Nikin Patel

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)

Printing patterns of biospecifically-adsorbed protein.

The advancement of elastomeric patterning techniques in recent years has significantly enhanced our ability to spatially control biomaterial surface chemistry at the micrometre level. The application of this technology to the patterning of biomolecules onto solid surfaces has created many potential applications including the development of advanced biosensors, combinatorial library screening and the formation of tissue engineering templates. In this paper, we describe the direct patterning of protein by microcontact printing. An important consideration for the fabrication of protein micropatterns intended for these applications is the nature of the protein immobilization to a substrate. To date, the patterning of proteins by direct microcontact printing (μCP) has relied on the non-covalent adsorption to a substrate. Ideally, the proteins need to be firmly anchored onto a surface without adversely effecting their activity. Here, the high affinity avidin-biotin receptor-ligand interaction has been exploited to form arrays of avidin molecules onto a polymeric substrate expressing biotin moieties. This has created a generic technique by which any biotinylated species can be subsequently immobilized into defined patterns. Utilizing atomic force microscopy (AFM), the patterned surfaces have been characterized to molecular resolution. The micropatterned sample supported cell adhesion when biotin-(G)11-GRGDS was bound to the avidin bearing arrays.

Identifying and mapping surface amorphous domains.

PurposeUndesirable amorphous material generation during formulation is implicated in a growing number of pharmaceutical problems. Due to the importance of interfacial properties in many drug delivery systems, it seems that surface amorphous material is particularly significant. Consequently, this study investigates a range of methods capable of detecting and mapping surface amorphous material.MethodsA micron-sized localized surface domain of amorphous sorbitol is generated using a novel localized heating method. The domain is subsequently investigated using atomic force microscopy (AFM) imaging, nanomechanical measurements, and Raman microscopy 3-D profiling.ResultsAFM phase and height images reveal nanoscale-order variations within both crystalline and amorphous sorbitol domains. Nanomechanical measurements are able to quantitatively distinguish the amorphous and crystalline domains through local Young’s modulus measurements. Raman microscopy also distinguishes the amorphous and crystalline sorbitol through variations in peak width. This is shown to allow mapping of the 3-D distribution of the amorphous phase and is hence complementary to the more surface sensitive AFM measurements.ConclusionsAFM and Raman microscopy map the distribution of amorphous material at the surface of a sorbitol crystal with submicron spatial resolution, demonstrating surface analysis methods for characterizing semicrystalline solids generated during pharmaceutical processing.

Nanoscale thermal analysis of pharmaceutical solid dispersions.

Formation of a solid solution of a drug in a water-soluble polymer is one of the primary techniques used to improve the dissolution rate and thus bioavailability of a poorly water-soluble drug. Understanding and detecting the state of the drug inside such a polymer matrix is critically important since issues such as drug stability, safety and efficacy can be greatly affected. In this study, two model formulations were prepared containing low and high levels of drug content. The heterogeneity of the formulations has been investigated using a novel nanothermal analysis technique. This technique has demonstrated a promising capability for imaging and quantitatively characterising the nanoscale properties of solid dispersion formulations.

The stability of solid dispersions of felodipine in polyvinylpyrrolidone characterized by nanothermal analysis.

Nanothermal analysis (NTA) supported by atomic force microscopy imaging has been used to study the changes that occur at the surfaces of solid dispersions of the drug felodipine and the water soluble polymer, polyvinylpyrrolidone (PVP) on exposure to standard pharmaceutical environmental stress conditions. Exposure to relative humidities above 75% (at 40 °C) was sufficient to achieve phase separation of the drug and polymer into areas which displayed a glass transition temperature consistent with pure drug and polymer over a period of a few days. Higher values of humidity at 25 °C (e.g. 95%RH) were also sufficient to cause such phase separation within a day. Extended studies of up to two months showed an eventual crystallization of the drug. NTA is shown to be effective at the early detection of instabilities in solid dispersions and the quantifiable identification of the relative composition of phase separated domains based upon their glass transition temperatures. The combined nanoscale analytical approach employed here is able to systematically study the influence of storage conditions and different drug loadings and to evaluate physical stability as a function of environmental conditions.

Morphological Development of β(1-40) Amyloid Fibrils

The Alzheimers disease-related peptide β(1-40) amyloid self-associates to form fibrils exhibiting a morphology characteristic of amyloidogenic proteins. The mechanism of this fibrillization process has yet to be fully elucidated. In this study we have immobilized the β(1-40) amyloid to flat gold surfaces using thiol-based self-assembled monolayers. Atomic force microscopy reveals the presence of spherical units of β(1-40) amyloid immediately following the initiation of fibrillization. Short fibrillar structures, termed nascent fibrils, which appear to be formed by the association of these units are also present at this time point. At later time points extended, branching networks of fibrils are observed. Some fibrils exhibit a more beaded appearance and greater axial periodicity than others. No nascent fibrils are seen to be present. We believe that these data identify an early fibril structure which could act as an intermediate in β-amyloid fibrillization. The oligomeric units of which these nascent fibrils are comprised are also determined.

Towards the understanding and prediction of material changes during micronisation using atomic force microscopy.

In this study we aim to explore the potential links between the mechanical properties, micronisation behaviour and surface energy of carbamazepine polymorphs using atomic force microscopy (AFM) measurements of material properties at the nanoscale. Carbamazepine Forms I, II and III were prepared and confirmed using X-ray powder diffraction (XRPD). AFM measurements of indentation hardness, Youngs modulus and surface energy were made on the starting material. In addition, the surface energy was measured immediately after micronisation and after storage for four weeks. Carbamazepine polymorphs could be ranked by Youngs modulus and hardness. Surface energy measurements showed an increase after micronisation in all cases, and a varying relaxation after storage for four weeks. Form I showed a smaller particle size distribution, indicating more complete micronisation. A promising correlation was observed between the hardness/Youngs modulus ratio and the micronisation behaviour, in terms of particle size reduction and surface energy change. The results show potential for the predictive capacity of such an approach, and help to provide a greater understanding of material behaviour and properties during micronisation.

Towards nanoscale metrology for biomolecular imaging by atomic force microscopy

Atomic force microscopy (AFM) provides three-dimensional images with resolution at or near the atomic level. Many researchers have addressed the metrological aspects of AFM imaging of physical samples, producing a range of recognized standards for calibrated spatial measurements. However, because of the complex interplay of forces between an AFM tip and biological samples, the dynamic environment in which such imaging takes place, and difficulties in immobilization, no satisfactory equivalents exist for the biological AFM community. Here an exploration of the effects of AFM imaging parameters on apparent biomolecular dimensions in aqueous environments is carried out for a three-component system comprising GroEL protein, plasmid DNA and gold nanoparticles. The biomolecules in this system have been chosen to present differing imaging challenges, while gold nanoparticles serve as an internal reference for tip performance. Concurrent immobilization of these entities requires optimization of sample preparation methodology, described here as it is hoped the system may act as a template for future bio-metrological standards. Investigation of differences in measured heights and lateral dimensions of DNA, GroEL and gold spheres under a range of imaging conditions and the implications of these results for accurate imaging of biological samples and as a potential bio-metrological standard are discussed.

Detection of Low Levels of Amorphous Lactose using H/D Exchange and FT-Raman Spectroscopy

PurposeTo demonstrate the potential of monitoring H/D exchange by FT-Raman spectroscopy as a tool for the detection and quantification of low levels of amorphous lactose in formulations.MethodsSamples containing different proportions of amorphous and crystalline lactose were prepared. H/D exchange was carried out by exposing the samples to a flow of D2O vapour. A calibration curve was constructed from the FT-Raman spectra of the deuterated samples by integrating the ν(OD) band and normalizing to an internal standard. This method was benchmarked against a conventional approach using Raman spectroscopy where the ratio of Raman bands associated with crystalline and amorphous lactose is used to estimate the amorphous content.ResultsThe H/D exchange method revealed a linear response over the entire composite range with an excellent correlation coefficient (R2 = 0.999). The sensitivity of this approach in detecting the amount of amorphous lactose present in a blend is significantly greater than that offered by conventional FT-Raman in the 0–10% level of amorphous material.ConclusionsA non-destructive method that is capable of providing reproducible measurements of low levels of amorphous material in lactose has been demonstrated and this method has enhanced sensitivity relative to approaches using Raman spectroscopy without deuteration.

A physical and chemical comparison of material from a conventional spray-dried system and a single particle spray-dried system.

When assessing the suitability of potential drug/polymer systems for improving drug bioavailability, substantial efficiency gains can be achieved through the development and application of rapid miniaturised screening methods. For this to be possible new methods of small-scale formulation manufacture that produce materials equivalent to full-scale manufacture are urgently required. In this work, we use Atomic Force Microscopy (AFM) and Confocal Raman Microscopy (CRM) to investigate the potential physical and chemical equivalence of individually dried particles generated using a DRYING KINETICS ANALYZER™ (DKA) with material from a conventional spray-drier. For our model system of griseofulvin (at loadings of 2.5%, w/w and 20%, w/w) and PEG 6000, the results demonstrate physicochemical equivalence between the two spray-drying methods for the same drug loading. Thus we suggest that single particle spray drying offers a viable and novel route to the production of materials for miniaturised methods of screening candidate drug/polymer formulations.