Design And Application Of Fluorinated Polymer-based MRNA Cancer Vaccine In Personalized Therapy

Cancer immunotherapy is to reactive the body’s anti-tumor immune response and strengthen the ability to recognize and kill tumor cells by activating the immune system,in order to achieve the purpose of eliminating tumors.Cancer vaccine is an attractive strategy for immunotherapy,which activates anti-tumor immune response and protects the body from tumor cell invasion by introducing tumor antigens.In the past few years,messenger RNA(mRNA)vaccine has attracted increasing attention as a promising vaccine.After mRNA vaccination,the antigen can be synthesized in the cytoplasm and presented to the cell surface by the major histocompatibility complex class I(MHC-I)to induce a cellular immune response,and an efficient cellular immune response is key factor in anti-tumor.However,mRNA is unstable and easily degraded by ribonuclease(RNase),and mRNA must cross lipid membrane barriers for entry into cells.These objective factors lead to less mRNA entering cells and insufficient protein expression in cells.Therefore,how to develop an efficient mRNA vaccine delivery system to protect mRNA from RNase degradation and promote the cytoplasmic delivery is an urgent problem to be solved.At present,lipid nanoparticles(LNPs)have achieved success as mRNA vaccine delivery systems.However,LNPs are usually composed by a variety of different lipid components with complicated compositions.Moreover,state-of-the-art microfluidic devices are required during the manufacturing process to produce mRNA vaccines.Developing new mRNA delivery carriers with simple components and easy preparation processes would still be of great significance for the development of mRNA vaccines.Fluorinated alkane chains are both hydrophobic and oleophobic,the polymer modified by fluorination can improve its affinity to cell membranes,promote cellular uptake and endosomal escape,and make fluoropolymers have good application prospects in gene and protein delivery.This paper synthesized a library of fluoroalkane-grafted polyethyleneimines(F-PEI)by grafting fluoroalkanes onto branched PEIs of varying molecular weights.Firstly,we evaluated the mRNA delivery effect of F-PEI,and obtained F13-3-PEI1.8k and F13-4-PEI1.8k with efficient mRNA delivery ability both in vivo and in vitro.Both F-PEIs could significantly improve the efficiency of mRNA entry into cells,activate the Toll-like receptor 4(TLR4)-mediated signaling pathway,and have the function of immune adjuvants,and compared with F13-4-PEI1.8k,F13-3-PEI1.8k showed stronger mRNA delivery efficiency and immune activation effect,which could be used to construct F-PEI-based mRNA nanovaccine platform.Subsequently,the above two F-PEIs were mixed with the model antigen ovalbumin(OVA)mRNA(mRNAOVA),and self-assembled to obtain the F-PEI/mRNAOVA cancer vaccine.The results showed that the F-PEI/mRNAOVA cancer vaccine coud effectively stimulate the maturation of dendritic cell(DC),significantly improve the efficiency of antigen presentation through MHC-I molecules,induce OVA-specific T cell immune responses,delay B 16-OVA tumor growth as a therapeutic cancer vaccine,and the mRNA cancer vaccine with F13-3-PEI1.8k as the carrier had better effect.When F-PEI was mixed with ovalbumin-expressing mRNA(mRNAOVA),the self-assembled F-PEI/mRNAOVA nanovaccine could be obtained.F-PEI/mRNAOVA nanovaccine could stimulate dendritic cell(DC)maturation;promote antigen presentation efficiency of MHC-I on DC;induce OVA-specific T cell immune responses;and delay B 16-OVA tumor growth as a therapeutic cancer vaccine.Therefore,using F13-3-PEI1.8k as a vector,a personalized mRNA cancer vaccine expressing MC38 neoantigen(F13-3-PEI1-8k/mRNAMC38)was further prepared for the mouse MC38 colon cancer model.By evaluating the immune response induced by the personalized neoantigen cancer vaccine,it was demonstrated that specific CD8+ T cells against the MC38 neoantigens were generated in mice.Combining F13-3-PEI1.8k/mRNAMC38 cancer vaccine with immune checkpoint inhibitors effectively inhibited mouse MC38 colon cancer growth,prolonged the survival rate and survival time of mice,and the combination therapy generated a strong immune memory effect,which could effectively protect the cured mice from re-challenge by the tumor.In conclusion,this paper developed a fluorinated polymer-based mRNA cancer vaccine platform,and used it for the development of personalized neoantigen cancer vaccines,which showed good application prospects in the field of cancer therapy.