Toward development of an autonomous network of bacteria-based delivery systems (BacteriaBots): spatiotemporally high-throughput characterization of bacterial quorum-sensing response.

Characterization of bacterial innate and engineered cooperative behavior, regulated through chemical signaling in a process known as quorum sensing, is critical to development of a myriad of bacteria-enabled systems including biohybrid drug delivery systems and biohybrid mobile sensor networks. Here, we demonstrate, for the first time, that microfluidic diffusive mixers can be used for spatiotemporally high-throughput characterization of bacterial quorum-sensing response. Using this batch characterization method, the quorum-sensing response in Escherichia coli MG1655, transformed with a truncated lux operon from Vibrio fischeri, in the presence of 1-100 nM exogenous acyl-homoserine lactone molecules has been quantified. This method provides a rapid and facile tool for high-throughput characterization of the quorum-sensing response of genetically modified bacteria in the presence of a wide concentration range of signaling molecules with a precision of ±0.5 nM. Furthermore, the quorum-sensing response of BacteriaBots has been characterized to determine if the results obtained from a large bacterial population can serve as a robust predictive tool for the small bacterial population attached to each BacteriaBot.

Directed transport of bacteria-based drug delivery vehicles: bacterial chemotaxis dominates particle shape.

Several attenuated and non-pathogenic bacterial species have been demonstrated to actively target diseased sites and successfully deliver plasmid DNA, proteins and other therapeutic agents into mammalian cells. These disease-targeting bacteria can be employed for targeted delivery of therapeutic and imaging cargos in the form of a bio-hybrid system. The bio-hybrid drug delivery system constructed here is comprised of motile Escherichia coli MG1655 bacteria and elliptical disk-shaped polymeric microparticles. The transport direction for these vehicles can be controlled through biased random walk of the attached bacteria in presence of chemoattractant gradients in a process known as chemotaxis. In this work, we utilize a diffusion-based microfluidic platform to establish steady linear concentration gradients of a chemoattractant and investigate the roles of chemotaxis and geometry in transport of bio-hybrid drug delivery vehicles. Our experimental results demonstrate for the first time that bacterial chemotactic response dominates the effect of body shape in extravascular transport; thus, the non-spherical system could be more favorable for drug delivery applications owing to the known benefits of using non-spherical particles for vascular transport (e.g. relatively long circulation time).

Data-driven statistical modeling of the emergent behavior of biohybrid microrobots.

Multi-agent biohybrid microrobotic systems, owing to their small size and distributed nature, offer powerful solutions to challenges in biomedicine, bioremediation, and biosensing. Synthetic biology enables programmed emergent behaviors in the biotic component of biohybrid machines, expounding vast potential benefits for building biohybrid swarms with sophisticated control schemes. The design of synthetic genetic circuits tailored toward specific performance characteristics is an iterative process that relies on experimental characterization of spatially homogeneous engineered cell suspensions. However, biohybrid systems often distribute heterogeneously in complex environments, which will alter circuit performance. Thus, there is a critically unmet need for simple predictive models that describe emergent behaviors of biohybrid systems to inform synthetic gene circuit design. Here, we report a data-driven statistical model for computationally efficient recapitulation of the motility dynamics of two types of Escherichia coli bacteria-based biohybrid swarms-NanoBEADS and BacteriaBots. The statistical model was coupled with a computational model of cooperative gene expression, known as quorum sensing (QS). We determined differences in timescales for programmed emergent behavior in BacteriaBots and NanoBEADS swarms, using bacteria as a comparative baseline. We show that agent localization and genetic circuit sensitivity strongly influence the timeframe and the robustness of the emergent behavior in both systems. Finally, we use our model to design a QS-based decentralized control scheme wherein agents make independent decisions based on their interaction with other agents and the local environment. We show that synergistic integration of synthetic biology and predictive modeling is requisite for the efficient development of biohybrid systems with robust emergent behaviors.

Patient-derived small intestinal myofibroblasts direct perfused, physiologically responsive capillary development in a microfluidic Gut-on-a-Chip Model.

The development and physiologic role of small intestine (SI) vasculature is poorly studied. This is partly due to a lack of targetable, organ-specific markers for in vivo studies of two critical tissue components: endothelium and stroma. This challenge is exacerbated by limitations of traditional cell culture techniques, which fail to recapitulate mechanobiologic stimuli known to affect vessel development. Here, we construct and characterize a 3D in vitro microfluidic model that supports the growth of patient-derived intestinal subepithelial myofibroblasts (ISEMFs) and endothelial cells (ECs) into perfused capillary networks. We report how ISEMF and EC-derived vasculature responds to physiologic parameters such as oxygen tension, cell density, growth factors, and pharmacotherapy with an antineoplastic agent (Erlotinib). Finally, we demonstrate effects of ISEMF and EC co-culture on patient-derived human intestinal epithelial cells (HIECs), and incorporate perfused vasculature into a gut-on-a-chip (GOC) model that includes HIECs. Overall, we demonstrate that ISEMFs possess angiogenic properties as evidenced by their ability to reliably, reproducibly, and quantifiably facilitate development of perfused vasculature in a microfluidic system. We furthermore demonstrate the feasibility of including perfused vasculature, including ISEMFs, as critical components of a novel, patient-derived, GOC system with translational relevance as a platform for precision and personalized medicine research.

Nanoscale Bacteria-Enabled Autonomous Drug Delivery System (NanoBEADS) Enhances Intratumoral Transport of Nanomedicine.

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.

Integrating nanofibers with biochemical gradients to investigate physiologically-relevant fibroblast chemotaxis.

Persistent cell migration can occur due to anisotropy in the extracellular matrix (ECM), the gradient of a chemo-effector, or a combination of both. Through a variety of in vitro platforms, the contributions of either stimulus have been extensively studied, while the combined effect of both cues remains poorly described. Here, we report an integrative microfluidic chemotaxis assay device that enables the study of single cell chemotaxis on ECM-mimicking, aligned, and suspended nanofibers. Using this assay, we evaluated the effect of fiber spacing on the morphology and chemotaxis response of embryonic murine NIH/3T3 fibroblasts in the presence of temporally invariant, linear gradients of platelet-derived growth factor-BB (PDGF-BB). We found that the strength of PDGF-mediated chemotaxis response depends on not only the gradient slope but also the cell morphology. Low aspect ratio (3.4 ± 0.2) cells on flat substrata exhibited a chemotaxis response only at a PDGF-BB gradient of 0-10 ng mL-1. However, high aspect ratio (19.1 ± 0.7) spindle-shaped cells attached to individual fibers exhibited maximal chemotaxis response at a ten-fold shallower gradient of 0-1 ng mL-1, which was robustly maintained up to 0-10 ng mL-1. Quadrilateral-shaped cells of intermediate aspect ratio (13.6 ± 0.8) attached to two fibers exhibited a weaker response compared to the spindle-shaped cells, but still stronger compared to cells attached to 2D featureless substrata. Through pharmacological inhibition, we show that the mesenchymal chemotaxis pathway is conserved in cells on fibers. Altogether, our findings show that chemotaxis on ECM-mimicking fibers is modulated by fiber spacing-driven cell shape and can be significantly different from the behavior observed on flat 2D substrata. We envisage that this microfluidic platform will have wide applicability in understanding the combined role of ECM architecture and chemotaxis in physiological and pathological processes.

Construction of Bacteria-Based Cargo Carriers for Targeted Cancer Therapy.

Despite significant recent progress in nanomedicine, drug delivery to solid tumors remains a formidable challenge often associated with low delivery efficiency and limited penetration of the drug in poorly vascularized regions of solid tumors. Attenuated strains of facultative anaerobes have been demonstrated to have exceptionally high selectivity to primary tumors and metastatic cancer, a good safety profile, and superior intratumoral penetration performance. However, bacteria have rarely been able to completely inhibit tumor growth in immunocompetent hosts solely by their presence in the tumor. We have developed a Nanoscale Bacteria-Enabled Autonomous Drug Delivery System (NanoBEADS) in which the functional capabilities of tumor-targeting bacteria are interfaced with chemotherapeutic-loaded nanoparticles, an approach that would amplify the therapeutic potential of both modalities. Here, we describe two biomanufacturing techniques to construct NanoBEADS by linking different bacterial species with polymeric theranostic vehicles. NanoBEADS are envisioned to significantly impact current practices in cancer theranostics through improved targeting and intratumoral transport properties.

Tissue Engineering the Vascular Tree.

A major hurdle in the field of tissue engineering and regenerative medicine remains the design and construction of larger (> 1 cm3) in vitro tissues for biological studies and transplantation. While there has been success in creating three-dimensional (3D) capillary networks, relatively large arteries (diameter >3-5 mm), and more recently small arteries (diameter 500 µm-1 mm), there has been no success in the creation of a living dynamic blood vessel network comprising of arterioles (diameter 40-300 µm), capillaries, and venules. Such a network would provide the foundation to supply nutrients and oxygen to all surrounding cells for larger tissues and organs that require a hierarchical vascular supply. In this study, we describe the different technologies and methods that have been employed in an effort to create individual vessels and networks of vessels to support engineered tissues for in vivo and in vitro applications. A special focus is placed on the generation of blood vessels with average dimensions that span from microns (capillaries) to a millimeter (large arterioles). We also identify major challenges while exploring new opportunities to create model systems of the entire vascular tree, including arterioles and venules.

Bacterial chemotaxis-enabled autonomous sorting of nanoparticles of comparable sizes.

High throughput sorting of micro/nanoparticles of similar sizes is of significant interest in many biological and chemical applications. In this work, we report a simple and cost-effective sorting technique for separation of similarly-sized particles of dissimilar surface properties within a diffusion-based microfluidic platform using chemotaxis in Escherichia coli bacteria. Differences in surface chemistry of two groups of similarly-sized nanoparticles in a mixture were exploited to selectively assemble one particle group onto motile E. coli, through either specific or non-specific adhesion, and separate them from the remaining particle group via chemotaxis of the attached bacteria. To enable optimal operation of the sorting platform, the chemotaxis behavior of E. coli bacteria in response to casamino acids, the chemoeffector of choice was first characterized. The chemical concentration gradient range within which the bacteria exhibit a positive chemotactic response was found to be within 0.25 × 10(-7)-1.0 × 10(-3) g ml(-1) mm(-1). We demonstrate that at the optimum concentration gradient of 5.0 × 10(-4) g ml(-1) mm(-1), a sorting efficiency of up to 81% at a throughput of 2.4 × 10(5) particles per min can be achieved. Sensitivity of the sorting efficiency to the adhesion mechanism and particle size in the range of 320-1040 nm was investigated.

Bacterial chemotaxis enabled autonomous sorting of micro-particles.

Autonomous manipulation and assembly at micro/nanoscale continues to be one of the main challenges of micro/nanorobotics. Biomotors are increasingly being considered as robust, versatile and cost-effective choices for a variety of micro/nanorobotic tasks. Here we propose utilization of motility and chemotaxis in flagellated bacteria for autonomous sorting of 6 µm and 10 µm micro-particles within a microfluidic platform. Difference in surface chemistry of the 6 µm and 10 µm particles are exploited to selectively assemble bacteria onto 6 µm particles and separate them from 10 µm particles via chemotaxis motility of the attached bacteria. It has been shown that within 1 hour, an increasingly larger number of 6 µm particles accumulate within a 600 µm radius, near the chemo-attractant source.

Computational and experimental study of chemotaxis of an ensemble of bacteria attached to a microbead.

Micro-objects propelled by whole cell actuators, such as flagellated bacteria, are being increasingly studied and considered for a wide variety of applications. In this work we present theoretical and experimental investigations of chemotactic motility of a 10 µm diameter microbead propelled by an ensemble of attached flagellated bacteria. The stochastic model presented here encompasses the behavior of each individual bacterium attached to the microbead in a spatiotemporally varying chemoattractant field. The computational model shows that in a chemotactic environment, the ensemble of bacteria, although constrained, propel the bead in a chemotactic manner with a 67% enhancement in displacement to distance ratio (defined as directionality) compared to nonchemotactic propulsion. The simulation results are validated experimentally. Close agreement between theory and experiments demonstrates the possibility of using the presented model as a predictive tool for other similar biohybrid systems.