Construction,Expression And Cell Evaluation Of Intelligent Albumin Fusion Protein Drug Delivery System

In this study,RGD-HSA-MMP-18His(RHMH18)fusion protein was used as the drug carrier,and the anti-tumor drug DOX and immune adjuvant Cp G ODNs were simultaneously loaded to prepare RHMH18-Cp G-DOX(RCD)nano-delivery system.Due to the up-regulated expression of ανβ 3 integrin specifically bound to RGD in tumor vascular endothelium,the fusion protein RHMH18 with RGD tag can actively target tumor tissue,resulting in the enrichment of therapeutic drugs in tumor site.And the responsive drug release of MMP-2 enzyme can be achieved in the tumor microenvironment.Subsequently,the dissociated RGD-HSA-DOX was internalized by tumor cells,and DOX could induce tumor cells apoptosis and the release of tumor-associated antigens.The release of His-loaded Cp G ODNs can trigger the expression of toll-like receptors 9 cells such as macrophages and dendritic cells such as antigen presenting cells,and start the signal cascade,proinflammatory cytokines,such as tumor necrosis factor(TNF-α),interleukin-6(IL-6)and interleukin-12(IL-12)such as expression,resulting in a strong anti-tumor immune response,so as to realize the synergistic effect of immunity and chemotherapy.The project is mainly divided into three stages:In the first part,the vector protein RHMH18 was prepared and characterized.RHMH18 fusion protein was expressed by Pichia pastoris.Western Blot was used to identify the fusion protein with RGD tag,HSA tag,His tag and MMP-2 enzyme responsiveness.The second part is the preparation and characterization of the delivery system.RHMH18,Cp G ODNs and DOX construct the RCD nano-delivery system at the ratio of 40 : 5 : 8,Cp G ODNs were loaded on the his end of RHMH18 by electrostatic adsorption,while DOX is loaded in the cavity of HSA.The results of UV-Vis spectra,Zeta potential and agarose gel electrophoresis showed that Cp G ODNs and DOX were successfully loaded.TEM and DLS showed that the average particle size of the nanoparticles was 40±5 nm.After incubated with MMP-2 enzyme for 24 h,the nanoparticles were digested into RGD-HSA-DOX and His-Cp G two parts by the enzyme.The results of drug release showed that under the condition of pH =6 and MMP-2 enzyme,the cumulative release amount of DOX at 72 h was 56.00%,which was about 5.8 times of the total release amount of DOX under the condition without stimulation.The cumulative release of Cp G ODNs at 72 h was 73.55%,which was about 4.1times of that under the non-stimulation condition.This indicates that the delivery system is pH responsive and MMP-2 responsive,showing good therapeutic selectivity.The third part of in vitro cell experiment,lung cancer cells(A549)with high expression of RGD and MMP-2 enzymes were used as tumor cell model,and macrophages(RAW 246.7)were used as normal cell model.Compared with free DOX group,the survival rate of RAW264.7 cells in RHMH18-DOX group and RCD group loaded with RHMH18 vector was significantly improved.When RHMH18 and RCD were at 700 μg/m L,the hemolysis rate of erythrocytes was still less than 2%.This shows that RHMH18 can effectively reduce the toxicity of antineoplastic drug DOX to normal cells,and the delivery system has good biocompatibility.The cytokine secretion of TNF-α and IF-6 in RCD treatment group was significantly higher than that in other groups.When A549 cells were co-cultured with RAW264.7 cells,the survival rate of A549 cells in RCD treatment group was significantly lower than that in other groups,indicating that the delivery system has RGD targeting and MMP-2 response,and can play a synergistic effect of chemotherapy and immunotherapy.