PH-sensitive Vector-mediated Immune Checkpoint Blockade Therapy And Immunogenic Cell Death To Achieve Anti-tumor Efficacy

Background:Tumor immunotherapy usually refers to the use of various means to restore or activate the response of immune cells,mainly T lymphocytes,to recognize and kill tumor cells.Unlike classic radiotherapy,cytotoxic chemotherapy or molecular targeted therapy,immunotherapy targets the immune cells rather than tumor cells,which is the most unique and novel feature of tumor immunotherapy.With the development of research on the immune system,especially the mechanism of tumor immune escape,tumor immunotherapy has gradually become the backbone of clinical cancer treatment.Immune checkpoint blockade therapy is currently one of the most common type of tumor immunotherapy,having shown excellent anti-tumor activity in various solid and blood tumors.Immune checkpoints are key proteins on inhibitory signaling pathways in the immune system,which play a crucial role in maintaining normal organs homeostasis and limiting excessive autoimmunity.Cancer cells use immune checkpoints to restrain the activity of T cells,which is one of the main mechanisms of tumor immune escape.Clinically,multiple immune checkpoint inhibitors(ICIs)has showed strong anti-tumor activity,the most confirmed efficacy of which are those targeting cytotoxic-T-lymphocyte associated protein 4(CTLA-4),programmed cell death protein 1(PD-1)and its ligand(PD-L1).However,the objective response rate(ORR)in cancer patients following ICIs is still low.For respond well tumor such as melanoma,non-small cell lung cancer,or urothelial cancer,the ORR is only 30～40%,the main reason of which is that the heterogeneity and instability of tumors leading to low immunogenicity.Even if the inhibitory pathway is blocked,tumor cells still cannot be recognized and eliminated by the body’s immune system.In addition,the currently approved ICIs are all monoclonal antibodies,which have limited blocking effect on immune checkpoints and are poor tissue permeability,also mainly contribute to the low response rate.When certain anthracycline drugs or radiotherapy induce apoptosis of tumor cells,the expression of immunogenic protein molecules such as calreticulin(CRT),high mobility group box protein 1(HMGB1)and adenosine triphosphate(ATP)could be up-regulated,promoting dendritic cells(DCs)maturation,and then activating T lymphocytes to specifically kill tumor cells.This process is called immunogenic cell death(ICD).The combination of immune checkpoint blockade therapy and ICD can play a synergistic effect to boost recognition and killing ability of immune system.Small interfering RNA(si RNA)is a type of double-stranded RNA with a length of 20～25 nt,which can recognize and cut specific sequence messenger RNA(m RNA).Compared with protein blocking therapy represented by antibodies,si RNA combines efficient degradation and specific recognition ability to significantly down-regulate the expression of target genes from the m RNA level.Short synthesis cycle,low cost and high gene silencing efficiency are also advantages,which give si RNA broad application prospects in the field of tumor gene therapy.Combining ICD mediated by doxorubicin(DOX)and immune checkpoint blockade mediated by si PD-L1 can complement each other and exert stronger anti-tumor efficacy.However,DOX has a strong cytotoxicity on normal tissues,and si PD-L1 is unstable as a small nucleic acid itself.Therefore,it is necessary to construct a safe and efficient co-delivery carrier to reduce DOX spillover and protect si PD-L1 from degradation.Polyethylenimine(PEI)is currently one of the most effective non-viral gene transfection vectors,and has become"gold standard"for measuring the transfection efficiency of other types of vectors in the field of non-viral vectors.Due to a large number of amino groups in PEI,a strong"proton sponge"effect under low p H conditions in lysosomes can be produced,which help gene molecules effective escape from lysosomes to cytoplasm.Although PEI has high gene transfection efficiency,its practical application is limited due to the disadvantages of high toxicity and no targeting effect on cells or tissues.Therefore,many researchers have devoted themselves to modifying the structure of PEI to improve its safety and transfection efficiency in vivo.Objective:Based on PEI,we designed a DOX and si PD-L1 co-delivery vector with high transfection efficiency and low cytotoxicity,realizing the synergistic cancer treatment of ICD and immune checkpoint blockade therapy.Content:1.PEI reacted with 3-maleimidopropionic acid hydrazide(BMPH)to synthesize PEI-BMPH(PM)through a sulfhydryl click reaction,and DOX combined with PM through a hydrazone bond to form PE1-BMPH-DOX(PMD).The structure was characterized by ~1H NMR,which confirmed the successful synthesis of PMD.The drug loading rate of DOX in the PMD was about 14%,and the release of DOX was acid-sensitive.Since the positive charge exhibited by PEI is masked after binding with BMPH,the PM carrier has lower cytotoxicity.Gel retardation experiment and RNase A protection experiment showed that PMD had a good ability to assemble and protect si PD-L1.By optimizing the transfection ratio,it was determined that PMD/si PD-L1was synthesized at a mass ratio of 8.After the nanocomplex was internalized by tumor cells,the hydrazone bond was broken in the acidic environment of the lysosome,and DOX was released and entered the nucleus to disturb the DNA synthesis.Si PD-L1could escape from the lysosome to the cytoplasm to degrade PD-L1 m RNA by virtue of the powerful"proton sponge"effect of PEI.By evaluating tumor cell apoptosis and proliferation inhibition ability,it was found that PMD/si PD-L1 had a strong anti-tumor activity.PMD/si PD-L1 could also significantly reduce PD-L1 expression and inhibit DOX-induced upregulation of PD-L1.Not only that,PMD/si PD-L1 could induce ICD like DOX,promoted CRT exposure,HMGB1 and ATP release,and mediated DCs maturation,T lymphocytes proliferation and cytokines production.2.In the mouse model of lung metastases,PMD used p H-sensitive hydrazone bonds to control the release of DOX,which significantly reduced damage to normal tissues,and could also maintain DOX at a high concentration in the tumor site for a long time to induce ICD and increase tumor immunogenicity.In addition,PMD could also well mediate the accumulation of si PD-L1 in tumor tissue,reduced the expression of PD-L1in tumor tissue,and blocked the immune checkpoint pathway.The synergistic effect of these two strategies resulted in the infiltration of more DCs,CD8~+T lymphocytes and cytokines in the tumor tissue of mice treated with PMD/si PD-L1,and then showed a good anti-tumor effect in vivo.Conclusions:In summary,this study constructed a co-delivery system of DOX and si PD-L1 with PEI derivatives as the carrier,which achieved efficient and stable delivery of si PD-L1and controlled release of DOX.Through immune checkpoint blockade and ICD worked synergistically to achieve a good tumor suppressive effect.The development of this co-delivery system builts a good platform for the design and evaluation of tumor immunotherapy carriers,and lays a solid foundation for the clinical application of new tumor immunotherapy strategies.