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Nicotinamide Adenine dinucleotide (NAD+) deficiency have shown to cause pathogenesis of age-related functional decline and diseases. Investigational studies have demonstrated improvements in age-associated pathophysiology and disease conditions. However, invasive methods such as immunohistochemistry, metabolic assays, and PCR currently used to measure cell metabolism render cells unviable and unrecoverable for longitudinal studies and are incompatible with in-vivo dynamic observations. We report a non-invasive optical technique to interrogate the upregulation of NAD+ in keratinocytes (both in-vitro and ex-vivo) upon administration of NMN. Our technique exploits intrinsic autofluorescence of cells and tissues using multiphoton microscopy. Keratinocytes (HaCat cells) was treated with four concentrations of NMN drug (50, 250, 500 and 1000 µg). Using multi-photon microscopy, we demonstrate that fluorescence of the endogenous NADH in the HaCat cells show a decreasing trend in both the average fluorescence lifetime (Tm) and the Free unbound NADH (T1), while we see an increasing trend in the NADH:NAD+ cellular redox ratio, with increasing dosage of NMN administration. A similar trend in the fluorescence of endogenous NADH was also seen in human ex-vivo skin upon delivery of NMN using NMN loaded dissolvable microneedle patches. NMN was successfully incorporated into the dissolvable microneedle patches using the solvent casting method. After successful delivery of 400 – 1600 µg of NMN from the dissolvable microneedle patches through human skin in-vitro (Franz cells), we show a promising, minimally invasive, alternative delivery system for the NAD+ precursor molecule that can enhance patient compliance and therapeutic outcomes.
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