Exosome/Stem Cell-based Nucleic Acid Drug Delivery Systems For The Treatment Of Colorectal Cancer And Limb Ischemia

Objectives:Nucleic acid‐based therapeutics as a new category has become the third revolution of biologics following small molecule drugs and antibody drugs.In this study,we designed new nucleic acid drug delivery systems based on endogenous biological carriers for the treatment of colorectal cancer and limb ischemia.First of all,we used siRNA as therapeutics and designed a targeted drug delivery system using endogenous exosomes as carriers for siRNA delivery.Based on the feature that tumor immune escape is mediated by multiple mechanisms,we proposed a combination therapeutic strategy that dually inhibiting FGL1 and TGF-β1 to block immune checkpoint and modulate the tumor microenvironment to realize the gene therapy of colorectal cancer.Secondly,to improve the therapeutic efficiency of stem cell transplantation in critical limb ischemia（CLI）,we used DNA as therapeutics and constructed CA-HIF-1αgene-modified CSC and transplanted it with fibrin gel to the ischemic region of CLI mouse to promote angiogenesis and realize the regenerative treatment of ischemic tissue.Methods and Results:1.Exosomes（Exo）were isolated from RAW 264.7 cell culture supernatants by ultracentrifugation and conjugated with cRGD targeting peptide by click chemistry,and then,siRNAs were loaded into Exo using chemical transfection reagent to form siRNA drug targeted delivery system（cRGD-Exo/siRNA）.The isolated exosomes showed complete structure,uniform particle size,and exosomal markers.Using Cy5-N₃ as a model drug verified that the targeting peptide was successfully conjugated to the surface of exosomes by click chemistry.siRNA was loaded into exosomes with an encapsulation efficiency higher than 95%,and the siRNA remained intact after incubation with nuclease when encapsulated by exosomes.2.The gene silence effect of Exo/siRNA was evaluated by q RT-PCR and ELISA,and the cell uptake-related behaviors were investigated by laser confocal microscope.The in vitro anti-tumor effect of combined inhibition of FGL1 and TGF-β1 was evaluated using CCK-8,flow cytometry and wound healing assay experiments.The results showed that siRNAs were efficiently up-taken by interest cells and silenced the expression of FGL1 and TGF-β1 m RNA and protein after loading into exosome.The modification of cRGD enhanced the uptake of exosomes in MC38 cells.cRGD-Exo and Exo were internalized by dynamin-mediated endocytosis and micropinocytosis.The siRNAs were partially co-localized with endosomes after uptake and then released into the cytoplasm over time.The exosome-mediated siRNA delivery system had no cytotoxicity to HUVEC and MC38 cells.Silencing FGL1 and TGF-β1 significantly inhibited the proliferation and migration of MC38 cells and promote their apoptosis.3.The biodistribution of cRGD-Exo was examined on the subcutaneous tumor-bearing mouse model by In Vivo Imaging System.Mice were treated with each group by tail vein injection and the gene silence efficiency of si FGL1 and si TGF-β1 in tumor tissues was investigated by q RT-PCR and immunohistochemistry staining.The anti-tumor effect was evaluated by monitoring the tumor size during treatment and checking the pathological section of tumors at the end of treatment.The anti-tumor mechanisms were explored by analyzing the changes of immune cells and cytokines in the tumor microenvironment.The safety of the drug delivery system was evaluated by detecting the weight of mice and pathological sections of main organs.The results showed that cRGD modification increased the accumulation of exosomes in tumors and significantly enhanced the gene silencing effect in vivo.Combined blocking of FGL1 and TGF-β1 induced tumor cell apoptosis and inhibited tumor growth by reshaping the tumor immune microenvironment and activating anti-tumor immunity.Besides,the exosome-mediated siRNA delivery system showed good biocompatibility.4.The feasibility of applying CSC to limb ischemia was preliminarily evaluated by detecting the expression level of proangiogenesis-related cytokines under hypoxic condition.CA-HIF-1αgene was transfected into CSCs by electroporation and the effect of overexpression of HIF-1αon the angiogenesis ability of CSC was evaluated by q RT-PCR,tube formation and wound healing assays.The paracrine effect of CSC was investigated by treating HUVEC with CSC culture medium.The feasibility of using fibrin（Gel）as cell transplantation scaffold was evaluated by observing the growth state of CSC in Gel.The results showed that the expressions of HIF-1αand proangiogenesis-related cytokines in CSC increased under hypoxic condition,which is conducive to ischemia treatment.After gene transfection,the expression levels of HIF-1αand pro-angiogenesis-related cytokines in CSC were significantly increased,and the overexpression of HIF-1αenhanced the endothelial differentiation,tube formation,migration ability and paracrine effect of CSC.5.The mouse critical limb ischemia model was established by ligating and severing the femoral artery,and then CA-HIF-CSC was co-transplanted with fibrin into the injury site.The therapeutic efficacy of CA-HIF-1α-modified CSC transplantation was evaluated by observing the blood perfusion,limb status,angiogenesis and muscle pathology.The results showed that CA-HIF-CSC/Gel transplantation promoted angiogenesis and blood flow reperfusion of ischemic limbs,and reduced the incidence of foot necrosis and limb loss.Conclusions:Herein,we focused on nucleic acid therapeutics and designed novel delivery systems using natural endogenous materials as carriers for the gene therapy of colorectal cancer and limb ischemia.cRGD-Exo/siRNA significantly inhibited tumor growth by reshaping the tumor microenvironment and reactivating the anti-tumor immune response.CA-HIF-CSC/Gel transplantation reduced the rate of amputation by promoting new vessel formation and blood flow recovery.This study provides new strategies for cancer immunotherapy and CLI regeneration therapy and provides a reference for the construction of biological gene delivery systems.