Nanotechnology: emerging tools for biology and medicine

Historically, biomedical research has been based on two paradigms. First, measurements of biological behaviors have been based on bulk assays that average over large populations. Second, these behaviors have then been crudely perturbed by systemic administration of therapeutic treatments. Nanotechnology has the potential to transform these paradigms by enabling exquisite structures comparable in size with biomolecules as well as unprecedented chemical and physical functionality at small length scales. Here, we review nanotechnology-based approaches for precisely measuring and perturbing living systems. Remarkably, nanotechnology can be used to characterize single molecules or cells at extraordinarily high throughput and deliver therapeutic payloads to specific locations as well as exhibit dynamic biomimetic behavior. These advances enable multimodal interfaces that may yield unexpected insights into systems biology as well as new therapeutic strategies for personalized medicineDamon Runyon Cancer Research Foundation (Merck Fellow, DRG-2065-10)Howard Hughes Medical Institute (Investigator)Lustgarten FoundationNational Institutes of Health (U.S.) (U54CA151884, , Massachusetts Institute of Technology-Harvard Center of Cancer Nanotechnology Excellence)National Institutes of Health (U.S.) (P41- EB002503, BIoMEMS Resource Center

Modeling host interactions with hepatitis B virus using primary and induced pluripotent stem cell-derived hepatocellular systems

Hepatitis B virus (HBV) chronically infects 400 million people worldwide and is a leading driver of end-stage liver disease and liver cancer. Research into the biology and treatment of HBV requires an in vitro cell-culture system that supports the infection of human hepatocytes, and accurately recapitulates virus–host interactions. Here, we report that micropatterned cocultures of primary human hepatocytes with stromal cells (MPCCs) reliably support productive HBV infection, and infection can be enhanced by blocking elements of the hepatocyte innate immune response associated with the induction of IFN-stimulated genes. MPCCs maintain prolonged, productive infection and represent a facile platform for studying virus–host interactions and for developing antiviral interventions. Hepatocytes obtained from different human donors vary dramatically in their permissiveness to HBV infection, suggesting that factors—such as divergence in genetic susceptibility to infection—may influence infection in vitro. To establish a complementary, renewable system on an isogenic background in which candidate genetics can be interrogated, we show that inducible pluripotent stem cells differentiated into hepatocyte-like cells (iHeps) support HBV infection that can also be enhanced by blocking interferon-stimulated gene induction. Notably, the emergence of the capacity to support HBV transcriptional activity and initial permissiveness for infection are marked by distinct stages of iHep differentiation, suggesting that infection of iHeps can be used both to study HBV, and conversely to assess the degree of iHep differentiation. Our work demonstrates the utility of these infectious systems for studying HBV biology and the virus’ interactions with host hepatocyte genetics and physiology.Skolkovo Institute of Science and Technology (Grant 022423-003)National Institutes of Health (U.S.) (Grant UH2 EB017103)National Institutes of Health (U.S.) (Grant DK085713)National Cancer Institute (U.S.) (Koch Institute Support. Grant P30-CA14051)American Gastroenterological Association (Research Scholar Award)National Institutes of Health (U.S.) (Grant 1K08DK101754)Hertz Foundation (Fellowship)National Science Foundation (U.S.). Graduate Research Fellowship Progra

A computational framework for identifying design guidelines to increase the penetration of targeted nanoparticles into tumors

Targeted nanoparticles are increasingly being engineered for the treatment of cancer. By design, they can passively accumulate in tumors, selectively bind to targets in their environment, and deliver localized treatments. However, the penetration of targeted nanoparticles deep into tissue can be hindered by their slow diffusion and a high binding affinity. As a result, they often localize to areas around the vessels from which they extravasate, never reaching the deep-seeded tumor cells, thereby limiting their efficacy. To increase tissue penetration and cellular accumulation, we propose generalizable guidelines for nanoparticle design and validate them using two different computer models that capture the potency, motion, binding kinetics, and cellular internalization of targeted nanoparticles in a section of tumor tissue. One strategy that emerged from the models was delaying nanoparticle binding until after the nanoparticles have had time to diffuse deep into the tissue. Results show that nanoparticles that are designed according to these guidelines do not require fine-tuning of their kinetics or size and can be administered in lower doses than classical targeted nanoparticles for a desired tissue penetration in a large variety of tumor scenarios. In the future, similar models could serve as a testbed to explore engineered tissue-distributions that arise when large numbers of nanoparticles interact in a tumor environment.Human Frontier Science Program (Strasbourg, France)David H. Koch Institute for Integrative Cancer Research at MIT (Marie D. and Pierre Casimir-Lambert Fund)National Institutes of Health (U.S.) (Grant U54 CA151884)National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051

HeLiVa platform: integrated heart-liver-vascular systems for drug testing in human health and disease

Our project team is developing an integrated microphysiological platform with functionally connected vascular, liver and cardiac microtissues derived from a single line of human pluripotent stem cells. The platform enables functional representation of human physiology in conjunction with real-time biological readouts (via imaging and homologous reporters for all three cell phenotypes) and compatibility with high-throughput/high-content analysis. In this paper, we summarize progress made over the first year of the grant

Macro-to-Micro Interface for the Control of Cellular Organization

The spatial organization of cellular communities plays a fundamental role in determining intercellular communication and emergent behavior. Few tools, however, exist to modulate tissue organization at the scale of individual cells, particularly in the case of dynamic manipulation. Micromechanical reconfigurable culture achieves dynamic control of tissue organization by culturing adherent cells on microfabricated plates that can be shifted to reorganize the arrangement of the cells. Although biological studies using this approach have been previously reported, this paper focuses on the engineering of the device, including the mechanism for translating manual manipulation to precise microscale position control, fault-tolerant design for manufacture, and the synthetic-to-living interface.National Science Foundation (U.S.) (Faculty Early Career Development Program)National Institute of Diabetes and Digestive and Kidney Diseases (U.S.)David & Lucile Packard FoundationNational Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service Awar

The impact of coping strategies of cancer caregivers on psychophysiological outcomes: an integrative review

A growing number of studies have explored the psychosocial burden experienced by cancer caregivers, but less attention has been given to the psychophysiological impact of caregiving and the impact of caregivers' coping strategies on this association. This paper reviews existing research on the processes underlying distress experienced by cancer caregivers, with a specific focus on the role of coping strategies on psychophysiological correlates of burden

Effect of blood storage on electrolyte levels

Background: Blood transfusion can be an immediate life saving measure in several acute conditions such as hemorrhage and anemia. However, various post transfusion complications are observed in patients which may be associated with the storage conditions of the collected blood. Electrolytes play a major role in maintaining homeostasis within the cells. Potassium is the most important extracellular cation responsible for maintenance of the cell integrity. Prolonged and improper storage of blood can lead to leakage of electrolytes, thus changing the cell morphology. This can adversely affect the patients who receive such blood. This study helps us analyze the effect of blood storage on electrolyte levels.Methods: For the study, 10ml of blood was collected from 30 blood bags containing CPDA-1 at the time of blood donation from 30 different volunteers. This blood containing the CPDA-1 was divided into 5 parts of 2ml and each 2ml sample was stored in plain bulbs. All the samples were stored at 4°C. Samples were tested to check for changes in the electrolyte (Na+, K+, Cl-) levels on day 0, 3, 7, 14 and 21. ANOVA was used to calculate the variance in the electrolyte levels.Results: Average sodium level on day 0 was 152.9±3.8 mEq/l. There was a significant decrease and it was measured at 139.5±4.8 mEq/l on day 21. Average potassium level on day 0 was 4.2±0.4 mEq/l. A significant spike was observed in potassium levels. The final reading of potassium level on day 21 was 15.2±0.7 mEq/l. Average chloride level on day 0 was 71.9±6.6 mEq/l which significantly declined to 67±5.9 mEq/l.Conclusions: Though blood is stored in proper conditions, a biochemical change occurs within the cells due to prolonged storage and thus affects its viability

Pluripotent stem cell-derived hepatocyte-like cells

Liver disease is an important clinical problem, impacting over 30 million Americans and over 600 million people worldwide. It is the 12th leading cause of death in the United States and the 16th worldwide. Due to a paucity of donor organs, several thousand Americans die yearly while waiting for liver transplantation. Unfortunately, alternative tissue sources such as fetal hepatocytes and hepatic cell lines are unreliable, difficult to reproduce, and do not fully recapitulate hepatocyte phenotype and functions. As a consequence, alternative cell sources that do not have these limitations have been sought. Human embryonic stem (hES) cell- and induced pluripotent stem (iPS) cell-derived hepatocyte-like cells may enable cell based therapeutics, the study of the mechanisms of human disease and human development, and provide a platform for screening the efficacy and toxicity of pharmaceuticals. iPS cells can be differentiated in a step-wise fashion with high efficiency and reproducibility into hepatocyte-like cells that exhibit morphologic and phenotypic characteristics of hepatocytes. In addition, iPS-derived hepatocyte-like cells (iHLCs) possess some functional hepatic activity as they secrete urea, alpha-1-antitrypsin, and albumin. However, the combined phenotypic and functional traits exhibited by iHLCs resemble a relatively immature hepatic phenotype that more closely resembles that of fetal hepatocytes rather than adult hepatocytes. Specifically, iHLCs express fetal markers such as alpha-fetoprotein and lack key mature hepatocyte functions, as reflected by drastically reduced activity (~ 0.1%) of important detoxification enzymes (i.e. CYP2A6, CYP3A4). These key differences between iHLCs and primary adult human hepatocytes have limited the use of stem cells as a renewable source of functional adult hepatocytes for in vitro and in vivo applications. Unfortunately, the developmental pathways that control hepatocyte maturation from a fetal into an adult hepatocyte are poorly understood, which has hampered the field in its efforts to induce further maturation of iPS-derived hepatic lineage cells. This review analyzes recent developments in the derivation of hepatocyte-like cells, and proposes important points to consider and assays to perform during their characterization. In the future, we envision that iHLCs will be used as in vitro models of human disease, and in the longer term, provide an alternative cell source for drug testing and clinical therapy.National Institutes of Health (U.S.) (Roadmap for Medical Research Grant 1 R01 DK085713-01))American Gastroenterological Association (Research Scholar Award

Quantum dots to monitor RNAi delivery and improve gene silencing

A critical issue in using RNA interference for identifying genotype/phenotype correlations is the uniformity of gene silencing within a cell population. Variations in transfection efficiency, delivery-induced cytotoxicity and ‘off target’ effects at high siRNA concentrations can confound the interpretation of functional studies. To address this problem, we have developed a novel method of monitoring siRNA delivery that combines unmodified siRNA with seminconductor quantum dots (QDs) as multi color biological probes. We co-transfected siRNA with QDs using standard transfection techniques, thereby leveraging the photostable fluorescent nanoparticles to track delivery of nucleic acid, sort cells by degree of transfection and purify homogenously-silenced subpopulations. Compared to alternative RNAi tracking methods (co-delivery of reporter plasmids and end-labeling the siRNA), QDs exhibit superior photostability and tunable optical properties for an extensive selection of non-overlapping colors. Thus this simple, modular system can be extended toward multiplexed gene knockdown studies, as demonstrated in a two color proof-of-principle study with two biological targets. When the method was applied to investigate the functional role of T-cadherin (T-cad) in cell–cell communication, a subpopulation of highly silenced cells obtained by QD labeling was required to observe significant downstream effects of gene knockdown