Development of a microneedle patch for delivery of mRNA-lipid nanoparticles

Abstract

mRNA delivered by microneedle patch (MNP) can enable painless delivery, reduced need for healthcare expertise, and improved thermostability. In this study, we investigated formulation and manufacturing approaches for developing MNPs that deliver mRNA-loaded lipid nanoparticles (LNPs) encoding luciferase as a reporter protein during MNP fabrication and storage, including mRNA-LNP concentration, formulation, pH, excipients, and backing material. MNPs were assessed for mRNA-LNP size, encapsulation efficiency, and protein expression in vitro and in vivo. MNPs fabricated with mRNA-LNPs initially prepared at a higher concentration yielded superior expression compared to mRNA-LNP concentration by centrifugation or tangential flow filtration. Acidic pH during MNP manufacturing enabled greater expression in vitro. However, no such correlation was observed in vivo. Polyvinyl alcohol (PVA) best stabilized mRNA-LNPs during the MNP manufacturing process amongst the tested polymers. Incorporating sugars in MNPs did not further improve stability. Low temperature drying (5 °C) preserved mRNA functionality better compared to drying at 25 °C and 40 °C. Though there was significant activity loss initially (87% loss in 2 days at 40 °C), mRNA expression was stabilized for extended subsequent periods even at accelerated conditions (10% additional loss after 28 days at 40 °C). Our systematic approach identified key parameters for successful formulation and manufacturing approaches to incorporate mRNA-LNPs into MNPs, which could expand access to mRNA-based medical interventions.

Introduction

mRNA emerged as a powerful technology with uses in vaccination and genetic therapy. Compared to other types of vaccines and biologics, mRNA design and manufacturing requires fewer steps, enabling rapid and cost-effective production of novel mRNA vaccines [1,2,3] and therapeutics, particularly in cancer treatment [4,5,6]. Despite these advantages, therapies using naked mRNA are limited by low transfection efficiency, degradation, and non-durable immune responses [7, 8]. Several delivery vehicles have been developed for mRNA to address these limitations, including cationic polymers, cell-penetrating peptides, liposomes, lipopolyplexes, cationic LNPs, and ionizable LNPs [9, 10]. Amongst the various platforms, the ionizable LNPs have demonstrated superior transfection efficiency, endosomal escape, and mRNA stability [11, 12].

Ionizable lipid nanoparticles (LNPs) are nanoparticles containing an ionizable lipid, cholesterol, a phospholipid, and a PEGylated lipid [13, 14]. The crucial component, ionizable lipid, is cationic at acidic pH and binds to negatively charged mRNA [15, 16]. Several ionizable mRNA-LNP products have received USFDA-approval, including patisiran (Onpattro®) for the treatment of hereditary transthyretin-mediated amyloidosis and BNT162b2 (Comirnaty®) and mRNA-1273 (Spikevax®) vaccines against SARS-CoV-2 [17]. mRNA vaccines gained significant scientific interest in the wake of the COVID-19 pandemic.

Microneedle patches (MNPs) contain micron-scale needle arrays that can be used to deliver drugs or vaccines into the skin [18,19,20,21]. Dissolvable MNPs contain drugs/vaccines embedded in a matrix made of water-soluble polymers and other excipients that dissolve upon application to the skin and release their cargo [22]. MNP technology possesses distinct advantages, including minimally invasive delivery, painless delivery, elimination of sharps waste, and simplified administration with minimal training [23,24,25]. Finally, as a dried formulation, MNPs can increase the thermostability of drugs and vaccines [26, 27]. However, MNP development has challenges. For example, dissolvable MNPs have a limited capacity and are not well-suited for drugs that require a large dose (e.g., > 10 mg) [28]. In addition, the MNP manufacturing process involves air-drying, which could be detrimental to fragile biomolecules, like mRNA. The development of a MNP for delivering mRNA-LNPs would need to overcome these challenges.

MNP technology that could deliver mRNA vaccines would be particularly useful in a pandemic scenario, when there is a need for a low-cost, thermostable, and easy-to-administer delivery method [29]. Additionally, as more mRNA technologies are developed, advancement of delivery methods such as MNPs will enable faster introduction and a wider reach of these new vaccines and therapeutics. Since the structure of mRNA molecules is relatively consistent, basic research on MNPs for mRNA delivery should be broadly applicable to mRNA products. While mRNA can encode any protein, it may still be stabilized by similar formulations, unlike protein-based drugs that may require different formulations for each product. However, the stability profile may vary depending on the length of the mRNA and specific modifications made during synthesis [30].

There has been limited work on delivering mRNA via MNP. In 2018, Koh et al. delivered naked mRNA in a dissolvable MNP [31]. In 2022, Yu et al. incorporated transfection agents, including polyethyleneimine and liposomes, into a cryomicroneedle for mRNA delivery [32]. In 2023 and 2025, Jaklenec et al. delivered mRNA-LNPs via a dissolvable MNP [33, 34]. In 2024, Rajesh et al. delivered liquid mRNA-LNPs via a lattice MNP [35]. Despite these initial demonstrations of mRNA delivery by MNP, there remains a need to broadly focus on the factors that affect mRNA-LNP stability in a dissolvable MNP in terms of formulation, manufacturing, and resulting properties, such as mRNA-LNP size, mRNA encapsulation efficiency, and reporter protein expression in vitro and in vivo. This study aims to systematically investigate these attributes of a mRNA-LNP MNP, identifying factors that influence mRNA-LNP characteristics and mRNA expression to guide continued development of MNP products for mRNA-LNP delivery.

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LNP fabrication

SM-102 ionizable lipid was obtained from BroadPharm (San Diego, CA). Cholesterol was obtained from Sigma-Aldrich (St. Louis, MO). 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) were obtained from Avanti Lipids (Alabaster, AL). LNPs were fabricated using a microfluidic chip as described previously [36]. Briefly, SM-102, cholesterol, DSPC, and DMG-PEG 2000 were dissolved in ethanol at a molar ratio 50:38.5:10:1.5, respectively, to make the ethanol phase. The aqueous phase consisted of PolyA/mRNA in 20 mM citrate buffer (100 mM, pH 3.0, Teknova, Hollister, CA diluted to 20 mM with RNase-free water). The SM-102:mRNA mass ratio was maintained at 15:1 or 20:1. The aqueous phase and ethanol phase were combined in a 3:1 flow rate ratio in a microfluidic chip, described previously [37]. For precise control of the flow rates, Pump 11 Elite syringe pumps (Harvard Apparatus, Holliston, MA) were used with FlowControl software (Harvard Apparatus).

Sakers, S.H., Reddy, B.P.K., Fiduccia, G. et al. Development of a microneedle patch for delivery of mRNA-lipid nanoparticles. Drug Deliv. and Transl. Res. (2025). https://doi.org/10.1007/s13346-025-01964-z