Hong Zhao, PhD - US grants

Atomically thin, 2D nanosheets are promising components for next-generation electronics. However, there is a lack of scalable manufacturing processes to fully showcase the superior properties of nanosheet materials. Specifically, no currently available technique has the requisite placement accuracy and topology control to build aligned stacks of unwrinkled nanosheets. This award supports fundamental research on a novel dual-droplet electrohydrodynamic printing process. Research results can enable the development of a unique additive manufacturing platform for patterning nanosheets, as well as other anisotropic colloidal particles (e.g., nanowires, and quantum dots). Such technology is crucial for the US to stay competitive in manufacturing and bring forth novel applications of nanosheets in high-performance printed electronics, sensors, actuators, and energy devices.

The new dual-droplet electrohydrodynamic printing process involves first depositing a support droplet which acts as a Langmuir-Blodgett trough, followed by a wetting droplet containing colloidal 2D nanosheets. Assembly of the 2D nanosheets will occur as the support droplet evaporates. The research objectives are (1) to understand the effects of solvent surface tensionand volume ratio of the support and wetting droplets on the spreading of the wetting droplet over the support droplet; (2) to understand the effects of nanosheet size and concentration, and substrate wetting properties on the alignment of nanosheets; and (3) to establish the structure-property relationships of the deposited nanosheets. Graphene and Molybdenum disulfide nanosheets will be used in this study. To achieve the first objective, the dual-droplet printing experiments will be conducted. Solvent surface tension will be varied between 30-50 mN/m by changing solvent composition, and volume ratio will be varied from 1 to 100 by changing the driving voltage and pulse width for both support and wetting droplets. The temporal change of spreading area will be measured by high-speed photography with a few tens of microseconds resolution. The second objective will be achieved by both experimental study and computer simulation. For dual-droplet printing experiments, nanosheet size will be varied between 0.2-10 µm in mean diameter, nanosheet concentration in the wetting droplet between 0.01-1 mg/mL, and the receding contact angle of the support droplet will be varied from about 0° with a pinned contact line up to ~90° with a depinned contact line. The nanosheet alignment in the assembly will be analyzed by microscopy characterization. A model of Lagrangian particle tracking will be created for prediction of nanosheet alignment, where molecular dynamics simulation will compute nanosheet dynamics under the evaporation-induced flow. Simulation predictions will be verified by experimental results in terms of nanosheet orientation and alignment. To achieve the third objective, the structure (in terms of topological roughness, sheet-to-sheet alignment, gaps or overlaps between nanosheets) of the deposited nanosheet assembly will be measured using electron microscopy and atomic force microscopy, and the property (conductivity) will be measured using four-point probe.

Inkjet printing as an additive fabrication method has been used in the manufacturing of printed electronics, 3-D object prototypes, solar cells, and light-emitting devices, as well as applications in tissue engineering and other biological and pharmaceutical fields. Unlike the more common method of piezo inkjet printing, which typically generates individual droplets of 10-50 micrometers in diameter, voltage-modulated electrohydrodynamic (EHD) jet printing has a demonstrated ability to produce sub-micrometer-sized droplets/fibers for the fabrication of patterns or features at nanometer scales. However, EHD jet printing has not been considered as a viable manufacturing tool because of the issues of nozzle clogging, ink accumulation at the nozzle exit, and low printing frequencies (resulting in a limited production rate). This project conducts fundamental research on a new form of EHD jet printing, using novel dual-channel printing nozzles and electrical current (instead of voltage) modulation. It is hypothesized that the circulation of liquid ink in the dual channels will eliminate nozzle clogging due to evaporation of carrier fluids or polymerization of ink, and the current modulation/control will enable high-speed, drop-on-demand EHD jet printing. This research could also lead to further improvements on current inkjet printing processes or devices in both industrial and household settings. Furthermore, the technique will be used in education and outreach activities geared toward students (at all levels) and workers in advanced manufacturing.

This project will investigate the fundamental science involved in a new current-modulated, drop-on-demand EHD printing method with novel dual-channel nozzles for the fabrication of high-resolution micro/nano patterns at high jetting frequencies (on the level of MHz). The proposed dual-channel printing nozzles use two concentric tubes, providing an annular channel around the inner tube. In the proposed nozzle configuration, one channel provides new ink and the other for extracts ink from the nozzle, thereby achieving fluid circulation within the dual tube nozzle. It is hypothesized that this fluid circulation will eliminate or greatly reduce the issues of nozzle clogging and ink accumulation associated with polymerization or carrier fluid evaporation at the nozzle outlet.  This project further hypothesizes that the ejection rate of droplets can be increased through current control, instead of the voltage control commonly used. Scientific understanding of fluid meniscus dynamics and droplet generation in the proposed EHD printing will be required to achieve robust current control, and needs to incorporate effects of fluid recirculaiton. The novelties of the proposed EHD jet printing technique are i) the proposed dual-channel nozzles that will resolve the technical issues often encountered in single-capillary inkjet printing; and ii) the modulation of frequencies with current rather than voltage in order to achieve reliable EHD jet printing at high jetting frequencies. The specific aims of this project are to: i) develop the fundamental science involved in liquid meniscus formation, jetting, and droplet ejection in a voltage-modulated EHD jet printing process with the dual-channel nozzles; ii) investigate the fundamental jetting mechanisms in current-modulated EHD jet printing process (particularly at high frequencies); iii) numerically model the jetting characteristics in the EHD jet printing technique to develop the underlying fundamental science and aide control strategies; and iv) provide a proof-of-concept of the proposed approach and validate the numerical models through parametric investigations of the quality (i.e. the size, uniformity, and resolution) of micro/nano-sized patterns created.

Although more than 1 in 4 men can be expected to develop symptomatic inguinal hernia, its mechanism is currently unknown. A subset of hernias may develop due to muscle fibrosis and myofiber atrophy leading to lower abdominal wall weakness. The long-term objective of this application is to determine the role of estrogen action in the etiology of lower abdominal muscle tissue (LAMT) fibrosis and atrophy associated with a subset of inguinal hernias. Aromatase, which converts testosterone to estradiol, is expressed only in the brain and testes of male mice. However, in men, aromatase is expressed in many additional tissues (muscle, fat) to provide physiologically necessary local quantities of estrogen. We generated transgenic humanized aromatase (Aromhum) mouse lines, each containing a single copy of the full-length human aromatase gene including its regulatory region, to mimic human patterns of estrogen production. Aromhum mice express the aromatase gene in peripheral tissues including the fibroblast component of the skeletal muscle tissue. LAMT has been found to be more sensitive to estradiol than the upper abdominal or quadriceps muscles, because the stroma of LAMT contains strikingly larger amounts of estrogen receptor-? (ER?)-expressing fibroblasts. Locally increased concentrations of estradiol in LAMT was associated with LAMT fibrosis characterized by progressive replacement of atrophic myocytes (muscle fibers) with ER?-rich fibroblasts and excessive extracellular matrix, resulting in formation of large inguinal hernias in >90% of Aromhum male mice by 24 weeks. However, there were no hernias observed in any of the wild-type (WT) littermates. Microarray expression analysis of LAMT at four weeks (before the appearance of hernias) showed activated profibrotic pathways in Aromhum vs. WT mice. We hypothesize that enhanced estrogen action caused by locally formed estradiol drives muscle fibrosis and myocyte atrophy, leading to the hernia phenotype affecting highly estrogen-sensitive portions of skeletal muscle tissue, which is LAMT in Aromhum mice. This resonates with the remarkable and parallel increases in inguinal hernia incidence and increased aromatase expression in skeletal muscle and fat in aging men. To ascertain the underlying mechanisms, we propose the following aims: 1. Determine whether treatment with an aromatase inhibitor, an estradiol antagonist, or a highly selective ER? antagonist prevents fibrosis, LAMT muscle atrophy, and hernia formation in Aromhum mice. The estradiol/ER?-mediated genomic mechanisms responsible for disordered proliferation of fibroblasts and extracellular matrix formation will be determined using integrative analysis of RNA-seq and ER?-ChIP-seq on LAMT and fibroblasts. 2. Determine whether the genetic disruption of ER? selectively in skeletal muscle fibroblasts affects LAMT fibrosis and hernia formation in Aromhum mice. In parallel, we will assess tissue steroid levels, aromatase and ER??expression, and estrogen responsive genes in abdominal muscle biopsies of men with or without hernia. We anticipate that this novel proposal will identify new drug targets and likely lead to the discovery of preventive approaches for hernia in high-risk populations.

As treatment outcomes of primary or systemic cancer sites improve, the clinical importance of brain metastasis (BM) is growing. Twenty-four to 45 percent of all cancer patients develop BM, the majority from lung, breast or melanoma primary cancers, but few patients with BM live longer than a year, and BM constitutes 20% of annual cancer deaths. Ironically, recent advancement in chemotherapy has further increased the incidence of BM because most therapeutic agents cannot effectively penetrate the blood-brain barrier (BBB) and tumor cells find the brain as a sanctuary. Therefore, it is of paramount importance to have a deeper understanding of mechanisms that promote BM growth, which could be specifically leveraged to overcome current limitations in therapy. As opposed to the molecular mechanisms involving cancer cell?host interactions shared by multiple cancer types that result in organ specific metastasis, a highly distinct set of structural, anatomic, physiologic and molecular factors regulate metastasis to the brain. Astrocytes, the most common glial cell comprising ~ 50% of all human brain cells, are a well characterized perilesional component of BM and recent discoveries, including ours, provide compelling evidence that molecular crosstalk between astrocytes and cancer cells is integral to BM development. Although seminal findings indicate that interactions with astrocytes occur at both early and late stages of tumor colonization process, our understanding of the reciprocal astrocyte-cancer cell crosstalk is limited. In preliminary studies, we have employed our Cell-Cell Communication Explorer (CCCExplorer), a unique computational modeling tool, in identifying the novel PCDH7-EGFR, IL6-IL6R, and CCL5-CCR5 astrocyte-tumor crosstalk signaling in regulating BM. Based on these observations and in view of the secretory nature of glial cells, we propose here to test the hypothesis that crosstalk with astrocyte-derived secreted factors is critical for tumor cell colonization in the brain. Given that an even more complicated paracrine signaling network may dynamically evolve at different stages of BM development, and the interactions could provide both anti- and pro-metastatic stimuli to cancer cells, we will test our hypothesis through the following aims: 1) to assess therapeutic potential of the PCDH7- EGFR, IL6-IL6R and CCL5-CCR5 paracrine signaling in BM mouse models employing gain and loss of function and pharmacologic approaches in syngeneic mouse and human cancer xenografts; 2) to assess the astrocyte secreted proteins in modifying the function of BBB and microglia/macrophage in early BM; 3) to further characterize the temporally evolved astrocyte-BM cell crosstalks in a cancer type specific fashion. Our study is highly innovative in that (i) this study integrates knowledge and methods from both neuroscience and cancer to identify and characterize pro- and anti-metastatic astrocyte molecular mechanisms, their evolution during disease progression, and their manipulation in order to provide a valuable means of targeting astrocyte-cancer cell interactions. (ii) This study leverages powerful predictive modeling of cell-cell communications (CCCExplorer) to investigate and delineate the complex network of tumor-astrocyte interactions holistically in an unbiased manner. (iii) This study will address whether there is any specific therapeutic window as to which time point during BM might represent the most effective point of modulating and targeting the vicious astrocyte-tumor crosstalk. (iv) Given the strong response of astrocytes to BM during the course of brain colonization, the identification of secreted molecules may represent putative biomarkers of early diagnosis or response to therapy. (v) Data generated in this study would form an extraordinary repository for comparative analyses between different brain disorders to interrogate common and different aspects of astrocyte biology in different scenarios as well as to evaluate the potential new therapeutic strategies such as drug repurposing and combinations. The outcome of our study will provide a paradigm shift in current understanding of the pathology of BM, while achieving a significant impact on future treatments for this devastating disease.