A microneedle vaccine printer for thermostable COVID-19 mRNA vaccines (2024)

Design, assembly and automation of the MNP device

The MVP was designed with the requirement to integrate and automate the necessary functions to convert ingredients necessary for fabricating MNPs into finished MNPs with minimal operator skill level and in a form factor suitable for transport. The resulting design consisted of three main functions: loading, dispensing and drying. These three functions were organized into the MVP (Extended Data Fig. 3f) and were programmed to function simultaneously. All programming related to MVP was implemented in the EPSON RC 7.0 SPEL programming language. The operator is required to load molds, fill the reservoirs for liquid formulation and polymer backing and unload MNPs.

A key operating function was developed such that, during the dispensing step, the liquid formulation is first dispensed above the MNPʼs mold and then drawn inside the negative mold cavity using vacuum underneath (taking approximately 15 min), and then the loading stage moves to the drying station where its atmosphere is sealed to accelerate the drying time.

The dispensing stage consists of a syringe pump (Harvard PHD 2000) and a robotic arm (Epson T3) holding a pair of nozzles (18-gauge needles for dye solutions, 25-gauge for vaccine printing) connected via tubing to the syringe pump. The motion of the robotic arm was programmed to be synchronized with the syringe pump. The motion of the robotic arm/pump was optimized to maximize the coverage of the MNP mold by the liquid formulation. A dispensing time gap was imposed between each two adjacent MNPs to minimize inter-patch dripping and vaccine waste.

Both processes were based on different sequences of applying vacuum on PDMS molds and dispensing the polymer solution (loaded with dye or biological agents), followed by vacuum drying. In the first process (de-gassing), vacuum was initially applied on empty PDMS molds, followed by dispensing polymer solution within a specific time (~7 min) right after vacuum application, eventually drying the polymer solution with vacuum. Vacuum de-gassing facilitated diffusion of polymer solution into the microneedle cavities in the PDMS mold (Extended Data Fig. 1b and Supplementary Video 1). In the second approach, polymer solution was dispensed on the PDMS mold, and vacuum was subsequently applied both from underneath and on top of the sheets (Extended Data Fig. 1a and Supplementary Video 2) to induce diffusion of polymer but also accelerate the drying time without bubble formation. When drying the solution using vacuum, it is important to maintain the pressure gradient through the PDMS mold by keeping a higher negative pressure below the PDMS sheet. By doing so, the air dissolved in the polymer–vaccine solution is driven downwards (toward the vacuum chamber below the PDMS mold), which avoids bubble formation during drying. Due to simplicity of automation, the second approach was further implemented into the device.

The loading stage was a custom-made 10 × 10 array of MNP molds, which was used to apply vacuum (−1 bar) below the PDMS sheets. The loading stage was fixed to a single-axis motion stage (Thorlabs) allowing transfer between the loading station and the drying station. Before dispensing the formulation, the alignment of the loading station with the dispensing station was calibrated.

The drying stage was a custom-made vacuum chamber that moved vertically via a single-axis motion stage (Thorlabs). The headcover comprised a vacuum connection as well as a desiccant bag (Extended Data Fig. 3c). The vacuum was set to −0.6 bar to accelerate drying. A single vacuum pump was used for both the loading and drying stages. The vacuum pump was directly connected to the loading stage, and a pressure controller was used to decrease the vacuum to −0.6 bar in the headcover. The lower vacuum in the headcover compared to inside the loading stage maintained a negative pressure gradient in the sheet of PDMS molds, avoiding bubble formation in the MNP solutions. Moreover, the pressure controller was used to automatically bring back the drying stage to atmospheric pressure after drying and release the vacuum chamber from the loading stage.

Patch coverage quantification

Patches (n = 100) were dispensed and dried with the MVP to study the effect of liquid formulation on patch coverage caused by different drying patterns on individual MNPs. The liquid formulation included a polymer formulation (PVP, PVA, PVP:PVA 1:1) dissolved at 20% (w/w) in PBS and mixed with dye. Patch coverage was defined as the number of microneedles formed successfully after drying divided by the total number of patches fabricable (100). The patch coverage was quantified individually for each patch under an optical microscope for each formulation. Drop area and circularity were quantified using image analysis after imaging.

Dispensing vaccines and other biological cargos

A two-step dispensing approach was followed for loading vaccine and other biological cargos. In the first step, vaccine solution was aspirated into the dispensing tube through the dispensing needles (25-gauge, BD Biosciences). The liquid formulation in the first step was composed of a certain concentration of the biological agent mixed with PVP:PVA 1:1, 4% (w/w). Approximately 36 ± 6 µl of vaccine solution (or other cargos) was then dispensed using a pair of 1-ml syringes (BD Biosciences) on the center of MNP molds on the dispensing tray. A time gap of 9 s was imposed between dispensing each two adjacent MNPs to minimize vaccine waste during the transfer of the dispensing robot from mold to mold. The backing solution (PVP:PVA 1:1, 20% (w/w)) was aspirated directly to a new dispensing syringe (10 ml-sized, BD Biosciences). An amount 48 ± 6 µl of the backing solution was dispensed in the second stage right on top of the previously dispensed (dried) vaccine spots. Before and after each dispensing round, the needles and the tubes were washed with ethanol (70%) followed by sterile water. In some experiments, a pair of cargos was co-dispensed such that each syringe/needle dispensed a different cargo. The same loading/dispensing procedure as in mono-dispensing was followed in this case, except that each syringe was loaded with a different cargo.

Computer-aided design and illustration

SolidWorks 2021 (Dassault Systèmes) was used for 3D illustrations of the MVP, robotic arm, dispensing station, vacuum devices, negative pressure chamber, sterile environment container, drying rack, conveyer belt and automatic microneedle patch demolding station. The Epson T3 robotic arm drawing was directly downloaded from the official Epson website. The XYZ plotter, drying rack and conveyer belt models were downloaded from GrabCAD. All other parts were custom designed in reference to the actual dimensions of the prototype parts.

Dispensing optimization

A design of experiments (DOE) approach was followed to systemically study the effect of various design parameters on the collective size of the drops, as the output response, dispensed on an individual PDMS array from the robotic nozzle (needle). To this end, an L₁₈ orthogonal array design of experiment (Taguchi DOE) was constructed in software (Minitab). The effect of the following design parameters was studied: (1) attachment of a 0.2-µm filter to the nozzle, (2) needle gauge, (3) dispensing height, (4) dispensing flow rate and (5) polymer viscosity as a function of polymer composition. The projected drop area of total drops at the end of each dispensing was quantified using ImageJ software. Results (n = 3–5) were analyzed in Minitab to find the mean response of drop area as a function of change in these parameters. A schematic illustration of the experimental setup is shown in Extended Data Fig. 3g. The total dispensed volume was considered constant and equal to 200 µl. Different flowrates were achieved by keeping the total dispensed volume constant (200 µl) but varying the dispensing duration.

Microneedle formation quantification

MNPs were printed using the automated MVP and imaged with an optical microscope to determine if microneedles were the full length and sharp (n = 30). Image analysis was used to estimate the needle height.

Scanning electron microscopy

Scanning electron microscopy (SEM) was used to image fabricated microneedles. Samples were initially coated by a thin layer of Au/Pd using a Hummer 6.2 Sputtering System (ANATECH) and then imaged using a JSM-5600LV SEM (JEOL) with an acceleration voltage of 5–10 kV.

PDMS molds fabrication

Steel MNP positives were used to generate negative molds made of PDMS (Sylgard 184, Dow Corning), which was mixed according to the manufacturer’s instructions and cured overnight at 60 °C. To create additional MNP positives, UV-curable Norland Optical Adhesive 61 was filled into the PDMS negative molds using a centrifuge at 3,234g for 1 min, placed in a UV-curing oven at room temperature for 20 min and manually removed.

To create a tray of negative molds, MNP positives were first aligned using a laser-cut 5 × 5 acrylic grid. Then, Norland Optical Adhesive 61 was poured between the individual resin positives replicates, UV cured at room temperature for 20 min and then held at 60 °C overnight. The resulting tray of evenly spaced 5 × 5 positive MNPs (Extended Data Fig. 3d) was used to fabricate a 5 × 5 PDMS sheet of negative molds (Extended Data Fig. 3e). PDMS was used to cover the aligned resin positives and create an additional 1-mm layer of PDMS above the positives. The sheet was leveled, cured overnight at 60 °C and removed using isopropanol to help separate the negative from the positive.

Dissolvable microneedle fabrication

MNPs were fabricated by loading and drying 200 µl of a 20% w/w PVP, PVA or PVP:PVA solution in a PDMS mold. When dye, LNPs, DNA or protein were loaded in the MNP, a two-step loading procedure was used. First, 200 µl of a solution in deionized (DI) water or PBS containing 0.8 mg, 1.6 mg, 2 mg or 8 mg of PVP:PVA and various amounts of LNPs (expressed as encapsulated mRNA mass), protein or DNA were loaded and dried. Then, a variable volume of PVP:PVA 20% w/w in DI water or PBS was used to bring the total MNP mass to 40 mg of PVP:PVA. That polymer solution was loaded and dried to create a thin polymer backing. All MNPs used in in vivo studies were fabricated using manual dispensing unless otherwise specified. All MNPs loaded with LNPs, DNA or protein were fabricated by applying vacuum through the mold using the pattern with lines at −1 bar gauge pressure and applying vacuum in the drying chamber at −0.6 bar gauge pressure.

For the one-step fabrication method, the 40 mg of PVP:PVA was mixed with various amounts of protein and added as one step.

Measurement of loading time

The time needed to load ink into the PDMS negative mold was evaluated by recording the loading with an electronic microscope (n = 3). The microscope was placed on the side of the PMDS mold and focused on the microneedles through the transparent PDMS.

Vacuum de-gassing loading method

Polymer solution was loaded in PDMS molds that were de-gassed under various vacuum values from 0 to −0.5 bar gauge pressure for various amounts of time. The amount of time between de-gassing and loading was varied from 5–10 min to 1 h. MNPs were then dried and removed from the PDMS mold. Optical images of the polymer–dye solution moving into the PDMS mold were acquired 0 min, 2 min, 5 min and 10 min after adding the solution to the mold. From these images, the needle height was estimated using image analysis.

Measurement of viscosity

Polymer solutions (n = 1) were characterized using a TA Instruments Discovery HR-2 Rheometer using a 40-mm parallel plate and a shear rate ramp from 0.01 s⁻¹ to 5,000 s⁻¹.

Protein quantification

Needles of an MNP loaded with 50 µg, 100 µg or 200 µg of BSA and 0.8 mg, 4 mg or 8 mg of PVP:PVA (for two-step tip loading) were cut and dissolved in DI water (n = 6). A bicinchoninic acid assay (BCA) (Thermo Fisher Scientific) was used to quantify the protein loading using the standard procedure for a 96-well plate. An internal standard of PVP and PVA was added to the calibration curve to account for their interference. The amount of PVP and PVA added to the calibration curve was calculated based on the needle volume (0.08 mm³), a density of 1.2 g/cm³ and the number of needles used for BSA quantification (adjusted to the dilution).

DNA quantification

For the loading efficiency comparison between manual and automated fabrication, needles of an MNP loaded with 100 µg of DNA and 1.6 mg of PVP:PVA (two-step tip loading) were cut and dissolved in Tris-EDTA (TE) buffer (n = 8). DNA was quantified using UV absorption at 260 nm and 280 nm using a NanoDrop. Because PVP also absorbs in UV range, the same amount of PVP was added to the calibration curve to account for its contribution. The amount of PVP to add was calculated the same way as described in protein quantification and assuming a 1:1 PVP-to-PVA ratio. This was repeated for the tray position heat map and loading efficiency calculation with DNA, but 10 µg of DNA and 2 mg of PVP:PVA were used per patch.

LNPs synthesis

Purified mRNAs were obtained with CleanCap AG cap, full N1-methyl-pseudouridine substitution and polyadenylated tail (120 A) (TriLink BioTechnologies). Various high-purity lipids were used for LNP synthesis, including 3,6-bis({4-[bis(2-hydroxydodecyl)amino]butyl})piperazine-2,5-dione (cKK-E12, Organix), heptadecane-9-yl 8-((2-hydroxyethyl)(8-nonyloxy)-8-oxoctyl)amino)octanoate (Lipid 5, Organix), 1-2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Avanti), cholesterol (Sigma-Aldrich) and 1-2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (C14-PEG2000, Avanti). LNPs were prepared using procedures previously described^34,35,36. Lipids were dissolved in ethanol at a molar ratio of 35:16:46.5:2.5 cKK-E12:DOPE:cholesterol:C14-PEG2000 or 38.4:12.3:47.4:1.9 Lipid 5:DOPE:cholestorol:C14-PEG2000 when using cKK-E12 or Lipid 5 as the ionizable lipid, respectively. To prepare the LNPs, the ethanoic solution was rapidly added to and mixed with an mRNA solution buffered with citrate at pH 3 at volume ratio 3:1 (aqueous:ethanol). When using cKKe12, the ionizable lipid-to-mRNA weight ratio was set to 10, and the final mRNA concentration was 0.1 mg ml⁻¹. When using Lipid 5, the ionizable lipid-to-mRNA weight ratio was set to 5, and the final mRNA concentration was 0.135 mg ml⁻¹. All nucleic acids were stored at −80 °C and allowed to thaw on ice before use. The LNPs were then dialyzed for at least 2 h in PBS at 4 °C in a 20,000 molecular weight cutoff (MWCO) cassette³⁴. For LNPs in DI water, the solution was dialyzed against DI water for an additional minimum of 2 h at 4 °C. When needed, the LNPs were concentrated on an Amicon filter by centrifuging at 3,000g⁴². All solutions were kept at 4 °C and used within 1 week.

Testing various polymer–vaccine inks for stabilizing dry LNPs

Polymers were dissolved in PBS or DI water at a concentration ranging from 10% to 30% w/w depending on their solubility (n = 4). These solutions were then weighed and mixed with LNP suspension to reach the appropriate polymer-to-mRNA mass ratio. The ink mixture was immediately dried in a LoBind Eppendorf tube in a desiccator under −0.5 bar vacuum. After 24 h drying, the ink pellet was redissolved in PBS and incubated for 10 min at 37 °C. PBS was used to adjust the volume so that 15 µl of dissolved ink contains 50 ng of encapsulated mRNA. This mixture was used to transfect cells. The ionizable lipid used was cKK-E12.

For the in vitro evaluation of MNPs loaded with LNPs, four MNPs were fabricated using the two-step loading, 10 µg of mRNA and 8 mg PVP:PVA for the tip loading. Two MNPs were used to quantify LNP tip loading, and two MNPs were used to transfect HeLa cells. Needles were cut and dissolved in 1 ml of TE buffer for the RNA quantification and in 1 ml of DMEM for the cell transfection. The procedure described in LNP synthesis was used to quantify mRNA, and HeLa cells were transfected as described hereunder. The ionizable lipid used was Lipid 5.

HeLa cells were cultured in high-glucose DMEM with phenol red (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic (Invitrogen). Then, 10,000 cells were seeded in wells of a white 96-well plate in full growth medium. Twenty hours after seeding, 15 µl of fresh LNPs or dissolved formulation, or 50 µl of fresh LNPs or dissolved MNPs, was added to the growth medium. In all cases, 50 ng of encapsulated mRNA was added to each well. Twenty-four hours after transfection, 100 µl of Bright-Glo Luciferase Assay Kit (Promega) reagent was added, and luminescence was measured in the following 2 min using a Tecan Infinite 200 PRO plate reader.

HeLa cell viability was measured in presence of 0.6 mg of polymer formulation and 50 ng of mRNA encapsulated in LNPs made with cKK-e12 as the ionizable lipid. Fifteen microliters of ink were used in each well. Cell viability was measured in a 96-well plate using a CKK8 assay as described by the manufacturer (ab228554).

mRNA spatial distribution in MNP

The MNP was fabricated using the two-step loading, using 10 µg of mRNA and 8 mg PVP:PVA in the first step. Each of the 100 needles of the MNP was manually cut at their base under a microscope and sorted based on their position on the array. RNA was quantified the same way as described in the LNP synthesis section. The ionizable lipid used was Lipid 5.

Particle size analysis of mRNA-LNPs from dissolved MNPs

FLuc mRNA-LNPs were used for all samples. LNPs were analyzed (1) after dialysis to PBS, (2) after concentrating to 320 μg ml⁻¹, (3) after diluting to 200 μg ml⁻¹ in 4% w/w polymer solution to form vaccine ink and (4) after drying to form MNPs. Samples were dissolved in 20 µl, vortexed for 2 s, diluted with an additional 200 µl and mixed. Then, 10 µl of reconstituted mRNA-LNPs was diluted in 490 µl of water, vortexed for 2 s and then analyzed using dynamic light scattering (DLS) on a Zetasizer Nano-NS (Malvern Instruments). Samples (n = 5 per group) were equilibrated for 60 s before measurement. Three technical replicates were performed for each sample.

TEM and cryogenic-TEM

FLuc mRNA-LNPs were used for all samples. LNPs were analyzed (1) after dialysis to PBS (2), after concentrating to 320 μg ml⁻¹ (3), after diluting to 200 μg ml⁻¹ in 4% w/w polymer solution to form vaccine ink and (4) after drying to form MNPs. Dissolved samples were added onto the carbon-coated copper TEM grids and blotted to remove the excess solution. Next, samples were stained using 1% phosphotungstic acid aqueous solution; the excess stain solution was removed; and samples were dried at room temperature before TEM imaging. For cryogenic-TEM imaging, samples were plunge-frozen using a 930 Gatan Cryoplunge 3. All the samples were imaged using a JEOL 2100 FEG microscope at 200-kV acceleration voltage.

Capillary electrophoresis

FLuc mRNA-LNPs were used for all samples. mRNA was analyzed (1) as provided by the manufacturer, (2) after mRNA-LNP synthesis and dialysis to PBS, (3) after diluting to 200 μg ml⁻¹ in 4% w/w polymer solution to form vaccine ink and (4) after drying to form MNPs. Then, 2 µl of dissolved samples was analyzed using an Agilent Femto Pulse using the Ultra Sensitivity RNA Kit (FP-1201). Samples were prepared using TE or TX buffer.

Water content analysis

The water content of the fabricated MNPs at different timepoints and stages of drying was quantified by thermogravimetric analysis (TGA) using a Pyris 1 Thermogravimetric analyzer (PerkinElmer) with a heating rate of 20 °C per minute from 50 °C to 600 °C under nitrogen flow (20 ml min⁻¹) (n = 3). The water content was evaluated by analyzing only the needles of the MNPs placed in ceramic pans and not the backing. The drying rate was estimated using a linear regression of water content measured over time throughout the drying process. All analyses were conducted in triplicate.

Compressive mechanical testing

For compression testing, a single MNP was mounted between compression platens (Instron 2501 Series) and compressed at a rate of 1 mm min⁻¹ using an Instron 5943 with a 500 N load cell (n = 10). The peak force before microneedle failure was reported and the slope of the linear region of initial compression. Microneedle failure mode was determined by imaging the patches after testing.

Microneedle skin penetration and dissolution assessment

MNPs were applied ex vivo on excised pig skin using a mini spring-loaded applicator (Micropoint Technologies) for 2 min, 5 min, 10 min or 30 min (n = 3). Subsequently, the skin samples were fixed into formalin for 48 h and then transferred to 70% ethanol and embedded in paraffin wax. Samples were sectioned and stained with hematoxylin and eosin.

For quantifying microneedle dissolution as a function of application time, microneedle patches were imaged before and after application on excised pig skin using a Leica DFC450. The patches were placed in a transverse manner for imaging using LAS version 4.7 software. Microneedle length was calculated using ImageJ for at least 10 microneedles from each patch, and these measurements were performed on three patches per timepoint.

Luciferase mRNA expression in mice

All animal procedures were approved and performed under the guidelines of the Massachusetts Institute of Technology Committee on Animal Care (1019-061-22). Six- to eight-week-old female C57BL/6 mice (Charles River Laboratories) were used and monitored for safety (n = 5). MNPs were fabricated with mRNA-LNPs encoding for FLuc (L-7010, TriLink BioTechnologies) using the two-step loading method described above. LNP dose and ionizable lipid chemistry were varied, maintaining at least a 100:1 polymer:mRNA mass ratio. Two ionizable lipids that were previously selected for IV or IM mRNA administration methods were studied, respectively: cKK-E12 (ref. ³⁴) and Lipid 5 (ref. ³⁵). MNPs were applied to either footpad with anesthesia. To more fully dissolve the full dose of LNPs carried in an MNP, when a 10 × 10 array was applied, MNPs were divided into two halves and applied consecutively to the same footpad, for 10 min per half. Hand pressure was applied for the first minute, and then the MNP was secured to the footpad using tape for the remaining 9 min. As a positive control, LNPs were administered to the caudal thigh muscle as a 40-µl suspension in PBS at matching doses. The footpad was selected because it has been previously used for IVIS imaging of luminescence after microneedle application³⁷ and because it is a well-studied site for ID administration of vaccines, allowing for isolation of the draining lymph node^62,63.

Twenty-four hours after MNP application, mice were imaged for bioluminescence in an IVIS kinetic imaging system (PerkinElmer). Then, 15 min before imaging, mice were injected intraperitoneally with RediJect D-Luciferin Ultra (PerkinElmer) at 150 mg kg⁻¹. Luminescence was quantified using LivingImage software (PerkinElmer). Comparison between luminescence results must be carefully considered because injection site light absorption, diffusion and depth of delivery can affect the signal measured using IVIS. For the kinetics experiment, imaging was performed at 2 h, 6 h, 24 h, 48 h and 72 h after administration.

RBD expression in HEK293

In total, 89,000 HEK293 cells were seeded per well of a BioCoat Poly-D-Lysine four-well culture slide (354577). The same growth medium as HeLa cells was used. Twenty-four hours after seeding, cells were transfected with 265 ng of mRNA per well from an LNP suspension made with Lipid 5 and encapsulating mRNA encoding for RBD. LNPs dissolved from an MNP were also used to demonstrate bioactivity after loading into MNPs. The same MNP as the ones used in the SARS-CoV-2 vaccination study was used. Needles were cut, dissolved in DMEM and used to transfect cells using the same mRNA mass as the control LNP suspension. The mRNA was quantified by dissolving needles of an independent replicate of the MNP in TE buffer and using the procedure described in LNP synthesis (PVP and PVA were also added as internal standards to the RNA calibration curve of the RiboGreen). Forty-eight hours after seeding, cells were washed two times with PBS at 37 °C. Cells were washed with PBS between all subsequent steps. Cells were left to equilibrate to room temperature for 15 min. Then, 1 ml of Image-iT Fixative Solution (Invitrogen) was added per well (FB002) and left for 15 min. Next, 1 mL of eBioscience Intracellular Fixation and Permeabilization Buffer (Invitrogen) was added and left to incubate for 10 min. Then, 1 ml of BSA 1% (w:v) in PBS was added for 1 h at room temperature. Then, 300 µl of SARS-CoV-2 Spike Protein (RBD) Recombinant Human Monoclonal Antibody (T01KHu, Thermo Fisher Scientific, 703958) diluted 100-fold in PBS was added to each well and left to incubate for 1 h. PBS-Tween was used to wash cells. Next, 300 µl of goat anti-human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, A-11013), diluted 400-fold in PBS was used as the secondary antibody and incubated for 1 h. Then, 300 µl of Rhodamine-Phalloidin (Invitrogen R415) staining diluted 400-fold in BSA 1% was used for cellular cytoskeleton imaging and incubated for 30 min. Cells were finally fixed using one drop of DAPI ProLong (Invitrogen, P36982). Cells were imaged on a confocal microscope (Olympus FV1000).

SARS-CoV-2 MNP vaccination with mRNA

All animal procedures were approved and performed under the guidelines of the Massachusetts Institute of Technology Committee on Animal Care (1019-061-22). Six- to eight-week-old female C57BL/6 mice (Charles River Laboratories) were used and monitored for safety (n = 5). MNPs were fabricated with human codon-optimized mRNA-LNPs encoding for SARS-CoV-2 RBD and a T4 trimerization motif, using the two-step loading method described above. Smaller MNP arrays were fabricated containing 1.5 µg of encapsulated mRNA, which was verified by mRNA quantification. Four MNPs with approximately 12 microneedles were applied to the left and right footpad of mice with anesthesia, with a 10-min application time for each, for a total encapsulated mRNA dose of 6.0 µg per mouse. Hand pressure was applied for the first minute, and then the MNP was secured to the footpad using tape for the remaining 9 min. As a positive control, LNPs were administered to the right caudal thigh muscle as a 40-µl suspension in PBS at 10.0 µg of encapsulated mRNA per mouse.

mRNA stability evaluation in mice

All animal procedures were approved and performed under the guidelines of the Massachusetts Institute of Technology Committee on Animal Care (1019-061-22). MNPs were fabricated with FLuc mRNA LNPs using the two-step loading method described above, at an mRNA dose of 1.0 µg per patch with a 500:1 polymer:mRNA mass ratio. Lipid 5 was used as the ionizable lipid. MNP size and application are identical to the above studies of fLuc expression (n = 5). MNPs were stored in a container with silica desiccant at various temperatures. As a positive control, sealed vials of suspension containing the same amount of fLuc mRNA in LNPs made with Lipid 5 were stored at various temperatures alongside MNPs.

SARS-CoV-2 anti-RBD binding titers

A SARS-CoV-2 Spike S1-RBD ELISA detection kit was used (L00845, GenScript). Plates were coated with purified recombinant SARS-CoV-2 Spike S1-RBD antigen. Plates were incubated with serial dilutions of heat-inactivated sera and incubated at 37 °C for 30 min. A 1:10,000 dilution of rabbit anti-mouse IgG-horseradish peroxidase conjugate (Abcam, ab6728) was used as a secondary antibody, and 3,5,3′,5′-tetramethylbenzidine (TMB) was used as a substrate. Interpolated endpoint titers were calculated as the dilution that emitted an optical density exceeding 3× background produced by serum from naive mice.

Electrochemiluminescence assay

ECLA plates (Meso Scale Discovery SARS-CoV-2 IgG, N05CA-1, Panel 7) were designed and produced with four antigen spots in each well. Antigens included were WA1/2020, B.1.1.7, P.1 and B.1.351 S1-RBD. Plates were blocked with 50 µl of 1% BSA solution for at least 30 min at room temperature with shaking at 700 r.p.m. During blocking, the serum was diluted 1:5,000 with Diluent 100 (Meso Scale Discovery). The plates were washed with 150 µl of wash buffer and blotted dry, and 50 µl of diluted samples was added in duplicate to the plates. The samples were incubated at room temperature with shaking at 700 r.p.m. for 2 h. Secondary antibody was prepared using Jackson ImmunoResearch rabbit anti-mouse IgG detection antibody (315-005-045) conjugated to the MSD GOLD SULFO-TAG by NHS-Ester chemistry per the manufacturer’s guidelines (R91AO-1). Plates were again washed three times, and 50 µl of SULFO-tagged anti-mouse IgG detection antibody diluted to 1× in Diluent 100 was added to each well and incubated for 1 h with 700 r.p.m. shaking. Plates were washed three times; 150 µl of MSD GOLD Read Buffer B was added to each well; and the plates were read on a MESO Quick-Plex SQ 120. MSD titers for each sample were reported as relative light units (RLU), which were calculated as sample minus blank for each spot and sample. The limit of detection was defined as 500 RLU for all assays.

Shelf-life study with SARS-CoV-2 mRNA MNPs

MNPs containing 1.5 μg of encapsulated SARS-CoV-2 mRNA were stored in a container with silica desiccant at various temperatures. As a positive control, sealed vials of suspension containing SARS-CoV-2 mRNA-LNPs at 200 µg ml⁻¹ were stored at various temperatures alongside MNPs. Six- to eight-week-old female BALB/c mice (Charles River Laboratories) were used and monitored for safety (n = 5). All mice received a prime dose of 10 µg of encapsulated mRNA in mRNA-LNPs administered to the right caudal thigh muscle as a 40-µl suspension in PBS. Mice were then boosted with various materials, including fresh controls and patches or suspension stored for 1 month or 3 months. For MNP groups, as in previous experiments, four MNPs with 12 microneedles were applied to the left and right footpad of mice with anesthesia, with a 10-min application time for each, for an estimated total mRNA dose of 6.0 µg per mouse. For IM groups, 4 μg of encapsulated mRNA in mRNA-LNPs was administered to the right caudal thigh muscle as a 40-µl suspension in PBS.

Statistical analyses

Statistical analyses were performed using GraphPad Prism software. P values are represented by: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.