| OjectiveBy constructing a nude mouse tumor model of ectopic subcutaneous xenografts,it was verified whether the co-loaded micelle delivery system also inhibited in vivo using co-loaded micelles coated with DTX and NS-siRNA designed and synthesized by a cooperative laboratory.The role of prostate cancer development,and its pharmacological effects in vivo and toxic side effects on the body.Methods1.Establishment of a heterotopic subcutaneous xenograft model in nude mice:routinely culture PC-3 cells,maintain the cells in logarithmic growth phase,adjust the viable cell concentration,and inoculate each nude mouse with 2×10~6 cells,dissolved in 150?L PBS.And 50?L of Matrigel,inoculated subcutaneously in the unilateral armpit,and observed the tumor formation in nude mice one week later.2.Administration of tail vein injection and observation of drug distribution and effects on tumors in nude mice:nude mices with successful tumors were randomly divided into 6 groups(n=3):saline group and treatment group:empty micelles Group,single-load DTX group,single-load NS-siRNA group,non-targeted drug-loaded drug group,and targeted co-loading drug group.According to the relevant literature and the dosage of docetaxel,the dose was determined to be DTX:10 mg/kg,NS-siRNA:1 mg/kg,and 5 times of injection was administered every 4 days through the tail vein to measure tumor volume.Changes in(V)and body weight of mice were observed for differences in growth and tumor growth of nude mice after continuous administration,and the results were statistically analyzed.3.In vivo pharmacodynamic study of co-loaded micelles:The ability of co-loaded micelles to target tumor tissues was observed by using the animal-in-vivo imaging system with Cy5.5 labeled co-loaded micelles;RNA was extracted by tumor tissue disruption The total protein,combined with qPCR and,was used to study the ability of co-loaded micelles to inhibit NS gene expression from protein expression levels,and the results were statistically analyzed.4.In vivo toxicity study of co-loaded micelles:After nude mice were killed according to medical ethics routine,important organs as heart,liver,spleen,kidney and lung were obtained,and toxicity of treatment groups observed by HE staining.Results1.By measuring the changes of tumor volume(V),mass and body weight of nude mice,the saline group and the empty micelle group were used as controls.The growth of tumors in the treatment group and the growth of weight in nude mice were affected to varying degrees.Compared with the other three groups,tumor-targeted co-loading group was more inhibited,and body weight(kg)of nude mice also had same trend.2.It was observed by animal in vivo imaging experiments that the signal value of the co-loaded micelles with targeting function was higher than that of the co-loaded micelles with no targeting function.3.We used IHC,the way of q-PCR and the WB,and the results analysis reveal the expression of NS was significantly lower in the tumor tissues of the other treatment groups except for the normal saline group and the empty micelle group.The targeted co-loading group had lower expression than other treatment groups,and non-targeted co-loading and the single-load NS-siRNA group had no difference.3.The results of HE staining by paraffin-embedded sections showed that the tissue structure of heart,liver,spleen,lung and kidney in the empty micelle group and the treated group did not change significantly with the saline group as the reference.Conclusion1.This co-loaded micelle transports docetaxel and NS-siRNA can inhibit the growth of castration-resistant prostate cancer cells,thereby exerting its anti-tumor effect and inhibiting the development of tumors in vivo.2.Co-loaded micelles with prostate cell membrane antigen targeting function can exert better ability to inhibit tumor development in nude mice.3.The co-loaded polymer micelles have less toxic side effects in nude mice,so the co-loaded micelles may be safer and have the prospect of being used in the treatment of castration resistant prostate cancer. |
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