Exosome-mediated Delivery Of DNAzyme And Cytochrome C For The Treatment Of Breast Cancer

Currently,common therapeutic methods for cancer include surgery,radiotherapy,and chemotherapy.With the development of molecular biology,biological therapies are receving more and more attention,especially in the field of cancer treatment.Biological therapy refers to the method of using biomacromolecule as therapeutic options.In cancer theatment,biological therapies are endowed with low toxicity and high specificity when compared with conventional therapies such as surgery,radiotherapy and chemotherapy.There are various biological therapies for cancer treatment,among which gene therapy and protein therapy are the most widely studied.Gene therapy refers to the treatment method by introducing exogenous genes into specific cells that have genetic defects and abnormalities associated with the disease.Selection of gene targets plays a decisive role in achieving desired therapeutic effects.SALL4 gene encodes a transcription factor that plays an important role in maintaining the self-renewal and pluripotency of embryonic stem cells during the embryonic stage.Studies have shown that the abnormally high expression of SALL4 is directly realeted to the occurance and development of tumors,and reducing the expression level of SALL4 is expected to inhibit tumorigenesis and development at the genetic level.Among the common gene therapy tools,deoxyribozymes(DNAzymes)have been shown to effectively catalyze the breakage of specific intracellular m RNAs to achieve efficient gene-knockdown at the post-transcriptioanl level.However,DNAzymes themselves do not have the ability to cross cell membrane,and their low molecular weight makes them easily cleared in vivo,thus being instable.Therefore,the construction of efficient and biocompatible vectors for the delivery of DNAzyme targeting SALL4 is of crucial importance for the realization of DNAzyme-based gene therapy in the treatment of breast cancer.Protein therapy is the technique to deliver biologically active proteins into diseased tissues or cells to replace proteins that have lost their function or have low or lost expression capacity,thereby achieving therapeutic effects.Protein therapy has attracted a lot of attention in the field of cancer treatment due to its many advantages.Studies have shown that the apoptosis can be effectively induced in tumor cells by delivering pro-apoptotic proteins into the tumor cells.Cytochrome c is a key initiator protein in the mitochondrial apoptosis pathway.By delivering cytochrome c to tumor cells,it can activate the mitochondrial apoptosis pathway and efficiently induce the apoptosis in tumor cells,thus achieving favorable suppression effect.However,cytochrome c itself does not have the ability to enter cells,and its positively charged nature also restricts the circulation of cytochrome c in blood.Thus,the construction of an ideal delivery vehicle is essential to achieve the in vivo circulation and intracellular delivery of cytochrome c.Non-viral delivery platforms such as cationic lipids,polymers and nanoparticles have shown their utility in nucleic acid-based and protein-based delivery.However,these aforementioned carriers still have drawbacks such as immunogenicity caused by excess cationic charge,innate toxicity and rapid clearance from body circulation.Exosomes are cell-shed,membrane-enclosed extracellular vesicles with a diameter ranging from 30 to 150 nm,they are natural carriers transferring biological cargo such as nucleic acids and proteins.Its endogenous origin,nanoscale size,stability and innate biosafety have been highlighted in the field of exosome-based delivery systems.Thus,exosomes could function as a novel and promising platform for DNAzyme-and cytochrome c-based delivery and promotes the exertion of enzyme-induced apoptosis.The results are summarized as follows:1.Exosome-mediated delivery of DNAzyme for the treatment of breast cancerCatalytic DNAzymes have been considered as one of the potent therapeutic approaches for gene therapy.They possess various catalytic activities such as RNA cleavage and flexibility for rational design,while their poor intracellular uptake and lack of targeting capability in vivo usually hinders their applications.Herein,HEK-293-derived exosomes were utilized as carriers to facilitate the intracellular delivery of previously estabilished-catalytic DNAzyme targeting Sal-like gene 4(SALL4),using MCF-7 cell and tumor-bearing nude mice as the model.Reverse Transcription-Polymerase Chain Reaction(RT-PCR)and Western Blot analysis demonstrated that the DNAzyme targeting SALL4(Dz-SALL4)could effectively decrease the expression of SALL4 at the m RNA as well as protein level.Furthermore,the efficient cellular uptake could be realized through the exosome-mediated delivery,as confirmed by confocal laser scanning microscope analysis and flow cytometry analysis.Meanwhile,the proliferation of MCF-7 cells was significantly impeded,as confirmed by MTT as well as cell colony formation analysis,which could be attributed to the apoptosis along with the cell cycle arrest induced by DNAzyme formulations.In addition,the inhibition of invasion and migration was also confirmed through would healing and transwell migration assays.Finally,the composite Dz-SALL4@Exo significantly inhibited the progression of tumor in a xenograft mouse model,and achieved similar therapeutic effect compared to that of doxorubicine,owing to the accumulation of DNAzymes in tumor tissue after the exosome-based delivery.These results suggest that DNAzymes,as catalytic nucleic acids,could be effectively incorporated into exosomes and achieve a significant knockdown of target gene and inhibition of cancer progression through catalyzing the cleavage of target m RNA.2.Exosome mediated delivery of cytochrome c for the treatment of breast cancer.Cytochrome c has received much attention in the fileld of protein-based tumor therapy as an important type of model pro-apoptotic proteins.The apoptotic effect of cytochrome c depends on its efficient delivery into cells.Tumor-derived exosomes are vesicles secreted by tumor cells,which have similar molecular composition as tumor cells,and studies have shown that tumor exosomes possess “homing”properties and can actively target the cell line from which they originate.Thus,it is promising to apply tumor exosomes as vehicles for the delivery of anti-tumor protein.Herein,MCF-7-derived exosomes were utilized as carriers for the delivery of pro-apoptotic protein cytochrome c.We prepared Cyt c@Exo nanocomplex by physical extrusion combined with the saponin incubation.First,we confirmed the successful loading of cytochrome c in exosomes by western blot as well as fluorescent labeling,and measured the encapsulation ratio and drug loading of cytochrome c.Then,we used the breast cancer cell line MCF-7 as a model to confirm the efficient cellular transfection of Cyt c@Exo nanocomplex,and systematically verified its ability to induce cytological effects such as the proliferation inhibition and apoptosis,after which the related mechanisms were thoroughly explored and verified.Finally,we evaluated the anti-tumor effect and tumor-targeting ability of Cyt c@Exo nanocomplex by using a tumor-bearing nude mouse modelIn summary,we have constructed a type of highly efficient and biocompatible platform for the delivery of anti-tumor enzyme molecules,namely DNAzyme and cytochrome c,using nature-derived exosomes.Our platform has successfully achieved the loading of DNAzyme and cytochrome c,respectively,and delivered them into MCF-7 cells.Meanwhile,our platform could induce obvious tumor inhibition in both cellular and well-established animal model.Thus,we conclude that our study not only shows a new method for the design and construction of nanomedicine for cancer therapy,but also provides new insights for understanding the signal transmission through exosomes between tumor cells and the mechanism of tumorigenesis and metastasis.