Preparation Of Tissue Factor Targeted Protein Nanoparticle And Its Application In Antithrombotic Therapy

Thrombotic disease are a class of serious harm to human health, it has become the main cause of mortality and disability in China and Western countries. It mainly containes arterial thrombosis and venous thrombosis disease, often reffers to multiple body organs, and involves in many clinical disciplines. Although great progress has been made in the anti-thrombosis drug research, but the risk of bleeding and restenosis makes the choice of antithrombotic drugs still controversial. In addition, when medical treatment for patients with cardiovascular and cerebrovascular diseases, formed thrombi often leads to embolism in vessel, and the treatment of these organs was noneeffective after ischemia. Thus, in the anti-thrombosis treatment, it holds great significance to develop low side effects and high specificity antithrombotic agents for the early treatment.Tissue factor is a transmembrane glycoprotein with a molecular weight of 47kD, it is served as a receptor of plasma coagulation factorⅦto trigger the extrinsic coagulation process. Some results show that, in addition to the effect in normal hemostasis, TF plays an important role in cerebral thrombosis, atherosclerosis, acute coronary syndrome, deep vein thrombosis, disseminated intravascular coagulation, inflammation and tumor. The TF-targeting stratey is very promising in the treatment of thrombotic disease. Currently, the studies on TF and its ligand FⅦhas found that the first epidermal growth factor-like domain(EGF1) of FⅦis important to mediate the combination between·TF and FⅦ, the EGF1 peptide has the binding ability with TF but no-procoagulant activity, so it can be used as a TF-targeting functional group based on the ligand/receptor interactions.Nanotechnology is one of the most popular technologies in targeted drug delivery study. In the nanoparticle-mediated strategy, nanoparticles are served as the transferring carrier to encapsulate different agents, including plasmid DNA, proteins, and low molecular weight compounds. Then different targeting moieties are put on the surface of the nanoparticles, which are then physiochemically or biologically directed to the targeted lesions where they can achieve relatively high concentrations. In the present study, the targeted nanoparticles mediated anti-tumor therapy has achieved significant results, while nanoparticles mediated anti-thrombotic therapy is not common, but it would become actual and possible to use nano-targeted therapy in the diagnosis and treatment of thrombotic diseases along with the maturity of nanotechnology.In this study, we try to apply nano technology and biomedical technology in the targeting treatment of thrombotic disease. A novel drug delivery system was developed by maleimide-mediated conjugation of PEG-PLA nanoparticles with TF-targeting protein. With the genetic intervention, we use the nanopraticles to deliver NF-κB decoy ODNs to the TF upstream promoter transcription factor, and to validat the bio-target effect in vitro and in vivo. In this way, we realize the purpose of targeting intervention of thrombi.Objective To construct the eukaryotic expression vector pET28a-EGFP-EGF1 and express EGFP labled EGFP-EGF1 fusion protein for further study on biological function of EGF1 peptide.Methods The EGF1 gene region was amplified from a rat liver by RT-PCR. A fusion expression vector named pET28a-EGFP-EGF1 was constructed by inserting the EGF1 gene into plasmid pET28a-EGFP.The expression of fusion protein was induced by IPTG. His-Ni+ chromatographic column was employed to purify the fusion protein. Western blotting and mass spectrometry were applied to identify EGFP-EGF1 protein.Results The EGF1 gene region was succesfully amplified, restriction digestion and PCR screening showed that EGF1 gene was cloned into pET28a-EGFP exactly. The EGFP-EGF1 proteins were efficiently expressed in E.coli. The protein yield in BL21 was about 1.2-1.6 g/L. Mass spectrometry determine the purity of protein was 84.6%. SDS-PAGE showed that the relative molecular mass of the fusion protein was about 36 kD.Conclusions The eukaryotic expression vector pET28a-EGFP-EGF1 was constructed successfully, and the EGFP-EGF1 fusion proteins waere efficiently expressed in E. coli BL21 cells.Objective To explore the binding function of EGFP-EGF1 protein with tissue factor and its ability of triggering coagulation. To set up the experimental base for tissue factor targeted drug delivery system.Methods The TF expression model in vitro was established by lipopolysaccharide induction. The binding ability of EGFP-EGF1 with TF-expressing cells was analyzed by using fluorescence microscope, confocal microscope and flow cytometer. The affinity of EGFP-EGF1 and rat soluble TF was quantitated by using a surface plasmon resonance instrument. The ability of EGFP-EGF1 in triggering coagulation was tested by prothrombin time assay using an automatic blood coagulation analyzer.Results The FCAS results showed rFⅦcould definitely depress the integration of EGFP-EGF1with rTF (68.65±3.86% VS 57.98±4.71%, P< 0.01). The SPR results indicated the association constant ka of EGFP-EGF1 proteins was higher when compared with rFⅦ(8.29±1.39 VS 3.75±0.32, P<0.01). However, the EGFP-EGF1 protein lost the activity of triggering coagulation when compared with that of blood plasma of normal SD rats (56.8±3.2 s VS 17.8±3.4 s, P<0.01).Conclusions The TF expression in vitro model was successfully established, it provides an experimental platform for binding ability study. EGFP-EGF1 protein can specifically bind to TF and competitive binding with rat coagulation with rFⅦ.The EGFP-EGF1 protein has no ability of triggering coagulation.Objective To construct EGFP-EGF1 protein conjugated nanoparticles and characterize its physical and chemical properties.Methods PEG-PLA nanoparticles were prepared by using the double emulsion and solvent evaporation method. The EGFP-EGF1 protein fractions were collected and introduced thiol groups. EGFP-EGF1-NPs were prepared by incubating the purified thiolated EGFP-EGF1 with the NPs. Tracer dye (Dir) and Oligonucleotides(decoy ODNs) were incorporated into the EGFP-EGF1 protein modified PEG-PLA nanoparticles. The mean diameter and zeta potential of the nanoparticles were determined by dynamic light scattering (DLS) by using a Zeta Potential/Particle Sizer NICOMPTM380 ZLS (Pss. nicomp particle size system, USA). The nanoparticles were morphologically examined by transmission electron microscopy. The encapsulation efficiency and release rate of nanoparticles were examined by centrifugation methods.The cytotoxicity of nanoparticles on RAOEC was evaluated by following the instructions of cell counting kit-8 (CCK-8).Results TEM photograph showed that the NPs, EGFP-EGF1-NPs, Dir-loaded EGFP-EGF1-NPs were generally spherical and of regular shape. The existence of EGFP-EGF1 on the surface of EGFP-EGF1-NP was verified by TEM observation. had an average diameter of about 87 nm and the diameter was increased to approximately 92 nm after EGFP-EGF1 conjugation. The zeta. potential values of blank NP and EGFP-EGF1-NP ranged from -10 mV to-20 mV. The DLE of Dir/ODNs loaded EGFP-EGF1-NP was 2.113±0.066%/0.16125±0.01375%. The release rate of Dir was 0.96±0.30% to 1.56±0.50% from 1 h to 4 h, the release rate of ODNs was 40.60±4.05% to 45.99±4.71% from 48 h to 96 h. There was no significant difference in the toxicity among the different groups at concentrations under 5 mg/ml (P>0.05). Cell viability was slightly reduced at higher concentrations.Conclusions In this study, a novel drug delivery system was developed by maleimide-mediated conjugation of PEG-PLA nanoparticles with EGFP-EGF1 protein. The nanoparticles with low cytotoxicity can be used to carry drugs(Dir or ODNs). Objective To evaluate the binding ability of EGFP-EGF1-NPs with TF-expressing cells in vitro and thrombosis regions in vivo. To explore the possibility of taking the nanoparticles as a TF-targeting delivery system.Methods TF expression model in vitro was established by lipopolysaccharide induction, Thrombosis model in vivo was induced by photochemically in young male rats. The binding ability of nanoparticles with TF-expressing cells was analyzed by using fluorescence microscope, confocal microscope and flow cytometer. The thrombosis targeting function and in vivo distribution of nanoparticles were carried out by fluorescence observation and In vivo multispectral fluorescent imaging.Results Cerebral thrombosis model was successfully established and TF expression in the thrombus regions were identified. EGFP-EGF1-NP could specifically bind to TF on the cell surface were certificated by fluorescence microscopy. FACS analysis also showed that the percentage of fluorescence ratio cell was about 65.23%, and the fluorescence intensity of the treated cells gradually increased with rising concentration of EGFP-EGF1-NPs. The animals injected with Dir-loaded NPs showed high fluorescence on heads, livers and lungs, while no fluorescence was detected in the model rats that were not injected with Dir-loaded NPs. The TF-expressing thrombus regions of the model rats injected with Dir-loaded EGFP-EGF1-NPs exhibited higher fluorescence than those of the control rats injected with Dir-loaded EGFP-EGF1-NPs and model rats treated with Dir-loaded EGFP-NPs.Conclusions The in vivo thrombosis formation model was successfully established. The EGFP-EGF1 modified PEG-PLA nanoparticles was certificated to be able to binding with TF-expressing cells in vitro and thrombosii region in vivo. The nanoparticles were distributed in thrombosis region, spleen, liver and lung. Objective To detect the expression of TF and thrombosis formation after being interfere by EGFP-EGF1 modified PEG-PLA nanoparticles mediated oligonucleotides.Methods The TF expression model in vitro was established by lipopolysaccharide induction. The expression of TF in cell nodels was measured by RT-PCR and Western blotting. Thrombosis model in vivo was induced by photochemically in young male rats. The effect of nanopartiles to thrombosis formation of was deternined by immunohistochemistry.Results The synthesized decoy ODNs was confirmed to be able to down-regulate the expression of TF mRNA and protein. The expression of TF was also down-regulation by incubating with EGFP-EGF1 modified PEG-PLA nanoparticles carrieded decoy ODNs. The cerebral thrombosis was significantly reduced by injecting with EGFP-EGF1 modified PEG-PLA nanoparticles carrieded decoy ODNs.Conclusions Decoy ODNs depressed the TF expression in vitro, and it's more significant to use the decoy ODNs carried by EGFP-EGF1 modified PEG-PLA nanoparticles. Prophylactic use of decoy ODNs carried by EGFP-EGF1 modified PEG-PLA nanoparticles can depress the cerebral thrombosis in rat models.