Stem Cells-based Novel Gene Delivery System And Its Application And Mechanism Studies In Tumor Treatment

Cancer gene therapy was emerged as a promising approach for the adjunctive treatment of anti-cancer therapy and has attracted wide attentions in recent years. Nevertheless, the success of this therapeutic strategy is still restricted by the currently available gene delivery methods. For instance, in suicide cancer gene therapy, to achieve an efficient therapeutic results, the therapeutic genes need to overcome physiological barriers to be delivered to the target tumor cells to obtain a high level of specific protein expression. Moreover, genes should be well protected to resist degradation in circulation before they reach the target tumor cells. Thus, high demands are made for the gene delivery system and a sophisticated carrier is required.To solve the inherent gene therapy delivery problems, we proposed a novel solution that by using the living cell as a "smart" bioactive carrier, which based on the concept of target drug deliverly system (TDDS). In the present studies, bone-marrow mesenchymal stem cell (BMSC) was chosed as the potential candidate carrier for delivering the therapeutic genes target to tumor sites. This is because BMSC possesses several unique natures, which make them extremely meet the high demands for the targeting delivery of genes. For example, the tumor-homing capacity of BMSC, enabling them to migrate toward and engraft into the tumor sites. Moreover, the non-immunogenic nature of BMSC allow them to avoid the attacks from the immune system and protect the carried genes in circulation. Therefore, the current studies explored the migration and penetration of BMSC to the melanoma and the feasibility and efficacy of using BMSC as a bioactive gene carrier for cancer gene therapy, as well as its safety evaluation.In order to enable BMSC to carry the theraputic gene efficiently and safely, several novel non-viral gene transfection vectors that previously reported by our group were tested and screened, as well as some commercial transfection agents. Results turned out that the spermine modified pullulan (SP) had the highest transfection efficiency and relative lower cytotoxicity on BMSC among all the tested non-viral gene transfection vectors. Further, the SP-based transfection system was optimized for further enhancing the gene expressing level. Additionaly, the cellular distribution of SP/pDNA complexes and the degradation of SP after endocytosis by BMSC were investigated to understand the mechanism and potential risks of SP for gene trasfection.Then, the tumor-tropism of BMSC to B16F10 mouse melanoma cells was investigated both in vitro and in vivo to ensure the feasibility of using BMSC as the carrier to deliver genes target to melanoma cells. Firstly, the in vitro transwell tests were applied to observe the migration of BMSC to B16F10 tumor cells. Then, BMSC was labled with a fluorescence probe and its distribution in tumor-bearing mice after systemic delivery was monitored, as well as its local distribution in tumor-bearing lungs, to demonstrate that BMSC has well tumor-homing capability after systemic delivery.In order to demonstrate the attractiveness and therapeutic efficacy of this cellular carrier in cancer gene therapy, the gene recombinant BMSC was applied as the theraputic gene carrier in a classical suicide gene therapy, which is consisted of herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV). Results turned out that BMSC was successfully tranfected to express HSV-TK through the SP-based non-viral gene transfection system. Additionally, this gene recombinant BMSC induced effective suicde effect on BMSC itself in the presence of GCV in a dose-dependent manner, and exerted a sufficient bystander effect on B16F10 tumor cells. Further studies found this bystander effect relied on the connect between BMSC and melanoma cells, and its efficacy was dependented on the cell number ratios between BMSC and B16F10 tumor cells. More recombinant BMSC means more effective bystander effect on the inhibition of B16F10 tumor cells’growth. In vivo studies further confirmed the well tumor inhibition effects after using BMSC as the carrier to deliver the suicde gene to tumor cells in a murine melanoma lung metastasis model.The present studies further designed a novel delivery strategy that co-administrates the gene recombinant BMSC with prodrug-encapsulated liposomes for synergistic anti-tumor effects. With this combination delivery strategy, both the suicide gene expressing level and the concentration of the prodrug in tumor sites were increased, which contributed to the enhancement of the therapeutic efficacy and reducing the potential side effects. The GCV-encapsulated liposomes were prepared through a modified reversed-phase evaporation method. Results demonstrated that the liposomes increased the locally GCV concentration in lungs, and could deliver the loaded drug to the area where BMSC and tumor cells were loacted. The in vitro theraputic effect of this delivery strategy was investigated in a 3D tumor spheroid model. The volume increases of tumor spheroids were significantly inhibited after the treatment with the gene recombinant BMSC and GCV-encapsulated liposomes. Further, the in vivo theraputic effect was investigated in a murine melanoma lung metastasis model, and results turned out that this co-targeting delivery strategy could avchieve an effective anti-tumor effect, which was two-times effective than using GCV solution with the gene recombinant BMSC in decreasing the tumor colonization in lungs. And this treatment also increased the mice’s survival rate significantly. Moreover, TUNEL-staining assays revealed that the highest apoptotic index was induced by the treatment with the co-targeting delivery strategy. Therefore, by delivering both the suicide gene and prodrug target to tumor sites can further enhance the theraputic efficency of this suicide gene therapy.Furthermore, the penetrability of BMSC into tumor tissues was investigated. This is because the accession of the therapeutic gene into the tumor tissues is also critical to achieve a successful cancer gene therapy. The present studies established an in vitro 3D multicellular tumor spheroid model to mimic the natural tumor tissue microenvironment, and this model was applied to evaluate the penetrability of BMSC by confocal laser scanning microscope. Results demonstrated a well penetrability of BMSC into the center of the tumor spheroid after being co-cultured for 72 h. While, for other kind of cells, like HEK-293 cells, despite being co-incubated with tumor spheroid for 72 h, these cells still can hardly penetrate into the tumor spheroid. Further, the green fluorescence expressing BMSC (GFP-BMSC) was used to monitor the penetrability of BMSC into tumor tissues in vivo. Green fluorescence signals were found in the inside of tumor tissues obviously 15 days after the initial injection of GFP-BMSC through the tail vein. Additionly, the iron oxide nanoparticles modified with ethylenediamine-pullulan (IONPs@ED-pul) was prepared, and these nanoparticles was applied for labeling BMSC. The labled BMSC can be stained by Prussian blue staining specially. Thus, the migration and penetration of BMSC into tumor tissues after systemic delivery can be clearly monitored. The stained tissue sections clearly demonstrated that the injected BMSC would migrate to and then penetrated into the tumor tissues gradually. These results further consolidated the penetrability of BMSC into tumor tissues.At last, the potential risks of using BMSC as a tumor-targted carrier were evaluated. The present studies demonstrated that the systemic delivery of large amount of unrecombinant BMSC (over 1×106 cells per mouse) in tumor-bearing mice may contribute to the tumor progress. Meanwhile, for the suicide gene recombinant BMSC, after the treatment with both GCV solution or GCV-encapsulated liposomes, the tumor growth was inhibited. Moreover, neither mice deaths nor significant body weight changes post the above treatments were found. Since the BMSC distributed mainly in lung and liver after systemic delivery, the pulmonary toxicity and hepatotoxicity were then investigated. No significant changes of serum alanine aminotransferase (ALT) or aspartate aminotransferase (AST) level were found before and after the treatments. Althoug mild inflammatory reactions were found in both lung and liver sections after the treatments, no significant morphological changes of lung cells or liver cells post the treatments were observed. Furthermore, the short-term and long-term side effects on lung cells post the treatments with gene recombinant BMSC and GCV solution or GCV liposomes were observed. No systemic toxicities were found post the treatments. And only mild damages on lung cells were found 2 days after removing the treatments, but these damages were hardly found 90 days after removing the treatments, which means such negative effects were reversible. Therefore, the present studies demonstrated that the risks for recruiting BMSC as the tumor-targeted gene delivery carrier were relative low.The current studies laid theoretical and experimental foundations for the development of a stem cell-based gene delivery system and its application in cancer gene therapy.