Clive J. Roberts

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.

Comparison of calibration methods for atomic-force microscopy cantilevers

Th es cientific community needs a rapid and reliable way of accurately determining the stiffness of atomic-force microscopy cantilevers. We have compared the experimentally determined values of stiffness for ten cantilever probes using four different methods. For rectangular silicon cantilever beams of well defined geometry, the approaches all yield values within 17% of the manufacturer’s nominal stiffness. One of the methods is new, based on the acquisition and analysis of thermal distribution functions of the oscillator’s amplitude fluctuations. We evaluate this method in comparison to the three others and recommend it for its ease of use and broad applicability.

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)

Surface plasmon resonance for real time in situ analysis of protein adsorption to polymer surfaces.

The adsorption of a range of plasma proteins to metal and polymer surfaces has been examined using surface plasmon resonance (SPR). The adsorption of proteins was initially studied on the SPR silver sensor surface, and then on a model polystyrene film spun coated directly onto this substrate. In both cases, reproducible adsorption profiles for albumin were attained which compared well with corresponding atomic force microscopy (AFM) and ellipsometry data on protein monolayer packing and thickness respectively. The SPR data revealed the influence of concentration on both protein adsorption kinetics and the time for formation of a monolayer coating. SPR data also highlighted different adsorption kinetics and final monolayer SPR angle shift values for three plasma proteins which have been interpreted in terms of their molecular dimensions and orientation at the polymer interface. AFM data confirmed the presence of a closely packed protein layer for all three protein systems. These studies are discussed in terms of employing SPR in the study of protein interactions at surfaces which are important in the design and evaluation of novel biomedical polymeric materials.

Desktop 3D printing of controlled release pharmaceutical bilayer tablets

Three dimensional (3D) printing was used as a novel medicine formulation technique for production of viable tablets capable of satisfying regulatory tests and matching the release of standard commercial tablets. Hydroxypropyl methylcellulose (HPMC 2208) (Methocel™ K100M Premium) and poly(acrylic acid) (PAA) (Carbopol(®) 974P NF) were used as a hydrophilic matrix for a sustained release (SR) layer. Hypromellose(®) (HPMC 2910) was used as a binder while microcrystalline cellulose (MCC) (Pharmacel(®) 102) and sodium starch glycolate (SSG) (Primojel(®)) were used as disintegrants for an immediate release (IR) layer. Commercial guaifenesin bi-layer tablets (GBT) were used as a model drug (Mucinex(®)) for this study. There was a favourable comparison of release of the active guaifenesin from the printed hydrophilic matrix compared with the commercially available GBT. The printed formulations were also evaluated for physical and mechanical properties such as weight variation, friability, hardness and thickness as a comparison to the commercial tablet and were within acceptable range as defined by the international standards stated in the United States Pharmacopoeia (USP). All formulations (standard tablets and 3D printed tablets) showed Korsmeyer-Peppas n values between 0.27 and 0.44 which indicates Fickian diffusion drug release through a hydrated HPMC gel layer.

Competitive protein adsorption as observed by surface plasmon resonance

The competitive nature of protein adsorption has been investigated in situ by surface plasmon resonance (SPR) analysis. The adsorption from blood plasma solutions of albumin, fibrinogen and immunoglobulin-G (IgG), to a polystyrene surface was investigated as part of concentration- and time-dependent studies, to observe the sequential adsorption of the three proteins at the surface. Adsorption of plasma solutions at a range of concentrations or incubation times was performed and the resulting surfaces were probed by the addition of an appropriate antibody to the protein surface. The process was repeated for each antigen leading to a surface concentration profile of each protein with respect to plasma concentration and plasma incubation time. The SPR was able to detect changes in the relative surface concentration of each component demonstrating that the proteins residence time at the interface was dependent upon its molecular weight, bulk concentration and surface affinity. All ri,hts reserved

3D printing of five-in-one dose combination polypill with defined immediate and sustained release profiles.

We have used three dimensional (3D) extrusion printing to manufacture a multi-active solid dosage form or so called polypill. This contains five compartmentalised drugs with two independently controlled and well-defined release profiles. This polypill demonstrates that complex medication regimes can be combined in a single personalised tablet. This could potentially improve adherence for those patients currently taking many separate tablets and also allow ready tailoring of a particular drug combination/drug release for the needs of an individual. The polypill here represents a cardiovascular treatment regime with the incorporation of an immediate release compartment with aspirin and hydrochlorothiazide and three sustained release compartments containing pravastatin, atenolol, and ramipril. X-ray powder diffraction (XRPD) and Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) were used to assess drug-excipient interaction. The printed polypills were evaluated for drug release using USP dissolution testing. We found that the polypill showed the intended immediate and sustained release profiles based upon the active/excipient ratio used.

Blind reconstruction of scanning probe image data

Scanning probe microscopy has proven to be an invaluable tool for the investigation of surface topography; however, the finite geometry of the imaging tip can often distort image data and complicate metrological investigations of surface features. Here, the derivation of a computational procedure for the estimation of the geometry of the scanning probe from the topographic image data alone is presented. The properties of the tip function extracted from such data permit an assessment of the sample‐related information content of an image. The technique is demonstrated by its application to simulated scanning probe microscopy image data, where its performance can be assessed, and by its application to experimental image data obtained from the scanning force microscope.

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.

Extracellular matrix-mediated osteogenic differentiation of murine embryonic stem cells

Embryonic stem cells (ESCs) are pluripotent and have the ability to differentiate into mineralising cells in vitro. The use of pluripotent cells in engineered bone substitutes will benefit from the development of bioactive scaffolds which encourage cell differentiation and tissue development. Extracellular matrix (ECM) may be a suitable candidate for use in such scaffolds since it plays an active role in cellular differentiation. Here, we test the hypothesis that tissue-specific ECM influences the differentiation of murine ESCs. We induced murine ESCs to differentiate by embryoid body formation, followed by dissociation and culture on ECM prepared by decellularisation of either osteogenic cell (MC3T3-E1) or non-osteogenic cell (A549) cultures, or on defined collagen type I matrix. We assessed osteogenic differentiation by formation of mineralised tissue and osteogenic gene expression, and found it to be significantly greater on MC3T3-E1 matrices than on any other matrix. The osteogenic effect of MC3T3-E1 matrix was reduced by heat treatment and abolished by trypsin, suggesting a bioactive proteinaceous component. These results demonstrate that decellularised bone-specific ECM promotes the osteogenic differentiation of ESCs. Our results are of fundamental interest and may help in tailoring scaffolds for tissue engineering applications which both incorporate tissue-specific ECM signals and stimulate stem-cell differentiation.