A New Anorganic Equine Bone Substitute for Oral Surgery: Structural Characterization and Regenerative Potential.

Different xenogeneic inorganic bone substitutes are currently used as bone grafting materials in oral and maxillo-facial surgery. The aim of the present study was to determine the physicochemical properties and the in vivo performance of an anorganic equine bone (AEB) substitute. AEB is manufactured by applying a process involving heating at >300 °C with the aim of removing all the antigens and the organic components. AEB was structurally characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray fluorescence (XRF), and Fourier-transformed infrared (FT-IR) spectroscopy and compared to the anorganic bovine bone (ABB). In order to provide a preliminary evaluation of the in vivo performance of AEB, 18 bone defects were prepared and grafted with AEB (nine sites), or ABB (nine sites) used as a control, in nine Yucatan Minipigs. De novo bone formation, residual bone substitute, as well as local inflammatory and tissue effects were histologically evaluated at 30 and 90 days after implantation. The structural characterization showed that the surface morphology, particle size, chemical composition, and crystalline structure of AEB were similar to cancellous human bone. The histological examination of AEB showed a comparable pattern of newly formed bone and residual biomaterial to that of ABB. Overall, the structural data and pre-clinical evidence reported in the present study suggests that AEB can be effectively used as bone grafting material in oral surgery procedures.

Polyphenolic characterisation and bioactivity of an Oxalis pes-caprae L. leaf extract.

The present work is focused on the characterisation of the polyphenolic content of an Oxalis pes-caprae L. leaf extract and on the evaluation of its bioactivity with particular interest on its vascular activity and antioxidant potential. The polyphenolic content was characterised by HPLC-DAD and LC-MS/MS. The vascular activity was evaluated according to the influence on the serotonergic and adrenergic systems of the human internal mammary artery (HIMA). Antioxidant and neuroprotective studies were also conducted. Several luteolin and apigenin derivatives were identified as main constituents of the extract, which did not present any contractile effect nor had any effect on the serotonergic system of HIMA. However, it showed antagonistic effect on the adrenergic system, inhibiting the contraction to noradrenaline (reduction of 58.44% of maximum contraction). The extract showed antioxidant activity and standardised luteolin and apigenin derivatives showed neuroprotective potential, particularly homoorientin.

Preparation, characterization and biocompatibility studies of thermoresponsive eyedrops based on the combination of nanostructured lipid carriers (NLC) and the polymer Pluronic F-127 for controlled delivery of ibuprofen.

CONTEXT: Nanostructured lipid carrier (NLC) dispersions present low viscosity and poor mucoadhesive properties, which reduce the pre-corneal residence time and consequently, the bioavailability of ocular drugs. OBJECTIVE: The aim of this study was to prepare thermoresponsive eyedrops based on the combination of lipid nanoparticles and a thermoresponsive polymer with mucomimetic properties (Pluronic® F-127). MATERIALS AND METHODS: NLCi dispersions were prepared based on the melt-emulsification and ultrasonication technique. Physicochemical and morphological characteristics of the colloidal dispersions were evaluated. The formulation was also investigated for potential cytotoxicity in Y-79 human retinoblastoma cells and the in vitro drug release profile of the ibuprofen was determined. RESULTS: NLCi showed a Z-average below 200 nm, a highly positive zeta potential and an efficiency of encapsulation (EE) of ∼90%. The gelification of the NLCi dispersion with 15% (w/w) Pluronic® F-127 did not cause significant changes to the physicochemical properties. The potential NLC-induced cytotoxicity was evaluated by the Alamar Blue reduction assay in Y-79 cells, and no relevant cytotoxicity was observed after exposure to 0-100 µg/mL NLC for up to 72 hours. The optimized formulations showed a sustained release of ibuprofen over several hours. DISCUSSION AND CONCLUSION: The strategy proposed in this work can be successfully used to increase the bioavailability and the therapeutic efficacy of conventional eyedrops.

New Thermoresponsive Eyedrop Formulation Containing Ibuprofen Loaded-Nanostructured Lipid Carriers (NLC): Development, Characterization and Biocompatibility Studies.

The low bioavailability and consequently the poor therapeutic response of traditional ophthalmic formulations is caused by reduced pre-corneal residence time of the formulation in contact with the ocular surface. The use of colloidal carrier systems, namely lipid nanoparticles in combination with in situ gelling polymers, is an excellent strategy which results in the exponential increase of the bioavailability of ophthalmic drugs. In the present study, we have developed thermoresponsive eyedrops prepared with nanostructured lipid carriers (NLC) dispersions for the controlled delivery of ibuprofen. Lipid solubility studies and DSC measurements have proved that the lipids solubilise ibuprofen and present a good compatibility. NLC were prepared based on the melt-emulsification and ultrasonication technique and lipid nanoparticles with a Z-average of 120-150 nm, polydispersity index below 0.3, highly positive zeta potential and an efficacy of encapsulation of ~87% were obtained. The cytotoxicity of NLC was evaluated by the Alamar Blue reduction assay using the Y-79 human retinoblastoma cell line, and no relevant toxicity was observed after exposure to 0-100 µg/mL NLC for up to 72 hours. The HET-CAM assay was used to assess the product eye compatibility, confirming that the developed product does not exhibit irritant potential. The in vitro release studies showed ibuprofen release over several hours.

Nanoparticles in Ocular Drug Delivery Systems for Topical Administration: Promises and Challenges.

The majority of pharmaceutical formulations for the treatment of ocular pathologies are for topical administration. However, this kind of ophthalmic formulations has disadvantages such as low bioavailability and, consequently, a reduced therapeutic effect. This happens due to the anatomical and physiological specificity of the eyeball (tissues with different characteristics, the presence of different defense mechanisms, etc.) effects, reducing the residence time of formulation in contact with the ocular surface and consequently fall dramatically the penetration ability of the formulation through the ocular tissues. The repeated administration of this type of ophthalmic formulations with the aim to produce the desired therapeutic effect leads to the appearance of side effects due to its systemic absorption. In order to overcome the weaknesses of this type of therapy is necessary to use different strategies. In this review article, we discuss some of these different strategies, with particular emphasis on the application of colloidal dispersions in ophthalmic formulations, particularly, the use of polymeric and lipid nanoparticles. In fact, the results of the published scientific research has demonstrated that the use of this type of strategy not only promotes the increase in the precorneal residence time of the ophthalmic formulation, but also the ability to penetrate through the ocular tissues, enhancing the drug bioavailability and the therapeutic efficacy of ophthalmic formulations. Finally, it is also given emphasis not only to the current state of the scientific research in this area, but also to the existing patents and the followed procedure to place on the market an ophthalmic formulation based on nanoparticles.

Antioxidant capacity automatic assay based on inline photogenerated radical species from L-glutathione-capped CdTe quantum dots.

This work aimed at the development of a methodology implemented in an automatic flow system for determination of the antioxidant capacity in food samples, based on the luminol oxidation by inline photogenerated radical species from cadmium telluride nanoparticles capped with L-glutathione. Radical species were generated inline by a high-power visible light obtained by Light Emitting Diodes (LEDs) assembled in a multipumping flow system (MPFS). The use of visible light instead of UV radiation allowed the development of a new methodology for antioxidant capacity determination, more environment friendly and to circumvent the risk for UV photo-induced degradation of sample antioxidant compounds. Additionally, the formation of superoxide radical species was theoretically predicted considering the variation of the redox potential with the size of CdTe QDs and the values of redox potential of the oxidizing and oxidable species present in the irradiated medium. The obtained results of trolox equivalent antioxidant capacity (TEAC) from the analysis of commercial beverages were compared with the results of ABTS and DPPH batch assays through Spearman's-Rho correlation coefficients and no correlation was found (for ABTS: ρ=0.2, p<0.6 and for DPPH: ρ=0.5, p<0.1) since the mechanism of action of the proposed methodology was based on the scavenging capacity of ROS species rather than the reduction of a colored oxidant. An analytical linear response range between 0.0001 and 0.005mmol L(-1) of trolox and a limit of detection of 0.00005mmol L(-1) was found. The QDs based MPFS methodology allowed a determination rate of about 79h(-1), a total waste generation of 20.5mL h(-1) and the consumption of 0.100mg h(-1) of QDs and 2.1mg h(-1) of luminol.

Immobilization of Distinctly Capped CdTe Quantum Dots onto Porous Aminated Solid Supports.

Immobilization of quantum dots (QDs) onto solid supports could improve their applicability in the development of sensing platforms and solid-phase reactors by allowing the implementation of reusable surfaces and the execution of repetitive procedures. As the reactivity of QDs relies mostly on their surface chemistry, immobilization could also limit the disruption of solution stability that could prevent stable measurements. Herein, distinct strategies to immobilize QDs onto porous aminated supports, such as physical adsorption and the establishment of chemical linking, were evaluated. This work explores the influence of QD capping and size, concentration, pH, and contact time between the support and the QDs. Maximum QD retention was obtained for physical adsorption assays. Freundlich and Langmuir isotherms were used to analyze the equilibrium data. Gibbs free energy, enthalpy, and entropy were calculated and the stability of immobilized QDs was confirmed.

Enhancing reactive species generation upon photo-activation of CdTe quantum dots for the chemiluminometric determination of unreacted reagent in UV/S2O8(2-) drug degradation process.

A new chemiluminescence (CL) flow method for persulfate determination was developed based on luminol oxidation by in-line generated radicals. Reactive oxygen species (ROS) generated by CdTe quantum dots (QDs) under a low energetic radiation (visible light emitted by LEDs) promoted the decomposition of persulfate ion (S2O8(2-)) into sulfate radical (SO4(∙-)), leading to subsequent radical chain reactions that yield the emission of light. Due to the inherent radical short lifetimes and the transient behavior of CL phenomena an automated multi-pumping flow system (MPFS) was proposed to improve sample manipulation and reaction zone implementation ensuring reproducible analysis time and high sampling rate. The developed approach allowed up to 60 determinations per hour and determine S2O8(2-) concentrations between 0.1 and 1 mmol with good linearity (R=0.9999). The method has shown good repeatability with relative standard deviations below 2.5% (n=3) for different persulfate concentrations (0.1 and 0.625 mmol L(-1)). Limits of detection (3σ) and quantification (10σ) were 2.7 and 9.1 µmol L(-1), respectively. The MPFS system was applied to persulfate determination in bench scale UV/S2O8(2-) drug degradation processes of model samples showing good versatility and providing real time information on the persulfate consumption in photo-chemical degradation methodologies.

A soft strategy for covalent immobilization of glutathione and cysteine capped quantum dots onto amino functionalized surfaces.

A novel strategy for immobilization of CdTe quantum dots (QDs) onto amino functionalized solid supports was developed. QDs capped with compounds holding an amino group were covalently bonded to the substrate under mild reaction conditions, exhibiting great stability and strong luminescence.

Evaluation of acetylcysteine promoting effect on CdTe nanocrystals photoluminescence by using a multipumping flow system.

A simple and straightforward quantification method integrated in a fully automated multi-pumping flow system (MPFS) using water-soluble mercaptopropionic acid (MPA)-capped CdTe quantum dots (QDs) was implemented for the fluorescence quantification of N-acetyl-L-cysteine (NAC) in pharmaceutical formulations. The developed approach was based on NAC ability to establish surface interactions that result in enhanced nanocrystals fluorescence intensity, proportional to analyte concentration. Size and concentration of QDs, ageing, composition, concentration and pH of the buffer solution revealed to have a noticeable effect on the enhancing efficiency affecting sensitivity and linear working range of the methodology. Under the optimal conditions, a linear working range was obtained for NAC concentrations ranging from 50 to 750µmolL(-1) (r=0.9978), with good precision (r.s.d.<1.6%; n=5) and a sampling rate of about 75hr(-1). The detection limit (LOD) was approximately 1.6µmolL(-1). The method was applied to pharmaceutical preparations and the results revealed good agreement with those obtained by the reference procedure with relative deviations between -2.1 and +4.2%. Advantages of the new procedure include speed, low consumption of reagents, minor waste generation, requiring also much less work than the recommended HPLC method. The mechanism for luminescence enhancement of CdTe QDs is discussed. FT-IR spectra revealed that sulphydryl groups of NAC have a high affinity with the nanocrystals.

Application of quantum dots as analytical tools in automated chemical analysis: a review.

Colloidal semiconductor nanocrystals or quantum dots (QDs) are one of the most relevant developments in the fast-growing world of nanotechnology. Initially proposed as luminescent biological labels, they are finding new important fields of application in analytical chemistry, where their photoluminescent properties have been exploited in environmental monitoring, pharmaceutical and clinical analysis and food quality control. Despite the enormous variety of applications that have been developed, the automation of QDs-based analytical methodologies by resorting to automation tools such as continuous flow analysis and related techniques, which would allow to take advantage of particular features of the nanocrystals such as the versatile surface chemistry and ligand binding ability, the aptitude to generate reactive species, the possibility of encapsulation in different materials while retaining native luminescence providing the means for the implementation of renewable chemosensors or even the utilisation of more drastic and even stability impairing reaction conditions, is hitherto very limited. In this review, we provide insights into the analytical potential of quantum dots focusing on prospects of their utilisation in automated flow-based and flow-related approaches and the future outlook of QDs applications in chemical analysis.

Photoactivation by visible light of CdTe quantum dots for inline generation of reactive oxygen species in an automated multipumping flow system.

Quantum dots (QD) are semiconductor nanocrystals able to generate free radical species upon exposure to an electromagnetic radiation, usually in the ultraviolet wavelength range. In this work, CdTe QD were used as highly reactive oxygen species (ROS) generators for the control of pharmaceutical formulations containing epinephrine. The developed approach was based on the chemiluminometric monitoring of the quenching effect of epinephrine on the oxidation of luminol by the produced ROS. Due to the relatively low energy band-gap of this chalcogenide a high power visible light emitting diode (LED) lamp was used as photoirradiation element and assembled in a laboratory-made photocatalytic unit. Owing to the very short lifetime of ROS and to ensure both reproducible generation and time-controlled reaction implementation and development, all reactional processes were implemented inline by using an automated multipumping micro-flow system. A linear working range for epinephrine concentration of up to 2.28×10(-6) mol L(-1) (r=0.9953; n=5) was verified. The determination rate was about 79 determinations per hour and the detection limit was about 8.69×10(-8) mol L(-1). The results obtained in the analysis of epinephrine pharmaceutical formulations by using the proposed methodology were in good agreement with those furnished by the reference procedure, with relative deviations lower than 4.80%.

Cadmium telluride nanocrystals as luminescent sensitizers in flow analysis.

A fully automated multipumping flow system (MPFS) using water-soluble CdTe quantum dots (QD) as sensitizers is proposed for the chemiluminometric determination of the anti-diabetic drugs gliclazide and glipizide in pharmaceutical formulations. The nanocrystals acted as enhancers of the weak CL emission produced upon oxidation of sulphite by Ce(IV) in acidic medium, thus improving sensitivity and expanding the dynamical analytical concentration range. By interacting with the QD, the two analytes prevented their sensitizing effect yielding a chemiluminescence quenching of the Ce(IV)-SO(3)(2-)CdTe QD system. The pulsed flow inherent to MPFS assured a fast and efficient mixing of all solutions inside the flow cell, circumventing the need for a reaction coil and facilitating the monitoring of the short-lived generated chemiluminescent species. QD crystal size, concentration and spectral region for measurement were investigated.

Quantum dots assisted photocatalysis for the chemiluminometric determination of chemical oxygen demand using a single interface flow system.

A novel flow method for the determination of chemical oxygen demand (COD) is proposed in this work. It relies on the combination of a fully automated single interface flow system, an on-line UV photocatalytic unit and quantum dot (QD) nanotechnology. The developed approach takes advantage of CdTe nanocrystals capacity to generate strong oxidizing species upon irradiation with UV light, which fostered a fast catalytic degradation of the organic compounds. Luminol was used as a chemiluminescence (CL) probe for indirect COD assessment, since it is easily oxidized by the QD generated species yielding a strong CL emission that is quenched in the presence of the organic matter. The proposed methodology allowed the determination of COD concentrations between 1 and 35 mg L(-1), with good precision (R.S.D.<1.1%, n=3) and a sampling frequency of about 33 h(-1). The procedure was applied to the determination of COD in wastewater certified reference materials and the obtained results showed an excellent agreement with the certified values.

Allergen-specific T-cell tolerance induction with allergen-derived long synthetic peptides: results of a phase I trial.

BACKGROUND: There is a need to improve the safety and efficacy of allergen-specific immunotherapy. Long synthetic peptide-based immunotherapy was proven safe, immunogenic, and protective in preclinical trials. OBJECTIVE: To evaluate the safety and immunogenicity of an allergen-derived long synthetic overlapping peptide (LSP) immunotherapy, we designed a double-blind, placebo-controlled phase I clinical trial in patients hypersensitive to bee venom. METHODS: Patients from the active group were injected at day 0 with a mixture of 3 LSPs mapping the entire PLA2 molecule, a major bee venom allergen, in a dose-escalating protocol to a maintenance dose of 100 microg per peptide repeated at days 4, 7, 14, 42, and 70. The control group was injected with human albumin. RESULTS: Whereas specific T-cell proliferation in the peptide group increased up to day 14, a sharp decline was observed thereafter, ending in specific T-cell hyporesponsiveness at day 80. Serum-specific IgG4 response was enhanced, in contrast to anti-PLA2 IgE. Specific T-cell cytokine modulation was marked by increased IL-10 and IFN-gamma secretion. LSP injections were well tolerated in all patients except for mild, late allergic reactions in 2 patients at day 70. CONCLUSIONS: The results of this short-term study demonstrate that LSP-based allergen immunotherapy was safe and able to induce T(H)1-type immune deviation, allergen-specific IL-10 production, and T-cell hyporesponsiveness. LSPs, which offer the advantage of covering all possible T-cell epitopes for any HLA genotype, can be considered candidates for a novel and safe approach of specific immunotherapy.