NAD+ modulation with nicotinamide mononucleotide coated 3D printed microneedle implants†

Abstract

Nicotinamide adenine dinucleotide (NAD⁺) deficiency has been 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 polymerase chain reaction 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 investigate the upregulation of nicotinamide adenine dinucleotide (NAD⁺) in keratinocytes (both in vitro and ex vivo) upon administration of nicotinamide mononucleotide (NMN) coated microneedle (μNDs) implants. Our technique exploits intrinsic autofluorescence of cells and tissues using multiphoton microscopy. Additionally, μND coating formulations to date have been evaluated using fluorescence microscopy to determine the coated amount, often an imprecise correlation between fluorescence intensity and the coated amount on the μND surface. We also show that rheomechanical attributes of the coating formulation (containing two different viscosity enhancers: sucrose and carboxy methyl cellulose) affect the flow mechanics of the coating formulation at micron scale, and thus the amount of drug coated on the μND surface. In vitro keratinocyte cells were investigated with four concentrations of NMN (50, 250, 500 and 1000 μg), and evaluated with time-dependent NMN (500 μg) treatment at 0, 5, 10, 30, 60, 360 and 1460 min. We demonstrate that intracellular keratinocyte fluorescence of the endogenous NADH shows a decreasing trend in both the average fluorescence lifetime (τ_m) and the free unbound NADH (τ₁), with increasing dosage of NMN administration. A similar trend in the average fluorescence lifetime (τ_m) of endogenous NAD(P)H was also seen in mouse ear skin ex vivo skin upon administration of NMN. We show a promising, minimally invasive, alternative delivery system for the NAD⁺ precursor molecule that can enhance patient compliance and therapeutic outcomes.