Sangeeta N. Bhatia

Probing the cytotoxicity of semiconductor quantum dots

With their bright, photostable fluorescence, semiconductor quantum dots show promise as alternatives to organic dyes for biological labeling. Questions about their potential cytotoxicity, however, remain unanswered. While cytotoxicity of bulk cadmium selenide (CdSe) is well documented, a number of groups have suggested that CdSe QDs are cytocompatible, at least with some immortalized cell lines. Using primary hepatocytes as a liver model, we found that CdSe-core QDs were indeed acutely toxic under certain conditions. Specifically, we found that the cytotoxicity of QDs was modulated by processing parameters during synthesis, exposure to ultraviolet light, and surface coatings. Our data further suggests that cytotoxicity correlates with the liberation of free Cd2+ ions due to deterioration of the CdSe lattice. When appropriately coated, CdSe-core QDs can be rendered non-toxic and used to track cell migration and reorganization in vitro. Our results inform design criteria for the use of QDs in vitro and especially in vivo where deterioration over time may occur.

Nanocrystal targeting in vivo

Inorganic nanostructures that interface with biological systems have recently attracted widespread interest in biology and medicine. Nanoparticles are thought to have potential as novel intravascular probes for both diagnostic (e.g., imaging) and therapeutic purposes (e.g., drug delivery). Critical issues for successful nanoparticle delivery include the ability to target specific tissues and cell types and escape from the biological particulate filter known as the reticuloendothelial system. We set out to explore the feasibility of in vivo targeting by using semiconductor quantum dots (qdots). Qdots are small (<10 nm) inorganic nanocrystals that possess unique luminescent properties; their fluorescence emission is stable and tuned by varying the particle size or composition. We show that ZnS-capped CdSe qdots coated with a lung-targeting peptide accumulate in the lungs of mice after i.v. injection, whereas two other peptides specifically direct qdots to blood vessels or lymphatic vessels in tumors. We also show that adding polyethylene glycol to the qdot coating prevents nonselective accumulation of qdots in reticuloendothelial tissues. These results encourage the construction of more complex nanostructures with capabilities such as disease sensing and drug delivery.

Biodegradable luminescent porous silicon nanoparticles for in vivo applications

Nanomaterials that can circulate in the body hold great potential to diagnose and treat disease. For such applications, it is important that the nanomaterials be harmlessly eliminated from the body in a reasonable period of time after they carry out their diagnostic or therapeutic function. Despite efforts to improve their targeting efficiency, significant quantities of systemically administered nanomaterials are cleared by the mononuclear phagocytic system before finding their targets, increasing the likelihood of unintended acute or chronic toxicity. However, there has been little effort to engineer the self-destruction of errant nanoparticles into non-toxic, systemically eliminated products. Here, we present luminescent porous silicon nanoparticles (LPSiNPs) that can carry a drug payload and of which the intrinsic near-infrared photoluminescence enables monitoring of both accumulation and degradation in vivo. Furthermore, in contrast to most optically active nanomaterials (carbon nanotubes, gold nanoparticles and quantum dots), LPSiNPs self-destruct in a mouse model into renally cleared components in a relatively short period of time with no evidence of toxicity. As a preliminary in vivo application, we demonstrate tumour imaging using dextran-coated LPSiNPs (D-LPSiNPs). These results demonstrate a new type of multifunctional nanostructure with a low-toxicity degradation pathway for in vivo applications.

Microfluidic organs-on-chips

An organ-on-a-chip is a microfluidic cell culture device created with microchip manufacturing methods that contains continuously perfused chambers inhabited by living cells arranged to simulate tissue- and organ-level physiology. By recapitulating the multicellular architectures, tissue-tissue interfaces, physicochemical microenvironments and vascular perfusion of the body, these devices produce levels of tissue and organ functionality not possible with conventional 2D or 3D culture systems. They also enable high-resolution, real-time imaging and in vitro analysis of biochemical, genetic and metabolic activities of living cells in a functional tissue and organ context. This technology has great potential to advance the study of tissue development, organ physiology and disease etiology. In the context of drug discovery and development, it should be especially valuable for the study of molecular mechanisms of action, prioritization of lead candidates, toxicity testing and biomarker identification.

Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues

In the absence of perfusable vascular networks, three-dimensional (3D) engineered tissues densely populated with cells quickly develop a necrotic core [1]. Yet the lack of a general approach to rapidly construct such networks remains a major challenge for 3D tissue culture [2–4]. Here, we 3D printed rigid filament networks of carbohydrate glass, and used them as a cytocompatible sacrificial template in engineered tissues containing living cells to generate cylindrical networks which could be lined with endothelial cells and perfused with blood under high-pressure pulsatile flow. Because this simple vascular casting approach allows independent control of network geometry, endothelialization, and extravascular tissue, it is compatible with a wide variety of cell types, synthetic and natural extracellular matrices (ECMs), and crosslinking strategies. We also demonstrated that the perfused vascular channels sustained the metabolic function of primary rat hepatocytes in engineered tissue constructs that otherwise exhibited suppressed function in their core.

Computationally Guided Photothermal Tumor Therapy Using Long-Circulating Gold Nanorod Antennas

Plasmonic nanomaterials have the opportunity to considerably improve the specificity of cancer ablation by i.v. homing to tumors and acting as antennas for accepting externally applied energy. Here, we describe an integrated approach to improved plasmonic therapy composed of multimodal nanomaterial optimization and computational irradiation protocol development. We synthesized polyethylene glycol (PEG)-protected gold nanorods (NR) that exhibit superior spectral bandwidth, photothermal heat generation per gram of gold, and circulation half-life in vivo (t(1/2), approximately 17 hours) compared with the prototypical tunable plasmonic particles, gold nanoshells, as well as approximately 2-fold higher X-ray absorption than a clinical iodine contrast agent. After intratumoral or i.v. administration, we fuse PEG-NR biodistribution data derived via noninvasive X-ray computed tomography or ex vivo spectrometry, respectively, with four-dimensional computational heat transport modeling to predict photothermal heating during irradiation. In computationally driven pilot therapeutic studies, we show that a single i.v. injection of PEG-NRs enabled destruction of all irradiated human xenograft tumors in mice. These studies highlight the potential of integrating computational therapy design with nanotherapeutic development for ultraselective tumor ablation.

Effect of cell–cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells

Heterotypic cell interaction between parenchymal cells and nonparenchymal neighbors has been reported to modulate cell growth, migration, and/or differentiation. In both the developing and adult liver, cell–cell interactions are imperative for coordinated organ function. In vitro, cocultivation of hepatocytes and nonparenchymal cells has been used to preserve and modulate the hepatocyte phenotype. We summarize previous studies in this area as well as recent advances in microfabrication that have allowed for more precise control over cell–cell interactions through ‘cellular patterning’ or ‘micropatterning’. Although the precise mechanisms by which nonparenchymal cells modulate the hepatocyte phenotype remain unelucidated, some new insights on the modes of cell signaling, the extent of cell–cell interaction, and the ratio of cell populations are noted. Proposed clinical applications of hepatocyte cocultures, typically extracorporeal bioartificial liver support systems, are reviewed in the context of these new findings. Continued advances in microfabrication and cell culture will allow further study of the role of cell communication in physiological and pathophysiological processes as well as in the development of functional tissue constructs for medical applications.— Bhatia, S. N., Balis, U. J., Yarmush, M. L., Toner, M. Effect of cell–cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells. FASEB J. 13, 1883–1900 (1999)

An extracellular matrix microarray for probing cellular differentiation

We present an extracellular matrix (ECM) microarray platform for the culture of patterned cells atop combinatorial matrix mixtures. This platform enables the study of differentiation in response to a multitude of microenvironments in parallel. The fabrication process required only access to a standard robotic DNA spotter, off-the-shelf materials and 1,000 times less protein than conventional means of investigating cell-ECM interactions. To demonstrate its utility, we applied this platform to study the effects of 32 different combinations of five extracellular matrix molecules (collagen I, collagen III, collagen IV, laminin and fibronectin) on cellular differentiation in two contexts: maintenance of primary rat hepatocyte phenotype indicated by intracellular albumin staining and differentiation of mouse embryonic stem (ES) cells toward an early hepatic fate, indicated by expression of a β-galactosidase reporter fused to the fetal liver-specific gene, Ankrd17 (also known as gtar). Using this technique, we identified combinations of ECM that synergistically impacted both hepatocyte function and ES cell differentiation. This versatile technique can be easily adapted to other applications, as it is amenable to studying almost any insoluble microenvironmental cue in a combinatorial fashion and is compatible with several cell types.

Targeting of drugs and nanoparticles to tumors.

The various types of cells that comprise the tumor mass all carry molecular markers that are not expressed or are expressed at much lower levels in normal cells. These differentially expressed molecules can be used as docking sites to concentrate drug conjugates and nanoparticles at tumors. Specific markers in tumor vessels are particularly well suited for targeting because molecules at the surface of blood vessels are readily accessible to circulating compounds. The increased concentration of a drug in the site of disease made possible by targeted delivery can be used to increase efficacy, reduce side effects, or achieve some of both. We review the recent advances in this delivery approach with a focus on the use of molecular markers of tumor vasculature as the primary target and nanoparticles as the delivery vehicle.

Controlling cell interactions by micropatterning in co‐cultures: Hepatocytes and 3T3 fibroblasts

The repair or replacement of damaged tissues using in vitro strategies has focused on manipulation of the cell environment by modulation of cell-extracellular matrix interactions, cell-cell interactions, or soluble stimuli. Many of these environmental influences are easily controlled using macroscopic techniques; however, in co-culture systems with two or more cell types, cell-cell interactions have been difficult to manipulate precisely using similar methods. Although microfabrication has been widely utilized for the spatial control of cells in culture, these methods have never been adapted to the simultaneous co-cultivation of more than one cell type. We have developed a versatile technique for micropatterning of two different cell types based on existing strategies for surface modification with aminosilanes linked to biomolecules and the manipulation of serum content of cell culture media. This co-culture technique allowed manipulation of the initial cellular microenvironment without variation of cell number. Specifically, we were able to control the level of homotypic interaction in cultures of a single cell type and the degree of heterotypic contact in co-cultures over a wide range. This methodology has potential applications in tissue engineering, implant biology, and developmental biology, both in the arena of basic science and optimization of function for technological applications.