(M1430-03-19) Antimicrobial and Angiogenic Silver Nanoparticles and Verapamil Double-Layered Electrospun/3D Printed Wound Healing Patch for Accelerated Wound Healing

Purpose: >The skin is considered to be the largest organ that act as the protecting reservoir for the anterior organs and tissues of the human body from the external environment including microbial infections. When skin is damaged or lost due to accidents, burns, surgeries, or wounds, the patient is highly susceptible to microbial infections that can dramatically progress into sepsis, gangrene or limb amputation^1,2. Accordingly, development of antimicrobial and drug-eluting wound healing patches that can act as a single-dose wound dressing represents a promising approach to in the prevention of microbial infections and treatment of complicated wounds through enhancing tissue regeneration rate while preventing microbial contamination.

Methods: The wound patch was designed to include two successive layers; each performing a specific role in wound treatment. The first layer adhering to the wound was designed to act as a mucoadhesive layer and angiogenic to help in increasing blood supply to the wound to propagate cell regeneration. First, verapamil loaded chitosan nanoparticles were successfully prepared using ionic gelation technique where glutamic acid was used as a crosslinking agent to formulate chitosan nanoparticles. The optimization of the nanoparticles was achieved by the manipulation of the medium pH with the aim of preparing the smallest size and polydispersity index while keeping the highest possible surface charge as well as entrapment efficiency. Once optimized, the nanoparticles were incorporated into PLLA-based nanofiberous matrices using electrospinning technique. The second layer was prepared through extrusion of silver sulfadiazine with PLLA pellets to prepare in situ reduced silver nanoparticles loaded PLLA filament which was then used in developing 3D printed layer using an FDM 3D printer. The second layer contained reduced silver nanoparticles that render the layer an antimicrobial cover for the wound. Finally, cell viability assay has been carried out to estimate the cell proliferation capability in the abundance of the wound healing patch.

Results: TEM images shown in Figure 1a confirmed the successful formation of verapamil loaded chitosan nanoparticles as spherical nanoparticles possessing as size range of 150 - 200 nm. This is slightly smaller than the size estimated by dynamic light scattering (DLS) technique owing to the hydrodynamic size attributing to the high swellability of the developed chitosan nanoparticles in aqueous solution. Furthermore, pH was found to play a major role in increasing the entrapment efficiency of verapamil within the developed chitosan nanoparticle. It has been noticed that slightly alkaline medium increases ionization of glutamic acid increasing its capability to crosslink chitosan chains in a more effective way to form nanoparticles, thus increasing entrapment efficiency. For instance, a pH of 6.8 led to an increase of the entrapment efficiency up to 85%. Finally, optimized nanoparticles were incorporated into PLLA nanofiberous matrices as shown in Figure 1b. Silver nanoparticles loaded PLLA filament has been successfully prepared through extrusion as shown in Figure 2a, where situ reduction of silver sulfadiazine into silver nanoparticles was accomplished within the fabrication process. Afterwards, the fabricated filaments have been used through FDM 3D printer to fabricate 3D printed layers with controlled shape and thickness as shown in Figure 2b. This layer was developed to be a supportive antimicrobial layer od the wound patch. Finally, cell viability assay showed the high biocompatibility towards human normal fibroblasts. Furthermore, it has been observed as demonstrated in Figure 3 that loaded wound patches led to noticeable increase of cell viability compared to the control and the unloaded patches, which confirms the ability of eth fabricated silver nanoparticles/verapamil wound patches to stimulate wound healing and tissue regeneration.

Conclusion: Wound patches developed from layers of PLLA based nanofibers matrices incorporating verapamil loaded chitosan glutamic acid nanoparticles as well as silver nanoparticles loaded 3D printed layers have been developed successfully and proven their efficiency as single dose patch for efficient wound healing.

References: (1) Ali IH, Khalil IA, and El-Sherbiny IM. ACS Appl Mater & Interface. 2016:8: 14453-14469.
(2) Ali IH, Khalil IA, and El-Sherbiny IM. Scientific Reports. 2023: 13(1): 3215.

Acknowledgements:This work was carried out in Nanomedicine Laboratory, Faculty of Pharmacy, University of Sadat City.

Figure 1. a) TEM of Verapamil Loaded Chitosan Glutamic Acid Nanoparticles, and b) SEM image of PLLA based Nanofibers

Figure 2. a) Silver nanoparticles Loaded PLLA Filament, and b) Silver Nanoparticles Loaded 3D Printed Layer.

Figure 3. Cell Viability and Proliferation of the Developed Wound Patches