| Objective: With the development of our economy and accelerated aging of society,cancer has become the primary threat to the life and health of people in our country.In 2020,it was estimated that there would be more than 456 million new cases of cancer and more than 3 million deaths resulting from cancer in our country.According to estimates by the World Health Organization in 2019,cancer has become the leading cause of death for people under 70 years of age in China.Therefore,research on cancer prevention and treatment has become an important breakthrough direction to improve people’s health and well-being.OVs is a kind of naturally existing or genetically modified virus that can effectively infect and kill tumor cells without causing excessive damage to normal non-cancer tissues.OVs can selectively replicate in tumor cells after administration and eventually dissolve tumor cells.The lysed tumor cells can release a large number of tumor-related antigens to induce the systemic anti-tumor immunity to kill tumor cells.OVs is a promising antitumor therapy.Currently,there are 4 types of oncolytic virus products approved for clinical use,all of which are intratumoral injectable drugs.However,the clinical value of local administration of OVs is limited by the operational difficulties of intratumoral injection,poor patient compliance and the poor efficacy of metastatic tumor therapy.Intravenous infusion of OVs is a better method for the treatment of metastatic tumors and tumors of deep organ origin.However,OVs are rapidly diluted and exposed to blood components immediately after intravenous infusion,and are easily phagocytized by innate antiviral immunity and presented to the adaptive immune system for clearance,which greatly limits the use of OVs for intravenous administration.The current research on improving the antitumor efficacy of OVs intravenous administration focuses on preventing OVs from binding to complement and antiviral neutralizing antibodies.These results indicated that modified OVs were well camouflaged in vitro,but the antitumor effect in vivo was not ideal.In addition,the poor response of patients lacking preconditioned adaptive antiviral immunity to initial intravenous viral therapy cannot be attributed to neutralizing antibodies in vivo.Therefore,we hypothesize that the current present study may have missed some key factors that may influence the rapid clearance of OVs in vivo after intravenous administration.In this study,inspired by the fact that Polyethylene glycol(PEG)liposomal coated biological nanoparticles can shield the surface antigens of nanoparticles to improve their cycling properties,we first tried to encapsulate adenovirus type 11(ad11)through liposomal to improve its circulation characteristics.However,we found that ad11 coated by liposome can shield its surface antigen,but does not prolong its circulation characteristics in vivo after intravenous administration.Further studies showed that viral corona formed on the surface of ad11 after co-incubation with serum.Different from the protective effect of protein corona on ordinary nanoparticles,the formation of viral corona has a great effect on viral cyclic clearance,and even a small amount of viral corona formation can lead to rapid clearance of OVs.Based on this discovery,we propose a viral corona replacement strategy to eliminate the formation of viral coronas by artificially creating viral coronas.The results showed that the modified ad11 eliminated the formation of viral corona,and its circulating time in peripheral blood was promoted by more than 30 times.Our study provides a new perspective on intravenously injected OVs,shifting the focus of future research from preventing OVs from binding to neutralizing antibodies and complement to preventing OVs from interacting with plasma proteins.Methods: 1.We first prepared PH-sensitive Liposome(PEG-Liposome,PEG-Lipo)with PEG seal by rotary evaporation method,and realized PEG-Lipo coating on ad11 by ultrasonic combined extrusion method to obtain Lip-ad11.Subsequently,we detected the metabolism of Lip-ad11 in vivo by q PCR.2.Transmission electron microscope(TEM)on the surface of ad11 viral corona,q PCR experiment and fluorescence confocal microscope to detect the influence of the formation of viral corona on the uptake of ad11 and Lip-ad11 by macrophages.3.The components of the protein corona and viral corona on the surface of bare ad11,Lipad11 and PEG-Lipo were detected by protein spectrum experiment,and the potential key proteins leading to the peripheral clearance of bare ad11 were obtained by correlation analysis.4.According to the results of protein spectrum,a series of ad11-related preparations including ad11-PEI-SA-Lip containing artificial viral corona were prepared by electrostatic adsorption method and ultrasonic method.5.We tested the antigen exposure of ad11-PEI-SA-Lip by fluorescence quenching experiment,antibody neutralization experiment and flow cytometry experiment.6.In vivo imaging of small animals and q PCR test were used to detect the intratumoral enrichment of each group of preparations,and the p H sensitivity of ad11-PEI-SA-Lip was detected by immunofluorescence technology.7.The antitumor ability of each group of preparations was tested by subcutaneous tumor and metastatic tumor models in mice,and the immune response caused by each group of preparations was tested by inflammatory factors and immune cell infiltration and other indicators.Results:1.We successfully prepared Lip-ad11 by ultrasonic combined extrusion method,and characterized it by TEM.Pharmacokinetic results showed that compared with bare ad11,the peripheral circulation ability of Lip-ad11 was not significantly enhanced.2.We characterized the viral corona formed by bare ad11 in plasma by TEM,and confirmed that the formation of viral corona can promote the uptake of bare ad11 and Lipad11 by macrophages by fluorescence confocal microscopy and q PCR.3.The components of viral corona and protein corona on the surface of bare ad11,Lipad11 and PEG-Lipo were identified by protein spectrometry,and a total of 21 proteins that may be associated with peripheral clearance of bare ad11 were obtained.4.According to the results of protein spectrum,we proposed the viral corona replacement strategy and prepared ad11-PEI-SA-Lip.Pharmacokinetic results showed that the peripheral circulation capacity of ad11-PEI-SA-Lip was 30 times higher than that of bare ad11.5.The results of fluorescence quenching experiment,antibody neutralization experiment and flow cytometry experiment showed that ad11-PEI-SA-Lip could completely shield the surface antigen of the virus and achieve complete masking of the surface antigen of the virus.6.The results of small animal imaging experiment and q PCR experiment showed that ad11-PEI-SA-Lip had stronger enrichment in tumor tissues compared with other groups.7.The pharmacodynamic results of mouse subcutaneous tumor model and metastatic tumor model showed that the antitumor effect of ad11-PEI-SA-Lip was significantly stronger than that of other groups.And ad11-PEI-SA-Lip was able to induce a stronger immune response than the other groups.Conclusion: After intravenously injecting the oncolytic virus,the viral corona formed on the surface of the virus will facilitate its elimination.In this study,a corona replacement strategy was proposed for the first time,and ad11-PEI-SA-Lip preparation was constructed,which achieved complete cover-up of the virus surface antigen and greatly enhanced the peripheral circulation ability of the virus.In vivo pharmacodynamic experiments confirmed that ad11-PEI-SA-Lip had the best anti-subcutaneous tumor and metastatic tumor efficacy compared with other experimental groups,which confirmed the effectiveness of the viral corona replacement strategy. |
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