Research

As an interdisciplinary team of classically trained biochemists, cell biologists and engineers, we look for approaches that combine answering fundamental questions with cutting-edge practical applications. Nowhere is this combined potential higher than in hydrogel research. Biological “living” hydrogels are self-assembled systems made from highly hydrated biopolymers. They coat all the wet surfaces in the human body, providing a selective barrier that allows nutrients and information in while keeping pathogens out. One important example of a hydrogel-based filter is the nuclear pore, the tiny channel that control what gets into and out of the cell nucleus. From an engineer’s perspective, we believe hydrogels can solve some vexing problems, such as passivating materials implanted into the human body, preventing bacterial biofilm formation on materials exposed to living organisms, and selectively filtering biomolecules. From a biochemist’s perspective, our goal is to elucidate the fundamental biophysical principles that allow biogels to act as selective barriers and filters, and the cellular mechanisms involved in building and regulating them.

One important and understudied hydrogel is mucus, the clear, slimy gel that lines an enormous surface area in our body (2000 square feet in the intestine alone). Mucus is an ideal system in which to elucidate general principles of bio-filtration by hydrogels. It is also of great practical importance to many engineering applications relating to the human body. Any device or drug in the gut, the reproductive tract or the eye is immersed in mucus, and its performance will be largely defined by this interaction. Moreover, mucus is home to trillions of microbes that form our microbiome and regulates their interactions with the host. Yet, mucus is little studied by biologists or engineers.

Over the last years my lab has developed a suite of quantitative methods and concepts to characterize passive transport and active migration of diverse particles and living cells through hydrogels, using mucus as a model system. The importance of mucus properties for health and disease has been recognized for quite some time, but this is the first systematic approach of its kind for the broad evaluation of hydrogel permeability, and it promises to extract general principles that govern selective filtering in biological hydrogels. This experimental and theoretical framework will not only help in understanding the basic principles that govern selective transport through the mucus barrier, but also will provide tools to predict the trafficking and fate of pathogens and drug delivery vehicles in the mucus barrier, which has significant implications for the prediction and treatment of infectious diseases. Our project is also innovative because it impacts our understanding of other hydrogel-based barriers that are relevant for disease and that generate an obstacle to delivery of genes and therapeutics, such as the extracellular matrix, nuclear pores, and the bacterial biofilm matrix.

Selective Permeability of Biological Hydrogels

One main mission of my laboratory is to determine the basic principles that govern selective permeability in biological hydrogels. A microfluidic system was developed (in collaboration with Prof. Jay Han), which enables us to measure particle uptake and transport (top). With this system we have begun to identify molecular properties that distinguish particles that are rejected by, or permeate, a defined mucus barrier (bottom).

Little is known about the detailed molecular properties that distinguish particles that permeate, or are rejected by, a defined mucu  s barrier. What is the relationship between a particle’s size, geometry and biochemistry, and its mobility across a mucus barrier? Can we build a physical model of hydrogels that predicts how their structure governs filtration properties? How do certain pathogens such as viruses or bacteria penetrate mucus barriers? To address these central questions we have developed new technology and concepts to quantify the uptake, transport and spatial distribution of particulates and cells in mucus. Our research shows that mucus gels provide an effective and selective barrier towards particles as diverse as protons, antibiotic molecules, peptides, and even toward a broad range of viruses. Our work suggests that the mucus hydrogel does not operate predominantly on the basis of size exclusion, a mechanism that is often suggested in text books, but rather as a sophisticated interaction filter in which binding of bypassing particles by the hydrogel polymer components is used to govern transport through the hydrogel. Specifically, we can conclude that commonly considered parameters such as net charge or lipophilicity are insufficient predictors for mucus permeability, and instead, the detailed spatial distribution of charged and hydrophobic groups can strongly modulate diffusivity. Over the next years we shall determine the rules for how the detailed biochemistry and structure of particles determine their passage through hydrogels. Mucus in the gastrointestinal, respiratory, and reproductive tracts are obvious sites for delivering therapeutics. Understanding the basic principles that govern its selectivity may enable the design of small molecules and vectors for drug delivery, and new approaches for preventing prevalent infectious diseases (e.g., HIV, Zika), or unwanted pregnancy. Our longterm aim is to provide the foundation for a theoretical framework that captures general principles governing selectivity in mucus, and likely also in other biological hydrogels such as the extracellular matrix, and bacterial biofilms.

Natural hydrogels have filtration properties that are unmatched by current synthetic filtration systems. We have begun to use the well-defined building block from the nuclear pore hydrogel to identify those parameters that govern selectivity, and to engineer synthetic hydrogels with tailored selectivity. This novel platform enables us to investigate crucial elements of hydrogel function, and how to manipulate those to gain full control over selectivity.

My laboratory also seeks to identify those parameters of hydrogel-forming polymers that are relevant for achieving selectivity. Mucus and other biological gels are built from polymers that contain regularly spaced repetitive domains, or ‘blocks’. Suitably configured repeat domains can produce incredibly effective filters as illustrated with the example of nuclear pores, the tiny channels inside the nuclear envelope that are filled with a hydrogel and regulate molecular transport between the nucleus and the cytoplasm. The conserved repeat domains within the nuclear pore hydrogel enable selected molecules with specificity for the hydrogel to pass through 100-1000 times faster than inert molecules of the same size. This is a new concept in filtration and fundamentally different from synthetic affinity chromatography systems, where molecules with binding specificity are retained inside the matrix. Understanding the key parameters within these repeat domains that govern selective filtering could enable us to build filters with tailored selectivity. Separating particles by filtration is enormously important in industry. In principle, we could build selective filters for proteins, cells, nanoparticles, perhaps even food products. To identify the elements of the repeat domains relevant for selectivity, my laboratory has begun to engineer sequences as modeled by the nuclear pore, where the character and length of the polymer domains have been varied, and where the charge density has been changed and reversed. Our work shows that even relatively short peptides with simple sequences can generate efficient filters, selectively translocating certain components while restricting passage of inert material. This novel platform enables us to investigate crucial elements of hydrogel function, and how to manipulate those to gain full control over selectivity.

Microbial Ecology in Mucus

We developed a 3D model based on purified mucin polymers to study the behavior of microbes inside mucus. Our work shows that mucins can suppress a range of virulence traits in microbes, such as surface attachment, the formation of biofilms, and horizontal gene transfer. Bottom right panels show a live culture of Pseudomonas aeruginosa bacteria grown in the absence of (left) or inside (right) a 3D mucin matrix. Our insights from the study of mucin-microbe interactions may inspire new strategies to target infections, neutralize microbial virulence, and the design of anti-biofilm coatings for implants.

An integral arm of our research is to better understand how the human body prevents infections by harmful microbes on mucosal surfaces. Mucus is one major ecological niche for the human microbiota – it accommodates an incredible 100 trillion microbes where densities approach 1011–1012 cells/mL, a record for any microbial ecosystem documented so far. Many of these microbes are important for our health because they help us build vitamins and digest food, and some can protect us from other damaging microbes. But in this crowd are also numerous potentially harmful pathogens that can cause infections. It is a wonder we are alive! It appears that over millions of years, the mucus has evolved the ability to control problematic pathogens, preventing them from causing damage, but studies that directly test this aspect of mucus are missing.

Over the past years my laboratory has developed laboratory models for mucosal surfaces to study the effects of mucus and mucins, the major gel-forming constituents of the mucus barrier, on the behavior of prevalent opportunistic pathogens, including Pseudomonas aeruginosa, Candida albicans, and Streptococcus mutans. We have discovered that the mucus has a profound influence on the behavior of microbes and specifically, their ability to swim, to settle, and to communicate with each other, and that it can suppress a range of virulence traits including surface attachment, biofilm formation, and horizontal gene transfer. Our work furthermore shows that the mucus creates an environment, which stabilizes the viability of microbes while preventing them from doing harm, analogous to the process of microbe domestication. This is important – mucus does not control pathogens by killing them, but instead appears to render them docile. Our work on this specific topic has been recognized with the Popular Science’s Brilliant 10 award (2014), and an invitation to present our work in an educational TEDEd video.

We would argue that our findings make a fundamental contribution to our understanding of host-microbe interactions as they are generating a paradigm shift from the textbook view of mucus as a simple catchall filter for particles, toward the understanding that is a sophisticated bioactive material with powerful abilities to manipulate microbial behavior. In addition, by introducing a well-defined in vitro system we can bring a big push to a field that lacks tools for the mechanistic analysis of microbe-mucus interactions.

The biophysical properties of cervical mucus (A, white arrow) such as extensibility (B) and permeability (C), can signify health and disease states of mucosal epithelia, as we have shown with the example of preterm birth. This technology will be applied broadly for the diagnostics also of other epithelia such as in the digestive tract, which I expect will have important implications for understanding disease progression, and directing the design of intervention strategies.

 

Mucus Biophysical Properties as Diagnostic Markers for Disease

Understanding the basic science of biological problems underlies all our endeavors, but we also strive to find cutting-edge practical applications for our work. One application with particularly high translational potential lies in using mucus as a non-invasive diagnostic for epithelial health. The rationale is that the physicochemical properties of mucus barriers are intricately related to health and disease, and the pathological onset of mucus barrier dysfunction can lead to a number of devastating pulmonary, gastrointestinal and urogenital conditions. Over the past years my laboratory has begun to unravel the strong diagnostic potential of mucus by interrogating the physicochemical properties of cervical mucus in the context of preterm birth. Preterm birth affects about 13 million babies worldwide each year, resulting in major pediatric morbidity and mortality, as well as significant health care costs. In a significant number of cases, preterm birth correlates with increased ascending bacterial infections from the female vaginal tract, suggesting that the cervical barrier to infection is weakened. Our study reveals that cervical mucus from women at high risk for preterm birth is more translucent, extensible, and permeable, than cervical mucus from women in a healthy pregnancy, suggesting that a weakening of the mucus barrier may be one main contributor to intrauterine infection. This discovery not only allows for patient stratification, but it also provides valuable mechanistic insights into the causes of preterm labor. Looking forward, we will apply our tools to diagnose diseases on other mucosal surfaces, including the oral cavity, the intestinal linings, and the lungs, with the goal to establish a structure-function relationship between mucus’ physicochemical properties and epithelial health. We expect that our work will have important implications for understanding disease progression on mucosal epithelia, may enable the more rapid development of diagnostic tools that utilize mucus as a source of biophysical indicators for health and disease, and possibly direct the design of intervention strategies.