Construction Of Intelligent Responsive Peptide Delivery Nanosystem And Its Study Of Tumour Immunotherapy

Peptide drugs have the advantages of strong biological activity,high selectivity and safety,and have attracted the attention of many researchers in the field of tumor immune checkpoint therapy.However,there are some limitations in the application of peptide drugs,such as poor chemical and physical stability and short circulating plasma half-life.To address these issues,targeted delivery of peptides using nano systems has become an effective strategy.This delivery method can not only avoid the rapid degradation of peptides in vivo,but also improve the efficacy and clinical safety of peptides.In this study,a multifunctional polymeric nanovesicle delivery system was constructed to improve the efficacy of a peptide with immunotherapeutic effects developed by our group.On the one hand,the delivery system can target the peptide to the tumor tissue and release the peptide in response to the weak acidic microenvironment of the tumor,thus prolonging the circulation time of the peptide in vivo and reducing the occurrence of immune adverse events（ir AEs）;on the other hand,the polymer itself acts as an activator of the c GAS-STING signaling pathway,promoting the production of type I interferon and other cytokines,activating immune cells and reshaping the tumor immunosuppressive microenvironment,thus enhancing the anti-tumor immune response of the peptide and achieving the effect of combination therapy.This article mainly includes the following five parts.PartⅠ.ReviewFirstly,a concise overview of polymeric carriers for drug delivery is presented,encompassing polymer micelles and polymersomes.Subsequently,the research progress of diverse types of stimulus-responsive polymeric carriers is summarized.Furthermore,a comprehensive review of active peptides for tumor therapy is provided,focusing on anticancer peptides and tumor-targeting peptides.Lastly,the current status of immunotherapy research,including immune checkpoint blockade therapy and pericyte therapy,is summarized.The review of these four subsections offers substantial theoretical and technical support for the current topic.PartⅡ.Synthesis and characterization of amphiphilic block polymersIn this chapter,the synthesis of p H-sensitive amphiphilic block polymer PEO-PC7A is detailed,employing activators regenerated by electron transfer atom transfer radical polymerization（ARGET ATRP）.The successful synthesis of copolymers is confirmed by nuclear magnetic resonance hydrogen spectroscopy and Fourier transform infrared spectroscopy.Finally,the critical aggregation concentration（CAC）of polymer PEO-PC7A is determined to be 1.41×10^-3mg/m L using pyrene fluorescence probing,further indicating that the synthesized polymer has good self-assembly properties.PartⅢ.Preparation and characterization of peptide-loaded polymersomeAn in vitro method for peptide analysis was established using high-performance liquid chromatography.The established standard curve exhibited good linearity（R²=0.9998）,and the precision,reproducibility,and recovery met the methodological requirements,making it suitable for in vitro peptide determination.The peptide-loaded polymersomes were prepared using the film hydration method,and the optimal formulation was determined through one-way testing.Under the optimal conditions,the encapsulation rate reached 74.08%,with a drug loading capacity of 12.35%.Subsequently,the particle size and morphological characteristics of the prepared peptide-loaded polymersomes were observed,and the p H responsiveness of the polymersomes was investigated.The results revealed that the polymersomes had a mean particle size of 91.16±3.45 nm,with good dispersion and a spherical or sphere-like shape.Upon co-incubation with PBS at p H 5.5,the particle size of the peptide-loaded polymersomes significantly increased,and a single-peak distribution was not observed.In vitro release results demonstrated that the peptide-loaded polymersomes exhibited excellent stability in a physiological environment,while the release of peptides from the polymer vesicles was significantly accelerated in an acidic environment in a p H-dependent manner.These experimental results indicate that the constructed peptide-loaded polymersomes has weak acid stimulus response.Part IV.In vitro antitumor activity study of peptide-loaded polymersomesThe cytotoxic effects of the blank polymer were evaluated using CCK-8 assay on MDA-MB-231 and 4T1 cells,and the results indicated that the polymers did not exhibit significant toxicity and showed good biocompatibility.To investigate the toxic effects of peptide-loaded polymersomes and free peptide drugs on MDA-MB-231cells,a co-culture system of Jurkat cells and MDA-MB-231 cells was established.The findings revealed that the peptide-loaded polymersomes significantly enhanced the killing effect of peptides on MDA-MB-231 cells.Moreover,the in vitro antitumor effect of peptide-loaded polymersomes was further increased,compared to the control group without the addition of Jurkat cells.These results suggest that peptide-loaded polymersomes have the potential to re-activate T-cell immune function and promote the killing of tumor cells.Additionally,the levels of IL-2 in Jurkat cells were measured in the co-culture cell supernatant using ELISA assay,and the results showed that peptide-loaded polymersomes significantly induced IL-2 secretion in Jurkat cells,Further validated the role of peptide-loaded polymersome in activating T cell immune function.Part V.In vivo antitumor activity study of drug-loaded polymersomeThe in vivo antitumor effect of the peptide-loaded polymersomes was further investigated by establishing a 4T1 tumor-bearing BALB/c mouse model.The results showed that the tumor volumes of mice in both the peptide-loaded polymersomes and free peptide groups was significantly reduced compared with the saline group.The tumor inhibition rate of the free peptide group was 46.9%,while the tumor inhibition rate of peptide-loaded polymersomes was 76.3%at the same dose,showing that the in vivo antitumor effect was better than that of the peptide.The level of IFN-γin tumor tissues of mice was measured by ELISA kit,and the results showed that the peptide-loaded polymersomes significantly induced the secretion of IFN-γ,and its secretion level was 3.3 times higher than that of saline group and 1.6 times higher than that of peptide group.In vivo tissue distribution of the peptide-loaded polymersomes was examined using in vivo imaging techniques.The results demonstrated that the peptide-loaded polymersomes had a significantly increased distribution in tumor tissues compared with free peptides,indicating certain targeting properties.These findings suggest that peptide-loaded polymersomes could enhance the accumulation of peptides in tumor tissues.Therefore,these results indicated that peptide-loaded polymersomes could achieve tumor targeting and peptide release in response to the stimulation of the tumor microenvironment and significantly enhance the anti-tumor immunotherapeutic efficacy of peptides by activating the STING signaling pathway.This study provides an experimental basis for further exploring the potential of peptide-loaded polymer vesicles in tumor immunotherapy.