Development Of Intravenous Delivery Technology Of Oncolytic Virus Coated With PH Sensitive Liposome Hybrid Erythrocyte Membrane And Its Role And Mechanism In Tumor Therapy

Objective:With the improvement of the global medical and health system and the increasing abundance of medical resources,the average life expectancy of the world has steadily increased.But as life expectancy increases,so does the incidence of aging-related diseases,such as malignant tumors,associated with an aging population.The latest statistics on malignant tumors in China show that there are about 3.925 million new cancer cases in China,and the age-standardized incidence rate is 186.39/100,000.Although traditional treatments such as surgery,chemotherapy and radiotherapy have made great strides in recent years,they have many side effects and limited efficacy.Therefore,it is of great significance and value to develop new diagnosis and treatment methods for malignant tumors.As a new immunotherapy,oncolytic virus therapy has attracted great attention in recent years.Many academic institutions and companies have made great efforts to promote the clinical translation of oncolytic viruses(OVs).Currently,OVs has a number of approved products on the market,two of the more well-known products are the US Food and Drug Administration-approved T-VEC and the Chinese Medical Products Administration-approved Ancourt.In addition,dozens of OVs products are undergoing clinical trials and are also expected to be approved for clinical oncology treatment.Despite the rapid progress of basic and clinical research on OVs,the clinical translation of OVs is still confronted with many difficulties.Among them,how to deliver OVs effectively to tumor site is a crucial problem.Currently,the most common method of OVs administration is intratumoral administration.Although intradermal injection of OVs can improve the viral concentration at the tumor site,due to dense tumor tissue structure and high interstitial pressure,the virus can not spread well in the tumor tissue after administration,which affects the anti-tumor efficacy of OVs.In addition,intradermal injection of OVs often requires the use of ultrasound,computed tomography,magnetic resonance imaging and other technologies to assist and guide the administration of OVs,which is complicated,risky and expensive.Moreover,single injection of OVs in primary tumor is difficult to achieve the therapeutic effect on metastatic tumor.Compared with intratumoral injection,intravenous OVs has two major advantages in the treatment of tumors:first,intravenous injection overcomes practical difficulties and poor patient compliance caused by intratumoral injection.Second,intravenous injection can effectively deliver the oncolytic virus throughout the body.Although the intravenous delivery of OVs has many advantages,the existing specific antiviral neutralizing antibodies in patients and the innate immune response caused by intravenous injection of OVs also make the intravenous delivery of OVs a bottleneck at present.Biomimetic nanomedicine delivery is to impart some characteristics of organisms or cells to nanomedicine so that nanomedicine can have corresponding functions.Previous studies have proved that by covering the surface of nanomaterials with erythrocyte membrane,nanomaterials can acquire the characteristics of red blood cells,reduce the phagocytosis of nanomaterials by the mononuclear macrophage system in vivo and non-specific binding with non-target organs,and significantly increase the presence time of nanomaterials in vivo.Therefore,we believe that biofilm encapsulation of oncolytic virus can also reduce phagocytosis by the mononuclear macrophage system in vivo and reduce neutralization of antibody complement,thus improving the delivery efficiency of oncolytic virus and improving efficacy.In this study,we constructed pH-sensitive liposome hybrid erythrocyte membrane based on artificial liposome and erythrocyte membrane,and encapsulated a new type of oncolytic adenovirus 11 subtype(ad11),and obtained a novel membrane-encapsulated virus preparation(Ery-ad11 for short)for intravenous delivery of oncolytic virus.In vivo and in vitro experiments confirmed that pH-sensitive liposome hybrid erythrocyte membranes have good shielding effect on the surface antigens of ad11.In subsequent pharmacodynamics experiments based on subcutaneous tumor model and metastatic tumor model,we proved that Ery-ad11 showed good antitumor activity.Our study leads to the development of a novel intravenously administered oncolytic virus preparation and provides a reference for other membrane-encapsulated biological nanoparticles.Methods:1.Firstly,pH-sensitive lipsome(Lip)and erythrocyte membrane(RBCs)were prepared by membrane hydration method and low osmotic centrifugation method respectively,and pH-sensitive liposome hybrid erythrocyte membrane(RM-PL)was further prepared.We then used ultra-high resolution confocal microscopy to explore the best way to prepare membrane-enveloped viruses and further prepared liposome enveloped ad11(Lip-ad11),erythrocyte membrane enveloped ad11(RBC-ad11)and RM-PL enveloped ad11(Ery-ad11)by this method.Subsequently,transmission electron microscopy and nanoparticle tracking analyzer were used to characterize each preparation.2.The shielding ability of various membranes to ad11 surface antigen was demonstrated by detecting the uptake amount of Lip-ad11,RBC-ad11,Ery-ad11 and natural non-coated ad11(bare ad11)by macrophages and binding amount of neutralizing antibody.3.Cell uptake assay and flow cytometry were used to verify the pH sensitivity of Ery-ad11.4.By detecting the circulation profile of Lip-ad11,RBC-ad11,Ery-ad11 and bare ad11 in peripheral blood of mice after intravenous injection,the shielding ability of various membranes to ad11 surface antigen was demonstrated in vivo.5.We used small animal imaging system and qPCR test to detect the organ distribution and tumor enrichment ability of each preparation.6.Finally,the anti-tumor ability of each preparation was verified in subcutaneous tumor model and lung metastatic tumor model mice,and immunological indexes such as inflammatory factors were detected to demonstrate the immune activation ability of each preparation.Results:1.We first prepared pH-sensitive liposome heterozygous erythrocyte membranes,and explored the best encapsulation method of membrane-enveloped viruses by ultra-high resolution confocal microscopy.The results showed that ultrasonic combined extrusion was the best way to prepare virus.Subsequently,Lip-ad11,RBC-ad11,Ery-ad11,bare ad11and RM-PL were characterized by transmission electron microscopy,which confirmed the successful preparation of each preparation.2.We compared the uptake of RBC-ad11,Lip-ad11,Ery-ad11 and bare ad11 by macrophages and their binding with anti-ad11-neutralizing antibodies in vitro.The results showed that the uptake amount of Ery-ad11 by macrophages and the binding amount of anti-ad11 neutralizing antibodies were lower than those of other groups,which proved that RM-PL had better shielding effect on the antigens on the surface of ad11 than liposomes and erythrocyte membranes.3.The results of qPCR assay and flow cytometry assay showed that Ery-ad11 had pH sensitivity.The results showed that RM-PL on the surface of Ery-ad11 could cleave at pH6.5 and release its coated ad11.4.Compared with Lip-ad11,RBC-ad11 and bare ad11,Ery-ad11 had longer peripheral circulation time.The results in vivo confirmed that RM-PL had better shielding effect on the antigens on the surface of ad11 than liposomes and erythrocyte membranes.5.We detected the enrichment of RBC-ad11,Lip-ad11,Ery-ad11 and bare ad11 in tumor-bearing mice by qPCR,and found that the enrichment of Ery-ad11 in liver and spleen was significantly lower than that in other groups,while the enrichment of Ery-ad11 in tumor was significantly higher than that in other groups.6.We verified the anti-tumor efficacy of each preparation in a mouse subcutaneous tumor model.The results showed that Ery-ad11 had the best antitumor effect.After Ery-ad11treatment,the contents of CD4~+and CD8~+T cells in the tumor were significantly increased,and the autophagy level and the expression levels of IFN-γ,TNF-α,IL-6 and PD-L1 in the tumor tissues were significantly increased.7.We further confirmed that Ery-ad11 had the best anti-metastatic effect in the mouse lung metastatic tumor model.These results further confirm the advantages of Ery-ad11 in tumor therapy.Conclusions:RM-PL had stronger shielding effect on the antigens on ad11 surface than liposomes and erythrocyte membranes.The encapsulation of RM-PL on ad11 effectively reduced the binding of neutralizing antibody and clearance of ad11 by macrophages caused by the antigen exposure of ad11,thus prolonging the circulation time of ad11 in peripheral blood.Ery-ad11 effectively increased the distribution of ad11 in tumors and reduced the enrichment of ad11 in liver and spleen.Ery-ad11 showed good antitumor efficacy in the treatment of subcutaneous tumor and metastatic tumor.In addition,the infiltration of CD4~+and CD8~+T cells in the tumor of the mice after Ery-ad11 treatment was significantly enhanced,confirming the strong immune activation ability of Ery-ad11.