Nanomaterials for mRNA‐based therapeutics: Challenges and opportunities

Abstract Messenger RNA (mRNA) holds great potential in developing immunotherapy, protein replacement, and genome editing. In general, mRNA does not have the risk of being incorporated into the host genome and does not need to enter the nucleus for transfection, and it can be expressed even in nondividing cells. Therefore, mRNA‐based therapeutics provide a promising strategy for clinical treatment. However, the efficient and safe delivery of mRNA remains a crucial constraint for the clinical application of mRNA therapeutics. Although the stability and tolerability of mRNA can be enhanced by directly retouching the mRNA structure, there is still an urgent need to improve the delivery of mRNA. Recently, significant progress has been made in nanobiotechnology, providing tools for developing mRNA nanocarriers. Nano‐drug delivery system is directly used for loading, protecting, and releasing mRNA in the biological microenvironment and can be used to stimulate the translation of mRNA to develop effective intervention strategies. In the present review, we summarized the concept of emerging nanomaterials for mRNA delivery and the latest progress in enhancing the function of mRNA, primarily focusing on the role of exosomes in mRNA delivery. Moreover, we outlined its clinical applications so far. Finally, the key obstacles of mRNA nanocarriers are emphasized, and promising strategies to overcome these obstacles are proposed. Collectively, nano‐design materials exert functions for specific mRNA applications, provide new perception for next‐generation nanomaterials, and thus revolution of mRNA technology.


| INTRODUCTION
As intermediate tunneling, messenger ribonucleic acid (mRNA) can transport genetic codes from DNA to ribosomes for protein expression, which has emerged as a highly appealing potential to change the vaccination, protein replacement therapy, and other treatments for human diseases. [1][2][3][4] The first study of successful mRNA therapeutics in the preclinical application was published in 1990. 5 In addition, due to the progress of mRNA manufacturing and intercellular delivery strategy, significant development and breakthrough have been made in the mRNA-based therapy. 6 In addition, mRNA-based therapeutics exhibit several advantages over traditional functional biomolecules and therapeutic proteins. 7 First, mRNA is endowed with perfect safety under physiological conditions because it is not transferred into the nucleus and does not integrate into the host genome. Therefore, mRNA-based therapeutics are transient, avoiding the potential risk of insertional mutagenesis. 8,9 Second, the initiation of protein translation is promoted once mRNA just reaches the cytoplasm. 10 Third, these theranostic mRNAs can achieve stability and controlled release through mRNA structural modification (such as 5 0 -Cap, 5 0 -UTR, ORF, 3 0 -UTR, and 3 0 -poly(A) tail) or nanomaterials delivery, such as lipidderived nanoparticles (LNPs), polymer-based nanoparticles, and hybrid nanoparticle, thus improving the pharmacokinetics of nucleic acid drugs. [11][12][13] Fourth, it is easy to design and manufacture mRNA using chemical synthesis and enzyme synthesis approaches. 14 Solid-phase chemical synthesis is generally suitable for the synthesis of shortchain RNAs (50-100 nt). Moreover, the synthesis yield using this method remains poor and impure with the increase of RNA length. 15 Recently, enzyme synthesis is a popular method based on RNA polymerase (usually T7) and linear DNA to obtain desired mRNA. However, the initial product of mRNA through this method is composed of targeted mRNA, untargeted mRNA, and oligodexynucleotides, which need to be purified for further application. 16 Despite its potential advantages, how to efficiently and stably perform intracellular delivery of mRNA is a significant barrier in the clinical application of mRNA as a therapeutic modality. 14,[17][18][19] Additionally, naked mRNA molecules are easily degraded by enzymes and are not easily taken up by the target cells. 20 Therefore, methods and vehicles for mRNA delivery, such as viral vectors, mechanical transfection, and nonviral vectors, have been developed. 11,21,22 Viral vectors are very efficiency in mRNA transfection and are applied in clinical trials, such as Kymriah for chimeric antigen receptor (CAR) T immunotherapy on lymphoblastic leukemia. 23,24 However, viral vectors have disadvantages of potential carcinogenic, high immunogenic, limited gene packaging ability, and low-volume production. 25,26 Mechanical transfection, such as injection of naked mRNA in conventional and self-amplifying forms, is not widely applied due to their extracellular exonucleases, inefficient cell uptake, unsuccessful endosomal release, or potential cytotoxicity. 27 Nonviral vehicles, including lipid or polymer-based nanoparticles, exosomes, and ligand-RNA conjugates, represent safe and efficacious technologies and allow repeated administrations. 28,29 Moreover, lipid-based nanoparticles have been the most extensively used for mRNA-based therapeutics in preclinical studies and undergoing clinical trials (Table 1). 49,50 In addition, some nonviral vehicles possess targeting effects at desired sites in mRNA-based therapeutics because of their organ-targeted properties. 50 Furthermore, nonviral nanoparticles are also substantially attractive in mRNA vaccines for virus outbreaks. 51 Here, we reviewed the latest developments in mRNA-based therapeutics, such as vaccination, genome editing, and protein replacement therapy. Moreover, we also emphasized the advanced delivery platforms for mRNA and their biomedical applications. In addition, we discussed the potential challenges of mRNA-based therapeutics and provided our future perspectives.

| mRNA FOR THERAPEUTIC APPLICATIONS
Over the past decades, mRNA-based therapeutics have been highly promising for treating various human diseases, including cancers, infections, and autoimmune diseases. [52][53][54] Recently, mRNA-based vaccines have become a promising alternative in booster immunizations. 1,55 In addition, mRNA-based protein replacement therapy provides a viable alternative and permanent cure for various diseases. 56,57 In 1961, as an intermediate hereditary substance, mRNA is first discovered. 58 As early as 1990, scientists have first reported that intramuscular injection of in vitro-transcribed mRNA into mouse skeletal muscle cells, results in in vivo expression of encoding UTR are located at the upstream and downstream domains of the mRNA coding region, respectively, and are essential to protect the mRNA from decomposition and degradation. 63 Moreover, the 5 0 UTR plays a crucial role in controlling protein translation efficiency due to its binding site for the preinitiation complex initiation of protein translation. 64 In addition, 3 0 UTR contains degradation signals of mRNA, and alpha-globin or beta-globin 3 0 UTR has been developed to improve the stability of heterologous mRNA in cells. 65 The ORF region contains protein-encoding nucleotide sequences with a start codon and ends with a stop codon. 66 For the production of mRNA, the in vitro transcription (IVT) is performed on a linear pDNA template, and the RNA polymerase of T7 DNA48 synthesizes multiple copies of the RNA transcript. 67 Once the RNA is capped at the 5 0 end and purified, it is ready for formulation. Three main types of RNA are currently being developed: self-amplifying RNA (saRNA), nonreplicating mRNA (nrRNA), and trans-amplifying mRNA (taRNA) 68,69 ( Figure 1). In this section, we summarized a detailed overview of mRNA-based clinical applications, and mRNA deliver system is available in part 3.

| mRNA-based protein replacement therapy
Over the past few years, evidence has revealed that mRNA-based therapeutics have gained immense interest and are demonstrating to be viable options in a wide range of diseases, including cancer, 70 infectious disease, 71 and rare genetic diseases. 72  Cardiovascular diseases (CVDs) lead to more than one-third of all deaths worldwide, mainly due to ischemic heart disease (IHD). 79,80 Recent evidence has demonstrated that mRNA-based management is promising to improve myocardial regeneration and prevent heart failure. 4,81,82 The main mechanisms of IHD therapy include inhibition of cardiomyocyte death, promotion of cardiac regeneration, maintenance of coronary plaque, enhancement of angiogenesis, and triggering of cardiac reprogramming. 81 For instance, pyruvate kinase muscle isoenzyme 2 (PMK2) is a critical enzyme in aerobic glycolysis, contributing to cell metabolic reprogramming. 83 Furthermore, PMK2 can induce cardiomyocyte proliferation and is primarily expressed at higher levels in regenerative fetal heart and neonatal cardiomyocyte but not in adult cardiomyocyte. 84 Therefore, in IHD therapeutics, the upregulation of PMK2-encoding mRNA can re-invigorate the proliferation of cardiomyocyte, resulting in cardiomyocyte regeneration. 84 Moreover, it has been reported that insulin-like growth factor-1 (IGF1) protects cardiomyocytes after ischemia and myocardial infarction (MI). 85 Therefore, promoting the expression of IGF1 in mouse hearts after MI can enhance cell survival and inhibit cell death under hypoxia-induced apoptosis. 85,86 In addition, for MI treatment, overexpression of an angiogenic factor, human vascular endothelial growth factor A (VEGFA)-encoding mRNA, in mice promotes the differentiation of F I G U R E 1 mRNA manufacturing process. In vitro transcribed mRNA encoding the gene of interest has been used to generate conventional mRNA. Use saRNA, which encodes a replicase that self-amplifies mRNA for positive-strand RNA viral genomes. The second utilizes a novel bipartite vector system of taRNA, in which the replicase is deleted to form a trans-replicon. Both protocols improved the half-life and translation efficiency of mRNA.
heart progenitor cells into endothelial cells, improves heart function, and prolongs long-term survival. 87 Notably, mRNA-based therapeutics play a vital role in injured hearts repair, angiogenesis, and cardiomyocyte regeneration.
mRNA-based therapeutics have also become a new pillar for liver disease treatment. Crigle-Najjar syndrome type 1 (CN1) is a rare metabolic disease characterized by the absence or decreased activity of UDP glucuronosyltransferase family 1 member A1 (UGT1A1), an enzyme required for glucuronidation of unconjugated bilirubin in the liver. 88 The mutation of UGT1A1 gene contributes to severe unconjugated hyperbilirubinemia and consequently develops jaundice and irreversible brain and muscular damage. 89 However, mRNA-based therapeutics can restore the hepatic expression of UGT1A1 and normalize the levels of bilirubin. 90 Moreover, α-1 antitrypsin (AAT) deficiency is a recessive disease caused by mutations in the serpin family A member 1 (SERPINA1) gene. 91 As a serine protease inhibitor, AAT neutralizes neutrophil plasminogen in the lung and liver to protect tissues from excessive proteinase activity. 92,93 The AAT deficiency can activate the intracellular injury cascade of apoptotic liver cell death, compensate hepatocellular proliferation, and lead to end-organ injury. 94 Nevertheless, supplement of AAT-encoding mRNA can significantly produce AAT protein and provide an alternative modality for liver AAT deficiency. 95 In addition, mRNA-based therapeutics are currently being tested in preclinical and clinical studies for other liver diseases, including thrombotic thrombocytopenic purpura (TTP), 96

| mRNA vaccines
Vaccines play a pivotal role in the public health and the quality of human life, which not only prevent various infectious diseases but also reduce and eradicate mostly fatal disease, such as tuberculosis, smallpox, ebola, meningitis, and malaria. 106,107 Traditionally, vaccines can be roughly subdivided into live attenuated vaccines, inactivated vaccines, subunit vaccines, and toxoid vaccines, which were commonly applied in clinical practice, lastingly provided protection for various diseases. [107][108][109] Although conventional vaccines can provide long term and durable protection against diseases, they have difficulty in fulfilling pandemic needs as some emerging infectious diseases require more development speed, low-cost manufacturing, and large-scale production. mRNA vaccines have emerged as promising alternatives to traditional vaccines due to their advantages of rapid development, high efficacy, and cost-efficient manufacturing. Furthermore, the mRNA vaccine is more likely to stimulate protective antibody and antigen-specific T-cell responses ( Figure 2). Notably, the innate immune response is significantly enhanced following secondary immunization. 110 In addition, conventional vaccines are not suitable for cancer vaccine development. In contrast, mRNA vaccines encoded using tumor-associated antigens (TAAs) can be developed more rapidly with cost-effective approaches. [111][112][113] Moreover, mRNA vaccines have several advantages over conventional vaccines. 114,115 First, mRNA vaccines are scalable due to modified mRNA sequence can satisfy all genetic information requirements to encode all protein. 55 Second, mRNA vaccines are comparatively effective and safe because they only target cytoplasmic delivery, avoiding the risk of genome integration. 116 Third, manufacturing mRNA vaccines on a large scale tends to be industrialized. 116 Therefore, mRNA vaccines have been widely studied in different kinds of diseases.
At present, there are several modes of production of RNA vaccines: template-directed synthesis, nonreplicating, and self-amplifying messenger RNA (samRNA) vaccines. 117 Nonreplicating vaccines represent a straightforward approach wherein administered mRNA is directly translated into the cytoplasm of transfected cells to encode the protein antigens of interest. 118 However, samRNA vaccines encode the RNA genome of a single-stranded RNA virus and contain additional sequences in the coding region for RNA replication. 118 However, both nonreplicating and samRNA vaccines have been proven effective against infectious diseases, and nonreplicating vaccines have strong potential to be used in cancer immunotherapy. 112,115,118,119 So far, most mRNA vaccines have been developed for infectious diseases or cancer immunotherapy. To our knowledge, most ongoing trials or FDA-approved mRNA therapeutics are based on typical linear mRNA, which can be produced by RNA polymerasemediated IVT. However, in contrast to typical linear mRNA, circular mRNA (circRNA) increases mRNA stability and has been found to contribute to robust and durable protein expression. 120 Therefore, the development of circRNA vaccines triggers higher and more durable effective neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and shows adequate protection against new SARS-CoV-2 variants. 121 In addition, these circRNA vaccines can strongly induce immune responses and are more durable than typical mRNA vaccines. However, the development of circRNA vaccines faces problems, such as efficient cyclization of intramolecular RNA, purification of circRNA, and high cost of IVT production reagents. 122  Moreover, mRNA-based vaccines can code tumor-specific antigens (TSAs), introduce them into APCs, and synthesize the required antigens, stimulating both humoral and cellular immunity to kill cancer cells. 115 In addition, mRNA-based vaccines can also encode and express TAAs and TSAs to specifically attack and eliminate cancer cells. 134 To date, some strategies have demonstrated that mRNA-based vaccines possess the feasibility and tremendous potential to be applied in cancer treatment. For instance, BI1361849, as an mRNA encoding non-small cell-associated tumor antigens, can promote the proliferation of functional tumor-specific CD4 and CD8 cell, killing the cancer cell and improving survival. 135 In addition, DCs-based mRNA vaccines also provide the necessary modalities to administer the tumor. DCs, as the most efficient APCs, play a vital role in the activation process of helper T and killer T cells through capturing, processing, and presenting antigens to T cells. 136 The DCs-based mRNA tumor vaccines are produced undergoing multiple procedures: DCs are extracted from the patient's peripheral blood, and the differentiation and maturation are stimulated using the granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4); then mRNA encoding TSAs is transfected; the mRNA-loaded DCs are finally reinfused into the patients, and the immune system is stimulated to combat cancer cells. 137 For example, the pp65-mRNA-loaded DCs vaccine is used to treat glioblastoma, and it can increase long-term progression-free survival and prolong overall survival. 138 All these studies show that mRNA-based vaccines may be safe and effective alternatives to cancer immunotherapy. In addition, many mRNA-based cancer vaccines have been evaluated for their effectiveness.

| Genome editing by mRNA
In recent years, genome editing has become the most potent strategy In addition, nanoparticle-mediated CRISPR/Cas9-encoding mRNA delivery is the most frequently used gene-editing technology ( Figure 3). Several studies have shown that in non-human primate models, CRISPR/Cas9-mediated CCR5 disruption is used to give resistance to HIV/SIV infection. 144 Therefore, mRNA-based genome editing will be a promising modality for gene therapy, and several of them are undergoing clinical studies.

| NANOPARTICLES FOR MRNA DELIVERY
mRNA must to enter the host cytoplasm and express specific antigens to remain functional. However, it is difficult for negatively charged mRNA macromolecules to penetrate the anionic lipid bilayer on the cell membrane. Moreover, the single-chain mRNA is fragile and easily degraded by extracellular ribonucleases in skin and blood. 9 Therefore, an ideal mRNA delivery system not only enhances efficient cellular uptake by host cells but also protects them from degradation. In the following sections, we describe various mRNA delivery systems developed and applied.

| Lipid and LNPs for mRNA delivery
The lipid can be cationic, neutral, and zwitterionic, which is an important class of vector material for mRNA delivery. 145 However, cationic lipids represent the widely applied and studied materials for mRNA delivery. 145 For example, N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA), as the first synthetic cationic lipids, is used to delivery mRNA to several cell lines. 146 Unfortunately, toxicity and immunogenicity may be the main obstacles to its clinical application. 147 The cationic lipids, combined with other components forming nanoparticles, are named LNPs. 148 LNPs are the most extensively investigated and clinically advanced delivery system for mRNA-based therapy. 13,149 LNPs are nanocarriers with a monolayer structure of phospholipids, which can protect the mRNA in the lipid core from deg- LNPs mediated delivery of Cas9 mRNA is to exert cancer immunotherapy or genome editing. [156][157][158] Despite their unprecedented potential, several studies have demonstrated that LNPs still have potential limitations that hinder them practical application. One limitation is the safety of LNPs due to their potential toxicological effects and the tissue distribution of their payloads. 159,160 Another limitation is immunogenicity of LNPs because LNPs constituents can induce immune response, promote neutrophil infiltration, and boost the secretion of proinflammatory factor and F I G U R E 3 Delivery of mRNA for genomic editing by nanoparticles. The CRISPR/Cas9 components (sgRNA, Cas9 mRNA) are encapsulated in nanocarriers, which need to escape from endocytosis/lysosomes after cellular uptake. Then, it will be translated directly into proteins in the cytoplasm and transported to the nucleus so that the CRISPR machinery can act on the genomic DNA of the cell. mRNA delivery and translation are transient and thus avoid the concern of permanent integration of CRISPR genes into the host genome.
reactive oxygen species (ROS). 161 Moreover, LNPs induced side effects, such as pain, redness, and fever, have been reported. 162,163 Therefore, further studies on the cytotoxicity and immunogenicity of LNPs delivery system are urgently needed.

| Lipopolyplex for mRNA delivery
The lipopolyplex (LPP) nano-delivery platform is a bilayer structure with a polymer-encapsulated mRNA molecule as the core, which is ing T-cell immunity. This approach provides a new way to explore effective and low inflammatory mRNA lipid polymorphs. 165  63% as a significant therapeutic approach in a mouse model of agerelated macular degeneration. Sequence results show that VLP-mRNA does not induce off-target effects. 167 These experimental results strongly support the potential of VLP for clinical application in delivering CRISPR gene therapy. 168

| Polymer-based delivery system
The polymer-based delivery system consists of another prominent family of mRNA delivery carrier and has excellent potential for mRNA delivery. 175 Moreover, a polymer-based delivery system is comprised three kinds of polymer, including cationic polymer, dendrimer, and polysaccharide polymer. 13 Cationic polymers have the advantages of easy synthesis and modification. In addition, cationic polymer can be complex with mRNA by electrostatic attraction and hydrophobic interaction, contributing to a more stable polyplex. 176 Polyethyleneimine (PEI) is one of the most widely used network polymers and has high efficiency of mRNA delivery. 177 However, PEI has the disadvantages of poor biodegradability and high toxicity, limiting its broad clinical application. 147 Fortunately, with the development of chemical modification, the toxicity of PEI is reduced while maintaining its protonatable properties and high delivery efficiency. 178  Interestingly, the delivery of therapeutic FAH mRNA based on dendritic molecules can restore liver function and prolong the survival time of HT-1 mice. 183 Therefore, these results provide a dendrimer polymer delivery system with great potential to improve mRNA delivery and treat genetic diseases.
Polysaccharides, as relatively common natural biomaterials, can be easily chemically modified for efficient delivery with high biocompatibility and little toxicity and are mainly composed of chitosan, alginate, dextran, and hyaluronic acid. 184 Chitosan is one of the most common polysaccharides to deliver mRNA. It has been reported that chitosan-based deliver therapeutic surfactant protein B (SP-B) mRNA can replace SP-B deficiency and experimental asthma in mouse models and prolonged life in treated mice. 185 Cystic fibrosis (Cf) is a genetic disorder that originates in an alteration in the cystic fibrosis transmembrane conduction regulator (CFTR) gene. 186,187 Fortunately, chitosan-based delivery of CFTR mRNA can restore CFTR function and provide great potential for treating CF. 188 Alginate, as an important polysaccharide, is derived from brown algae and bacteria and consists of a-L-guluronic acid and b-D-mannuronic acid building blocks linearly linked by 1,4-glycosidic linkages. 189 Moreover, alginate has biodegradable, nontoxic, and abundant features. 190 It has been reported that delivery of alginate-based neurotrophin-3 (NT-3) mRNA can enhance nerve regeneration. 191 Dextran, as a water-soluble, naturally degradable polysaccharide, originates from bacterial metabolites and consists of (1,6)-a-D-glucose with various ratios of linkages and branches. 190 It has been demonstrated that dextran-based mRNA delivery can induce high gene expression and low cytotoxicity and provide a promising strategy to treat breast cancer. 192 Hyaluronic acid, as a nonsulfated glycosaminoglycan, is composed of glucuronic acid and N-acetyl-D-glucosamine bound linked beta-linkages with good bio-compatibility and biodegradability. 193  Detailed toxicological assessments (genotoxicity and oxidative stress) are therefore needed. It is crucial to develop biodegradable and scavengable inorganic nanomaterials to minimize the short-and longterm cytotoxicity of inorganic nanoparticles. Besides, these inorganic nanomaterial carriers must be targeted to specific tissues or organs to minimize side effects. responses. 205 Collectively, these results reveal that exosomes can be utilized as deliver carrier for mRNA against COVID-19.  The production of RNA-loaded exosomes can be further promoted by mechanically extruding the vesicles through a micrometer pore filter using cells expressing of the desired mRNA. 215 In addition to exosomes as emerging carriers for mRNA drug delivery, surface functionalization of exosomes can achieve target-specific delivery. 216

| CONCLUSION AND FUTURE PERSPECTIVES
The rapid development of mRNA therapy has brought unprecedented prospects and opportunities in the biomedical field. We comprehensively reviewed the latest progress in mRNA delivery, which gives essential value for designing mRNA delivery systems.
Addressing To this purpose, the commonly used carriers for mRNA delivery, including lipid nanoparticles, LNP, polymer nanoparticles, exosomes, and MVs, are evolved as a promising class of nanomaterials for RNA delivery. We summarized the strengths and weaknesses of various nanomaterials in mRNA delivery (Table 3).
mRNA must cross several tissue barrier, extracellular barrier, intracellular endosomal escape, and intracellular immunity before it arrives at the intracellular target site. Therefore, it remains challenging to deliver mRNA precisely into the target cells. Most of the existing LNPs are accumulated in the liver after being injected into the body.
Therefore, how to precisely delivery sufficient mRNA to target organs when using intramuscular LNP-RNA is of great importance. This problem can be overcome by selective organ targeting (SORT) strategies.
The key to organ-specific delivery is manipulating the internal or external charges of LNPs. For example, altering the disulfide bonds between the long lipid chains leads to selective accumulation of mRNA formulations in the liver. 230  One of the leading ongoing challenges for exosomes is the commercially available methods. However, their active substances in exosomes are relatively complex and face a certain degree of inherent biological variation, which may lead to product heterogeneity between different batches. Cell culture is currently one of the main sources of exosomes, while its production and scale are still challenging. In terms of scale-up production, several strategies have been developed, including the use of bioreactors, 233 while the cost/ yield is still economically unsuitable for clinical application. Alternative sources, including blood, milk, and plant, to obtain exosomes or exosome-like vesicles may allow for lower production costs and shorter production times. 234,235 In the future, exosomes are expected to bring breakthroughs in the field of mRNA drugs when technical difficulties are solved.
For the clinical application of nano-mediated mRNA delivery, it is also necessary to optimize the production and purification methods to

CONFLICT OF INTEREST
The authors declare no competing interests.

DATA AVAILABILITY STATEMENT
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