(T1330-05-26) Transdermal Delivery of Nicotinamide Mononucleotide Using Dissolvable Microneedles as a Possible Treatment for Aging-Related Diseases

Purpose: Nicotinamide adenine dinucleotide (NAD) is a vital cellular metabolic redox cofactor which plays an important role in various biological enzymatic reactions. By regulating NAD⁺ sensing enzymes, NAD⁺ controls key cellular metabolic processes, from energy metabolism to cell survival, increase or reduced levels of NAD⁺. Evidence from multiple surveillance studies show that NAD⁺ levels steadily decline with age, resulting in altered cellular metabolism and increased disease susceptibility, causing neurodegenerative diseases such as arthritis and Alzheimer. Among the NAD⁺ precursors, nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN) are two promising metabolites that have shown fewer unfavourable side effects. Even though they are available in oral doses, their effect was shown to be variable or no effect when compared to intraperitoneal injection. Therefore, the objective of this study was the controlled transdermal delivery of NAD+ precursor molecule NMN using dissolvable microneedles (µNDs)

Methods: NMN-µNDs was fabricated using micro-moulding technique, from female moulds with negative 392 conical shape indentations on poly-dimethyl siloxane polymer. 25% (w/v) Methyl Vinyl ether Maleic acid (PMVE/MA) and 6 NMN load (0.039, 0.0856, 0.1113, 0.3707, 0.5092 and 1.0390 mg) were used to make the drug polymer matrix. Following NMN-µND fabrication using mould casting, µND’s dimensional analysis was characterised using light microscopy. The µND mechanical properties assessed using a texture analyser, whereby the µND arrays were subjected to pressures of up to 35N against a metallic block. We also assessed µNDs insertion on in-vitro human skin at controlled pressure (35N), embedding Rose Bengal as a model dye into the µND polymer matrix, to visualise the embedded molecule skin diffusion using confocal laser scanning microscopy (n=3), and the insertion depth visualised using scanning electron microscopy (n=3). Fourier transform Infra-Red (FTIR) was performed to confirm the interaction between NMN and polymer matrix. In-vitro release test was performed to study the drug release kinetics at 37C, NMN-µNDs wrapped in snakeskin membrane and dipped in PBS, replenishing PBS every two minutes for 1 hr. In-vitro permeation test on dermatomed human skin (thickness 500µm) using a flow-through system was performed to study the drug permeation kinetics in skin for 24 hr (n=3, only arrays containing 0.3707, 0.5092 and 1.0390 mg NMN load/array). Additionally, NAD+ synthesis in rat ex-vivo skin as a result of skin treatment with NMN- µND array (0.5092 and 1.032 mg NMN load/array, n=3) was performed using a fluorometric assay kit (NAD+/NADH detection). Further to this, using a stereolithography 3D printer, we fabricated µND mould with high accuracy and reproducibility as an alternative fabrication technique to existing mould fabrication technique such as laser drilling.

Results: Following NMN- µND array fabrication using mould casting, the NMN µNDs arrays consisted of µNDs shaft height of 606.75 ± 37.25 µm, and a base diameter of 369.25 ± 52.75 µm, yielding an aspect ratio (height: base diameter) of approximately 2:1 (n=92 µNDs). The µND's mechanical properties were assessed using a texture analyser, being subjected to pressures of up to 35N causing a µND tip height displacement/buckling of 270µm (n=9). We also assessed µNDs insertion on in-vitro human skin at controlled pressure (20 and 35N) and demonstrated that our dissolvable µNDs pierced the stratum corneum and into the dermis, causing a puncture at a depth of 100 ± 48 µm (n=3), confirmed using scanning electron microscopy. While confocal laser scanning microscopy confirmed the presence of Rose Bengal dye at 75 µm into the human in-vitro skin. FTIR studies confirmed the successful incorporation of NMN into the polymer matrix of the dissolvable µNDs. Our in-vitro release test show that at higher NMN load (0.3707, 0.5092 µg and 1.039 mg) per µND array, approximately 80% of NMN was released from the polymer matrix. While our in-vitro permeation test on dermatomed human skin, the NMN- µND array demonstrated 208 ± 45 µg (n=3) from 0.5092 mg NMN per µND array and 400 ± 15.3 µg (n=3) permeation from 1.039 mg NMN per µND array over 24h. Additionally, after treating rat ex-vivo skin with NMN- µND array (1.039 mg NMN load, n=3), we have demonstrated the synthesis of NAD+ from the application of 1.039 mg NMN-µNDs array when compared to negative control (µNDs without NMN load). Finally, we have successfully fabricated µND moulds with increased accuracy and reproducibility with height of 1011 ± 25.90 µm (n=19) and tip diameter of 53.45 ± 13.98 µm (n=19), demonstrating facile and non-expensive technique for µND fabrication.

Conclusion: Our studies demonstrate that dissolvable µNDs have the capability to puncture human skin, delivery embedded NMN molecule into the skin, and illicit NAD+ synthesis in rat skin. Therefore, dissolvable µND arrays can serve as a potential alternative form of NMN delivery. Further to this, we were also successful in the facile fabrication of high aspect ratio µNDs mould using 3D printing. Future work will assess delivery of NMN in µNDs array in-vivo.


Graphical presentation of nicotinamide mononucleotide embedded in dissolvable microneedle and it's pharmacodynamics within skin fauna