Patrick G. Hartley

Lactose surface modification by decantation: Are drug-fine lactose ratios the key to better dispersion of salmeterol xinafoate from lactose-interactive mixtures?

AbstractPurpose. The role of fine lactose in the dispersion of salmeterol xina- foate (SX) from lactose mixtures was studied by modifying the fine lactose concentration on the surface of the lactose carriers using wet decantation. Methods. Fine lactose was removed from lactose carriers by wet decantation using ethanol saturated with lactose. Particle sizing was achieved by laser diffraction. Fine particle fractions (FPFs) were determined by Twin Stage Impinger using a 2.5% SX mixture, and SX was analyzed by a validated high-performance liquid chromatography method. Adhesion forces between probes of SX and silica and the lactose surfaces were determined by atomic force microscopy. Results. FPFs of SX were related to fine lactose concentration in the mixture for inhalation grade lactose samples. Reductions in FPF (2- tp 4-fold) of Aeroflo 95 and 65 were observed after removing fine lactose by wet decantation; FPFs reverted to original values after addition of micronized lactose to decanted mixtures. FPFs of SX of sieved and decanted fractions of Aeroflo carriers were significantly different (p < 0.001). The relationship between FPF and fine lactose concentration was linear. Decanted lactose demonstrated surface modification through increased SX-lactose adhesion forces; however, any surface modification other than removal of fine lactose only slightly influenced FPF. Conclusions. Fine lactose played a key and dominating role in controlling FPF. SX to fine lactose ratios influenced dispersion of SX with maximum dispersion occurring as the ratio approached unity.

Self-assembly of ciprofloxacin and a tripeptide into an antimicrobial nanostructured hydrogel

This work reports the self-assembly of a sparingly soluble antibiotic (ciprofloxacin) and a hydrophobic tripeptide ((D)Leu-Phe-Phe) into supramolecular nanostructures that yield a macroscopic hydrogel at physiological pH. Drug incorporation results in modified morphology and rheological properties of the self-assembled hydrogel. These changes can be correlated with intermolecular interactions between the drug and the peptide, as confirmed by spectroscopic analysis (fluorescence, circular dichroism, IR). The drug appears bound within the hydrogel by non-covalent interactions, and retains its activity over a prolonged release timescale. Antimicrobial activity of the ciprofloxacin-peptide self-assembled hydrogel was evaluated against Staphylococcus aureus, Escherichia coli, and a clinical strain of Klebsiella pneumoniae. Interestingly, the peptide hydrogel alone exhibited a mild anti-bacterial activity against Gram-negative bacteria. While toxic to bacteria, no major cytotoxicity was seen in haemolysis assays of human red blood cells or in mouse fibroblast cell cultures. This new approach of drug incorporation into the nanostructure of a simple tripeptide hydrogel by self-assembly may have important applications for cost-effective wound dressings and novel antimicrobial formulations.

A Simple Microfluidic Chip Design for Fundamental Bioseparation

A microchip pressure-driven liquid chromatographic system with a packed column has been designed and fabricated by using poly(dimethylsiloxane) (PDMS). The liquid chromatographic column was packed with mesoporous silica beads of Ia3d space group. Separation of dyes and biopolymers was carried out to verify the performance of the chip. A mixture of dyes (fluorescein and rhodamine B) and a biopolymer mixture (10 kDa Dextran and 66 kDa BSA) were separated and the fluorescence technique was employed to detect the movement of the molecules. Fluorescein molecule was a nonretained species and rhodamine B was attached onto silica surface when dye mixture in deionized water was injected into the microchannel. The retention times for dextran molecule and BSA molecule in biopolymer separation experiment were 45 s and 120 s, respectively. Retention factor was estimated to be 3.3 for dextran and 10.4 for BSA. The selectivity was 3.2 and resolution was 10.7. Good separation of dyes and biopolymers was achieved and the chip design was verified.

Unzipping the role of chirality in nanoscale self-assembly of tripeptide hydrogels

Change of chirality is a useful tool to manipulate the aqueous self-assembly behaviour of uncapped, hydrophobic tripeptides. In contrast with other short peptides, these tripeptides form hydrogels at a physiological pH without the aid of organic solvents or end-capping groups (e.g. Fmoc). The novel hydrogel forming peptide (D)Leu-Phe-Phe ((D)LFF) and its epimer Leu-Phe-Phe (LFF) exemplify dramatic supramolecular effects induced by subtle changes to stereochemistry. Only the d-amino acid-containing peptide instantly forms a hydrogel in aqueous solution following a pH switch, generating long fibres (>100 μm) that entangle into a 3D network. However, unexpected nanostructures are observed for both peptides and they are particularly heterogeneous for LFF. Structural analyses using CD, FT-IR and fluorescent amyloid staining reveal anti-parallel beta-sheets for both peptides. XRD analysis also identifies key distances consistent with beta-sheet formation in both peptides, but suggests additional high molecular order and extended molecular length for (D)LFF only. Molecular modelling of the two peptides highlights the key interactions responsible for self-assembly; in particular, rapid self-assembly of (D)LFF is promoted by a phenylalanine zipper, which is not possible because of steric factors for LFF. In conclusion, this study elucidates for the first time the molecular basis for how chirality can dramatically influence supramolecular organisation in very short peptide sequences.

The in vivo performance of an enzyme-assisted self-assembled peptide/protein hydrogel

We demonstrate the distribution of the important extracellular matrix protein laminin in a novel biomaterial consisting of a hydrogel underpinned by nanofibrillar networks. These are formed by the immobilised enzyme mediated self-assembly of fmoc-L(3) (9-fluorenylmethoxycarbonyl-tri-leucine). The peptide assembly yields nanofibrils formed of β-sheets that are locked together via π-stacking interactions. This ordering allows the localisation of the peptide sidechains on the surface, creating a hydrophobic environment. This induces the formation of bundles of these nanofibrils producing a clear hydrogel. This mechanism enables the three dimensional distribution of laminin throughout the network via supramolecular interactions. These forces favour the formation and improve the order of the network itself, as observed by spectroscopic and mechanical testing. In order to test the stability and suitability of this class of material for in vivo applications, we utilise microinjection to deliver the biomaterial under fine spatial control into a dystrophic zebrafish model organism, which lacks laminin as a result of a genetic mutation. Using confocal and transmission electron microscopy, we confirm that the biomaterial remains stable structurally, and is confined spatially to the site of injection.

Salt Induced Lamellar to Bicontinuous Cubic Phase Transitions in Cationic Nanoparticles

The development of improved methods to allow the low energy production of cubic phase forming nanoparticles (cubosomes) is highly desired. The lamellar to hexagonal and cubic phase change of these lipid nanoparticles has previously been induced via the lowering of pH and the addition of calcium ions to anionic lipid nanoparticles. We have developed a method to produce low polydispersity cubosomes without the requirement of high energy input such as shear, sonication or homogenization under physiological conditions. We have found that the simple addition of phosphate buffered saline solution to aqueous dispersions of cationic liposome vesicles made with phytantriol results in the spontaneous formation of cubosomes after vortex mixing. This finding demonstrates the potential of utilizing this technique to incorporate shear and temperature sensitive compounds into cubosomes under extremely mild conditions for biomedical and nanotechnological applications.

The influence of dipalmitoyl phosphatidylserine on phase behaviour of and cellular response to lyotropic liquid crystalline dispersions

Lyotropic liquid crystalline nanoparticles (cubosomes) have the potential to act as amphiphilic scaffolds for the presentation of lipids and subsequent application in, for example, bioseparations and therapeutic delivery. In this work we have formulated lyotropic liquid crystalline systems based on the synthetic amphiphile 1,2,3-trihydroxy-3,7,11,15-tetramethylhexadecane (phytantriol) and containing the lipid dipalmitoyl phosphatidylserine (DPPS). We have prepared a range of DPPS-containing phytantriol cubosome formulations and characterized them using Small Angle X-ray Scattering and Cryo-transmission electron microscopy. These techniques show that increased DPPS content induces marked changes in lyotropic liquid crystalline phase behaviour, characterized by changes in crystallographic dimensions and increases in vesicle content. Furthermore, in vitro cell culture studies indicate that these changes correlate with lipid/surfactant cellular uptake and cytotoxicity. A model cell membrane based on a surface supported phospholipid bilayer was used to gain insights into cubosome-bilayer interactions using Quartz Crystal Microgravimetry. The data show that mass uptake at the supported bilayer increased with DPPS content. We propose that the cytotoxicity of the DPPS-containing dispersions results from changes in lipid/surfactant phase behaviour and the preferential attachment and fusion of vesicles at the cell membrane.

A Surface Masking Technique for the Determination of Plasma Polymer Film Thickness by AFM

A method for the determination of coating film thicknesses at nanometer resolution based on surface masking and atomic force microscopy (AFM) is described. A polymeric mask is used to cover part of a substrate during the deposition of thin polymeric coatings by plasma polymerization, allowing the production of well defined polymer steps of heights of a few tens of nanometers. Tapping mode AFM has been employed to analyze the topography of these steps at high resolution. This method has also allowed accurate measurement of the kinetics of the deposition of plasma polymer films over a range of exposure times. XPS analysis of different substrate surfaces following mask removal found barely detectable residues, suggesting that the underlying surface chemistry remains unchanged, and accessible for further modification. In combination with quartz crystal microgravimetry, the method has been applied to the measurement of the density of plasma polymer coatings in the thickness range 4–50 nm.

Glycerol Monooleate-Based Nanocarriers for siRNA Delivery in Vitro

We present studies of the delivery of short interfering ribonucleic acid (siRNA) into a green fluorescent protein (GFP) expressing cell line, using lipid nanocarriers in cubic lyotropic liquid crystal form. These carriers are based on glycerol monooleate (GMO) and employ the use of varying concentrations of cationic siRNA binding lipids. The essential physicochemical parameters of the cationic lipid/GMO/siRNA complexes such as particle size, ζ otential, siRNA uptake stability, lyotropic mesophase behavior, cytotoxicity,and gene silencing efficiency were systematically assessed. We find that the lipid nanocarriers were effectively taken up by mammalian cells and that their siRNA payload was able to induce gene silencing in vitro. More importantly, it was found that the nonlamellar structure of some of the lipid nanocarrier formulations were more effective at gene silencing than their lamellar structured counterparts. The development of cationic lipid functionalized nonlamellar GMO-based nanostructured nanoparticles may lead to improved siRNA delivery vehicles.

Development of a high pressure automated lag time apparatus for experimental studies and statistical analyses of nucleation and growth of gas hydrates.

Nucleation in a supercooled or a supersaturated medium is a stochastic event, and hence statistical analyses are required for the understanding and prediction of such events. The development of reliable statistical methods for quantifying nucleation probability is highly desirable for applications where control of nucleation is required. The nucleation of gas hydrates in supercooled conditions is one such application. We describe the design and development of a high pressure automated lag time apparatus (HP-ALTA) for the statistical study of gas hydrate nucleation and growth at elevated gas pressures. The apparatus allows a small volume (≈150 μl) of water to be cooled at a controlled rate in a pressurized gas atmosphere, and the temperature of gas hydrate nucleation, T(f), to be detected. The instrument then raises the sample temperature under controlled conditions to facilitate dissociation of the gas hydrate before repeating the cooling-nucleation cycle again. This process of forming and dissociating gas hydrates can be automatically repeated for a statistically significant (>100) number of nucleation events. The HP-ALTA can be operated in two modes, one for the detection of hydrate in the bulk of the sample, under a stirring action, and the other for the detection of the formation of hydrate films across the water-gas interface of a quiescent sample. The technique can be applied to the study of several parameters, such as gas pressure, cooling rate and gas composition, on the gas hydrate nucleation probability distribution for supercooled water samples.