Research Highlights
Genetic Engineering Through Click Chemistry Highlighted in C&E News
Yousaf group has published a new way to transfect cells with nucleic acids in ACS Central Science. By combining liposome fusion, bio-orthogonal chemistry and cell surface engineering a new rapid transfection technology was created termed SnapFect. The technology is being commercialized by a new company called OrganoLinX.
Engineering Cell Surfaces via Liposome Fusion
Highlighted in C&E News
The Yousaf group has published in Bioconjugate Chemistry a new method to tether chemoselective ketone and oxyamine groups from cell surfaces by liposome delivery toward the goal of rewiring the cell surface. Alkyl ketone and oxyamine molecules spontaneously insert into lipid vesicles upon synthesis. When mixed, chemical recognition occurs, producing stable oxime bonds under physiological conditions. The reaction is also bio-orthogonal and thus, does not interfere with any other chemical moieties in its cellular surroundings. The synthetic ketone and oxyamine molecules fused on the cell membrane serve as cell-surface receptors, providing tools for the attachment of other functional materials, biomolecules, and probes on the cell surface.
OrganoLinX Launched
A new company founded by Prof. Yousaf is launched in Toronto, Canada that will commercialize new innovative Life Science Reagents and Technology.
*Snapfect - a new transfection reagent based on bio-orthogonal chemistry
*ViaGlue - a rapid scaffold free way to assemble cells into co-culture spheroids or tissues for autocrine and paracrine studies
* Aydin Reagents - a suite of new click chemistry molecules
*R-Live 3D Tissue - a scaffold free way of making complex organ tissue
*Microfluidix Tech - a microfluidic technology to engineer cells in flow.
Yousaf Research Highlighted in Science
As highlighted in Science the Yousaf Group has developed a new strategy to induce specific and stable cell-cell contacts in 3D through chemoselective cell-surface engineering based on liposome delivery and fusion to display bioorthogonal functional groups from cell membranes.
Published in JACS, this strategy uses liposome fusion for the delivery of ketone or oxyamine groups to different populations of cells for subsequent cell assembly via oxime ligation. The method can be used for several applications including the delivery of reagents to cells for fluorescent labeling and cell surface engineering, the formation of small 3D spheroid cell assemblies, and the generation of large and dense 3D multilayered tissue-like structures for tissue engineering applications.
Redox Switchable Peptide Structure
Published in JACS, the Yousaf Group describes a general surface chemistry strategy for the development of new switchable materials. The method modulates a surface immobilized molecules structure by using two orthogonal click reactions based on Huisgen cycloaddition and oxime chemistry where the oxime linkage is redox active and switchable.
Liposome fusion was characterized by matrix-assisted laser-desorption/ionization mass spectrometry (MALDI-MS), dynamic light scattering (DLS), fluorescence resonance energy transfer (FRET), transmission electron microscopy (TEM), fluorescence-activated cell sorting (FACS), and fluorescence microscopy analyses.
Tailored Carbon Nanotubes for Controlling Cell Behaviour
The Yousaf group reports a strategy for tailoring and patterning carbon nanotubes (CNTs) for biospecific cell behaviour studies. This research was featured on the cover of Nanotechnology.
The Yousaf Group demonstrates this strategy by developing a non-invasive, biocompatible, in-situ surface chemistry that is able to modulate the affinity of a cell adhesive peptide (RGD) to cell integrin receptors to study dynamic cell adhesion and cell migration in real-time and as a new hide and reveal strategy for application in new types of smart biofouling biomaterials.
This work demonstrates the integration of a new functionalization strategy to immobilize a variety of ligands to CNTs for a range of potential drug delivery, tissue imaging and cellular behavior studies and a microfluidic patterning strategy for generating complex high-throughput surfaces for biotechnological and cell based assay applications.
Yousaf Named Founding Editor in Chief of Bioorthogonal Chemistry
The new international journal of Bioorthogonal Chemistry has selected York university Chemistry Professor Muhammad Yousaf as its founding Editor in Chief. The journal will be launched in 2012. This distinction recognizes his expertise in surface chemistry, biology and material science.
Renewable 'Green' Microarrays
Featured in the journal Chemical Communications in a special issue highlighting emerging investigators the Yousaf Group has developed a novel renewable microarray technology to immobilize and release carbohydrates and proteins from electroactive surface materials.
Bioorthogonal Chemistry is an open access journal that provides an interdisciplinary platform for scientific exchange among the biology, chemistry, physics, and materials sciences communities. It offers a discussion forum for rapid dissemination of scientific theories, results, and interpretations. The journal serves as a global vehicle for the chemical biology community and encourages dialog between scientists and the public with respect to cogent policy issues.
Tissue Morphing on Patterned Surfaces
Highlighted in C&E News
Model surfaces presenting gradient arrays of ligands have been used for the first time to study tissue shape morphing and directed tissue migration. As highlighted in C&E News, and published in JACS, theYousaf Group has developed a strategy for preparing dynamic bioactive and chemoselective substrates for investigating cancer cell behavior.
This method may be applied to a variety of research fields for use in biosensor technology and the generation of renewable and tailored ligand density microarrays for biospecific proteomic and cell-based assays.
Microfluidic Printing for Cell Polarity
To study complex cell behavior on model surfaces requires biospecific interactions between the interfacing cell and material. Developing strategies to pattern well-defined molecular gradients on surfaces is difficult but critical for studying cell adhesion, polarization and directed cell migration. The Yousaf Group introduces a new strategy, microfluidic SPREAD ,Solute PeRmeation Enhancement And Diffusion, for inking poly(dimethylsiloxane) ,PDMS, microfluidic cassettes with a gradient of alkanethiol.
The Yousaf team used a combined photo- and electroactive surface technology to generate precisely controlled ligand patterns and ligand gradient patterns with defined density and slope on a surface. After photo and electrochemical treatments, the surface can be functionalized via oxyamine linkages with RGD, a tripeptide that promotes cell migration and adhesion. By controlling the size, shape, and concentration gradient of the RGD regions with patterned photomasks, the team caused cells and tissues to migrate to the RGD regions and form new patterns. In addition, they measured the rate of directed tissue morphing and its dependence on the slope and direction of the gradient.
Microarray for Stem Cell Differentiation
Featured on the cover of the Journal of Colloids and Interface Science, the Yousaf Group has developed an integrated microarray technology to present well-defined surfaces for high-throughput investigations of cell-surface interactions.
This system is able to control the composition, ligand density, and spatial distribution of molecules on a surface to probe chemical effects on stem cell differentiation.
Yousaf Highlighted in JACS Select
The ability to pattern surfaces with monolayers and multilayers is a central focus of numerous chemical studies because of the implications in fields ranging from sensor design to microelectronics. Goals vary from patterning single molecules at the sub-nanometer scale to forming vast assemblies over macroscopic areas. Moreover, this chemistry can range from simple homogeneous systems to mixed monolayers with complex patterns. As illustrated in JACS Select, the Yousaf Group's research in surface chemistry and specifically developing tailored substrates for biotechnology applications and as model substrates for studies of cell behavior is highlighted.
Using SPREAD, an oxyamine-terminated alkanethiol is able to permeate into a PDMS microfluidic cassette, creating a chemical gradient, which can subsequently be transfer printed onto a gold surface to form the corresponding chemoselective gradient of oxyamine-alkanethiol self-assembled monolayer, SAM. The immobilization of a cell adhesive Arg-Gly-Asp (RGD)-ketone peptide to the SPREAD stamped oxyamine-alkanethiol SAMs provides a stable interfacial oxime linkage for biospecific studies of cell adhesion, polarity and migration.
Dynamic Surfaces for Cell Behavior
Cells exist in a complex, dynamic, and highly evolving environment, a key component of which is the extracellular matrix.
Published as the cover story on ChemBioChem, the Yousaf Group details recent advances in the design and utility of dynamic molecular surfaces for the analysis of cell adhesion, division, migration, polarization and co-cultures.
Model Substrates for Cell Mobility
Cells do not live in static surroundings, they exist in highly evolving dynamic environments. During cell adhesion and migration, cells adapt and communicate to their environment by numerous methods ranging from differentiation, gene expression, growth and apoptosis. How and when cells determine to adhere, polarize and migrate is important to a number of fundamental biological processes such as wound healing, metastasis, inflammation and development.
In order to elucidate the spatial and temporal mechanisms of these important complex processes on a molecular basis, the Yousaf Group has generated model substrates that can present nanopatterned ligands, well defined gradients of ligands and surfaces that can be dynamically modulated where the interaction between cell and material is defined at the molecular level.
Tailoring ITO Surfaces for Cell Biology
Indium tin oxide (ITO) is a transparent conductor used for applications ranging from solar cells to neurobiology. Tailoring ITO surfaces with a range of functional groups is challenging due to the difficulty in synthesizing phosphonate or siloxane terminated molecules. As reported in Advanced Materials, the Yousaf Group has developed a chemoselective immobilization strategy to tailor ITO surfaces by selectively oxidizing hydroxyl-terminated phosphonate SAMs to aldehydes, using microfluidics, followed by reaction with oxyamine-containing ligands. This rapid, inexpensive, and selective, on-chip activation allows for a wide range of ligands to be tethered onto ITO. Electrochemistry, contact angle, and XPS characterize the alcohol oxidation and subsequent reactivity. They also show control of ligand density and patterned cells on the newly generated aldehyde-terminated ITO surface.
This methodology allows for the generation of patterned complex surface chemistry on ITO surfaces from a simple hydroxyl-terminated SAM surface. The ability to generate complex surfaces with simple starting materials and minimal to no synthesis may have wide-ranging utility for numerous applications in molecular electronics and biotechnology including co-culture and cell arrays.
Controlled Microfluidic Surface Oxidation
Members of the Yousaf Group have recently developed a new rapid and inexpensive strategy to generate two chemistries from one surface composition for dual-ligand immobilization using microfluidic oxidation on indium tin oxide surface. Through chemical oxidation of an alcohol-terminated self-assembled monolayer, aldehyde and carboxylic acid groups are formed with density and spatial control. These surface groups may then react with oxyamine- and amine-tethered ligands to generate covalent oxime and amide linkages, respectively.
This patterning strategy circumvents multi-step syntheses and is applicable to tailoring a variety of other materials. Ongoing research includes exploring multiple ligand immobilization for co-culture studies, cell migration studies, and for generating high-throughput ligand microarrays on other metal-oxide surfaces.
Spin-Down for Cell Co-Cultures
As reported in Angewandte Chemie International Edition, Devin Barrett of the Yousaf Group reports a new methodology that combines surface chemistry patterning with soft lithography and centrifugation for the rapid, inexpensive, and complete patterning of cells and cell co-cultures on surfaces with spatial and temporal control.
Although, the patterning of one cell type to a range of materials has become routine, the ability to pattern multiple cell lines with spatial and temporal control of cell population interactions remains technically challenging and impractical for the larger biological community to access. Until now, a simple, fast and inexpensive strategy to generate multiple cell-patterned arrays would greatly expand the current scope of cell biological research and generate new co-culture array screens, tissue patterning materials and cell based devices.
By employing micro-contact printing in conjunction with poly(dimethylsiloxane) (PDMS) masks and centrifugation, we report the patterning of co-cultures on substrates with feature sizes as small as 30 μm. This strategy can routinely and rapidly immobilize single cells that are separated by as little as 100 μm, which provides exquisite spatial control for autocrine and paracrine signalling studies. In addition, we show the patterning of co-cultures of fibroblast cells and GFP transfected Drosophila cells to show the generality of the method for cell biological applications.
Photo-Electroactive Surface Strategy
As published in Molecular BioSystems, the Yousaf Group reports a combined photochemical and electrochemical method to pattern ligands and cells in complex geometries and gradients on inert surfaces. Their work demonstrates: (1) the control of density of immobilized ligands within overlapping photopatterns, and (2) the attached cell culture patterned onto ligand defined gradients for studies of directional cell polarity. Our approach is based on the photochemical activation of benzoquinonealkanethiols. Immobilization of aminooxy terminated ligands in selected region of the quinone monolayer resulted in patterns on the surface.
This approach is unique in that the extent of photochemical deprotection, as well as ligand immobilization can be monitored and quantified by cyclic voltammetry in situ. Furthermore, complex photochemical patterns of single or multiple ligands can be routinely generated using photolithographic masks. Finally, this methodology is completely compatible with attached cell culture and we show how the subtle interplay between cell-cell interactions and underlying peptide gradient influences cell polarization. The combined use of photochemistry, electrochemistry and well defined surface chemistry provides molecular level control of patterned ligands and gradients on surfaces.
Microfluidic Lithography for Cell Migration
As reported in Langmuir, Brian Lamb, Devin Barrett, and Nathan Westcott of the Yousaf Group report a straightforward, flexible, and inexpensive method to create patterned self-assembled monolayers (SAMs) on gold using microfluidics – microfluidic lithography. Using a microfluidic cassette, alkanethiols were rapidly patterned on gold surfaces to generate monolayers and mixed monolayers. The patterning methodology is flexible and, by controlling solvent conditions and thiol concentration, permeation of alkanethiols into the surrounding PDMS microfluidic cassette can be advantageously used to create different patterned feature sizes and to generate well-defined SAM surface gradients.
To demonstrate the utility of microfluidic lithography, multiple cell experiments were conducted. By patterning cell adhesive regions in an inert background, a combination of selective surface masking and centrifugation achieved spatial and temporal control of patterned cells, enabling the design of both dynamic surfaces for directed cell migration and contiguous co-cultures. Cellular division and motility resulted in directed, dynamic migration, while the centrifugation-aided seeding of a second cell line produced contiguous co-cultures with multiple sites for heterogeneous cell-cell interactions.
Tailoring of Biodegradable Polyketoesters
The Yousaf lab has recently generated a straightforward and inexpensive method to synthesize and etch biodegradable poly(1,2,6-hexanetriol £\-ketoglutarate) films for tissue engineering applications, Langmuir, 9861–9867, 2008. Microfluidic delivery of the etchant, a solution of NaOH, can create micron-scale channels through local hydrolysis of the polyester film. In addition, the presence of a ketone in the repeat unit allows for prior or post chemoselective modifications, enabling the design of functionalized microchannels.
Delivery of oxyamine tethered ligands react with ketone groups on the polyketoester to generate covalent oxime linkages. By thermally sealing an etched film to a second flat surface, poly(1,2,6-hexanetriol £\-ketoglutarate) can be used to create biodegradable microfluidic devices. In order to determine the versatility of the microfluidic etch technique, poly(£'-caprolactone) was etched with acetone. This strategy provides a facile method for the direct patterning of biodegradable materials, both through etching and chemoselective ligand immobilization.
The JACS Select issue on surface chemistry and biomaterials provides a taste for the state-of-the-art in a field that is rapidly moving and highly interdisciplinary. Remarkably, surface functionalization, patterning, and characterization borrows fro m virtually every field of chemistry and ties directly into engineering, materials science, biology, physics, and medicine.
Yousaf Selected for Marcus Lecture
Prof. Muhammad N. Yousaf has been selected to conduct the 2011 Marcus Lecture at the University of Washington at St. Louis. This annual lecture award highlights Dr. Yousaf's pioneering work in interfacing Organic, Bioanalytical and Biomaterials research to study Cell behavior and his development of new biophysical tools For tissue engineering applications. Recent Marcus Lecturers Include Professors Thomas Cech, George Whitesides, Richard Zare, Robert Langer, Peter Schultz and Larry Overman.
Yousaf named to Editorial Board
The journal Biointerphases has selected Carolina Chemistry Assistant Professor Muhammad Yousaf to its Editorial Board. This distinction recognizes his expertise in surface chemistry, biology and material science.
Biointerphases is an open access journal that provides an interdisciplinary platform for scientific exchange among the biology, chemistry, physics, and materials sciences communities. It offers a discussion forum for rapid dissemination of scientific theories, results, and interpretations. The journal serves as a global vehicle for the biomaterials interface community and encourages dialog between scientists and the public with respect to cogent policy issues.
Microarrays for Stem Cell Differentiation
Stem cells possess the ability to self-replicate to give rise to identical daughter cells and they can also undergo a complex differentiation process to generate new cell lineages. While stem cells hold much promise as an unlimited source of cells for transplantation therapies, and for treating numerous cancers and diseases, the precise control of the differentiation process is challenging and little is known about the complex interplay of the multitude of crucial factors ranging from signaling molecules to the cell microenvironment that influences stem cell differentiation.
As reported in JACS, the Yousaf Group develops and applies for the first time a quantitative electroactive microarray strategy that can present a variety of ligands with precise control over ligand density to study human mesenchymal stem cell (hMSC) differentiation on transparent surfaces with a new method to quantitate stem cell differentiation. They found that both the ligand composition and ligand density influence the rate of adipogenic differentiation from hMSC's. Furthermore, this new analytical biotechnology method is compatible with other biointerfacial characterization technologies and can also be applied to investigate a range of protein-ligand or cell – material interactions for a variety of systems biology studies or cell behavior based assays.
Electrochemistry in Microfluidics
To generate model substrates for cell adhesion, the Yousaf Group has developed two different biocompatible strategies based on self-assembled monolayers (SAMs) of alkanethiolates on gold terminated with latent ketones and aldehydes. Under spatial control, the hydroquinone and alcohol terminated SAMs can be oxidized to allow for oxyamine ligand patterning on the surface with microfluidic cassettes. These immobilization strategies were characterized by electrochemistry and fluorescence microscopy. By utilizing a cell adhesive peptide, cell patterns were also generated.
These methods are of broad utility to the research community as an easily accessible chemoselective strategy to immobilize ligands to surfaces. Previous immobilization strategies require multistep synthesis to generate the reactive head group on the surface. The Yousaf method requires either a simple synthesis or commercially available materials. The many different functional groups compatible with carbonyl chemistry allow for a range of ligands to be immobilized. In the future, the ability to oxidize hydroxy terminated SAMs may be extended to tailor a broad range of materials for molecular electronic and biological sensing applications.
Spatio-Temporal Control of Cell Interactions
The Yousaf Group reports a combined photochemical and electroactive self-assembled monolayer (SAM)-based substrate strategy to generate a co-culture platform with spatial and temporal control of cell-cell interactions. These dynamic substrates possess the ability to present a variety of ligands on the surface for biospecific interactions between the ligands and cell surface receptors.
Furthermore, the photo-patterning step enables the ligands to be immobilized in patterns and even gradients. This feature provides additional flexibility to study the role of ligand pattern (geometry) and presentation (gradient) on co-culture interactions on complex dynamic surfaces.
Microfluidic Gold Etching
As published in Analytical Chemistry, Nathan Westcott and Brian Lamb in the Yousaf Group report a combined microfluidics and electrochemical approach to generate complex surfaces for studies of mechanistic cell adhesion and cell migration. A variety of surfaces were generated including gradients of gold height, completely etched gold/glass hybrids, and partially etched gold surfaces for pattern visualization.
The integration of microfluidics and the electrochemical and chemical etching methods allow for the creation of gold/glass hybrids surfaces for numerous biointerfacial studies. In particular, the gradient surfaces allow for the study of cell migration on varying slopes of gold height with tailored surface chemistry. In the future, the etched gold surfaces will be used to simulate the varying nanotopology experienced by migrating cells in vivo.
Tailored Electroactive Nanorods
The Yousaf Group reports a strategy for the fabrication of tailored electroactive nanorod substrates for biospecific studies of cell adhesion and stem cell differentiation. To control the interfacial properties of the nanorods they formed self-assembled monolayers of an electroactive hydroquinone group that is able to chemoselectively immobilize oxyamine tethered ligands.
These tailored nanorods may be used to study how topology and surface chemistry influence cell behavior.
Yousaf Earns NSF CAREER Award
The National Science Foundation has awarded Carolina Chemistry Assistant Professor Muhammad Yousaf its prestigious CAREER Award, in the amount of $600,000 over five years. The award recognizes his well known pioneering work in applying surface chemistry to cell biology for studies of cell adhesion, polarization and migration. Yousaf’s group will develop and integrate new surface chemistries, live cell high resolution fluorescence microscopy techniques and microfluidic lithography based approaches to generate new cell based microarrays and a class of dynamic surfaces to study the internal and external cues that are critical for cell polarization and directed cell migration.
The Faculty Early Career Development Program is a foundation-wide activity that offers the National Science Foundation's most prestigious awards in support of junior faculty who exemplify the role of teacher-scholars through outstanding research, excellent education and the integration of education and research within the context of the mission of their organizations.
Biodegradable Polyketoesters
As reported in Macromolecules, researchers in the Yousaf Group report the design of several elastomers based on the thermal polycondensation of α-ketoglutaric acid and one of three triols: glycerol, 1,2,4-butanetriol, or 1,2,6-hexanetriol. By varying the curing temperature and the duration of the curing process, a wide range of mechanical properties was achieved.
The values of the Young’s modulus (0.1 - 657.4 MPa), ultimate stress (0.2 – 30.8 MPa), and ultimate strain (22 - 583 %) encompass the mechanical properties of many biological materials, allowing for potential use of poly(triol α-ketoglutarate) as a biomaterial. Furthermore, the poly(triol α-ketoglutarate) series hydrolytically degraded in as fast as 2 days and as long as 28 days in phosphate-buffered saline solutions. For post-polymerization modifications, the repeat units contain ketones, which are capable of reacting with a variety of oxyamine-terminated molecules to generate stable oxime linkages. Finally, the versatility and utility of these elastomers were demonstrated by creating micro-patterned structures and films for biospecific cell scaffold supports.
Dynamic and Catalytic Surfaces for Cell Culture
The Yousaf Group has developed an electroactive and catalytic dynamic substrate strategy that captures and subsequently releases ligands and cells in-situ via an electrochemical potential. The surface is catalytic for multiple rounds of immobilization and release with a quantitative functional group transformation from an oxyamine group to a primary alcohol upon mild electrochemical potential that is pH dependent.
Combining this strategy with a photochemical approach, the Yousaf Group shows the capture and release of peptide ligands that mediate biospecific cell attachment on defined surface gradient patterns.