Co-deliveey of Oxygen and Nitric Oxide using a dissolvable Microneedle Array helps in Diabetic Wound healing.

Introduction: Diabetic wounds pose significant healthcare challenges, requiring prolonged healing and facing infection risks. Nitric oxide (NO) and oxygen show promise in diabetic wound healing due to their angiogenic and antibacterial properties. We have developed a GelMA microneedle array patch for simultaneous delivery of NO and oxygen. Mechanical testing confirmed adequate strength for transdermal drug delivery. The patch exhibited sustained release of NO and oxygen over 180 minutes. In vitro studies revealed no significant cytotoxicity of both blank and dual release GelMA microneedles in human dermal fibroblast cell lines, suggesting their potential in diabetic wound management.

Materials and

Methods: GelMA was synthesized using standard protocol7. Similarly, calcium peroxide nanoparticles were synthesized using NH3OH and CaCl2.2H2O8. Nitric oxide releasing agent, S-nitrosoglutathione (GSNO), was purchased from sigma Aldrich.
A visible light crosslinked microneedle array was created by centrifuging GelMA with therapeutic agents, Ruthenium and Sodium persulfate (SPS) into a mold, crosslinking with a torch, then drying and detaching the dissolvable microneedle.
Scanning electron microscopic (SEM) images confirmed the formation of microneedle array. Compression test using dynamic mechanical analyzer (DMA) was conducted to check the skin penetrability by exerting 0-35 N of force at a rate of 0.01 mm/sec. Release studies using Griess reagent (NO detection) and oxygen microsensor were conducted to confirm the presence of GSNO and CPO, as well as to quantify the rate of release of these agents. Finally, Live/Dead assay was utilized to evaluate MNs associated cytotoxicity.

Results, Conclusions, and Discussions: SEM images in Figure 1 show the morphological characteristics of the 10x10 blank GelMA microneedle array-based patch, which distinctly portray the formation of needles, infused with GSNO and CPO NPs for nitric oxide and oxygen delivery. These pyramidal needles, 640µm long and 250µm wide, have sharp tips for seamless insertion into the wound bed.
Compression testing (Figure 2) assessed microneedle array’s strength for skin penetration, showing resilience with forces per needle below 100 mN, remaining elastic. Deformation occurred beyond 100 mN, yet within safe skin penetration parameters (45 mN force required for skin penetration).
Nitric oxide release from GelMA microneedles demonstrated a gradual increase over 180 minutes, reaching 1.7 µM and 3.3 µM for GSNO concentrations of 100 µg/ml and 200 µg/ml (Figure 3A), respectively. Oxygen release showed a peak of 1.8 PPM after 180 minutes, with nearly 50% released within 30 minutes (Figure 3B).
The Live/Dead assay evaluated cytotoxicity of oxygen and nitric oxide releasing GelMA microneedle arrays (Figure 4). GelMA/GSNO-1 and GelMA/GSNO-2 (100 and 200 µg/ml) showed no cytotoxicity. GelMA/CPO-2 (0.5 mg/ml) exhibited significant cytotoxicity, unlike GelMA/CPO-1 (0.25 mg/ml). Interestingly, the co-release microneedle patch formulation, integrating both CPO and GSNO at concentrations of 0.25 mg/ml and 100 or 200 µg/ml respectively (designated as GelMA/GSNO/CPO-1 and GelMA/GSNO/CPO-2), exhibited no cytotoxic effects on the neonatal fibroblast cell line. This finding suggests a potential synergistic effect between CPO and GSNO, mitigating cytotoxicity while maintaining the intended oxygen and nitric oxide release capabilities of the microneedle array patch.
In conclusion, the developed GelMA microneedle patch shows promise for diabetic wound care with strong mechanical strength and controlled nitric oxide and oxygen release. Despite concentration-dependent cytotoxicity observed with Calcium Peroxide (CPO), its integration with S-nitrosoglutathione (GSNO) in a co-release formulation showed no adverse effects. This indicates a possible synergistic effect between CPO and GSNO, highlighting the patch's safety and therapeutic efficacy. These findings underscore the significance of this innovative approach in advancing diabetic wound care, warranting further exploration in clinical settings.

Acknowledgements (Optional): This article was made possible by the NPRP12S-0310–190276, grant funded by the Qatar National Research Fund (a part of the Qatar Foundation).