The effect of pH on PAMAM dendrimer-siRNA complexation:endosomal considerations as determined by molecular dynamics simulation

Intracellular degradation of genes, most notably within the endo-lysosomal compartment is considered a significant barrier to (non-viral) gene delivery in vivo. Previous reports based on in vitro studies claim that carriers possessing a mixture of primary, secondary and tertiary amines are able to buffer the acidic environment within the endosome, allowing for timely release of their contents, leading to higher transfection rates. In this report, we adopt an atomistic molecular dynamics (MD) simulation approach, comparing the complexation of 21-bp siRNA with low-generation polyamidoamine (PAMAM) dendrimers (G0 and G1) at both neutral and acidic pHs, the latter of which mimics the degradative environment within maturing 'late-endosomes'. Our simulations reveal that the time taken for the dendrimer-gene complex (dendriplex) to reach equilibrium is appreciably longer at low pH and this is accompanied by more compact packaging of the dendriplex, as compared to simulations performed at neutral pH. We also note larger absolute values of calculated binding free energies of the dendriplex at low pH, indicating a higher dendrimer-nucleic acid affinity in comparison with neutral pH. These novel simulations provide a more detailed understanding of low molecular-weight polymer-siRNA behavior, mimicking the endosomal environment and provide input of direct relevance to the "proton sponge theory", thereby advancing the rational design of non-viral gene delivery systems.

The activity of pH membrane disruptive pseudopeptides and their subcellular fate in mammalian cells cultured in-vitro

This thesis describes investigations upon pseudopeptides which were conducted to improve our understanding of the fate of synthetic macromolecules in cells and to develop approaches to influence that fate. The low uptake of molecules across the external cellular membrane is the principal barrier against effective delivery of therapeutic products to within the cell structure. In nature, disruption of this membrane by amphiphilic peptides plays a central role in the pathogenesis by bacterial and toxin infections. These amphiphilic peptides contain both hydrophobic and weakly charged hydrophilic amino acid residues and upon activation they become integrated into the lipid bilayers of the extracellular or endosomal membranes. The architectures of the pseudopeptides described here were designed to display similar pH dependent membrane rupturing activity to that of peptides derived from the influenza virus hemagglutinin HA-2. This HA protein promotes fusion of the influenza virus envelope with the cell endosome membrane due to a change in conformation in response to the acidic pH of the endosome lumen (pH 5.0-6.0). The pseudopeptides were obtained by the copolymerisation of L-lysine and L-lysine ethyl-ester with various dicarboxylic acid moieties. In this way a linear polyamide comprising of alternating pendant carboxylic acids and pendant hydrophobic moieties was made. At physiological pH (pH 7.4), electrostatic repulsion of pendant anionic carboxyl groups along the polymer backbone is sufficient to overcome the intramolecular association of the hydrophobic groups resulting in an extended conformation. At low pH (typically pH 4.8) loss of charge results in increased intramolecular hydrophobic association and the polymer chain collapses to a compact conformation, leading to precipitation of the polymer. Consequently, a conformation dependent functional property could be made to respond to small changes in the environmental pH. Pseudopepides were investigated for their cytoxicity towards a well known cell line, namely C26 (colorectal adenocarcinoma) and were shown through the use of a cell viability assay, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) to be well tolerated by C26 cells over a range of concentrations (2-500,μg/ml) at physiological pH (pH 7.4). A modified version of a shorter 30-minute coupled enzymatic assay, the LDH (lactate dehydrogenase) assay was used to evaluate the ability of the pseudopeptides to disrupt the membrane of two different cell lines (COS-1; African green monkey, kidney and A2780; human ovarian carcinoma) at low pH (pH 5.5). The cell membrane disruption property of the pseudopeptides was successfully demonstrated for COS-I and A2780 cell lines at this pH (pH 5.5). A variety of cell lines were chosen owing to limited availability and to compare the cytotoxic action of these pH responsive psudopeptides towards normal and tumorogenic cell lines. To investigate the intracellular delivery of one of the pseudopeptides, poly (L-lysine iso-phthalamide) and its subcellular location, a Cy3 bisamine fluorophore was conjugated into its backbone, at ratios of dye:lysine of 1:20, 1:30, 1:40, 1:60 and 1:80. Native polyacrylacrylamide gel electrophoresis (PAGE) and high voltage paper electrophoresis (HVPE) studies of the polydyes were conducted and provided evidence that that the Cy3 bisamine fluorophore was conjugated into the backbone of the polymer, poly (L-lysine iso-phthalamide). The subcellular fate of the fluorescentlylabelled "polydye" (hereafter PD20) was monitored by laser scanning confocal microscopy (LSCM) in CHO (Chinese hamster ovary) cells cultured in-vitro at various pH values (pH 7.4 and 5.0). LSCM images depicting time-dependent internalisation of PD20 indicated that PD20 traversed the extracellular membrane of CHO cells cultured in-vitro within ten minutes and migrated towards the endosomal regions where the pH is in the region of 5.0 to 6.0. Nuclear localisation of PD20 was demonstrated in a subpopulation of CHO cells. A further study was completed in CHO and HepG2 (hepatocellular carcinoma) cells cultured in-vitro using a lower molecular weight polymer to demonstrate that the molecular weight of "polydye" could be tailored to attain nuclear trafficking in cells. Prospective use of this technology encompasses a method of delivering a payload into a living cell based upon the hypercoiling nature of the pseudopeptides studied in this thesis and has led to a patent application (GB0228525.2; 20(2).

The pH-induced swelling and collapse of a polybase brush synthesized by atom transfer radical polymerization

We have used neutron reflectometry to characterize the swelling behaviour of brushes of poly[2-(diethyl amino)ethyl methacrylate], a polybase, as a function of pH. The brushes, synthesized by the "grafting from" method of atom transfer radical polymerization, were observed to approximately double their thickness in low pH solutions, although the pK is shifted to a lower pH than in dilute solution. The composition-depth profile obtained from the reflectometry experiments for the swollen brushes reveals a region depleted in polymer between the substrate and the extended part of the brush.

Expression of heterologous aquaporins for functional analysis in Saccharomyces cerevisiae

In this study the yeast Saccharomyces cerevisiae, which is a genetically tractable model for analysis of osmoregulation, has been used for analysis of heterologous aquaporins. Aquaporin water channels play important roles in the control of water homeostasis in individual cells and multicellular organisms. We have investigated the effects of functional expression of the mammalian aquaporins AQP1 and AQP5 and the aquaglyceroporins AQP3 and AQP9. Expression of aquaporins caused moderate growth inhibition under hyperosmotic stress, while expression of aquaglyceroporins mediated strong growth inhibition due to glycerol loss. Water transport was monitored in protoplasts, where the kinetics of bursting was influenced by presence of aquaporins but not aquaglyceroporins. We observed glycerol transport through aquaglyceroporins, but not aquaporins, in a yeast strain deficient in glycerol production, whose growth depends on glycerol inflow. In addition, a gene reporter assay allowed to indirectly monitor the effect of AQP9-mediated enhanced glycerol loss on osmoadaptation. Transport activity of certain aqua(glycero)porins was diminished by low pH or CuSO 4, suggesting that yeast can potentially be used for screening of putative aquaporin inhibitors. We conclude that yeast is a versatile system for functional studies of aquaporins, and it can be developed to screen for compounds of potential pharmacological use. © Springer-Verlag 2006.

The geochemistry of the sedimentary rocks of the Harlech Dome, North Wales

Bedrock geochemical analysis, coupled with detailed data analysis, was carried out on some 260 samples taken from two areas of 'the Harlech Dome, near Dolgellau, North Wales. This was done to determine if rocks from mineralised and non-mineralised areas could be distinguished, and to determine mineralisation types and wall rock alterations. The Northern Area, near Talsarnau, has no recorded mineralisation, while the Southern Area, near Bontddu, has been exploited for gold. The rocks sampled, in both areas, were from the Cambrian Gamlan Flags, Clogau Shales, Vigra Flags, later vein materials, and igneous intrusions. All samples were analysed, using a new rapid, atomic absorption spectrophotometric technique, for Si, AI, Fe, Cu, Ni, Zn, Pb, Sr, Hg, and Ba. In addition 60 samples were analysed by X-ray fluorescence for Mn, Ti, Ca, K, Na, P, Cr, Ce, La, S, Y , Rh, and Th. Total CO2 was determined, on selected samples, using a combustion technique. Elemental distributions, for each rock type, in each area, were· plotted, and means, standard deviations, and enrichment indices were calculated. Multivariate statistical analysis on the results distinguished a Cu-type mineralisation in the Northern area, and both Cu and Pb/Zn types in the Southern Area. It also showed the Northern Area to be less strongly mineralised than the Southern one in which both mineralisation types are associated with wall rock alteration. Elemental associations and trends due to sedimentary processes were distinguished from those related to mineralisation. Hg is related to mineralisation, and plots of factor scores, on the sampling grid, produced clusters of mineralisation related factors in areas of known mineralisation. A double Fourier Trend Analysis program, with a wavelength search routine, was developed and used to recognise sedimentary trends for Sr. Y., Rb, and Th. These trends were interpreted to represent areas of low pH and reducing conditions. They also indicate that the supply of sediment remained constant over Gamlan, Clogau, and Vigra times. The trend surface of Hg showed no association with rock type. It is shown that analysis of a small number of samples, for a carefully selected number of elements, with detailed data analysis, can provide more useful information than analysis of a large number of samples for many elements. The mineralisation is suggested to have been the result of water solutions leaching ore metals from the sedimentary rocks and redepositing them in veins.

Formulation design considerations for oral vaccines

Whilst oral vaccination is a potentially preferred route in terms of patient adherence and mass vaccination, the ability to formulate effective oral vaccines remains a challenge. The primary barrier to oral vaccination is effective delivery of the vaccine through the GI tract owing to the many obstacles it presents, including low pH, enzyme degradation and bile-salt solubilization, which can result in breakdown/deactivation of a vaccine. For effective immune responses after oral administration, particulates need to be taken up bythe M cells however, these are few in number. To enhance M-cell uptake, particle characteristics can be optimized with particle size, surface charge, targeting groups and bioadhesive properties all being considerations. Yet improved uptake may not translate into enhanced immune responses and formulating particulates with inherent adjuvant properties can offer advantages. Within this article, we establish the options available for consideration when building effective oral particulate vaccines.

Tail gas catalyzed N2O decomposition over Fe-beta zeolite. On the promoting role of framework connected AlO6 sites in the vicinity of Fe by controlled dealumination during exchange

A novel route to prepare highly active and stable N2O decomposition catalysts is presented, based on Fe-exchanged beta zeolite. The procedure consists of liquid phase Fe(III) exchange at low pH. By varying the pH systematically from 3.5 to 0, using nitric acid during each Fe(III)-exchange procedure, the degree of dealumination was controlled, verified by ICP and NMR. Dealumination changes the presence of neighbouring octahedral Al sites of the Fe sites, improving the performance for this reaction. The so-obtained catalysts exhibit a remarkable enhancement in activity, for an optimal pH of 1. Further optimization by increasing the Fe content is possible. The optimal formulation showed good conversion levels, comparable to a benchmark Fe-ferrierite catalyst. The catalyst stability under tail gas conditions containing NO, O2 and H2O was excellent, without any appreciable activity decay during 70 h time on stream. Based on characterisation and data analysis from ICP, single pulse excitation NMR, MQ MAS NMR, N2 physisorption, TPR(H2) analysis and apparent activation energies, the improved catalytic performance is attributed to an increased concentration of active sites. Temperature programmed reduction experiments reveal significant changes in the Fe(III) reducibility pattern with the presence of two reduction peaks; tentatively attributed to the interaction of the Fe-oxo species with electron withdrawing extraframework AlO6 species, causing a delayed reduction. A low-temperature peak is attributed to Fe-species exchanged on zeolitic AlO4 sites, which are partially charged by the presence of the neighbouring extraframework AlO6 sites. Improved mass transport phenomena due to acid leaching is ruled out. The increased activity is rationalized by an active site model, whose concentration increases by selectively washing out the distorted extraframework AlO6 species under acidic (optimal) conditions, liberating active Fe species.

High permeability of the anionic form restricts accumulation of indomethacin by cultured gastric surface epithelial cells exposed to low apical pH

The 'ion-trapping' hypothesis suggests that the intracellular concentration of acidic non-steroidal anti-inflammatory drugs in gastric epithelial cells could be much higher than in the gastric lumen, and that such accumulation could contribute to their gastrotoxicity. Our aim was to examine the effect of the pH of the apical medium on the apical to basal transfer of the acidic drug indomethacin (pK a 4.5) across a gastric mucous epithelial cell monolayer, and to determine whether indomethacin accumulated in cells exposed to a low apical pH. Guinea-pig gastric mucous epithelial cells were grown on porous membrane culture inserts (Transwells®) for 72 h. Transfer and accumulation of [ 14C] indomethacin were assessed by scintillation counting. Transfer of [ 3H]mannitol and measurement of trans-epithelial electrical resistance were used to assess integrity of the monolayer. Distribution of [ 14C] urea was used to estimate the intracellular volume of the monolayer. The monolayer was not disrupted by exposure of the apical face to media of pH ≥ 3, or by indomethacin. Transfer of indomethacin (12 μM) to the basal medium increased with decreasing apical medium pH. The apparent permeability of the undissociated acid was estimated to be five times that of the anion. The intracellular concentration of indomethacin was respectively 5.3, 4.1 and 4.3 times that in the apical medium at pH 5.5, 4.5 and 3.0. In conclusion, this study represents the first direct demonstration that indomethacin accumulates in gastric epithelial cells exposed to low apical pH. However, accumulation of indomethacin was moderate and the predictions of the ion-trapping hypothesis were not met, probably due to the substantial permeability of anionic indomethacin across membranes. © 2006 Elsevier B.V. All rights reserved.

Biosensor based on excessively tilted fiber grating in thin-cladding optical fiber for sensitive and selective detection of low glucose concentration

We report a highly sensitive, high Q-factor, label free and selective glucose sensor by using excessively tilted fiber grating (Ex-TFG) inscribed in the thin-cladding optical fiber (TCOF). Glucose oxidase (GOD) was covalently immobilized on optical fiber surface and the effectiveness of GOD immobilization was investigated by the fluorescence microscopy and highly accurate spectral interrogation method. In contrast to the long period grating (LPG) and optical fiber (OF) surface Plasmon resonance (SPR) based glucose sensors, the Ex-TFG configuration has merits of nearly independent cross sensitivity of the environmental temperature, simple fabrication method (no noble metal deposition or cladding etching) and high detection accuracy (or Q-factor). Our experimental results have shown that Ex-TFG in TCOF based sensor has a reliable and fast detection for the glucose concentration as low as 0.1~2.5mg/ml and a high sensitivity of ~1.514nm·(mg/ml)−1, which the detection accuracy is ~0.2857nm−1 at pH 5.2, and the limit of detection (LOD) is 0.013~0.02mg/ml at the pH range of 5.2~7.4 by using an optical spectrum analyzer with a resolution of 0.02nm.