Saul J. B. Tendler

Surface plasmon resonance analysis of dynamic biological interactions with biomaterials

Surface plasmon resonance (SPR) is an optical technique that is widely gaining recognition as a valuable tool to investigate biological interactions. SPR offers real time in situ analysis of dynamic surface events and, thus, is capable of defining rates of adsorption and desorption for a range of surface interactions. In this review we highlight the diversity of SPR analysis. Examples of a wide range of applications of SPR are presented, concentrating on work relevant to the analysis of biomaterials. Particular emphasis is given to the use of SPR as a complimentary tool, showing the broad range of techniques that are routinely used alongside SPR analysis.

Spatially controlled cell engineering on biodegradable polymer surfaces

Controlling receptor‐mediated interactions between cells and template surfaces is a central principle in many tissue engineering procedures (1–3). Biomaterial surfaces engineered to present cell adhesion ligands undergo integrin‐mediated molecular interactions with cells (1, 4, 5), stimulating cell spreading, and differentiation (6–8). This provides a mechanism for mimicking natural cell‐to‐matrix interactions. Further sophistication in the control of cell interactions can be achieved by fabricating surfaces on which the spatial distribution of ligands is restricted to micron‐scale pattern features (9–14). Patterning technology promises to facilitate spatially controlled tissue engineering with applications in the regeneration of highly organized tissues. These new applications require the formation of ligand patterns on biocompatible and biodegradable templates, which control tissue regeneration processes, before removal by metabolism. We have developed a method of generating micron‐scale patterns of any biotinylated ligand on the surface of a biodegradable block copolymer, polylactide‐poly(ethylene glycol). The technique achieves control of biomolecule deposition with nanometer precision. Spatial control over cell development has been observed when using these templates to culture bovine aortic endothelial cells and PC12 nerve cells. Furthermore, neurite extension on the biodegradable polymer surface is directed by pattern features composed of peptides containing the IKVAV sequence (15, 16), suggesting that directional control over nerve regeneration on biodegradable biomaterials can be achieved.—Patel, N., Padera, R., Sanders, G. H. W., Cannizzaro, S. M., Davies, M. C., Langer, R., Roberts, C. J., Tendler, S. J. B., Williams, P. M., and Shakesheff, K. M. Spatially controlled cell engineering on biodegradable polymer surfaces. FASEB J. 12, 1447–1454 (1998)

Poly(l-lysine)–GRGDS as a biomimetic surface modifier for poly(lactic acid)

The immobilization of adhesion peptide sequences (such as RGD) at the surfaces of poly(alpha-hydroxyacid)s, including poly(lactic acid) (PLA), is complicated by an absence of functional groups to support covalent attachment. We demonstrate a method to overcome this problem, by attaching the peptide to poly(L-lysine) (PLL), which immobilizes the sequence through adsorption at the poly(alpha-hydroxyacid) surface. When coated using a 0.01% w/v solution of PLL-GRGDS, bovine aortic endothelial cells seeded upon the modified PLA showed a marked increase in spreading over unmodified PLA. However, inhibition of the cell-spreading effect occurred when using higher concentrations of PLL-GRGDS, which we attribute to the PLL component. This inhibitory effect can be challenged by increasing the amount of GRGDS attached to each PLL molecule. Potentially, this is a flexible method of surface modification that can engineer many different types of tissue engineering scaffolds with a variety of biomolecules, thus allowing initial cell adhesion to be controlled.

Liver Tissue Engineering: A Role for Co-culture Systems in Modifying Hepatocyte Function and Viability

A major limitation in the construction of a functional engineered liver is the short-term survival and rapid de-differentiation of hepatocytes in culture. Heterotypic cell-cell interactions may have a role to play in modulating long-term hepatocyte behavior in engineered tissues. We describe the potential of 3T3 fibroblast cells in a co-culture system to modulate function and viability of primary isolated rat hepatocytes. Over an 18-day period after isolation, hepatocytes in pure culture rapidly declined in viability, displayed sparse bile canaliculi, and lost two function markers, the secretion of albumin and ethoxyresorufin O-dealkylase (EROD) activity. In comparison, the hepatocytes within the co-cultures maintained viability, possessed well-formed canalicular systems, and displayed both functional markers. Fixed 3T3 cells or 3T3 cell conditioned medium did not substitute for the viable 3T3 cell co-culture system in preserving hepatocyte viability and functionality.

Immunological and structural features of the protein core of human polymorphic epithelial mucin

The protein core of high mol. wt polymorphic epithelial mucin (PEM--approximately 400 kDa glycoprotein) which is associated with breast carcinomas, consists of a repeating 20 amino acid peptide motif [Gendler et al. (1988) J. biol. Chem. 263, 12,820-12,823]. Monoclonal antibodies C595 (anti-urinary mucin) and NCRC-11 (anti-breast carcinoma cells), and other antibodies against human milk fat globule membranes, were found to recognize determinants present within this 20 amino acid peptide. A model of the peptide was developed based on hydropathicity and structure prediction calculations and these indicated that the repeated structure is dominated by a hydrophilic domain of seven amino acids, extending into two flanking beta turns. NMR analysis of the 20 amino acid peptide was undertaken to probe the secondary structure. Epitope mapping experiments involving solid phase synthesis of overlapping heptapeptides in the repeat unit identified the minimum structures for antibody binding as Arg-Pro-Ala-Pro and Arg-Pro-Ala for the C595 and NCRC-11 antibodies, respectively. These determinants were found within the predicted hydrophilic turn region domain of the peptide. The epitopes for six other PEM-reactive monoclonal antibodies were also determined to reside within the predicted hydrophilic turn domain. This evidence is in accord with the disposition of this region of the PEM peptide core being at the exterior of the glycoprotein where it would be accessible to antibody recognition and binding events.

A novel biotinylated degradable polymer for cell‐interactive applications

We describe the development of a novel biodegradable polymer designed to present bioactive motifs at the surfaces of materials of any architecture. The polymer is a block copolymer of biotinylated poly(ethylene glycol) (PEG) with poly(lactic acid) (PLA); it utilizes the high-affinity coupling of the biotin-avidin system to undergo postfabrication surface engineering. We show, using surface plasmon resonance analysis (SPR) and confocal microscopy that surface engineering can be achieved under aqueous conditions in short time periods. These surfaces interact with cell surface molecules and generate beneficial responses as demonstrated by the model study of integrin-mediated spreading of endothelial cells on polymer surfaces presenting RGD peptide adhesion sequences.

Anion exchange in co-ordination polymers: a solid-state or a solvent-mediated process?

Interconversion of chain co-ordination polymers {[Ag(4,4′-bipy)](X)}∞ (X = NO3− or BF4−) in aqueous media has been studied by IR and 1H NMR spectroscopy, transmission electron (TEM) and atomic force (AFM) microscopies and by X-ray powder diffraction (PXRD). The exchange leads to the formation of a pure crystalline phase of a new co-ordination polymer, and detailed TEM and AFM studies indicate a solvent-mediated rather than a solid-state mechanism for the exchange process.

Characterization of Particle-Interactions by Atomic Force Microscopy: Effect of Contact Area

AbstractPurpose. The purpose of this work was to compare adhesion forces, contact area, and work of adhesion of salbutamol sulphate particles produced using micronization and a supercritical fluid technique (solution-enhanced dispersion by supercritical fluids - SEDS™) using atomic force microscopy (AFM). Methods. Adhesion forces of individual particles of micronized and SEDS™ salbutamol against a highly orientated pyrolytic graphite surface were acquired in a liquid environment consistent with that of a pressurized metered dose inhaler. The forces were then related to contact area and work of adhesion. Results. The raw adhesion force data for the micronized and SEDS™ material were 14.1 nN (SD 2.5 nN) and 4.2 nN (SD 0.8 nN), respectively. After correction for contact area, the forces per unit area were 13 mN/μm2 (SD 2.3 mN/μm2) and 3 mN/μm2 (SD 0.6 mN/μm2). The average work of adhesion was calculated using the Johnson-Kendall-Roberts theory and was found to be 19 mJm−2 (SD 3.4 mJm−2) for the micronized particle and 4 mJm−2 (SD 0.8 mJm−2) for the SEDS™ particle. Conclusions. It is possible to produce a three-dimensional representation of the contact area involved in the interaction and make quantitative comparisons between different particles. There was a lower force per unit area and work of adhesion observed for the SEDS™ material, possibly because of its lower surface free energy.

An Atomic Force Microscopy Study of the Effect of Nanoscale Contact Geometry and Surface Chemistry on the Adhesion of Pharmaceutical Particles

AbstractPurpose. To understand differences in particle adhesion observed with increasing humidity between samples of salbutamol sulfate prepared by two different methods. Methods. Atomic force microscopy (AFM) force measurements were performed as a function of humidity (<10% to 65% RH) using two systems. The first system used clean AFM tips against compressed disks of micronized and solution enhanced dispersion by supercritical fluid (SEDS) salbutamol. The second system involved particles of both salbutamol samples mounted onto the apexes of AFM cantilevers, and force measurements being performed against a highly orientated pyrolytic graphite (HOPG) substrate. Following these measurements, the contact asperities of the tips were characterized. Results. The first system showed a maximum in the observed adhesion at 22% relative humidity (RH) for the SEDS salbutamol compared to 44% RH for the micronized salbutamol. The second system showed a mix of peaks and continual increases in adhesion with humidity. The predicted Johnson-Kendall-Roberts forces were calculated and divided by the actual forces in order to produce a ratio. Conclusions. By relating the nature of the asperities to the force measurements, we propose a model in which adhesion scenarios range from single asperity nanometer-scale contact in which peaks in the adhesion were observed, to multiasperity contact where a continuous increase in adhesion was seen with humidity.

Formaldehyde at Low Concentration Induces Protein Tau into Globular Amyloid-Like Aggregates In Vitro and In Vivo

Recent studies have shown that neurodegeneration is closely related to misfolding and aggregation of neuronal tau. Our previous results show that neuronal tau aggregates in formaldehyde solution and that aggregated tau induces apoptosis of SH-SY5Y and hippocampal cells. In the present study, based on atomic force microscopy (AFM) observation, we have found that formaldehyde at low concentrations induces tau polymerization whilst acetaldehyde does not. Neuronal tau misfolds and aggregates into globular-like polymers in 0.01–0.1% formaldehyde solutions. Apart from globular-like aggregation, no fibril-like polymerization was observed when the protein was incubated with formaldehyde for 15 days. SDS-PAGE results also exhibit tau polymerizing in the presence of formaldehyde. Under the same experimental conditions, polymerization of bovine serum albumin (BSA) or α-synuclein was not markedly detected. Kinetic study shows that tau significantly misfolds and polymerizes in 60 minutes in 0.1% formaldehyde solution. However, presence of 10% methanol prevents protein tau from polymerization. This suggests that formaldehyde polymerization is involved in tau aggregation. Such aggregation process is probably linked to the taus special “worm-like” structure, which leaves the ε-amino groups of Lys and thiol groups of Cys exposed to the exterior. Such a structure can easily bond to formaldehyde molecules in vitro and in vivo. Polymerizing of formaldehyde itself results in aggregation of protein tau. Immunocytochemistry and thioflavin S staining of both endogenous and exogenous tau in the presence of formaldehyde at low concentrations in the cell culture have shown that formaldehyde can induce tau into amyloid-like aggregates in vivo during apoptosis. The significant protein tau aggregation induced by formaldehyde and the severe toxicity of the aggregated tau to neural cells may suggest that toxicity of methanol and formaldehyde ingestion is related to tau misfolding and aggregation.