N17 - Antibiotic-loaded Extracellular Vesicles from Staphylococcus aureus Biofilm as "Trojan-Horse" Antimicrobial Agents

Introduction: : Biomaterials have become a popular alternative in bone repair, however the formation of bacterial biofilms on implants pose a significant barrier due to aggressive pathogenesis, which halts regeneration and prolongs healing time. Biofilms are difficult to penetrate with traditional antibiotics, therefore we need a new drug delivery method to treat these types of infections. Extracellular vesicles (EVs) are nanometer sized, lipid bilayer vesicles produced by all cell types, and are essential cell-cell signaling and have been implicated in biofilm formation. EVs isolated from mesenchymal stem cells (MSC) have previously been studied as drug carriers, but EVs derived from bacteria liquid culture (liquid-EVs) or the biofilm microenvironment (biofilm-EVs) have not been thoroughly assessed. Since EVs are decorated with surface markers of their "origin cell", we hypothesize that biofilm-EVs loaded with antibiotics will most effectively penetrate the biofilm environment and deliver antibiotics, preventing infection of biomaterials.

Materials and

Methods: : Extracellular vesicles were isolated from conditioned cell medium and extracellular matrix of mesenchymal stem cells (MSC liquid-EV, MSC matrix-EV, respectively) and conditioned cell medium and biofilm of Staphylococcus aureus (Bacteria liquid-EV, Bacteria matrix-EV, respectively). EVs were isolated using ultracentrifugation and confirmed by Nanoparticle Tracking Analysis, protein quantification, and transmission electron microscopy prior to downstream applications. To determine the impact of EVs on MSC health prior to loading, MSCs were dosed with EVs (1 x 10^4 - 1 x 10^8 total particles) and after 24hrs an Alamarlue assay quantified final cell count. To determine the impact of EV on bacterial health, planktonic bacteria were dosed with EVs (1 x 10^4 total particles - 1 x 10^7 total particles) and CFU/mL were quantified at various time points (0, 2, 23 hours). We incorporate antibiotics into EVs using sonication, electroporation, direct incubation, and freeze-thaw methods,. We then add antibiotic-loaded EVs to MSC and bacteria and evaluate their response as stated above.

Results, Conclusions, and Discussions::

Results: All EV samples had average particle sizes between 30 - 200 nm, and confirmed EV morphology following TEM. Statistical analysis of EV isolated from bacteria had no significant difference on MSC viability compared to our no EV control, highlighting that bacterial-derived EVs at 1 x 10^8 total particles are potentially non-toxic to human cells (Fig. 1). Data is forthcoming on antibiotic loading and impact of bacteria and stem cells on exposure to antibiotic-loaded EVs. Our study will be one of the first to examine how gram-negative biofilm EVs can deliver antibiotics, and if this ability changes based on EV origin (liquid, matrix).

Discussion: EV samples showed consistent morphology and expected size of current literature, overall supporting EV isolation from both bacteria and mammalian cell cultures. While bacteria-derived EV often carry cargo to support bacterial growth and viability, our results indicate that even at substantial dosages, MSC health is not significantly affected by the increasing dosage of bacterial EV. We will soon load our EV with antibiotics to explore the effect of these systems as drug carriers. Further utilizing EV as optimal drug delivery systems offers a potentially advantageous therapeutic for implant-associated infections.

Conclusions: The use of biomaterials and other implants in medicine is often set back due to high rates of infection and biofilm formation. Extracellular vesicles, due to their cell-specific surface markers, are a potential therapeutic due to their essential functions and effective cargo-delivery applications. We showcase one of the first instances of the ability to isolate EVs from the biofilm of gram-negative bacteria. Our results indicate that bacterial EV cargo is non-cytotoxic to MSC health (up to a dose of 1 x 10^8). The development of a drug delivery vehicle able to penetrate biofilms without negatively impacting mammalian cell health would dramatically change our way of treating implant-associated infections.

Acknowledgements and/or References (Optional):: Acknowledgements to the Dewey Lab for their support and to Marley Dewey for being such an amazing role model.