Nose-to-brain delivery of enveloped RNA - cell permeating peptide nanocomplexes for the treatment of neurodegenerative diseases.

Direct nose-to-brain (N-to-B) delivery enables the rapid transport of drugs to the brain, while minimizing systemic exposure. The objective of this work was to engineer a nanocarrier intended to enhance N-to-B delivery of RNA and to explore its potential utility for the treatment of neurological disorders. Our approach involved the formation of electrostatically driven nanocomplexes between a hydrophobic derivative of octaarginine (r8), chemically conjugated with lauric acid (C12), and the RNA of interest. Subsequently, these cationic nanocomplexes were enveloped (enveloped nanocomplexes, ENCPs) with different protective polymers, i.e. polyethyleneglycol - polyglutamic acid (PEG-PGA) or hyaluronic acid (HA), intended to enhance their stability and mucodiffusion across the olfactory nasal mucosa. These rationally designed ENCPs were produced in bulk format and also using a microfluidics-based technique. This technique enabled the production of a scalable nanoformulation, exhibiting; (i) a unimodal size distribution with a tunable mean size, (ii) the capacity to highly associate (100%) and protect RNA from degradation, (iii) the ability to preserve its physicochemical properties in biorelevant media and prevent the premature RNA release. Moreover, in vitro cell culture studies showed the capacity of ENCPs to interact and be efficiently taken-up by CHO cells. Finally, in vivo experiments in a mouse model of Alzheimer's disease provided evidence of a statistically significant increase of a potentially therapeutic miRNA mimic in the hippocampus area and its further effect on two mRNA targets, following its intranasal administration. Overall, these findings stress the value of the rational design of nanocarriers towards overcoming the biological barriers associated to N-to-B RNA delivery and reveal their potential value as therapeutic strategies in Alzheimer's disease.

Enhancing cutaneous delivery with laser technology: Almost there, but not yet.

Preclinical research has shown the potential of different laser-based strategies (direct ablation, photothermolysis and mechanical waves) to overcome the stratum corneum and facilitate the cutaneous delivery of drugs. However, specific protocols for the routine use of these strategies have not been stablished yet. The aim of this review has been to provide the readers with a view of the translational prospects of the different laser technologies with regards to their utility for enhancing the penetration of drugs. For this, we have comparatively analyzed the preclinical research disclosed for laser-assisted delivery of classical small molecules as well as new biologics with the studies performed at the clinical level. In addition, we present the future perspectives of laser technological developments considering the evolution of the global laser market.

Synthetic nanocarriers for the delivery of polynucleotides to the eye.

This review is a comprehensive analysis of the progress made so far on the delivery of polynucleotide-based therapeutics to the eye, using synthetic nanocarriers. Attention has been addressed to the capacity of different nanocarriers for the specific delivery of polynucleotides to both, the anterior and posterior segments of the eye, with emphasis on their ability to (i) improve the transport of polynucleotides across the different eye barriers; (ii) promote their intracellular penetration into the target cells; (iii) protect them against degradation and, (iv) deliver them in a long-term fashion way. Overall, the conclusion is that despite the advantages that nanotechnology may offer to the area of ocular polynucleotide-based therapies (especially AS-ODN and siRNA delivery), the knowledge disclosed so far is still limited. This fact underlines the necessity of more fundamental and product-oriented research for making the way of the said nanotherapies towards clinical translation.

Modular microfluidic valve structures based on reversible thermoresponsive ionogel actuators.

This paper reports for the first time the use of a cross-linked poly(N-isopropylacrylamide) ionogel encapsulating the ionic liquid 1-ethyl-3-methylimidazolium ethyl sulphate as a thermoresponsive and modular microfluidic valve. The ionogel presents superior actuation behaviour to its equivalent hydrogel. Ionogel swelling and shrinking mechanisms and kinetics are investigated as well as the performance of the ionogel when integrated as a valve in a microfluidic device. The modular microfluidic valve demonstrates fully a reversible on-off behaviour without failure for up to eight actuation cycles and a pressure resistance of 1100 mbar.

A simple and portable device for the quantification of TNF-α in human plasma by means of on-chip magnetic bead-based proximity ligation assay.

There is a general need in healthcare systems all around the world to reduce costs in terms of time and money without compromising patients outcome. Point-of-Care Testing (POCT) is currently being used in some applications (e.g. POC coagulation devices) as an alternative to already established standard central laboratory tests to overcome sample transportation and long turnaround times. The main objective of this investigation was to quantify Tumour Necrosis Factor-alpha (TNF-α) on-chip within the clinical relevant range of 5-100 pg/mL in human pooled plasma. The novel solid-phase assay developed in this study was a magnetic bead-based proximity ligation assay (PLA) in which one of the assay proximity probes was directly immobilised onto streptavidin-coated magnetic beads. The portable device was based on a disposable and single-use cyclo-olefin polymer (COP) microfluidic chip interfaced with a quantitative real-time polymerase chain reaction (qPCR) device previously developed in-house. Sample volume was 10 µL and total assay time under 3 h. The POC device and assay developed offer portability, smaller reagent and sample consumption, and faster time-to-results compared with standard ELISAs. Determination and monitoring of TNF-α therapy at the point-of-care will help to improve clinical and/or economical outcome in governmental healthcare budgets.

A microfluidic anti-Factor Xa assay device for point of care monitoring of anticoagulation therapy.

The development of new point of care coagulation assay devices is necessary due to the increasing number of patients requiring long-term anticoagulation in addition to the desire for appropriate, targeted anticoagulant therapy and a more rapid response to optimization of treatment. The majority of point of care devices currently available for hemostasis testing rely on clot-based endpoints which are variable, unreliable and limited to measuring only certain portions of the coagulation pathway. Here we present a novel fluorescence-based anti-Factor Xa (FXa) microfluidic assay device for monitoring the effect of anticoagulant therapy at the point of care. The device is a disposable, laminated polymer microfluidic strip fabricated from a combination of hydrophobic and hydrophilic cyclic polyolefins to allow reagent deposition in addition to effective capillary fill. Zeonor was the polymer of choice resulting in low background fluorescence (208.5 AU), suitable contact angles (17.5°± 0.9°) and capillary fill times (20.3 ± 2.1 s). The device was capable of measuring unfractionated heparin and tinzaparin from 0-0.8 U ml(-1) and enoxaparin from 0-0.6 U ml(-1) with average CVs < 10%. A linear correlation was observed between the device and the fluorescent assay in the plate for plasma samples spiked with UFH, with an R(2) value of 0.99, while correlations with tinzaparin and enoxaparin resulted in sigmoidal responses (R(2) = 0.99). Plasma samples containing UFH resulted in a linear correlation between the device and a standard chromogenic assay with an R(2) value of 0.98, with both LMWHs resulting in sigmoidal relationships (R(2) = 0.99).

Effects of four commercially available factor Xa proteins on the fluorogenic anti-factor Xa assay when monitoring unfractionated heparin.

Four commercially available factor Xa (FXa) reagents were evaluated in a fluorogenic anti-FXa assay. The four reagents - of which three were of human origin and the fourth was bovine - were compared in terms of the resulting assay dynamic ranges, lag times, coefficient of variation and R2 values, as well as their sensitivity to unfractionated heparin within the therapeutic range of 0-1.2 U/ml. Similar performance of reagents in the fluorogenic anti-FXa assay was observed independent of the source of the reagent or its physical state, which may assist in the standardization of coagulation assays in clinical settings.

Comparison of the anticoagulant response of a novel fluorogenic anti-FXa assay with two commercial anti-FXa chromogenic assays.

INTRODUCTION: Fast and accurate monitoring is crucial in the successful regulation of coagulation therapy. For the treatment of venous thromboembolism, both unfractionated heparin (UFH) and low molecular weight heparins (LMWHs) are commonly administered. The chromogenic anti-factor Xa (FXa) assay is currently considered the 'gold standard' assay for monitoring LMWH. However different commercial chromogenic methods often differ when tested with the same samples. Fluorogenic anti-FXa assays have the potential to offer greater benefits over chromogenic assays in terms of greater specificity, sensitivity and they are not so influenced by sample opacity or turbidity. MATERIALS AND METHODS: Commercial plasmas were spiked with pharmacologically relevant concentrations (0-1 U/ml) of UFH, enoxaparin, and tinzaparin. The fluorogenic assay was carried out using previously optimized concentrations of 12 nM FXa and 2.7µM fluorogenic substrate, in addition to 6µl of 100mM CaCl(2) and 44µl of plasma. The Biophen® and Coamatic chromogenic assays were carried out according to the manufacturer's instructions. Reaction rates and endpoint values were analyzed and statistical analysis by means of one-way analysis of variance (ANOVA) was performed. RESULTS: The fluorogenic anti-FXa assay was found to have the broadest therapeutic range of 0-1 U/ml with CVs of<5% for UFH and tinzaparin and CVs<9% for enoxaparin. Despite their limited measuring range, good assay reproducibility was observed with both chromogenic kits. CONCLUSIONS: This study indicated that the fluorogenic assay is the most sensitive assay with the broadest dynamic range for monitoring LMWH therapy when compared with standard chromogenic assays.

Quantification of unfractionated heparin in human plasma and whole blood by means of novel fluorogenic anti-FXa assays.

Novel and sensitive plate-based fluorogenic anti-factor Xa (FXa) assays were investigated to quantify unfractionated heparin (UFH) in human plasma and whole blood within the therapeutic ranges of 0-1.6 U/mL and 0-0.8 U/mL, respectively. Two fluorogenic anti-FXa assay methods were defined for low (0-0.6 U/mL) and high (0.6-1.2 U/mL) pharmacologically relevant UFH concentration ranges in pooled human plasma. In both cases significant differences were observed at intervals of 0.2 U/mL (P<0.05). The semi-logarithmic plots of the calibration curves in the low and high UFH range were both fitted to linear regressions with correlation coefficients of 0.96 and >0.99, respectively. The assay was also optimized for whole blood which was capable of differentiating UFH concentrations at intervals of 0.2 U/mL (P<0.05) in the range of 0-0.4 U/mL. The statistically different results were fitted to a linear regression with a correlation coefficient of >0.99. The results obtained in this study could assist diagnostic laboratories towards improved monitoring of UFH therapy.

Comparison of a fluorogenic anti-FXa assay with a central laboratory chromogenic anti-FXa assay for measuring LMWH activity in patient plasmas.

INTRODUCTION: Low molecular weight heparins (LMWHs) are used worldwide for the treatment and prophylaxis of thromboembolic disorders. Routine laboratory tests are not required due to the predictable pharmacokinetics of LMWHs, with the exception of pregnant patients, children, patients with renal failure, morbid obesity, or advanced age. Anti-Factor Xa (anti-FXa) plasma levels are most often employed in the assessment and guidance of accurate dosing in these patient cohorts. MATERIALS AND METHODS: A LMWH calibration curve was generated using citrated human pooled plasma spiked with pharmacologically relevant concentrations (0-1.2U/ml) of two low molecular weight heparins; enoxaparin and tinzaparin. Least squares analysis determined the best curve fit for this set of data which returned low sum of squares (SS) values for the log linear fit with an R(2) value of 0.98. 30 patient samples were tested in the fluorogenic assay and concentrations were determined using the log linear regression equation and correlated with a standard chromogenic assay used for heparin monitoring. RESULTS: A statistically significant correlation was found between the fluorogenic and the chromogenic anti-FXa assays for 30 patient samples, with a slope of 0.829, offset of 0.258 and an R(2) value of 0.72 (p<0.0001). CONCLUSIONS: In the study presented here, a fluorogenic anti-FXa assay was correlated with a standard laboratory chromogenic anti-FXa assay using samples from patients on LMWH therapy. Significant correlations between the values derived by the fluorogenic and chromogenic anti-FXa assays were found for the patient cohort tested in this study.

Comparative study of Factor Xa fluorogenic substrates and their influence on the quantification of LMWHs.

Low molecular weight heparins (LMWHs) are recognised as the preferred anticoagulants in the prevention and treatment of venous thromboembolism. Anti-Factor Xa (anti-FXa) levels are used to monitor the anticoagulant effect of LMWHs and such assays are routinely employed in hospital diagnostic laboratories. In this study, a fluorogenic anti-FXa assay was developed using a commercially available fluorogenic substrate with an attached 6-amino-1-naphthalene-sulfonamide (ANSN) fluorophore and was used for the determination of two LMWHs, enoxaparin and tinzaparin and the heparinoid, danaparoid. The assay was based on the complexation of heparinised plasma with 100 nM exogenous FXa and 25 µM of the fluorogenic substrate Mes-D-LGR-ANSN (C(2)H(5))(2) (SN-7). The assay was tested with pooled plasma samples spiked with anticoagulant concentrations in the range 0-1.6 U mL(-1). The statistically sensitive assay range was 0-0.4 U mL(-1) for enoxaparin and tinzaparin and 0-0.2 U mL(-1) for danaparoid, with assay variation typically below 10.5%. This assay was then compared with a previously published fluorogenic anti-FXa assay developed with the peptide substrate, methylsulfonyl-D: -cyclohexylalanyl-glycyl-arginine-7-amino-4-methylcoumarin acetate (Pefafluor FXa). Both assays were compared in terms of fluorescence intensity, lag times and sensitivity to anticoagulants.

Development of a fluorescent anti-factor Xa assay to monitor unfractionated and low molecular weight heparins.

Fluorogenic assays have many potential advantages over traditional clot-based and chromogenic assays such as the absence of interference from a range of factor deficiencies as well as offering the possibility of assays in platelet rich plasma or whole blood. A fluorogenic anti-factor Xa (anti-FXa) assay has been developed for the determination of unfractionated heparin (UFH), low molecular weight heparins (LMWHs), namely enoxaparin and tinzaparin, and the synthetic heparinoid danaparoid, in commercial human pooled plasma. The assay was based on the complexation of heparin-spiked plasmas with exogenous FXa at a concentration of 4nM in the presence of 0.9microM of the fluorogenic substrate methylsulfonyl-D-cyclohexylalanyl-glycyl-arginine-7-amino-4-methylcoumarin acetate (Pefafluor FXa). Pooled plasma samples were spiked with concentrations of anticoagulants in the range 0-1.6U/ml. The assay was capable of the measurement of UFH and danaparoid in the range 0-1U/ml, and enoxaparin and tinzaparin in the range 0-0.8 and 0-0.6U/ml, respectively. Correlation coefficients generated by linear regression of the log/lin data analysis were between 0.93 and 0.96 for the anticoagulants tested. Assay percentage coefficients of variation were typically below 7%.