Nanotechnology Based mRNA Vaccines: Technological Innovations, Delivery Systems, Clinical Outcomes, and Safety Profiles

Messenger RNA vaccines are a new type of nucleic acid based vaccination approach which works by introducing synthetic mRNA that encodes a particular antigen into host cells and is translated into protein and presented to the immune system.[1] This is the main opposite to the classical vaccine systems (vaccines made out of live attenuated viruses, inactivated pathogens, protein subunit vaccines, or viral vectors) that inject ready-made antigens or replicating genetic material within the body. The theoretical basis of mRNA vaccines is the utilization of nature biological mechanism of translation without the threat of genomic integration or infection [2].

The theoretical usefulness of mRNA as a modality of therapy was appreciated several decades prior and the initial experiments showed that exogenous mRNA could induce protein expression in mammalian cells. Although this was promised, the initial mRNA based mechanisms were fraught with significant technical issues limiting their clinical implementation.[3] Unbound mRNA is unstable by nature, very fragile to degradation by ubiquitous ribonucleases, and cannot easily go through cell membranes because of its size and negative charge. Moreover, unmodified mRNA is a potent stimulator of innate immune sensors that results in uncontrolled inflammatory reactions and protein translation inhibition [4].These limitations were slowly overcome by major advancement in mRNA chemistry and molecular engineering. Innate immune recognition was decreased, and translational efficiency was increased by the introduction of chemically modified nucleosides like pseudouridine and that of 1 methylpseudouridine. Codon optimization and untranslated regions also made the protein expression and mRNA stability better [3]. Nevertheless, optimization of mRNA constructs resulted in optimal delivery to target cells, which was still the main barrier to clinical success.[5]

Figure 1. Conceptual framework and technological evolution of mRNA vaccines in comparison with traditional vaccine platforms.

Nanotechnology has been the enabler of the moment and saw mRNA vaccines through as an experimental idea and be an experimentally validated platform. Systems of nanoparticle based delivery can prevent the enzyme degradation of mRNA and enable the entry of the nucleic acid into the cells and also the release of the nucleic acid within intracellular space in a controlled manner.[6] Lipid nanoparticles have become the most well-recognized and successful delivery vehicle of mRNA vaccines among other nanocarriers [7].

Lipid nanoparticles are usually stipulated by four main components, namely, ionizable lipids, phospholipids, cholesterol and polyethylene glycol conjugated lipids.[8, 9] All components are involved in stability, biocompatibility and delivery efficacy. Ionizable lipids are especially essential since they combine with negatively charged mRNA and facilitate endosomal escape after cellular uptake. These lipids are mostly neutral at physiological pH, which minimizes the toxicity to the system, whereas at the acidic endosomal pH, the lipids are positively charged and destabilize the endosomal membrane, releasing mRNA into the cytoplasm .[10]

The clinical effectiveness of lipid nanoparticle encapsulated mRNA vaccines against SARS CoV 2 indicated that nanotechnology based delivery systems would be safe to be administered globally. The world has received billions of doses of mRNA vaccines and has acquired unprecedented real world experience on the safety and efficacy of nanoparticle based gene delivery [11]. This success put nanomedicine on the foundation of contemporary vaccinology and gave impetus to the development of nanoparticle carriers in a large variety of therapeutic uses.

1.1 Naked mRNA has Several Limitations in Its History.

The intrinsic biological limitations to the clinical failure of early naked mRNA approaches are attributed to a number of biological limitations. First, mRNA is chemically unstable and it is quickly broken down by extracellular and intracellular ribonucleases. Second, it is highly polyanionic and is large in size, which means that it cannot easily penetrate lipid bi-layers leading to a low cellular uptake.[12, 13] Third, unmodded mRNA induces the activation of pattern recognition receptors, including Toll like receptor 3, Toll like receptor 7, and Toll like receptor 8 and induces type I interferon responses that suppress translation and result in systemic inflammation [6]. The limitations are countered by the nanoparticle based delivery systems in a number of ways. The mRNA is also encapsulated in nanoparticles to protect against nucleases and minimize immune responses by the immune system. The mRNA and cationic or ionizable materials can interact through an electrostatic process that allows the internalization of cells through endocytosis. Moreover, the carriers of nanoscale allow the targeted intracellular delivery and release of mRNA and guarantee the arrival of the drug at the cytoplasm where it is translated into protein. Lipid nanoparticles among the available nanocarriers have since proved to have better performance in clinical environments than polymer based or liposomal systems.[14] Their capacity to reconcile efficacy of delivery with tolerable safety profiles has seen them become the platform of choice of mRNA vaccines [15].

1.2 Rapid Development of the mRNA Vaccines in the COVID 19 Pandemic.

The COVID 19 pandemic was a key point in the history of vaccines development. The speed of antigen design was made possible because the SARS CoV 2 genome sequence was rapidly published at the beginning of 2020, one of the greatest opportunities of mRNA platforms: speed. The mRNA vaccines can be prepared and produced within few weeks once the genetic sequence of a pathogen is known [16].Nanotechnology also increased this benefit by offering a standardized delivery platform, which could be quickly modified to new sequences of mRNA without any change in formulation chemistry. The familiarity of the regulatory system with lipid nanoparticle based systems, through the prior approval of small interfering RNA therapeutics, simplified expedited regulatory process and emergency use approval.[17]

The pandemic also created a large amount of post marketing surveillance data, which provides special knowledge about the long term safety and population level impact of nanoparticle based mRNA vaccines.[18] This practical experience has transformed the regulatory and translational processes of nanomedicine and minimized skepticism about large scale clinical implementation [19]. Scope, Objectives and Timeframe of the Review (2019 2024). The reviewed nanotechnology based mRNA vaccines were developed and tested between 2019-2024. The goals include a critical examination of technological advances in mRNA design and nanoparticle delivery systems, assessment of clinical outcomes of approved vaccines and clinical trials, provide a critical analysis of safety profiles based on clinical and post-marketing data, and underlying challenges and future opportunities in the science. This review will serve as a foundation of up-to-date peer reviewed papers and regulatory reports to become a reliable reference resource to researchers, clinicians, and policy formulators working in mRNA vaccine development. [20]

2. mRNA Vaccine Technology

It is a novel technology developed by the company to replace the flu vaccine. MRNA Vaccine Technology is a new technology that the company has developed to take the place of the flu vaccine. Prior to the 1990s, mRNA vaccines were developed against typhoid fever caused by Salmonella typhimurium in animals. [21, 22]

2.1 Early Development of mRNA Vaccines

Pre-1990s mRNA vaccines were developed against Salmonella typhimurium-caused typhoid in animals. Until 2019, the research of messenger RNA vaccines was conducted solely at the experimental and early clinical development phases. Preliminary work aimed at showing that synthetic mRNA could be expressed effectively in vivo and result in generation of antigen specific immunity. The initial targets were influenza virus, rabies virus, Zika virus, cytomegalovirus and multiple tumor associated antigens to cancer immunotherapy [23, 24]. Although they found that biological feasibility was achieved in these studies, there was scanty clinical translation. The significant challenges of this step were connected to the unstable nature of mRNAs, poor intracellular delivery, and overactivation of innate immunity. Extracellular ribonucleases were capable of rapidly degrading naked mRNA and thus minimal production of antigen could be achieved after the administration. Also, the negative negative charge and high molecular weight of mRNA inhibited its extracellular diffusion across cell membranes leading to the ineffective cellular uptake. High doses were consequently necessary to obtain measurable immune responses and therefore enhanced reactogenicity and decreased tolerability.[25] The other limitation that was critical was the stimulation of natural immune sensors like Toll like receptor 3, Toll like receptor 7, and Toll like receptor 8 using unmodified mRNA. This was an activation induction of type I interferon responses that inhibited the expression of proteins and reduced the effects of vaccines [26, 27]. Even though some background level of innate immune vaccination is considered advantageous to vaccine adjuvanticity, too much activation was detrimental. Nevertheless, background innovations were made before 2019. Nucleoside modified mRNA was introduced, which decreased the innate immune recognition and increased the translational efficiency. Improvements in the technology of in vitro transcription enhanced the purity of the mRNAs by reducing the contamination of the target samples with the double stranded RNA. Meanwhile, advances in lipid based nanocarriers of small interfering RNA therapeutics like patisiran were an invaluable lesson on scalable and clinically viable systems of nanoparticle delivery [28, 29]. Such progressive changes prepared the groundwork of fast progress in situations when the world faced a health crisis that required faster vaccine development.

2.2 Major Technological Advances.

The 2019-2024 period will be a transformational period of mRNA vaccine technology. The onset of the COVID 19 pandemic established a pressing need to develop a vaccine as quickly as possible, which is why there was a new level of cooperation between academia, business, and regulatory bodies. In this scope, mRNA vaccines were developed as experimental platforms and globally distributed medical countermeasures within one year [30]. The lipid nanoparticle based mRNA delivery through clinical validation at an unprecedented scale was one of the most important technological innovations. The approval of BNT162b2 and mRNA 1273 indicated that lipid nanoparticles were safe and effective in delivering mRNA in billions of doses across the world. This practical demonstration erased all the fears of scalability and the safety of nanotechnology based gene delivery systems on a population level. At the same time, significant breakthroughs had been made in lipid chemistry. New ionizable lipids were designed so as to maximize pKa values so as to be minimally charged at physiological pH, highly charged under endosomal acidic conditions. This property increased endosomal membrane destabilization and cytosolic release of mRNA which enhanced antigen expression significantly [31, 32]. Further optimization of polyethylene glycol lipid composition was used to improve the stability and circulation of nanoparticles. The technologies of manufacturing also developed quite fast. Microfluidic mixing systems allowedAssemblyMicrofluidic mixing systems allowed assemblyMicrofluidic mixing systems allowed the assembly of narrow mRNA lipid nanoparticles with high encapsulation efficiencies. These technologies enabled high scaling up and even product quality which is a key issue to approval by the regulatory bodies and distribution worldwide [33].

2.3 Analysis of Conventional Vaccines and mRNA Vaccines.

The mRNA vaccines and the conventional vaccines differ in their basic designs, development and immunological processes. Conventional vaccines are based on entire pathogens or protein antigens that are manufactured by cell culture, fermentation, or expression of a recombinant protein. Such methods tend to be time consuming and demand a lot of biosafety measures [34]. In comparison, mRNA vaccines are engineered by cell free in vitro transcription methods wherein the virus is quickly made upon having the genetic sequence of a particular pathogen. After vaccination, antigen production takes place directly in host cells, much close to natural infection, and favors a strong humoral and cellular immune response [35].

Table 1. Comparison of Conventional Vaccines and mRNA Vaccines

Compared to conventional vaccine platforms and mRNA vaccines, the table 1 identifies several distinctions in the development speed, manufacturing regimens, and biological processes, which can be mainly attributed to the cell free manufacturing and temporary expression.[36]

2.4. The benefits of mRNA Platforms in Rapid Vaccine Development are as follows.

The speed is the most unique benefit of mRNA vaccine platforms. After the genome of a pathogen has been sequenced, antigen encoding mRNA constructs can be designed and produced within days. This ability was crucial in the COVID 19 pandemic and remains to be used in the rapid reaction to new variants and other pathogens. The other benefit is platform modularity. The lipid nanoparticle formulation may be reused, but the mRNA sequence only changed, making it easier to develop and review regulatory requirements.[38, 39] This modularity is also useful in supporting personalized medicine apps, such as personalized cancer vaccines using patient specific tumor neoantigens. mRNA vaccines have also positive safety properties. Since mRNA does not penetrate the nucleus and no longer becomes a part of the host genome, the threat of insertional mutagenesis is low. Also, the mRNA intrinsically decays following translation and leads to short-lived antigen expression [40].

An immunological point of view is that mRNA vaccines cause humoral and cellular immunity. The presence of endogenous antigens also supports the presentation of major histocompatibility complex, class I and II, followed by the healthy production of CD8 positive and CD4 positive T cells as well as neutralization of antibodies [41, 42]. When combined, these properties make mRNA vaccines a very versatile and potent vaccine development platform in the future.

3. NANOTECHNOLOGY PLATFORMS APPLICABLE IN mRNA VACCINATIONS.

The key to the clinical success of mRNA vaccines lies in nanotechnology, which allows the protection, delivery, uptake by cells and cytosolic delivery of flimsy mRNA molecules.

. Because naked mRNA is highly unstable, rapidly degraded by extracellular ribonucleases, and incapable of crossing cellular membranes, advanced nanoscale delivery systems are essential. Between 2019 and 2024, several nanotechnology platforms were optimized, clinically validated, and expanded beyond infectious disease vaccines. This section critically examines the major nanocarrier systems used for mRNA vaccine delivery, with emphasis on their structural design, biological performance, advantages, and limitations.[43, 44]

3.1 Lipid Nanoparticles

The composition and structural components are related to the state of the subject, which is linked to the variations in variables that affect the subject's characteristics.

3.1.1 Composition and Structural Components

The composition and structural components refer to the condition of the subject, which is connected to the differences in variables that influence the characteristics of the subject.The most common and clinically approved nanocarriers to mRNA vaccines are lipid nanoparticles.[45] They are usually between 60 and 120 nanometers in diameter and comprise four basic constituents namely ionizable lipids, helper phospholipids, cholesterol and polyethylene glycol lipids [46]. The core functional component is made of ionizable lipids and electrostatically interacts with negatively charged mRNA in the formulation at acidic pH. The lipid bilayer structure is stabilized by helper phospholipids like distearoylphosphatidylcholine and membrane fusion is promoted by them. Cholesterol enhances the rigidity and structural integrity of nanoparticles whereas polyethylene glycol lipids provide a hydrophilic corona that prevents aggregation and increases colloidal stability in storing and systemic circulation.[47]

The mRNA itself is enclosed by the lipid matrix as opposed to being adsorbed on the surface, which offers structural protection against enzyme degradation and mechanical forces. The research on cryo electron microscopy has revealed that mRNA is packed into lipid RNA complexes that are tightly packed by the nanoparticle core and contributes to high encapsulation efficiency of greater than 90 percent [48].

3.1.2 Ionizable Lipids and their contribution to Endosomal Escape.

The ionizable lipids are made to be specifically neutral at physiological pH but positively charged in acidic environment of endosomes after cellular uptake. This pH sensitive activity reduces the systemic toxicity and allows the ionizable lipids to engage with anionic endosomal membrane lipids, which destabilize the membrane by forming non bilayer architectures.When the ionizable lipids are protonated in endosomes, the lipids interact with the anionic lipids of the endosomal membranes leading to the destabilization of the membranes through non bilayer architecture. The result of this process is a temporary pore formation or fusion of membranes which enable mRNA to escape into the cytosol prior to lysosomal degradation [49, 50]. The rate limiting step in the endosomal delivery of mRNA has been endosomal escape, and as of 2019, Deliveries of mRNA were greatly enhanced by advances in ionizable lipid chemistry.

3.1.3 PEGylation and Stability Improval.

Polyethylene glycol lipids are very important in stabilizing the lipid nanoparticles by decreasing the interactions between the surface charges and further by preventing aggregation of the lipid nanoparticles during preparation and storage. PEGylation also extends the time of circulation of nanoparticles by decreasing opsonization and mononuclear phagocyte system uptake. But a cellular uptake and endosomal escape inhibitor may happen when PEG density is too high, resulting in a decrease in transfection efficiency. In addition, some individuals have linked repeated exposure to PEGylated nanoparticles to anti PEG antibodies and faster blood clearance [51]. Thus, after 2020 recipes maximized PEG lipid chains length and molar ratios to strike the balance between stability and biological performance.

3.1.4 Commercial Examples

The most notable commercial BNT162b2 and mRNA 1273 are both approved to be used in emergencies and full-approved during the COVID 19 pandemic, but they are based on lipid nanoparticles. These formulae displayed large scale manufacturability, quality control consistency and good benefit risk profile across a variety of populations [52]. The lipid nanoparticles have been solidified as the gold standard of mRNA vaccine delivery through their success.

3.2 Polymer Based Nanocarriers

Another methodology of mRNA delivery is the use of polymer based nanocarriers, especially in the preclinical and early clinical studies. These methodologies generally rely on cationic or ionizable polymers which are electrostatic complexed with mRNA called polyplexes [53]. Some of the polymers that have been studied in common are polyethyleneimine, poly beta amino esters, chitosan derivatives, and biodegradable polyesters. They can be tuned very finely in molecular weight, charge density and degradation rate due to their modular chemistry. The main benefit of the polymer based carriers is the chemical diversification and biodegradability. Polymers are capable of responding to particular intracellular conditions (pH or redox) unlike lipid nanoparticles can be engineered to respond to specific stimuli. Nevertheless, the cationic polymers tend to be more cytotoxic and inflammatory than lipid systems, which confines their translation to clinical use [54].

3.3 Liposomes and Nanoemulsions.

Liposomes are vesicles that are spherical, that are made up of phospholipid bilayers that surround an aqueous core and that have been used in delivering drugs and nucleic acids historically. Although structurally related to lipid nanoparticles, the typical classical liposomes have a lower mRNA encapsulation efficiency and reduced in vivo stability. Nanoemulsions are oil in water suspensions stabilized with surfactants and have been investigated, mainly in self amplifying RNA vaccines. They have the potential to cause potent immune responses, but in most cases, they need increased doses and demonstrate increased reactogenicity relative to lipid nanoparticles [55]. Nevertheless, liposomes and nanoemulsions can still be used in mucosal and intranasal delivery of vaccines, where local activation of immunity is required.

3.4 Systems of emerging nanocarriers.

3.4.1 Hybrid Nanoparticles

Hybrid nanoparticles consist of lipid and polymer to fuse the benefits of these two systems. The goals of these platforms are to increase stability, decrease toxicity, and increase the efficiency of endosomal escape [56]. It has shown enhanced expression of antigens and immune effects over single component carriers.

3.4.2 Exosome Based Delivery

Exosomes are naturally occurring extracellular vesicles released by cells and have become of interest as being biocompatible mRNA carriers. They have been attractive targets of next generation vaccines because of their innate capability to bind to recipient cells and avoids immune clearance [57]. Nevertheless, difficulties associated with being able to produce and load cargo in large quantities are not resolved.

3.4.3 Nanostructures Self Assembling.

Self assembling nanostructures take advantage of sequence specific reactions between mRNA and fabricated nanomaterials to create stable delivery systems that do not require significant formulation procedures. These strategies provide simplified production though they remain mostly experimental [58].

Table 2. Comparison of Nanotechnology Platforms for mRNA Vaccine Delivery

The table 2, summarizes the main nanotechnology platforms used for mRNA vaccine delivery, comparing their efficiency, development stage, strengths, and limitations.

Figure 2. Schematic representation of lipid nanoparticle mediated mRNA delivery