Introduction

With the emergence of SARS-CoV-2 in 2020, mRNA vaccines have garnered global attention. Leveraging years of research investigating mRNA vaccines as therapeutic strategies against cancer in clinical trials, COVID-19 vaccines have been swiftly developed and manufactured [1]. The US Food and Drug Administration (FDA) has granted approval to two mRNA vaccines, namely mRNA-1273 and BNT162b2, which have been administered to hundreds of millions of individuals [2]. These vaccines have demonstrated remarkable efficacy with consistent protection rates exceeding 90% [2]. In comparison to other vaccine types, mRNA vaccines possess several distinct advantages. (1) High safety: It is achieved by the cytoplasmic functionality of mRNA, eliminating the risk of integration into the host genome unlike DNA-based approaches [3, 4]. (2) Superior efficiency: The stability and translation efficiency of mRNA can be significantly improved through appropriate modification and sequence optimization [5, 6]. Additionally, an efficient delivery system has been developed to facilitate rapid uptake of mRNA and its expression in the cytoplasm. (3) Short development cycle: The mRNA encoding the antigenic protein can be designed as soon as the pathogen's genome sequence is determined [7, 8]. Subsequently, in vitro transcription (IVT) of the mRNA can be performed without cell expansion, thereby significantly enhancing production speed. (4) High flexibility: The original vaccine may become ineffective if a mutation occurs in the virus. In such cases, new mRNA vaccines can be rapidly redesigned and manufactured based on updated viral sequences. (5) The application scope is extensive: The mRNA technology, with its advanced principles and efficient development and production processes, has achieved significant breakthroughs in the treatment of infectious diseases, liver diseases, cancer, diabetes, among others.

The development of effective vaccines against the COVID-19 pandemic, namely BNT162b2 (Pfizer) and mRNA-1273 (Moderna), were rooted in a decade-long pioneering effort by those who recognized the therapeutic potential of mRNA. Since the initial report of mRNA by Francois Jacob et al. in 1961 [9], mRNA has been acknowledged as a pivotal intermediary between DNA coding sequences and the production of functional proteins. Subsequently, in 1990, Wolff et al. [10] established the groundwork for utilizing mRNA as a therapeutic agent through direct gene transfer in vivo by injecting naked mRNA containing reporter genes into the skeletal muscle of mice, thus providing proof of principle. Over the subsequent decades, substantial advancements have been made in comprehending the properties and potential applications of mRNA as a therapeutic agent [11].

The components essential for the expression of the encoded antigen and induction of adaptive immunity in humans in a typical mRNA vaccine comprised two main elements: the mRNA and its delivery system. The mRNA encoding the desired protein or peptide serves as the molecular foundation for inducing specific immunity against the pathogen. Technically, mRNA vaccines are typically generated through in vitro transcription (IVT) from DNA templates containing T7 RNA polymerase [12]. Additionally, the delivery system plays a crucial role in mRNA vaccines [13]. The delivery system not only safeguards the mRNA against degradation in the extracellular environment but also facilitates its translocation into the cytoplasm of antigen presenting cells (APCs), potentially enhancing the expression of encoded antigens. The optimization of mRNA and its delivery systems has resulted in diverse clinical applications of mRNA vaccines. Various materials, such as lipids, lipid-like compounds, polymers, and protein derivatives, have been developed for mRNA delivery [14,15,16,17]. Among these advanced delivery systems, lipid nanoparticles (LNPs) represent a promising approach to enhance the efficacy and stability of mRNA vaccines [18, 19]. LNPs are composed of ionizable/cationic lipids, helper lipids, cholesterol, and polyethylene glycol (PEG)-coupled lipids. Ionizable/cationic lipids play a crucial role in determining the delivery and expression efficacy of mRNA vaccines. Both helper lipids and cholesterol contribute to the stabilization and promotion of membrane fusion in LNPs. Lastly, PEG-modified lipids are essential for enhancing stability in vivo applications due to stealth effects [20, 21].

While the SARS-CoV-2 mRNA vaccines have undoubtedly achieved remarkable success, there are still many challenges ahead. Despite individuals receiving vaccination against SARS-CoV-2 through mRNA vaccines, instances of confirmed COVID-19 cases have been reported [22]. Therefore, it is crucial to enhance the efficacy of mRNA vaccines by optimizing both the mRNA and its delivery system. Furthermore, when formulating future mRNA vaccines, considerations should be given to storage requirements and antigen mutation [23].

This review focuses on the advancements in mRNA delivery system, specifically highlighting the current status of LNPs and providing insights for future optimization. Additionally, we discussed the potential prospects of LNPs as a clinical treatment for mRNA delivery.

Lipid nanoparticles (LNPs)

The current FDA-approved LNPs formulations consist of four lipids: ionizable cationic lipids, helper lipids, cholesterol, and polyethylene glycol (PEG) [24, 25] (Fig. 1). These constituents facilitate the formation of monodisperse nanoparticles, enhance nanoparticle stability, promote efficient nucleic acid encapsulation, facilitate cellular uptake, and support nucleic acid release. Figure 2 illustrates three ionizable lipids, namely those employed in Onpattro®, Comirnaty®, and Spikevax® for clinical applications, all of which have received FDA approval.

Fig. 1
figure 1

The schematic illustrations of the LNPs for mRNA delivery

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Fig. 2
figure 2

Structure and molar ratio of the component lipids in the three clinical applications of LNPs

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The role of ionizable lipids

Ionizable lipids are amphiphilic compounds comprising three distinct domains: a polar head group, a hydrophobic tail region, and a linker connecting the two domains [26], as shown in Fig. 3. The head groups of ionizable lipids typically exhibit a positive charge. The size and charge density of the head group play crucial roles in nucleic acid wrapping, LNPs stabilization, cell membrane interaction, and endosome escape [27]. Common ionizable lipids typically possess a singular head group, although instances of multiple head groups can also be observed. Prominent examples of these head groups encompass amine, guanidine, and heterocyclic moieties [28] (Fig. 3). The ionizable lipids currently available for clinical use, namely DLin-MC3-DMA, ALC-0315, and SM-102, all feature tertiary amine head groups and represent the sole FDA-approved cationic lipids for RNA delivery [17, 29, 30] (Fig. 2).

Fig. 3
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Schematic representation of ionizable lipids and their components (headgroup, linker, and tails)

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The linker fragments establish a connection between the head and tail of LNPs, occasionally concealing themselves within the tail region, as observed in SM-102 and ALC-0315 (Fig. 1). This phenomenon significantly impacts the stability, biodegradability, cytotoxicity, and transfection efficiency of lipid nanoparticles [27, 28]. Ionizable lipids may contain one or more linker fragments (Fig. 3). However, the majority of ionizable lipids typically consist of a single type of linker fragment [31]. In general, linker fragments can be categorized as non-biodegradable (ethers and carbamates) or biodegradable (esters, amides, and thiols) [31]. The incorporation of biodegradable lipids enhance the tolerance of LNPs, facilitating rapid metabolism while maintaining mRNA delivery efficacy. The biodegradability of lipids can be enhanced by the incorporation of ester motifs. For instance, DLin-MC3-DMA, ALC-0315, and SM-102 encompass fragments bonded by esters [32, 33].

Ionizable lipids typically consist of 1 to 4 hydrophobic tails, each containing 8 to 20 carbon atoms [27, 28]. These tails can be saturated or unsaturated lipid chains, and the degree of unsaturation may impact nucleic acid delivery by modulating properties associated with membrane instability [28, 34] (Fig. 3). DLin-MC3-DMA features two linoleic acid tails, while ALC0315 and SM-102 have two branched saturated tails that are believed to possess a conical geometry and promote endosomal membrane destabilization for intracytoplasmic release of nucleic acid [35].

Currently, the primary constituents employed in LNPs formulation predominantly consist of ionizable lipids possessing adjustable pKa values, which can enhance nucleic acid loading efficiency and impact the stability and toxicity of LNPs, thereby further augmenting the efficacy of gene therapy [36]. Conventional cationic lipids with permanent charge, such as 1,2-dioxane-3-trimethylammonium-propane (DOTAP), previously employed for nucleic acid delivery, exhibit facile interaction with negatively charged serum proteins and tend to accumulate in the bloodstream [29, 36]. Consequently, the rapid clearance of LNPs by the monocyte-macrophage system ensues, resulting in a diminished half-life within the bloodstream [29]. Moreover, the relatively elevated hemolytic activity of cationic lipids amplifies the potential for toxic side effects, such as hemoglobin release resulting from impairment to the erythrocyte membrane [37]. To address these challenges, ionizable cationic lipids with pKa values (typically 6.0–7.0) have been developed to ensure efficient nucleic acid encapsulation under acidic conditions (e.g., pH 4.0, where the lipids are protonated) and reduced toxicity during circulation under physiological conditions (pH 7.4, where the lipids are nearly neutral) [38]. Upon entering the endosome (with a lower pH than the lipid pKa), the amine groups of ionizable lipids become protonated and interact with anionic groups on the endosomal membrane, facilitating nucleic acid release from the endosome [39]. Notably, LNPs with pKa values of 6.2–6.5 and 6.6–6.9 were found to be optimal for intrahepatic siRNA delivery and intramuscular mRNA vaccination, respectively [40, 41].

The role of PEGylated lipids

PEGylated lipids constitute a crucial component of LNPs, albeit accounting for the smallest molar percentage (typically 1.5 mol%). They exert significant influence on various key properties of LNPs, including particle size and dispersibility [42,43,44], as well as the stability of nanoparticles during preparation and storage. Moreover, polyvinyl alcohols also impact nucleic acid encapsulation efficiency [42], in vivo distribution [45], transfection efficiency [42], and immune response [46]. All these properties are intricately linked to the molar ratio of polyvinyl alcohols, as well as the structure and length of both the polyvinyl alcohol chain and lipid tail (alkyl/dialkyl chain).

PEGylated lipids comprise two distinct domains: PEGylated lipids forming the surface layer of the lipid particles, and lipid domains encapsulated within the core of the particles, with PEG domains extending outward from the surface. The spatial barrier effect of PEG polymers prevents plasma protein binding, thereby avoiding rapid clearance by the reticuloendothelial system (RES). Consequently, they have found extensive application in liposome systems to effectively prolong in vivo circulation time [26].

Polyvinyl alcohol enhances LNPs self-assembly by creating a hydrophilic spatial barrier on the surface of LNPs [43]. This spatial barrier also plays a crucial role in preventing nanoparticle aggregation, thereby contributing to their stability [42, 43]. Lokugamage et al. [47] demonstrated that formulations lacking polyvinyl alcohol resulted in unstable and polydisperse LNPs with diameters exceeding 200 nm. Conversely, studies have shown that incorporating just 0.5 mol% of polyethylene glycol lipid can produce stable and homogeneous LNPs with diameters below 80 nm [48]. The stability of mRNA-LNPs were maintained for up to three weeks at 4 °C through monitoring particle size, PDI, Zeta potential, and encapsulation efficiency during storage [42, 49]. Furthermore, these studies indicated that different compositions of polyvinyl alcohol chains can adequately provide the necessary repulsive forces for particle formation without compromising long-term stability [42].

The particle size of LNPs must be strictly controlled during the preparation process, as it plays a pivotal role in determining the pharmacokinetics and biodistribution of the nanoparticles [50]. Kulkarni et al. [48] investigated the impact of varying the fixed molar ratio on lipid structure using cryo-electron microscopy, assuming that PEGylated lipids were exclusively located at the surface of LNPs [45]. Consequently, increasing the molar percentage of PEGylated lipids led to an augmented specific surface area, thereby reducing nanoparticle size.

While PEG is valuable for enhancing the stability and bio-conjugation of LNPs, its desorption plays a pivotal role in facilitating cell transfection [51, 52]. In a previous study conducted by Mui et al. [44], the desorption rates of three PEGylated lipids in siRNA-LNPs were tested, which had dialkyl chains consisting of 14, 16 or 18 carbons. It was observed that shorter carbon chains in PEGylated lipids led to faster desorption rates and better effects. The efficiency of LNPs in delivering nucleic acids is enhanced by reducing the peg-lipid content, as they exhibit increased binding affinity towards ApoE [38, 53, 54]. Additionally, the length of the PEGylated lipids anchor plays a crucial role in determining desorption kinetics [26]. In addition to length, the molecular weight of the PEG fragment also influences the delivery efficiency of LNPs [55].

The role of cholesterol

Cholesterol, as a constituent of the cell membrane, plays a crucial role in enhancing the stability of LNPs and facilitating cell membrane fusion [17, 20]. Therefore, optimizing the structural characteristics of cholesterol can effectively enhance the delivery efficiency of LNPs and confer them with unique functionalities. Paunovska et al. [56] demonstrated that incorporating esterified cholesterol into LNPs significantly improved their delivery efficiency. Furthermore, cholesterol has been exploited to enhance drug delivery systems. Patel et al. [57] conducted a screening study on various natural cholesterol analogues and discovered that β-sitosterol LNPs exhibited remarkably enhanced transfection efficiency.

Among LNPs formulations containing nucleic acids, lipid preparations incorporating cholesterol yielded two key findings: (1) Cholesterol is a readily exchangeable molecule capable of accumulating within liposomes during circulation [58], and (2) incorporation of cholesterol significantly diminishes the presence of surface-restricted proteins while enhancing the circulating half-life [59].

Lipids, including cholesterol, play a crucial role in the encapsulation of nucleic acids. By increasing membrane stiffness, cholesterol effectively reduces liposome drug leakage [17]. A recent study [44] demonstrated that the presence of helper lipids above a certain threshold was essential for achieving stable encapsulation. Specifically, a minimum concentration of 40 mol% cholesterol (in the absence of any phospholipids) was required to achieve nearly complete siRNA encapsulation.

The role of phospholipids

Phospholipids serve as helper lipids facilitating the formation of LNPs and promoting endosomal escape [60]. Moreover, they possess the ability to enhance liposome stability and improve internal circulation, thereby exerting a significant impact on the efficacy of mRNA-LNPs [61]. In preclinical studies and clinical applications, the commonly employed phospholipids include DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and DOPE (dioleoylphosphatidylethanolamine). Presently, commercial LNPs systems exclusively incorporate DSPC, likely owing to its stability within liposomes used in commercial settings as well as its efficient intima disruption facilitated by ionizable lipids. Kauffman et al. [48, 61] reported that their DSPC-based LNPs exhibited a higher mRNA encapsulation rate compared to those containing DOPE. However, the latter demonstrated superior protein expression. Regarding safety, Oberli et al. [55] observed inflammation at the injection site in 20% of mice 5–10 days after administration of LNPs containing either DSPC or DOPE.

The LNPs-induced immune response

The human clinical trials of the Pfizer/BioNTech and Moderna vaccines have reported side effects such as pain, swelling, fever, and sleepiness [62,63,64]. These are typical symptoms associated with inflammation induced by cytokines such as IL-1β and IL-6 [65, 66]. Some clinicians and public health advocates interpreted the observed acute side effects as indicative of the vaccine's efficacy in eliciting an adaptive immune response. The observed side effects, however, were more in line with the acute inflammatory response induced by the vaccine [67]. Many studies [64, 68, 69] have shown that LNPs used for many preclinical studies are highly inflammatory. This could explain their potent adjuvant activity and their superiority, compared with other adjuvants, in supporting the induction of adaptive immune responses. Ionizable lipids were developed to mitigate the inflammatory properties associated with permanently charged cationic lipids [25]. However, studies have revealed that the proprietary ionizable lipid components of these LNPs induce an inflammatory response. The inflammation triggered by LNPs or their complexes with mRNAs is independent of the route of administration and is primarily characterized by neutrophil infiltration. The specific inflammatory pathways activated by these LNPs or their ionizable lipid components are currently not well understood. In theory, LNPs could activate various pathways or only one pathway, thereby triggering an inflammatory cascade. Some cationic/ionizable lipids bind and activate TLRs [29, 70, 71].

Relevant clinical interventions for LNPs therapy

Therapeutic mechanism of mRNA-LNPs

Ionizable lipids play a pivotal role in the formation of LNPs and in vivo transfection. Initially, ionizable lipids form electrostatic complexes with negatively charged mRNA molecules, thereby enhancing the stability of mRNA molecules [60, 72]. The pH neutrality of LNPs, attributed to the presence of ionizable lipids and PEGylated lipids, effectively mitigates nonspecific interactions with serum proteins in vivo [20, 21, 73]. After PEG lipids dissociation, LNPs are taken up by cells through an ApoE-dependent pathway. Upon reaching the cell membrane, mRNA-LNPs trigger fusion with the negatively charged cell membrane via an ionizable lipid, leading to endocytosis and cellular absorption [52]. Subsequently, lysosomes containing various hydrolytic enzymes create an acidic environment causing a decrease in pH values. At low pH values, protonated ionizable lipids induce the formation of a hexagonal phase structure that disrupts the bilayer structure of LNPs. This disruption facilitates the release of mRNA into the cell where it binds to ribosomes responsible for protein production and undergoes translation into viral proteins [52]. Antigens are released extracellularly or degraded by proteasomes, thereby exposing antigenic epitopes. Peptides are subsequently loaded onto major histocompatibility complex (MHC) class I to activate antigen-specific CD8+ T cells [74]. Exogenous proteins that are released early on are presented on MHC class II to stimulate CD4+ T cells activation [75]. CD4+ T cells can also co-stimulate B cells specific to the protein, leading to B cell maturation and subsequent antibody secretion [76] (Fig. 4).

Fig. 4
figure 4

Delivery mechanism of mRNA-LNPs. ① Endocytosis of mRNA-LNPs. ② Protonation of ionizable lipid. ③ Endocytosis mediates the internalization of post-translational mRNA, which then escapes from the endosomes into the cytosol. ④ The host cell ribosomes facilitate the translation of mRNA into the desired antigen protein intracellularly. ⑤ The major histocompatibility complex (MHC) class I presents peptide antigens on the cell surface for antigen presentation. ⑥ MHC class I presents peptide epitopes to CD8+ T cells. ⑦ Endocytosis of exogeneous protein released early. ⑧ Exogenous protein is degraded and presented by MHC class II epitope. ⑨ Exogeneous protein is presented on MHC class II to activate CD4+ T cells. CD4+ T cells can co-activate protein-specific B cells, leading to the maturation of B cells and the release of antibodies

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RNA therapy

Gene therapy has emerged as a prominent avenue of clinical investigation in recent years, with LNPs serving as nucleic acid carriers for various gene therapeutic modalities including antisense oligonucleotide (ASO), short interfering RNA (siRNA), microRNA (miRNA), and mRNA, each exerting distinct mechanisms of action [77]. The target mRNA is hybridized and cleaved by ASO through the action mediated by RNAse-H. SiRNA and miRNA inhibit mRNA translation through the RISC-mediated RNAi pathway, thereby preventing protein synthesis. In contrast, mRNA binds to ribosomes via capsids and poly(A)-binding proteins, leading to the translation of therapeutic proteins. This is the underlying mechanism of mRNA-LNPs vaccines [77].

mRNA vaccines could induce strong and robust immune responses in both preclinical models and clinical trials. Adjuvants are used to stimulate specific components of the immune system to enhance the immunogenicity of the vaccine. Huang et al. [78] found that combining mRNA-encoded genetic adjuvants with mRNA-encoded antigens and adding them to lipid nanoparticle vaccines can enhance immune responses against viral and tumor antigens. A research team immunized mice with.

LNP(poly(I:C)) adjuvanted recombinant Spike protein from the SARS-CoV-2 virus yielded robust antigen-specific antibody titers [79]. Their study findings suggest that LNP formulation is a feasible and scalable approach that can greatly enhance the adjuvant properties of poly(I:C) by activating TLR3 and RLRs, and altering its pharmacokinetic and pharmacodynamic properties in vivo. Pamela T Wong’s group [80] developed a rationally designed IN adjuvant consisting of a combined nanoemulsion (NE)-based adjuvant and an RNA-based RIG-I agonist (IVT DI) to drive more robust, broadly protective antibody and T cell responses.

Given the remarkable safety and efficacy of mRNA vaccines employing LNPs as delivery vectors for COVID-19 prevention, the mRNA-LNPs delivery system has gained widespread utilization in the treatment of diverse ailments including cardiovascular diseases, cancers, and infectious diseases [81] (Fig. 5).

Fig. 5
figure 5

The schematic illustrations of clinical applications of mRNA-LNPs

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Application of mRNA-LNPs in infectious diseases

Since the first vaccine was used to treat cowpox in 1796 [82], researchers have developed vaccines to prevent and control many infectious diseases. Traditional vaccines, which consist mainly of inactivated pathogens, have had great success in preventing more than 30 infectious diseases worldwide, and even eradicated many of them. However, traditional vaccines against some of the more challenging infectious diseases have still failed to achieve high levels of protection. RNA vaccine technology is widely acknowledged as an advanced immunization approach capable of effectively inducing a robust humoral and cellular immune response. Given the rapid global dissemination of COVID-19, two mRNA vaccines targeting SARS-CoV-2, namely mRNA-1273 and BNT162b2, received emergency use authorization from the FDA in 2021 [83,84,85]. Furthermore, numerous vaccines designed for chronic or recurrent viral infections such as HIV were currently undergoing clinical trials [77]. These promising findings underscore the pivotal role of mRNA vaccines in shaping the future landscape of infectious disease prevention and control.

New or re-emerging infectious viruses are characterized by the emergence of novel pathogens or the resurgence of previously undetectable pathogens. These viruses pose a significant threat to human health due to their abrupt and uncontrollable transmission [86]. Firstly, traditional vaccines may not offer the expeditious progress required during a pandemic, owing to the imperative demand for efficacious vaccines in controlling novel or resurgent viruses [87]. Furthermore, the highly variable nature of re-emerging viruses like influenza presents significant challenges in developing a broadly effective vaccine [88]. mRNA vaccines offer immense potential for rapid, cost-effective, and scalable good manufacturing practices, making them an ideal platform for the development of highly efficacious and timely vaccines against emerging infections.

mRNA vaccines can be utilized for both prophylactic and therapeutic immunization purposes, rendering them highly versatile in their application. Moreover, their rapid production time enables swift response to emerging viral threats. Notably, the respiratory syncytial virus fusion glycoprotein (RSV-F) represents a conservative target for neutralizing antibodies and stands as the most promising antigen in RSV vaccine development. Recently, Espeseth et al. [89] have developed a lipid nanoparticle mRNA-LNPs vaccine by encapsulating chemically modified mRNA within LNPs, demonstrating robust immunogenicity and protective efficacy against respiratory syncytial virus (RSV). mRNA-LNPs vaccine elicited significant CD4 + and CD8 + T cell responses in mice compared to protein-based vaccines and triggered a strong cellular immune response against RSVF. This mRNA-LNPs vaccine expressing RSVF protein has the potential to be used safely and effectively to prevent RSV diseases [77]. Similarly, Moderna is also very active in the field of mRNA-LNPs vaccines for many infectious diseases.

An OspA-encoding mRNA-LNPs vaccine was designed and synthesized by Matthew et al. [90], which demonstrated superior immunogenicity and protective efficacy compared to an aluminum adjuvanted OspA protein subunit vaccine. Single immunization with OspA mRNA-LNPs induced potent humoral and cell-mediated immune responses in mice, resulting in protection against bacterial infection.

mRNA-LNPs vaccines can also be employed in veterinary medicine for the prevention of infectious diseases in animals. Saxena et al. [91] utilized a self-amplifying mRNA vaccine encoding the glycoprotein of rabies virus to elicit immune responses against canine rabies. Recently, VanBlargan et al. [92] have developed an mRNA-LNPs vaccine encoding the prM and E genes of deer Powassan virus and demonstrated its immunogenicity and efficacy in mice.

The mRNA vaccines (vaccine candidates) currently undergoing preclinical and clinical trials for infectious diseases are documented in Table 1.

Table 1 List of clinical trials evaluating mRNA vaccines against infectious diseases
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Application of mRNA-LNPs in cardiovascular diseases

Heart failure is partially attributed to the activation of cardiac fibroblasts, which respond to myocardial injury and inflammation by excessively synthesizing fibrous material, leading to myocardial sclerosis and impairment of cardiac function [77]. Therefore, the scientists proposed the development of CAR-T cell therapy that targets activated cardiac fibroblasts. Typically, current CAR-T therapy involves isolating T cells from patients and modifying them in vitro to express CARs that recognize cancer cell surface antigens. These modified T cells are then amplified before being infused back into the patient to effectively eliminate cancer cells [77].

However, due to the pivotal role of fibroblasts in the process of wound healing, significant hurdles arise when employing CAR-T cell therapy for heart failure or other fibrotic disorders [77]. CAR-T cells can persist in patients for an extended duration, ranging from months to years, thereby exerting a prolonged inhibitory effect on fibroblast growth and potentially leading to complications in wound healing. The sustained presence of CAR-T cells is advantageous for cancer treatment as it ensures a durable therapeutic impact. However, in the case of patients with cardiac fibrosis who experience injuries subsequent to receiving conventional CAR-T therapy, the persistent existence of CAR-T cells may impede normal wound healing processes and pose safety concerns. To address this issue, researchers [93] engineered an mRNA that encodes a chimeric antigen receptor (CAR) capable of specifically binding to fibroblast activation protein expressed on the surface of fibroblasts. Subsequently, they encapsulated the mRNA within LNPs, which were further conjugated with an antibody targeting CD5 + T cells. This innovative approach enabled the generation of intact therapeutic CAR-T cells in vivo. Remarkably, experimental studies conducted in a murine model of heart failure demonstrated the efficacy of this treatment strategy in reducing cardiac fibrosis and restoring cardiac function [77].

Application of mRNA-LNPs in liver diseases

Rizvi et al. [94] developed a LNP-encapsulated nucleoside-modified mRNA that exhibited transient and sustained expression of hepatocyte growth factor (HGF) and epidermal growth factor (EGF) in murine hepatocytes. The liver-specific targeting of mRNA-LNPs were also demonstrated through intravenous administration of mRNA-LNPs encoding firefly luciferase, resulting in sustained protein expression for approximately 3 days. Furthermore, HGF mRNA-LNPs effectively induced hepatocyte proliferation. In murine models of chronic liver injury and nonalcoholic fatty liver disease, as well as in mice with acetaminophen-induced acute liver injury, administration of mRNA-LNPs containing hepatocyte growth factor (HGF) and epidermal growth factor (EGF) exhibited significant amelioration of steatosis, prompt activation of the hepatocyte regeneration pathway, and accelerated recovery of hepatic function following injury.

Application of mRNA-LNPs in cancer

Due to its rapid synthesis, mRNA has emerged as a suitable option for developing personalized vaccines, particularly in the context of highly heterogeneous diseases like cancer. The mRNA-LNPs vaccines have demonstrated the ability to elicit immune cell-mediated responses, thereby generating robust CD8 + T cell responses for effective eradication or reduction of tumor cells [77]. Consequently, they are currently regarded as highly promising therapeutic interventions for cancer [95,96,97,98,99]. Cancer mRNA vaccines can express tumor-associated antigens and elicit cell-mediated immune responses, thereby facilitating eradication or inhibition of cancer cells. Consequently, these vaccines are increasingly employed as therapeutic agents rather than prophylactic measures [100]. Recent studies have also demonstrated the development of mRNA vaccines with potent anticancer properties, eliciting robust and efficacious T cells and humoral immune responses. Lee et al. [101] employed tripalmitoyl-S-glycero-cysteine linked to a pentapeptide (PAM3CSK4, Pam3) as an adjuvant for encoding ovalbumin-bound mRNA in LNPs, thereby developing a Pam3-doped mRNA/Pam3-LNPs vaccine. Upon cellular internalization through endocytosis, the mRNA/Pam3-LNPs vaccine undergoes dissociation and release mRNA in acidic environments, which is subsequently recognized by TLR7/8 on the endosomal membrane. Through the synergistic effect of Pam3 and mRNA targeting different TLR subclasses, Pam-LNPs exhibit potent immune stimulation within cells and elicit robust antigen-specific CD8 + T cell responses, thus greatly augmenting the therapeutic efficacy of mRNA vaccines for cancer prevention [101].

Jamile et al. [102] compared three different mRNA vaccine modes in mice with HPV-16 infection-associated tumors. They prepared self-amplifying mRNA encapsulated in LNPs and unmodified and nucleoside-modified nonreplicating mRNA vaccines encoding a chimeric protein fused by HPV-16 E7 oncogene and herpes simplex virus type 1 (gDE7). The results showed that a single low-dose immunization with any of the gDE7 mRNA vaccines induced e7-specific CD8 + T cell activation and produced a memory T cell response that prevented tumor recurrence and eradicated subcutaneous tumors at different growth stages.

The development of therapeutic vaccines for cancer treatment has been a time-consuming process spanning several decades. Despite the approval of only one dendritic cell-based vaccine (sipuleucel-T) by the FDA for hormone-refractory prostate cancer, its effectiveness remains unsatisfactory [103]. The efficacy of cancer vaccines can be influenced by various factors, such as the limited specificity of tumor-associated antigens (TAAs), immune evasion mechanisms employed by cancer cells, and immunosuppressive conditions within the tumor microenvironment [104]. mRNA vaccines were demonstrated promising efficacy in clinical trials for anticancer treatment. However, neoantigen-based cancer vaccines face two primary challenges: limited neoantigen immunogenicity and high production costs [105]. mRNA vaccines have gained significant popularity as a promising approach for cancer treatment [106]. On one hand, mRNA vaccines offer advantages such as rapid development, cost-effective manufacturing, and safe administration, thereby alleviating the economic burden associated with neoantigen vaccines. On the other hand, mRNA vaccines capable of efficiently expressing multiple antigens in both in vivo and in vitro antigen-presenting cells (APCs) can elicit robust T cell responses against neoantigens.

In contrast to delivery systems employed for mRNA vaccines against pathogenic infections, therapeutic mRNA vaccines designed for cancer treatment necessitate a robust CD8 + and CD4 + T cell response [107]. Activation of type I interferon (IFN) has been demonstrated to play a crucial role in the development of cytotoxic T cell response [108]. Consequently, diverse strategies have been investigated to augment the activation of type I IFN. In addition to augmenting the immune response via delivery systems, injectable mRNA cancer vaccines have witnessed a transition from tumor-associated antigens (TAAs) to neoantigens. These neoantigens are characterized by their highly specific somatic mutations occurring randomly in individual tumor cells, which distinguishes them from normal cells. Consequently, they are regarded as ideal candidates for the development of cancer vaccines [109]. While traditional peptide-based vaccines are constrained by their limited immunogenicity and challenging physicochemical properties, mRNA vaccines offer greater design flexibility and have been demonstrated enhanced immune responses [106]. Sahin et al. [110] reported the first injectable mRNA cancer vaccine, which effectively elicited T-cell responses against multiple neoantigens in all patients with advanced melanoma through intranodal administration of patient-specific encoded neoantigens. Moreover, Moderna has successfully developed a range of mRNA cancer vaccines encoding neoantigens that specifically target multiple types of cancers. These innovative mRNA-based vaccines have demonstrated the ability to elicit T-cell responses against up to nine predicted neoantigens in a single patient. BioNTech has also developed a personalized mRNA cancer vaccine, known as RO7198457, which encodes a novel antigen specifically targeting advanced or metastatic solid tumors larger than 20 inches.

The rapid advancement in delivery systems and novel antigens has sparked a revolutionary transformation in mRNA cancer vaccines, with accumulating evidence substantiating the immense potential of individualized mRNA-encoded cancer vaccines. The mRNA vaccines (vaccine candidates) currently undergoing preclinical and clinical trials for cancer is documented in Table 2.

Table 2 List of clinical trials evaluating mRNA vaccines against cancers
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Future directions and prospects of mRNA-LNPs

The rapid development of mRNA COVID-19 vaccines, facilitated by advancements in mRNA technology and LNPs delivery systems, showcases the clinical potential of mRNA-LNPs formulations and provides a formidable tool to combat the SARS-CoV-2 pandemic. With the continuous exploration of the structural characteristics of ionizable lipids and auxiliary lipids, an increasing number of delivery systems with remarkable efficiency have been developed, leading to successive advancements in mRNA vaccines against various infectious diseases. Nevertheless, progress in clinical research on mRNA vaccines for cancer remains sluggish. Based on the insights gained from clinical studies, there is scope for further enhancement of mRNA-LNPs formulations.

mRNA-LNPs vaccines exhibit high instability and rapid degradation at room temperature, necessitating ultra-low temperature storage. Consequently, addressing the challenges associated with long-term preservation and long-distance transportation of mRNA-LNPs assumes paramount importance. Furthermore, the emergence of pathogen variants and immunosuppression of antigens pose persistent obstacles in the development of efficacious vaccines.

Encouragingly, the success of mRNA-LNPs vaccines in the treatment of COVID-19 has revealed their effectiveness. It is believed that mRNA-LNPs formulations will be more widely used and become a new breakthrough point in biopharmaceuticals.