In Situ Construction Of Self-assembled Vaccines With Dual Switchable Nano-delivery Systems For Tumor Immunotherapy

Most of the traditional therapeutic tumor vaccines are targeted to specific antigens,but their therapeutic efficacy are not ideal due to low antigen coverage and other reasons.Whole tumor cell vaccines can induce the body to target most of the antigens expressed by the tumor cells,but whole tumor cell vaccines have problems such as immune rejection(allogeneic),not suitable for difficult-to-operate patients(autologous),and so on.In situ tumor vaccines have attracted much attention due to their advantages of wide antigen coverage,no immune rejection,wide selection of tumor and patient types,simple administration,and so on.However,there are still few studies on in situ vaccines,and there are many problems that need to be solved urgently:1)Insufficient uptake of tumor antigens by antigen presenting cells(APCs);2)After being taken up by APCs,most tumor antigens are restricted in lysosomes,which are difficult to be presented by MHC I molecules,resulting in insufficient activation of CD8+T cells.;3)There is a phenomenon of tumor immune escape,which make it difficult for effector T cells to exert anti-tumor efficacy.Therefore,how to overcome the shortcomings of existing in situ vaccines while retaining the advantages of in situ vaccines is the focus of existing in situ vaccines.In consideration of the shortcomings of the existing in situ vaccines,we proposed the following solutions:1)This study intended to use the immunogenic cell death(ICD)effect of doxorubicin(DOX)to accelerate the production of tumor antigens.Then,the liposomes with surface-modified Mal groups were used to capture the antigens,and the modification of the targeting ligand mannose enhanced the phagocytosis of liposomes by dendritic cells(DCs),and finally increased the uptake of the tumor antigens by DCs.2)Using DLin-MC3-DMA as the main component of the liposomes,endowed the liposomes with lysosomal escape ability and improved the rate of antigen presentation by MHC I.At the same time,STING agonist(cGAMP)was added as an immune adjuvant,thereby accelerating the maturation of DCs and promoting the activation of CD8+T cells.3)Adding 1-methyl-D-tryptophan(1MT)to inhibit the activity of indoleamine-2,3-dioxygenase(IDO),thereby reducing the content of Regulatory T cell(Treg)and increasing the activity of effector T cells,thus reversing the tumor immunosuppressive microenvironment and improving the therapeutic effect of the vaccine.The realization of the above solutions required the efficient delivery of the above mentioned drugs and liposomes into tumor tissues.Due to the complex physiological structure of tumor tissue,nanomedicines mainly accumulate in the periphery of the tumor tissue,and it is difficult to reach the deep layers of the tumor tissue.Poor tumor penetration has become one of the main reasons for the low clinical transformation rate of nanomedicines.Among the factors affecting tumor penetration of nanoparticles,size and surface charge are considered to be the most critical factors affecting tumor penetration of nanoparticles.Studies have shown that small size(<50 nm),positively charged nanoparticles are more likely to penetrate deep into tumors.Based on the above discussion,the main research contents of this study were as follows:Part 1,the sudy of small morph nanoparticles for deep tumor penetrationFirst,this study verified the deep tumor penetration ability of small-sized,positively charged nanoparticles.Considering that the positively charged nanoparticles are easily cleared by the reticuloendothelial system in the blood circulation,this part used dimethylmaleic anhydride with pH-sensitive properties to modify the small size nanoparticles(PAMAM)to obtain the small morph nanoparticles(PAMAM-Dimethylmaleic anhydride,PDMA).PDMA maintained the negative surface charge in the blood circulation,but they could realize charge reversal in response to the slightly acidic environment of the tumor tissue,thereby achieving deep tumor penetration.In this part,four small morph nanoparticles were successfully synthesized and their pH-sensitive charge reversal properties were investigated.The results showed that PDMA had excellent pH-responsive charge reversal ability,and could responsively transform into small-sized,positively charged nanoparticles at pH 6.5.In vitro cellular uptake and tumor penetration results showed that PDMA was more readily taken up by tumor cells and had better tumor penetration at pH 6.5 than pH 7.4.The results of in vivo experiments showed that PDMA had excellent tumor tissue accumulation ability and deep tumor penetration ability.The tumor penetration mechanism of PDMA was investigated,and the results showed that PDMA achieved deep tumor penetration mainly through caveolae-mediated transcytosis.Finally,the antitumor efficacy and safety of DOX-loaded nanoparticles PDMA/DOX were evaluated.The results showed that PDMA/DOX had excellent tumor inhibiting effect and good preliminary biological safety.Part 2,in situ construction of self-assembled vaccines with dual switchable nano-delivery systems for tumor immunotherapyAccording to the improved strategies proposed by this study for the existing problems of in situ vaccines,combined with the study of the tumor penetration mechanism in Part 1.In this part,a size-shrinkable,charge-reversible dual switchable nano-delivery system(NGR-modified LCL/DM and MCL/G co-loaded nanoparticles,NLM/DMG)was designed for the in situ construction of self-assembled vaccines(ICSV),to enhance the antitumor effect of in situ vaccines.Firstly,NLM/DMG could actively target the tumor tissue under the mediation of NGR.In tumor microenvironment(TME),NLM/DMG could disassemble responding to the slightly acidic TME to release LCL/DM(LHRH-modified cationic liposome loaded with DOX and 1MT)and MCL/G(mannose-modified cationic liposome loaded with cGAMP),and the deep tumor penetration was achieved due to the small size(<50 nm)and positive charge of LCL/DM and MCL/G.LCL/DM could actively targeted to tumor cells mediated by LHRH(luteinizing hormone-releasing hormone analogs),and released DOX and 1MT.In TME,①DOX exerted an ICD effect to promote the release of tumor antigens,which was the initiation phase of ICSV.And the ICD effect of DOX could promote the maturation of DCs.② Subsequently,MCL/G assembled into ICSV through capturing the tumor antigens via the Maleimide(Mal)group.ICSV could promote the uptake of tumor antigens by DCs,enhance the lysosomal escape of antigens and cGAMP,increase the rate of tumor antigen presentation by MHC I,thereby promoting the maturation of DCs and the activation of CD8+T cells.③ During the antitumor stage of effector T cells,1MT could inhibit the activity of IDO,reverse the tumor immunosuppressive microenvironment and improve the therapeutic effect of the vaccine.This part successfully synthesized the targeted materials and successfully assembled NLM/DMG.The characterization results showed that the NLM/DMG had uniform particle size,negative charge,and had the dual switch ability of pH-responsive size reduction and charge reversal,and had excellent deep tumor penetration ability.The in vitro and in vivo targeting investigations showed that NLM/DMG had excellent tumor active targeting ability and differ-targeting delivery ability.Investigating the assembly mechanism of ICSV,the results showed that MCL/G had strong antigens capture ability,could successfully assemble into ICSV,and could improve the uptake of antigens by DCs,promoted the lysosomal escape of antigens and cGAMP,and finally successfully promoted the maturation of DCs.Immunity assay showed that NLM/DMG could exert the ICD effect,activate the cGAS-STING pathway,effectively inhibit the activity of IDO,and ultimately play a positive regulatory role in TME.The in vivo therapeutic effect evaluation showed that NLM/DMG could effectively inhibit tumor growth,had excellent inhibitory effect on postoperative tumor recurrence and lung metastasis.And NLM/DMG could stimulate the generation of effector memory T,inhibit the growth of rechallenge tumor,and had potential long-term immune antitumor effect.In conclusion,1)this study confirmed that small-sized,positively charged nanoparticles had excellent deep tumor penetration ability,and they achieved deep tumor penetration mainly via caveolae-mediated transcytosis;2)The NLM/DMG constructed in this study could achieve charge and size dual switch,had the ability of deep tumor penetration and differ-targeting delivery ability,realize the self-assembly of in situ tumor vaccines.3)this study proposed a new strategy(ICSV)for tumor vaccine construction.Combined with 1MT and cGAMP,the anti-tumor immune effect of ICSV was enhanced.