Study On Degradable Nanocomplex Co-delivery Photosensitizer And TLR7/8 Agonist As In Situ Tumor Vaccine

Immunotherapy has revolutionized cancer treatment and emerged as a major therapeutic modality in oncology.In recent years,therapeutic tumor vaccines aimed at treating existing tumors or preventing tumor recurrence have been widely used in the field of tumor immunotherapy as a direct method to induce effective immune response.A successful tumor vaccine needs tumor antigens and appropriate adjuvants to stimulate DCs maturation,which then achieves the best T cell activation effect.Traditional treatment methods such as photothermal therapy(PTT)and photodynamic therapy(PDT)could directly kill tumor cells,while induce immunogenic cell death(ICD).ICD can be characterized by the release of damage-associated molecular patterns(DAMPs)and tumor-associated antigens(TAAs)from dying tumor cells.The in situ vaccination strategy,which can release TAAs locally at the tumor site to activate its own DCs,has attracted considerable attention.As a photosensitizer,IR780 has both photothermal conversion efficiency and photodynamic property,and could preferentially accumulate in tumor cells.Therefore,IR780 can be applied to in situ vaccine strategy.Toll like receptor(TLR)agonists can promote DCs maturation,which are well immune adjuvants.Imidazoquinoilines(IMDQ)are potent small molecule agonists of TLR7/8,which can induce activation of the MyD88 dependent classical TLR signaling pathway to trigger immune reaction.Here,an in situ vaccination strategy was studied,which combined ICD and release of TAAs inducted by PTT/PDT with concomitant innate immune activation triggered by TLR7/8 agonist triggering,to promote the induction of tumor specific immunity.The biodegradable polypeptide polymer polyaspartic acid(PASP)was used as the carrier material to conjugate IMDQ and IR780 respectively through amide condensation,which could effectively prevent the leakage of drugs.Among them,IR780 could preferentially accumulates in tumor cells.To increase uptake of IMDQ by DCs,mannose was linked to PASP-IMDQ(denoted as pIM),to target the mannose receptor on the surface of DCs.Due to a large number of carboxyl groups in PASP,it could be crosslinked with calcium ions to form a pH-responsive nanocomplex(denoted as NanopIR/mpIM).Upon intratumoral injection,NanopIR/mpIM dissociated gradually in the slightly acidic microenvironment of the tumor.PASP-IR780(denoted as pIR)could induce PTT and PDT under 808 nm near-infrared laser irradiation to directly kill tumor cells,trigger ICD and release TAAs.PASP-IMDQ-mannose(denoted as mpIM)could promote maturation of DCs and stimulate the proliferation and differentiation of initial T cells.Main researches and results of the study are as follows:1.Determination methods for IR780 and IMDQMethods for the determination of IR780 and IMDQ were respectively established through UV-vis spectrophotometry.The methodological test results could meet the requirements.2.Preparation and characterization of co-loaded IR780 and IMDQ nanocomplexIR780-NH2,pIR and mpIM were successfully synthesized.The prepared NanopIR/mpIM had regular spherical morphology and pH sensitive characteristics,which could be dissociated under pH 6.5(simulating tumor acidic microenvironment).And it had good photothermal and photodynamic properties.3.In vitro anti-tumor effects evaluation of NanopIRCT26 colorectal cancer cells were selected to evaluate the antitumor effect of NanopIR in vitro.It was proved that NanopIR could be better absorbed by tumor cells,and had good biocompatibility and superior performance of phototherapy to kill tumor cells.NanopIR could cause ICD after laser irradiation,which provided a basis for combining immune adjuvants to activate antitumor immune response.4.The ability of mpIM to target and activate DCs in vitro and in vivoThe uptake and the maturation of DCs were characterized by flow cytometry and ELISA kit.It was proved that modification with mannose derivative improved the targeting of mpIM to DCs and promoted the maturation of DCs in vitro and in vivo.5.In vivo anti-tumor effects evaluation of NanopIR/mpIMA bilateral tumor model was established to evaluate the antitumor effect of NanopIR/mpIM in vivo.It was proved that NanopIR/mpIM had good tumor accumulation ability and higher stability,and could significantly inhibit the growth of primary and distal tumors.And NanopIR/mpIM had good preliminary safety in vivo.6.Study on anti-tumor mechanism of NanopIR/mpIMNanopIR/mpIM treatment could effectively promote the maturation of DCs and repolarization of tumor associated macrophages,and further effectively activate T cell immune response.Moreover,NanopIR/mpIM treatment significantly increased the proportion of effector memory T cells and had a long-term protective effect.