Construction Of Multifunctional Micelles Containing Doxorubicin And Its Evaluation Of Breast Cancer Bone Metastasis In Vivo And In Vitro

Objective: In this paper,the multifunctional mixed micelles system of P123-ALN/P123-DP-8 was prepared to study the therapeutic effect on bone metastasis of breast cancer,where pluronic was the carrier,alendronate and(DMPGTVLP 8 peptide)DP-8 peptide were targeting ligands and doxorubicin was the drug.Methods:P123-ALN/P123-DP-8@DOX was prepared by thin film hydration method,and the preparation process of micelles was screened and characterized by Central composite design-response surface method.The MTT method,laser confocal microscope,flow cytometry,Western blot and other experimental techniques were used to determine the toxicity,uptake,apoptosis,cycle and caspase-3 of P123-ALN/P123-DP-8@DOX to MDA-MB-231 cells.A 3D metastasis model was established in vitro,and the effects of P123-ALN/P123-DP-8@DOX on bone resorption,osteoclast activity and bone resorption area were observed by scanning electron microscopy,neutral red staining and silver nitrate counterstaining.The bone metastasis model was established by in-situ tibial injection,and diferent therapeutic drugs were used for tail vein injection.In vivo imaging was used to observe the distribution of DOX in tumor-bearing nude mice,and to determine the body weight and tumor volume of tumor-bearing nude mice,HE staining and immunohistochemical analysis of the body and bone tissue to evaluate the anti-tumor activity of P123-ALN/P123-DP-8@DOX in vivo.Results: The best prescription of micelles was screened by the Central composite design-response surface method: the dosage was 5.48 mg,the volume of organic solvent methanol was 7.28 ml,the hydration volume was 8.58 ml,and the micelle particle size was 122.97 nm,PDI 0.408,Zeta potential is-12.60 mv,encapsulation efficiency is 76.87%,drug loading is 3.44%,spherical appearance,sustained release and good in vitro targeting.The results of cell experiments in vitro show that P123-ALN/P123-DP-8@DOX has obvious cytotoxicity to MDA-MB-231 cells,enhances the uptake of tumor cells,and significantly increases intracellular fluorescence intensity,which can enhance cell apoptosis induction,It can significantly increase the uptake of drugs in the G2/M phase,and activate the expression of Caspase-3 protein,and promote the apoptosis of MDA-MB-231 cells.In vitro 3D cancer bone metastasis model found that P123-ALN/P123-DP-8@DOX exerts a good anti-bone resorption and anti-tumor effect by inhibiting the activity of osteoclasts.The results of in vivo experiments show that P123-ALN/P123-DP-8@DOX has a good anti-tumor effect in vivo and can significantly reduce the cardiotoxicity of DOX.Conclusion: In summary,in this article the P123-ALN/P123-DP-8@DOX prepared meets the preparation conditions of nanomicelles to tail vein injection we need,increases the uptake of tumor cells,has strong cytotoxicity,and can significantly induce tumor cell growth.Apoptosis provides a theoretical and experimental basis for the treatment of breast cancer bone metastases.