Monolayer detection of ion binding at a crown ether-functionalised supramolecular surface via an integrated optical Bragg grating.

There have been significant recent developments in the field of integrated optical Bragg grating sensors for use in the biological domain, where changes in the thickness of a surface layer upon specific binding of biological targets allows quantitative detection. However in the chemical domain less work has been reported. We present here an integrated optical Bragg grating sensor, capable of evanescently detecting small changes in refractive index down to 10(-6) RIU at infrared wavelengths, within a microfluidic system. The high spectral fidelity of the Bragg gratings combined with precise thermal compensation enables direct monitoring of the surface throughout the experiment. This allows the sensor to probe surface changes in situ and in real-time, from preparation through to chemical modification of the surface, so that the progress of dynamic surface-localized interactions can be followed. Here we describe confirmatory studies to validate this approach, including a comparison with the modelled optical system, before assessing the ability to detect binding of Group I cations at a crown ether-functionalised supramolecular surface. Unlike larger biological entities, for these small chemical species, simple additive changes in film-thickness no longer prevail.

Investigation of dendriplexes by ion mobility-mass spectrometry.

Highly branched polyamidoamine (PAMAM) dendrimers presenting biological activities have been envisaged as non-viral gene delivery vectors. They are known to associate with nucleic acid (DNA) in non-covalent complexes via electrostatic interactions. Although their transfection efficiency has been proved, PAMAMs present a significant cytotoxicity due to their cationic surface. To overcome such a drawback, different chemical modifications of the PAMAM surface have been reported such as the attachment of hydrophobic residues. In the present work, we studied the complexation of DNA duplexes with different low-generation PAMAM; ammonia-cored G0(N) and G1(N) PAMAM, native or chemically modified with aromatic residues, i.e., phenyl-modified-PAMAM G0(N) and phenylalanine-modified-PAMAM G1(N). To investigate the interactions involved in the PAMAM/DNA complexes, also called dendriplexes, we used electrospray ionization (ESI) coupled to ion mobility spectrometry-mass-spectrometry (IM-MS). ESI is known to allow the study of non-covalent complexes in native conditions while IM-MS is a bidimensional separation technique particularly useful for the characterization of complex mixtures. IM-MS allows the separation of the expected complexes, possible additional non-specific complexes and the free ligands. Tandem mass spectrometry (MS/MS) was also used for the structural characterization. This work highlights the contribution of IM-MS and MS/MS for the study of small dendriplexes. The stoichiometries of the complexes and the equilibrium dissociation constants were determined. The [DNA/native PAMAM] and [DNA/modified-PAMAM] dendriplexes were compared.

Effects of PEGylation and acetylation of PAMAM dendrimers on DNA binding, cytotoxicity and in vitro transfection efficiency.

Poly(amidoamine) (PAMAM) dendrimers are promising multipotent gene delivery vectors, providing favorable DNA condensation properties also in combination with the possibility of conjugation of different targeting ligands to their surface. They have been used for transfection both in vitro and in vivo, but their application is currently somewhat limited due to inherent cytotoxicity. In this work we investigate how two types of surface modification, acetylation and PEGylation, affect the DNA binding characteristics, the cytotoxicity and the in vitro transfection efficiency of generation 4 and 5 PAMAM dendrimers. Particularly, we address how the morphology of DNA-dendrimer complexes, formed under low salt conditions, changes upon dilution in cell growth medium, an event that inevitably occurs before the complexes reach the cell surface in any transfection experiment. We find that acetylation and PEGylation essentially eliminates the inherent dendrimer cytotoxicity. However, the transfection efficiency of the modified dendrimers is lower than that of the corresponding unmodified dendrimers, which can be rationally understood by our observations that DNA is less condensed when complexed with these modified dendrimers. Although small DNA-dendrimer particles are formed, the availability for ethidium intercalation and nuclease degradation is significantly higher in the modified DNA-dendrimer complexes than in unmodified ones. Dilution in cell growth medium has a drastic effect on these electrostatically assembled complexes, resulting in increase in size and DNA availability. Our results strongly add to the notion that it is of importance to perform a biophysical characterization under conditions as close to the transfection situation as possible, to enable conclusions regarding structure-activity relations of gene delivery vectors.

Engineering multi-layered tissue constructs using acoustic levitation.

Engineering tissue structures that mimic those found in vivo remains a challenge for modern biology. We demonstrate a new technique for engineering composite structures of cells comprising layers of heterogeneous cell types. An acoustofluidic bioreactor is used to assemble epithelial cells into a sheet-like structure. On transferring these cell sheets to a confluent layer of fibroblasts, the epithelial cells cover the fibroblast surface by collective migration maintaining distinct epithelial and fibroblast cell layers. The collective behaviour of the epithelium is dependent on the formation of cell-cell junctions during levitation and contrasts with the behaviour of mono-dispersed epithelial cells where cell-matrix interactions dominate and hinder formation of discrete cell layers. The multilayered tissue model is shown to form a polarised epithelial barrier and respond to apical challenge. The method is useful for engineering a wide range of layered tissue types and mechanistic studies on collective cell migration.

Light harvesting in silicon(111) surfaces using covalently attached protoporphyrin IX dyes.

We report the photosensitization of crystalline silicon via energy transfer using covalently attached protoporphyrin IX (PpIX) derivative molecules at different distances via changing the diol linker to the surface. The diol linker molecule chain length was varied from 2 carbon to 10 carbon lengths in order to change the distance of PpIX to the Si(111) surface between 6 Å and 18 Å. Fluorescence quenching as a function of the PpIX-Si surface distance showed a decrease in the fluorescence lifetime by almost two orders of magnitude at the closest separation. The experimental fluorescence lifetimes are explained theoretically by a classical Chance-Prock-Silbey model. At a separation below 2 nm, we observe for the first time, a Förster-like dipole-dipole energy transfer with a characteristic distance of R0 = 2.7 nm.

Ex-vivo models of the Retinal Pigment Epithelium (RPE) in long-term culture faithfully recapitulate key structural and physiological features of native RPE.

The Retinal Pigment Epithelium (RPE) forms the primary site of pathology in several blinding retinopathies. RPE cultures are being continuously refined so that dynamic disease processes in this important monolayer can be faithfully studied outside the eye over longer periods. The RPE substrate, which mimics the supportive Bruch's membrane (BrM), plays a key role in determining how well in-vitro cultures recapitulate native RPE cells. Here, we evaluate how two different types of BrM substrates; (1) a commercially-available polyester transwell membrane, and (2) a novel electrospun scaffold developed in our laboratory, could support the generation of realistic RPE tissues in culture. Our findings reveal that both substrates were capable of supporting long-lasting RPE monolayers with structural and functional specialisations of in-situ RPE cells. These cultures were used to study autofluorescence and barrier formation, as well as activities such as outer-segment internalisation/trafficking and directional secretion of key proteins; the impairment of which underlies retinal disease. Hence, both substrates fulfilled important criteria for generating authentic in-vitro cultures and act as powerful tools to study RPE pathophysiology. However, RPE grown on electrospun scaffolds may be better suited to studying complex RPE-BrM interactions such as the formation of drusen-like deposits associated with early retinal disease.

Biocompatibility of poly(2-alkyl-2-oxazoline) brush surfaces for adherent lung cell lines.

Development of synthetic surfaces that are highly reproducible and biocompatible for in vitro cell culture offers potential for development of improved models for studies of cellular physiology and pathology. They may also be useful in tissue engineering by removal of the need for biologically-derived components such as extracellular matrix proteins. We synthesised four types of 2-alkyl-2-oxazoline polymers ranging from the hydrophilic poly(2-methyl-2-oxazoline) to the hydrophobic poly(2-n-butyl-2-oxazoline). The polymers were terminated using amine-functionalised glass coverslips, enabling the synthetic procedure to be reproducible and scaleable. The polymer-coated glass slides were tested for biocompatibility using human epithelial (16HBE14o-) and fibroblastic (MRC5) cell lines. Differences in adhesion and motility of the two cell types was observed, with the poly(2-isopropyl-2-oxazoline) polymer equally supporting the growth of both cell types, whereas poly(2-n-butyl-2-oxazoline) showed selectivity for fibroblast growth. In summary, 2-alkyl-2-oxazoline polymers may be a useful tool for building in vitro model cell culture models with preferential adhesion of specific cell types.

Experiences with a researcher-centric ELN.

Electronic Laboratory Notebooks (ELNs) are progressively replacing traditional paper books in both commercial research establishments and academic institutions. University researchers require specific features from ELNs, given the need to promote cross-institutional collaborative working, to enable the sharing of procedures and results, and to facilitate publication. The LabTrove ELN, which we use as our exemplar, was designed to be researcher-centric (i.e., not only aimed at the individual researcher's basic needs rather than to a specific institutional or subject or disciplinary agenda, but also able to be tailored because it is open source). LabTrove is being used in a heterogeneous set of academic laboratories, for a range of purposes, including analytical chemistry, X-ray studies, drug discovery and a biomaterials project. Researchers use the ELN for recording experiments, preserving data collected, and for project coordination. This perspective article describes the experiences of those researchers from several viewpoints, demonstrating how a web-based open source electronic notebook can meet the diverse needs of academic researchers.

Direct TLC/MALDI-MS coupling for modified polyamidoamine dendrimers analyses.

Polyamidoamine (PAMAM) are synthetic dendrimers which present attractive properties for the biological and biomedical fields, as they proved to be efficient drug and gene carriers. In order to increase their transfection efficiency, chemical modifications of the amino end-groups had been reported. In this work, the synthesis of the ammonia-cored G1(N) PAMAM and the consecutive chemical modification with glycine or phenylalanine amino-acids were monitored using the coupling of thin layer chromatography (TLC) with matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). Thus, the monitoring of the PAMAM synthesis included the identification of the by-products such as defective structures of PAMAM dendrimers as well as the study of phenylalanine-grafted PAMAM oligomer distribution.

Improving cellular adhesion on scaffolds for transplantation: synthesising a poly(MMA-co-PEGM) network.

Electrospun fibrous matrices prepared from methacrylate-based copolymers are investigated as a tool for retinal pigment epithelium (RPE) transplantation in the treatment of degenerative retinal diseases. Human RPE cells were used to probe the cell-surface interactions on these copolymer matrices. For the first time, simple changes in chemical functionality have been found to induce gel formation of these methacrylate backbone copolymers in vitro. This effect is shown to significantly improve RPE cell adhesion and survival.

An investigation into dispersion upon switching between solvents within a microfluidic system using a chemically resistant integrated optical refractive index sensor.

A planar Bragg grating device has been developed that is capable of detecting changes in the refractive index of a wide range of fluids including solvents, acids and bases. The integration of this high precision refractive index sensor within a chemically resistant microfluidic flow system has enabled the investigation of diverse fluid interactions. By cycling between different solvents, both miscible and immiscible, within the microfluidic system it is shown that the previous solvent determines the nature of the refractive index profile across the transition in composition. This solvent dispersion effect is investigated with particular attention to the methanol-water transition, where transients in refractive index are observed that are an order of magnitude larger in amplitude than the difference between the bulk fluids. The potential complications of such phenomenon are discussed together with an example of a device that exploits this effect for the unambiguous composition measurement of a binary solvent system.

Developing methacrylate-based copolymers as an artificial Bruch's membrane substitute.

Age-related macular degeneration (AMD) is the most common cause of blindness in the developed world. There is currently no treatment for the cellular loss, which is characteristic of AMD. Transplantation of retinal pigment epithelium (RPE) cells represents a potential therapy. Because of AMD-related pathology in the native support, Bruch's membrane, transplanted RPE cells require a scaffold to reside on. We present here the development of an electrospun fibrous scaffold derived from methyl methacrylate and poly(ethylene glycol) (PEG) methacrylate for novel application as an RPE scaffold. Scaffolds were chemically modified to improve cell adhesion by functionalization not previously reported for this type of copolymer system. A human RPE cell line was used to investigate cell-scaffold interactions for up to two weeks in vitro. Scanning electron microscopy was used to characterize the fibrous scaffolds and confirm cell attachment. By day 15, cell area was significantly (p < 0.001) enhanced on scaffolds with chemical modification of the PEG chain terminus. In addition, significantly, less-apoptotic cell death was demonstrable on these modified surfaces.

The chemistry of retinal transplantation: the influence of polymer scaffold properties on retinal cell adhesion and control.

Age-related macular degeneration is the most common cause of blindness in the UK. Cellular replacement of retinal pigment epithelium cells is a potential therapeutic option to treat the cellular loss and dysfunction which is characteristic of age-related macular degeneration and other progressive retinopathies. A supportive scaffold, natural or artificial, may be required to facilitate cell delivery to the eye. Research to improve the biomimetic properties of such scaffolds, in order to optimise cell attachment and functionality following implantation, is ongoing. This short review will focus on the potential of biomaterials for ocular tissue engineering and how surface modification and the physical properties of these scaffolds can be tailored to help realise the full clinical potential of retinal pigment epithelium cell transplantation.

Optimisation of polymer scaffolds for retinal pigment epithelium (RPE) cell transplantation.

AIM: To evaluate a variety of copolymers as suitable scaffolds to facilitate retinal pigment epithelium (RPE) transplantation. METHODS: Five blends of poly(l-lactic acid) (PLLA) with poly(d,l-lactic-glycolic acid) (PLGA) were manufactured by a solid-liquid phase separation technique. The blends were 10:90, 25:75, 50:50, 75:25 and 90:10 (PLLA:PLGA). All blend ratios were validated by nuclear magnetic resonance spectroscopy. Samples of polymer blends were coated with laminin. Coated and uncoated blends were seeded with a human RPE cell line. Cell attachment, viability and retention of phenotype were assessed. RESULTS: As the lactide unit content increased pore size generally became smaller. The 25:75 PLLA:PLGA blend was the most porous (44%) and thinnest (134 µm) scaffold produced. ARPE-19 cells retained an appropriate phenotype with minimal cell death for up to 4 weeks in vitro. Cell density was maintained on only one of the fabricated ratios (25% PLLA:75% PLGA). A consistent decrease in apoptotic cell death with time was observed on coated samples of this blend. A decrease in polymer thickness concomitant with an increase in porosity characteristic of degradation was observed with all polymer blends. CONCLUSIONS: This study demonstrates that a 25:75 copolymer blend of PLLA:PLGA is a potentially useful scaffold for ocular cell transplantation.

Modified dipicolinic acid ligands for sensitization of europium(III) luminescence.

The effect of substitution at the 4 and 3,5 positions in the pyridine ring of europium(III) pyridine-2,6-dicarboxylate complexes has been investigated with particular emphasis on sensitization of the Eu3+ ion. Sensitization of the Eu3+ 615-nm emission was achieved through excitation of the ligands in which the 4 substituent was -H, -OH, and -Cl and the 3,5 position was -H. In these cases, the ligand-to-Eu3+ ratio was confirmed as being 3:1. The sensitization was found to increase following substitution of the 4 position in the order Cl > H > OH. This is attributed to energy transfer occurring from the ligands into different Eu3+ intra-atomic energy levels, with spin selection rules governing the efficiency of this process. The Eu3+ luminescence lifetime was measured and found to vary from 1.16 to 2.90 ms depending on the excitation energy, ligand, and solvent. For the case of the 3,5-dibromo-4-hydroxy derivative, no sensitization was observed and a ligand-to-Eu3+ ratio of 1:1 was found. The solubility of these complexes in water and their long emission lifetime make them attractive for use as probes in biological systems.

Modeling novel radiopharmaceuticals: mono-C6-substituted PnAO ligands (PnAO = 3,3,9,9-tetramethyl-4,8-diazaundecane-2,10-dione dioxime).

The solid-state behavior of six novel 6-substituted PnAO (propylene amine oxime) complexes (6-11) involving Tc(V), Co(III), and Cu(II) salts is reported. Each of the Tc complexes 6-8 has the C6-substituent located equatorially in a six-membered chelate ring involving a Tc=O unit which has the expected boat geometry. The C6-substituent therefore has little effect on the conformational behavior of the PnAO complex and thus provides an attractive site for further modification. The Co(III) complex 9 has the expected octahedral geometry, while the Cu(II) complexes 10 and 11 form square-based pyramids capped by water molecules. One Cu(II) system (10) contains two unique complexes in the asymmetric unit which are associated via multiple hydrogen bonds to a BF(4) anion, the remaining BF(4) anion being loosely hydrogen bonded to a coordinating water molecule. The cobalt and copper complexes 9-11 each exhibit a chair conformation for the six-membered chelate ring.