Frances J. Harding

Biochemical and pharmacological characterization of isatin and its derivatives: from structure to activity

Isatin, 1H-indole-2,3-dione, is a heterocyclic compound of significant importance in medicinal chemistry. It is a synthetically versatile molecule, a precursor for a large number of pharmacologically active compounds. Isatin and its derivatives have aroused great attention in recent years due to their wide variety of biological activities, relevant to application as insecticides and fungicides and in a broad range of drug therapies, including anticancer drugs, antibiotics and antidepressants. The purpose of this review is to provide an overview of the pharmacological activities of isatin and its synthetic and natural derivatives. Molecular modifications to tailor the properties of isatin and its derivatives are also discussed.

Evaluation of mesoporous silicon/polycaprolactone composites as ophthalmic implants.

The suitability of porous silicon (pSi) encapsulated in microfibers of the biodegradable polymer polycaprolactone (PCL) for ophthalmic applications was evaluated, using both a cell attachment assay with epithelial cells and an in vivo assessment of biocompatibility in rats. Microfibers of PCL containing encapsulated pSi particles at two different concentrations (6 and 20 wt.%) were fabricated as non-woven fabrics. Given the dependence of Si particle dissolution kinetics on pSi surface chemistry, two different types of pSi particles (hydride-terminated and surface-oxidized) were evaluated for each of the two particle concentrations. Significant attachment of a human lens epithelial cell line (SRA 01/04) to all four types of scaffolds within a 24h period was observed. Implantation of Si fabric samples beneath the conjunctiva of rat eyes for 8 weeks demonstrated that the composite materials did not cause visible infection or inflammation, and did not erode the ocular surface. We suggest that these novel composite materials hold considerable promise as scaffolds in tissue engineering with controlled release applications.

Exploring the mesenchymal stem cell niche using high throughput screening

In the field of stem cell technology, future advancements rely on the effective isolation, scale-up and maintenance of specific stem cell populations and robust procedures for their directed differentiation. The stem cell microenvironment - or niche - encompasses signal inputs from stem cells, supporting cells and from the extracellular matrix. In this context, the contribution of physicochemical surface variables is being increasingly recognised. This paradigm can be exploited to exert control over cellular behaviour. However, the number of parameters at play, and their complex interactions, presents a formidable challenge in delineating how the decisions of cell fate are orchestrated within the niche. Additionally, in the case of mesenchymal stem cells (MSC), more than one type of stem cell niche has been identified. By employing high throughput screening (HTS) strategies, common and specific attributes of each MSC niche can be probed. Here, we explore biological, chemical and physical parameters that are known to influence MSC self-renewal and differentiation. We then review techniques and strategies that allow the HTS of surface properties for conditions that direct stem cell fate, using MSC as a case study. Finally, challenges in recapturing the niche, particularly its three dimensional nature, in surface-based HTS formats are discussed.

Insights into Cellular Uptake of Nanoparticles

Nanomaterials promise to improve disease diagnosis and treatment by enhancing the delivery of drugs, genes, biomolecules and imaging agents to specific subcellular targets. In order to optimize nanomaterial design for this purpose, a comprehensive understanding of how these materials are taken up and transported within the cell is required. In this review, we discuss the endocytic pathways employed by different types of nanoparticles with emphasis on the influence of nanoparticle surface modification. The use of pharmacological inhibition to probe internalization and intracellular trafficking pathways of nanoparticles is critically evaluated. Finally, approaches to target-specific delivery of therapeutics via nanoparticles into the cytoplasm and nucleus are addressed.

Assessing embryonic stem cell response to surface chemistry using plasma polymer gradients

The control of cell-material interactions is the key to a broad range of biomedical interactions. Gradient surfaces have recently been established as tools allowing the high-throughput screening and optimization of these interactions. In this paper, we show that plasma polymer gradients can reveal the subtle influence of surface chemistry on embryonic stem cell behavior and probe the mechanisms by which this occurs. Lateral gradients of surface chemistry were generated by plasma polymerization of diethylene glycol dimethyl ether on top of a substrate coated with an acrylic acid plasma polymer using a tilted slide as a mask. Gradient surfaces were characterized by X-ray photoelectron spectroscopy, infrared microscopy mapping and profilometry. By changing the plasma polymerization time, the gradient profile could be easily manipulated. To demonstrate the utility of these surfaces for the screening of cell-material interactions, we studied the response of mouse embryonic stem (ES) cells to these gradients and compared the performance of different plasma polymerization times during gradient fabrication. We observed a strong correlation between surface chemistry and cell attachment, colony size and retention of stem cell markers. Cell adhesion and colony formation showed striking differences on gradients with different plasma polymer deposition times. Deposition time influenced the depth of the plasma film deposited and the relative position of surface functional group density on the substrate, but not the range of plasma-generated species.

Nitric oxide-releasing porous silicon nanoparticles

In this study, the ability of porous silicon nanoparticles (PSi NPs) to entrap and deliver nitric oxide (NO) as an effective antibacterial agent is tested against different Gram-positive and Gram-negative bacteria. NO was entrapped inside PSi NPs functionalized by means of the thermal hydrocarbonization (THC) process. Subsequent reduction of nitrite in the presence of d-glucose led to the production of large NO payloads without reducing the biocompatibility of the PSi NPs with mammalian cells. The resulting PSi NPs demonstrated sustained release of NO and showed remarkable antibacterial efficiency and anti-biofilm-forming properties. These results will set the stage to develop antimicrobial nanoparticle formulations for applications in chronic wound treatment.

Surface Engineering for Long-Term Culturing of Mesenchymal Stem Cell Microarrays

The cell microarray format can recreate a multitude of cell microenvironments on a single chip using only minimal amounts of reagent. In this study, we describe surface modifications to passivate cell microarrays, aiming to adapt the platform to the study of stem cell behavior over long-term culture periods. Functionalization of glass slides with (3-glycidyloxypropyl) trimethoxysilane enabled covalent anchoring of extracellular matrix proteins on microscale spots printed by a robotic contact printer. Subsequently, the surface was passivated by bovine serum albumin (BSA) or poly(ethylene glycol)bisamine (A-PEG) with molecular weights of 3000, 6000, and 10 000 Da. Cloud-point conditions for A-PEG grafting were attained that were compatible with protein deposition. Passivation strategies were assessed by culturing mesenchymal stem cells on the microarray platform. While both BSA and A-PEG passivation initially blocked cell adhesion between the printed spots, only A-PEG grafting was able to maintain cell pattern integrity over the entire culture period of 3 weeks.

Versatile Particle-Based Route to Engineer Vertically Aligned Silicon Nanowire Arrays and Nanoscale Pores.

Control over particle self-assembly is a prerequisite for the colloidal templating of lithographical etching masks to define nanostructures. This work integrates and combines for the first time bottom-up and top-down approaches, namely, particle self-assembly at liquid-liquid interfaces and metal-assisted chemical etching, to generate vertically aligned silicon nanowire (VA-SiNW) arrays and, alternatively, arrays of nanoscale pores in a silicon wafer. Of particular importance, and in contrast to current techniques, including conventional colloidal lithography, this approach provides excellent control over the nanowire or pore etching site locations and decouples nanowire or pore diameter and spacing. The spacing between pores or nanowires is tuned by adjusting the specific area of the particles at the liquid-liquid interface before deposition. Hence, the process enables fast and low-cost fabrication of ordered nanostructures in silicon and can be easily scaled up. We demonstrate that the fabricated VA-SiNW arrays can be used as in vitro transfection platforms for transfecting human primary cells.

Surface Bound Amine Functional Group Density Influences Embryonic Stem Cell Maintenance

Gradient surfaces are highly effective tools to screen and optimize cell- surface interactions. Here, the response of embryonic stem (ES) cell colonies to plasma polymer gradient surfaces is investigated. Surface chemistry ranged from pure allylamine (AA) plasma polymer on one end of the gradient to pure octadiene (OD) plasma polymer on the other end. Optimal surface chemistry conditions for retention of pluripotency were identified. Expression of the stem cell markers alkaline phosphatase (AP) and Oct4 varied with the position of the ES cell colonies across the OD-AA plasma polymer gradient. Both markers were more strongly retained on the OD plasma polymer rich regions of the gradients. The observed variation of expression across the plasma polymer gradient increased with duration of stem cell culture. While maximum cell adhesion to the gradient substrate occurred at a nitrogen- to-carbon (N/C ratio) of approximately 0.1, Oct4 and AP expression was best retained at an N/C ratio < 0.04. Stem cell marker expression correlated with colony size and morphology: more compact, multilayered colonies with prominent F-actin staining arose as the N/C ratio decreased. Disruption of actin polymerization using Y-27632 ROCK inhibitor resulted in a collapse of the multilayer colony structure into monolayers with limited cell-cell contact. A corresponding decrease in expression of AP and Oct4 was observed. Oct4 expression along with 3D colony morphology was partially rescued on the OD plasma polymer rich regions of the gradient.

Microplasma arrays: a new approach for maskless and localized patterning of materials surfaces

“Maskless” microplasma treatment of passivated surfaces has been developed for the micropatterning of materials surfaces. The micropatterned surfaces are used in the fabrication of arrays for protein and cell-based assays. The advantage of this micropatterning approach is that it can be easily integrated into current manufacturing practices and the resultant micropatterned surfaces used with existing life sciences techniques and instrumentation.