Lipid-based delivery
Lipid nanoparticles (LNPs) have been widely used as carriers to deliver mRNAs [
27,
108,
109]. The first conducted clinical trial of mRNA vaccines is LNP-delivered. LNPs are mature negatively-charged nucleic acid delivery platforms characterized as ionizable amino lipids, polyethylene glycol, phospholipids and cholesterol. The essential ionizable amino lipids facilitate mRNA to escape from the endosome by interacting with ionizable amino lipids and the endosomal membrane [
110]. Polyethylene glycol is another essential component to prolong LNP circulation time because it spatially hinders the binding of mRNA and plasma proteins, which accelerates the clearance by reticuloendothelial (ER). Phospholipids and cholesterol can stably integrate the LNP structure [
111]. To be specific, LNPs have two advantages as an mRNA vaccine vector. On the one hand, LNPs defend mRNA from degradation by enzymes from the endosome [
112]. The characteristic guarantees high encapsulation efficiency [
113]. On the other hand, LNPs have good biocompatibility through a series of biological processes to deliver mRNAs for expression. The first process involves the apolipoprotein E (ApoE)-low density lipoprotein receptor (LDLR) pathway. This endogenous pathway is a targeting foundation for efficient delivery [
113]. Then, the TLR4-mediated endocytosis takes up LNPs, forming a vesicle to fuse with endosomes [
114]. After escaping endosomes, LNPs release mRNA into the cytoplasm to synthesize vaccine antigens.
The delivery efficacy of LNPs relies on multiple lipid components and lipid-related modifications. Foremost, the primary substance is cationic or ionizable lipids. Various cationic or ionizable lipids are used to deliver RNA, such as N-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA) [
115], dilinoleylmethyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), N,N-Dimethyl-2,3-bis[(9Z,12Z)-octadeca-9,12-dienyloxy]propan-1-amine (DLinDMA) [
116], 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE) [
117], 1,2-dioleoyloxy-3-trimethylammonium propane chloride (DOTAP) [
118] and N
1,N
3,N
5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide (TT3) [
119]. These lipids are good delivery helpers because they are positively charged at a certain pH. Thus the negatively charged mRNA interacts with those lipids electrostatically for delivery. Due to cellular membrane structure, mRNA-encapsulating lipids easily fuse with the targeted cellular membrane [
120]. Subsequently, the endocytosis of LNPs triggers proton-pump-mediated pH reduction. At this pH, the ionizable cationic lipid becomes more positively charged. Then, endogenous anionic lipids remove the cationic lipids by binding to the cationic lipids to generate a non-bilayer structure. Such a process results in disrupting the endosomal membrane and releasing mRNAs into the cytoplasm [
121]. Secondly, the lipids’ head and tail are important modification sites for enhancing delivery efficacy. YSK12-C4 is a pH-sensitive cationic lipid. On the one hand, the hydrophilic head of YSK12-C4 determines the acid dissociation constant (pKa), an indicator of intrahepatically distributional conditions and the endosomal escape. On the other hand, the hydrophilic tail is a non-pKa-dependent structure and shares similar functions with the hydrophilic head [
122]. Thirdly, Anderson et al. developed an isocyanide-containing lipid library. Isocyanide is a linker of di-hydroimidazole. They added di-hydroimidazole to LNPs for the optimization of mRNA delivery. Consistently, they added the STING (stimulator of interferon genes) agonist to LNPs for internalization. The adjuvant stimulation increases the mRNA efficiency. Additionally, they used LNPs to deliver antigen-specific mRNA vaccines in several mouse models for vaccination. The results showed an increased survival rate [
123].
The administration methods determine the distribution and expression kinetics of lipid-based mRNA vaccines in vivo. Local delivery is realized by subcutaneous (SC) administration, intramuscular (IM) administration, intradermal (ID) administration and intranodal (IN) administration. Scientists applied these methods to APCs and other immune cells, eliciting locally strong and long-lasting immune responses. So local delivery is used to initiate the stimulatory reaction in the small area. For example, the lipid-polymer-RNA lipopolyplexes (LPRs) containing a tri-antenna of α-d-mannopyranoside (triMN-LPR) were administrated intradermally (ID) to C57BL/6 mice. The triMN-LPR increased the local inflammatory responses several days later and recruited the activated DCs to the lymph nodes around the intradermal injection site. When injecting triMN-LPR encapsulating vaccine antigens to tumor-bearing mouse models intradermally, Moignic et al. found a more robust stimulatory immune response to fight against cancer [
124]. Another example is that mannosylated LNPs delivering influenza (hemagglutinin) encoded saRNA vaccine generated faster antigen-specific CD8
+ T cell responses by intradermal administration [
125]. Subcutaneous administration facilitated the PEGylated LNPs to be uptaken by the DCs in the lymph nodes and allowed the rapid release of mRNA vaccines after cellular internalization of LNPs [
126]. Intravenous (IV) administration is a kind of systematic delivery. Compared with local delivery, intravenously administered mRNA antigen vaccines generate extensive and effective immunity [
127]. IV administration causes the accumulation of mRNA-LNPs in the liver, resulting in the tremendous translational activity of proteins and the productive protein-synthesizing activity [
128]. Immunologically, intravenously administered LNPs encapsulating mRNA antigen vaccines can mature DCs and activate antigen-specific T cells both in vivo and in vitro. When scientists intravenously immunized mice with OVA-mRNA-encapsulating LNPS, mice with lymphoma were presented with the inhibition of tumor growth and the recovery of abnormal hemogram [
129]. Therefore, the IV administration of LNPs is commonly used in systematic diseases, like hematological diseases.
In short, the LNP is currently a potential mRNA-delivering candidate for good biocompatibility, high delivering efficacy and so on. Except for the above preclinical investigation, phase I (NCT04064905) and phase II clinical trials (NCT03897881) are undergoing for evaluating LNP-based mRNA antigen vaccines.
Polymer-based delivery
Polymeric materials are less clinically investigated as ionizable lipids do. However, they coat mRNA without suffering from degradation and promote protein expression. The disadvantages of polymeric materials are polydispersity and the clearance of large molecules [
130]. To improve the therapeutic effect, scientists have added lipid chains, expand branch structures and construct biodegradation-promoting domains [
131‐
133].
The classification of polymers contains the cationic polymer and the anionic polymer. As for the cationic polymer, polyethylenimine (PEI) [
134], polyamidoamine (PAMAM) dendrimer [
135] and polysaccharide [
136] are the three members. Since saRNA is sensitive to the RNase and is taken up inefficiently by DCs, condensing mRNA into the PEI-polyplex vehicle tackles the problem. An mRNA encoding hemagglutinin of influenza virus and nucleocapsid are both coated with the PEI-polyplex. The cytosolic complex facilitates the mRNA translation and induces both humoral and cellular responses [
78]. In another study, the polymer-based intranasal mRNA vaccination system is designed to overcome the difficulty that the nasal epithelium serves as a barrier to hinder delivering antigens to nasal associated lymphoid tissue (NALT). The cyclodextrin-polyethylenimine 2 k conjugate (CP 2 k) with HIV gp120 mRNA undoes tight junctions for paracellular delivery. Both the paracellular delivery and intracellular delivery can extend the residential time in the nose and initiate immense anti-HIV responses by producing cytokines and a balanced Th1/Th2/Th17 type [
137]. Next, the dendrimer is a potential delivery vector because it has multiple functional groups with high tolerability. PAMAM dendritic polymers with NH
2 and OH end functionalities respectively enter A549 human lung epithelial carcinoma cells faster than hyperbranched polymers [
138]. PAMAM dendrimers were once used to construct antigen-encoding saRNA to protect mice from Toxoplasma gondii, Ebola and H1N1 influenza [
139]. Taken mice with Zika virus for example, premembrane (prM)- and envelope (E)-encoding saRNA with dendrimer formulation enhanced IgG concentration and induced CD8
+ T cell-dominating responses [
140]. Subsequently, Shi et al. used PAMAM (generation 0) dendrimer complexed with ceramide-PEG and poly(lactic-co-glycolic acid) (PLGA) to transfect phosphatase and tensin homolog (PTEN) mRNA for the restoration of tumor-growth suppression [
141]. However, due to the spatial conformation of PAMAM, the biodegradation rate is limited, resulting in toxic accumulation. Hence, such a limitation hinders clinical development. Finally, chitosan is a polysaccharide substance involved in nanoparticulate delivery vehicles (nanogel-alginate (NGA)). Similarly, influenza virus hemagglutinin- and nucleoprotein-encoding mRNA are delivered to DCs by the chitosan-based nanoparticle. The vector promoted the successful translation of the two foreign antigen genes [
77]. The delivery system favors the naked RNA passing through the cellular membrane and surviving in the biological condition.
Although cationic polymers are the dominant material, anionic ones are also sometimes used to deliver. The most commonly used anionic molecule is poly D, L-lactide-co-glycolide. Since the negatively charged mRNA is uneasy to be delivered by anionic polymers, the addition of cationic lipid in a PGLA complex would assist in establishing an efficient RNA-encapsulating system [
142]. In recent years, PLGA-incorporating nanoparticles coated with LNPs have validated its delivery efficiency for up to 80%. The complex system rapidly induced the protein translation, reaching a peak in a short time and vanishing after 48 h. The mRNA characteristic is in accordance with such a phenomenon [
143]. Consistently, researches from Sharifnia et al. [
144] and Zhao et al. [
145] both favor the advantage of the PLGA nanoparticle system. In lymphoma-bearing mouse models, lipid-assisted nanoparticles (CLAN) encapsulating ovalbumin (OVA) mRNA reduced the tumor growth rate [
129].
Collectively, the polymer-based delivery system is a promising platform for its mRNA-delivering efficacy. However, the investigation is still in the early stage of preclinical trials. More problems need to be further illustrated.
Peptide-based delivery
Due to the electrostatic interaction, the negatively charged mRNA is easily delivered by the cationic peptide. The reason why peptides are positively charged is the positively charged amino groups. For example, the lysine residue and the arginine residue bring positive charges to the amino acid, enabling electronegative mRNA to adsorb onto the cationic peptide tightly [
146]. The amount of loaded mRNA is positively correlated with N/P (negative/positive) ratios [
147]. Additionally, an increased ratio of charged amino/ phosphate groups can increase zeta potential and minimize the particle size, increasing encapsulating efficiency [
147].
Protamines are one of the cationic peptides to deliver mRNA. Two aspects make it a potential vector. On the one hand, protamines can protect mRNA from RNase-mediated degradation in the serum [
148]. Stitz et al. used CureVac’s RNActive technology, in which the protamine-formulated RNA is an initiator of immunity. The protamine stabilized the immunogenicity at the changing temperature without affecting the antigen-encoding mRNA vaccine [
149]. If not the RNActive vaccine platform, the protamine-formulated RNA alone would inhibit the translational process, further affecting the vaccine efficacy [
150]. The reason for it is the excessively tight combination of the protamine and mRNAs [
151]. On the other hand, protamine is an adjuvant. A study demonstrated that the protamine-formulated mRNA activated DCs and monocytes, secreting TNF-α and IFN-α. It also activated immune cells by the TLR-7/TLR-8-mediated recognition of the protamine-formulated mRNA. The protamine-formulated mRNA shared some structural similarities with condensed RNA in the nucleocapsids of RNA viruses [
152]. Another study proved its antitumor priority over naked nucleic acid adjuvants in glioblastoma mouse models [
153]. Clinically, of all the peptide-based carriers, only the protamine is undergoing evaluations [
154‐
156].
Cationic cell-penetrating peptides (CPPs) are another kind of small peptides containing 8-30 amino acids. They are excellent delivery vehicles because they are not only equipped with low charge densities, but also able to disrupt membrane for endosomal escape. The latter reason is essential for synthesizing proteins [
6,
147]. Coolen et al. compared the three-CPP mRNA platform, namely RALA (WEARLARALARALARHLARALARALRACEA), LAH4 (KKALLALALHHLAHLALHLALALKKA) and LAH4-L1 (KKALLAHALHLLALLALHLAHALKKA). The three peptide/mRNA complexes were all introduced into DCs and generated innate immunity. Mechanically, this process was PRR-mediated and fostered adaptive immune responses. Meanwhile, the uptake process and their intracellular activities involve clathrin-mediated endocytosis and phagocytosis. Among the three, the LAH4-L1/mRNA complex showed the optimal protein expression [
157]. Another investigation combined the cationic characteristic and the cell-penetrating characteristic by formulating the fused protamine-CPP protein. The fused protein delivered reporting genes to human cell lines [
158].
Like polymer-based delivery, anionic peptides are also conjugated to a positively charged substance because two negatively charged substances repel each other. For example, the addition of a positively charged copolymer p(HPMA-DMAE-co-PDTEMA-co-AzEMAm) (pHDPA) is to encapsulate the OVA-mRNA. Then, the azide groups on pHDPA conjugate an anionic peptide GALA (N-WEAALAEALAEALAEHLAEALAEALEALAA-OH-C). Such a formulated complex, as a vector, showed enhanced EGFP-mRNA transfection in RAW 246.7 macrophages and DCs. It entered DCs by sialic acid-mediated endocytosis, regardless of the maturation of DCs. Introducing GALA contributed to cell uptake and mRNA release from the endosome by integrating with the sialic acid group on the DC surface. The uptaking process is more efficient than the lipofectamine. Based on the effective transfection, the OVA-mRNA-encapsulating complex triggered prominent immune responses [
159].
Virus-like replicon particle
Virus-like Replicon Particles (VRPs) can encapsulate antigen-encoding saRNA for delivering into the cytosol, which is like a virus-infecting manner. The viral structure proteins are synthesized in vitro, followed by encapsulating designated antigen-encoding saRNA. Some attenuated viruses maintain the ability of self-replication [
160]. In multiple virus types, the enhancement of vaccine potency has been validated. Most recently, alphavirus-derived replicon RNA encoding the SARS-CoV-2 spike (S) protein has been encapsulated in lipid inorganic nanoparticles (LIONs). The formulated vaccine was intramuscularly injected into mice and primates, indicating the increased level of anti-SARS-CoV-2 S protein IgG antibody [
161]. In another study, VRP was based on an HIV-derived mRNA encoding clade C envelope glycoprotein. A VRP packaged the mRNA. In rhesus macaques, the complex provoked cellular immune responses [
91]. In Venezuelan equine encephalitis VRP, the mRNA encoded two kinds of E antigen of dengue virus (subviral particles [prME] and soluble E dimers [E85]). The immunization of such a VRP induced protective efficacy and E85-VRP had priority in speed and magnitude of immunity [
162]. Another example is the flavivirus Kunjin based mRNA encoding granulocyte colony-stimulating factor (G-CSF). The VRP with the mRNA inhibited the growth of subcutaneous CT26 colon carcinoma and B16-OVA melanomas for half by inducing CD8
+ T cells [
163]. Lundstrom summarized viral saRNA replicon particles against viral diseases (Influenza, HIV, SIV, Ebola, Lassa, SARS-CoV, MERS-CoV, RSV, MPV, Dengue, HBV and CMV), bacterial diseases (
P. falciparum, M. tuberculosis, C. botulinum,
B. abortus, B. antracis, malaria,
L. monocytogenes, prion and staphylococcus) and cancer, respectively [
164].
Even if VRP had a therapeutic effect on viral diseases, bacterial diseases and cancer, there are two limitations. First, large-scale production has not been realized. The current time-consuming production process is limited by producing VRPs from cell lines [
165]. The second one is that the complex would promote anti-vector antibodies’ production, impeding the ongoing clinical trials [
166]. Hence, we should lower the immunogenicity of VRPs.
Cationic nanoemulsion
Cationic Nanoemulsion (CNE) is a non-viral delivery system, which potentiates the mRNA vaccines by binding to saRNAs. One of the most essential components is the cationic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOTAP). DOTAP has been utilized in clinical trials for its positive charges emulsified with the same component of the emulsion adjuvant MF59. Except for clinical use, DOTAP has priority in availability, squalene solubility and the cationic feature at a certain pH [
92]. Cationic lipids can form a pH-dependent nano-sized emulsion [
167].
Brito et al. investigated CNE-delivered saRNA in animals (rabbit, mouse, nonhuman primate). Their saRNA encoded multiple vaccine antigens, such as the fusion (F) glycoprotein of respiratory syncytial virus (RSV), the envelope glycoprotein B (gB) of human cytomegalovirus (hCMV), a fusion protein (pp65-IE1) of phosphoprotein 65 (pp65) and immediate early protein 1 (IE-1) of hCMV and gp140 envelope glycoprotein (env) of the human immunodeficiency virus (HIV). The results demonstrated good CNE efficacy, the induction of immune responses by the adjuvant subunit and the low dose of CNE complex [
92]. Consistently, it is validated that the cellular immune responses induced by saRNA-delivered CNE were more robust than that by saRNA-delivered VRP. The dose is as low as 50 μg, which is safe enough for immunogenicity [
91]. Similarly, to fight against the venezuelan equine encephalitis virus and Zika virus, scientists developed the saRNA vaccine delivered by CNE, both inducing robust protective immunogenicity [
168,
169].
Based on the above preclinical studies, CNE has the potentials in human clinical evaluations.