymer-PTX micelles,(M(FA/PTX)) are expected to have multiple functions, including water solubility, prolonged circulation, biodegradability, folatereceptor targeting and rapid cell-uptake.Objective: To study the therapeutic efficacy of M(PTX) and(M(FA/PTX)) in EMT-6breast cancer in vitro and in vivo, including anti-tumor activity, tumor cell apotosis and survival experiment.Methods: Two kinds of PTX conjugate micelles, one of which(M(PTX)) contained 25 wt.% of PTX and the other(M(FA/PTX)) contained 22.5 wt.% of PTX and 1.4 wt.% of folate(FA), were prepared for cell apoptosis and antitumor activity evaluation on EMT-6mice breast cancer cell line and models in comparison with 0.9 wt.% saline(control) and equivalent PTX.(1) The cytotoxic effect of PTX, M(PTX) and(M(FA/PTX) on EMT-6 breast cancer cell was evaluated by MTT at different time in different concentration. Cell apoptosis was analyzed by flow cytometry with 1ug/ml equivalent PTX. Inverted microscope was used to observe to the morphologic change of tumor cell between before and after drug administration. Hoechst 33258 fluorescence staining was used to detect the apotosis index with 1ug/ml equivalent PTX at 72 hours after drug administration.(2) EMT-6 breast cancer model was prepared. On day 5 after drug administration, at an equivalent paclitaxel dose of 20 mg/kg, mice were killed. Tumor inhibition rate was calculated. Cell apoptosis was analyzed by flow cytometry. Breast tumors were examined histologically with H&E staining and immunohistochemically by examining Bax and Bcl-2expression. And at an equivalent paclitaxel dose of 26.7mg/kg survival status of tumor-bearing mice with different treatments was also examined.(3) The results were analyzed using statistical software SPSS 18.0, each set of data was given as mean ± SD. Difference comparison between groups was carried out by one-way ANOVA. The χ2 test was used to test for associations between factors and the odds ratio.Life-tables were calculated according to the Kaplan–Meier method. Survival curves were compared with the log-rank test. The level of statistical significance was set as p< 0.05.Results:(1) In vitro experiment, MTT results showed that the inhibition effect of M(PTX) and M(FA/PTX) was best at 72 hours with1ug/ml equivalent PTX after drug administration.With the prolonged administration time, the inhibition raqte of each drug on EMT-6 breast cancer cell line was also increased. Flow cytometry results showed that the cell apotosis rate of control group, PTX, M(PTX) and M(FA/PTX) group was 14.4%, 20.7%, 19.7%, 25.7%.respectively. the cell apotosis rate of M(FA/PTX) group was the highest(p<0.05).With inverted microscope the tumor cell morphologic change of PTX, M(PTX) and M(FA/PTX)group was significant. Hoechst 33258 fluorescence staining showed that the cell apotosis rate of control group, PTX, M(PTX) and M(FA/PTX) group was 5.8%,15.8%, 15.2% and21%, respectively.(2) In vivo, The PTX group mice were dysphoric and screamed from time to time during drug injection. Mice of other groups had no obvious adverse reactions. On day 5 after drug administration, the average tumor masses were 0.49, 0.33, 0.22, and 0.18 g for the control, PTX, M(PTX) and M(FA/PTX) groups, respectively. The inhibition rates of tumor growth calculated for the three drug groups were 32.6%, 51.6% and 62.3%, respectively.H&E staining results showed that the necrosis area in M(FA/PTX) micelle group is significantly increased compared with other groups. Bcl-2 expression difference is statistically significant between PTX and M(FA/PTX) groups and control group(p<0.05).Bax expression difference is statistically significant between drug groups and control group(p<0.05). The percentage of cell apoptosis based on flow cytometry was 1.0%, 36.6%,55.9% and 66.1%, respectively, which showed statistically significant differences(P<0.05)between three drug groups and the control group. At an equivalent paclitaxel dose of 26.7mg/kg, the average survival time was 33 days, 31 days, 34 days and 42 days, respectively,and the survival rate at 30 days was 50%, 55.6%, 72.2% and 77.8%, respectively(p<0.05).Conclusion: In vitro, M(PTX) and M(FA/PTX) demonstrated the similar anti-tumor activity as PTX on EMT-6 breast cancer cell line,and the effect of M(FA/PTX) was more better. In vivo, the M(FA/PTX) have better anti-tumor activity on EMT-6 breast cancer model and can prolong the survival time of tumor-bearing mice. the M(FA/PTX) are promising in treatment of human breast cancers.