Monitoring Drug Crystallization in Percutaneous Penetration Using Localized Nanothermal Analysis and Photothermal Microspectroscopy.

Drug crystallization on and in the skin has been reported following application of topical or transdermal formulations. This study explored novel probe-based approaches including localized nanothermal analysis (nano-TA) and photothermal microspectroscopy (PTMS) to investigate and locate drug crystals in the stratum corneum (SC) of porcine skin following application of simple ibuprofen (IBU) formulations. We also conducted in vitro skin permeation studies and tape stripping. The detection of drug crystals in the SC on tape strips was confirmed using localized nano-TA, based on the melting temperature of IBU. The melting of IBU was also evident as indicated by a double transition and confirmed the presence of drug crystals in the SC. The single point scans of PTMS on the tape strips allowed collection of the photothermal FTIR spectra of IBU, confirming the existence of drug crystals in the skin. The combined methods also indicated that drug crystallized in the SC at a depth of ∼4-7 µm. Future studies will examine the potential of these techniques to probe crystallization of other commonly used actives in topical and transdermal formulations.

Designing Peptide/Graphene Hybrid Hydrogels through Fine-Tuning of Molecular Interactions.

A recent strategy that has emerged for the design of increasingly functional hydrogels is the incorporation of nanofillers in order to exploit their specific properties to either modify the performance of the hydrogel or add functionality. The emergence of carbon nanomaterials in particular has provided great opportunity for the use of graphene derivatives (GDs) in biomedical applications. The key challenge when designing hybrid materials is the understanding of the molecular interactions between the matrix (peptide nanofibers) and the nanofiller (here GDs) and how these affect the final properties of the bulk material. For the purpose of this work, three gelling ß-sheet-forming, self-assembling peptides with varying physiochemical properties and five GDs with varying surface chemistries were chosen to formulate novel hybrid hydrogels. First the peptide hydrogels and the GDs were characterized; subsequently, the molecular interaction between peptides nanofibers and GDs were probed before formulating and mechanically characterizing the hybrid hydrogels. We show how the interplay between electrostatic interactions, which can be attractive or repulsive, and hydrophobic (and π-π in the case of peptide containing phenylalanine) interactions, which are always attractive, play a key role on the final properties of the hybrid hydrogels. The shear modulus of the hydrid hydrogels is shown to be related to the strength of fiber adhesion to the flakes, the overall hydrophobicity of the peptides, as well as the type of fibrillar network formed. Finally, the cytotoxicity of the hybrid hydrogel formed at pH 6 was also investigated by encapsulating and culturing human mesemchymal stem cells (hMSC) over 14 days. This work clearly shows how interactions between peptides and GDs can be used to tailor the mechanical properties of the resulting hydrogels, allowing the incorporation of GD nanofillers in a controlled way and opening the possibility to exploit their intrinsic properties to design novel hybrid peptide hydrogels for biomedical applications.

Development and Biological Evaluation of Inkjet Printed Drug Coatings on Intravascular Stent.

Inkjet-printing technology was used to apply biodegradable and biocompatible polymeric coatings of poly(d,l-lactide) with the antiproliferative drugs simvastatin (SMV) and paclitaxel (PCX) on coronary metal stents. A piezoelectric dispenser applied coating patterns of very fine droplets (300 pL) and inkjet printing was optimized to develop uniform, accurate and reproducible coatings of high yields on the stent strut. The drug loaded polymeric coatings were assed by scanning electron microscopy (SEM), atomic force microscopy (AFM), and transition thermal microscopy (TTM) where a phase separation was observed for SMV/PLA layers while PCX showed a uniform distribution within the polymer layers. Cytocompatibility studies of PLA coatings showed excellent cell adhesion with no decrease of cell viability and proliferation. In vivo stent implantation studies showed significant intrastent restenosis (ISR) for PCX/PLA and PLA plain coatings similar to marketed Presillion (bare metal) and Cypher (drug eluting) stents. The investigation of several cytokine levels after 7 days of stent deployment showed no inflammatory response and hence no in vivo cytotoxicity related to PLA coatings. Inkjet printing can be employed as a robust coating technology for the development of drug eluting stents compared to the current conventional approaches.

Spatial characterization of hot melt extruded dispersion systems using thermal atomic force microscopy methods: the effects of processing parameters on phase separation.

PURPOSE: In this study we explore the use of nano-scale localized thermal analysis (LTA) and transition temperature microcopy (TTM) as a novel combined approach to studying phase separation in HME dispersions of cyclosporine A in Eudragit EPO. METHODS: Modulated temperature differential scanning calorimetry (MTDSC), attenuated total reflectance FTIR spectroscopy, nano-LTA and TTM were performed on raw materials and dispersions prepared by hot melt extrusion (HME) and spin coating. For samples prepared by HME, two mixing temperatures (110°C and 150°C) and residence times (5 and 15 min) were investigated. RESULTS: Spin coated samples showed an intermediate T g for the mixed systems consistent with molecular dispersion formation. The HME samples prepared at 110°C showed evidence of inhomogeneity using MTDSC and FTIR, while those produced at 150°C h showed evidence for the formation of a single phase system using MTDSC. The nanothermal methods, however, indicated the presence of phase separated cyclosporine A at the higher preparation temperature while the TTM was able to map regions of differing penetration temperatures, indicating the presence of compositionally inhomogeneous regions in all but the high processing temperature/high residence time samples. CONCLUSIONS: TTM is a potentially important new method for studying phase separation and that such separation may remain undetected or poorly understood using conventional bulk analytical techniques.

Early stage phase separation in pharmaceutical solid dispersion thin films under high humidity: improved spatial understanding using probe-based thermal and spectroscopic nanocharacterization methods.

Phase separation in pharmaceutical solid dispersion thin films under high humidity is still poorly understood on the submicrometer scale. This study investigated the phase separation of a model solid dispersion thin film, felodipine-PVP K29/32, prepared by spin-coating and analyzed using probe-based methods including atomic force microscopy, nanothermal analysis, and photothermal infrared microspectroscopy. The combined use of these techniques revealed that the phase separation process occurring in the thin films under high humidity is different from that in dry conditions reported previously. The initial stage of phase separation is primarily initiated in the bulk of the films as amorphous drug domains. Drug migration toward the surface of the solid dispersion film was then observed to occur under exposure to increased humidity. PVP cannot prevent phase separation of felodipine under high humidity but can minimize the crystallization of amorphous felodipine domains in the solid dispersion thin films. This study demonstrates the unique abilities of these nanocharacterization methods for studying, in three dimensions, the phase separation of thin films for pharmaceutical applications.

Development of photothermal FTIR microspectroscopy as a novel means of spatially identifying amorphous and crystalline salbutamol sulfate on composite surfaces.

Photothermal Fourier transform infrared (FTIR) microspectroscopy (PTMS), involving the combination of FTIR spectroscopy with atomic force microscopy, has been used to examine compacts of amorphous and crystalline salbutamol sulfate in order to assess the ability of the technique to distinguish between different physical forms in a multicomponent material. Samples of amorphous and crystalline material were assessed using modulated temperature differential scanning calorimetry (DSC), atomic force microscopy, microthermal analysis, and conventional FTIR. Mixed compacts were then prepared such that verification of the location of the forms present was possible via topography and localized thermal analysis. PTMS studies were then performed on selected interrogation points, with spectra obtained which were largely intermediate between those corresponding to the two individual forms. Calculation of the thermal diffusivity indicated a resolution for the technique corresponding to a hemisphere of a major diameter in the region of 40 µm, which is large in relation to the particle sizes involved. However, distinction into amorphous, crystalline, and indeterminate categories was possible using chemometric analysis (hierarchical cluster analysis and principal component analysis). Good agreement was found between the identification methods for the mixed systems. The study has therefore shown the potential, as well as identifying the limitations, of using PTMS as a means of spatially identifying components in complex materials.

Analysis of single particle photodegradation using photothermal infrared microspectroscopy.

The increasing use of high throughput methods, coupled with the need to develop approaches to anticipate long term stability issues, has necessitated the introduction of testing approaches whereby extremely small samples may be rapidly analysed. A novel method is described whereby the UV light-induced degradation of single particles of a model drug, nifedipine, may be rapidly and simply monitored using photothermal infrared microspectroscopy (PTMS). The technique involves the contact attachment of individual particles to a heated probe tip composed of a modified Wollaston wire which enables temperature fluctuations to be measured. Application of a focused IR beam to excite the sample allows measurement and subsequent Fourier transformation of the resultant interferogram to produce an IR spectrum which is in good agreement with that obtained from conventional IR methods. By application of a UV source to the assembly for specified time periods, we demonstrate that it is possible to monitor the appearance of peaks associated with degradation products as a function of time. The technique is critically evaluated in terms of practical issues associated with volatilization, particle size effects and orientation to the light source as well as more general issues associated with the sensitivity, resolution and quantitative interpretation of data from the PTMS technique. Overall the method has been shown to be capable of rapid measurement of photo-instability on individual particles, with important implications for development of the approach as a rapid screening or high throughput technique, although there are practical and theoretical limitations to reliable quantitative analysis at the present time.

Time-Lapse In Situ 3D Imaging Analysis of Human Enamel Demineralisation Using X-ray Synchrotron Tomography.

Caries is a chronic disease that causes the alteration of the structure of dental tissues by acid dissolution (in enamel, dentine and cementum) and proteolytic degradation (dentine and cementum) and generates an important cost of care. There is a need to visualise and characterise the acid dissolution process on enamel due to its hierarchical structure leading to complex structural modifications. The process starts at the enamel surface and progresses into depth, which necessitates the study of the internal enamel structure. Artificial demineralisation is usually employed to simulate the process experimentally. In the present study, the demineralisation of human enamel was studied using surface analysis carried out with atomic force microscopy as well as 3D internal analysis using synchrotron X-ray tomography during acid exposure with repeated scans to generate a time-lapse visualisation sequence. Two-dimensional analysis from projections and virtual slices and 3D analysis of the enamel mass provided details of tissue changes at the level of the rods and inter-rod substance. In addition to the visualisation of structural modifications, the rate of dissolution was determined, which demonstrated the feasibility and usefulness of these techniques. The temporal analysis of enamel demineralisation is not limited to dissolution and can be applied to other experimental conditions for the analysis of treated enamel or remineralisation.

In situ nanoscale imaging reveals self-concentrating nanomolar antimicrobial pores.

Host defence peptides are critical factors of immune systems in all life forms. Considered for therapeutic development in the post-antibiotic era, these molecules rupture microbial membranes at micromolar concentrations. Here we report a self-concentrating mechanism of membrane disruption, which occurs at therapeutically more relevant nanomolar concentrations. Induced by a four-helix bacteriocin the mechanism manifests in a multi-modal disruption pattern. Using in situ atomic force microscopy we show that the pattern and its kinetic profiles remain the same in a range of nano-to-micromolar concentrations. We reveal that the bacteriocin creates its own boundaries in phospholipid bilayers in which it self-concentrates to promote transmembrane poration. The findings offer an exploitable insight into nanomolar antimicrobial mechanisms.

Quantitative nanomechanical properties evaluation of a family of ß-sheet peptide fibres using rapid bimodal AFM.

Self-assembling peptides have become important building blocks for materials design (e.g. hydrogels) and play a crucial role in a range of diseases including Alzheimer and Parkinson. In this context, accessing the nanomechanical properties of ubiquitous ß-sheet rich nanofibres (e.g.: amyloids) is key to the formulation of materials and design of therapies. Although the bulk mechanical properties of hydrogels can easily be accessed using common techniques and equipment, the mechanical properties of their constituent fibres, in particular if with radii in the nanometre scale, are more challenging to measure and estimate. In this work we show for the first time how the rapid nanomechanical mapping technique: amplitude modulation-frequency modulation (AM-FM), can be used to determine the heights, Young's moduli and viscosity coefficients of a series of ß-sheet peptide nanofibres with high statistical confidence. Our results show how peptide sequence and in particular length, charge and interaction with the substrate affect the viscoelastic properties of the peptide fibres.

Compositional analysis of metal chelating materials using near-field photothermal Fourier transform infrared microspectroscopy.

Photothermal-Fourier transform-infrared (PT-FT-IR) microspectroscopy employs a thermal probe mounted in a scanning probe microscope (SPM). By placement of the tip of the probe on the surface of a solid sample, it can obtain localized IR spectra of a wide range of samples. A second mode of analysis is also available; a sample can be taken from the selected location using a technique called thermally assisted nanosampling (TAN), then a spectrum can be obtained of the nanosample while the probe is remote from the surface. We report a novel method of local compositional analysis that combines both of these types of measurement; a reagent is attached to the tip using TAN, then the reagent is placed in contact with analyte. IR spectroscopy can then be used to analyze any interaction between the reagent and surface it is placed in contact with. All of these modes of analysis were illustrated using a metal chelating agent. In the surface mode, changes to a solid bead of a chelating resin were measured using standard PT-FT-IR. In the nanosampling mode of analysis, a particle of a chelating polymer was attached to the tip of the probe using TAN and this was placed in contact with a concentrated calcium solution. Strong spectral changes were observed that mirrored those found when exposing the surface bound chelating resin bead to a solution of the same ion. A semiquantitative simulation of the PT spectrum for a chelating resin bead was achieved using a thermal diffusion model derived from photoacoustic spectroscopy indicating that semiquantitative or quantitative measurements will be possible in such a system.

Resistance to extended-spectrum cephalosporins in Escherichia coli and other Enterobacterales from Canadian turkeys.

The goal of this study was to determine the frequency of resistance to extended-spectrum cephalosporins (ESCs) in Escherichia coli and other Enterobacterales from turkeys in Canada and characterize the associated resistance determinants. Pooled fecal samples were collected in 77 turkey farms across British Columbia, Québec, and Ontario. Isolates were obtained with and without selective enrichment cultures and compared to isolates from diagnostic submissions of suspected colibacillosis cases in Ontario. Isolates were identified using MALDI-TOF and susceptibility to ESCs was assessed by disk diffusion. The presence of blaCMY, blaCTX-M, blaTEM, and blaSHV was tested by PCR. Transformation experiments were used to characterize blaCMY plasmids. Genome sequencing with short and long reads was performed on a representative sample of blaCTX-M-positive isolates to assess isolates relatedness and characterize blaCTX-M plasmids. For the positive enrichment cultures (67% of total samples), 93% (587/610) were identified as E. coli, with only a few other Enterobacterales species identified. The frequency of ESC resistance was low in E. coli isolates from diagnostic submission (4%) and fecal samples without selective enrichment (5%). Of the ESC-resistant Enterobacterales isolates from selective enrichments, 71%, 18%, 14%, and 8% were positive for blaCMY, blaTEM, blaCTX-M, and blaSHV, respectively. IncI1 followed by IncK were the main incompatibility groups identified for blaCMY plasmids. The blaCTX-M-1 gene was found repeatedly on IncI1 plasmids of the pMLST type 3, while blaCTX-M-15, blaCTX-M-55, and blaCTX-M-65 were associated with a variety of IncF plasmids. Clonal spread of strains carrying blaCTX-M genes between turkey farms was observed, as well as the presence of an epidemic blaCTX-M-1 plasmid in unrelated E. coli strains. In conclusion, Enterobacterales resistant to ESCs were still widespread at low concentration in turkey feces two years after the cessation of ceftiofur use. Although blaCMY-2 is the main ESC resistance determinant in E. coli from Canadian turkeys, blaCTX-M genes also occur which are often carried by multidrug resistance plasmids. Both clonal spread and horizontal gene transfer are involved in parallel in the spread of blaCTX-M genes in Enterobacterales from Canadian turkeys.

Thermal probe based analytical microscopy: thermal analysis and photothermal Fourier-transform infrared microspectroscopy together with thermally assisted nanosampling coupled with capillary electrophoresis.

In this study, we have demonstrated that a scanning probe microscope (SPM) can be used for thermally assisted nanosampling (TAN) with subsequent analysis by capillary electrophoresis (CE). Localized thermomechanical analysis (L-TMA) and photothermal Fourier-transform infrared (PT-FTIR) microspectroscopy can also be employed using the same probe, thus illustrating how a single instrument can carry out a number of different complementary analytical measurements. Benzoic acid and 4-hydroxybenzoic acid were manipulated with a heated Wollaston wire probe and successfully deposited onto the surface of a piece of CE capillary tubing. The deposited samples were then separated with CE. L-TMA and PT-FTIR were also used to characterize these materials. We have also demonstrated how a nanosample of a nonparticulate material can be taken and then deposited onto the surface of an inert matrix. TAN of a nonparticulate material was explored using polyethylene as the analyte and fluorene as the matrix. These examples show that thermal probe techniques provide a versatile "tool box" of modes of analysis with the potential to analyze a wide range of samples in a spatially resolved way.

The fate of cellulose nanocrystal stabilised emulsions after simulated gastrointestinal digestion and exposure to intestinal mucosa.

It is well recognised that the average UK diet does not contain sufficient fibre. However, the introduction of fibre is often at the detriment of the organoleptic properties of a food. In this study on the gastrointestinal fate of nanoparticles, we have used cellulose nano-crystals (CNCs) as Pickering stabilising agents in oil in water emulsions. These emulsions were found to be highly stable against coalescence. The CNC and control emulsions were then exposed to simulated upper gastrointestinal tract digestion and the results compared to those obtained from a conventional protein stabilised emulsion. Finally the digested emulsions were exposed to murine intestinal mucosa and lipid and bile absorption was monitored. Importantly, the results show that the CNCs were entrapped in the intestinal mucus layer and failed to reach the underlying epithelium. This entrapment may also have led to the reduced absorption of saturated lipids from the CNC stabilised emulsion versus the control emulsion. The results show the potential of CNCs as a safe and effective emulsifier.

Targeting discriminatory SNPs in Salmonella enterica serovar Heidelberg genomes using RNase H2-dependent PCR.

We report a novel RNase H2-dependent PCR (rhPCR) genotyping assay for a small number of discriminatory single-nucleotide polymorphisms (SNPs) that identify lineages and sub-lineages of the highly clonal pathogen Salmonella Heidelberg (SH). Standard PCR primers targeting numerous SNP locations were initially designed in silico, modified to be RNase H2-compatible, and then optimized by laboratory testing. Optimization often required repeated cycling through variations in primer design, assay conditions, reagent concentrations and selection of alternative SNP targets. The final rhPCR assay uses 28 independent rhPCR reactions to target 14 DNA bases that can distinguish 15 possible lineages and sub-lineages of SH. On evaluation, the assay correctly identified the 12 lineages and sub-lineages represented in a panel of 75 diverse SH strains. Non-specific amplicons were observed in 160 (15.2%) of the 1050 reactions, but due to their low intensity did not compromise assay performance. Furthermore, in silico analysis of 500 closed genomes from 103 Salmonella serovars and laboratory rhPCR testing of five prevalent Salmonella serovars including SH indicated the assay can identify Salmonella isolates as SH, since only SH isolates generated amplicons from all 14 target SNPs. The genotyping results can be fully correlated with whole genome sequencing (WGS) data in silico. This fast and economical assay, which can identify SH isolates and classify them into related or unrelated lineages and sub-lineages, has potential applications in outbreak identification, source attribution and microbial source tracking.

Time dependent decomposition of ammonia borane for the controlled production of 2D hexagonal boron nitride.

Ammonia borane (AB) is among the most promising precursors for the large-scale synthesis of hexagonal boron nitride (h-BN) by chemical vapour deposition (CVD). Its non-toxic and non-flammable properties make AB particularly attractive for industry. AB decomposition under CVD conditions, however, is complex and hence has hindered tailored h-BN production and its exploitation. To overcome this challenge, we report in-depth decomposition studies of AB under industrially safe growth conditions. In situ mass spectrometry revealed a time and temperature-dependent release of a plethora of NxBy-containing species and, as a result, significant changes of the N:B ratio during h-BN synthesis. Such fluctuations strongly influence the formation and morphology of 2D h-BN. By means of in situ gas monitoring and regulating the precursor temperature over time we achieve uniform release of volatile chemical species over many hours for the first time, paving the way towards the controlled, industrially viable production of h-BN.

Publisher Correction: Time dependent decomposition of ammonia borane for the controlled production of 2D hexagonal boron nitride.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Visualisation of xanthan conformation by atomic force microscopy.

Direct visual evidence obtained by atomic force microscopy demonstrates that when xanthan is adsorbed from aqueous solution onto the heterogeneously charged substrate mica, its helical conformation is distorted. Following adsorption it requires annealing for several hours to restore its ordered helical state. Once the helix state reforms, the AFM images obtained showed clear resolution of the periodicity with a value of 4.7nm consistent with the previously predicted models. In addition, the images also reveal evidence that the helix is formed by a double strand, a clarification of an ambiguity of the xanthan ultrastructure that has been outstanding for many years.

Rapid epitaxy-free graphene synthesis on silicidated polycrystalline platinum.

Large-area synthesis of high-quality graphene by chemical vapour deposition on metallic substrates requires polishing or substrate grain enlargement followed by a lengthy growth period. Here we demonstrate a novel substrate processing method for facile synthesis of mm-sized, single-crystal graphene by coating polycrystalline platinum foils with a silicon-containing film. The film reacts with platinum on heating, resulting in the formation of a liquid platinum silicide layer that screens the platinum lattice and fills topographic defects. This reduces the dependence on the surface properties of the catalytic substrate, improving the crystallinity, uniformity and size of graphene domains. At elevated temperatures growth rates of more than an order of magnitude higher (120 µm min(-1)) than typically reported are achieved, allowing savings in costs for consumable materials, energy and time. This generic technique paves the way for using a whole new range of eutectic substrates for the large-area synthesis of 2D materials.