key: cord-0989676-hrop3nwa authors: Li, Mingyuan; Li, Yuan; Li, Shiqin; Jia, Lin; Wang, Haomeng; Li, Meng; Deng, Jie; Niu, Ali; Ma, Liqiao; Li, Weihong; Yu, Peng; Zhu, Tao title: The nano delivery systems and applications of mRNA date: 2021-10-08 journal: Eur J Med Chem DOI: 10.1016/j.ejmech.2021.113910 sha: df5c763720735ee0b0150b5fe1399f3afd6849a9 doc_id: 989676 cord_uid: hrop3nwa The current COVID-19 epidemic has greatly accelerated the application of mRNA technology to our real world, and during this battle mRNA has proven it's unique advantages compared to traditional biopharmaceutical and vaccine technology. In order to overcome mRNA instability in human physiological environments, mRNA chemical modifications and nano delivery systems are two key factors for their in vivo applications. In this review, we would like to summarize the challenges for clinical translation of mRNA-based therapeutics, with an emphasis on recent advances in innovative materials and delivery strategies. The nano delivery systems include lipid delivery systems (lipid nanoparticles and liposomes), polymer complexes, micelles, cationic peptides and so on. The similarities and differences of lipid nanoparticles and liposomes are also discussed. In addition, this review also present the applications of mRNA to other areas than COVID-19 vaccine, such as infectious diseases, tumors, and cardiovascular disease, for which a variety of candidate vaccines or drugs have entered clinical trials. Furthermore, mRNA was found that it might be used to treat some genetic disease, overcome the immaturity of the immune system due to the small fetal size in utero, treat some neurological diseases that are difficult to be treated surgically, even be used in advancing the translation of iPSC technology et al. In short, mRNA has a wide range of applications, and its era has just begun. Structure of LNP [7] . LNPs are prepared through rapid mixing, often facilitated by microfluidic devices [34] [35] [36] [37] . A particularly popular microfluidic preparation method is ethanol dilution, referring to the rapid condensation of lipids into nanodroplets when their ethanol solution is added to the excess of aqueous media [38] . The resulting LNPs may be considered a kinetic product and typically yield considerable encapsulation of nucleic acid. There are currently two 'conventional' mRNA COVID-19 vaccines in use that delivering S-mRNA through LNPs. These are the mRNA-1273 vaccine by Moderna and the BNT162b2 by BioNTech/Pfizer (Table 1) . Table 1 . Information about three mRNA-LNP products that are presently in use [39] . Aguado et al. [40] developed four kinds of LNPs with different composition for mRNA delivery (as shown in Table 2 ). In the comparison of DOTAP, DODAP and DOBAQ (as shown in Fig 2. ), DOTAP as the only cationic lipid showed good stability after 7 months, and the stability was improved with the addition of polysaccharides. Table 2 Solid lipid nanoparticles with different formulations [40] Number Davies et al. [41] found that subcutaneous injection of LNPs containing mRNA, can result in measurable secreted proteins in plasma exposure, but will be affected by dose limit of related inflammation. In order to overcome this limitation, the LNPs composed of MC3 amino-lipid and L608 amino-lipid (Fig 3.) were constructed, showing extended protein expression duration, which can realize system level of therapeutic proteins for chronic disease. Different LNPs also have different targeting and administration effects. Zhang et al. [45] used a microflow controller to prepare atomizable lipid nanoparticles for the delivery of mRNA to the lungs. During in vitro evaluations, high protein expression was detected. In addition, the luciferase protein was found to be highly expressed in the lungs of mice after the inhalation of four lead preparations. The development of mRNA delivery by LNP is very fast. At present, many companies are developing mRNA LNP products for different diseases. Some of the mRNA-LNP technology in development or on the market was shown in Table 3 . Although LNP is one of the most effective means for mRNA delivery, there is a virtually endless parameter space that can be modified in order to achieve a highly efficient, nontoxic, and tissue, organ, or cell-selective LNP formulation. In addition, the poor stability makes mRNA-LNP expensive to transport and store. The long-term storage of it is a significant yet underexplored part of the LNP lifecycle. Liposomes (Fig. 5 .) are spherical enclosed vesicles formed by phospholipid bilayer [46] [47] . Liposome was first discovered by A. D. Bangham in 1965 and used for small molecule drug delivery for a long time, with particle size range from 20 nm to 1000 nm [48] [49] . In addition to the encapsulation of small molecule chemotherapeutic drugs, more and more studies focus on the encapsulation and delivery ability of liposomes for gene drugs (including mRNA, pDNA, siRNA et al.), protein drugs, hormone drugs and so on. Cationic liposomes are positively charged, mainly composed of cationic lipids, which are able to efficiently concentrate nucleic acids in a targeted manner [50] [51] . In addition, good pharmacokinetic properties can be obtained in vivo by changing the physical and chemical properties of cationic liposomes, such as adjusting the size of particle size and modifying the surface of cationic liposomes [52] . The commonly used phospholipid materials for mRNA delivery liposomes was shown in Table 4 . In general, the preparation methods of liposomes include thin film dispersion, solvent injection, freeze drying, pH gradient method, etc. J o u r n a l P r e -p r o o f Table 4 The commonly used lipid materials for mRNA delivery Thirdly, as a delivery system, liposomes are not subject to host restrictions. Finally, phospholipid double layered membrane structure highly simulates cell membrane. It is a stable structure known from biological evolution theory and has excellent long-term storage stability. Michel et al. [53] prepared cationic liposome by thin film dispersion method, and However, although mRNA has been successfully delivered through liposomes, liposomes have some disadvantages. Compared with LNP, the production process of liposome preparation and mRNA encapsulation is much more complex. In general, LNP and liposome are both lipid nanoparticles. They have many similarities, as well as great differences (as shown in Table 5 ). Although less explored, polymer based delivery systems can also be used. Polymer materials are high molecular weight (usually up to 10~10 6 ) compounds, which are repeatedly connected by covalent bonds from simple structural units, such as chitosan, polyethyleneimine, polyurethane, and so on. Most polymer materials used for mRNA delivery require modification to improve their transfection efficiency and stability [58] . Polyethylenimine (PEI) systems successfully delivered mRNA to cells [59] and intranasally [60] . Additionally, PEI-based systems improved the response to sa-mRNA vaccines in skin explants [61] and in mice [62] . Soliman et al. [63] prepared nanoparticles containing mRNA by electrostatic complexation, which were composed of different degrees of deacetylation and sulfonation. The results showed that the polymer length and charge density of hyaluronic acid and chitosan directly affected the transfection efficiency by regulating the mRNA affinity, and the mixture concentration of N:P:C ratio trehalose and the nucleic acid dose also affected the transfection efficiency. Choia et al. [64] reported an Micelles refer to ordered aggregates of molecules that begin to form in large quantities after surfactant concentration reaches a certain value in aqueous solution. In micelles, hydrophobic groups of surfactant molecules aggregate to form the core of micelles, and hydrophilic polar groups form the outer layer of micelles [66] . Roloff [67] et al. studied the assembly of a novel RNA-polymer amphiphilic molecule into spherical micelle (as shown in Fig 7) with diameters of about 15-30 nm, demonstrating that they can efficiently enter living cells without the use of transfection reagents. Chan et al. [68] propose the use of specially tailored polyplex nanomicelles for the intravenous delivery of mRNA into the brain of mice. In brief, along the backbone of a polyaspartamide polymer that is terminated with a 42k Polyethylene glycol chain (PEG), aminoethylene-repeating groups (two, three, and four units, respectively) were conjugated to side-chains to promote electrostatic interactions with mRNA. This structural configuration would ultimately condense into a polyplex nanomicelle ranging between 24 and 34 nm. Then the luciferase (Luc2) mRNA as a reporter gene through in vitro transcription (IVT) and subsequently infused the polyplex nanomicelles into mouse brains via an intracerebroventricular (ICV) injection to bypass the blood-brain barriers (BBB). Data revealed that PEGylated polyplex nanomicelles possessing four repeating units of aminoethylene groups had exhibited the best Luc2 mRNA delivery efficiency with no significant immune response registered, and may be applied in the treatment of brain diseases. vivo [70] , modulate innate immune response and enhance protein expression in both DC and human cancer cells in vitro [71, 72] . mRNA polyplexes conjugated with an anion peptide, exhibited an increase in cellular uptake without inducing cytotoxicity in DC cells [73] . Qiu et al. [74] studied a novel RNA delivery vector, PEG12KL4, which synthesized cationic In view of the great application potential of mRNA, more and more researches are advanced for mRNA nano delivery systems, and are bound to invent novel delivery vectors with stronger transfection efficiency, lower toxicity and better stability. The specific delivery of mRNA is an excellent alternative to plasmid DNA, due to the latter's potential risk for random integration into the host genome. With the development of mRNA modification technology and delivery technology, its application prospect is more and more widespread. In addition, mRNA vaccine could activate both cellular and humoral immunity, achieving high protection rate. The which is the main site for neutralizing antibodies and prone to mutation [77, 78] . N501Y mutation can enhance the infectivity by enhancing the binding force with host ACE2 [79] , while the variant G614 virus gradually replaced the originally discovered D614 as the main epidemic strain, and the patients with G614 will release more viral nucleic acid [80] . The variant strains with reduced antibody sensitivity will become the main strain, such as N439K is not only more infectious, but also has the ability to resist a variety of antibodies, including a neutralizing antibody authorized by the FDA for emergency use [81] . Highly mutated viruses pose a great threat to the effectiveness of existing vaccines, while mRNA vaccines can be rapidly applied by updating their sequences based on the mutated genes of the mutant strains. As shown in Fig 9, after entering the cytoplasm, S-mRNA could be translated to S protein, which were produced by the host cell and could induce immune response. Meanwhile, the mRNA vaccine also shortened time by using the body's own molecular mechanisms. Martinon et al. [87] Pardi et al. [92] proposed a bivalently modified mRNA vaccine that encodes the foremembrane and envelope glycoproteins of the Zika virus strain in the 2013 outbreak. A single dose of the vaccine, encapsulated in LNPs and delivered intradermally, was sufficient to protect mice from viral attack two weeks or five months after vaccination and was sufficient to protect non-human primates five weeks after vaccination. Using the same antigen, Moderna has developed an unmodified, encapsulated mRNA-1893 vaccine against Zika, which received rapid FDA approval and was undergoing phase I trials to evaluate its safety, tolerability, and immunogenicity [93] . Importantly, mRNA-1893 prevented congenital transmission of the virus in a mouse model of congenital infection. John et al. [94] have In addition to enhancing active immunity, mRNA can also be used for passive immunity. The J o u r n a l P r e -p r o o f candidate drug mRNA-1944 is a good example of mRNA therapy that encodes human monoclonal neutralizing antibodies. The mRNA-1944 is designed to provide passive protection against chikungunya infection [95] . The super-potent antibody was isolated from B cells of natural infection survivors, and its sequence was encoded into mRNA molecules, encapsulated in LNPs, and delivered to mice by infusion. After mRNA delivery, CHKV-24, a human monoclonal antibody, was found to be expressed at immune-related levels and its protective ability was evaluated in a chikungunya mouse model. Treatment with CHKV-24 mRNA 2 days after inoculation reduced viremia to undetectable levels and protected mice from death. Further studies in non-human primates have also shown that the mRNA-1944 has long-lasting immunogenic effects [96] . In general, preclinical data encourage first human trials. Latourette II et al. [97] evaluated the protection of mRNA-LNP and protein vaccine against neonatal herpes by immunizing female mice before copulation, then compared the levels of IgG and neutralizing antibodies in mothers and newborns. Both vaccines protected first and second borns from disseminated infection, and proved effective in preventing neonatal herpes. The purpose of mRNA tumor vaccine is to prompt the cell-mediated response, such as the typical T lymphocyte response, so as to achieve the aim of remove or reduce tumor cell without harming normal cells [98] . STING is considered to be the center of the innate immunity and adaptive immunity adjustment factor. When stimulated, STING will induce type I interferon. The expression of cytokines and T cell recruiting factors leads to the natural effector cells of macrophages dendritic cells (such as NK) and the tumor specific T cells, which can not only express antigen in vivo, but also activate STING signaling pathway, and significantly enhance the tumor antigen-specific immune response. This also means that mRNA vaccines can be combined with other oncology therapies, such as checkpoint inhibitors and immune agonists, to achieve a more comprehensive oncology therapeutic effect [99] . Moderna, BioNtech and CureVac AG are three tycoon committed to mRNA technology, and invested a lot in the application of mRNA technology in tumor vaccine (as shown in Table 6 ). Several mRNA tumor vaccines are currently in clinical trials. CV9202 is a self-modifying mRNA vaccine that expresses six antigens commonly expressed in non-small cell lung cancer. The mRNA vaccine enhances the antiviral and antitumor effects of the host by enhancing the antigenic reactivity of T cells [105] . Some exogenous gene expression products directly act on immune cells and promote the growth and proliferation of immune cells. Thus, they could enhance the host's anti-tumor and antiviral capabilities. There is a growing body of studies demonstrating utility of RNA for targeting previously 'undruggable' pathways involved in development and progression of cardiovascular disease. Despite significant advances in treatment options, cardiovascular disease remains the number one cause of death in the world [106] . One of the earliest nucleic acid therapies for cardiovascular disease was Mipomersen, a This ASO can be injected subcutaneously and functions by binding APOC3 mRNA and promoting its degradation. In a Phase II study, volanesorsen, decreased Apoc-II (-80%) and triglycerides (-71%), and increased HDL-cholesterol levels (46%) [108] . Another approach to lowering cholesterol is to target mRNA encoding the enzyme proprotein convertase subtilisin/kexin type 9 (PCSK9) which is predominantly produced in the liver [109] . Beyond drugs inhibiting endogenous mRNA, the first cardiovascular-related mRNA drug to have J o u r n a l P r e -p r o o f reached clinical trials, is the mRNA encoding vascular endothelial growth factor (VEGF)-A mRNA (Moderna, Cambridge, Massachusetts, USA). In a Phase I study, intradermal administration of VEGF-A mRNA led increased local VEGF-A protein expression (as assessed by cutaneous microdialysis) and increased skin blood flow in men with type 2 diabetes [110] . Based on these results, a Phase 2a clinical trial will determine if this mRNA therapeutic restores ischemic but viable myocardial regions in patients with coronary artery disease, as assessed by ejection fraction [111] . This is a randomized, double-blind, placebo-controlled, multicenter study in patients with moderately impaired systolic function undergoing coronary artery bypass surgery. Patients are randomized to doses of 0, 3, or 30 mg of VEGF A mRNA in a citrate buffer by epicardial injections. If this programmatic effort is successful, it would provide evidence that direct injection of mRNA into an ischemic tissue may improve perfusion and function. Patel et al. [112] also used the microflow controller to prepare 11 lipid nanoparticles and tested Riley et al. [113] studied the use of LNPs in utero to overcome the immaturity of the immune system due to the small fetal size. They developed a library of LNPs for mRNA delivery to mouse fetuses in utero. The LNPs for luciferase mRNA delivery were first screened and formulations that could accumulate in the fetal liver, lung and intestine compared to the benchmark delivery systems with higher efficiency and safety, demonstrating that LNPs can deliver mRNA to induce liver production of therapeutic secretory proteins. Dhaliwal et al. [114] developed an mRNA cationic liposome for the treatment of chronic diseases based on nucleic acid therapy, in which the liposome was composed of DOTAP, DPPC and cholesterol. The potential of intranasal delivery to the brain in mouse model has been evaluated. The results demonstrated the feasibility of brain-specific non-viral mRNA delivery in the treatment of various neurological diseases, as shown in Fig. 11 . The synthetic mRNA undergoes transient protein expression after delivered to cytoplasm and can be completely degraded via physiological metabolic pathways, which can avoid the risk of genomic integration. This transient feature meets the need for many applications which require protein expression for only limited periods of time, such as gene editing, cell reprogramming, and some immunotherapies [115] . Different mRNA delivery system has different delivery mechanisms. Taking lipid nano delivery system for example, LNPs entrap mRNA in the core through J o u r n a l P r e -p r o o f microfluidic preparation method, and delivery mRNA to cells mainly by endocytosis, lysosomal escape pathway; however, mRNA attach inside and outside liposomes electrostatically, and delivery mRNA to cells mainly by membrane fusion. Besides lipid nano delivery systems, the other nano delivery systems also have their own mRNA entrapping strategy and delivery features, such as polymer complexes, micelles, cationic peptides, and so on. However, the synthetic mRNA-based therapeutics also suffer from some drawbacks such as inefficient delivery and instability [116] . At present, hampered by limited endosomal escape of nano delivery system, only a small amount of mRNA (0.01%) could successfully enter the cytoplasm and express the protein [117] . Therefore, high dose administration is still normal and will bring great side effects. In the nearest future, lyophilization or other pharmaceutical processing methods may help to resolve these problem and even enable nasal, oral, or respiratory administration [118] . The administration routes of mRNA nano delivery systems is very important to determine the metabolism of the mRNA vaccine in vivo and the efficiency of the translation of the target antigen protein. For example, if exposed mRNA is given intravenously without any treatment, it is rapidly degraded by nucleases in the blood. Currently, mRNA vaccines are administered systematically or locally, depending on where the antigenic protein needs to be expressed. Prophylactic vaccines are usually administered locally subcutaneously and intramuscularly to induce a strong immune response, while therapeutic mRNA vaccines are usually administered intraperitoneally or intravenously. Moreover, innovative versatile materials may be another solution to the challenge of mRNA applications. In conclusion, the COVID-19 pandemic pushed mRNA technologies to the world, which showed their unique advantages at the critical moment. These technologies have been developed through years of painstaking work by scientists in academia and industry. Although not perfect, it is undeniable that mRNA therapeutics are ready for its time to shine, and the transition to a full-scale industrial revolution. The authors report no conflflicts of interest. The authors alone are responsible for the content and writing of this paper. 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