Poster W15 - E. coli Source of Outer Membrane Vesicles Influences Properties Relevant to Oral Delivery Applications

Introduction: Oral delivery of drugs, vaccines, and gene therapies offers ease of administration without medically trained personnel, simple repeated dosing, and the potential for high patient compliance compared to parenteral routes [1]. Not only does oral delivery give direct access to gastrointestinal (GI) tissue for targeted therapies, but the highly vascularized nature of the GI tract also allows for systemic distribution of therapeutic cargo. However, oral drug and gene delivery is complicated by variations in pH, an abundance of degrading enzymes, and the presence of a mucosal barrier throughout the GI tract [1]. Consequently, protective nanocarrier platforms have been extensively investigated to prevent degradation of orally administered drugs and other bioactive cargo and to enhance cellular targeting and internalization. Bacterial outer membrane vesicles (OMVs) are nanosized extracellular vesicles (EVs) secreted by gram-negative bacteria that facilitate bacterial cell-to-cell interactions, as well as bacterial-host communication and immune modulation within the gut [2-3]. Due to various innate properties, OMVs are being investigated for use as nanocarriers for oral drug and gene delivery. Here, we screened a collection of OMVs isolated from 30 human commensal GI-tract derived E. coli strains for features that could impact the function of OMVs as an oral delivery vehicle, including morphological features, immune modulation, and internalization into intestinal epithelial cells (IECs) and macrophages.

Materials and

Methods: All 30 E. coli strains were cultured as previously described [4], and OMVs were isolated using a Vivaspin centrifugal concentrator. To confirm isolation and characterize the vesicles, OMVs were sized using nanoparticle tracking analysis, nano-flow cytometry, and transmission electron microscopy. OMV cytotoxicity was determined in both J774 macrophage cell line and the Caco2 intestinal epithelial cell line using the LIVE/DEAD viability/cytotoxicity kit. OMVs were labeled with the lipophilic fluorescent dye dioctadecyloxacarbocyanine perchlorate (DiO) and then administered to Caco2 and J774 cells for 24 hr; internalization of labeled OMVs into cells was determined using flow cytometry. To determine innate immune response, OMVs were administered to Caco2 cells as described, and then cells were assessed using a Human Toll Like Receptor RT2 Profiler PCR Array. For immunomodulatory properties of OMVs in J774 cells, after OMV administration to cells for 24 hrs, supernatants were collected and analyzed neat for IL-10, IL-1β, IL-6 and TNF-α. As early proof of delivery platform potential, select OMVs were loaded with plasmid DNA and tested for transgene expression in HEK 293T cells.

Results, Conclusions, and Discussions: Results and

Discussion: Regardless of producer strain from which they were isolated, E. coli OMVs were nanoscale in size, with no significant strain-specific differences in diameter observed and were biocompatible with both epithelial cells and macrophages (cell viability remained over 95% after treatment with OMVs from all strains). While E. coli OMVs were internalized by both cell types, there were significant strain-dependent differences in the percentage of the epithelial cell population (Caco2) which internalized OMVs, whereas macrophages (J774) showed internalization of OMVs at similar levels regardless of producer strain (Fig. 1 A and B). E. coli OMVs induced a spectrum of mRNA expression changes in epithelial cells for genes associated with TLR innate immune signaling and cytokine responses from macrophages. Few correlations between internalization and cytokine production were found indicating that native factors such as surface receptors and/or cargo are potentially responsible for the differences in cytokine production observed among the strains in this screen. From the screen we have identified lead candidates that display high internalization into Caco2 and J774 cells and a range of innate signaling responses. Lead candidate OMVs are being used to formulate DNA-OMVs nanocarriers and are being tested for DNA loading efficiency, gastric protection of DNA cargo, and transfection efficiency.

Conclusions: These results demonstrate that OMVs can be successfully isolated from all 30 commensal E. coli strains in our collection without strain-specific differences in their size or particle concentration. There are strain-specific differences in cellular internalization and innate responses that can allow for selection of appropriate OMVs for further development as DNA-OMV nanocarriers to promote oral gene delivery, with immunomodulatory properties that act synergistically with the delivered transgene, thus allowing for customization of this platform for a variety of oral delivery applications.

Acknowledgements (Optional):

References: [1] Farris E. et al., Curr Opin Biomed Eng 2018. [2] Kaparakis-Liaskos M. et al., Nat Rev Immunol 2015. [3] Schwechheimer C. et al., Nat Rev Micro 2015. [4] Kittana H. et al., Front Immunol 2018.

Funding: NIH/NIBIB (1 DP2 EB025760-01, 3DP2EB025760-01S1; 4 DP2 EB025760-02), NIH/NCATS (1R21TR005069-01).