Extended Data Fig. 7: Safety evaluation of QD-PMMA loaded MNPs.

(a) The cytotoxicity of the OPMR dye (QD-PMMA) was analyzed using HeLa cells. (n = 3, S.D., cytotoxicity test performed once.) When different concentrations of QD-PMMA microparticles were incubated for 20 hours, there was no effect on the cell growth % (b) The cytotoxicity of the QD-PMMA was also analyzed using adult human dermal fibroblasts that were incubated with QD-PMMA for 20 hours and assessed for toxicity via: Live/Dead assay, which uses green-fluorescent calcein-AM to indicate live cells and red-fluorescent ethidium homodimer-1 to stain dead cells, and the CCK-8 assay, which quantifies the metabolic activity of viable cells. (Scale bar=100 µm) (c) All QD-PMMA concentrations exhibited cell viability above 85%, indicating that the QD-PMMA microparticles are not cytotoxic, n = 5, S.D. (d) Grading criteria used for microscopic lesions seen in histopathological pig skins after MNP applications. (e) Skin sections were retrieved 3, 30, and 70 days post OPMR-MNP application and were processed and stained with hematoxylin and eosin for biocompatibility analyses for histopathological evaluation, showing mild to moderate subacute inflammation in the superficial dermis at day 3 post-application, and this dermatitis was minimal at day 30 and 70 post-application. At day 3 post-application, the dermis at the sites of microneedle injections was often infiltrated by histocytes, eosinophils, neutrophils, and occasional multinucleated foreign body giant cells, scattered around blood vessels. The epidermis at the sites of injections was slightly hyperplastic and/ or hyperkeratotic, containing nucleated keratin flakes, necrotic cellular debris (depicted at day 30 post-application). Groups: untreated skin, 3 days, 30 days, n = 3 biological replicates (samples from 3 different pigs), group 70 days, n = 4 biological replicates (samples from 4 different pigs), 6 tissue samples per biological replicate. (f) Quantification of CC3 staining of pig tissue with no treatment, with MNP applications loaded with just PVA/PVP polymer, blank PMMA microparticles, and QD-PMMA microparticles were applied 3 days prior to skin excision. To study if the OPMR system activates cell apoptotic mechanisms, tissue samples were stained with cleaved caspase-3 (CC3). And quantitative analysis showed no differences in the apoptotic cell % between the control and experimental groups, indicating there was no signal of immunoreactivity in the skin sections. n = 4 biological replicates (samples from 4 different pigs), 6 tissue samples per biological replicate, S.D. (g) To confirm the QD clearance from skin tissue, the zinc (Zn) content in pig skin 7 days and 70 days post OPMR-MNP application was evaluated using inductively coupled plasma optical emission spectrometer (ICP-OES, Agilent ICP-OES 5100 VDV) analysis after excising the tissue and dissolving it in Aqua Regia medium (nitric acid: hydrochloric acid 1:3). As a result, we observed 41 (±12.77) % of QD clearance at 70 days post-application as shown below. This reduction in the measured Zn content was conforming with the amount of signal reduction of the applied patch, which suggests the signal decrease over time is attributed to the QD clearance from the skin tissue. n = 3, biological, S.D.