Application Of Targeting Peptide TMTP1 Modified Nanomicelles Synergizing Photothermal Therapy And Immunotherapy In Ovarian Cancer

PurposeOvarian cancer remains the most lethal of the gynecologic malignancies.However,the current treatment options cannot significantly improve the poor outcomes of the patients.In this study,PEG-PLGA nanocarrier was used to encapsulate photosensitizer ICG and the immune adjuvant MPLA,and modified with tumor targeting peptide TMTP1,thus cTMTP1@PEG-PLGA-ICG/MPLA(referred as TP1-IM)nanomicelles were constructed.By combination of TP1-IM nanomicelles and immune checkpoint inhibitor,this study aimed to synergize photothermal therapy and immunotherapy for the inhibition of ovarian cancer.Methods1.The TP1-IM nanomicelles were prepared by nanoparticle-precipitation method,PEG-PLGA nanocarrier was modified with TMTP1 peptides,and encapsulated photosensitizer ICG and the immune adjuvant MPLA.The physicochemical properties of nanomicelles were evaluated in vitro.2.CCK-8 assay and Calcein/PI staining assay were performed to determine the cytotoxicity of TP1-IM nanomicelles on ovarian cancer cells under NIR laser irradiation in vitro.3.Cellular immunofluorescence staining assay and flow cytometry assay were conducted to detect the immune activation effect of TP1-IM nanomicelles under NIR laser irradiation in vitro.4.The model mice bearing unilateral subcutaneous tumor were constructed to detect the tumor targeting ability of TP1-IM nanomicelles in vivo.5.The model mice bearing unilateral subcutaneous tumor were established to detect the immune activation effect of TP1-IM nanomicelles under NIR laser irradiation in vivo.6.The model mice bearing unilateral subcutaneous tumor were constructed to determine the therapeutic effect on ovarian cancer by TP1-IM nanomicelles-mediated photothermal therapy and immunotherapy.7.The bilateral subcutaneous tumor model mice and ovarian cancer intraperitoneal metastasis model mice were established to evaluate the inhibition effect on ovarian cancer metastasis by combination of TP1-IM nanomicelles and anti-PD-L1.8.To explore the mechanism of combination of TP1-IM nanomicelles and anti-PD-L1 in inhibiting ovarian cancer metastasis,immune cells in the secondary tumor tissues,tumor draining lymph nodes and spleen of the bilateral subcutaneous tumor model mice were detected and analysed.9.The ovarian cancer recurrence model mice were constructed and applied to determine the inhibition effect on ovarian cancer recurrence by combination of TP1-IM nanomicelles and anti-PD-L1.And the mechanism was further investigated.Results1.The TP1-IM nanomicelles were successfully synthesized.The diameter of the micelles was about 160 nm.The micelles kept the NIR fluorescence property and heat generating ability of ICG.Moreover,the micelles could specifically target ID8 cells in vitro.2.TP1-IM micelles could effectively kill ID8 cells under NIR irradiation in vitro.3.TP1-IM micelles could induce immunogenic cell death of ID8 cells and stimulate the maturation of BMDCs under NIR irradiation in vitro.4.TP1-IM micelles could specifically target ID8-bearing subcutaneous tumors in vivo.5.TP1-IM micelles could stimulate DCs activation and promote the secretion of cytokines TNF-α,IL12p70 and IFN-γ in serum under NIR irradiation in vivo.6.TP1-IM micelles-mediated photothermal therapy and immunotherapy could significantly inhibit the growth of ovarian cancer and prolong the survival time of ID8-bearing mice.7.The combination of TP1-IM micelles and anti-PD-L1 could significantly inhibit ovarian cancer metastasis.8.The combination of TP1-IM micelles and anti-PD-L1 could promote the infiltration of cytotoxic T cells into tumor metastases,and reduce the proportion of Treg cells in them.Moreover,the strategy could also significantly activate systemic anti-tumor immune responses.9.The combination of TP1-IM micelles and anti-PD-L1 could effectively inhibit the recurrence of ovarian cancer.Further mechanism studies have shown that the strategy could increase the proportion of immune memory T cells in the spleen.ConclusionsTP1-IM micelles could specifically target ovarian cancer.Under NIR irradiation,the micelles-mediated photothermal therapy could induce immunogenic cell death of ID8 cells and formed an endogenous tumor vaccine in situ with the assistance of MPLA,which could activate the anti-tumor immune response and effectively inhibit the growth of ovarian cancer.Moreover,the combination of TP1-IM micelles and anti-PD-L1 could significantly inhibit the metastasis and recurrence of ovarian cancer.In summary,this work suggested that TP1-IM micelles provided a new option for the treatment of ovarian cancer.