Applications of microfluidics in mRNA vaccine development: A review

Abstract

The transformative potential of microfluidics in the development of mRNA vaccines is explored in this review, highlighting its pivotal role in enhancing easy-to-use functionality, efficacy, and production efficiency. Moreover, we examine the innovative applications of microfluidics in biomedical research, including its contribution to the rapid and cost-effective synthesis of lipid nanoparticles for mRNA delivery and delve into the advantages of mRNA vaccines, such as targeted delivery and controlled expression. Furthermore, it outlines the future prospects of microfluidic devices, their cutting-edge examples in both research and industry, and the potential to revolutionize vaccine formulation and production. The integration of microfluidics with mRNA vaccine development represents a significant advancement in public health and disease prevention strategies.

I. INTRODUCTION

Nucleic acid (NA) therapy is predicated upon the utilization of nucleic-acid-derived substances as pharmaceutical agents for disease mitigation at the genetic scale.¹ This form of therapy boasts numerous benefits when compared to conventional approaches, notably in their capacity for targeted delivery to specific tissues.² The journey of mRNA from its initial discovery to its current role in cutting-edge technologies like vaccines spans several decades, marked by significant breakthroughs that have shaped both scientific knowledge and practical applications.

The concept of mRNA was first proposed in the early 1960s when François Jacob and Jacques Monod introduced the idea of an intermediary molecule that conveys genetic information from DNA to protein-synthesizing machinery in cells.³ This hypothesis was supported by the experimental work conducted by Sydney Brenner and colleagues, who demonstrated that mRNA acts as the template for protein synthesis in bacteria (Escherichia coli) infected by bacteriophages, confirming its pivotal role in gene expression.⁴

Throughout the 1970s and 1980s, further advancements were made in understanding mRNA's role in eukaryotic cells. In 1977, Richard J. Roberts and Phillip A. Sharp independently discovered the process of mRNA splicing, a breakthrough that revealed the complex modifications eukaryotic mRNA undergoes before being translated into proteins. This discovery was monumental as it highlighted how a single gene could produce multiple proteins through alteRNAtive splicing.⁵ A major milestone in the therapeutic application of mRNA occurred in 1989 when researchers demonstrated that mRNA could be introduced into cells in vivo to produce proteins, an essential step in developing mRNA-based therapies.⁶ However, it was not until the 2000s, through the pioneering work of Katalin Karikó and Drew Weissman, that critical obstacles related to mRNA's instability and immunogenicity were overcome. By incorporating modified nucleosides into mRNA, they significantly improved its stability and reduced immune reactions, making mRNA a viable therapeutic platform.⁷

The concept of utilizing mRNA as a therapeutic agent gained momentum in 1989, and the following benefits were also compared to widely applicable techniques for their transfection.^8,9 This breakthrough laid the groundwork for the subsequent proposition of mRNA as a vaccine platform. The appeal of mRNA in this context lies in its unique amalgamation of immunological attributes. It combines the advantageous features of live attenuated vaccines, such as the expression of antigens within the body and the induction of T cell responses, with the favorable characteristics of killed or subunit vaccines, including a precise and controlled composition and a higher safety profile.^10,11 This blend of qualities positions mRNA vaccines as potentially ideal candidates in the realm of immunization strategies.^12,13

The rapid development of mRNA vaccines during the COVID-19 pandemic marked a major application of decades of research. The mRNA vaccines developed by Pfizer-BioNTech and Moderna, based on lipid nanoparticle (LNP) technology, leveraged mRNA's ability to instruct cells to produce viral proteins that elicit an immune response.^14,15 This breakthrough demonstrated the versatility and safety of mRNA as a vaccine platform.

After their successful impact during the COVID pandemic, mRNA vaccines have garnered substantial interest for their potential in combating infectious diseases and various cancers.^1,16 These vaccines offer distinct advantages in terms of safety and efficacy over traditional live attenuated and DNA-based vaccines. A key feature of mRNA vaccines is their minimalistic genetic architecture, which includes only the necessary elements for expressing the target protein. This streamlined structure significantly reduces the risk of genomic integration, as mRNA, being a single-stranded RNA molecule, does not typically interact with the host genome, thus averting the potential hazards of genetic alteration.^12,17 Additionally, the transient nature of mRNA, characterized by a short half-life, allows for a controlled level of in vivo expression. This attribute is particularly advantageous for managing therapy-related adverse effects as it enables a more regulated and safer therapeutic approach.¹ Fig. 1 provides a detailed, informative, and step-by-step overview of the mRNA vaccine mechanism.

FIG. 1.

General overview of how mRNA vaccine works. In the first step, the mRNA-loaded LNPs should be injected into the muscle cells of the body (usually the upper arm) (1). Once mRNA-loaded LNPs enter the body (2), muscle cells and immune cells take up LNPs from the injection site (3). In the next step, inside the cells, the LNPs are broken down, releasing the mRNA into the cytoplasm (4). Ribosomes in the cytoplasm read the mRNA sequence and translate it into the target protein, such as the spike protein of the SARS-CoV-2 virus (5). The newly made viral protein (or a portion of it) is processed and presented on the cell surface (6). Finally, immune cells produce target antibodies for capturing translated protein (7). These antibodies can neutralize the virus by preventing it from infecting cells if the person is later exposed to the virus. Note that the immune system's response to the mRNA vaccine mimics the natural infection process but without causing disease. This trains the immune system to recognize and combat the real virus if exposed.

Another notable aspect of mRNA vaccines is their production methodology. The synthesis of mRNA in a cell-free system through the in vitro transcription of a DNA template containing the desired mRNA sequence eliminates the need for microorganisms and/or cultured cells. This approach simplifies the purification process and facilitates a more rapid and cost-effective manufacturing pipeline.^1,18 Consequently, mRNA vaccines represent a promising and innovative direction in modern therapeutic strategies, offering a blend of safety, control, and efficiency.

RNA delivery technology, including mRNA vaccines, has been globally researched for around three decades.¹⁹ Vaccines are vital for public health and are key in controlling infectious diseases. mRNA vaccines are emerging as a promising alternative to traditional vaccines because of their accuracy, safety, and adaptable manufacturing methods. mRNA vaccines are being studied in the clinic to treat a number of diseases, including cancer, HIV, influenza, the COVID-19 pandemic, and even genetic disorders.²⁰ This review outlines the latest progress in mRNA vaccines, highlighting how to convert this innovative vaccination technology into a rapid, economical, and effective solution for tackling emerging health crises using microfluidic devices.

In recent decades, microfluidic chips or devices, referred to as lab-on-a-chip or micro-total analysis systems (μTASs), have been considerably taken into consideration due to their wide range of applications and can be used in many fields such as biology, chemistry, fluid dynamics, microelectronics, and so on.^21–24 Microfluidic chips are a set of models and etched micro-channels and tunnels for the flow of fluid. Microfluidics focuses on the behavior of fluids at the microscale level. In other words, less reagent is required due to their small sample size. In addition, not only does utilizing microfluidic devices increase the opportunities for research because of the extremely low volume of analytes but multiple analytes can also be processed simultaneously.^22–26 In Sec. II, we will deeply understand how microfluidics has deeply transformed and enhanced the development of mRNA vaccines.

After reading this review, while gaining a profound understanding of the mRNA vaccines and their mechanism and microfluidics, one can comprehensively understand the role of microfluidics in the fast-evolving and fascinating world of mRNA vaccine development. Beyond the theoretical explanations, an extensive overview of the state-of-the-art recent industrial vaccine developer microfluidic chips is given, and the forthcoming potentials of utilizing microfluidics in vaccine development are discussed.

II. MICROFLUIDICS AND THEIR APPLICATIONS IN BIOMEDICAL RESEARCH

Microfluidics refers to the field of science and technology dedicated to handling or controlling minute volumes of fluids, typically ranging from 10⁻⁹ to 10⁻¹⁸ l. This involves the utilization of channels with dimensions spanning from tens to hundreds of micrometers.²⁷ They enable precise manipulation of fluids in microchannels with dimensions in the micrometer range. It offers advantages such as controlled mixing, parallelization, minimal reagent usage, improved mass and heat transfer, rapid system response, enhanced reproducibility, portability, low power consumption, safety, and cost reduction.^1,28 One of the most important characteristics of microfluidics is the laminar flow due to the system's microscopic size. This feature means that the fluids do not mix convectively, and the flow is transparent, allowing the visual detection of the particles passing through them (i.e., under optical microscopes), which is crucial in almost all microfluidic-based detection devices.

Microfluidic systems, with their ability to be designed for diverse tasks such as detecting airborne toxins and analyzing DNA and protein sequences, hold significant potential for enhancing the efficiency of drug discovery and diagnostics techniques. Specific microfluidic systems capable of various DNA-type analyses have been developed, including an integrated system for PCR analysis that minimizes sample processing and handling.²⁹ This system enables DNA typing from whole blood samples using capillary microfluidics and capillary array electrophoresis, allowing blood to serve directly as the sample template for PCR amplification analysis;²⁹ see Fig. 2.

FIG. 2.

General schematic of the whole blood capillary flow direct PCR analysis with samples being manipulated within the microfluidic channels.³⁰ [Adapted with permission from Zhang et al., Anal. Chem. 71, 1138–1145 (1999). Copyright 1999 American Chemical Society²⁹.]

Moreover, microfluidics technology demonstrates promise in detecting very low numbers of DNA molecules, potentially even individual molecules. Electrophoretic mobility shift assays for DNA–protein interactions have been conducted in a microfluidic chip environment, resulting in benefits such as reduced sample volumes, avoidance of labeling procedures, and decreased analysis times.³¹ Additionally, microfluidic technology has been integrated into the analysis of proteins and peptides, particularly when linked with mass spectrometric analysis.^32,33 This allows peptides to be adsorbed onto hydrophobic membranes, desalted, and eluted in a controlled manner through microfluidics, facilitating direct mass spectrometric analysis of picomole amounts of peptides using electrospray ionization mass spectrometry procedures.^30,34

Microfluidics finds its roots in four distinct origins: molecular biology, molecular analysis, microelectronics, and biodefense.²⁷ Before becoming an independent discipline, it was intricately interwoven with various fields, each contributing significantly to developing its techniques and materials.³⁵ However, we focus on microfluidics molecular analysis and microelectronics in this context. Notably, the genesis and progression of microfluidics owe much to the Human Genome Project (HGP), an international scientific research program.³⁵ Commencing in 1990, this research field spurred intense interest among scientists in studying and sequencing nucleic acids, leading to the development of sequencing devices capable of handling minuscule samples while delivering high throughput and sensitive readouts.^36,37

In recent times, the integration of microfluidics into biomedical research has surged, driven by its manifold advantages and applications in this domain. Microfluidics enables biomedical assays on a miniature scale, facilitating rapid screening and precise fluid flow control.³⁵ This integration has spurred significant advancements in understanding intricate biological processes, devising novel therapeutic strategies, and enhancing healthcare outcomes. With ongoing technological advancements, microfluidic devices are poised to assume an increasingly pivotal role in fostering innovation in biomedicine. Point-of-care devices, human/organ-on-a-chip models, and mRNA vaccines exemplify the diverse applications of microfluidics in the biomedical realm.^35,38,39

III. mRNA VACCINES: AN OVERVIEW

The evolution of vaccination strategies has significantly advanced with the development of RNA-based vaccines. Unlike traditional methods, RNA vaccines offer a versatile and efficient approach to induce potent immune responses against various pathogens and diseases, including infectious agents and cancers.⁴⁰ Notably, the World Health Organization highlights the importance of immunization coverage in preventing millions of deaths annually.⁴¹

The development and therapeutic implications of childhood vaccination programs have been extensively discussed, emphasizing the global and domestic impact on public health.⁴⁰ Furthermore, the historical perspective of vaccines, spanning four centuries, underscores the continuous evolution and significant milestones in vaccine development.⁴² The shift toward mRNA vaccines represents a pivotal development, offering the potential to address previously unmet medical needs through a targeted and personalized approach.⁴³ Additionally, mRNA vaccines represent a paradigm shift in vaccine development, leveraging the process by which cells produce proteins to trigger an immune response against various pathogens, including viruses and bacteria. This innovative approach has rapidly come to the forefront of medical research, particularly highlighted by its critical role in addressing the COVID-19 pandemic.

Recent advancements in gene transfer techniques, specifically through the use of non-viral vector-based delivery systems, have paved the way for the development of mRNA vaccines.⁴⁴ These vaccines utilize lipid nanoparticles (LNPs) for the delivery of mRNA, encoding the antigen of interest, directly into host cells, thereby inducing a robust immune response.⁴⁵ This approach offers a high degree of flexibility in vaccine design and circumvents the limitations associated with traditional vaccine platforms.

The immunogenicity of mRNA vaccines has been further enhanced by incorporating adjuvants, such as liposomes, to improve the formulation strategies for antigen presentation and immune stimulation.⁴⁶ This has significantly improved vaccine efficacy, as demonstrated through preclinical and clinical studies targeting various infectious diseases and cancers.⁴⁷

Moreover, the development of nucleoside-modified mRNA vaccines has shown promising results in terms of increased translational capacity, biological stability, and reduced immunogenicity, making them highly effective for both prophylactic and therapeutic applications.⁴⁸ Clinical trials have already demonstrated the protective efficacy of mRNA vaccines against challenging pathogens, marking a significant milestone in vaccine technology.⁴⁷

In conclusion, mRNA-based vaccines represent a revolutionary advancement in the field of immunization, offering a powerful tool against a wide array of diseases. With ongoing research and clinical trials, mRNA vaccines promise to reshape the future of public health and disease prevention.⁴³

A. Pharmacological mechanisms of adaptive immune responses induced by mRNA–LNP vaccines

The advent of mRNA–LNP (lipid nanoparticle) vaccines has introduced a novel paradigm in immunization strategies, showcasing a unique mechanism of action to elicit potent adaptive immune responses, as seen in Fig. 3. The process commences with synthesizing in vitro transcribed mRNA, encoding the antigenic protein specific to a target pathogen. This mRNA is then encapsulated within lipid nanoparticles, which serve as a protective envelope and a delivery system, ensuring the mRNA's stability and facilitating its efficient entry into host cells.⁵⁰

FIG. 3.

Diagram illustrating the step-by-step pharmacological mechanism of adaptive immune responses induced by mRNA–LNP vaccines. The process begins with the encapsulation of in vitro transcribed mRNA into lipid nanoparticles (LNPs). These mRNA–LNP complexes are then transfected into host cells via specialized lipids. Following transfection, the LNPs undergo endocytosis, and the mRNA is released into the cytosol through endosomal escape. The mRNA is translated into the target antigen protein by host cell ribosomes. This antigenic protein may either be secreted outside the cell or processed intracellularly, where it is degraded by the proteasome, revealing antigenic sites. These sites are presented on the cell surface via major histocompatibility complex I (MHC I) molecules to CD8+ T cells. Additionally, any exogenously released proteins can be processed and presented on MHC II molecules, which are recognized by B cells, leading to B cell activation and maturation. From P. K. Gote et al., Int. J. Mol. Sci. 24(3), 2700 (2023).⁴⁹

Upon administration, the mRNA–LNP complexes are taken up by host cells through a series of steps, beginning with the transfection facilitated by specialized lipids on the LNPs' surface. This interaction promotes the uptake of mRNA–LNP into the cells via endocytosis, a cellular process characterized by the engulfing and internalization of the mRNA–LNP into endosomes.⁵⁰ Crucial to the vaccine's efficacy is the subsequent endosomal escape of mRNA into the cytosol, mediated by the lipid components of the LNP, thereby preventing mRNA degradation by endosomal enzymes.

In the cytosol, the released mRNA undergoes translation by host cell ribosomes, completing the synthesis of the target antigenic protein. This intracellular antigen production leads to two primary pathways of immune activation: First, the antigenic protein can be processed and presented on the cell surface via major histocompatibility complex class I (MHC I) molecules, effectively presenting epitopes to CD8+ T cells. This interaction is pivotal for activating cytotoxic T lymphocytes, which play a key role in directly targeting and eliminating pathogen-infected cells.⁵⁰ Second, some of the synthesized antigens can be secreted from the cell, where they may be taken up by antigen-presenting cells (APCs), processed, and presented via MHC II molecules to CD4+ T cells. This pathway is essential for helper T cell activation, which supports cellular and humoral immune responses, including the activation and maturation of B cells into antibody-secreting plasma cells.⁵¹

The orchestrated activation of these immune pathways facilitates immediate response to the pathogen and aids in developing long-term immunity through the generation of memory T and B cells. Such memory cells are primed to respond more rapidly and effectively upon subsequent exposures to the pathogen, thereby providing lasting protection against the disease.

Recent studies have further elucidated the pharmacological mechanisms by which mRNA–LNP vaccines modulate adaptive immune responses. Research demonstrates that the use of ionizable lipids in LNPs not only enhances mRNA delivery but also plays a critical role in modulating the immune environment to favor a stronger response from both CD8+ and CD4+ T cells, contributing to robust cellular immunity. This modulation is largely due to the ability of ionizable lipids to promote endosomal escape and reduce mRNA degradation, ensuring higher antigen expression levels that are crucial for effective immune activation.^52–54

Additionally, studies by Zhu et al. highlighted the importance of the innate immune signaling pathways triggered by mRNA–LNP vaccines. The activation of pattern recognition receptors (PRRs) like Toll-like receptor 7 (TLR7) and retinoic acid-inducible gene I (RIG- I) has been shown to initiate a cascade of immune responses that amplify the recruitment and activation of antigen-presenting cells (APCs). This innate activation is essential for enhancing the downstream adaptive immune responses, including the activation of T follicular helper (Tfh) cells and B cells, which are critical for long-term antibody production and memory B cell formation.⁵⁵

Moreover, another study by Wu et al. explored the role of mRNA–LNP vaccines in inducing a balanced Th1/Th2 response. Their findings suggest that the formulation of LNPs can be fine-tuned to favor a Th1-biased response, which is typically associated with a more effective antiviral immunity through the production of cytokines like interferon-gamma (IFN-γ). This balance between Th1 and Th2 responses is pivotal for not only controlling the initial infection but also preventing immune-related side effects that might arise from skewed immune activation.⁵⁶ These studies underscore the multifaceted pharmacological interactions at play in the adaptive immune responses elicited by mRNA–LNP vaccines, highlighting their capacity to induce both cellular and humoral immunity through precisely controlled mechanisms.

In summary, mRNA–LNP vaccines represent a significant advancement in vaccine technology, employing a sophisticated mechanism to achieve effective and durable immunity. Through the targeted delivery and expression of antigenic proteins, these vaccines harness the body's cellular machinery to simulate an infection and activate a comprehensive, adaptive immune response, underscoring their potential to prevent infectious diseases.

B. Conventional production methodology for mRNA vaccines

The traditional process of mRNA vaccine production involves a sequence of critical phases: upstream production, downstream purification, and the final formulation, with each stage playing a pivotal role in ensuring the vaccine's efficacy and safety. During the upstream production phase, mRNA transcripts are synthesized from a plasmid DNA template through in vitro transcription (IVT).⁵⁷ This procedure typically utilizes RNA polymerase enzymes such as T7, SP6, or T3, which catalyze the creation of mRNA from a linearized DNA template. Critical to the IVT reaction are enzymes like RNA polymerase, which converts DNA to RNA; inorganic pyrophosphatase, which enhances the yield; guanylyl transferase and Cap 2′-O-methyltransferase that are involved in capping the mRNA; and DNase I for removing any contaminating genomic DNA from the samples.⁵⁸ Although this method is fundamentally effective, it can exhibit significant variability in mRNA yield and quality due to the batch-dependent nature of the enzymatic reactions and sensitivity to minute changes in reaction conditions.⁵⁸ Figure 4 illustrates the IVT method's inputs and output products.

FIG. 4.

The in vitro transcription (IVT) reaction includes both input and output components, as well as potential impurities. On the left side, the inputs to the IVT reaction are listed, comprising the linear DNA template (such as linearized plasmid and PCR product), nucleoside-triphosphates (NTPs) including N1-methylpseudouridine- 5′-triphosphate (m1Ψ), and RNA polymerase (RNA Pol II). On the right side, the outputs of the reaction include the mRNA (drug substance) and various IVT byproducts. In the center, potential impurities that may arise from raw materials or be introduced during the production process are indicated. [From Lenk et al., Front. Mol. Biosci. 11, 1426129 (2024). Copyright 2024 Author(s), licensed under a Creative Commons Attribution (CC BY) license⁵⁹.]

In the downstream purification phase, the newly synthesized mRNA is purified to remove various impurities, including residual NTPs, enzymes, and misformed mRNAs. Traditional approaches, such as DNase enzyme digestion followed by lithium chloride precipitation, are commonly employed.⁶⁰ However, these methods often do not fully eliminate all aberrant mRNA species, potentially diminishing the quality and immunogenicity of the final product. More advanced methods, such as reverse-phase HPLC, have been shown to significantly improve the purity of modified mRNA, which in turn enhances translational efficiency and reduces immune activation.⁶¹

The final stage, formulation, involves encapsulating the mRNA in lipid nanoparticles (LNPs) to protect the mRNA from degradation and to facilitate effective delivery into cells. Conventionally, this is achieved by mixing the mRNA with lipids in bulk, which can result in LNPs with inconsistent sizes and variable encapsulation efficiencies.

Another approach for the production of mRNA vaccines is cell-based systems. Cell-based systems for mRNA vaccine production utilize live eukaryotic cells, such as human embryonic kidney (HEK293) or Chinese hamster ovary (CHO) cells, to transcribe and translate DNA into mRNA. The process begins with the design of a DNA template that encodes the target antigen, typically cloned into a plasmid vector with essential regulatory elements. This plasmid is introduced into cultured cells through transfection techniques like lipofection, electroporation, or viral transduction, enabling the cells to utilize their machinery for mRNA synthesis.⁶²

Once transfected, the cells transcribe the plasmid DNA into mRNA and subsequently translate it into the desired protein. After sufficient mRNA has been produced, it is isolated and purified through methods such as phenol-chloroform extraction or column purification. The purified mRNA is then formulated into lipid nanoparticles (LNPs) to protect it from degradation and facilitate delivery into host cells. Rigorous quality control ensures that the mRNA–LNP formulation meets the required safety and efficacy standards before further use.⁶³

Despite their effectiveness, cell-based systems face several challenges. The production process is resource-intensive and costly, involving significant expenses for cell culture media, reagents, and specialized equipment. Additionally, the timeline for mRNA production can be lengthy, taking days to weeks, which may hinder rapid responses to emerging infectious diseases. While scaling up cell culture processes is possible, it can introduce variability in yield and quality. Moreover, live cell cultures are susceptible to contamination risks, which can compromise the final product. Regulatory hurdles also pose challenges as cell-based processes often require comprehensive documentation to meet stringent safety and efficacy standards.⁶⁴

A cell-free system is another conventional method for synthesizing the mRNA vaccine. Cell-free systems for mRNA vaccine production utilize in vitro transcription (IVT) to synthesize mRNA from DNA templates without the need for live cells. The process begins with the design of a DNA template, which is typically cloned into a plasmid vector. This vector contains necessary regulatory sequences, such as a promoter and terminator, to facilitate efficient transcription. The plasmid is then linearized, usually by restriction enzyme digestion, to prepare it for transcription.⁶⁵

The core of the cell-free system is the use of an enzyme, often T7 RNA polymerase, to transcribe the linearized DNA template into mRNA. This reaction takes place in a controlled environment, typically involving a buffer solution that provides the necessary nucleotides, salts, and cofactors required for transcription. The resulting mRNA can be engineered with modifications, such as a 5′ cap and a poly-A tail, to enhance stability and translation efficiency in target cells. After transcription, the mRNA is purified to remove any residual enzymes and byproducts, often using methods like phenol-chloroform extraction or affinity chromatography.²⁰

One of the primary advantages of cell-free systems is their ability to produce mRNA quickly and efficiently. This method allows for rapid optimization and scaling of production processes, making it particularly advantageous during public health emergencies when rapid vaccine development is crucial. Additionally, because there are no living cells involved, the risk of contamination from cell culture is eliminated, and the process can be more easily controlled and standardized.

However, there are notable challenges associated with cell-free mRNA production. One significant issue is the cost of reagents and enzymes, which can be higher than traditional cell-based methods. The requirement for high-quality plasmid templates and the need for complex reaction conditions can also increase costs and production time. Additionally, while cell-free systems can achieve high yields, there can be variability in the quality and integrity of the mRNA produced, which may affect its efficacy as a vaccine. The optimization of transcription conditions is crucial for maximizing yield and ensuring that the mRNA produced is correctly processed and functional.⁶⁶

Microfluidic technologies have revolutionized this phase by enabling precise control over the mixing process, thereby producing LNPs with uniform size distributions and high encapsulation efficiencies.²⁰ Microfluidic systems offer several advantages over traditional methods in the production of mRNA vaccines. These systems provide a controlled environment that allows for the accurate manipulation of fluids at microscale levels, thereby enhancing the reproducibility and scalability of mRNA production. The precise control of reaction conditions through microfluidic devices leads to higher yields and more consistent product quality compared to conventional methods. In the purification phase, microfluidic devices can facilitate continuous, on-chip separation of mRNA from impurities, which improves purity and reduces batch-to-batch variability. For formulation, the rapid and controlled mixing of mRNA with lipids under microfluidic conditions results in the efficient production of LNPs with desired characteristics.⁶⁷

While conventional methods have been instrumental in the development and production of mRNA vaccines, they are often hampered by scale dependence, variability in product quality, and inefficiencies in the purification processes. These limitations can lead to increased costs and extended development times, which are impractical in urgent scenarios such as pandemic responses. Microfluidic technologies, by contrast, offer substantial improvements in efficiency, scalability, and consistency of mRNA vaccine production, which are crucial as the use of mRNA technology in vaccine development and other therapeutic areas continues to grow.

IV. APPLICATIONS OF MICROFLUIDICS IN mRNA VACCINE DEVELOPMENT

A. LNP production and synthesis

Several traditional techniques for producing LNPs exist, such as lipid film hydration and ethanol injection.^68,69 However, these methods often yield large, heterogeneous particles with low encapsulation efficiency. To address this issue and produce smaller LNPs, further modifications like extrusion and sonication are typically required.⁶⁹ Microfluidic devices have emerged as a promising alternative to conventional methods, allowing for precise mixing of different fluids within micro-scale channels. They offer advantages such as controlled manipulation, parallelization, minimal reagent consumption, enhanced mass transfer, lower costs, continuous and reproducible production of small-sized LNPs, and scalability.^1,28,68–74

Typically, microfluidic devices designed for preparing LNPs feature two separate inlets: one for the solution containing lipids and the other for the aqueous solution containing therapeutic agents. These solutions are mixed within the microfluidic path, forming LNPs, which are then collected at the outlet.^75,76 The schematic of a typical microfluidic device is shown in Fig. 5. Microfluidic LNP production is highly versatile, allowing for adjustments in channel sizes and the utilization of various materials such as silicon, polymers, or glass. This versatility enables the use of different lipid formulations, resulting in the production of uniformly sized particles. However, microfluidic systems face challenges, particularly the dominance of laminar flow within the channels, which can impede the efficient mixing of fluids.^{28,68–74,77} Micromixers are employed to reduce diffusion lengths and improve fluid mixing to address this issue. Popular micromixer designs include the staggered herringbone micromixer (SHM), segmented flow micromixer, high-pressure micromixer, and flow-focusing micromixer.⁷⁰ These enhancements contribute to the effectiveness and reliability of microfluidic LNP production.

FIG. 5.

A schematic of a microfluidic device preparing LNPs including two inlets for introducing a lipid solution, often containing lipids dissolved in ethanol or another organic solvent and an aqueous solution, often containing the therapeutic agent to be encapsulated into the microfluidic device. As a product in the outlet, we have formed LNPs with different sizes.

Several structures are currently used for microfluidic devices. T-junction is the most basic one, with two inlets intersecting and flowing through the outlet. In the intersection, two fluids from the inlet have high momentum, leading to chaotic mixing.⁷⁹ HFF is the most typical method for continuous production. Despite others, this method has three inlets. One of them, a lipid-contained liquid, would be injected into the inlet with a low flow rate. Then, the aqueous phase would be injected into the other two inlets.⁸⁰ The most popular and widely used one is SHM, which allows for the perfect mixing of fluids and LNP properties like their size due to the herringbone structure of the channels.^{73,74,77,81,82} Also, because of their unique structure, they provide mixing in narrow channels and are easy to use. However, some disadvantages are low throughput and probable channel clogging.^73,74,81,83 Figure 6 illustrates the schematic of an SHM microfluidic device in detail and the conceptual patterns of other mentioned microfluidic device structures.

FIG. 6.

(a) Different structures used for microfluidic devices. From left to right: T-junction, HFF, SHM, Baffle, and bifurcation.¹ (b) Top view of the staggered herringbone micromixer microfluidic device. (c) The 3D view and dimensions of the channel of SHM.⁷⁸ In this structure, the channel width is around hundreds of μm, and the width of each groove is selected to be around tens of μm based on different applications and desired LNP sizes.

Baffles are structures placed within the microfluidic channel that disrupt the flow of fluid. Depending on the desired effect, they can take various forms, such as ridges, grooves, or obstacles. The primary function of baffles is to induce mixing or enhance mixing efficiency within the microfluidic channel. When fluid flows past baffles, it experiences velocity and direction changes due to these obstacles. This disruption in flow promotes the mixing of different components within the fluid.⁸⁴ Bifurcation is splitting a single fluid stream into two or more separate channels within a microfluidic device. This splitting can occur passively, through geometric features of the microfluidic channel, or actively, using external forces such as pressure or electric fields. In passive bifurcation, the geometry of the microfluidic channel is designed such that when fluid flows through it, and it naturally divides into multiple branches. Active bifurcation involves the use of external forces to control the splitting of the fluid stream.⁸⁵

Briefly, the mechanism of forming LNPs in microfluidic devices is that a solution containing lipids and a solution containing nucleic acids are injected into two inlets and meet at an intersection. Several stages occur through the microfluidic path for forming LNPs. First, lipid molecules aggregate, and an intermediate disk-like structure forms. Then, the formed structure diffuses, and finally, LNPs enclose.⁸⁶ Figure 7 shows this process from the beginning to the end.

FIG. 7.

siRNA-LNP formation process from the first step, mixing lipids and nucleic acids, to the last step, where LNPs are completely formed in the outlet of the microfluidic device. [From Chen et al., J. Am. Chem. Soc. 134, ja301621z (2012). Copyright 2024 Author(s), licensed under a Creative Commons Attribution (CC BY) license⁸⁷.]

B. mRNA-loaded LNP using a microfluidic device

Current methods for producing mRNA lipid nanoparticles (LNPs) primarily use microfluidic or T-junction mixing to swiftly combine an ethanol phase containing hydrophobic lipids with an aqueous phase containing mRNA in a buffer, such as acetic acid. Microfluidic mixing allows for the mixing of very small volumes of lipids in ethanol with mRNA in aqueous solutions (tens of microliters), facilitating the screening of numerous components and formulation parameters. The LNP formation process is driven by hydrophobic and electrostatic forces. Initially, the four lipids (ionizable lipid, DSPC, cholesterol, PEG-lipid) are soluble in ethanol, with the ionizable lipid being unprotonated and electrically neutral. Typically, one volume of the lipid-ethanol solution is mixed with three volumes of mRNA in a pH 4 acetate buffer. When the lipids contact the aqueous buffer, they become insoluble, and the ionizable lipid becomes protonated and positively charged, binding electrostatically to the negatively charged mRNA phosphate backbone. The insoluble lipids form a lipid particle encapsulating the mRNA in an aqueous suspension.

The PEG-lipid plays a key role in this process, as its hydrophilic PEG chain coats the particle, determining its final stable size. A critical observation is that LNP structure and size continue to evolve after mixing when the mRNA LNP suspension is diluted in aqueous buffer or dialyzed to raise the pH and remove ethanol. Initial mixing produces a pH of around 5.5, protonating the ionizable lipid, which facilitates mRNA binding and encapsulation. Raising the pH further neutralizes the ionizable lipid, making it less soluble and forming larger hydrophobic lipid domains, driving LNP fusion and increasing size. The core of the LNP becomes an amorphous electron-dense phase, mainly containing the ionizable lipid bound to mRNA.⁸⁸ Figure 8 visually represents the procedure of creating the mRNA-loaded LNP produced using a microfluidic device.

FIG. 8.

The procedure of mRNA-loaded LNP production. This process takes place in four steps. In the first phase, a solution of ethanol containing four lipids mixes with another buffer containing mRNAs. Next, ionizable lipids get protonated and become charged positively, which leads to the attachment of this positively charged lipid to the backbone of mRNA. The final objective of this step is to encapsulate mRNAs. By diluting the aqueous solution, pH increases. This neutralizes the ionizable lipid, making it more hydrophobic, which drives the fusion of vesicles and leads to the further sequestration of the ionizable lipid with mRNA into the interior of the solid lipid nanoparticles. Finally, the PEG-lipid content prevents further fusion by creating a hydrophilic exterior for the LNP, thus establishing its thermodynamically stable size. Just beneath this PEG-lipid layer, the DSPC forms a bilayer. From D. Buschmann et al., Vaccines 9(1), 65 (2021).⁸⁸

Recent developments in microfluidic-based LNP production have focused on optimizing the lipid composition to enhance the stability and delivery efficiency of the encapsulated mRNA. Studies by Kulkarni et al. explored the use of ionizable lipids specifically tailored for mRNA delivery, which significantly improved the transfection efficiency and reduced off-target effects. This advancement is particularly important in developing mRNA vaccines, where precise delivery to target cells can determine the efficacy of the immune response.⁸⁹ In another novel study, researchers developed a 3D-printed Omnidirectional Sheath-flow Enabled Microfluidics (OSEM) device for the high-throughput production of lipid nanoparticles (LNPs) encapsulating SARS-CoV-2 spike protein mRNA. The device features a four-way hydrodynamic flow focusing region and a staggered herringbone mixer (SHM), which enhance the formation of LNPs with low polydispersity and high mRNA encapsulation efficiency. This design enables a fivefold higher throughput rate compared to commercial micromixers while maintaining low production costs, making it a cost-effective and scalable solution for LNP manufacturing.⁹⁰

Moreover, Shepherd et al. present a novel scalable approach for producing lipid nanoparticles (LNPs) that encapsulate RNA therapeutics. The authors designed a parallelized microfluidic device (PMD) with 128 mixing channels operating simultaneously, achieving over 100-fold higher production rates compared to conventional microfluidic devices while maintaining desirable LNP properties like small size and potency. They addressed the challenges of scaling LNP production for clinical applications without sacrificing the precision and efficacy offered by microfluidic methods. This approach enables scalable production from small bench-scale experiments to large-scale clinical manufacturing. Furthermore, the LNPs produced with this method demonstrated improved delivery of RNA therapeutics in vivo, with enhanced gene silencing and protein expression in mice compared to bulk mixing methods.⁹¹

V. STATE-OF-THE-ART ADVANCEMENTS OF MICROFLUIDIC DEVICES

The role of microfluidic devices in mRNA vaccine development has been redefined, contributing to increased precision, scalability, and efficiency in production. Innovations in microfluidic technology enable controlled handling of fluids at a scale that is decreasing rapidly each year with the introduction of new devices, manufacturing technologies, etc., allowing precise manipulation of flow rates, mixing, and particle sizes. Modern devices, such as staggered herringbone mixers (SHMs) and 3D-printed designs, optimize mixing and encapsulation processes for lipid nanoparticles (LNPs), achieving high encapsulation efficiency with minimal reagent usage. These advances also support high-throughput production and reproducibility, which is critical for scaling vaccine formulation to meet global industrial demands. Integrating artificial intelligence (AI) with microfluidics further enhances process control, adapting real-time adjustments to maintain product quality. The industrial application of these devices allows rapid response capabilities for emerging health crises, illustrating microfluidic technology's transformative impact on the biopharmaceutical landscape.

A. Research advances

In recent years, lots of research groups have developed several innovative devices that enhance efficiency, precision, and scalability, pushing the limits of microfluidics even further. The SCALAR Microfluidic Chip, for instance, incorporates 256 parallelized mixing units with a branching design that reduces operating pressure by 40%, achieving a production rate of 17 l /h. This high-throughput capability, along with the use of silicon and glass materials for enhanced solvent compatibility makes SCALAR particularly valuable for pandemic response scenarios requiring large-scale, precise vaccine production.⁹² Similarly, the Omni-directional Sheath-flow Enabled Microfluidics (OSEM) device, equipped with a four-way hydrodynamic flow focusing region and staggered herringbone mixer, facilitates cost-effective, high-throughput LNP production with low polydispersity and high mRNA encapsulation efficiency, addressing key production needs for SARS-CoV-2 mRNA vaccines.⁹⁰ The Parallel Microfluidic Device (PMD) also supports large-scale mRNA vaccine formulation by utilizing arrayed staggered herringbone mixers with clog-resistant architecture, achieving uniform LNP production suited for high-demand applications.⁹¹

Integrating advanced technology, the continuous-flow, AI-enhanced microfluidic system merges process analytical technology (PAT) sensors with AI to provide real-time process optimization, enabling on-demand, high-throughput mRNA production. This system addresses the need for precise control in large-scale applications, although its dependency on automated control and AI presents a cost challenge.⁶⁷ Another noteworthy development is the nanoemulsion-based microfluidics platform, which uses nanoemulsion techniques to achieve stable, uniform LNPs with high mRNA encapsulation efficiency. This system is particularly promising for scalable mRNA production across various therapeutic applications but faces challenges in fabrication and scale-up.⁹³ Finally, the optimized microfluidic platform for gene editing demonstrates the versatility of microfluidics by integrating optimized lipid excipients and flow conditions to improve mRNA–LNP delivery, with a particular focus on mRNA-based CRISPR-Cas9 platforms. This device shows significant potential for gene therapy and vaccine applications requiring precise targeting and efficiency.⁹⁴ Together, these innovations exemplify how microfluidic research is advancing the field of mRNA therapeutics, enhancing both the production capabilities and clinical applicability of mRNA vaccines. Table I summarizes the key aspects of these contemporary, novel microfluidic devices in detail.

TABLE I.

Summary of recent advances and developments in microfluidic devices for mRNA vaccine formulation.

Device/technology	Recent innovations	Applications and impact on mRNA vaccine development	Limitations
SCALAR microfluidic chip92	Incorporates 256 parallelized mixing units on silicon and glass, enabling a 17 l/h production rate, ladder, and branching design reduces operating pressure by 40%, and uses silicon and glass materials for enhanced solvent compatibility and sterilization capability	High-throughput, scalable mRNA LNP production with uniform properties at various scales, enables large-scale, precise mRNA vaccine production; useful in rapid response scenarios, and potent SARS-CoV-2 mRNA vaccine delivery with consistent physical properties across scales	Susceptibility to fouling, requiring periodic cleaning for long-term use, the complexity of initial fabrication and high-pressure requirements, and expensive compared to polymer alteRNAtives; scalability limitations
3D-printed omnidirectional sheath-flow enabled microfluidics (OSEM) device90	Omnidirectional sheath flow and staggered herringbone mixer	High-throughput, cost-effective LNP production with low polydispersity; enhanced encapsulation for mRNA vaccines like SARS-CoV-2	Limited scalability; durability under industrial conditions needs validation
Parallel microfluidic device (PMD)91	Arrayed SHMs with clog-resistant architecture and integrated filters	Uniform, scalable LNP production; can meet demands for high-precision and large-scale manufacturing	Complex multi-layer design and high initial fabrication costs
Continuous-flow, AI-enhanced microfluidic system67	Integrates AI with PAT sensors for real-time optimization	Enables on-demand, high-throughput production with enhanced process control	Costly setup; dependency on automated control and AI systems for efficiency.
Nanoemulsion-based microfluidics for LNPs93	Incorporates nanoemulsion techniques to create uniform, stable LNPs with efficient mRNA encapsulation, achieving a high degree of stability and encapsulation efficiency	Enables high-throughput LNP production for scalable mRNA vaccine deployment, potentially applicable to various mRNA therapeutics	Complex fabrication process; scale-up challenges
Optimized microfluidic formulation for enhanced LNP gene editing94	This microfluidic platform integrates optimized lipid excipients and flow conditions to improve the delivery efficiency of mRNA-loaded LNPs for gene editing applications. Enhanced formulations demonstrated higher encapsulation and delivery efficiency, focusing on mRNA-based CRISPR-Cas9 platforms	Though primarily designed for gene editing, the innovations in LNP encapsulation and formulation optimization hold significant promise for mRNA vaccine delivery, especially where precise targeting and efficiency are critical	The need for intricate lipid optimization, limiting generalizability across various RNA types and formulations

B. Industrial advances

In recent years, the pharmaceutical industry has experienced rapid advancements in vaccine formulation technologies, particularly with the emergence of mRNA vaccines, which require novel and efficient production methods. To address these needs, several companies have developed ready-to-use microfluidic platforms that significantly streamline the process of mRNA vaccine formulation. These innovations are crucial as they offer a scalable solution for rapidly producing vaccines, especially during global health crises such as the COVID-19 pandemic.

Table II provides a detailed overview of key players in the industry that have rolled out these advanced microfluidic systems. Understanding the contributions of these companies is essential for this paper as it highlights the intersection of biotechnology and engineering innovations, illustrating how these platforms can enhance vaccine development. By comparing different technologies, we can assess their impact on the efficiency, scalability, and speed of vaccine production, informing future vaccine formulation and deployment strategies.

TABLE II.

Summary of current available technologies developing microfluidic platforms for mRNA–LNP vaccine formulation.

Technology	Key innovations	Applications and industry impact	Critique
NanoFabTx™ kits, mRNA vaccine formulation kits by MilliporeSigma95	Ready-to-use microfluidic platform for rapid screening and synthesis of lipid-encapsulated mRNA	Streamlining mRNA vaccine formulation, enhancing efficiency in preclinical assessments, contributing to rapid vaccine development during the COVID-19 pandemic	Limited customization options for specific research needs could hinder some advanced applications
Microfluidic control systems by Fluigent96	Advanced microfluidic control solutions for precise manipulation of fluids in vaccine development	Supporting high-throughput screening and precise control in vaccine development, focusing on size, concentration, and compartmentalization in microenvironments	The high cost of systems may be prohibitive for smaller research institutions or startups
Aurora TX, Apollo., by Red-ShiftBio97	Utilizes microfluidic modulation spectroscopy (MMS) for de tailed RNA and protein structure analysis with high precision and minimal sample requirements	Providing critical insights into biomolecular structures and stability, enhancing therapeutic development with applications extending beyond traditional spectroscopic techniques	The complexity of data interpretation may require users to have higher technical expertise
NxGen™ microfluidic devices by Cytiva98	Microfluidic mixing technologies to enable large-scale, efficient production of lipid nanoparticles	Production of RNA vaccines through scalable microfluidic techniques, optimizing nanoparticle characteristics for improved drug delivery and efficacy	Scale-up processes face challenges in ensuring uniformity across large batches
Microfluidizer® high shear processors by Microfluidics InteRNAtional Corporation99	Advanced high-shear fluid processors designed for scalable vaccine manufacturing and RNA vaccine delivery systems	Facilitating scalable vaccine production with Microfluidizer® technology, ensuring consistent particle size distribution and efficient nanoemulsion production	The high energy input required may affect sensitive biological materials adversely
NanoGenerator™ synthesis system by Precigenome LLC100	High-performance nanoparticle synthesis system for precise, scalable, and reproducible production of lipid nanoparticles	Enhancing the scalability of nanoparticle production from laboratory to GMP production, with applications in mRNA LNP vaccine development and other precision medicine areas	The requirement for high technical expertise and initial investment may limit accessibility for some labs

These platforms facilitate the rapid synthesis of lipid-encapsulated mRNA vaccines and ensure higher reproducibility and quality control, which are vital for meeting regulatory standards and achieving high efficacy. Furthermore, the ability to swiftly adapt these platforms to emerging pathogens is paramount in the ongoing effort to mitigate public health threats. Therefore, discussing these technologies provides a comprehensive view of current capabilities and sets the stage for future improvements in vaccine technology.

VI. FUTURE PROSPECTS

This review has highlighted the integration of microfluidic technologies in the development and production of mRNA vaccines, highlighting its pivotal innovation, especially evident in the rapid deployment of vaccines during the COVID-19 pandemic. Microfluidics offers significant improvements in the precision, scalability, and efficiency of vaccine production processes, however, challenges remain. The precise control over fluid handling at the microscale minimizes reagent use and reduces waste, enhancing cost-effectiveness and environmental sustainability. Furthermore, the ability of microfluidic devices to generate consistent and reproducible lipid nanoparticles (LNPs) ensures the stability and efficacy of the mRNA vaccine, which is crucial for maintaining vaccine integrity during large-scale production. Despite these advantages, challenges such as upscaling microfluidic production for global vaccine distribution, integrating microfluidic systems into existing pharmaceutical manufacturing frameworks, and the need for skilled personnel pose significant obstacles. Future developments will likely focus on enhancing scalability, integrating with automation, and addressing the environmental impact of microfluidic manufacturing to align with sustainable practices. Beyond mRNA vaccines, microfluidic technologies hold promises for developing personalized medicine, such as vaccines tailored to individual genetic profiles or specific tumor antigens in cancer patients. Addressing current limitations through continued research, regulatory innovation, and training is essential for realizing the full potential of microfluidics in improving public health outcomes, with lessons learned from the rapid development of COVID-19 vaccines serving as a foundation for future advancements in vaccine development and other therapeutic areas.

VII. CONCLUSION

The integration of microfluidic technologies in mRNA vaccine development marks a pivotal advancement in biomedical engineering, revolutionizing traditional vaccine production methods and setting new standards for efficiency, precision, and scalability. This review has detailed the role of microfluidics in synthesizing and purifying mRNA, as well as in the production of lipid nanoparticles (LNPs), demonstrating how these technologies streamline the vaccine development process and enhance the capability for rapid response to emerging health crises. The COVID-19 pandemic, serving as a real-world application, exemplifies how microfluidics can expedite vaccine development, ensuring timely availability and deployment on a global scale. Furthermore, microfluidic systems’ utility extends beyond production to include roles as innovative detection devices, offering robustness and versatility in handling pandemics. However, as we advance, it is crucial to address the challenges associated with scaling these technologies for mass production and integrating them into established manufacturing protocols without compromising quality or efficacy. Moving forward, the continued evolution of microfluidic platforms promises to not only impact public health positively by enhancing vaccine accessibility and response times but also to expand into other areas of medicine, potentially transforming approaches to disease prevention and therapy. This confluence of technology and health underscores a significant shift toward more dynamic and adaptive biomedical solutions, promising a future where microfluidics plays a central role in advancing healthcare technologies.

AUTHOR DECLARATIONS

Conflict of Interest

The authors have no conflicts to disclose.

Author Contributions

Ali Fardoost and Koosha Karimi contributed equally to this paper.

Ali Fardoost: Conceptualization (equal); Investigation (equal); Project administration (equal); Supervision (equal); Writing – original draft (equal); Writing – review & editing (equal). Koosha Karimi: Conceptualization (equal); Investigation (equal); Project administration (equal); Supervision (equal); Writing – original draft (equal); Writing – review & editing (equal). Harshitha Govindaraju: Writing – original draft (supporting). Pegah Jamali: Writing – original draft (supporting). Mehdi Javanmard: Funding acquisition (lead); Supervision (equal).

DATA AVAILABILITY

Data sharing is not applicable to this article as no new data were created or analyzed in this study.