Study On New Cationic Liposomes As MRNA Vaccine Delivery Carrier

Since the end of 2019,there has been a global outbreak of COVID-19,and it is extremely contagious,so it has been a serious threat to public health.Currently,there are more than 4000 novel Coronavirus variants.In order to deal with many mutant strains,mRNA vaccines have a significant advantage in dealing with such risks due to their rapid application characteristics,which can quickly update the mRNA sequence according to the mutant gene of the mutant strain.However,the poor stability of mRNA vaccines,low mRNA expression efficiency,easy to be degraded by nuclease,strong toxic and side effects and other problems restrict its popularization and application.Therefore,in order to solve the above problems,this paper focuses on the study of new cationic lipid membrane material and new delivery carrier based on the characteristics of mRNA vaccines.This topic is divided into three research stages:In the first stage,in view of the high toxicity of cationic liposome(CTL),positive charge,strong immunogenicity and non-toxic cell penetrating peptide-sterol conjugates were independently designed and synthesized,and used as the main membrane material to construct new CTL.In the second stage,to solve the problem of poor stability of mRNA vaccines,the lyophilization process was optimized to investigate its stability at 2-8 ℃ storage.In the third stage,to solve the problem of low mRNA expression efficiency and easy to be degraded by nuclease,the mRNA vaccines were evaluated in vitro and in vivo.The main research work is as follows:1.Cell penetrating peptide CGYKK was independently designed and synthesized by Fmoc solid-phase synthesis method,and the MS value of the synthesized peptide chain CGYKK was detected by MALDI-TOF mass spectrometry,the purity was detected by HPLC,and the cytotoxicity was detected by MTT method.2.The extraction and purification process of ergosterol from yeast were studied.Firstly,the determination method of ergosterol content was established,then the optimum extraction conditions were determined by orthogonal experiment,finally the extracted ergosterol was purified and it was detected by 1H-NMR and HPLC.3.A new type of lipid material was synthesized,and the peptide chain CGYKK,cholesterol and ergosterol were linked by disulfide bond,respectively,to obtain chol-CGYKK and ergo-CGYKK,which were detected by MALDI-TOF mass spectrometry and HPLC.4.CTL was prepared by thin film dispersion method,thermogravimetry of phase transition temperature was measured,the mixing ratio of CTL and S-mRNA was determined by gel block experiment,and the freeze-drying process of mRNA vaccine formulation was optimized.5.CTL-mRNA-chol vaccines were prepared by freeze-drying method using chol-CGYKK as main membrane material.The particle size were about 150 nm,the potential were about 35 mV,and the encapsulation rate were more than 90%.It had a spherical appearance and good stability.The protein expression level of prescription 1 was significantly higher than that of prescription 2～4.Uptake experiments confirmed that CTL entered cells by membrane fusion.Compared with Lipofectamine?2000(lipo2000),the transfection efficiency was 66.7%.In vitro toxicity results showed that CGYKK had no toxicity to cells in the range of 0.0125-0.20 mg/mL within 24 h.In the range of 0.25-4.00μg/mL,the vaccines had no toxicity to cells within 24 h.In vivo immunoassay results showed that prescriptions 1～4 had better immune effect compared with saline injection,and there was no significant difference between them.6.CTL-mRNA-ergo vaccines were prepared by freeze-drying method using ergo-CGYKK as main membrane material.The particle size were about 200 nm,and the potential were about 35 mV,showing positive charge.And the encapsulation rate were more than 90%.The appearance were spherical and evenly distributed.The protein expression of prescription 1 was significantly higher than prescription 2～4.Uptake experiments confirmed that CTL entered cells by membrane fusion.In vitro toxicity results showed that CTL-mRNA-ergo and CTL-mRNA-chol had no toxicity to cells in the range of 0.25-4.00μg/mL within 24 h.However,CTL-mRNA-ergo still had no toxicity to cells within 48 h,which was significantly different from CTL-mRNA-chol.In vivo immune results showed that prescriptions 1～4 had better immune effect compared with normal saline injection.