Silk Fibroin Microneedle Patches: A Cheap, Modular Drug Delivery System for Uncommon and Challenging Therapeutics

Introduction: Microneedle patches (MNs) are an effective, minimally invasive and painless alternative to many other drug administration routes such as injections or oral administrations. These patches are theoretically able to transdermally deliver a variety of therapeutic substances utilizing dermal circulation while avoiding the harsh conditions present throughout the digestive process (Fig. 1A). However, current silk MNs cannot penetrate human skin or human skin analogues, as they have only been tested on rat or mouse epidermis. As silk can stabilize hydrophobic molecules and undergo a wide variety of potential chemical modifications, it is an ideal biomaterial for delivering biologics that is currently underutilized as its mechanical properties do not support human skin penetration1. In an effort to expand the potential of silk MNs, we developed silk fibroin microneedle film patches capable of transdermally delivering an arctic fish derived Antifreeze Protein (AFPs), a small peptide that prevents ice crystallization, into porcine skin, which is equivalent to human skin in terms of thickness and mechanical properties. We chose AFPs as they have emerging applications in extending solid transplant organ shelf-life, towards addressing the global transplant organ shortage. We hypothesized that reducing water content and increasing silk weight by volume during manufacturing would make the patches strong enough to penetrate pig skin.

Materials and

Methods: Microneedles were fabricated from Bombyx mori silk cocoons as previously described2. Raw silk was solubilized and combined with arctic fish derived Antifreeze Proteins (AFP) (0 to 500μg) to form silk (10% w/v) AFP solutions 3. These solutions were then degassed and centrifuged into custom polydimethylsiloxane (PDMS) microneedle mold using a novel optimized protocol and custom 3d printed adaptors. Once degassed, the filled molds were allowed to air dry in a fume hood creating a shelf stable a 1.7 × 1.7 cm2 patch with an array of 400 microneedles measuring 620 μm tall and with base and tip diameters of 360 and 17 μm, respectively (Fig 1B.). Patches were evaluated for their delivery potential and mechanical properties. A Cellscale Univert compression testing device and a custom bioreactors system was used to evaluate these patches' mechanical properties. The silk fibroin microneedle film patches loaded with green fluorescent protein tagged AFPs were compressed into porcine dermal tissue using custom 3D printed clamps to observe the transfer of the therapeutic into skin and evaluate its effectiveness using fluorescence imaging.

Results, Conclusions, and Discussions: The optimized manufacturing process of these silk microneedle patches resulted in vastly improved patch quality and proper microneedle formation validated through Scanning Electron Microscopy (SEM) (Fig. 1C) and Digital imaging analysis via a Keyence VHX 7000 Digital Microscope (Fig. 1D). Additional quantification of the microneedles mechanical properties found the Young's modulus to be 278 kPA and concluded these devices were capable of piercing human epidermal tissue through validation using porcine skin as a tougher analog along with visual confirmation (Fig. 1E). Patches were able to dissolve in water saturated environments releasing an aqueous solution of silk and AFPs (Fig. 1F). Additionally, fluorescent imaging revealed these AFP-loaded SF patches were able to deposit and transfer AFPs into the epidermal layer even without an active immunomodulatory response or vascular transport (Fig. 1G). To our knowledge, our MNs are the first silk transdermal device shown to penetrate a human skin analogue and deposit meaningful quantities of encapsulated small molecules.

Silk microneedles have significant potential as therapeutic platforms for the delivery of challenging therapeutics such as small peptides. Silk microneedle patches and their potential as an alternative drug delivery system need to be further explored with other therapeutics, such as small molecule inhibitors. Ongoing work is exploring the delivery of hydrophobic substances as well as live genetically modified microbiotics to better judge the its effectiveness and versatility. Additionally, the effectiveness of these AFP-loaded SF MN patches will be evaluated in tissue for their ability to prevent frost damage. The initial successful transfer of AFPs into porcine skin supports further development of these promising drug delivery system to effectively deliver bioactive doses of therapeutics with challenging biochemical characteristics.

Acknowledgements (Optional): References.
1. Vaishya, R., Khurana, V., Patel, S. & Mitra, A. K. Long-term delivery of protein therapeutics. Expert Opin. Drug Deliv. 12, 415–440 (2015).
2. Yavuz, B. Chambre, L. Harrington, K. Kluge, J. Valenti, L. and Kaplan, D. Silk Fibroin Microneedle Patches for the Sustained Release of Levonorgestrel. ACS Applied Bio Materials. 3 (8), 5375-5382 (2020).
3. Meister, K., DeVries, A. L., Bakker, H. J., & Drori, R. Antifreeze Glycoproteins Bind Irreversibly to Ice. Journal of the American Chemical Society, 140(30) (2018).