Noah Malmstadt, Ph.D. - US grants

DESCRIPTION (provided by applicant): The capacity of lipid molecules in cell membranes to separate into multiple liquid phases, forming so- called lipid rafts, has in the past decade been identified as an important physiological process. Lipid rafts are a control element in the cell membrane, and they take part in numerous molecular pathways with implications for human health, including signal transduction and viral entry. In vitro models of the cell membrane built from synthetic bilayers have played an important role in elucidating the fundamental mechanisms underlying raft formation. Existing artificial bilayer models, however, lack some properties that are inherent to the cell plasma membrane and that are likely important in reproducing the physiological behavior of lipids. These properties include the mechanical attachment of the bilayer to an underlying polymeric cytoskeleton and the compositional asymmetry of the cell membrane. This proposal calls for the fabrication of novel artificial bilayer constructs that will better mimic the structure of the cell plasma membrane and serve as research platforms for investigating lipid raft behavior. This will be accomplished by building vesicular bilayer structures (so called giant unilamellar vesicles, or GUVs) that are filled with a polymer hydrogel. This hydrogel will serve as a biomimetic cytoskeleton, and the membrane will be physically anchored to it via chemical conjugation. One major shortcoming of existing artificial bilayer models of the cell membrane is their inability to properly recapitulate the nanometer scale of lipid rafts found in actual cells, producing instead micrometer-sized lipid domains. There is significant evidence that the size of rafts in cells is limited by the mechanical attachment of the membrane to the cytoskeleton. Building a biomimetic cytoskeleton will allow for precise control over the nature and density of membrane-cytoskeleton attachments and therefore a detailed investigation of the relationship between cytoskeletal attachment and raft size. It will also provide a versatile research platform that can be used to investigate a wide variety of lipid structure-related questions. Investigation of lipid structures at the nanoscale requires the development of analytical techniques that can address these tiny structures. This proposal outlines a set of techniques based on total internal reflection fluorescence microscopy and Fvrster resonance energy transfer that will allow for the detection and evaluation of nanoscale rafts with spatial and temporal resolution. Also proposed is a microfluidic technology for assembling bilayers on GUVs in a layer-by-layer fashion, allowing for the composition of each layer to be controlled and facilitating the fabrication of asymmetric bilayers like those that compose the plasma membrane. Together with the hydrogel cytoskeleton, this technology will allow for a new type of artificial cell that mimics accurately most important properties of the eukaryotic plasma membrane. PUBLIC HEALTH RELEVANCE: Lipid nanostructures in cell membranes help control how cells interact with their environments and are therefore central actors in many disease states, including type-2 diabetes and viral infection. While synthetic lipid bilayers modeling the cell membrane have been important tools for elucidating the molecular mechanisms that underlie lipid structure formation, these systems fail to reproduce important properties of real cell membranes. The new artificial cell constructs proposed here mimic both the cytoskeletal attachment and compositional asymmetry found in cell membranes, allowing them to serve as research platforms for understanding how lipid nanostructures behave and how novel therapeutic approaches can alter lipid- mediated processes.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).
As novel applications for nanocrystalline materials continue to emerge, there is an increasing need for new approaches to manufacture these materials efficiently, at high yield, and with minimal damage to the environment. In this project, two green chemistry technologies?ionic liquid solvents and microfluidic reactors?are combined to engineer new approaches to nanocrystal synthesis. Ionic liquids are ideal for nanofabrication reactions because they can act both as solvents and as surface passivation ligands. Microfluidic systems are ideal because nanocrystal nucleation and growth processes are exquisitely sensitive to local conditions that can be engineered and controlled in microfluidic flows.
Beyond the immediate advantages of green manufacture of nanomaterials, this work promises to provide novel insights to the role that mixing plays in controlling nanofabrication reactions. This system will allow for precise control of the timing of the nucleation and growth phases of nanocrystal formation, allowing for improved crystal homogeneity and better control over final morphology. Metal oxide nanocrystals are used as photocatalysts, piezoelectric and photovoltaic materials, and pigments. The green principles developed in this proposal are broadly applicable to manufacturing these materials, and will have correspondingly broad impacts. The proposed work includes an education and outreach program. At the high school level, the PI will work with the USC Center for Engineering Diversity to provide laboratory experiences for high school teachers from local schools. The co-PI will undertake an experimental outreach program to local community colleges.

DESCRIPTION (provided by applicant): Over the past half century, as molecular biology and biochemistry have developed to inform our understanding of disease, and as this understanding has driven the search for treatments in biotechnology and molecular medicine, the dominant theoretical scaffold for interpreting molecular behavior has been the structure-function relationship. Our understanding of molecular biology's structure-function relationships is largely limited, however, to the molecules of the central dogma: proteins and oligonucleotides. The vast variety of molecular structure outside of the central dogma, particularly among lipids and carbohydrates, suggests that these, too, can be understood in terms of structure driving function. In particular, there has been intense interest in the functional role that lipids might play in the plasma membrane. This project deploys a new class of synthetic lipid bilayers to begin drawing connections between the structure of lipid molecules-both in terms of individual molecular structure and supermolecular organization-and the function of the plasma membrane. The research tools developed and deployed here, called asymmetric giant unilamellar vesicles (AGUVs), are designed to uniquely mimic the cell membrane, capturing properties such as compositional asymmetry and molecular crowding better than other existing synthetic lipid bilayers. One of the many questions that AGUVs can help answer involves passive transport across the cell membrane. Passive transport is an important route for drug delivery and passage of environmental toxins into cells. Recent results show that the mechanism of this transport is complex, and highly dependent on lipid behavior. This project deploys an AGUV-based technique for systematically measuring the dynamics of solute molecules interacting with and penetrating lipid bilayers, yielding richer mechanistic data than other approaches are capable of delivering. AGUVs can also be used to study the mechanical properties of the cell membrane. These properties- particularly resistance to bending-control protein function and are important in a range of physiological processes. While synthetic lipid bilayers have been used to probe these properties, little is known about the effects of bilayer asymmetry on them. This project uses AGUVs to discover these effects. Lipid interactions with integral membrane proteins are likely a major mode by which lipids influence cell behavior. Very little is known, however, about the origins or controlling parameters of these interactions. This project begins to untangle this problem in AGUVs by using fluorescence microscopy to probe how peptides modeling the transmembrane regions of various proteins associate with segregated lipid domains. Finally, AGUVs can facilitate the systematic study of the effects of macromolecular crowding in the cell interior. The microfluidic technique by which AGUVs are formed allows for the inclusion of arbitrary molecules within them, leading to unique molecularly crowded structures. PUBLIC HEALTH RELEVANCE: Cells are surrounded by membranes that consist of two layers of lipid molecules, and the chemical composition is different in these two layers. In this project, a new type of artificial cell membrane that is uniquely capable of mimicking this asymmetry is deployed to study a range of important biological processes, including drug transport into cells, mechanical deformation of the cell membrane, and interactions between lipid molecules and receptor proteins involved in cancer treatment and diabetes.

The research objective of this award is to combine experiment and computer simulation to investigate the relationships between cell membrane composition, organization, and mechanical behavior. The nanoscale response of cell membranes to mechanical stress is an essential aspect of important biophysical processes such as endo- and exocytosis, viral fusion and budding, and intracellular trafficking. The joint experimental-simulation studies conducted under this award will probe how molecular processes such as phase separation into liquid-ordered and liquid-disordered domains by membrane lipids and cholesterol flip-flop across the cell membrane bilayer modulate the nanomechanical response of membranes. Newly developed computational tools include a petascale computational framework to perform multimillion-atom MD simulations embedded in coarse-grained MD simulations.

These studies will add significantly to biophysical understanding of bending mechanics of cell membranes, which has profound implications for viral infection and neuron communication. The educational plan focuses on a dual-degree program in which students fulfill Ph.D. requirements within their own discipline while studying towards an M.S. in computer science. Dual-degree students will help organize computational science workshops for underrepresented groups, which are held regularly at the University of Southern California. The educational outreach plan also integrates high-school students into experimental research.

1067021
Malmstadt

Passive transport through the cell membrane represents a major route by which drugs enter cells. It is the primary route by which orally delivered drugs enter systemic circulation. Environmental toxins can also enter the body passively. Understanding the mechanistic details of passive transport is essential to understanding how and why certain molecules make good drugs or dangerous toxins. Unfortunately, current approaches to measuring passive transport across membranes face severe limitations and have failed to yield reproducible results. This project applies a new approach to measuring passive transport that minimizes measurement artifacts by: 1.) Facilitating transient, rather than steady-state measurements, thereby minimizing the formation of transport artifacts and 2.) Allowing for complete characterization of the spatial concentration profile of a transported molecule, allowing for precise fitting of a transport model. This approach is based on the observation of the transport of molecules into giant unilamellar lipid vesicles (GUVs) via spinning-disc confocal microscopy (SDCM). SDCM allows for rapid imaging of the concentration profile on both sides of the GUV membrane at any instant in time. This, in turn, will allow researchers to establish the time course of the evolution of the concentration profile. Simple modeling of early experimental results shows that membrane permeability can be determined easily from transient concentration profile data. This technique can answer a series of important research questions: 1. What are the limits of SDCM in measuring passive transport? 2. What molecular properties control passive transport. 3. How do membrane composition and charge state modulate passive transport. The goal of this project is to develop and perfect a new technology for measuring how easily drugs and toxins can enter cells. This new technology will improve on existing techniques by providing researchers with real-time images of drug and toxin molecules crossing cell membranes. A detailed picture of the process of cell entry will be a valuable tool for designing effective next-generation drugs.

Intellectual Merit

The proposed work develops new engineering tools to address central shortcomings of techniques currently used to measure the passive transport of molecules across lipid bilayer membranes. Most existing tools for observing passive transport can only access bulk concentrations at steady state, leading to highly limiting measurement artifacts. The tools developed here will allow for the transient observation of the full concentration profile, which will allow for precise measurement of the parameters that govern passive transport.These precise measurements will provide a foundation for a quantitative study of the relationship between molecular structure and membrane permeability. Two aspects of molecular structure will be investigated: the structure of molecules permeating the bilayer and the structure of the lipids that make up the bilayer itself. Molecules crossing the bilayer will be investigated by systematically varying lipophilicity, molecular weight, and hydrogen-bonding groups. Lipid molecules will be investigated by varying tail length, charge state, hydrogen bonding capacity, and membrane phase state. Developing a quantitative relationship between molecular structure and passive transport will facilitate mechanistic insight into this process. Such an insight is key to developing a comprehensive theory of how drug molecules enter cells.

Broader Impacts

Passive transport across the cell membrane is of critical importance to how drugs behave in the body. Drugs that are able to pass through the cell membrane without activation of the cellular machinery have high oral bioavailability. A thorough mechanistic understanding of passive transport is essential to understanding how small molecules interact with the human body, a fundamental question with implications ranging from drug development to environmental toxicology. This research project is integrated with a comprehensive education and outreach plan that focuses on five areas: undergraduate research, laboratory module development for undergraduates, graduate curriculum development, outreach to high school students, and outreach to underrepresented graduate students. The primary objective of the high school outreach program is to facilitate intensive research experiences for students drawn from the diverse population of the Los Angeles Unified School District (LAUSD). The primary broader impact of this outreach program will be to provide unique lab-based experiential education opportunities to the LAUSD population, which contains a larger proportion of disadvantage students and minority groups that are underrepresented in science and engineering. The graduate level outreach program involves participation in a workshop at the Graduate Institute of the annual Society of Hispanic Professional Engineers conference.

Nanoparticles made of precious metals (gold, silver, platinum, etc.) are useful for biomedical therapies, energy generation, and improving the efficiency of chemical reactions. Current techniques for manufacturing these nanoparticles, however, rely on slow and costly laboratory-scale chemical reactions, making metal nanoparticles cost-prohibitive for their most promising applications. Reactions performed in small batches in the lab guarantee high particle quality and uniformity. This quality and uniformity would be lost if the reaction volumes were increased to the industrial scale. This award supports research in the development of microfluidic chemical reactors for generating nanoparticles. Microfluidic chemical reactors handle very small volumes of chemicals in channels that are smaller than a millimeter across. Because of their small size, they can produce nanoparticles with the same quality and uniformity as the laboratory-scale reactions currently used. The goal of the research supported by this award is to understand how to operate many microfluidic reactors simultaneously, allowing for large quantities of nanoparticles to be produced in an automated, continuous manner while maintaining the quality of these particles. The technology developed here will facilitate the efficient and sustainable industrial-scale production of metal nanoparticles. This project will be integrated with an educational outreach program that incorporates high school and community college students as active participants in the research.

While nanofabrication in microreactors is an established technology, there are several challenges that need to be overcome to make it a sustainable industrial-scale process. This research will accomplish four objectives to overcome these challenges. (1) Development of sustainable ionic-liquid based chemistry for the fabrication of Pt and Rh nanoparticles. (2) Design of microfluidic surface coatings for maximized throughput of droplet flows. (3) Implementation of new droplet merger techniques to facilitate multistep (e.g., seeded growth) nanoparticle fabrication reactions. (4) Feedback control of highly parallelized systems of microreactors to facilitate industrial-scale yields. This award will advance knowledge at the intersection of diverse fields of study, facilitating the application of techniques from chemistry, chemical engineering, and microfabrication technology to develop fundamental principles for the scale up of nanoparticle manufacturing in continuous-flow microreactors. It will lead to the development of broadly adaptable schemes for the rational assembly and control of massively parallel microfluidic reactors and techniques for performing sustainable chemistry in these systems. The goal is a science-based approach to the design of industrial-scale, sustainable microreactor manufacturing systems.

Diagnosing and treating cancer requires having a reliable set of affinity reagents that can specifically and strongly bind to cancer-related protein targets. These reagents are the necessary molecular tools that will enable next-generation technologies for studying, diagnosing, and fighting cancer. Current approaches to producing such reagents, however, are unreliable, expensive, and slow. Existing reagent generation methods (e.g., hybridoma technology, phage display, yeast display) are not readily adapted to leverage high throughput library sequencing. Where reagents do exist, they are often both extremely expensive and poorly characterized. It is these important shortfalls that this research project aims to address. This project combines two powerful existing technologies: 1) mRNA display and 2) Modular Microfluidic and Instrumentation Components (MFIC) to dramatically speed target-directed, renewable reagent development. mRNA display is a molecular selection technology that is uniquely capable of searching libraries of more than a trillion unique compounds to develop ultrahigh affinity reagents against cancer-relevant targets. While this technology has an impressive demonstrated track record of producing such reagents, it has so far been limited to the laboratory scale. This project will adapt it to a true high-throughput format by integrating it into a continous flow microfluidic system based on MFIC technology. By automating mRNA display, this project will make it broadly accessible to the research community while decreasing its cost and increasing its throughput. Once the automated system is developed, we will use it to produce affinity reagents that target two key cancer screening protein markers?PSA and CA125. These markers are broadly used (they were the primary tools used in 154,000 patient Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO)) and renewable, inexpensive reagents that recognize these biomarkers would clearly be of utility. An automated microfluidic mRNA display system will be used to develop novel, specific, and high affinity reagents for these targets. Developing a microfluidic approach for mRNA display selection will involve implementing a magnetophoretic separation system that can both perform affinity selections and purify the products of preparative biochemical reactions. An automated system for preparing mRNA reagents will be combined with an automated target selection system. The DNA-encoded products of this selection will be amplified in a microfluidic PCR system. The entire process workflow will be implemented in a closed loop to enable multiple rounds of selection and amplication, producing an optimal high affinity binding reagent. Each step of the automated mRNA display will be benchmarked for quality assurance by developing affinity reagents for the cancer marker Bcl-xL; standard manual mRNA display has proved resoundingly successful at producing such reagents and there is plentiful existing data against which to benchmark.

The unique biological, optical, and chemical properties of metal nanoparticles have driven several decades of research into their many potential applications. If these applications are to be realized at a level that will make a significant societal impact, cost-effective techniques for producing industrially relevant quantities of nanoparticles must be developed. Today, chemical manufacturing techniques for high quality nanoparticle fabrication remain at small production scales, with a cost that reflects the limited throughput and labor intensity of a by-hand process. This is because only small-scale chemical reactions can achieve uniform mixing conditions and uniform temperatures throughout the reaction vessel, which are essential conditions for producing uniform, high-quality nanoparticles. Standard, large-volume industrial chemical reactors lack uniform mixing and temperature distribution tend to produce low-quality particles and are therefore an inappropriate route to the scale-up of high-quality nanoparticle manufacturing, for example, for catalysis. This award investigates continuous-flow chemical micron scale reactors as a means to maintain the small-scale conditions necessary to make high-quality nanoparticles while allowing for continuous processing that can be automated and operated around the clock. Further, to scale these microreactors to industrially relevant conditions, this research investigates massive parallelization, i.e., the controlled operation of many microreactors at once to produce large quantities of high-quality nanoparticles. This research effort is coordinated with an outreach program that integrates community college students into research, and makes science and engineering careers accessible to these students, especially women and minority students.

High quality nanoparticles for commercial purposes are still prepared at the lab scale, essentially by hand. The limit to scale-up is the fact that in solution-phase chemical techniques, the size and monodispersity of the resulting nanoparticles are extremely sensitive to the reaction temperature and reagent mixing conditions. It is impossible to maintain the necessary uniformity in current industrial-scale reactors even with stirring. Microfluidic reactors, however, have inherently good thermal uniformity and droplet microfluidic systems allow for rapid mixing and homogenization. This research approach relies on ionic liquid (IL)-based nanoparticle synthesis in microfluidic reactors. In these reactors, droplets of IL are separated in a fluorocarbon oil-based carrier stream. The microfluidic system developed in this research will operate at remarkably high colloid concentrations, nearly 50-100 mg nanoparticles/mL reaction solvent, compared to nearly 2 mg/mL for traditional solution phase approaches. In the nanomanufacturing system studied here, a microreactor system is scaled to sixteen parallel channels. The funded work is a science-based investigation of the key system parameters, such as, process monitoring and feedback control, that must be addressed to scale such a parallel system to an arbitrarily large capacity.