Kinetic Model of Fluorescein Release through Bioprinted Polylactic Acid Membrane.

Polylactic acid (PLA)-based cylindrical membranes for the controlled release of fluorescein sodium salt (FS) were prepared by bioprinting on systems with an initial FS concentration of 0.003763 gdm-3 and 37.63 gdm-3, and the drug release process was monitored in a bath at 37 °C. Photographs, acquired at regular intervals during the process, revealed marked osmotic swelling of the polymer. Osmotic swelling consists in the enlargement of the polymer structure and due to the influx of water molecules across the membrane. The cylindrical PLA membrane starts to significantly swell once a certain threshold range is crossed. Important amounts of FS can dissolve under these radically changed circumstances, and the dissolved FS molecules are mobile enough to diffuse out of the cylinder, thus allowing drug release. As a matter of fact, in this investigation, we ascertained that polymer swelling promotes the mass transport phenomenon by altering the conditions for drug dissolution and diffusion, hence facilitating FS release after a specific lag time. Furthermore, in order to compare the release kinetics, the half-release time, t0.5, was taken into consideration. The data of this study evidence that, while increasing the initial concentration of FS by three orders of magnitude, the time parameter, t0.5, is only reduced by 5/6. In addition, the yield of the release process is drastically reduced due to the strong aggregation ability of the dye. Finally, it is demonstrated that a compressed exponential kinetic model fits the experimental data well despite the varying physical conditions.

Effects of Anionic Liposome Delivery of All-Trans-Retinoic Acid on Neuroblastoma Cell Differentiation.

All-trans-retinoic acid (ATRA) has long been known to affect cell growth and differentiation. To improve ATRA's therapeutic efficacy and pharmacodynamics, several delivery systems have been used. In this study, free ATRA and anionic-liposome-encapsulated ATRA were compared for their effects on SK-N-SH human neuroblastoma cell growth and differentiation. Anionic liposomes made of L-α-phosphatidylcholine (PC) and L-α-phosphatidic acid (PA), empty (PC-PA) and loaded with ATRA (PC-PA-ATRA), were characterized by dynamic light scattering (DLS) and electrophoretic mobility measurements, and drug entrapment efficiency (EE%) was measured to evaluate the applicability of the new colloidal formulation. The results of brightfield microscopy and cell growth curves indicated that ATRA, whether free or encapsulated, reduced growth and induced differentiation, resulting in SK-N-SH cells changing from epithelioid to neuronal-like morphologies, and producing a significant increase in neurite growth. To further characterize the neuro-differentiation of SK-N-SH cells, the expression of ßIII-Tubulin and synaptophysin and mitochondria localization were analyzed via immunofluorescence. Increased expression of neuronal markers and a peculiar localization of mitochondria in the neuritic extensions were apparent both in ATRA- and PC-PA-ATRA-differentiated cells. As a whole, our results strongly indicate that ATRA treatment, by any means, can induce the differentiation of parent SK-N-SH, and they highlight that its encapsulation in anionic liposomes increases its differentiation ability in terms of the percentage of neurite-bearing cells. Interestingly, our data also suggest an unexpected differentiation capability of anionic liposomes per se. This work highlights the importance of developing and carefully testing novel delivery nanocarriers, which are a necessary first "step" in the development of new therapeutic settings.

Excess Absorbance as a Novel Approach for Studying the Self-Aggregation of Vital Dyes in Liquid Solution.

In the present paper, a simple method for analyzing the self-aggregation of dyes in a solution by a UV-visible absorption measurements is proposed. The concept of excess absorbance is introduced to determine an equation whose coefficients determine the parameters of the aggregation equilibrium. The computational peculiarities of the model are first discussed theoretically and then applied to sodium fluorescein in polar protic and aprotic solvents, as well as in aqueous solutions of methylene blue, which is a cationic dye. Although the experimental responses are very different, the model appears to work equally well in both cases. The model reveals that the trimer is the most likely configuration in both solvents. Furthermore, aggregation is strongly favored for the protic solvent. Interestingly, the model establishes that in aqueous solutions of methylene blue, the tetramer is the predominant form, which has long been assumed and recently demonstrated with sophisticated computational techniques.

Targeting fibrosis in the failing heart with nanoparticles.

Heart failure (HF) is a clinical syndrome characterized by typical symptoms and signs caused by a structural and/or functional cardiac abnormality, resulting in a reduced cardiac output and/or elevated intracardiac pressures at rest or during stress. Due to increasing incidence, prevalence and, most importantly mortality, HF is a healthcare burden worldwide, despite the improvement of treatment options and effectiveness. Acute and chronic cardiac injuries trigger the activation of neurohormonal, inflammatory, and mechanical pathways ultimately leading to fibrosis, which plays a key role in the development of cardiac dysfunction and HF. The use of nanoparticles for targeted drug delivery would greatly improve therapeutic options to identify, prevent and treat cardiac fibrosis. In this review we will highlight the mechanisms of cardiac fibrosis development to depict the pathophysiological features for passive and active targeting of acute and chronic cardiac fibrosis with nanoparticles. Then we will discuss how cardiomyocytes, immune and inflammatory cells, fibroblasts and extracellular matrix can be targeted with nanoparticles to prevent or restore cardiac dysfunction and to improve the molecular imaging of cardiac fibrosis.

Nicotinic acid adenine dinucleotide phosphate activates two-pore channel TPC1 to mediate lysosomal Ca2+ release in endothelial colony-forming cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most recently discovered Ca2+ -releasing messenger that increases the intracellular Ca2+ concentration by mobilizing the lysosomal Ca2+ store through two-pore channels 1 (TPC1) and 2 (TPC2). NAADP-induced lysosomal Ca2+ release regulates multiple endothelial functions, including nitric oxide release and proliferation. A sizeable acidic Ca2+ pool endowed with TPC1 is also present in human endothelial colony-forming cells (ECFCs), which represent the only known truly endothelial precursors. Herein, we sought to explore the role of the lysosomal Ca2+ store and TPC1 in circulating ECFCs by harnessing Ca2+ imaging and molecular biology techniques. The lysosomotropic agent, Gly-Phe ß-naphthylamide, and nigericin, which dissipates the proton gradient which drives Ca2+ sequestration by acidic organelles, caused endogenous Ca2+ release in the presence of a replete inositol-1,4,5-trisphosphate (InsP3 )-sensitive endoplasmic reticulum (ER) Ca2+ pool. Likewise, the amount of ER releasable Ca2+ was reduced by disrupting lysosomal Ca2+ content. Liposomal delivery of NAADP induced a transient Ca2+ signal that was abolished by disrupting the lysosomal Ca2+ store and by pharmacological and genetic blockade of TPC1. Pharmacological manipulation revealed that NAADP-induced Ca2+ release also required ER-embedded InsP3 receptors. Finally, NAADP-induced lysosomal Ca2+ release was found to trigger vascular endothelial growth factor-induced intracellular Ca2+ oscillations and proliferation, while it did not contribute to adenosine-5'-trisphosphate-induced Ca2+ signaling. These findings demonstrated that NAADP-induced TPC1-mediated Ca2+ release can selectively be recruited to induce the Ca2+ response to specific cues in circulating ECFCs.

Reaction-diffusion model as framework for understanding the role of riboflavin in "eye defence" formulations.

Analysis of UV-visible spectra, performed on commercial riboflavin-based eye drops, showed that absorbance is a saturating function of vitamin concentration. This implies a threshold concentration, C t, such that for riboflavin concentration > C t the absorbance remains constant and the effectiveness of the eye drops is independent of the dose used. These experimental results were combined with a diffusion-reaction model to elucidate the mechanism of action within the cornea. The model predicts that the eye drops have a low effectiveness on UVB and UVC, while they have a good performance for UVA. Indeed, at the center of the cornea the transmittance is significantly reduced and after 1 h it is reduced by about 70% compared to a cornea devoid of eye drops.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Induces Intracellular Ca2+ Release through the Two-Pore Channel TPC1 in Metastatic Colorectal Cancer Cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP) gates two-pore channels 1 and 2 (TPC1 and TPC2) to elicit endo-lysosomal (EL) Ca2+ release. NAADP-induced EL Ca2+ signals may be amplified by the endoplasmic reticulum (ER) through the Ca2+-induced Ca2+ release mechanism (CICR). Herein, we aimed at assessing for the first time the role of EL Ca2+ signaling in primary cultures of human metastatic colorectal carcinoma (mCRC) by exploiting Ca2+ imaging and molecular biology techniques. The lysosomotropic agent, Gly-Phe ß-naphthylamide (GPN), and nigericin, which dissipates the ΔpH which drives Ca2+ refilling of acidic organelles, caused massive Ca2+ release in the presence of a functional inositol-1,4,5-trisphosphate (InsP3)-sensitive ER Ca2+ store. Liposomal delivery of NAADP induced a transient Ca2+ release that was reduced by GPN and NED-19, a selective TPC antagonist. Pharmacological and genetic manipulations revealed that the Ca2+ response to NAADP was triggered by TPC1, the most expressed TPC isoform in mCRC cells, and required ER-embedded InsP3 receptors. Finally, NED-19 and genetic silencing of TPC1 reduced fetal calf serum-induced Ca2+ signals, proliferation, and extracellular signal-regulated kinase and Akt phoshorylation in mCRC cells. These data demonstrate that NAADP-gated TPC1 could be regarded as a novel target for alternative therapies to treat mCRC.

Determination of bisphenol A in red wine using a double vortex-ultrasound-assisted microextraction assay: Role of the interfacial properties.

Bisphenol A (BPA) is a synthetic compound broadly used in medical devices as well as in packaging of food and drinks. Recently, BPA toxicity has become of concern to environmental public health. Red wine that is susceptible to BPA contamination is an alcoholic beverage made from yeast fermentation of grapes in the presence of grape skins so as to extract phenolic compounds. The aim of this study was to validate an efficient, low cost, and time-saving method for BPA determination in red-wine beverage. To this end, a rapid and simple microextraction method is here proposed consisting in liquid-liquid separation assisted by a vortex-ultrasound-vortex procedure combined with gas chromatographic analysis (GC-Fid or GC-IT/MS). By means of a comparative study between real red-wine matrix and synthetic hydroalcoholic solutions, different parameters related to the microextraction steps were investigated. The minimal amount of extraction solvent for a given volume of sample was calculated for both the systems. It was demonstrated that for red-wine matrix, the extent of phase separation is strongly affected by some wine constituents and that separation can be tuned by varying the amount of the extraction solvent. This double vortex-ultrasound-assisted method achieved high recovery of BPA and enrichment factor compared with other microextraction methods.

Glutamate triggers intracellular Ca2+ oscillations and nitric oxide release by inducing NAADP- and InsP3 -dependent Ca2+ release in mouse brain endothelial cells.

The neurotransmitter glutamate increases cerebral blood flow by activating postsynaptic neurons and presynaptic glial cells within the neurovascular unit. Glutamate does so by causing an increase in intracellular Ca2+ concentration ([Ca2+ ]i ) in the target cells, which activates the Ca2+ /Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca2+ ]i and NO production. The current study assessed whether and how glutamate drives Ca2+ -dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca2+ ]i , which was maximally activated at 200 µM and inhibited by α-methyl-4-carboxyphenylglycine, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca2+ oscillations were triggered by rhythmic endogenous Ca2+ mobilization and maintained over time by extracellular Ca2+ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca2+ release was mediated by InsP3 -sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) gated two-pore channel 1. Constitutive store-operated Ca2+ entry mediated Ca2+ entry during ongoing Ca2+ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca2+ signals. Of note, glutamate induced Ca2+ -dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca2+ oscillations and NO release have the potential to impact on neurovascular coupling in the brain.

Temperature Effect on Rheological Behavior of Silicone Oils. A Model for the Viscous Heating.

The rheological behavior of silicone oils, (CH3)3SiO-[Si(CH3)2O]n-Si(CH3)3, and their mixtures is studied. Shear-stress measurements, in the temperature range of 293-313 K, reveal that this polymer family is a group of shear-thinning liquids with a yield stress below which no flow occurs. Experimental diagrams, i.e., shear stress versus shear rate, are satisfactorily described by the Casson fluid model over a wide range of shear rates. In order to monitor the effect of temperature on fluid properties, Casson's rheological model is reformulated using the fictitious shear rate, γ̇f, and the infinite-shear viscosity, η∞, as constitutive parameters. Due to low intermolecular forces and high chain flexibility, γ̇f varies very little when the temperature increases. For this reason, the apparent material viscosity depends on temperature only through η∞, which exponentially decreases until high shear rates are reached, and there is more alignment possible. Interestingly, the temperature sensitivity of this pseudoplastic behavior is the same for all of the silicone oils investigated; therefore, they can be classified according to their tendency to emulsify. Experimental results are then used to model the flow of silicone oils in a cylindrical pipe and estimate the temperature increase due to viscous heating. Numerical results show that the normalized temperature, i.e., ratio of fluid temperature to wall temperature, increases approximately 23%, and the apparent viscosity decreases drastically, going toward the center of the tube. The non-Newtonian nature of fluid is reflected in the presence of a critical region. In this region, the velocity and temperature gradients vanish. Since silicon oil is a surgical tool, we hope that the acquired physicochemical information can provide help to facilitate the removal of this material during surgical procedures.

Liposomes as a Putative Tool to Investigate NAADP Signaling in Vasculogenesis.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the newest discovered intracellular second messengers, which is able to release Ca2+ stored within endolysosomal (EL) vesicles. NAADP-induced Ca2+ signals mediate a growing number of cellular functions, ranging from proliferation to muscle contraction and differentiation. Recently, NAADP has recently been shown to regulate angiogenesis by promoting endothelial cell growth. It is, however, still unknown whether NAADP stimulates proliferation also in endothelial progenitor cells, which are mobilized in circulation after an ischemic insult to induce tissue revascularization. Herein, we described a novel approach to prepare NAADP-containing liposomes, which are highly cell membrane permeable and are therefore amenable for stimulating cell activity. Accordingly, NAADP-containing liposomes evoked an increase in intracellular Ca2+ concentration, which was inhibited by NED-19, a selective inhibitor of NAADP-induced Ca2+ release. Furthermore, NAADP-containing liposomes promoted EPC proliferation, a process which was inhibited by NED-19 and BAPTA, a membrane permeable intracellular Ca2+ buffer. Therefore, NAADP-containing liposomes stand out as a promising tool to promote revascularization of hypoxic/ischemic tissues by favoring EPC proliferation. J. Cell. Biochem. 118: 3722-3729, 2017. © 2017 Wiley Periodicals, Inc.

Nanotechnology-Based Cardiac Targeting and Direct Cardiac Reprogramming: The Betrothed.

Cardiovascular diseases represent the first cause of morbidity in Western countries, and chronic heart failure features a significant health care burden in developed countries. Efforts in the attempt of finding new possible strategies for the treatment of CHF yielded several approaches based on the use of stem cells. The discovery of direct cardiac reprogramming has unveiled a new approach to heart regeneration, allowing, at least in principle, the conversion of one differentiated cell type into another without proceeding through a pluripotent intermediate. First developed for cancer treatment, nanotechnology-based approaches have opened new perspectives in many fields of medical research, including cardiovascular research. Nanotechnology could allow the delivery of molecules with specific biological activity at a sustained and controlled rate in heart tissue, in a cell-specific manner. Potentially, all the mediators and structural molecules involved in the fibrotic process could be selectively targeted by nanocarriers, but to date, only few experiences have been made in cardiac research. This review highlights the most prominent concepts that characterize both the field of cardiac reprogramming and a nanomedicine-based approach to cardiovascular diseases, hypothesizing a possible synergy between these two very promising fields of research in the treatment of heart failure.

The Effect of Temperature Changes in Vitreoretinal Surgery.

PURPOSE: Recent studies on temperature control in biology and medicine have found the temperature as a new instrument in healthcare. In this manuscript, we reviewed the effects of temperature and its potential role in pars plana vitrectomy. We also examined the relationship between intraocular pressure, viscosity, and temperature in order to determine the best balance to manipulate the tamponades during the surgery. METHODS: A literature review was performed to identify potentially relevant studies on intraocular temperature. Physics equations were applied to explain the described effects of temperature changes on the behavior of the endotamponades commonly used during vitreoretinal surgery. We also generated an operating diagram on the pressure-temperature plane for the values of both vapor-liquid equilibrium and intraocular pressure. RESULTS: The rapid circulation of fluid in the vitreous cavity reduces the heat produced by the retinal and choroidal surface, bringing the temperature toward room temperature (22°C, deep hypothermia). Temperature increases with endolaser treatment, air infusion, and the presence of silicone oil. The variations in temperature during vitreoretinal surgery are clinically significant, as the rheology of tamponades can be better manipulated by modulating intraocular pressure and temperature. CONCLUSIONS: During vitreoretinal surgery, the intraocular temperature showed rapid and significant fluctuations at different steps of the surgical procedure inside the vitreous cavity. Temperature control can modulate the rheology of tamponades. TRANSLATIONAL RELEVANCE: Intraoperative temperature control can improve neuroprotection during vitreoretinal surgery, induce the vaporization of perfluorcarbon liquid, and change the shear viscosity of silicone oil.

Method for Determining the Activation Energy Distribution Function of Complex Reactions by Sieving and Thermogravimetric Measurements.

A method for studying the kinetics of thermal degradation of complex compounds is suggested. Although the method is applicable to any matrix whose grain size can be measured, herein we focus our investigation on thermogravimetric analysis, under a nitrogen atmosphere, of ground soft wheat and ground maize. The thermogravimetric curves reveal that there are two well-distinct jumps of mass loss. They correspond to volatilization, which is in the temperature range 298-433 K, and decomposition regions go from 450 to 1073 K. Thermal degradation is schematized as a reaction in the solid state whose kinetics is analyzed separately in each of the two regions. By means of a sieving analysis different size fractions of the material are separated and studied. A quasi-Newton fitting algorithm is used to obtain the grain size distribution as best fit to experimental data. The individual fractions are thermogravimetrically analyzed for deriving the functional relationship between activation energy of the degradation reactions and the particle size. Such functional relationship turns out to be crucial to evaluate the moments of the activation energy distribution, which is unknown in terms of the distribution calculated by sieve analysis. From the knowledge of moments one can reconstruct the reaction conversion. The method is applied first to the volatilization region, then to the decomposition region. The comparison with the experimental data reveals that the method reproduces the experimental conversion with an accuracy of 5-10% in the volatilization region and of 3-5% in the decomposition region.

Fractal dimension as a new tool to analyze optic nerve head vasculature in primary open angle glaucoma.

AIM: To investigate the features of optic nerve head (ONH) microvasculature in primary open angle glaucoma using fractal geometry analysis. PATIENTS AND METHOD: ONH blood flow was analyzed at the level of the lamina cribrosa by means of confocal scanning laser Heidelberg Doppler flowmetry (HRF) in medically-controlled early and advanced glaucoma. Fractal dimension D of vasculature map was calculated using the Box Counting. RESULTS: Our data demonstrated that, in patients with advanced glaucoma, fractal dimension D was significantly lower than in controls, whereas, in the early stage of disease, its value was similar. Fractal dimension D of microcirculation was significantly and negatively correlated with the cup-disk area ratio in both early and advanced glaucoma groups, whereas linear cup-disk ratio of the disk, cup shape measure and nerve fiber layer thickness, where correlated only in advanced stage of the disease. CONCLUSION: These findings demonstrate that fractal dimension D of ONH appeared significantly reduced in advanced glaucoma and correlated with the optic disc damage.

Corneal epithelial wound healing promoted by verbascoside-based liposomal eyedrops.

Different liposomal formulations were prepared to identify those capable of forming eyedrops for corneal diseases. Liposomes with neutral or slightly positive surface charge interact very well with the cornea. Then these formulations were loaded with verbascoside to heal a burn of corneal epithelium induced by alkali. The cornea surface affected involved in wound was monitored as a function of time. Experimental results were modeled by balance equation between the rate of healing, due to the flow of phenylpropanoid, and growth of the wound. The results indicate a latency time of only three hours and furthermore the corneal epithelium heals in 48 hours. Thus, the topical administration of verbascoside appears to reduce the action time of cells, as verified by histochemical and immunofluorescence assays.

Some physicochemical remarks on spontaneous emulsification of vitreal tamponades.

The importance of gravitational instability in determining the emulsification of vitreal tamponades is discussed. Theoretical results and numerical simulations indicate that the spontaneous formation of water-silicon oil is a rare event and that the very low concentration of surface active agents cannot justify the systematic formation of emulsions. The gravitational instabilities seem to play the main role. Our theoretical results seem in agreement with the experimental evidences; furthermore they indicate a future research line for the improvement of endotamponades. Indeed, the use of biodegradable antifoam may avoid the formation of bubbles and delay the formation of emulsions.

The role of microemulsions in lipase-catalyzed hydrolysis reactions.

The kinetics of the p-nitrophenyl butyrate hydrolysis reaction, catalyzed by Candida rugosa lipase in the water-in-oil microemulsion cetyltrimethylammonium bromide/water/pentanol/hexane, was investigated. The results described in the present manuscript reveal two peculiar characteristics of the reaction: (i) the initial rate of hydrolysis is very fast and (ii) by decreasing the water content of the microemulsion, the reaction rate approaches the typical behavior of reactions performed in aqueous solution. In particular, for microemulsion systems with a high water content, the end points of the reactions are dictated by the shape stability of the microemulsion. For these systems, our methodological approach shows that the process follows a second-order kinetics equation, indicative of the dual role played by water, which is involved both as a component of the microemulsion, i.e., relevant for the microemulsion stability and as a reagent of the hydrolysis reaction. In contrast, for microemulsions containing a small amount of water, after the hydrolysis reaction the system seems to fall in the no existence range of the microemulsion. Accordingly, the kinetics results are more complex: in the initial stage, the reaction follows a zero-order kinetics equation, while for longer reaction times a first-order kinetics equation fits the experimental data, as would be expected for an enzymatic reaction in a homogeneous system.

Ocular tissues and fluids oxidative stress in hares fed on verbascoside supplement.

The influence of a prolonged diet supplemented with the powerful antioxidant verbascoside on the oxidative state of 20 healthy hares eye fluids and tissues has been studied. Verbascoside was dosed at 2, 3, 4 mg/die and the impact on the oxidative state of ocular tissues and fluids was tested by TBARS (thio barbituric acid reactive substances) and TEAC (trolox equivalent antioxidant capacity) assays. The percentage of change in antioxidant activity increased largely in retina and lenses at a daily verbascoside dose of 3 mg, whereas for optic nerve and vitreous humor the higher antioxidant capacity was measured at 4 mg/die verbascoside dose. The present findings demonstrate that verbascoside supplementation is able to protect ocular tissue and fluids from naturally occurring oxidation and that its protective effect depends on the daily dose, being maximum up to 3 mg/die.