| The Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and its variants have caused millions of deaths worldwide.Currently,commercial vaccines against SARS-COV-2 include inactivated vaccines,adenovirus vector vaccines,recombinant protein subunit vaccines,and mRNA vaccines,of which mRNA vaccines have the highest protection efficiency.Due to the easy degradation of mRNA,the development of an effective delivery system becomes the key to the clinical application of mRNA vaccines.Lipid nanoparticles(LNPs)can efficiently encapsulate mRNA,and possess good delivery efficiency and biocompatibility.Actually,both Moderna and Pfizer/BioNTech’s mRNA vaccines are mediated by LNPs as delivery systems.However,LNPs still face challenges,such as poor thermal stability and low lysosomal escape efficiency after internalization.LNPs are commonly composed of ionizable lipid,cholesterol,1,2-distearoyl-sn-glycerol-3-phosphate choline(DSPC),and PEG lipid.Ionizable lipids are the most important component of LNPs,and their performance determines the efficiency of the mRNA vaccine.In this work,two ionizable lipids(HEAH and BBAD)were synthesized to construct LNP delivery systems,which were applied to the development of the COVID-19 mRNA vaccine.The main contents are as follows:(1)In the first part,a LNP delivery system based on a new ionizable lipid 2-hexyldecyl 6-(ethyl(3-((2-hexyldecyl)oxy)-2-hydroxypropyl)amino)hexanoate(HEAH)was constructed.Unlike the commercial lipid ALC-0351 containing two easily degradable ester bonds,HEAH has one ether bond and one ester bond to improve stability and ensure biodegradability.Besides,a hydroxyl group was added to the branch chain of HEAH to enhance the delivery efficiency of LNPs.SARS-CoV-2 RBD mRNA was synthesized by in vitro transcription,and mixed with HEAH,DSPC,cholesterol,and PEG lipid using a microfluidic mixer to prepare an mRNA vaccine.The vaccine solution presented light blue opalescence with good physicochemical properties(particle size: 106.22 nm,PDI: 0.14,potential: +11.50 m V)and storage stability.Moreover,in vitro transfection,Western Blot,and cytotoxicity experiments demonstrated that the delivery efficiency and biocompatibility of HEAH-derived LNPs were slightly better than those of the commercial lipid ALC 0315-derived LNPs.(2)In the second part,a new mRNA vaccine based on the HEAH-derived LNPs was developed to fight against the Delta strain.The surface morphology and size of the mRNA vaccine were characterized by transmission electron microscopy(TEM)and dynamic laser scattering(DLS).CCK-8 test showed that the HEAH-derived LNPs(cell viability: 94.77%)had lower cytotoxicity than that of ALC 0315-derived LNPs(cell viability: 87.12%).Elisa and Western Blot assays proved that the mRNA vaccine can efficiently transfect HEK 293 T cells and express Delta RBD protein.In vivo distribution tests showed that the mRNA vaccine had liver targeting function.Besides,the muscle and the upper abdomen of mice showed the strongest fluorescence signals12 hours after injection.Furthermore,a secondary immune strategy was carried out to determine the immune function of the Delta RBD mRNA vaccine.Mice were injected with the Delta RBD mRNA vaccine mediated by HEAH derived-LNPs on Days 0 and14.The Ig G antibody,neutralizing antibody and cellular immune levels were analyzed using Elisa,pseudovirus,and flow cytometry experiments.The results showed the Delta RBD mRNA vaccine successfully induced humoral and cellular immunity of BALB/c mice with excellent safety.(3)In the third part,an efficient LNP delivery system based on a new ionizable lipid BBAD was constructed to develop a COVID-19 vaccine against Omicron variants.First,Omicron RBD mRNA was synthesized by in vitro transcription,and mixed with BBAD,DSPC,cholesterol,and PEG-lipid to obtain the Omicron RBD mRNA vaccine.The physicochemical properties of the mRNA vaccine were characterized by DLS(particle size: 109.05 nm,PDI: 0.12,zeta potential: +3.04 m V).The BBAD-derived LNPs mediated mRNA vaccine was stored at 4℃,-20℃,and-80℃ for 30 days.DLS assay showed that the particle size and PDI of the vaccine did not change significantly,indicating excellent stability.Besides,Elisa analysis showed that the delivery effect of the BBAD-derived LNPs in HEK 293 T cells was about 9.41 times that of ALC 0315-derived LNPs.In addition,the lysosomal escape ability and biocompatibility of the former were significantly better than those of the latter.Furthermore,The humoral and cellular immune levels of the mRNA vaccine were evaluated using ELISA,pseudovirus,and flow cytometry assays.Seven days after BALB/c mice received the second immunization,the specific Omicron Ig G antibody in the BBAD group was about 10.95 times that of the ALC 0315 group.Importantly,the lipid BBAD-mediated mRNA vaccine did not cause any systemic toxicity to mice,suggesting that BBAD had excellent biological safety.(4)Given the immune escape ability of the SARS-CoV-2 variants,a novel trivalent mRNA vaccine was developed based on ionizable lipid BBAD-derived LNPs.Firstly,SARS-CoV-2,Delta,and Omicron RBD mRNAs were encapsulated into BBADderived LNPs in a mass ratio of 1:1:1 by using a microfluidic mixer.The trivalent vaccine solution was light blue opalescent and possessed a remarkable Tyndall effect.After that,Elisa and Western Blot tests demonstrated the expression of three RBD proteins in cells.To further verify the delivery ability of BBAD,the commercial reagent Lipo 3000 with a superior delivery effect than ALC 0315-derived LNPs was selected as a control.The results showed the BBAD-based LNPs possessed better lysosomal escape ability and transfection effect in vitro than the Lipo 3000.In addition,twentyone days after initial immunization,three specific Ig G antibodies in the high-dose group against SARS-CoV-2,Delta,and Omicron approached ～1/1,833,333,～1/1,866,667,and ～1/925,000,respectively.The results showed that a high dose(mRNA = 15 μg)of trivalent vaccine induced stronger immune responses in the BALB/c mice against multiple epidemic SARS-CoV-2 strains.In summary,the LNP delivery systems based on new ionizable lipids(HEAH and BBAD)were designed and constructed,which had high delivery efficiency,good thermal stability,and excellent biosafety.Importantly,a series of mRNA COVID-19 mRNA vaccines were developed,which showed satisfactory cellular and humoral immune effects in mice.They had potential application fighting against infection of SARS-CoV-2,Delta,and Omicron strains. |
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