(A-37) Encapsulation and delivery of live probiotic microbes using silk fibroin microneedles

Introduction:: Microneedle patches are an emerging minimally invasive and painless alternative to oral or injection-based drug administration routes. Microneedle patches transdermally deliver a variety of therapeutic substances, utilizing dermal circulation and immune stimulation, while avoiding the harsh conditions of the digestive system. The acidity of the digestive tract decreases medication efficiency and requires large drug doses, resulting in side effects. However, many materials currently used in microneedle patches lack sufficient biocompatibility and appropriate release profiles. Silk from the domesticated silkworm, Bombyx mori, addresses all of these challenges, as it is a bio- and cytocompatible material that can be chemically modified to enhance its affinity for various drug payloads and provide controllable drug release¹. Silk has never been used to deliver a living organism. Living microbes have recently been explored as treatments for a variety of chronic or incurable diseases, but the acidic conditions of the digestive tract render oral probiotics inactive or dead. We hypothesized that silk could transdermally deliver genetically modified (GMO) living microbes. To test this hypothesis, we encapsulated living Lactococcus lactis bacteria that were genetically modified to produce γ-aminobutyric acid (GABA) within silk microneedles. Our novel patch supports the first known microneedle storage and delivery system for living bacteria. GABA-producing L. lactis survived encapsulation and storage in the silk, suggesting that our novel silk microneedle patch can successfully deliver live microbes and probiotics by bypassing the harsh environment of the digestive tract. GMO microbes delivered transdermally may revolutionize therapeutic interventions for many previously untreatable conditions.

Materials and Methods:: Microneedles were fabricated from B. mori silk cocoons as previously described¹. Briefly, raw silk was solubilized and combined with live L. lactis suspended in a 5% weight/volume (w/v) sucrose buffer. Bacteria (9% w/v) and silk (7, 8, 9, and 10% w/v) solutions were combined with gentle mixing and poured into a custom polydimethylsiloxane (PDMS) microneedle mold. The aqueous solution was placed under vacuum to fill the voids of the mold, creating a 1.5 × 1.5 cm² patch with an array of 400 microneedles measuring 700 μm tall and with base and tip diameters of 360 and 17 μm, respectively. The filled molds were centrifuged, frozen at - 80 °C, and lyophilized for 24 hours. The patches were removed from the lyophilizer and stored at - 80 °C until further testing. To assess bacteria viability, the patches were cut into four equal sized pieces and dissolved in phosphate buffered saline (PBS) or lysogeny broth (LB) and grown on standard agar plates for 48 hours to assess bacterial colony formation. Patches were formulated to dissolve readily in LB broth by minimizing the silk crosslinking. Iterations for implantation into ongoing work in murine models will be formulated to dissolve within 3 days.


Results, Conclusions, and Discussions:: The manufacturing process of the silk microneedle patches is shown in Figure 1 A-H. The procedure resulted in consistent microneedle formation (Fig1 I). Scanning electron microscopy (SEM) of the microneedle patches showed rounded microneedle structures with well-formed tips. Bacteria encapsulated in the microneedles survived the encapsulation, freezing, lyophilization, and storage processes, as evidenced by robust bacterial colony formation when patches were dissolved and cultured on agar plates (Fig. 1J). Although very small amounts of bacteria were encapsulated in the patches (40 μL/patch), 64 and 78 colonies formed from one quarter of a patch in the 9% and 10% silk fibroin groups, respectively, resulting in a colony formation rate of roughly 178 colonies per mL of bacterial solution. Ongoing work suggests the GMO L. lactis produce normal amounts of GABA (data not shown).

These results indicate that small amounts of delivered living microbes may be sufficient for producing therapeutic levels of GABA; the precise number of living microbes required is the subject of ongoing work in our labs. The microbes survived being frozen at - 80 °C as well as being put under vacuum during lyophilization. These findings support our hypothesis that silk microneedle patches will facilitate high degrees of probiotic survivability and result in the successful delivery of these living microbes in vivo. Future work will test the microneedle patches in a mouse model of multiple sclerosis (MS), a disease that may respond favorably to GABA treatment (Fig. 1 K). Future GMO living microbes that are harmless to human hosts, such as other GMO versions of L. lactis, may be designed to treat a wide variety of conditions. The utility of silk for the controlled release of microbiotics and probiotics will continue to be evaluated in follow up studies in vivo.

For the first time, we have demonstrated the encapsulation and survival of a living organism within silk microneedles. Silk microneedles have significant and untapped potential as therapeutic platforms for the delivery of living organisms. Silk microneedle patches have multiple potential applications in the delivery of GMO probiotics that target neurotransmitter-based conditions such as MS, PTSD, and stress disorders.


Acknowledgements (Optional): : The authors thank Calvin Jones for assistance with the PDMS molds, and Dr. David Estrada for the use of the lyophilizer.

References (Optional): :

1. 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. 2020, 3 (8), 5375-5382