An Efficient And Safe Delivery Platform Of MicroRNA,for Multistage Targeted Cancer Therapy

In recent years,RNA has broken the limitations of traditional anticancer drugs due to its high efficiency and easy amplification,and has promoted the continuous development of RNA-targeted gene therapy in the field of precision medicine.However,nucleic acid is limited in cancer treatment due to its easy degradation,low cell uptake efficiency and lack of targeting.In order to achieve efficient targeted delivery of nucleic acid drugs,it is difficult to develop gene drugs that can target target sites,improve cellular uptake and maintain stable expression of intracellular RNA.Currently,magnetic particle carriers can significantly increase the drug concentration at sites due to their unique advantages of rapid magnetic response and precise localization.Polyethyleneimine-based cationic gene carriers can condense nucleic acids into stable complexes and release nucleic acids through endosome escape.They are the most important non-viral vectors for gene delivery.However,its high charge density is beneficial for nucleic acid loading and transfection while producing cytotoxicity.Introducing electrostatic,hydrogen bonding,and hydrophobic multiple interactions between polymers and nucleic acids(or cell membranes)is expected to improve transfection efficiency and reduce cytotoxicity.Based on this,this study constructed a multistage targeted micro RNA(mi RNA)delivery platform based on magnetic particles and cationic polymers to achieve mi RNA tumor site aggregation,promote drug uptake by tumor cells,increase gene expression in the nucleus,and achieve multistage targeted tumor therapy.First,cationic polymers(PER)with guanidine and hydrophobic groups and magnetic particles(MPS)were prepared by amide reaction and ATRP.MPS/PER/mi RNA magnetic complexes were prepared by self-assembly.The drug binding efficiency,surface potential,morphology,cytotoxicity and magnetic response properties of the magnetic complexes were analyzed.The magnetic complex with a mass ratio of 100:2.5:1 was screened out with high nanomedicines loading rate,good biocompatibility and magnetic response performance.Secondly,the active targeting properties of the magnetic complexes were investigated by interacting with in vitro cells.A microfluidic breast cancer chip was constructed to simulate the tumor in vivo environment and evaluate the effect of MPS/PER/mi RNA targeted drug delivery.The results showed that the magnetic complexes rapidly aggregated around the MDA-MB-231 breast cancer cells,with a targeting efficiency as high as 50%,and increasing the drug concentration at the tumor site.In a microfluidic breast cancer model that simulates blood flow in vivo,MPS/PER/mi RNA magnetically guided precise targeting to the tumor site,and its efficiency was 13.1 times higher than that without a magnetic field.The results show that MPS/PER/mi RNA can regulate its motion direction under the guidance of magnetic field,indicating that MPS/PER/mi RNA has good magnetic targeting regulation performance.Further,the cellular uptake of MPS/PER/mi R-7-5p in breast cancer cells was observed by fluorescence microscopy and the transfection efficiency was analyzed by flow cytometry.The mi R-7-5p efficient delivery and breast cancer therapeutic effect of MPS/PER/mi R-7-5p complexes were investigated by breast cancer cell viability detection and immunofluorescence protein analysis.The results showed that the MPS/PER/mi R-7-5p magnetic complex achieved the first-stage active targeting of the tumor site under the magnetic guidance;and then multiple interactions between the PER/mi R-7-5p nanomedicine and cells can improve the efficient uptake of tumor cells to achieve the second-stage cell uptake of mi RNA delivery.Finally,the PER proton sponge effect increases the escape of mi R-7-5p and exerts the gene regulation effect of mi R-7-5p.Preliminary mechanism discussion: efficiently delivery of mi R-7-5p upregulated mi R-7-5p expression in breast cancer cells,down-regulated the expression of related EGFR,TGF-α oncogenes and EGFR protein,reduced the activity of cancer cells,and killed 50% of the cancer cells.In conclusion,MPS/PER/mi R-7-5p gene drugs can target tumor sites,increase the uptake of tumor cells,further regulate the expression of targeted genes in the nucleus,and achieve efficient and precise tumor treatment.Multistage targeted gene therapy with RNA and other nucleic acid drugs provides new ideas and directions.