Nanoparticles and the blood-brain barrier: advancing from in-vitro models towards therapeutic significance.

The blood-brain barrier is a unique cell-based restrictive barrier that prevents the entry of many substances, including most therapeutics, into the central nervous system. A wide range of nanoparticulate delivery systems have been investigated with the aim of targeting therapeutics (drugs, nucleic acids, proteins) to the brain following administration by various routes. This review provides a comprehensive description of the design and formulation of these nanoparticles including the rationale behind individual approaches. In addition, the ability of currently available in-vitro BBB models to accurately predict the in-vivo performance of targeted nanoparticles is critically assessed.

Orthogonal identification of gunshot residue with complementary detection principles of voltammetry, scanning electron microscopy, and energy-dispersive X-ray spectroscopy: sample, screen, and confirm.

Field-deployable voltammetric screening coupled with complementary laboratory-based analysis to confirm the presence of gunshot residue (GSR) from the hands of a subject who has handled, loaded, or discharged a firearm is described. This protocol implements the orthogonal identification of the presence of GSR utilizing square-wave stripping voltammetry (SWSV) as a rapid screening tool along with scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) to confirm the presence of the characteristic morphology and metal composition of GSR particles. This is achieved through the judicious modification of the working electrode of a carbon screen-printed electrode (CSPE) with carbon tape (used in SEM analysis) to fix and retain a sample. A comparison between a subject who has handled and loaded a firearm and a subject who has had no contact with GSR shows the significant variations in voltammetric signals and the presence or absence of GSR-consistent particles and constituent metals. This initial electrochemical screening has no effect on the integrity of the metallic particles, and SEM/EDX analysis conducted prior to and postvoltammetry show no differences in analytical output. The carbon tape is instrumental in retaining the GSR sample after electrochemical analysis, supported by comparison with orthogonal detection at a bare CSPE. This protocol shows great promise as a two-tier detection system for the presence of GSR from the hands of a subject, whereby initial screening can be conducted rapidly onsite by minimally trained operators; confirmation can follow at the same substrate to substantiate the voltammetric results.

In vitro investigations of the efficacy of cyclodextrin-siRNA complexes modified with lipid-PEG-Octaarginine: towards a formulation strategy for non-viral neuronal siRNA delivery.

PURPOSE: Development of RNA interference based therapeutics for neurological and neurodegenerative diseases is hindered by a lack of non-viral vectors with suitable properties for systemic administration. Amphiphilic and cationic cyclodextrins (CD) offer potential for neuronal siRNA delivery. We aimed to improve our CD-based siRNA formulation through incorporation of a polyethyleneglycol (PEG) shielding layer and a cell penetrating peptide, octaarginine (R8). METHODS: CD.siRNA complexes were modified by addition of an R8-PEG-lipid conjugate. Physical properties including size, charge and stability were assessed. Flow cytometry was used to determine uptake levels in a neuronal cell model. Knockdown of an exogenous gene and an endogenous housekeeping gene were used to assess gene silencing abilities. RESULTS: CD.siRNA complexes modified with R8-PEG-lipid exhibited a lower surface charge and greater stability to a salt-containing environment. Neuronal uptake was increased and significant reductions in the levels of two target genes were achieved with the new formulation. However, the PEG layer was not sufficient to protect against serum-induced aggregation. CONCLUSIONS: The R8-PEG-lipid-CD.siRNA formulation displayed enhanced salt-stability due to the PEG component, while the R8 component facilitated transfection of neuronal cells and efficient gene silencing. Further improvements will be investigated in the future in order to optimise stability in serum and enhance neuronal specificity.

Solid-state Forensic Finger sensor for integrated sampling and detection of gunshot residue and explosives: towards 'Lab-on-a-finger'.

Increasing security needs require field-deployable, on-the-spot detection tools for the rapid and reliable identification of gunshot residue (GSR) and nitroaromatic explosive compounds. This manuscript presents a simple, all-solid-state, wearable fingertip sensor for the rapid on-site voltammetric screening of GSR and explosive surface residues. To fabricate the new Forensic Fingers, we screen-print a three-electrode setup onto a nitrile finger cot, and coat another finger cot with an ionogel electrolyte layer. The new integrated sampling/detection methodology relies on 'voltammetry of microparticles' (VMP) and involves an initial mechanical transfer of trace amounts of surface-confined analytes directly onto the fingertip-based electrode contingent. Voltammetric measurements of the sample residues are carried out upon bringing the working electrode (printed on the index finger cot) in direct contact with a second finger cot coated with an ionogel electrolyte (worn on the thumb), thus completing the solid-state electrochemical cell. Sampling and screening are performed in less than four minutes and generate distinct voltammetric fingerprints which are specific to both GSR and explosives. The use of the solid, flexible ionogel electrolyte eliminates any liquid handling which can resolve problems associated with leakage, portability and contamination. A detailed study reveals that the fingertip detection system can rapidly identify residues of GSR and nitroaromatic compounds with high specificity, without compromising its attractive behavior even after undergoing repeated mechanical stress. This new integrated sampling/detection fingertip strategy holds considerable promise as a rapid, effective and low-cost approach for on-site crime scene investigations in various forensic scenarios.

Rapid field identification of subjects involved in firearm-related crimes based on electroanalysis coupled with advanced chemometric data treatment.

We demonstrate a novel system for the detection and discrimination of varying levels of exposure to gunshot residue from subjects in various control scenarios. Our aim is to address the key challenge of minimizing the false positive identification of individuals suspected of discharging a firearm. The chemometric treatment of voltammetric data from different controls using Canonical Variate Analysis (CVA) provides several distinct clusters for each scenario examined. Multiple samples were taken from subjects in controlled tests such as secondary contact with gunshot residue (GSR), loading a firearm, and postdischarge of a firearm. These controls were examined at both bare carbon and gold-modified screen-printed electrodes using different sampling methods: the 'swipe' method with integrated sampling and electroanalysis and a more traditional acid-assisted q-tip swabbing method. The electroanalytical fingerprint of each sample was examined using square-wave voltammetry; the resulting data were preprocessed with Fast Fourier Transform (FFT), followed by CVA treatment. High levels of discrimination were thus achieved in each case over 3 classes of samples (reflecting different levels of involvement), achieving maximum accuracy, sensitivity, and specificity values of 100% employing the leave-one-out validation method. Further validation with the 'jack-knife' technique was performed, and the resulting values were in good agreement with the former method. Additionally, samples from subjects in daily contact with relevant metallic constituents were analyzed to assess possible false positives. This system may serve as a potential method for a portable, field-deployable system aimed at rapidly identifying a subject who has loaded or discharged a firearm to verify involvement in a crime, hence providing law enforcement personnel with an invaluable forensic tool in the field.

Simultaneous electrochemical measurement of metal and organic propellant constituents of gunshot residues.

The simultaneous electrochemical measurement of heavy-metal and organic propellants relevant to gunshot residues (GSRs) is demonstrated. Cyclic voltammetry (CV) and cyclic square-wave stripping voltammetry (C-SWV) are shown to detect, in a single run, common propellants, such as nitroglycerin (NG) and dinitrotoluene (DNT), along with the heavy metal constituents of GSR, antimony (Sb), lead (Pb), zinc (Zn) and barium (Ba). The voltammetric detection of the stabilizer diphenylamine (DPA) along with inorganic constituents has also been examined. The resulting electrochemical signatures combine -in a single voltammogram- the response for the various metals and organic species, based on the reduction and oxidation peaks of the constituents. Cyclic square-wave voltammetry at the glassy carbon electrode (GCE), involving an intermittent accumulation at the reversal potentials of -0.95 V (for Sb, Pb, DNT and NG) and -1.3 V (for Sb, Pb, Zn and DPA) is particularly useful to offer distinct electrochemical signatures for these constituents of GSR mixtures, compared to analogous cyclic voltammetric measurements. Simultaneous voltammetric measurements of barium (at thin-film Hg GCE) and DNT (at bare GCE) are also demonstrated in connection to intermittent accumulation at the reversal potential of -2.4 V. Such generation of unique, single-run, information-rich inorganic/organic electrochemical fingerprints holds considerable promise for 'on-the-spot' field identification of individuals firing a weapon, as desired for diverse forensic investigations.

A click chemistry route to 2-functionalised PEGylated and cationic ß-cyclodextrins: co-formulation opportunities for siRNA delivery.

A new approach to the synthesis of amphiphilic ß-cyclodextrins has used 'click' chemistry to selectively modify the secondary 2-hydroxyl group. The resulting extended polar groups can be either polycationic or neutral PEGylated groups and these two amphiphile classes are compatible in dual cyclodextrin formulations for delivery of siRNA. When used alone with an siRNA, a cationic cyclodextrin was shown to have good transfection properties in cell culture. Co-formulation with a PEGylated cyclodextrin altered the physicochemical properties of nanoparticles formed with siRNA. Improved particle properties included lower surface charges and reduced tendency to aggregate. However, as expected, the transfection efficiency of the cationic vector was lowered by co-formulation with the PEGylated cyclodextrin, requiring future surface modification of particles with targeting ligands for effective siRNA delivery.

Strategies to Mitigate and Treat Orthopaedic Device-Associated Infections.

Orthopaedic device implants play a crucial role in restoring functionality to patients suffering from debilitating musculoskeletal diseases or to those who have experienced traumatic injury. However, the surgical implantation of these devices carries a risk of infection, which represents a significant burden for patients and healthcare providers. This review delineates the pathogenesis of orthopaedic implant infections and the challenges that arise due to biofilm formation and the implications for treatment. It focuses on research advancements in the development of next-generation orthopaedic medical devices to mitigate against implant-related infections. Key considerations impacting the development of devices, which must often perform multiple biological and mechanical roles, are delineated. We review technologies designed to exert spatial and temporal control over antimicrobial presentation and the use of antimicrobial surfaces with intrinsic antibacterial activity. A range of measures to control bio-interfacial interactions including approaches that modify implant surface chemistry or topography to reduce the capacity of bacteria to colonise the surface, form biofilms and cause infections at the device interface and surrounding tissues are also reviewed.

Modification and implications of changes in electrochemical responses encountered when undertaking deoxygenation in ionic liquids.

Physicochemical changes and substantially modified electrochemical behavior have been reported when ionic liquids are degassed with nitrogen. In conventional experiments in aqueous and organic media, degassing with N(2) is commonly used to remove the electroactive dissolved oxygen. However, in hydrophilic ionic liquid media, degassing with N(2) removes not only the dissolved oxygen but also a significant amount of the adventitious water present. Given the low viscosity of water, this in turn leads to a dramatic change of the viscosity of the degassed ionic liquid and hence mass transport properties that influence voltammetric responses. In the widely used and relatively viscous room temperature ionic liquid, 1-n-butyl-3-methylimidazolium tetrafluoroborate (BMIMBF(4)) containing the redox probe tetracyanoquinodimethane (TCNQ) and 9% (v/v) deliberately added water, 1 h degassing with very dry N(2) under benchtop conditions results in a dramatic decrease of the TCNQ reduction current obtained under steady-state conditions at a 1 mum diameter microdisc electrode. This is reflected by a change of diffusion coefficient of TCNQ (D(TCNQ)) from 2.6 x 10(-7) to 4.6 x 10(-8) cm(2) s(-1). Karl Fischer titration measurements show that almost complete removal of the deliberately added 9% water is achieved by degassing under benchtop conditions. However, displacement of oxygen by nitrogen in the ionic liquid solution results in the decrease of electrochemical reduction current by 6%, implying that dissolved gases need not be inert with respect to solvent properties. Oxygen removal by placing the BMIMBF(4) ionic liquid in a nitrogen-filled glovebox or in a vacuum cell also simultaneously leads to removal of water and alteration of voltammetric data. This study highlights that (i) important physicochemical differences may arise upon addition or removal of a solute from viscous ionic liquids; (ii) degassing with dry nitrogen removes water as well as oxygen from ionic liquids, which may have implications on the viscosity and structure of the medium; (iii) particular caution must be exercised when deoxygenation is applied in ionic liquid media as part of the protocol used in electrochemical experiments to remove oxygen; (iv) gases such as oxygen, argon, and nitrogen dissolved in ionic liquids need not be innocent with respect to the properties of an ionic liquid. The use of vacuum based techniques to eliminate all volatile solutes, including water and oxygen, is advocated.

Toward membrane-free amperometric gas sensors: a microelectrode array approach.

Room temperature ionic liquids (RTILs) have been applied to a microelectrode array and been demonstrated to form effective, membrane-free amperometric gas sensors. Determining the RTIL [P(6,6,6,14)][FAP] as the most appropriate choice for extended use, the amperometric quantification of oxygen has been demonstrated. The response of the sensor was quantified by both cyclic voltammetry and chronoamperometry. A range of O(2) contents (2-13% v/v) and RTIL layer thicknesses (from ca. 6 to 125 mum) have been investigated. The combination of microelectrode array and RTIL, as well as the absence of membrane and volatile solvent, results in an elegant, easy to calibrate gas sensor with potential utility in standard and nonstandard conditions.

Microarrays of ring-recessed disk electrodes in transient generator-collector mode: theory and experiment.

The fabrication, characterization, and use of arrays of ring-recessed disk microelectrodes are reported. These devices are operated in generator-collector mode with a disk acting as the generator and the ring as the collector. We report experiments and simulations relating to time-of-flight experiments in which material electrogenerated at a disk is diffusionally transported to the ring. Analysis of the current transient measured at the latter when it is potentiostatted at a value to ensure diffusionally controlled "collection" is shown to sensitively reflect the diffusion coefficients of the species forming the redox couple being driven at the generator electrode. The method is applied to the ferrocene/ferrocenium couple in the room temperature ionic liquid [N(6, 2, 2, 2)][NTf(2)], and the results are found to agree with independent measurements.

Microelectrode arrays for electrochemistry: approaches to fabrication.

Microelectrode arrays have unique electrochemical properties such as small capacitive-charging currents, reduced iR drop, and steady-state diffusion currents. These properties enable the use of microelectrode arrays and have captured much interest in the field of electrochemistry. Techniques for the fabrication of such arrays are reviewed. The relative features and merits of different techniques are also discussed.

Biophysical and structural characterisation of nucleic acid complexes with modified cyclodextrins using circular dichroism.

Modified cyclodextrins (CDs) have shown great promise as non-viral gene and siRNA delivery vectors in a range of in vitro and in vivo studies. In the current study, structural and biophysical characterisation of selected CDs was carried out to enhance our understanding of their interaction with nucleic acids. The methods used for such characterisation were dynamic light scattering, zeta potential measurements and circular dichroism. Variations in the chemistries of individual CDs and in the type of formulation were shown to affect key properties of complexes such as size, surface charge and nucleic acid conformation. Furthermore, the effects of temperature and pH on the conformation of nucleic acids were investigated. pH studies were intended to mimic the conditions encountered by cationic complexes during endocytosis. Circular dichroism studies revealed that changes occurred in DNA and siRNA conformation upon complexation with CDs and when exposed to increasing temperature and decreasing pH. Overall, siRNA appeared to be more susceptible to conformational changes although complexation of siRNA with CDs tended to have a stabilising effect.

Amphiphilic polyallylamine based polymeric micelles for siRNA delivery to the gastrointestinal tract: in vitro investigations.

Targeting the gastrointestinal (GI) tract represents a promising strategy for local or systemic delivery of nucleic acid-based therapeutics. The development of a nano-carrier for siRNA delivery via the GI tract would enable localised therapy for a range of gastrointestinal diseases. Previously, nano-sized polymeric micelles (PM) formed by a variety of amphiphilic poly(allylamine) (PAA) derivatives have shown potential for oral delivery of hydrophobic drugs and bioactive peptides. The aim of this study was to evaluate the ability of these amphiphilic PAA-based PM for siRNA delivery via the GI tract. The physicochemical characteristics of PAA·siRNA transfection complexes and their biological efficacy in vitro were investigated. Physicochemical profiles demonstrated that PAAs and siRNA self-assembled into complexes with nano-sized diameters (150-300 nm) and cationic surface charge (+ 20 to 30 mV). The PAA·siRNA complexes were stable in the presence of salt solutions and simulated gastric/intestinal fluids. In undifferentiated Caco-2 cells, PAA·siRNA complexes achieved up to 35% cellular uptake, with successful siRNA release from endosomes/lysosomes and significant luciferase gene knockdown. These results highlight the potential of these nano-sized PM for siRNA oral delivery via the GI tract for treatment of gastrointestinal diseases.

Cationic and PEGylated Amphiphilic Cyclodextrins: Co-Formulation Opportunities for Neuronal Sirna Delivery.

Optimising non-viral vectors for neuronal siRNA delivery presents a significant challenge. Here, we investigate a co-formulation, consisting of two amphiphilic cyclodextrins (CDs), one cationic and the other PEGylated, which were blended together for siRNA delivery to a neuronal cell culture model. Co-formulated CD-siRNA complexes were characterised in terms of size, charge and morphology. Stability in salt and serum was also examined. Uptake was determined by flow cytometry and toxicity was measured by MTT assay. Knockdown of a luciferase reporter gene was used as a measure of gene silencing efficiency. Incorporation of a PEGylated CD in the formulation had significant effects on the physical and biological properties of CD.siRNA complexes. Co-formulated complexes exhibited a lower surface charge and greater stability in a high salt environment. However, the inclusion of the PEGylated CD also dramatically reduced gene silencing efficiency due to its effects on neuronal uptake. The co-formulation strategy for cationic and PEGylated CDs improved the stability of the CD.siRNA delivery systems, although knockdown efficiency was impaired. Future work will focus on the addition of targeting ligands to the co-formulated complexes to restore transfection capabilities.

Non-viral nanosystems for gene and small interfering RNA delivery to the central nervous system: formulating the solution.

The application of gene and RNAi-based therapies to the central nervous system (CNS), for neurological and neurodegenerative disease, offers immense potential. The issue of delivery to the target site remains the single greatest barrier to achieving this. There are challenges to gene and siRNA (small interfering RNA) delivery which are specific to the CNS, including the post-mitotic nature of neurons, their resistance to transfection and the blood-brain barrier. Viral vectors are highly efficient and have been used extensively in pre-clinical studies for CNS diseases. However, non-viral delivery offers an exciting alternative. In this review, we will discuss the extracellular and intracellular barriers to gene and siRNA delivery in the CNS. Our focus will be directed towards various non-viral strategies used to overcome these barriers. In this regard, we describe selected non-viral vectors and categorise them according to the barriers that they overcome by their formulation and targeting strategies. Some of the difficulties associated with non-viral vectors such as toxicity, large-scale manufacture and route of administration are discussed. We provide examples of optimised formulation approaches and discuss regulatory hurdles to clinical validation. Finally, we outline the components of an "ideal" formulation, based on a critical analysis of the approaches highlighted throughout the review.

Gastrointestinal gene delivery by cyclodextrins--in vitro quantification of extracellular barriers.

Local gene delivery represents a promising therapeutic approach for diseases of the intestine. However, the gastrointestinal tract poses significant challenges to successful gene delivery. Cyclodextrins (CDs) have been extensively investigated as non-viral vectors. Here, we assessed the suitability of an amphiphilic cationic CD for intestinal gene transfer, with particular focus on extracellular barriers. Stability and transfection efficiency of CD·DNA complexes were assessed post incubation in simulated gastric and intestinal fluids, bile salts and mucin, or with intestinal enzymes to represent extracellular barriers to intestinal gene delivery. Stability was determined by gel electrophoresis and transfection was measured by luciferase expression in intestinal epithelial cells (Caco-2). Transfection efficiency of CD·DNA complexes was enhanced after incubation in bile salts but was reduced after incubation in gastric and intestinal fluids and mucin. CD·DNA complexes were stable after incubation with pancreatic enzymes and with a model lower intestinal enzyme. Furthermore, the CD protected pDNA from degradation by DNase. In summary, physiologically relevant in vitro models were established and used to quantify the barriers posed by the intestinal extracellular environment to gene delivery. This systematic assessment identified the advantages and limitations of the CD vector and facilitated the proposal of formulation strategies to overcome these barriers.

Simultaneous identification and quantification of nitro-containing explosives by advanced chemometric data treatment of cyclic voltammetry at screen-printed electrodes.

The simultaneous determination of three nitro-containing compounds found in the majority of explosive mixtures, namely hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), 2,4,6-trinitrotoluene (TNT) and pentaerythritol tetranitrate (PETN), is demonstrated using both qualitative and quantitative approaches involving the coupling of electrochemical measurements and advanced chemometric data processing. Voltammetric responses were obtained from a single bare screen-printed carbon electrode (SPCE), which exhibited marked mix-responses towards the compounds examined. The responses obtained were then preprocessed employing discrete wavelet transform (DWT) and the resulting coefficients were input to an artificial neural network (ANN) model. Subsequently, meaningful data was extracted from the complex voltammetric readings, achieving either the correct discrimination of the different commercial mixtures (100% of accuracy, sensitivity and specificity) or the individual quantification of each of the compounds under study (total NRMSE of 0.162 for the external test subset).