Mitochondria-based Carrier For The Therapy Of Colorectal Cancer

Objective:More than one million cases of colorectal cancer(CRC)are diagnosed each year in the world.In recent years,the incidence and mortality of colorectal cancer in China rank in the top five among all tumors.Although the surgical techniques have been developed,the recurrence rate of colorectal cancer is nearly 40%.Besides,many patients with colorectal cancer have advanced diagnosis missing the chance of surgery.Therefore,chemotherapy plays an important role in the treatment of colorectal cancer.However,the toxicity side effects of chemotherapy drugs limit their further application.Drug delivery system can improve the physicochemical properties of drugs by various drug carriers,optimize pharmacokinetics and pharmacodynamic properties,and the like.This will hopefully achieve a more efficient and accurate killing of tumors by chemotherapy drugs and reduce the side effects of drugs.However,drug delivery systems still have some potential dangers,such as biosafety.In recent years,the precise medical concept of “treating its own diseases with its own substances” has gradually attracted the attention of researchers.Some substances derived from cells have shown good application prospects in drug delivery,such as exosomes and extracellular vesicles,etc.The success of mitochondrial transplantation in the treatment of mitochondrial defects deseases indicates that exogenous mitochondria can enter the cell,which is consistent with the requirements of the drug carrier.Therefore,we designed a drug delivery system that used the mitochondria derived from normal colonic epithelial cells carrying chemotherapy drugs which could response to tumor microenvironment,and we explored the effect of the drug delivery system in the treatment of colorectal cancer.Methods:First,we isolated and purified mitochondrias from cells.Next,we constructed a stable cell line of NCM460 colonic epithelial cells stably expressing GFP in mitochondria,and isolated the mitochondrias,then co-cultured with LoVo cells,detected whether the mitochondrias can enter the colorectal cancer-derived LoVo cells by confocal fluorescence microscopy and flow cytometry;then,we used the Annexin V-FITC/PI apoptosis kit and the EdU cell proliferation kit combined with flow cytometry to detect the influence of mitochondrias and DOX-Mitos(mitochondrias carring Doxrubicin)on apoptosis and proliferation of LoVo cells.Finally,we optimized the drug-loading system.On the one hand,we modified the isolated mitochondrias with the cell membrane penetrating peptide Pep-1 to enhance the mitochondrias' ability to enter the cell;on the other hand,we connected the triphenylphosphine(TPP)and DOX by the condensation reaction of the amine group and the carboxyl group,and synthesized TPP-DOX,a deriative of DOX,which could target at mitochondrias,and demonstrated whether TPP could enhance the capability of mitochondrial drug-loading by fluorescence quantification and fluorescence confocal imaging experiments.We designed an acid-sensitive bond between TPP and DOX to modify the drug delivery system,and applyed it to the treatment of colorectal cancer.Results: We isolated mitochondrias,chracterized them by transmission electron microscopy,potential analysis,particle size detection,etc.Confocal fluorescence microscopy and flow cytometry results showed that our extracted mitochondrias could enter NCM460 cells and LoVo cells in a dose-dependent manner.We found that mitochondria had no significant effect on apoptosis and proliferation of LoVo cells by flow cytometry,the Annexin V-FITC/PI apoptosis kit,and the EdU cell proliferation kit,while mitochondria-loaded DOX significantly strengthen DOX-induced LoVo cell apoptosis and enhanced the ability of DOX to inhibit LoVo cells proliferation.Intracellular distribution experiments showed that TPP-DOX can target at mitochondria,and the ability of mitochondria to carry TPP-DOX is about two times over than that of DOX.Conclusion: We constructed a mitochondrial drug-loading system equipped with DOX,and proved that the mitochondria-loaded drugs enhance the ability of drugs to kill tumor cells,which indicates that the mitochondria as a drug carrier has some application prospects in the treatment of colorectal cancer.