Preliminary research has shown that in vivo nude mRNA delivery can produce an immunotherapeutic reaction in mice [8]. Currently, administration methods for mRNA encompass subcutaneous, intradermal, intranodular, intramuscular, intravenous, and intratumoral injections, which are vital for stimulating antigens and initiating a response from the immune system [33, 71, 72]. Phua et al. [73] found that subcutaneous nude mRNA injection in mice surpassed the efficacy of nanoparticle-based delivery of mRNA. Lint et al. [74] discovered that when mRNA is directly injected into the tumor through intratumoral injection, it provokes a suitable response from the immune system and is considered a potentially feasible vaccine delivery technique in the coming years. Currently, the direct injection of unencapsulated mRNA is mostly employed for the treatment or prevention of viral infections [75]. Nonetheless, despite the potential to produce a response from the immune system, the efficacy of this transport route is comparatively limited, and nude mRNA is frequently subject to fast degradation post-injection. The direct injection of nude mRNA is overly simplistic and rudimentary for application in human patients; it is frequently utilized as a method to administer modified mRNA vaccines in conjunction with alternative delivery techniques to enhance vaccination efficacy.

The efficacy of antigen release by mRNA is enhanced using many established physical techniques, including microneedles, electroporation, and gene gun, among others. Electroporation possesses an adjuvant effect, capable of recruiting pro-inflammatory cells and stimulating cytokine synthesis at the site of administration, hence enhancing the immune response of the mRNA vaccine [76]. It also enhances mRNA transport effectiveness without requiring additional receptor modalities, minimizing unwanted immunological responses [77]. Callis et al. [78] discovered that transfection efficacy in plant and animal cells can be increased by the electroporation of the mRNA. In 2017, the electroporation approach increased the mRNA transfection efficacy (50–90%) in dendritic cells (DCs) [79]. The gene gun technique employs pressurized He gas as an accelerating force to propel gold particles coated with mRNA into host cells, serving as an effective way for the delivery of mRNA [80]. Qiu et al. [80] employed the gene gun technique on mouse skin for the introduction of human alpha-1 antitrypsin mRNA, effectively inducing an antibody release. Peking et al. [81] treated hereditary skin disorders by mRNA-based formulation, with mRNA successfully delivered to mice targeted skin layer using the gene gun method. However, the gene gun technique is hardly employed in humans or large animals. Physical methods of delivery of mRNA may influence the function of cells and their physiological structure and potentially result in aberrant cell death. Consequently, using physical mRNA delivery in humans poses significant risks [33, 76].

DCs are among the most powerful immune system stimulants for antibody release. DCs use the MHC for the delivery of processed antigen to CD4+ and CD8+ T cells, hence initiating cellular immune response [33, 82]. Concurrently, DCs can deliver antigens to B cells, triggering a humoral immune response [83]. The delivery of mRNA involves transfecting mRNAs that encode proteins, peptides, or other immune stimulants in DCs, followed by the host organism being infected with DCs carrying mRNA to initiate the specific response from the immune system based on the antigen [84]. This method operates independently of additional carrier molecules and achieves great delivery efficiency. It is extensively utilized in clinical research, animal models, and pre-clinical trials [85–88]. This method has mostly been utilized in the treatment of cancer due to the predominance of eliciting cellular immune responses [89]. Nonetheless, the mRNA transfection rate is rather poor when relying just on DC endocytosis, prompting the frequent utilization of electroporation to enhance the rate of transfection of the mRNA [90]. Gay et al. [90] employed electroporation to DCs carrying HIV-specific antigens encoded in mRNA for HIV therapy. Followed by at least twice intradermal injection, cytotoxic T-lymphocytes (CTLs) CD28+/CD45RA–CD8+ immune responses were induced in HIV patients. A significant obstacle to the practical implementation of DC-loaded mRNA is the time and cost-intensive manufacturing procedure, which limits the scalability of DC-based mRNA vaccines. Furthermore, within hours of transfection, the immune system produced a diminished response, resulting in a decreased therapeutic response [91]. Considering these factors, diseases necessitating substantial mRNA vaccine administration in the immediate future should prioritize delivery methods with rapid manufacturing capabilities.

The polymer-based delivery system uses a cationic polymer as a vector for mRNA delivery [92, 93]. Frequently utilized polymer delivery polymers are poly(beta-amino esters) (PBAEs) and polyethylenimine (PEI), which are a few examples. PEI is the most extensively utilized polymer. It is a water-soluble cationic polymer recognized by branched, linear, or dendritic structures, commonly used for mRNA transfection [94, 95]. Linear PEI is marketed as jetPEI^TM was previously utilized for siRNA (short interfering RNA) and DNA transfection and can be used for transfection of mRNA [92, 96]. Nonetheless, PEI possesses inherent cytotoxicity that is resistant to degradation; hence, researchers frequently alter low-molecular-weight PEI with fatty chains to mitigate its toxicity [94, 97, 98]. PBAEs are biodegradable polymers initially created for DNA transfection [99]. Kaczmarek et al. [100] created a delivery system based on a polymer by combining lipid-PEG with PBAEs, achieving high effectiveness and stable mRNA transport, after successfully injecting intravenously. In 2007, research showed that PBAEs can facilitate mRNA delivery, achieving elevated mRNA transfection levels in vitro in the absence of serum proteins [101]. The ease of synthesis has led to the creation of numerous PBAEs, some of which improve in vivo mRNA stability [92, 102, 103]. Lipids and PBAEs can be combined to enhance the stability of mRNA in blood. Compared to lipids, polymer materials are strong contenders for mRNA transport. However, the cytotoxicity of polymer complexes, similar to cationic lipids, has hindered wider application [104]. In addition to modifying polymer-based vectors with other materials to enhance their capabilities, optimizing branching patterns and molecular weight appears to be a promising approach.

LNPs are the sole nano-delivery technology for mRNA vaccines that are clinically successful and have received approval for human use. The Pfizer and Moderna SARS-CoV-2 mRNA vaccination uses LNPs to transport the mRNA to the target. Cationic liposomes are frequently utilized as RNA carriers, since lipids are positively charged and RNA is negatively charged, which creates electrostatic complexes, hence enhancing mRNA transport efficacy [105]. The mRNA-lipid combination, along with other excipients, may produce LNP ranging from 80 to 200 nm, which can reach the cell cytoplasm through endocytosis. Their most common composition comprises cholesterol, natural phospholipids, ionizable cationic lipids, and PEG [76]. The mRNA is self-aggregated due to cationic lipids forming approximately 100 nm particles and, through endocytosis, reaches the cytoplasm, gets ionized, and releases mRNA. Cholesterol stabilizes the LNP, PEG increases its half-life, and phospholipids help form the lipid bilayer [33, 92]. LNP encapsulates the mRNA in the core, which safeguards it from degradation. The lipophilic characteristics of the LNP component facilitate the transport of mRNA lipid complex across the membrane of the cell through endocytosis [76, 91]. LNP is frequently utilized as a delivery method for siRNA in initial studies [106].

Currently, LNP is extensively utilized in the delivery of mRNA. Geall et al. [107] employed an mRNA-lipid complex to administer a self-amplifying RNA, resulting in elevated mRNA expression levels when compared to naked mRNA in mice, while also efficiently producing CD4+ and CD8+ T cell immunological responses. Various delivery routes can elicit distinct immune stimulation responses from mRNA LNPs, potentially fulfilling the therapeutic requirements for diverse illnesses. Pardi et al. [108] discovered that administering mRNA LNPs at the correct dosage via intradermal, intramuscular, and subcutaneous routes might facilitate localized gene translation. Common mRNA-LNP delivery methods are tracheal inhalation, intraperitoneal injection, and intravenous injection, the latter showing the highest systemic delivery efficiency [109]. However, the methods by which mRNA escapes from electrostatic complexes to achieve a free state for functionality in the cytoplasm remain inadequately comprehended. The in vivo ambient pH alters the ionization state of lipid-carrying mRNA is considered crucial to the release mechanism [104]. Simultaneously, more investigation is needed into decreasing the toxicity, and the management of immunogenicity in cationic lipid-based delivery systems is critically required.

Cellular transport and encapsulation can be enhanced by using phospholipids in LNP formulation. To extend the circulation duration for LNP, the cholesterol level should be increased while decreasing the level of phospholipid. Some studies indicate that higher phospholipid molar ratios enhance LNP delivery efficiency. Zwitterionic phospholipids may help stabilize electrostatic interactions between mRNA, lipids, and surrounding water molecules. However, the precise role of phospholipids in mRNA transport through LNPs remains unclear [110].

Protamines are positively charged proteins with resinous structures. The combination of protamine with mRNA in various ratios of mass can produce electrostatic mRNA-protamine complex nanoparticles of various sizes [111]. This compact conjugate structure may efficiently safeguard mRNA from degradation by RNase enzyme, and this complex can produce a robust immunological response from immune cells, including DCs, neutrophils, NK cells, B cells, and monocytes [112–114]. It suggests that protamine may serve as both an mRNA transporter and an immunological activator. In 1961, protamine was among the initial materials investigated for the delivery of RNA [115].

Fotin-Mleczek et al. [116] administered a vaccine that has protamine as a carrying agent of an mRNA to the tumor, effectively inducing a comprehensive anti-tumor-specific response. At the mass ratio of mRNA to protamine of 2:1, the resultant complex measures around 300 nm, exhibiting relative stability and eliciting robust immunological activation alongside elevated cytokine levels, but with a considerable inhibition of protein production. Consequently, it is widely believed that while mRNA-protamine complexes induce immune responses, translation efficiency may be limited [116, 117]. In recent years, mRNA-protamine formulations have been extensively utilized in clinical studies, demonstrating significant therapeutic efficacy in conditions such as rabies [110].

Influenza virus vaccines have been utilized for over 80 years, although they remain a significant focus of investigation-based study and a considerable demand on community health resources due to the necessity for frequent updates to address the continually evolving viruses [122]. The inaugural influenza vaccine was employed in the mid-1930s, following the recent discovery of influenza A viruses (IAVs) a few years earlier at Mill Hill. Influenza immunization originally entailed subcutaneous administration of formalin-inactivated viruses cultured in murine lungs; subsequently, egg-cultivated viruses that are inactivated were created by Jonas Salk and Thomas Francis Jr. for the military of the USA. In the following decades, both IAVs and IBVs were encompassed in influenza vaccination and are now generally advocated for yearly administration. These vaccinations are crucial instruments for managing seasonal influenza and addressing influenza pandemics [123–125].

Notwithstanding the available vaccine alternatives, viruses of seasonal influenza are projected to result in more than a few million infections and are linked to around 650,000 fatalities annually worldwide [126, 127]. Vaccine policy and coverage differ significantly among nations: in 2019, immunization rates for those aged 65 and older ranged from around 6% to over 70% of the population. In nations with substantial vaccination coverage, the virus of seasonal influenza exerts a significant illness burden, which can be largely ascribed to the deficiencies of existing vaccines [126, 128].

A limited number of pre-clinical and early clinical experiments for mRNA-based influenza vaccines have been performed or are underway. A phase I experiment was conducted to assess the safety of mRNA vaccines carried by liposomes containing H7N9 or H10N8, the full-length IAV, avian viruses with pandemic potential, can be delivered either intradermally or intramuscularly [129]. The immunizations were well handled and produced elevated, enduring antibody titers, comparable to current vaccines. The adverse effects documented in the 2019 research, including discomfort and edema at the site of administration, myalgia, and exhaustion, are analogous to those associated with mRNA vaccines for coronavirus [130]. The participants are recruited for trials by Moderna to evaluate a quadrivalent seasonal formulation of mRNA-1010, and preliminary findings after its first phase clinical trial in old and adults indicate it is safe and generates antibody responses [34]. The phase II clinical study performed by Pfizer of mRNA vaccines targeting seasonal influenza in people above 65 years [131]. Multiple firms are exploring the feasibility of a multivalent vaccination incorporating antigens from both the influenza virus and influenza and SARS-CoV-2. ‘Novavax’ is a nanocarrier-based vaccine, while Moderna has also declared to merge SARS-CoV-2 and influenza antigens into a unified vaccine [132]. Although influenza mRNA vaccines have been under research for some years, the efficacy of mRNA vaccines against SARS-CoV-2 has elevated their clinical application to a prominent prospect. The ability to manufacture substantial volumes of tailored, precisely regulated vaccinations in a comparatively short timeframe is appealing in relation to existing vaccine platforms. The regulatory inquiries and logistical challenges associated with the implementation of novel technology for the deployment of coronavirus vaccines which is expected to facilitate the introduction of next-generation influenza vaccines soon [133]. There is growing interest and investment in mRNA-based influenza vaccines, with several candidates in or completing phase III trials. It’s also crucial to monitor efforts like the WHO/MPP mRNA Technology Transfer Programme to track how low and middle-income countries are advancing in influenza vaccine development [134].

The initial human case of Zika virus infection was identified in 1952 in Nigeria. It is spread by Aedes spp. mosquitoes, remained inconspicuous for years until epidemics transpired from 2007 to 2017 in America, French Polynesia, and Yap Island. After the 2015 Brazil epidemic, which marked the initial instance of the indigenous Zika virus spread, initiatives started to emphasize the Zika virus investigation and the formulation of vaccines for the Zika virus. The worldwide concern for pregnant women stemmed mostly from the adverse effects on fetuses infected with the Zika virus in Brazil and other nations globally [135]. Neutralizing antibodies are essential agents of shield against infections and have been associated with the effectiveness of vaccinations [75, 136, 137]. Despite global interest in Zika resulting in several vaccine candidates, there is still no approved vaccine for the disease.

Despite the extensive range of vaccine platforms for the Zika virus, only two mRNA technology-based vaccine candidates have undergone clinical testing. The mRNA vaccination technology is a secure and efficacious method for developing protective immune responses. In contrast to existing vaccination candidate platforms, mRNA vaccines eliminate the danger of insertional and infection mutagenesis, while also circumventing the potential for antivector protection, so permitting recurrent delivery [33]. The effectiveness of an mRNA vaccine candidate in an AG129 murine model. A single administration of Zika virus prM-E mRNA-LNP conferred protection to rats following a deadly Zika virus infection. In comparison to the placebo group, vaccinated animals exhibited no clinical symptoms or weight reduction and had decreased virus loads. Finally, the BALB/c mice vaccination, accompanied by the isolated splenocytes analysis for T cells, revealed specific antigens for CD4+ and CD8+ T-cell responses. This study facilitates the usage of liposomes for mRNA vaccine delivery by liposome [138].

Since the initial clinical identification of acquired immunodeficiency syndrome (AIDS) and the subsequent discovery of the retrovirus HIV in the early 1980s, the HIV/AIDS epidemic remains a significant global health issue despite four decades of rigorous study. In 2019, there were 1.7 million new infections and 690,000 AIDS-related fatalities worldwide; in 2020, 38 million individuals were living with HIV. Contemporary prevention and treatment modalities for HIV/AIDS encompass the administration of antiretroviral medications for pre-exposure prophylaxis (PrEP) and antiretroviral therapy (ART), which have facilitated the transition of AIDS from a life-threatening condition to a manageable chronic disease [139]. Nonetheless, the drugs are costly, need rigorous compliance with dose protocols for effectiveness, and induce adverse effects. Furthermore, many HIV-infected patients acquire treatment resistance, necessitating alterations in their pharmaceutical regimen. Access to treatment is a significant obstacle, especially in low- and middle-income countries, as indicated by the consistently elevated rates of new infections over the past decade [140]. Consequently, preventive vaccination is a fundamental element of a comprehensive approach to eradicating the pandemic. Creating an effective HIV vaccine has, however, been shown to be exceptionally challenging. As the pandemic approaches its fifth decade, there are no authorized vaccines for HIV, with just one promising clinical research (RV144 trial) exhibiting a low effectiveness of 31% [141].

The mRNA vaccine utilizes mRNA that encodes an immunogen, which is converted into protein once delivered to host cells. In 1989, it was discovered that mRNA contained in cationic liposomes could transfect mouse cell lines [142]. The protein expression and translation were shown following the injection of nude mRNA into the skeletal muscle of the mouse [8, 143]. Nonetheless, employing mRNA for vaccines was impractical due to the instability of RNA molecules, insufficient delivery mechanisms, challenges in the large-scale production of mRNA, and unregulated activation of innate immunity via RNA sensors [143, 144]. Current technical advancements and enhanced delivery systems have mitigated these challenges, leading to the emergence of mRNA vaccines as a potential platform for antigen delivery. The mRNA-based vaccines provide several advantages compared to conventional vaccination platforms, such as enhanced safety, effectiveness, and streamlined manufacturing processes. The absence of effective delivery mechanisms for molecules of mRNA was a significant impediment to the advancement of mRNA vaccines. The optimal delivery mechanism will enhance cellular uptake with minimal toxicity and safeguard mRNA against destruction [145].

Rabies is a deadly neurological illness caused by the neurotropic rabies virus, affecting infected animals and humans. It is an ssRNA virus belonging to the Lyssavirus family, transmitted by several animal vectors; dog bites are the cause of more than 95% of rabies cases in humans, while additional carriers include bats, skunks, raccoons, and coyotes [146]. The true impact of rabies is challenging to assess due to case under-reporting and insufficient monitoring data; nonetheless, the risk of exposure is estimated to be more than 3 billion people worldwide, with 59,000 fatalities annually attributed to canine rabies. Regrettably, rabies continues to claim numerous lives, despite Louis Pasteur’s introduction of the first effective human vaccination against the disease over 130 years ago [147, 148].

The application of mRNA technology for the efficient synthesis of mRNA that is precisely tailored to the encoded antigen offers several benefits compared to current vaccination technologies in the creation and manufacture of preventive vaccines for infectious diseases. The mRNA vaccines do not incorporate any living viral components, unlike attenuated or live vaccinations, hence eliminating the risk of consequent infection and reversion to pathogenicity [118]. The prophylactic mRNA vaccine, which was first developed for rabies, has been advanced through in vivo animal models before human evaluations. The choice to prioritize rabies was influenced by many considerations. The rabies genome is well-defined and uncomplicated, as is the immune response target, the RABV-G protein antigen [149]. Numerous licensed vaccinations are available and established standardized assays for assessing the RABV-G antigen immune response, allowing for straightforward comparisons of rabies mRNA vaccine candidates with licensed and commercially available rabies vaccines [150].

Recent reports have detailed a technological framework for the versatile and precise synthesis of protein antigens in mRNA, which has been utilized for rabies. The investigations at the preclinical level have shown that intradermal or intramuscular administration of mRNA can produce cellular and humoral immune responses against the protein antigen [151].

COVID-19 is an illness induced by the SARS-CoV-2, which is a positive ssRNA virus enveloped by protein. It belongs to the Betacoronavirus genus within the Coronaviridae family. The whole genome comprised 29,881 nucleotides in the ancestral SARS-CoV-2 strain, with a 3' poly(A) tail and methylated 5' cap, encoding 9,860 amino acids that represent 4 structural proteins, 9 accessory proteins, and 16 nonstructural proteins. More than 220 countries have been devastated by COVID-19, resulting in more than 400 million incidence cases and 5.75 million fatalities [152–154].

The initial group of vaccines for COVID-19 is mRNA vaccines undergoing clinical testing. According to the WHO report, the number of COVID-19 vaccines exceeded 300, including 47 mRNA vaccines [155]. Moderna (mRNA-1273), Pfizer-BioNTech (BNT162b2), and CureVac mRNA vaccines represent the swiftest developed vaccine in medical history [156, 157]. The initial two received permission from various regulatory bodies for emergency use in China, the USA, and Canada [158]. The FDA approved official marketing for the Pfizer-BioNTech COVID-19 vaccine in August 2021, making it the first vaccine to get such approval. Additionally, on October 29, 2021, it became the first vaccine authorized for children under the age of 11 years. Kitano et al. [159] did a risk assessment of the mRNA vaccine for COVID-19 in children from 6 months to 4 years and found that the benefits of the mRNA vaccine far outweigh any negative effects. The Pfizer-BioNTech vaccine has a dose of 30 μg and is 95% effective, and the Moderna vaccine is 94.5% effective with a dose of 50 μg [160]. After inoculation with homologous SARS-CoV mRNA vaccines, individuals have a superior antibody response than other vaccines [161], and spike antibody assay of the antibody is preferred over nucleoside antibody [162].

Because of the intricate relationships between SARS-CoV-2, its variants, and societal dynamics, environmental factors are crucial to the spread of COVID-19 and make predictive models more difficult to understand [163, 164]. While higher COVID-19 vaccination rates are essential, they alone are not sufficient to significantly reduce mortality; additional health policies and interventions are also necessary [165]. Higher healthcare spending and better medical infrastructure are linked to lower COVID-19 fatality rates, with Western European countries showing stronger outcomes compared to Eastern Europe due to greater per capita health investment [166]. Organizations like WHO, Coalition for Epidemic Preparedness Innovations (CEPI), Global Alliance for Vaccines and Immunization, and International Vaccine Institute (IVI) are crucial in pandemic preparedness, working together to enhance vaccine access and strengthen global responses to infectious diseases [167].

The 2019 pandemic has catalyzed the growth of platforms for mRNA vaccines for the treatment and prevention of several contagious infections, leading to the progressive introduction of a new generation of vaccinations for the general public, which has garnered amplified attention. The vaccines based on mRNA, in contrast to conventional vaccinations, enable the modification of the design of antigens and provide the integration of sequences from many variations to address novel changes in the viral genome. In the future, the mRNA-based vaccination will facilitate the management and prevention of viral diseases and the treatment of other conditions. The foremost vaccination approved by the FDA for COVID-19 was an mRNA-based vaccine because of its benefits, rapid development cycle, absence of cell culture requirements, and excellent immunogenicity [168]. Even machine learning can be applied to data like the number of infections, the turnout in the participation in the vaccination campaign, the respective vaccination plan, and the doses administered to reduce side effects and fatality rate [169]. The COVID-19 pandemic demonstrated that urgent health crises catalyze problem-driven innovation, resulting in rapid breakthroughs such as mRNA vaccines. Addressing these challenges not only advances industrial competitiveness but also contributes significantly to humanity’s ability to overcome pandemic situations [170, 171]. Table 2 lists a few examples of mRNA-based vaccines for viral diseases under clinical trials [172].

This review explains how molecular design, delivery methods, and immune modulation interact to influence vaccine efficacy, adding to the expanding theoretical framework surrounding mRNA vaccine development. The necessity for quick, scalable, and efficient solutions to pandemic emergencies and resistant viral diseases led to the development of mRNA vaccines, which are highlighted as a problem-driven innovation. The study deepens our knowledge of how nucleotide modifications and nanoparticle-based delivery systems affect mRNA stability, immunogenicity, and expression efficiency by combining findings from preclinical and clinical investigations.

Additionally, this study offers a novel conceptual framework for vaccine development that integrates translational medicine, immunology, and nanotechnology. It suggests that mRNA vaccines are not merely an alternative to traditional platforms but a foundational shift in how we approach global immunization strategies, with implications that extend into oncology and other therapeutic areas.

This review highlights the strategic importance of investing in mRNA infrastructure from a managerial and policy perspective, especially for pandemic preparedness and global health equity. The effectiveness of mRNA vaccines during COVID-19 has shown that they can be developed and implemented quickly, which is essential for halting the spread of newly emerging infections. Funding and regulatory support for cold-chain innovations, scalable manufacturing, and region-specific vaccine platforms that target regional disease burdens should be given top priority by policymakers.

The pharmaceutical industry and health authorities around the globe must also adopt flexible regulatory frameworks that can take into account the modular nature of mRNA platforms and their quicker development schedules. In addition, the integration of mRNA technology into routine vaccination programs and public health policy could revolutionize how we prevent and respond to infectious disease outbreaks, especially in resource-limited settings.

Even though the available data is encouraging, the science behind mRNA vaccines has not yet reached its full potential. Future studies ought to concentrate on:

Recognizing the long-term immune reactions brought on by mRNA vaccines, including any possible impacts on immune memory and innate immunity.

Finally, expanded research on multi-antigen mRNA constructs and self-amplifying RNA platforms may pave the way for next-generation vaccines that are more potent, cost-effective, and durable. As the field matures, collaborative efforts between academia, industry, and government will be crucial to unlocking the full potential of mRNA technologies for global health transformation.