Hyunjoon Kong - US grants

ID: MPS/DMR/BMAT(7623) 0847253 PI: Kong, Hyunjoon ORG: Illinois-UC

Title: CAREER: Biomaterials and Biology for Control of Cell Function in 3D Matrices

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

INTELLECTUAL MERIT: The extracellular matrix (ECM) in human tissues plays a critical role in protecting and nourishing cells by providing cell adhesion sites and sequestering growth factors. Artificially constructed ECM-simulating biomaterials are often combined with cells and growth factors for use in biological studies and clinical treatments of tissue defects and several chronic diseases. However, the biological role played by growth factors remains unclear due to limited understanding about complex cellular microenvironment. This lack of understanding also limits the therapeutic efficacy of growth factors. In order to establish a more thorough understanding of the role of growth factors in regulating cell function, the proposed research goal is to create a cell-encapsulating hydrogel that allows researchers to regulate growth factor-induced signal transduction in cells by engineering the cellular microenvironment. The PI specifically hypothesizes that the hydrogel stiffness modulates the extent of growth factor binding to cells and subsequently affects the efficacy of the growth factors in stimulating cell growth and differentiation. Experiments to examine this hypothesis will require decoupling the currently interdependent gel stiffness and permeability to nutrients by building hydrogels with nearly constant chain density but with varying crosslink density. It is expected that changes in crosslink density will not materially affect diffusion through the gel at a given chain density.

BROADER IMPACTS: A successful demonstration that matrix elasticity and growth factor efficacy can be decoupled would open the way to advances in tissue engineering and regenerative medicine. The proposed educational goal is to create a cross-disciplinary biomaterials curriculum for college students plus a variety of outreach programs designed to fill the knowledge gap with motivated and diverse scientists. The educational plan will leverage existing institutional outreach programs for pre-college students, including women and minorities, and science teachers to ensure successful completion. The goals of this CAREER proposal will be accomplished by implementing four research and educational projects: (Project 1) designing and creating a biomaterial that decouples the interdependency between biomaterial stiffness and nutrient transport; (Project 2) establishing a set of integrative biomaterial design principles that describe growth factor-induced signal transduction with biomaterial stiffness; (Project 3) creating a cross-disciplinary course entitled ?Biomaterial-Biological Systems Interactions in Biology and Medicine? and providing students with research opportunities to serve as a model that other institutions may adopt, adapt, and build upon; and (Project 4) introducing pre-college students and teachers to the science between biomaterials and biology to inspire them and increase the number and diversity of students in the biomaterial field.

DESCRIPTION (provided by applicant): Ischemia in myocardial and peripheral tissues is a leading cause of heart failure and tissue necrosis in the United States. Ischemic diseases are clinically treated with drug administration and surgery, which still meet many challenges for treatment on a permanent basis. Recently, revascularization therapy to rebuild the vascular network of ischemic tissue via angiogenesis, vasculogenesis or both is being extensively studied to restore blood perfusion in various tissues. A variety of stem and progenitor cells are promising revascularization medicines in conjunction with several angiogenic cytokines and growth factors. Commonly, these cells are transplanted via intracoronary injection, but the therapeutic efficacy of transplanted cells is greatly reduced by a significant loss of cells due to the absence of the signals to guide the cells to the injured endothelium. The objectives of this proposed study are to develop a nano-sized cell guidance molecule and attach it to the transplanted cells, so the transplanted cells can pinpoint the injured endothelium and subsequently improve blood perfusion of ischemic tissue. We hypothesize that a hyper-branched poly(glycerol) linked with both epitopes binding with transplanted cells and those binding with vascular cell adhesion molecules (VCAM)-1 will precisely guide transplanted cells to the injured endothelium because the endothelial injury stimulates endothelial cells to over-express VACM-1. Ultimately, this tuning of cell guidance will significantly improve restoration of blood perfusion in the ischemic tissue. We will examine this hypothesis using endothelial progenitor cells (EPCs) derived from a porcine cord blood. The oligopeptide containing RGD sequence (RGD peptide) will be used as the EPC-binding epitope and that containing VHSPNKK sequence (VHSPNKK peptide) will be used as the VCAM1- binding epitope. The oligopeptide structure will be varied to improve the binding affinity to cells and VCAM-1. These two oligopeptides will be chemically linked to the poly(glycerol). The degree of oligopeptides substitution to poly(glycerol) will be further optimized with in vitro analysis. Specifically, we will use a fluorescence resonance energy transfer (FRET) technique we previously developed to quantify the number of poly(glycerol) bound to EPCs. We will complete this proposed study by first functionalizing poly(glycerol) with RGD peptides [RGD- poly(glycerol)] and analyzing the amount of poly(glycerol) bound with EPCs (Aim 1), secondly modifying RGD-poly(glycerol) with VHSPNKK peptides [RGD-poly(glycerol)-VHSPNKK] and analyzing its ability to guide EPCs to the synthetic endothelium (Aim 2) and finally demonstrate the function of bioactive poly(glycerol) in vivo using the immunodeficient mouse with an ischemic hindlimb (Aim 3). This study will be performed through the interdisciplinary collaboration between a tissue engineer (Kong, investigator), chemist (Zimmerman) and biologist (Schook). Kong and Zimmerman's groups are responsible for the synthesis of bioactive poly(glycerol) and evaluation of its ability to enhance the transplanted cell adhesion to the target ischemic tissue in vitro and in vivo. The cell isolation from a cord blood and characterization will be evaluated by the Schook group. We believe that the successful completion of this proposed study will significantly minimize the loss of transplanted cells and improve the therapeutic potency of EPCs for repairing ischemic tissue. Results from our in vitro and in vivo studies will be readily translated into the large scale preclinical and clinical trials, and aid the expedition of cell-based neovascularization therapies to the clinical setting. Finally, this design strategy of a cell guidance system and quantitative analysis of the molecular binding with cells and target tissue will be widely applicable to a broad array of stem and progenitor cells for the treatment of many diseases. PUBLIC HEALTH RELEVANCE: The successful completion of this proposed study will create a precision cell guidance system that will greatly improve the regenerative efficacy of therapeutic cells and expedite the use of cells in clinical treatment of ischemic disease. Specifically, the through in vitro and in vivo analysis of cell guidance system will expedite the translation of the results of this study into the clinical trials. In the end, this study will aid saving a number of patients who suffer from the ischemic disorders of myocardial and peripheral tissues.

1033906
Boppart

Despite important advances within the past decade, current tissue engineered skin equivalents represent little more than a fragile, short-term dressing for patients that need viable skin replacements. The major weakness in current skin equivalents is that the constituent cells are cultured and applied under conditions that are very different from that of natural skin. It is believed that this is principally due to our limited understanding of the roles that 3-D scaffold topography and mechanical stimuli have on the intercellular organization, connectivity, and communication of engineered tissues. In this project an advanced integrated microscope capable of simultaneous optical coherence and multi-photon microscopy, and optical coherence elastography is utilized to uniquely visualize the structural and functional relationships of cells within 3-D engineered skin constructs, and measure the evolving biomechanical properties. Second, this project investigates and longitudinally images in 3-D the growth of engineered skin constructs on varying microtopographic substrates. This will provide fundamental insight into the mechanical influences at the dermal-epidermal junction on the keratinocytes and fibroblasts. Third, the effects of mechanical stimuli on these constructs will be investigated, defining how varying stimuli affect the 3-D cell dynamics and tissue organization over time. such stimulation effects will provide a more physiologically-relevant culture condition. Finally, this project will longitudinally image the cell and tissue responses in vivo following the grafting of the skin constructs to host pre-clinical models, contributing significantly to our understanding of how engineered tissue grafts interface with biological hosts.

DESCRIPTION (provided by applicant): The objective of this proposed study is to synthesize and validate multifunctional 3T (targeting, tracking, and treating) nanocells for repair of blood vessels damaged by acute renal ischemic- reperfusion injury. For this study, nanocells are defined as nano-sized drug-encapsulating polymersomes, structurally similar to biological cells. Clinical studies suggest that certain antibodies and cytokines that bind to endothelial cells can be used as drugs that induce vascular normalization and ultimately improve treatments of various acute, chronic and malignant diseases. It has been often proposed that such vascular normalization therapies can be significantly improved by combining these drugs with carriers capable of targeting and tracking to the target blood vessels. However, the development of such multifunctional drug carriers has been plagued by difficulties in independently controlling targeting, tracking and treatment functions. We hypothesize that the 3T function of nanocells can be independently tuned by (1) integrating into the nanocell via self- assembly process, a targeting module, a poly (glycerol) substituted with varying numbers of alkyl chains and leaky endothelium-targeting oligopeptides, and (2) further incorporating into the nanocell via in situ encapsulation, surface-engineered super paramagnetic iron oxide nanoparticles that enable tracking of the nanocell via magnetic resonance imaging (MRI). The resulting 3T nanocells will allow us to significantly improve the vascular normalization while monitoring 3T nanocells'therapeutic activity using MRI. We will accomplish our goals, first, by modifying and validating the nanocells with targeting modules via self-assembly [Aim 1];second, by encapsulating iron oxide nanoparticles in the nanocell created in the Aim 1 study and validating its tracking function [Aim 2];and finally incorporating drugs that normalize leaky blood vessels, specifically Angiopoietin 1, in the nanocells created in the Aim 2 study and evaluating its function to treat porcine renal arteries damaged by acute ischemia-reperfusion injury [Aim 3]. In this study, polymersomes of alkyl-substituted poly (2-hydroxy ethyl aspartamide) (PEHA) filled with biodegradable poly (ethylene glycol) nanogels will be used as nanocells. This proposed study will be implemented through an extensive interdisciplinary collaboration between a biomaterials group [Kong, University of Illinois (UI)];organic and polymer synthesis group [Zimmerman, UI];and bioimaging and vascular medicine group [Misra, Mayo Clinic]. The results of this proposed study are expected to significantly impact research in bioengineering and clinical strategies in medicine, because it will not only create an innovative strategy for assembling multifunctional drug carriers, but also validate its functionality to improve vascular normalization. PUBLIC HEALTH RELEVANCE: The successful completion of this proposed study will create an innovative strategy for assembling a multifunctional drug carrier, 3T nanocell, which allows independent tuning of targeting, tracking and treating functions. Ultimately, this study will create a novel drug delivery system which will significantly improve the therapeutic efficacy of drugs that induce vascular normalization. Overall, this study will greatly contribute to improving peoples'quality of life who is suffering from a wide array of acute, chronic and malignant diseases related to leaky blood vessels.

DESCRIPTION (provided by applicant): The objective of this proposed study is to synthesize and validate multifunctional 3T (targeting, tracking, and treating) nanocells for repair of blood vessels damaged by acute renal ischemic- reperfusion injury. For this study, nanocells are defined as nano-sized drug-encapsulating polymersomes, structurally similar to biological cells. Clinical studies suggest that certain antibodies and cytokines that bind to endothelial cells can be used as drugs that induce vascular normalization and ultimately improve treatments of various acute, chronic and malignant diseases. It has been often proposed that such vascular normalization therapies can be significantly improved by combining these drugs with carriers capable of targeting and tracking to the target blood vessels. However, the development of such multifunctional drug carriers has been plagued by difficulties in independently controlling targeting, tracking and treatment functions. We hypothesize that the 3T function of nanocells can be independently tuned by (1) integrating into the nanocell via self- assembly process, a targeting module, a poly (glycerol) substituted with varying numbers of alkyl chains and leaky endothelium-targeting oligopeptides, and (2) further incorporating into the nanocell via in situ encapsulation, surface-engineered super paramagnetic iron oxide nanoparticles that enable tracking of the nanocell via magnetic resonance imaging (MRI). The resulting 3T nanocells will allow us to significantly improve the vascular normalization while monitoring 3T nanocells' therapeutic activity using MRI. We will accomplish our goals, first, by modifying and validating the nanocells with targeting modules via self-assembly [Aim 1]; second, by encapsulating iron oxide nanoparticles in the nanocell created in the Aim 1 study and validating its tracking function [Aim 2]; and finally incorporating drugs that normalize leaky blood vessels, specifically Angiopoietin 1, in the nanocells created in the Aim 2 study and evaluating its function to treat porcine renal arteries damaged by acute ischemia-reperfusion injury [Aim 3]. In this study, polymersomes of alkyl-substituted poly (2-hydroxy ethyl aspartamide) (PEHA) filled with biodegradable poly (ethylene glycol) nanogels will be used as nanocells. This proposed study will be implemented through an extensive interdisciplinary collaboration between a biomaterials group [Kong, University of Illinois (UI)]; organic and polymer synthesis group [Zimmerman, UI]; and bioimaging and vascular medicine group [Misra, Mayo Clinic]. The results of this proposed study are expected to significantly impact research in bioengineering and clinical strategies in medicine, because it will not only create an innovative strategy for assembling multifunctional drug carriers, but also validate its functionality to improve vascular normalization.

PI: Kong, Hyunjoon
Proposal Number: 1403491
Institution: University of Illinois at Urbana-Champaign
Title: Interrogating Cadherin/Matrix Rigidity Dependent Neural Differentiation and Neuromuscular Junction Formation of Multipotent Stem Cells

Understanding and recreating neural networks represents a major scientific and engineering challenge with ramifications ranging from fundamental biology to clinical translation. Stem cells residing in bone and fat tissue possess the potential to be converted to neurons that constitute neural networks. This cell conversion process is stimulated when cells contact each other and are cultured on a substrate that is not ?too hard? or ?too soft?, but that is ?just right?. These neurons will help control muscle tissue movement, thus serving to create various biomedical tools used for fundamental and applied bioscience studies. The studies in this proposal use a Jello-like hydrogel system linked with proteins involved in cell-cell communication and tuned to present a ?just right? softness. The successful completion of this project will therefore create an advanced cell culture platform that will be broadly useful for studying a wide array of stem cells and for exploiting this in neural tissue engineering. The proposed work will also uncover the coordinate effects of cell-cell adhesions and matrix softness on stem cell fates, including differentiation into neurons. Finally, the results of the proposed study will be highly useful in creating various neural implants more efficiently than previously possible and intelligent, biological robots. Utilization of the results of this study as an educational module will have broad impacts by attracting future young scientists and encouraging them to pursue engineering careers. This proposal is co-funded by the Biomedical Engineering Program in the Chemical, Bioengineering, Environmental and Transport Systems Division, and by the Biomaterials Program in the Division of Materials Research.

There have been numerous attempts to reproduce neural networks and neuromuscular junctions in vitro for both fundamental and applied neuroscience studies that used neuronal cells differentiated from multipotent stem cells. Therefore, it is crucial to regulate neural differentiation of stem cells in an elaborate manner, in terms of differentiation levels and glial-to-neural cell population. The proposed research seeks to systematically understand and modulate the potential interplay between cell-cell adhesion and cell-matrix adhesion in regulating the neural differentiation of stem cells. To this end, the goals of this proposed study are (1) to modulate the neural differentiation of multipotent, bone marrow stromal cells (BMSCs) by combining the effects of cadherin, a cell-cell adhesion protein, and matrix stiffness; and (2) to use the differentiated cholinergic neurons to build a functional, neuromuscular junction in vitro. This goal will be accomplished by culturing BMSCs on a hydrogel chemically linked with a specific number of recombinant N- or E-cadherin, and further tailored to present controlled stiffness. The integrative effects of cadherin and matrix rigidity on cellular differentiation, cell traction force, and Rac1-GTPase signal activation will be analyzed. Finally, differentiated cholinergic neurons will be co-cultured with skeletal myoblasts to form a neuromuscular junction and evaluate neural control over muscular contraction and relaxation. Successful completion of the proposed studies is expected to result in significant contributions in neuroscience and neuroengineering and stem cell technology.

PROJECT SUMMARY/ABSTRACT Currently, 8-10 million people in United States suffer from vascular disease, and a large number of those affected succumb to death and major amputation. Development of methods to recreate microvascular networks and increase perfusion is necessary to prevent tissue damage. Mesenchymal stem cells (MSCs) derived from a patient?s bone marrow or adipose tissue are increasingly utilized in the clinic as a result of their potential to secrete multiple pro-vascular and regenerative factors. These factors include vascular endothelial growth factor, plasminogen activator inhibitor, and matrix metalloproteinase-9. However, it is a challenging task to sustainably regulate the secretory activity within an ischemic tissue. The goals of this proposed project are therefore to develop a nanostimulator that can associate with MSCs and regulate stem cell stromal capacity, and to use it to improve angiogenesis and physiological outcomes. We hypothesize that a nanostimulator assembled to sustainably deliver insoluble and soluble signals, two of which separately involve in cellular secretome, can sustainably stimulate MSCs to release therapeutic molecules in the ischemic tissue. To test this hypothesis, we will use interferon-gamma (IFN?) as a soluble stimulatory factor and CD44-binding hyaluronic acid (HA) as an insoluble stimulatory factor. These molecules will be spatially organized in the 400 nm-diameter poly(ethylene glycol)diacrylate gel/liposome core/shell nanoparticles. We will accomplish our goal by first modifying the core/shell particle surface with HA and examining the extent to which the resulting nanoparticle associates with MSCs and regulates cellular secretion activities (Aim 1). Second, we will introduce IFN? into the core/shell nanoparticles and evaluate the extent to which the sustained molecular release modulates cellular secretion activities (Aim 2). Finally, we will determine the extent to which MSCs loaded with the optimized core/shell nanostimulator can promote reperfusion of ischemic muscle and minimize tissue damage using a mouse ischemic hindlimb model (Aim 3). The significance of this study lies in the ability to (1) enhance the therapeutic capacity of MSCs and (2) improve quality of ischemic muscular treatment using a reduced number of stem cells. Accordingly, the innovation components include (1) control of the nanostimulator stability in physiological condition, (2) spatial organization of the HA and IFN? in the nanoparticle, (3) in situ orthogonal tailoring of cell?s secretory activities, and (4) the multidisciplinary team established to conduct this study (Kong, PI, biomaterials; Boppart, Co-I, stem cell and muscle biology).

This National Science Foundation Research Traineeship award to the University of Illinois at Urbana-Champaign will address the next frontier in biotechnology: to engineer, and then decipher and harness, the living three-dimensional brain. The program will provide doctoral students with the skills and knowledge base to develop and utilize miniature brain machinery in an effort to understand and regulate brain activities. To achieve the goals of developing cross-disciplinary researchers, trainees will learn diverse fundamentals in biology, mathematics, engineering, and cognitive science, relevant to miniature brain machinery. The training grant anticipates providing a unique and comprehensive training opportunity for sixty (60) PhD students, including thirty four (34) funded trainees. Trainees will be recruited from neuroscience, cell and developmental biology, molecular and integrative physiology, chemistry, chemical and biomolecular engineering, bioengineering, electrical and computer engineering, and psychology. The training program will foster a culture of innovation and translational research, and will produce a new generation of scientists and engineers prepared to tackle major problems in brain studies that can improve the quality of human life.

The research and training program will bridge two dominant, non-overlapping brain research paradigms: i) cognitive and behavioral studies, focused principally on understanding of adaptation, decision-making, psychology, and learning of an individual using bioimaging and computational tools vs. ii) cell and tissue studies, focused on activities of multiple neuronal cells by altering their internal and external microenvironments comprised of biomolecules, extracellular matrix, and external stimuli. The goal of this NRT training program is to unite these two dominant paradigms in brain science studies and bridge the expertise of cell and molecular biologists, physiologists, chemists, nano/micro technologists, and cognitive neuroscientists. This training program prepares students for studies that enable control over the networks producing behavior and thus to study causal relations. The overarching goal of the program is to provide students with an interdisciplinary curriculum grounded in problem-based learning and an immersive research experience that blends techniques from multiple disciplines. A second goal is to increase the participation of women, underrepresented minorities, and students with disabilities in neuroscience, life sciences, chemical sciences, and engineering fields. A third goal is to train students in communication skills with the public. Evaluative studies conducted throughout this research traineeship project will explore the dynamics and efficacy of interdisciplinary collaboration by students in this program. Project outcomes will be a demonstrated, evaluated model for transformative graduate training that is effective in developing broadly trained professionals.

The NSF Research Traineeship (NRT) Program is designed to encourage the development and implementation of bold, new potentially transformative models for STEM graduate education training. The Traineeship Track is dedicated to effective training of STEM graduate students in high priority interdisciplinary research areas, through comprehensive traineeship models that are innovative, evidence-based, and aligned with changing workforce and research needs.

Muscle is a unique tissue that can contract, allowing for movement and essential involuntary actions such as breathing, heart pumping, and digestion in humans and animals. Muscle can also secrete various chemicals, called "myokines," which are involved in proper function of the immune system and brain. Neurons are responsible for transmitting signals between the brain and muscle tissues that control muscle contraction and secretion activity. These signals travel through neurons and then transferred to the muscle through the neuromuscular junction. Many injuries and muscle diseases are due to the loss of connection between neurons and muscle. Therefore, reproducing muscle connected to neurons in vitro (in the lab) would enable better understanding of the role/importance of the neuromuscular junction, and this understanding could lead to better treatments for muscle injuries and diseases. Thus there is an urgent need for creating an in vitro physiologically relevant model of muscle connected with/innervated by neurons. To this end, the investigators aim to engineer and validate muscle that contracts and secretes myokines in response to bioelectrical signals from neurons. The project proposes that these neuron-induced muscular activities depend on size, number, and alignment of the muscle fibers in the engineered muscle. This hypothesis will be studied by co-culturing neuron-forming cells on engineered muscle tissue. The quality of neuron-innervated muscle will be evaluated by monitoring contractions and myokine secretion of muscles in response to a neural impulse. In parallel, the investigators will utilize the research program to train undergraduate and graduate students who are involved in bioengineering-related research. The research program will be incorporated into various outreach activities that aim to attract future young scientists and engineers to the biomedical area. Overall, the project will significantly impact efforts to recreate biologically functional muscle tissues and also educate the next generation of biologists and biomedical engineers in diverse ways.

The project is focused on engineering and validating motor neuron-innervated human skeletal muscle that contracts and, in turn, secrets myokines in response to neurotransmitters (e.g., glutamate). Experiments are designed to test the hypothesis that controlling the expression of acetylcholine receptors on engineered muscle is key to enhancing formation of the neuromuscular junction and that the topology and softness of a matrix on which skeletal myoblasts form muscle fibers modulate acetylcholine expression of muscle fibers. The hypothesis will be examined via three aims. The FIRST Aim is to assess the extent that topology of myoblasts-adhered substrates modulates neural innervation and contraction of muscle in response to neural impulse. Human skeletal myoblast cells will be cultured on grooved substrates with grooves (2 to 100 micrometers in width) patterned onto a collagen conjugated PEDGA gel. Studies are designed to answer to what extent the groove-patterned substrate modulates maturity and expression of acetylcholine receptors of myofibers, modulates the alignment and innervation of motor neurons, and influences the neurotransmitter-respondent muscle contraction. The SECOND Aim is to study the effects of softness of the myoblast-adhered substrate on the neural innervation into muscle. The elastic modulus of the gel will be varied from 5 to 40 kPa. Studies are designed to answer to what extent the gel softness and topology orchestrate maturity of muscle and expression of acetylcholine receptors, modulate neural innervation into muscle fibers, influence the neurotransmitter respondent muscular contraction and if neural innervation is related to the mechanotransduction. The THIRD Aim is to evaluate the extent that neuron-innervated muscle produces myokines in response to a neural impulse. Motor neuron progenitor cells will be plated on the myofibers formed on the grooved substrates and differentiated to motor neurons. The focus will be on analyzing mRNA expression and protein secretion levels that lead to increased secretion of myokines. Studies are designed to answer to what extent the neural impulse increases protein and myokine expression by the innervated muscle, to what extent the innervated muscle increases glucose uptake and fat oxidation in response to the neural impulse and to what extent the neuromuscular junction serves to increase the volume of the innervated muscle. The end result is expected to be a neuron-innervated muscle that will actively express and secrete myokines and, in turn enhance metabolic activity and increase muscle volume over time when the muscle is regularly contracted.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

PART 1: NON-TECHNICAL SUMMARY

Dense microbial films called ?biofilm? often foul medical tools, household items, and infrastructures such as an endoscopes, bathroom tiles, and water supply pipes, and can lead to dangerous infections. These biofilms are slimy aggregates of bacterial cells surrounded by scaffolds adhering to anything they touch. About 80 percent of all medical infections originate from biofilms that invade the inner workings of clinical devices and implants inside patients. Cleaning of biofilms in such a hard-to-reach area is extremely difficult because traditional disinfectants and antibiotics cannot penetrate a biofilm's tough scaffolds. How can we let antibacterial reagents cross over such a barrier of biofilms? This proposed study attempts to develop a small particle that can penetrate and destroy tough scaffolds by generating oxygen bubbles. The particle named ?self-propelling microbubbler? would be prepared by loading an oxygen-generating chemical on diatoms ? the tiny skeletons of algae. As a consequence, this system would improve the delivery of antibacterial deathblow to the bacterial cells living inside. While tuning the oxygen bubble generation rate and subsequent propulsion speed of the micrububblers, this proposed study will examine extents that the microbubblers can penetrate biofilm clinging to a material with complex topology, damages the scaffold of biofilms, kill bacterial cells protected by the scaffolds, and, ultimately, prevent the return of biofilm formation. In parallel, for broad impacts, the unique microbubblers will be used as education and training tools for a new generation of bioscientists and bioengineers. Overall, this project will serve to improve people?s health, safety, and life quality against infectious diseases and fouling.

PART 2: TECHNICAL SUMMARY

Biofilms composed of microbial cell colonies and surrounding extracellular polymer substances (EPS) are major causes of medical infection and material deterioration, thus threatening both human health and sustainability. A variety of disinfectants were developed to date, but none of these systems are active in removing biofilms forming in confined spaces. To this end, this proposed study aims to assemble and analyze a ?self-propelling microbubbler? that can invade biofilms grown in the hard-to-reach area and, subsequently, clean out both bacterial colonies and EPS. This study hypothesizes that diatom particles doped with zinc oxide (ZnO) or manganese dioxide (MnO2) catalysts decomposing hydrogen peroxide (H2O2) eject oxygen bubbles and, in turn, act as the self-propelling microbubbler in the antiseptic 3% H2O2 solution. After the invasion, the microbubblers would continue to generate oxygen bubbles that fuse to produce a wave of mechanical energy capable of destroying the biofilms. The self-propulsion of microbubblers will be studied by analyzing the activation energy for H2O2 decomposition, the H2O2 decomposition rate, and the kinetic energy. In parallel, the extent that the microbubblers remove biofilm in the micro-grooved substrate will be examined by monitoring invasion of particles into the biofilm and subsequent changes in stiffness, adhesion force, and chemical composition of the biofilms formed by Escherichia coli and Pseudomonas aeruginosa. Finally, the efficacy of microbubblers to killing biofilm bacteria will be evaluated by monitoring the increase of intracellular oxidative stress, the reduction of viable cells, and the recurrence of biofilm. The successful completion of this study will elucidate the unique cleaning mechanism attained by chemical-to-mechanical energy conversion and transform current disinfection strategies that rely on chemicals mostly. In parallel, this project will make broad impacts by incorporating the proposed research modules into several educational programs developed for students at various educational levels and also disseminating the research outcomes to a broad spectrum of communities.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.