Fabrication and Characterization of PLGA Nanoparticle-Bacteria Conjugate Nanobeads for Increased Tumor Penetration

Cancer is the second cause of overall mortality in the world and systemic chemotherapy is a major therapeutic approach for nearly all types and stages of cancer. Grand challenges of limited efficacy and systemic toxicity of existing chemotherapeutic treatments can be addressed through better drug carrier targeting and enhanced penetration of drug carriers. Attenuated strains Salmonella Typhimurium have previously been shown to have the ability to preferentially colonize tumor tissues. However, clinical success in immunocompetent hosts has been rare due to insufficient tumor colonization by bacteria^1–3. We hypothesize that a combinatorial therapy approach based on integrating bacteria with chemotherapeutics-loaded nanoparticles will amplify both forms of therapy. Thus, we have developed a Nanoscale Bacteria-Enabled Drug Delivery System (NanoBEADS)⁴ in which the functional capabilities of bacteria are interfaced with and augmented by chemotherapeutic-loaded nanoparticles. Each NanoBEADS agent is constructed by interfacing a Salmonella Typhimurium VNP20009 (i.e. a live engineered tumor-targeting bacterium) with an ensemble of poly(lactic-co-glycolic acid) nanoparticles loaded with chemotherapeutics for the purpose of using the bacterium to transport the particle deep into the tumor. To create the NanoBEADS, S. Typhimurium VNP20009 bacteria were cultured in Luria-Bertani broth (1% tryptone, 0.5% yeast extract and 0.5% sodium chloride) and coated with biotin-conjugated rabbit polyclonal anti-S. Typhimurium antibody. The nanoparticles were fabricated using a modified nanoprecipitation method⁵ and fluorescently tagged with 6,13-Bis(triisopropylsylylethynyl)pentacene (TIPS pentacene). With a size of approximately 110 nm in diameter and a zeta potential of about -30 mV, the carboxyl end groups allow for EDC coupling to streptavidin. The biotin-coated bacteria were then mixed with streptavidin coated nanoparticles for 30 minutes at room temperature to promote self-assembly of the particles to bacteria. Scanning electron microscopy (SEM) images of NanoBEADS show the average number of nanoparticles attached to each bacterium to be 35. In-vitro assays were carried out to compare the penetration of passively diffusing PLGA nanoparticles and NanoNBEADS in multicellular tumor spheroids.

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