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.

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.

FIG. 2.

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

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).

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) license.]

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.

FIG. 6.

(a) Different structures used for microfluidic devices. From left to right: T-junction, HFF, SHM, Baffle, and bifurcation. (b) Top view of the staggered herringbone micromixer microfluidic device. (c) The 3D view and dimensions of the channel of SHM. 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.

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) license.]

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).