Research On Construction Of Gene And Drug Co-delivery Platform Based On Conjugated Oligonucleotides

Cancer, as one of the great threats to human health, has induced a mortality rate of about 13 percent globally. And the morbidity and mortality are still rising remarkably. How to develop safe and efficient strategies for cancer treatment has become one great challenge for people worldwide. It has been demonstrated that the combination of chemotherapy and gene therapy has been reported to be an effective strategy for cancer treatment and could potentially achieve the synergistic effects of the two treatments, including enhanced gene transfection efficiency and anti-tumor effect of chemotherapeutic drugs.RNA interference (RNAi) is a powerful method for the regulation of gene expression and is utilized by injecting double-stranded RNA (dsRNA) into the body. RNAi induces the degradation of the corresponding messenger RNA (mRNA) and blocks the translation of mRNA into protein. RNAi, which developed new area for gene researchers and was emphasized by Nobel Prize Winners, has been widely applied in biomedical research for various disease types, such as viral hepatitis, cardiovascular diseases, HIV, and cancer. Because of high silencing efficiency induced by siRNA, combination therapy of RNAi and chemotherapy has become a hot topic recently.One of the greatest challenges for co-delivery is the construction of a platform to load the drug and siRNA simultaneously. Step-by-step assembly is the main method to construct the co-delivery platform, which is based on different loading mechanisms of drug and gene. Compared with step-by-step assembly, one-step assembly has good potential in simplifying the preparation process of co-delivery system. In this study, oligodeoxynucleotides with CGA repeating units (CGA-ODNs) were selected as carriers of the anticancer drug Doxorubicin (Dox). Based on the specific interaction between Dox and CGA-ODNs, we prepared the Dox loaded CGA-ODNs (CGA-ODNs-Dox). After obtaining the mixture of siRNA and CGA-ODNs-Dox, we employed cationic polymer polyethylenimine (PEI) to condense the mixture, obtaining the co-loaded system with one self-assemble step, abbreviated as PEI/CGA-ODNs-Dox&siRNA (PDR). The negatively charged copolymer o-carboxymethyl-chitosan-PEG-aspargine-glycine-argi-nine (CMCS-PEG-NGR, CPN) could be further coated on the surface of PDR, obtaining the CPN modified PDR (CPN-PDR).In this subject, vascular endothelial growth factor (VEGF) targeted siRNA and Dox were selected as the model gene and drug to prepare PDR and CPN-PDR. The preparation process, physical and chemical properties, cellular uptake and intracellular distribution of PDR and CPN-PDR would be characterized, respectively. After that, the cytotoxicity of materials and blank carriers, the in vitro cytotoxicity and the preliminary safety evaluation in vivo would be explored, supporting the in vivo research with some data. The main methods and results are as follows:1. Determination of Dox content in PBS with different pH valuesThe content of Dox in PBS with pH5.0, pH6.0 and pH7.4 was determined by fluorescence spectrophotometry in this part. The methodology was examined. The results showed that this method was easy, convenient and correct to determine the Dox concentration in vitro.2. Construction and characterization of the gene and drug co-delivery platformVEGF targeting siRNA and Dox was chosen as model gene and drug in this part. At first, CGA-ODNs-Dox was prepared by incubating CGA-ODNs solution with Dox solution. Fluorescent photometry was employed to explore the loading ratio and stability of CGA-ODNs-Dox. MTT assays were carried out to investigate the cytotoxicity of CGA-ODNs-Dox. Agarose gel retardation assays were carried out to explore the proper weight ratio of PEI to the ODNs&siRNA and weight ratio of CPN to the ODNs&siRNA, respectively. The morphology optimized PDR and CPN-PDR were characterized by transmission electron microscope (TEM). The particle sizes and zeta potentials were detected using laser particle size and potential analyzer, respectively. The release of Dox from CPN-PDR was assayed in PBS buffer with a pH of 5.0,6.0 and 7.4 using a dialysis bag diffusion technique.The optimized ratio of CGA-ODNs to Dox was measured to be 1:5. There was good stability of CGA-ODNs-Dox in 4℃ for 12h and instability in pH5.0, shown by the results of stability tests. The cytotoxicity of CGA-ODNs-Dox was confirmed to be comparable with free Dox on tested concentrations against A549 and HepG2 cells. The optimized weight ratios of PEI to ODNs&siRNA and CPN to ODNs&siRNA were 1:1 and 4:1, respectively. TEM images of both PDR and CPN-PDR displayed a type of analogous spherical shape and compacted structure with a narrow distribution. Size measurements showed an average diameter of (91.9±2.6) nm of PDR, which was (144.4±3.9) nm of CPN-PDR. The zeta potentials of PDR and CPN-PDR were (12.56±0.94) mV and (-7.04±1.72) mV, respectively. Additionally, the PDI of PDR and CPN-PDR was less than 0.3. The results of in vitro drug release showed that incubation of CPN-PDR at pH 5.0 led to a rapid release of Dox and the cumulative release of Dox at pH 7.4 was significantly lower than that of pH 5.0 (P<0.05).3. Evaluation of intracellular delivery of drug and geneFAM-labeled negative control siRNA and Dox were chosen as model gene and drug in this part. At first, flow cytometry was used to assess the expression level of CD 13 receptor on the surface of A549 and HepG2 cells, respectively. MTT assays were carried out to investigate the cytotoxicity of materials and blank carriers. Then, cellular internalization of PDR was observed by reverse fluorescence microscopy and quantified by flow cytometry. Media of different pH was prepared and the cell nuclei were labeled with Hoechst 33342 to explore the cellular uptake of CPN-PDR. The competitive targeted cellular uptake of CPN-PDR in A549 cells were visualized using confocal laser scanning microscopy (CLSM). Finally, lysosomes were labeled to investigate the intracellular distribution of Dox and FAM-siRNA.According to the results of flow cytometry, A549 cells were confirmed as CD 13 receptor positive cell line with expression level at 91.52% and HepG2 cells were confirmed as CD 13 receptor negative cell line with expression level at 2.24%. MTT assays verified that CGA-ODNs and CPN were biocompatible and non-toxic to cells. However, PEI was confirmed to be toxic to cells. Moreover, bPDR was tested to be much more toxic than bCPN-PDR, especially on the high concentrations. The quantitative data of flow cytometry demonstrated that Dox and FAM-siRNA could be co-delivered into both A549 cells and HepG2 cells by PDR with a high efficiency of 39.52% and 36.78%, respectively. The mean intensity of both green and red fluorescence of targeting cellular uptake were much higher than that of suppressed group, which were 1.90 times and 1.77 times, respectively. Dox was observed to escape lysosomes successfully and start to accumulate into cell nuclei at 1h and almost all enter cell nuclei at 2h. However, FAM-siRNA was observed to be most overlaid with lysosomes and start to escape lysosomes at 2h.4. In vitro cytotoxicity and preliminary safety in vivoMCF-7 cells were chosen as the model cell in this part. The cytotoxicity of materials and blank carriers were tested on MCF-7 cells using MTT assays. RealTime-PCR and western blot were employed to assess the expression level of VEGF mRNA and VEGF protein. In vitro cytotoxicity was investigated based on MTT assays. And finally, hemolysis test and tissue section method were displayed to evaluate the safety of nanoparticles in vitro and in vivo preliminarily, respectively.MTT assays witnessed that there was no obvious cytotoxicity of CGA-ODNs and CPN in 72h. The cytotoxicity of PEI was obvious, especially on high concentrations. And there was no remarkable difference between the cell viability of bPDR and bCPN-PDR at 24h, while, bPDR was observed to be toxic with time prolonging. The cell viability of bPDR was calculated to be remarkable lower than that of bCPN-PDR (P<0.05). The expression level of VEGF mRNA confirmed that the delivery of VEGF targeting siRNA could induce significant VEGF mRNA silencing effect. What’s more, it was calculated to be no arresting difference among Lipo/siRNA, CPN-PR and CPN-PDR. The image taken in western blot assay reflected that a remarkable reduction of VEGF protein compared with that of Control and bCPN-PDR, which was in accordance with results of RealTime-PCR. The in vitro cytotoxicity of CPN-PDR was significantly higher than that of Dox and CPN-PD, confirming the potential good anti-tumor effect of CPN-PDR. Hemolysis test implied that CPN-PDR was safe to intravenous injection without hemolysis. And the image of tissue section illustrated that there was no pathological change of the organs of mice which were administrated with CPN-PDR.In conclusion, with the conjugated oligonucleotides CGA-ODNs as drug carrier, we constructed the drug and gene co-loading platform based on one-step assembly. The co-loading nanoparticles PDR and CPN-PDR could co-load and co-deliver siRNA and Dox intracellular, and siRNA was observed to accumulate in cytoplasm and Dox into cell nuclei as expected. The one-step assembly technique could simplify preparation process of drug and gene co-delivery system. The obtained co-delivery system was confirmed with enhanced cytotoxicity against tumor cells. The preliminary evaluation of safety testified CPN-PDR with good biocompatibility, which has the great potential of further exploitation and clinical application prospects. With the development of aptamer technology, the promising application prospects of this novel oligodeoxynucleotides based co-loading platform will further increase.