Sarah P. Hudson

Proteins in Mesoporous Silicates

Mesoporous silicates (MPS) have an ordered pore structure with dimensions comparable to many biological molecules. They have been extensively explored as supports for proteins and enzymes in biocatalytic applications. Since their initial discovery, novel syntheses methods have led to precise control over pore size and structure, particle size, chemical composition, and stability, thus allowing the adsorption of a wide variety of biological macromolecules, such as heme proteins, lipases, antibody fragments, and proteases, into their structures. This Review discusses the application of ordered, large-pore, functionalized mesoporous silicates to immobilize proteins for biocatalysis.

The biocompatibility of mesoporous silicates

Micro- and nano-mesoporous silicate particles are considered potential drug delivery systems because of their ordered pore structures, large surface areas and the ease with which they can be chemically modified. However, few cytotoxicity or biocompatibility studies have been reported, especially when silicates are administered in the quantities necessary to deliver low-potency drugs. The biocompatibility of mesoporous silicates of particle sizes approximately 150 nm, approximately 800 nm and approximately 4 microm and pore sizes of 3 nm, 7 nm and 16 nm, respectively, is examined here. In vitro, mesoporous silicates showed a significant degree of toxicity at high concentrations with mesothelial cells. Following subcutaneous injection of silicates in rats, the amount of residual material decreased progressively over 3 months, with good biocompatibility on histology at all time points. In contrast, intra-peritoneal and intra-venous injections in mice resulted in death or euthanasia. No toxicity was seen with subcutaneous injection of the same particles in mice. Microscopic analysis of the lung tissue of the mice indicated that death may be due to thrombosis. Although local tissue reaction to mesoporous silicates was benign, they caused severe systemic toxicity. This toxicity might be mitigated by modification of the materials.

Injectable in situ cross-linking hydrogels for local antifungal therapy.

Invasive fungal infections can be devastating, particularly in immunocompromised patients, and difficult to treat with systemic drugs. Furthermore, systemic administration of those medications can have severe side effects. We have developed an injectable local antifungal treatment for direct administration into existing or potential sites of fungal infection. Amphotericin B (AmB), a hydrophobic, potent, and broad-spectrum antifungal agent, was rendered water-soluble by conjugation to a dextran-aldehyde polymer. The dextran-aldehyde-AmB conjugate retained antifungal efficacy against Candida albicans. Mixing carboxymethylcellulose-hydrazide with dextran-aldehyde formed a gel that cross-linked in situ by formation of hydrazone bonds. The gel provided in vitro release of antifungal activity for 11 days, and contact with the gel killed Candida for three weeks. There was no apparent tissue toxicity in the murine peritoneum and the gel caused no adhesions. Gels produced by entrapment of a suspension of AmB in CMC-dextran without conjugation of drug to polymers did not release fungicidal activity, but did kill on contact. Injectable systems of these types, containing soluble or insoluble drug formulations, could be useful for treatment of local antifungal infections, with or without concurrent systemic therapy.

Combinatorial Extracellular Matrices for Human Embryonic Stem Cell Differentiation in 3D

Embryonic stem cells (ESCs) are promising cell sources for tissue engineering and regenerative medicine. Scaffolds for ESC-based tissue regeneration should provide not only structural support, but also signals capable of supporting appropriate cell differentiation and tissue development. Extracellular matrix (ECM) is a key component of the stem cell niche in vivo and can influence stem cell fate via mediating cell attachment and migration, presenting chemical and physical cues, as well as binding soluble factors. Here we investigated the effects of combinatorial extracellular matrix proteins on controlled human ESC (hESC) differentiation. Varying ECM compositions in 3D markedly affects cell behavior, and optimal compositions of ECM hydrogels are identified that facilitate specific-lineage differentiation of stem cells. To our knowledge, this is the first combinatorial analysis of ECM hydrogels for their effects on hESC differentiation in 3D. The 3D matrices described herein may provide a useful platform for studying the interactive ECM signaling in influencing stem cell differentiation.

A Prototype Antifungal Contact Lens

PURPOSE To design a contact lens to treat and prevent fungal ocular infections. METHODS Curved contact lenses were created by encapsulating econazole-impregnated poly(lactic-co-glycolic) acid (PLGA) films in poly(hydroxyethyl methacrylate) (pHEMA) by ultraviolet photopolymerization. Release studies were conducted in phosphate-buffered saline at 37°C with continuous shaking. The contact lenses and their release media were tested in an antifungal assay against Candida albicans. Cross sections of the pre- and postrelease contact lenses were characterized by scanning electron microscopy and by Raman spectroscopy. RESULTS Econazole-eluting contact lenses provided extended antifungal activity against Candida albicans fungi. Fungicidal activity varied in duration and effectiveness depending on the mass of the econazole-PLGA film encapsulated in the contact lens. CONCLUSIONS An econazole-eluting contact lens could be used as a treatment for fungal ocular infections.

Quantitative TEM analysis of a hexagonal mesoporous silicate structure

TEM analysis of mesoporous materials is generally undertaken to give qualitative results. Accurate quantitative analysis is demonstrated in this study. A systematic image analysis of a powder form of a hexagonal mesoporous material known as KIT-6 is conducted using a transmission electron microscope (TEM). Three types of image contrast typically appear in this material (a hexagonal honeycomb structure, wide and narrow parallel lines). The honeycomb face is used to characterise this material in terms of a conventional 2-D hexagonal structure and the d-spacings for the (100) and (110) planes are experimentally measured in varying focus conditions. A tilting experiment is conducted to determine how the angle of tilt affects the line spacing and their visibility. Tilting has very little effect on the line spacing, whereas it affects the visibility of both the wide and narrow lines by limiting an angle range of visibility. The hexagonal lattice structure parameter determined by TEM method is found to be approximately 7% lower than that calculated by low-angle X-ray diffraction. Thus we conclude that TEM data can be used to determine the geometry and dimensions of hexagonal mesoporous silica materials, with a small error in the hexagonal lattice parameter.

Investigating the solubility and cytocompatibility of CaO–Na2O–SiO2/TiO2 bioactive glasses

This study aims to investigate the solubility of a series of titanium (TiO2 )-containing bioactive glasses and their subsequent effect on cell viability. Five glasses were synthesized in the composition range SiO2 -Na2 O-CaO with 5 mol % of increments TiO2 substituted for SiO2 . Glass solubility was investigated with respect to (1) exposed surface area, (2) particle size, (3) incubation time, and (4) compositional effects. Ion release profiles showed that sodium (Na(+) ) presented high release rates after 1 day and were unchanged between 7 and 14 days. Calcium (Ca(2+) ) release presented a significant change at each time period and was also composition dependent, where a reduction in Ca(2+) release is observed with an increase in TiO2 concentration. Silica (Si(4+) ) release did not present any clear trends while no titanium (Ti(4+) ) was released. Cell numbers were found to increase up to 44%, compared to the growing control population, with a reduction in particle size and with the inclusion of TiO2 in the glass composition.

Enhancement and restriction of chain motion in polymer networks.

Sevelamer carbonate, a polymeric drug, adsorbs phosphate ions from the gastro intestine of patients suffering from chronic kidney disease. Polymer chain mobility becomes critical during its manufacture and storage. How the polymer chain mobility in sevelamer carbonate is quantitatively controlled by small molecular species, in this case by water molecules and bicarbonate anions, is demonstrated here. Spin-lattice relaxation times of the protons in the hydrogel, detected by solid state NMR, are indicative of mobility within the polymer. They decreased with increasing water content but increased as the bicarbonate anion content increased. As the water content increased, the glass transition temperature decreased but increasing the bicarbonate anion content had the opposite effect. FTIR analysis indicated that the anions were involved in bonding while the water molecules were not. The stability and physicochemical properties of polymers during storage and formulation depend on the polymeric structure and the dynamic behaviour of the polymer chains.

Carrier particle design for stabilization and isolation of drug nanoparticles.

Nanoparticles of poorly water-soluble drugs were prepared in suspension via antisolvent precipitation in order to improve their dissolution behaviour. Insoluble, surface-functionalized, micron-range, clay carrier particles were employed for the dual purpose of stabilizing the nanoparticles in suspended state, and facilitating their unhindered isolation to solid state; often a challenging step in nanoparticle production. The carrier particles, which were functionalized with an optimal level of cationic polymer (protamine), attracted negatively-charged nanoparticles to their surface as a uniform and segregated nanoparticle layer, at drug loadings up to 9% w/w. By using carrier particles to stabilise the nanoparticles on their surface, the traditionally used solubilised nanosuspension stabilisers could be eliminated, thus avoiding time-consuming stabiliser screening tests. The carrier particle system facilitated stabilisation of nanoparticles in suspension, isolation of nanoparticles to the solid state via filtration, and preservation of fast nanoparticle-induced dissolution rates of the dried nanoparticle-carrier composites, indicating preservation of their high surface area during drying. The process was validated with two poorly water-soluble BCS Class II drugs, fenofibrate and mefenamic acid, both of which demonstrated negative surface charge in aqueous suspension.

Preparation and characterization of amorphous ciprofloxacin-amino acid salts

Graphical abstract Figure. No Caption available. ABSTRACT The amorphization of the poorly soluble drug ciprofloxacin (CIP) may be facilitated by the use of a suitable stabilizer. In this study seven amino acids, with various side chain properties, were evaluated in this regard. Solid dispersions were prepared by ball milling 1:1 molar ratios of CIP with the amino acids, and their solid‐state and pharmaceutical properties were then examined. Fully X‐ray amorphous solid dispersions were obtained with aspartic acid, glutamic acid, cysteine and arginine. In each case, evidence of salt formation between the drug and amino acids was found via Fourier transform infrared spectroscopy and solid‐state nuclear magnetic resonance. In contrast, semi‐crystalline solid dispersions were obtained with serine, alanine and glycine. The glass transition temperatures of the amorphous salts were significantly higher than those of the starting materials, and they remained fully X‐ray amorphous during long‐term stability studies. Significant improvements in the solubility of CIP were also observed with the amorphous salts in water and simulated biological fluids, over and above that of the corresponding physical mixtures. In permeability studies on the other hand, the amorphous aspartate and glutamate salts were found to be less permeable than the pure drug, whereas formulation as an amorphous salt containing cysteine or arginine increased the permeability of CIP. Therefore, while amorphous salt formation with amino acids appears to be a suitable means of improving the thermal stability and solubility of CIP, in some cases this is associated with a decrease in permeability.