Extended Data Fig. 9: Characterization of SARS-CoV-2 MNP.

(a) Schematic of SARS-CoV-2 RBD sequence of the mRNA constructs used in this paper. The signal sequence (SS) is colored orange, RBD domain is colored blue, and the T4 trimerization motif is colored green. (b-c) LNPs encapsulating mRNA that encodes for fLuc were mixed with various water-soluble polymers and dried. LNPs were either dialyzed in PBS or DI water before their mixing with the polymer matrix. Protein expression in HeLa cells was measured after transfection with re-dissolved polymer to screen for a combination of polymers that produces protein expression comparable to LNPs in suspension (n = 4 technical replicates). (d-E) Droplet size is critical for high vaccine loading efficiency. Due to the Marangoni effect, a larger droplet size (d) has lower loading efficiency than a small droplet (e) dispensed in the center of a mold. (f) HEK cells transfected with LNP suspension made with Lipid 5 and encapsulating mRNA encoding for RBD. LNPs encapsulating mRNA encoding for RBD were dissolved from a MNP and added to demonstrate bioactivity after loading into MNPs. RBD is shown in green (Alexa Fluor 488 used as secondary antibody), actin is shown in red (rhodamine phalloidin) and nucleus in blue (DAPI). (g) MNP tip-loading (h) and loading efficiency of different mRNA-LNP constructs used for in vivo studies (n = 3 independent samples). Four MNPs were administered for in vivo studies at 1.5 μg per patch to yield a theoretical 6 μg dose. (i) Of the 6 μg mRNA used for MNP fabrication, we assessed the percent mRNA recovered in various regions of the MNP. Approximately 25% were found in the microneedles, matching the loading efficiency measurement, and the remaining mRNA was found in the backing. The total reported is the sum of needles and backing, showing that all mRNA was accounted for in the MNP (n = 5 independent samples). Data represent mean ± s.d.