Date Published: December 05, 2018
Publisher: John Wiley and Sons Inc.
Author(s): SeungBeum Suh, Ami Jo, Mahama A. Traore, Ying Zhan, Sheryl L. Coutermarsh‐Ott, Veronica M. Ringel‐Scaia, Irving C. Allen, Richey M. Davis, Bahareh Behkam.
Cancer drug delivery remains a formidable challenge due to systemic toxicity and inadequate extravascular transport of nanotherapeutics to cells distal from blood vessels. It is hypothesized that, in absence of an external driving force, the Salmonella enterica serovar Typhimurium could be exploited for autonomous targeted delivery of nanotherapeutics to currently unreachable sites. To test the hypothesis, a nanoscale bacteria‐enabled autonomous drug delivery system (NanoBEADS) is developed in which the functional capabilities of the tumor‐targeting S. Typhimurium VNP20009 are interfaced with poly(lactic‐co‐glycolic acid) nanoparticles. The impact of nanoparticle conjugation is evaluated on NanoBEADS’ invasion of cancer cells and intratumoral transport in 3D tumor spheroids in vitro, and biodistribution in a mammary tumor model in vivo. It is found that intercellular (between cells) self‐replication and translocation are the dominant mechanisms of bacteria intratumoral penetration and that nanoparticle conjugation does not impede bacteria’s intratumoral transport performance. Through the development of new transport metrics, it is demonstrated that NanoBEADS enhance nanoparticle retention and distribution in solid tumors by up to a remarkable 100‐fold without requiring any externally applied driving force or control input. Such autonomous biohybrid systems could unlock a powerful new paradigm in cancer treatment by improving the therapeutic index of chemotherapeutic drugs and minimizing systemic side effects.
Nanoparticle delivery systems have the potential to significantly enhance cancer therapy through improved targeting, reduced systemic toxicity, regulation of therapeutic residence time in circulation, and preservation of therapeutics’ bioavailability. Optimization of nanotherapeutics with respect to vesicle size, geometry, binding, cellular uptake, degradation, and release rates continues to enhance their efficacy.1 However, several physical and biological barriers limit delivery of systemically administered nanoparticles to tumors as well as extravascular transport of nanotherapeutics within the tumor, thereby impeding therapy.2, 3, 4, 5, 6, 7 In particular, highly irregular structure and function of the tumor microvasculature coupled with impaired lymphatic drainage elevates interstitial fluid pressure to levels comparable with intravascular pressure and diminishes the interstitial pressure gradient.2, 5 This combined with a densely structured extracellular matrix, an unusually high fraction of stromal cells, accumulated compressive tissue stress, and expanded intercapillary spaces impose formidable barriers to convective transport of macromolecular chemotherapeutic drugs within the tumor interstitium, precluding therapeutic delivery to cells distal from functioning blood vessels.4, 8, 9, 10 To eradicate tumors, therapeutic agents must disperse throughout the cancerous tissue in sufficiently high concentrations and eliminate every malignant cell. Overcoming the aforementioned transport barriers would significantly improve the efficacy of nanomedicine for cancer therapy.
Efficacious delivery of nanomedicine to all malignant cells in adequate concentrations is strongly dependent on the nanotherapeutics extravascular transport properties. This important challenge in cancer treatment has received little attention up to now (for a recent review of the topic, refer to ref. 4). Moreover, there has been a burgeoning interest in microbial‐mediated cancer therapy as an alternative approach for hard to treat cancers.18 Thus far, bacteria monotherapy has not been successful, and bacteria therapy protocols rely on adjuvant radiation or chemotherapy, which lead to systemic exposure and the associated side effects. The NanoBEADS platform provides a simple, versatile, and clinically applicable strategy to enhance the efficacy of bacteria while at the same time augmenting the specificity and transport of nanoparticles into sites poorly accessible from circulation without the need for any externally applied driving force (e.g., magnetic field) or genetic modification of bacteria. We showed that nanoscale drug delivery vectors can be stably bound to the bacteria without toxicity or statistically significant impediment in the bacteria intratumoral transport properties. Thus, NanoBEADS agents robustly surpass the convection/diffusion limits, primarily through intercellular self‐replication and translocation, and effectively carry nanoscale loads. The transport quantitation metrics (PI, CI, and DI) defined herein could serve as a framework for the quantitative evaluation of the spatial distribution of other therapeutics within tissue and an added measure for evaluating the efficacy of new therapeutic compounds for the treatment of solid tumors.
Bacteria Culture: All experiments in this study were performed using the tumor targeting Salmonella enterica serovar Typhimurium strain VNP2000925 (ATCC 202165, American Type Culture Collection, Manassas, VA). Bacteria were transformed with a plasmid encoding a red fluorescent protein (RFP) for constitutive expression. The plasmid was constructed from BioBrick (iGEM Foundation, Cambridge, MA) parts. BBa_J04450, encoding mRFP1 expression, was assembled in the high copy number pSB1C3 vector conferring resistance to chloramphenicol. Lysogeny broth (LB; 1% w/v of tryptone, 1% w/v of NaCl, 0.5% w/v of yeast extract, supplemented with 35 µg mL−1 of chloramphenicol, pH 7.0) was inoculated with a single bacterial colony and shaken overnight at 37 °C and 100 rpm. Overnight bacteria culture was diluted 100‐fold in LB supplemented with chloramphenicol and shaken at 37 °C and 100 rpm until the optical density at 600 nm (OD600) reached 1.0. A 2 mL aliquot of the culture was then centrifuged at 1700 × g for 5 min at room temperature and suspended in 1 mL of freshly prepared motility buffer (MB; 6.4 × 10−3m K2HPO4, 3.5 × 10−3m KH2PO4, 0.1 × 10−3m ethylenediaminetetraacetic acid (EDTA), 1 × 10−6m L‐methionine, 10 × 10−3m DL‐lactate, 2 × 10−3m MgSO4, 2 × 10−3m CaCl2, pH 7.0) and washed once more in MB prior to use in the biomanufacturing of the NanoBEADS.
The authors declare no conflict of interest.