EGFP-EGF1 Protein Conjugated Stealth Nanoparticle For Cerebral Thrombi Targeting Drug Delivery

Tremendous mortality and disability rate of cerebral thrombosis disease brought great sufferings of mankind. There are two main kinds of stategies currently:periphery intravascular administration and catheter-directed thrombolytic therapy. Both therapeutic options accompany of discouragingly high incidence of intracerebral hemorrhage and restenosis. Consequently, innovations for therapy of cerebral thrombosis have focused on safe, thrombus-specific and noninvasive anti-thrombotic agents.It is generally accepted that tissue factor (TF) is the key trigger of the coagulation cascade and TF interacts with either plasma coagulation factor FVII or FVIIa, and the resulting fibrin serves as a trigger for thrombosis formation in acute vascular events such as ischemic stroke and myocardial infarction. The interact domains of FVII are the first epidermal growth factor-like domian (EGF1) and the SP domain, the EGF1 has binding ability with TF and without procoagulant activity. In the previous research, We have produced EGFP-EGF1 fusion protein according to the structure of EGF1 and conjugated with PEG-PLA nanoparticle.It has shown cerebral thrombus targeting property by advantage of theTF-targeting property of EGFP-EGF1 fusion protein. In vivo multispectral fluorescent imaging demonstrated that EGFP-EGF1-NP had high specificity and sensitivity in targeting TF.It had been recently reported that NPs formulated using PLGA polymer demonstrated greater gene transfection than those formulated using PLA polymer. In this study, we developed a new anti-thrombotic drug delivery system, EGF1-EGFP protein-conjugated PEG-PLGA nanoparticles——GF1-EGFP-NP. The surface modified EGF1-EGFP can enhance the nanoparticles’infarction cortex entrance via receptor-mediated transcytosis; the biodegradable nanoparticle served as drug carrier, which can endow the drug delivery system with good safety and increase its drug loading capacity; the double-emulsion and solvent evaporation preparation method facilitated encapsulation of macromolecules such as proteins, peptides and genes, and can mask their physico-chemical characteristics instead of nanoparticle properties; the stealthy pegylated nanoparticle can avoid the uptake by reticuloendothelial system, prolong their plasma half-life, and enhance area under the concentration-time curve (AUC).The first part described the construction and characterization of EGF1-EGFP-NP. The pegylated poly lactide-poly glycolide nanoparticle, whose surface was co-modified with maleimide-polyethylene glycol (MAL-PEG) and methoxy-polyethylene glycol (MPEG), was made by double-emulsion and solvent evaporation method and covalently conjugated with thiolated EGFP-EGF1 via its maleimide function group and the ratio of Maleimide-PEG-PLGA:MPEG-PLGA was 1:10. The average particle size of EGF1-EGFP-NP was below 100 nm, and its zeta potential was around-15 mV. Immuno-gold staining result showed that bioactive EGFP-EGF1 was covalently coupled to the nanoparticle’s surface, and the surface protein number per nanoparticle was around 112.To evaluate the TF-targeted delivery characteristics and toxicity of EGF1-EGFP-NP, the second part cultured primary BCEC and stimulated TF expression with TNF-a. A lipophilic fluorescent dye, coumarin-6, serving as nanoparticle probe, was incorporated in EGF1-EGFP-NP to investigate its BCEC delivery characteristics. The accumulative release of coumarin-6 from EGF1-EGFP-NP and NP in pH 7.0 PBS after 24 h were all below 3.62%, which proved that coumarin-6 is an ideal nanoparticle probe that can indicate accurately the in vitro behavior of EGF1-EGFP-NP and NP. EGF1-EGFP-NP was uptaken more efficiently by the TF abnormal expression BCEC than NP, which probably was transfected depending largely on the combination of EGF1 and TF. CCK-8 assay illustrated that there were no significant difference in IC20 and IC50 between EGF1-EGFP-NP and NP. All the in vitro results showed that EGF1-EGFP-NP is a good TF-targeted delivery system with very low toxicity.The third part employed photochemical cortex thrombosis rats for further investigation.At first, coumarin-6 serving as nanoparticle probe, and the cerebral infarction region delivery property of EGF1-EGFP-NP was evaluated by pharmacokinetics studies with NP as control. The biodistribution result in rats revealed the brain drug targeting index of EGF1-EGFP-NP was 2.58 compared with NP. The distribution of EGF1-EGFP-NP to the lung and kidney was increased, and decreased in the liver compared with NP. A near infrared (NIR) lipophilic fluorescent dye, Dir, serving as nanoparticle probe, was incorporated in EGF1-EGFP-NP to investigate its distribution characteristics in vivo. In vivo multispectral fluorescent imaging demonstrated that the TF-expressing regions of the model rats injected with EGF1-EGFP-NP exhibited higher fluorescence than those of the model rats treated with NP, which was around 2 times more than the NP groups during the 24 h after i.v. administration, and the highest difference was 2.66 times at 1 h. The EGF1-EGFP-NP was predominantly distributed in the areas adjacent to the illuminated regions and only existed in the vessels of the infarction cortex, which were restricted to the tissue factor on the vessel wall. TEM of microvaculature proved that it was not only exhibited in the microvilli, but also transfected in the microvesicles and further entered the nuclei of endothelial cell. High dose of EGF1-EGFP-NP didn’t interfere the coagulogram of SD rats in the acute toxicity test. Immunostaining of monocyte-macrophage demonstrated that high dose of EGF1-EGFP-NP didn’t induce the increase amount of macrophage in cerebrum, heart and liver in SD rats, only had light toxicity to lung, spleen and kidney, and its acute toxicity was transient. These results proved the cerebral thrombi delivery property and safety of EGF1-EGFP-NP in vivo.To achieve the targeting therapy to the cerebral thrombosis disease, the fourth part described that EGF1-EGFP-NP was used as cerebral thrombi drug delivery carrier to encapsulated the NFκB DECOY oligonucleotides (ODNs), termed as EGF1-EGFP-NP-ODNs gene therapy system. The characteristic examination proved its volume-based particle size was 105.5±10.02 nm; Zeta potential was-17.23±1.49 mV; drug loading capacity was 0.23% and entrapping efficiency was 55.75%. The preparation procedure cannot cause significant fragmentation of ODNs. By using coumarin-6 and CY3-ODNs as the fluorescent probe, the investigation result of uptake of EGF1-EGFP-NP-ODNs and NP-ODNs by TF abnormal expression BCEC revealed that the uptake of EGF1-EGFP-NP-ODNs was higher than NP-ODNs and the ODNs was delivered into the nuclei. The result of Real Time PCR showed that EGF1-EGFP-NP-ODNs can more efficiently intefere the TF m RNA expression induce by TNF-αcompared with the nake ODNs and NP-ODNs, which was consequence with the result of Western blotting. By using Dir as the fluorescent probe, the mean fluorescence intensity of EGF1-EGFP-NP-ODNs group was more than 4 times that of the NP-ODNs group at the cerebral infarction regions.Immunostaining of TF demonstrated that EGF1-EGFP-NP-ODNs notably inhibited the endothelium TF expression and more effective than the control and NP-ODNs (P=0.002 and P=0.023, respectively) at 6 h after illuminatin and drug adminstration. MRI analysis showed that EGF1-EGFP-NP-ODNs remarkably palliated the cerebral infaction and was more advantageous than the control and NP-ODNs (P=0.004 and P=0.015, respectively). Therefore, EGF1-EGFP-NP-ODNs obvious interfere cerebral thrombosis by advantage of its TF targeting ability, and it may also be applied in the treat of a variety of TF-related diseases.