Construction Of Targeted Drug Delivery System Based On Dunaliella Salina Exosome And Its Effects On Esophageal Cancer

Esophageal cancer is one of the common malignant tumors,and its lethality ranks sixth among other malignant tumors.China as a high incidence area of esophageal cancer,accounting for half of the world’s total incidence.Although esophageal cancer patients have been treated with a combination of surgery,radiotherapy and chemotherapy,but the average survival still lower.The poor prognosis of esophageal cancer is closely related to the aggressiveness of malignant tumor cells and the toxic and side effects of these non-specific therapies.Almost all patients died after recurrence or metastasis of residual tumor cells.Therefore,there is an urgent need to find new and effective treatments to improve the treatment effect of esophageal cancer patients and prolong their survival.The emergence of mi RNA provides a new breakthrough for solving this problem.mi RNAs act as oncogenes or tumor suppressors by regulating cell differentiation,proliferation,and apoptosis.In recent years,people have achieved excellent antitumor effects by up-regulating or down-regulating mi RNAs expression in tumor.Studies have shown that mi R-375 as one of the significantly down-regulated mi RNAs in esophageal cancer,and the restoration of mi R-375 expression can significantly inhibit the proliferation,invasion and metastasis of esophageal cancer cells.Which shows that mi R-375 is expected to become a candidate molecule for mi RNA gene therapy,but mi RNA have disadvantages including easily degraded,negatively charged,difficult to reach the tumor site and be absorbed by tumor cells in the complex environment.Therefore,urgent need to develop a safe and efficient targeted delivery system to deliver therapeutic genes / drugs for tumor treatment.As a natural nanocarrier,exosome has increasingly shown great potential for drug delivery.However,how to obtain numerous,uniform,stable,multi-component controllable exosomes become a obstacle that limits its further application.This subject innovatively uses natural single-cell algae-Dunaliella salina for large-scale preparation of exosomes.With the natural advantages of exosomes in drug delivery,it utilizes the high affinity of esophageal cancer cells for RGD polypeptides to construct high expression.The exosomes can targeted esophageal cancer cells and then loaded with therapeutic mi R-375 and DOX to achieve drug combined therapy targeted at esophageal cancer.Our further studies focus on the exosomes modification with RGD polypeptide,and loading / release of mi R-375 / DOX to esophageal cancer cells;in addition,the general rules of recognition,interaction and intracellular transport with esophageal cancer cells were revealed,and the problem of targeting in vitro were solved;and further investigate the ability of Dunaliella salina exosome to target codelivery and tumor suppression mechanism in a mouse esophageal cancer model.The results are as follows: Transmission electron microscopy and nanometer particle size analyzer characterization show that the size of Dunaliella salina exosomes extracted by ultracentrifugation is mainly distributed in the range of 122 ± 5 nm,and the "disc" morphology consistent with typical exosomes;the zeta potential was-1.51 ±0.23 m V,and the successful coupling of the RGD polypeptide to the Dunaliella salina exosome was confirmed by fluorescence colocalization technology.Next,the electroporation method was used to load the mi R-375 plasmid and the chemotherapeutic drug DOX onto the exosome;the best load rate of the plasmid and DOX were both at a voltage of 300 V and a capacitance of 250 μF: the mi R-375 plasmid The loading rate is 2.11 ± 0.17%,and the DOX loading rate is 2.24 ± 0.08%.The transmission electron microscopy and nanometer particle size analyzer results of the engineered Dunaliella salina exosomes after loading showed that the successful loading of peptides and plasmids slightly increased the size of the Dunaliella salina exosomes,but did not destroy the shape of Dunaliella salina exosomes.At the cell level,the DIO fluorescent dye-labeled Dunaliella salina exosomes modified with RGD peptides were observed to enriched in the cells,indicating the Dunaliella salina exosomes successful modification of RGD polypeptide can easier uptaked by esophageal cancer KYSE-150 cells;MTT results show that engineered exosomes(R-DExo/mi R-375/DOX)have a significant inhibitory effect on the proliferation of esophageal cancer cells;The cell migration and transwell assay results show that the engineered exosome delivery system significantly inhibits cell migration and invasion,which is significantly reduced compared to the control group.Further flow cytometry results showed that the cell cycle of KYSE-150 treated with RDExo/mi R-375/DOX was arrested in G1 phase,which resulting in a decrease in G2 phase and thus inhibiting the normal cell cycle.And the results of the apoptosis experiment also indicate that the engineered exosomes promote the apoptosis of esophageal cancer cells.Finally,we used Western Blot to analyze the expression level of cyclin E2(CCNE2),and found that the CCNE2 expression in R-DExo/mi R-375/DOX experimental group was significantly reduced.Next,we established ectopic esophageal cancer tumors in Balb/c nude mice to further research the tumor targeting and anti-tumor effects of engineered exosomes in vivo.The results show that RGD-modified exosomes can be efficiently enriched in tumor sites,indicating that engineered exosomes have excellent targeting ability in vivo.The monitoring results of tumor growth curve,tumor weight and mouse survival curve confirmed that R-DExo/mi R-375/DOX also had a significant inhibitory effect on esophageal cancer tumors.At the same time,pathological analysis of various organs in mice shows that the delivery system has good biocompatibility.In summary,this study successfully constructed a targeted drug delivery system based on Dunaliella salina exosomes through chemical coupling technology,and proved that the delivery system had tumor-specific targeting function and a good biological phase at the cellular and animal levels.Preliminary mechanistic studies have shown that mi R-375 could inhibit tumor cell cycle-related proteins to achieve synergistic effects with DOX.As an effective drug delivery vehicle,exosomes have the advantages of good biocompatibility and no immune rejection compared with other known drug delivery systems.However,how to prepare exosomes that are uniform,stable,controllable,and low in production cost is the bottleneck restricting its further application.This research provides a new idea for mi RNA drug delivery,and it could be more effective and specific for targeted therapy when combined with engineering exosomes.