Tissue Factor Targeted Nanopartcles Mediated Sirna Targeting Delivery

Tissue factor (TF) is best known as the primary initiator of blood coagulation. After vessel injury, the TF/FVII a complex activates the coagulation protease cascade, which leads to fibrin deposition and activation of platelets. In addition, aberrant TF expression within the vasculature initiates fetal thrombosis in various diseases, such as sepsis, atherosclerosis, and cancer. Recent studies have revealed a non-hemostatic role of TF in the generation of coagulation proteases and subsequent activation of protease activated receptors (PARs) on vascular cells. This TF-dependent signaling contributes to a variety of biological processes, including inflammation, metastasis, and cell migration. Previous studies had been showed the fusion protein EGFP-EGF1was affinity to TF, and EGFP-EGF1conjugated PLGA nanoparticles had the ability of targeting to TF.Small interfering RNAs (siRNAs) are known to guide sequence-specific gene silencing of target mRNAs to which they are perfectly complementary by directing the RNA induced silencing complex (RISC) to mediate site-specific cleavage, and, then, destruction of the targeted mRNA. They have been shown to effectively down-regulate gene expression in human cells, giving them potential to cure disease. However, the naked siRNA molecule, with negative charges, is susceptible to serum nucleases, renal clearance, and non-targeted bio-distribution, making cellular target sites more difficult to access. Thus, it has many limitations, such as poor stability, short half-life, and low efficiency. The major hurdle faced by current RNAi therapeutic strategies is the efficiency of the delivery system. Recently, viral delivery, such as lentivirus and adenovirus delivery, is highly efficient delivery systems for nucleic acids, but the potential for mutagenicity, limited loading capacities, and most importantly, safety risks caused by their inflammatory and immunogenic effects severely limit the applicability of viruses. Thus, the direct, systemic, non-viral administration of siRNA molecules that allows for therapeutic use is most desirable, that are capable of delivering nucleic acids, including genes, siRNA, or antisense RNA into cells. The non-viral siRNA delivery systems include chemical modification of siRNA, liposomes, nanoparticles for siRNA delivery, cell-penetrating peptides, and targeted delivery. With the progress of material synthesis and the rise of nanotechnology, the synthesis of nanoparticles with specific functions has become possible. The development of nanotechnology in the biological area has made nano-delivery systems popular. Due to their low toxicity, biodegradability, and biocompatibility, many nanomaterials are used as transfection carriers, such as chitosan, polyethyleneimine (PEI), poly(lactic-co-glycolic acid)(PLGA), magnetic nanoparticles, and carbon nanotubes. Because of its high nuclease resistance and mucoadhesive properties, has become popular siRNA vectors. Targeted RNAi therapy is a relatively new approach that can be used to silence genes in vivo by selective targeting. Targeting the diseased cell, organ, or tissue will increase the silencing potency of a given dose of siRNA. Specific cell targeting will also prevent side effects by avoiding other cells. Antibodies, small peptides, and other ligands that bind to the signature molecules with high specificity and affinity have been studied extensively for their ability to guide siRNA to the target tissues and cells.The double emulsion method has been used into synthesis of the EGFP-EGF1-PLGA nanoparticles (ENP), it is suitable to entrapment of water-solubility gene drugs. It can not only protect gene drugs, but also targeted delivery the gene drugs to intend tissue or cells. In view of above, the ENP is chosen for the vector of siRNA or shRNA, and targeted delivery the gene drugs to the injured cells and down-regulation of the gene expression. Due to the previous studies were about rat models, we want to verify whether the ENP has the ability to mice model of TF high expression in this study.Part1. The construction and characteristic of tissue factor specific siRNA loaded EGFP-EGF1conjugated PLGA nanoparticles.ObjectivesScreening out the best tissue factor specific siRNA (TF-siRNA); Entrapment the fragment into the EGFP-EGF1conjugated PLGA nanoparticles. Then, we test the characteristic of TF-siRNA-ENP.MethodsThe rat glioma cell line C6is chosen for screening out the siRNAs, because of the cells express TF natively. Transfecting cells use the transfection regnant lipo2000. And the TF mRNA and protein expression was tested by real-time PCR and western-blot respectively. Analysis of the expression of TF, the best siRNA was selected.The TF-siRNA-ENP was constructed by the double emulsion method. The zeta potential and particle size were tested by the particle size and zeta potential analyzer. The shape of nanoparticle was observed by normal electron microscopy, and the conjugated protein was observed by immune-electron microscopy. The inside siRNAs were extracted and the Drug Loading Capability (DLC) was calculated. The release rate of siRNAs in vitro was calculated in same way.ResultsSiRNA-638was the best one of the rat TF-siRNAs.The size of TF-siRNA-ENP was about100nm, the appearance was rounding and uniform. The zeta potential was about-11mV. The results of immune-electronic microscopy showed the EGFP-EGF1protein was covalently coupled to the nanoparticles’surface. The Entrapment Efficiency of siRNA was81.33%. The release of siRNA was no difference in the environment of different pH, and release quickly in6hours that the rate was about42%.ConclusionThe nanoparticle size of TF-siRNA-ENP was about100nm, and the protein was combined to nanoparticles’surface. The characteristic of drug loading capability and release in vitro was suitable to subsequent experiments. Part2. The targeting characteristic and in vitro evaluation of tissue factor specific siRNA loaded EGFP-EGFl conjugated PLGA nanoparticles.ObjectivesConfirm the targeting characteristic of the TF-siRNA-ENP. To evaluate the TF expression and TF activity of TNFa induced Brain microvascular endothelial cells (BMECs). And to evaluate the cytotoxity of the TF-siRNA-ENP in BMECs.MethodsThe primary BMECs from rats were extracted through double enzyme digestion method. The TNFa was chosen to be a inducer to establish injured endothelial cells model in vitro. The coumarin-6serving as nanoparticle probe, was entrapped in ENP to investigate its BMECs delivery characteristic. At the same time, CY3labeled siRNA to investigate its distribution in BMECs. The real-time PCR, flow cytometry and western-blot were detected to evaluate the level of TF expression. TF activity determined to evaluate the TF activity of intervened cells. The cytotoxity was determined by CCK-8.ResultsThe TNFa induced BMECs high expressed TF. The TF-siRNA-ENP was uptake better by the injured BMECs. Down-regulation of TF expression and TF activity in the process of injury. The TF-siRNA-ENP had almost no effect on cells’activity.ConclusionTF-siRNA-ENP is an effective delivery system, and can target delivery TF-siRNA into the injured BMECs. It has great safety and effective gene silencing.Part3. The target delivery of the EGFP-EGFl conjugated PLGA nanoparticles in atherosclerosis mice model.ObjectivesTo confirm the target delivery of the EGFP-EGF1conjugated PLGA nanoparticles in atherosclerosis mice model in vivo and in vitro. MethodsSynthesis of coumarin-6or Dir loaded ENPs through the double emulsion method. And their DLC was detected. MCP-1was chosen for inducing mice vascular smooth muscle cells (VSMCs), oxLDL was chosen for inducing mice mononuclear macrophage, the AS model of high TF expression was established in vitro. The AS mice model was established through feed with high fat diet for14weeks continuously. The fluorescence microscope and flow cytometry were determined to evaluate the targeting of ENP in vitro. To verify the AS mice model established, the oil O and immunofluorescence were carried out. Dir loaded ENP (Dir-ENP) was injected into AS model mice, and though organ imaging and confocal laser scanning microscope carried out to determine the targeting of Dir-ENP in vivo.ResultsThe two types of cells were induced to establish the AS model in vitro, and they expressed TF highly. ENP could be uptake better in the induced cells. There is statistical difference between the ENP and NP groups.The serum cholesterol in the mouse that fed with high fat diet was upgrade. The thickening of intima and deposition of the plaques could be seen in aorta. The organ imaging showed the injured position could uptake ENP better. The section results showed the ENP could coincidence with position of plaques and high TF expression.ConclusionThe EGFP-EGF1conjugated PLGAnanoparticles can target delivery to atheromatous plaque in atherosclerosis mice model in vivo and in vitro.Part4. The study of CCR2-shRNA loaded EGFP-EGF1conjugated PLGA nanoparticles in vitroObjectivesTo verify the CCR2-shRNA loaded EGFP-EGF1conjugated PLGA nanoparticles (CCR2-shRNA-ENP) could down-regulate the expression of CCR2in AS model in vitro, and inhibit the cell migration.MethodsTo determine the TF and CCR2expression in aorta of AS model mice through the double immunofluorescence and real-time PCR. To determine the best concentration and time of operation of inducer to establish the AS model in vitro through real-time PCR. To evaluate effect of CCR2-shRNA-ENP in these induced cells, the real-time PCR and western-blot were to be in progress. The cytotoxity of CCR2-shRNA-ENP was detected by CCK-8kit. The cells migration experiment were determined to evaluate the effect of CCR2-shRNA-ENP in cell migration.ResultsTF and CCR2expressed highly in aorta of mice AS model, and higher expressed at14weeks. lOng/ml MCP-1induced VSMCs for2hours, and50μg/ml oxLDL induced Raw264.7cells for1hour, to establish the AS model in vitro. The CCR2-shRNA-ENP could down-regulate the CCR2expression in these induced cells at the levels of mRNA and protein. And the CCR2-shRNA-ENP had almost no effect on these cells. At the same time, the migration of the cells was downgrade.ConclusionThe CCR2-shRNA-ENP could be an effective and selective delivery of CCR2-shRNA. It could be down-regulation of the CCR2expression and migration in AS model in vitro. Laying the groundwork for the following experiments.