Combination Of Camptothecin Nanotherapy And Immune Checkpoint Inhibitors For The Treatment Of Malignancies

Background:Tumors have been threatening human life and health for a long time.In recent years,cancer treatment consisting of chemotherapy,radiotherapy,molecular targeted therapy and immunotherapy have developed rapidly.However,there are a lot of limitations in monotherapy.For example,only few patients can benefit from immunotherapy.So people began to explore combined therapy of these treatments to achieve better anti-tumor effects.In addition,the nano-delivery systems have excellent performance as drug carriers.Nano-drugs can be enriched in the tumor tissue,increasing the anti-tumor efficacy and reducing toxic side effects.Designing an effective combined therapy strategy and establishing a reasonable nano-drug delivery system to obtain nano-drugs with better efficacy and lower toxicity,will help more patients benefit from anti-tumor therapy,which was of great clinical importance.Aim:In this research,nano-drug delivery systems were used as carriers to combine the chemotherapy drug camptothecin with immune checkpoint inhibitors,aiming to obtain new systems of high-efficiency and low-toxicity anti-tumor medicines.Methods:First,the 7-ethyl-10-hydroxycamptothecin(SN38)was covalently conjugated to oligo-ε-caprolactone(oligo CL)via an ester bond to form esterase-responsive polyprodrugs(oligo CL-SN38).The prodrugs were further encapsulated with polymer matrices to form SN38-loaded nanoparticles(oligo CL-SN38 NPs).We then characterized the size and morphology of nanoparticles,and verified their esterase responsiveness.Cell toxicity experiments,colony forming,cell proliferation staining and flow cytometry were conducted to detect nanomedicines’toxicity and effects on different tumor cell lines.Second,we compared the anti-tumor efficacy of oligo CL-SN38 NPs combined with programmed cell death protein 1(PD-1)antibody to that of monotherapy in a mouse melanoma subcutaneous xenograft model.The safety of the combined therapy was evaluated in healthy mice through H&E staining and peripheral blood examinations.Third,the polylactic acid-modified SN38 prodrugs were encapsulated with polymer matrices to form the SN38-loaded nanoparticles.Then these nanoparticles were modified with programmed cell death protein ligand 1(PD-L1)antibody,termed PDL1-SN38 NPs.Cell toxicity experiments were conducted to detect the toxicity of nanoparticles on different tumor cell lines.The targeting of nanomedicines was verified by in vitro cell uptake experiments and in vivo tumors’drug concentration detection.We explored the anti-tumor efficacy of PDL1-SN38 NPs in a mouse melanoma subcutaneous xenograft model.We further established a tumor resection-rechallenging model to track the survival rate of mice,and analyzed tumor infiltrating lymphocytes by flow cytometry.Results:Section 1:oligo CL-SN38 could co-assemble with polymer matrices to form stable nanoparticles,with a hydrated particle size of 99.33±1.16 nm.The amount of SN38released from oligo CL-SN38 NPs in 24 hours reached 25.16±1.10%in an esterase environment at 37°C,which was significantly higher than that in an esterase-free environment(12.31±0.88%,p<0.001).Cell toxicity experiments showed that the half maximal inhibitory concentrations of oligo CL-SN38 NPs in different cell lines were similar to that of free SN38,while nearly 100-fold lower than that of irinotecan.Colony forming experiments and cell proliferation staining indicated that oligo CL-SN38 NPs have a stronger ability to inhibit tumor cell proliferation than irinotecan.Flow cytometry demonstrated that the efficacy of oligo CL-SN38 NPs in inducing cell apoptosis was remarkably superior to irinotecan.Section 2:In animal experiments,it was found that the tumor inhibition rate of oligo CL-SN38 NPs combined with PD-1 antibody reached 70.11%,which was markedly higher than that of monotherapy groups(p<0.05).Serum interferon-γlevel in the combination group(45.53±10.78 pg/m L)was remarkably higher than post-administration(18.36±4.97 pg/m L,p<0.05),and a significant increase was also discovered compared with the control group(21.00±1.79 pg/m L,p<0.05).No obvious changes in peripheral blood examinations were observed by the combined therapy.Section 3:We successfully prepared the SN38 and PD-L1 co-loaded nanoparticles(PDL1-SN38 NPs),which has a targeting effect on tumor cells.The PDL1-SN38 NPs group showed the best efficacy in vivo.The relative tumor volume(2.29±0.48)and tumor weight(0.39±0.26 g)at the end of treatment were significantly smaller than those of other groups(p<0.01).The tumor resection-rechallenging model showed that PDL1-SN38 NPs greatly improved the survival rate.Flow cytometry indicated that compared with the control group,PDL1-SN38 NPs increased CD8~+(9.88±2.46%,p<0.01)and CD4~+(18.57±8.14%,p<0.01)T cells proportion in tumor infiltrating lymphocytes.Conclusion:Esterase-activatable SN38 nanomedicines were constructed,and they can significantly enhance the anti-tumor efficacy compared with irinotecan.When combined with PD-1 antibody,the therapeutic effectiveness was superior than monotherapy.Moreover,a drug delivery system co-delivering SN38 and PD-L1antibody was further constructed to improve the targeting of nanoparticles.This new nanomedicine can ameliorate the tumor microenvironment,and achieve better anti-tumor effects.