Micropatterned Janus Biobot design, characterization and in vivo validation for theranostic (therapy+diagnosis) applications.
During my PhD dissertation, I designed nanocluster integrated micropatterned platforms to understand how mammalian and bacterial cells receive and respond to information in their environment. In my post-doctoral work, by utilizing the tools of microcontact printing and cell biology, I studied the influence of geometry and force control on complex cell function (adhesion, chiral alignment and apoptosis). I want to design biohybrid microbots using bacterial cell surface engineering and microfabrication for drug, and gene delivery vectors, smart prosthetics, and lab-on-a-chip devices.
PhD: Medical Nanotechnology, European School of Molecular Medicine (SEMM), Milan, Italy, 2011
Cell Death \& Disease, 9(3):348, March 2018 (article)
The effect of redox metals such as iron and copper on multiple sclerosis and amyotrophic lateral sclerosis has been intensively studied. However, the origin of these disorders remains uncertain. This review article critically describes the physiology of redox metals that produce oxidative stress, which in turn leads to cascades of immunomodulatory alteration of neurons in multiple sclerosis and amyotrophic lateral sclerosis. Iron and copper overload has been well established in motor neurons of these diseases' lesions. On the other hand, the role of other metals like cadmium participating indirectly in the redox cascade of neurobiological mechanism is less studied. In the second part of this review, we focus on this less conspicuous correlation between cadmium as an inactive-redox metal and multiple sclerosis and amyotrophic lateral sclerosis, providing novel treatment modalities and approaches as future prospects.
Cancer cells have the capacity to synthesize nanoparticles (NPs). The detailed mechanism of this process is not very well documented. We report the mechanism of biomineralization of aqueous gold chloride into NPs and microplates in the breast-cancer cell line MCF7. Spherical gold NPs are synthesized in these cells in the presence of serum in the culture media by the reduction of HAuCl4. In the absence of serum, the cells exhibit gold microplate formation through seed-mediate growth albeit slower reduction. The structural characteristics of the two types of NPs under different media conditions were confirmed using scanning electron microscopy (SEM); crystallinity and metallic properties were assessed with transmission electron microscopy (TEM) and x-ray photoelectron spectroscopy (XPS). Gold-reducing proteins, related to cell stress initiate the biomineralization of HAuCl4 in cells (under serum free conditions) as confirmed by infrared (IR) spectroscopy. MCF7 cells undergo irreversible replicative senescence when exposed to a high concentration of ionic gold and conversely remain in a dormant reversible quiescent state when exposed to a low gold concentration. The latter cellular state was achievable in the presence of the rho/ROCK inhibitor Y-27632. Proteomic analysis revealed consistent expression of specific proteins under serum and serum-free conditions. A high-throughput proteomic approach to screen gold-reducing proteins and peptide sequences was utilized and validated by quartz crystal microbalance with dissipation (QCM-D). Statement of significance Cancer cells are known to synthesize gold nanoparticles and microstructures, which are promising for bioimaging and other therapeutic applications. However, the detailed mechanism of such biomineralization process is not well understood yet. Herein, we demonstrate that cancer cells exposed to gold ions (grown in serum/serum-free conditions) secrete shock and stress-related proteins with specific gold-binding/reducing polypeptides. Cells undergo reversible senescence and can recover normal physiology when treated with the senescence inhibitor depending on culture condition. The use of mammalian cells as microincubators for synthesis of such particles could have potential influence on their uptake and biocompatibility. This study has important implications for in-situ reduction of ionic gold to anisotropic micro-nanostructures that could be used in-vivo clinical applications and tumor photothermal therapy.
PloS One, 12(4):e0175428, Public Library of Science, April 2017 (article)
The considerable morbidity associated with hospitalized patients and clinics in developed countries due to biofilm formation on biomedical implants and surgical instruments is a heavy economic burden. An alternative to chemically treated surfaces for bactericidal activity started emerging from micro/nanoscale topographical cues in the last decade. Here, we demonstrate a putative antibacterial surface using copper nanowhiskers deposited by molecular beam epitaxy. Furthermore, the control of biological response is based on hydrophobic pinning of water droplets in the Wenzel regime, causing mechanical injury and cell death. Scanning electron microscopy images revealed the details of the surface morphology and non-contact mode laser scanning of the surface revealed the microtopography-associated quantitative parameters. Introducing the bacterial culture over nanowhiskers produces mechanical injury to cells, leading to a reduction in cell density over time due to local pinning of culture medium to whisker surfaces. Extended culture to 72 hours to observe biofilm formation revealed biofilm inhibition with scattered microcolonies and significantly reduced biovolume on nanowhiskers. Therefore, surfaces patterned with copper nanowhiskers can serve as potential antibiofilm surfaces. The topography-based antibacterial surfaces introduce a novel prospect in developing mechanoresponsive nanobiomaterials to reduce the risk of medical device biofilm-associated infections, contrary to chemical leaching of copper as a traditional bactericidal agent.
Raymond, M. J., Ray, P., Kaur, G., Fredericks, M., Singh, A. V., Wan, L. Q.
Cellular and Molecular Bioengineering, 10(1):63-74, 2017 (article)
Intrinsic cell chirality has been implicated in the left--right (LR) asymmetry of embryonic development. Impaired cell chirality could lead to severe birth defects in laterality. Previously, we detected cell chirality with an in vitro micropatterning system. Here, we demonstrate for the first time that chirality can be quantified as the coordination of multiaxial polarization of individual cells and nuclei. Using an object labeling, connected component based method, we characterized cell chirality based on cell and nuclear shape polarization and nuclear positioning of each cell in multicellular patterns of epithelial cells. We found that the cells adopted a LR bias the boundaries by positioning the sharp end towards the leading edge and leaving the nucleus at the rear. This behavior is consistent with the directional migration observed previously on the boundary of micropatterns. Although the nucleus is chirally aligned, it is not strongly biased towards or away from the boundary. As the result of the rear positioning of nuclei, the nuclear positioning has an opposite chirality to that of cell alignment. Overall, our results have revealed deep insights of chiral morphogenesis as the coordination of multiaxial polarization at the cellular and subcellular levels.
Biohybrid cell-driven microsystems offer unparalleled possibilities for realization of soft microrobots at the micron scale. Here, we introduce a bacteria-driven microswimmer that combines the active locomotion and sensing capabilities of bacteria with the desirable encapsulation and viscoelastic properties of a soft double-micelle microemulsion for active transport and delivery of cargo (e.g., imaging agents, genes, and drugs) to living cells. Quasi-monodisperse double emulsions were synthesized with an aqueous core that encapsulated the fluorescence imaging agents, as a proof-of-concept cargo in this study, and an outer oil shell that was functionalized with streptavidin for specific and stable attachment of biotin-conjugated Escherichia coli. Motile bacteria effectively propelled the soft microswimmers across a Transwell membrane, actively delivering imaging agents (i.e., dyes) encapsulated inside of the micelles to a monolayer of cultured MCF7 breast cancer and J744A.1 macrophage cells, which enabled real-time, live-cell imaging of cell organelles, namely mitochondria, endoplasmic reticulum, and Golgi body. This in vitro model demonstrates the proof-of-concept feasibility of the proposed soft microswimmers and offers promise for potential biomedical applications in active and/or targeted transport and delivery of imaging agents, drugs, stem cells, siRNA, and therapeutic genes to live tissue in in vitro disease models (e.g., organ-on-a-chip devices) and stagnant or low-flow-velocity fluidic regions of the human body.
Advanced Drug Delivery Reviews, 106, pages: 27-44, Elsevier, November 2016 (article)
The use of bacterial cells as agents of medical therapy has a long history. Research that was ignited over a century ago with the accidental infection of cancer patients has matured into a platform technology that offers the promise of opening up new potential frontiers in medical treatment. Bacterial cells exhibit unique characteristics that make them well-suited as smart drug delivery agents. Our ability to genetically manipulate the molecular machinery of these cells enables the customization of their therapeutic action as well as its precise tuning and spatio-temporal control, allowing for the design of unique, complex therapeutic functions, unmatched by current drug delivery systems. Early results have been promising, but there are still many important challenges that must be addressed. We present a review of promises and challenges of employing bioengineered bacteria in drug delivery systems and introduce the biohybrid design concept as a new additional paradigm in bacteria-based drug delivery.
Advanced Healthcare Materials, 5(18):2306-2306, September 2016 (article)
On page 2325, Ajay Vikram Singh and Metin Sitti propose a facile surface patterning technique and a specific, strong biotin–streptavidin bonding of bacteria on patterned surfaces to fabricate Janus particles that are propelled by the attached bacteria. Such bacteria-driven Janus microswimmers could be used for future medicine in targeted drug delivery and environmental remediation.
Advanced Healthcare Materials, 5(18):2325-2331, May 2016 (article)
A surface patterning technique and a specific and strong biotin–streptavidin bonding of bacteria on patterned surfaces are proposed to fabricate Janus particles that are propelled by the attached bacteria. Bacteria-driven Janus microswimmers with diameters larger than 3 μm show enhanced mean propulsion speed. Such microswimmers could be used for future applications such as targeted drug delivery and environmental remediation.
Current Pharmaceutical Design, 22(11):1418-1428, Bentham Science Publishers, March 2016 (article)
Miniature untethered medical robots have been receiving growing attention due to technological advances in microactuation, microsensors, and microfabrication and have significant potential to reduce the invasiveness and improve the accessibility of medical devices into unprecedented small spaces inside the human body. In this review, we discuss therapeutic and diagnostic applications of untethered medical microrobots. Wirelessly controlled milli/microrobots with integrated sensors are revolutionizing micromanipulation based medical interventions and are enabling doctors to perform minimally invasive procedures not possible before. 3D fabrication technologies enabling milli/microrobot fabrication at the single cell scale are empowering high-resolution visual imaging and in vivo manipulation capabilities. Swallowable millirobots and injectabale ocular microrobots allow the gastric ulcer imaging, and performance of vitreoretinal microsurgery at previously inaccessible ocular sites. Many invasive excision and incision based diagnostic biopsy, prostrate, and nephrolgical procedures can be performed minimally or almost noninvasively due to recent advancements in microrobotic technology. Advances in biohybrid microrobot systems are pushing microrobotic systems even smaller, using biological cells as on-board microactuators and microsensors using the chemical energy. Such microrobotic systems could be used for local targeted delivery of imaging contrast agents, drugs, genes, and mRNA, minimally invasive surgery, and cell micromanipulation in the near future.
Our goal is to understand the principles of Perception, Action and Learning in autonomous systems that successfully interact with complex environments and to use this understanding to design future systems