Muhammad N. Yousaf

Using electroactive substrates to pattern the attachment of two different cell populations

This report describes the development of an electroactive mask that permits the patterning of two different cell populations to a single substrate. This mask is based on a self-assembled monolayer of alkanethiolates on gold that could be switched from a state that prevents the attachment of cells to a state that promotes the integrin-mediated attachment of cells. Monolayers were patterned into regions having this electroactive monolayer and a second set of regions that were adhesive. After Swiss 3T3 fibroblasts had attached to the adhesive regions of this substrate, the second set of regions was activated electrically to permit the attachment of a second population of fibroblast cells. This method provides a general strategy for patterning the attachment of multiple cell types and will be important for studying heterotypic cell-cell interactions.

Turning On Cell Migration with Electroactive Substrates

Herein we describe an electroactive substrate that was designed to turn on the migration of mammalian cells. The migration of cells is important in many developmental and disease processes that are temporally regulated.[1] Mechanistic studies of cell migrationÐwhich depend on specific interactions of cell-surface receptors with ligands of the extracellular matrix[2]Ðare complicated by the large number of ligands present in the matrix and the changes in ligand activity over isomer in 81 % yield. Protection as the ethoxyethyl (EE) ether followed by desilylation and oxidation of the resulting alcohol with TPAP/NMO provided ketone 37. Exposure of 37 to EtSH in the presence of Zn(OTf)2 gave mixed thioketal 38. Finally, radical reduction[18] of 38 furnished the target GHIJKLM ring system 2 in 56 % overall yield from 36. The configuration of 2 was unambiguously determined by NOE experiments. In conclusion, we have developed a highly convergent synthetic route to the GHIJKLM ring system 2 of ciguatoxin. The present synthesis demonstrates the general applicability of a strategy based on B-alkyl Suzuki coupling to the convergent synthesis of a polyether system. Progress toward the completion of the total synthesis of ciguatoxins is underway. Received: September 18, 2000 [Z 15822]

An Electroactive Catalytic Dynamic Substrate that Immobilizes and Releases Patterned Ligands, Proteins, and Cells

The ability to spatially and temporally control the attachment and detachment of molecules and cells on solid supports is important to research areas ranging from fundamental cell biology to biomaterial development and heterogeneous catalysis. Various approaches have been developed to promote or inhibit cell attachment by altering the macroscopic properties of the materials in situ, including mechanical stretching, photochemical illumination, electrochemical modulation, and thermal activation. Alternatively, other strategies have focused on manipulating the cell–surface interactions at the molecular level by incorporating specific chemistries that can alter ligand presentation for attached cell culture through a noninvasive external switch. To access more sophisticated and complex cell behavior studies, a surface strategy that can dynamically modulate attached cell culture at the molecular level catalytically, with spatial and temporal control in patterns and gradients, would greatly extend the utility of these model surfaces for a variety of cell motility, cell signaling, and cell– cell communication studies. These surfaces may also lead to the development of renewable surfaces for synthetic organic chemistry applications ranging from new solid-phase peptide synthesis resins to heterogeneous catalysis. Herein, we report an electroactive quinone-terminated self-assembled monolayer (SAM) on gold that captures and subsequently releases ligands, proteins, and cells in situ through an electrochemical potential. We also show that the surface is catalytic for multiple rounds of immobilization and release that are pH dependent. From a synthetic organic chemistry perspective, a clean and quantitative functionalgroup transformation occurs from an oxyamine group to a primary alcohol upon mild electrochemically applied potential. Furthermore, by extending this strategy with a photochemical approach, we demonstrate the immobilization and release of peptide ligands that mediate cell attachment in defined gradient patterns on inert surfaces. Our approach is based on a redox-active hydroquinoneterminated SAM that can be electrochemically oxidized to the corresponding quinone (Figure 1). The resulting quinone

Synthetic chemoselective rewiring of cell surfaces: generation of three-dimensional tissue structures.

Proper cell-cell communication through physical contact is crucial for a range of fundamental biological processes including, cell proliferation, migration, differentiation, and apoptosis and for the correct function of organs and other multicellular tissues. The spatial and temporal arrangements of these cellular interactions in vivo are dynamic and lead to higher-order function that is extremely difficult to recapitulate in vitro. The development of three-dimensional (3D), in vitro model systems to investigate these complex, in vivo interconnectivities would generate novel methods to study the biochemical signaling of these processes, as well as provide platforms for tissue engineering technologies. Herein, we develop and employ a strategy to induce specific and stable cell-cell contacts in 3D through chemoselective cell-surface engineering based on liposome delivery and fusion to display bio-orthogonal functional groups from cell membranes. 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. We demonstrate how this 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.

Design and Applications of Biodegradable Polyester Tissue Scaffolds Based on Endogenous Monomers Found in Human Metabolism

Synthetic polyesters have deeply impacted various biomedical and engineering fields, such as tissue scaffolding and therapeutic delivery. Currently, many applications involving polyesters are being explored with polymers derived from monomers that are endogenous to the human metabolism. Examples of these monomers include glycerol, xylitol, sorbitol, and lactic, sebacic, citric, succinic, α-ketoglutaric, and fumaric acids. In terms of mechanical versatility, crystallinity, hydrophobicity, and biocompatibility, polyesters synthesized partially or completely from these monomers can display a wide range of properties. The flexibility in these macromolecular properties allows for materials to be tailored according to the needs of a particular application. Along with the presence of natural monomers that allows for a high probability of biocompatibility, there is also an added benefit that this class of polyesters is more environmentally friendly than many other materials used in biomedical engineering. While the selection of monomers may be limited by nature, these polymers have produced or have the potential to produce an enormous number of successes in vitro and in vivo.

Asymmetric Peptide Nanoarray Surfaces for Studies of Single Cell Polarization

We report the production of symmetric and asymmetric cell adhesive peptide nanoarrays for the study of single cell polarization. Dip pen nanolithography is used to pattern nanometer-sized spots of hydroquinone terminated alkanethiol on gold substrates. After electrochemical activation of the surface, the corresponding quinone can then undergo a reversible chemoselective reaction with an oxyamine terminated ligand. Substrates presenting both symmetric and asymmetric nanoarrays of immobilized linear Arg-Gly-Asp (RGD) peptides and cyclic (RGD) peptides are used to examine the effect of the spatial distribution of cell adhesive ligands on the polarization of adhered Swiss Albino 3T3 fibroblasts by determining the directional vector between the nucleus centroid, centrosome centroid, and Golgi center. This methodology is extended to investigate the effect of spatial arrangement of immobilized ligands, affinity of ligands, and nanospot size on polarity and focal adhesion formation within the adherent cells on th...

An Interfacial Oxime Reaction To Immobilize Ligands and Cells in Patterns and Gradients to Photoactive Surfaces

We report a molecularly controlled interfacial chemoselective methodology to immobilize ligands and cells in patterns and gradients to self-assembled monolayers on gold. This strategy is based on reacting soluble ketone or aldehyde tethered ligands to surface-bound oxyamine alkeanethiols to generate a covalent oxime linkage to the surface. We characterize the kinetic behavior of the reaction on the surface with ferrocenecarboxaldehyde (FcCHO) as a model ligand. The precise extent of immobilization and therefore surface density of FcCHO on the SAM is monitored and determined by cyclic voltammetry, which shows a peudo-first-order rate constant of 0.13 min(-1). In order to generate complex surface patterns and gradients of ligands on the surface, we photoprotected the oxyamine group with nitroveratryloxycarbonyl (NVOC). We show that ultraviolet light irradiation through a patterned microfiche film reveals the oxyamine group and we characterize the rate of deprotection by immobilization of ketone containing redox active groups. Finally, we extend this strategy to show biospecific cell attachment of fibroblast cells by immobilizing ketone-GRGDS peptides in patterns. The interfacial oxime reaction is chemoselective and stable at physiological conditions (pH 7.0, 37 degrees C) and may potentially be used to install ligands on the surface in the presence of attached cells to modulate the cell microenvironment to generate dynamic surfaces for monitoring changes in cell behavior in real time.

Geometric control of stem cell differentiation rate on surfaces.

We develop a general methodology to create a patterned surface array that allows for the study of how the cell population, surface adhesion area, and pattern geometry combine to influence stem cell differentiation. We use a simple microfabrication technique to pattern human mesenchymal stem cells (hMSCs) on transparent surfaces and develop a new method to quantitate adipogenic differentiation. We found that the pattern geometry and therefore the cell population, rather than the cell adhesive area, influence the rate of adipogenic differentiation from hMSCs. Furthermore, the cells within the pattern behave more characteristically of a tissue than do individual cells because a certain critical threshold cell density is required to induce differentiation.

Engineering cell surfaces via liposome fusion.

In this study, we have rewired cell surfaces with ketone and oxyamine molecules based on liposome fusion for applications in cell-surface engineering. Lipid vesicles, functionalized with ketone and oxyamine molecules, display complementary chemistry and undergo recognition, docking, and subsequent fusion upon covalent oxime bond formation. Liposome fusion was characterized by several techniques including matrix-assisted laser-desorption/ionization mass spectrometry (MALDI-MS), light scattering, fluorescence resonance energy transfer (FRET), and transmission electron microscopy (TEM). When cultured with cells, ketone- and oxyamine-containing liposomes undergo spontaneous membrane fusion to present the respective molecules from cell surfaces. Ketone-functionalized cell surfaces serve as sites for chemoselective ligation with oxyamine-conjugated molecules. We tailored and fluorescently labeled cell surfaces with an oxyamine-conjugated rhodamine dye. As an application of this cell-surface engineering strategy, ketone- and oxyamine-functionalized cells were patterned on oxyamine- and ketone-presenting surfaces, respectively. Cells adhered, spread, and proliferated in the patterned regions via interfacial oxime linkage. The number of ketone molecules on the cell surface was also quantified by flow cytometry.

Tailored Electroactive and Quantitative Ligand Density Microarrays Applied to Stem Cell Differentiation

The ability to precisely control the interactions between materials and mammalian cells at the molecular level is crucial to understanding the fundamental chemical nature of how the local environment influences cellular behavior as well as for developing new biomaterials for a range of biotechnological and tissue engineering applications. In this report, we develop and apply 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 adipogenic differentiation. We found that both the ligand composition and ligand density influence the rate of adipogenic differentiation from hMSCs. Furthermore, this new analytical biotechnology method is compatible with other biointerfacial characterization technologies (surface plasmon resonance, mass spectrometry) 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.