Imprinted hydrogels with LbL coating for dual drug release from soft contact lenses materials.

A combined strategy to control the release of two drugs, one anti-inflammatory (diclofenac sodium, DCF) and one antibiotic (moxifloxacin hydrochloride, MXF), from a soft contact lens (SCL) material, was assessed. The material was a silicone-based hydrogel, which was modified by molecular imprinting with MXF and coated by the layer-by-layer (LbL) method using natural polyelectrolytes: alginate (ALG), poly-l-lysine (PLL) and hyaluronate (HA), crosslinked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Imprinting was used to increase the amount of MXF loaded and to sustain its release, while the LbL coating acted as a diffusion barrier for DCF and improved the surface properties. The drugs were loaded by soaking in a DCF + MXF dual solution. High hydrostatic pressure (HHP) was successfully applied in the sterilization of the drug-loaded hydrogels. The transmittance, refractive index, wettability and ionic permeability of the hydrogels remained within the required levels for SCLs application. The concentrations of the released DCF and MXF stayed above the IC50 and the MIC (for S. aureus and S. epidermidis) values, for 9 and 10 days, respectively. No ocular irritancy was detected by the HET-CAM test. NIH/3T3 cell viability demonstrated that the drug-loaded hydrogels were not toxic, and cell adhesion was reduced.

Moxifloxacin-imprinted silicone-based hydrogels as contact lens materials for extended drug release.

Contact lenses may act as drug release platforms for the treatment of ocular infections, but there is still the need for extending their typical release periods and enhancing ocular bioavailability. The present study aimed to develop a molecularly imprinted silicone-based hydrogel to be used in the manufacturing of contact lenses that can be loaded efficiently and be able to release the antibiotic moxifloxacin hydrochloride (MXF) in a sustained way. A set of hydrogels was prepared by the molecular imprinting method using acrylic acid (AA) as the functional monomer for the specific recognition of MXF. The modified hydrogels loaded a higher amount of MXF, which was released for a longer time. In vitro experiments, using a microfluidic cell to mimic the ocular surface fluid turnover, showed that the imprinted hydrogel TRIS(300)-I prepared with the highest content in AA led to MXF concentrations in the release medium which were effective against S. aureus and S. epidermidis for about 2 weeks. Furthermore, some important properties such as water uptake, wettability, transmittance, ionic permeability, and Young´s modulus of the modified hydrogel remained within the range of values recommended for contact lenses. No cytotoxicity and no potential ocular irritancy effect were detected. Such hydrogel seems to be a promising alternative to the current options for the treatment of ocular infections.

Diclofenac sustained release from sterilised soft contact lens materials using an optimised layer-by-layer coating.

A layer-by-layer (LbL) coating was designed using ionic polysaccharides (chitosan, sodium alginate, sodium hyaluronate) and genipin (crosslinker), to sustain the release of diclofenac sodium salt (DCF) from soft contact lens (SCL) materials. The coating was hydrophilic, biocompatible, non-toxic, reduced bacterial growth and had minor effects on the physical properties of the material, such as wettability, ionic permeability, refractive index and transmittance, which remained within the recommended values for SCLs. The coating was applied on a silicone-based hydrogel and on commercial SofLens and Purevision SCLs. The coating attenuated the initial drug burst and extended the therapeutic period for, at least, two weeks. Relevantly, the problems of sterilizing drug loaded SCLs coated with biopolymers, using classic methods that involve high temperature or radiation, were successfully solved through high hydrostatic pressure (HHP) sterilization.

Effects of Low-Moderate Load High-Velocity Resistance Training on Physical Performance of Under-20 Futsal Players.

Resistance training (RT) is an effective methodology to improve physical performance of athletes. However, up to now, no studies have addressed the RT benefits in under-20 futsal players. The purpose of this study was to evaluate the effects of six weeks of RT with high-velocity movements, low-to-moderate loads, and low volume on physical performance of under-20 futsal players. A total of 21 players were divided into two groups: A control group (CG, n = 10) and a RT group (RTG, n = 11). The RTG performed two weekly training sessions constituted by leg-press, jumps, and sprints, along with three futsal training sessions, while the CG only performed the futsal training. Before and after the intervention, the sprint time in 0⁻10 m (T10), 10⁻20 m (T10⁻20), and 0⁻20 m (T20), the countermovement jump (CMJ) height, the T-Test time, the kicking ball speed (KBS), and the maximum dynamic strength in the leg-press, were assessed. In post-test, significant improvements in CMJ, T-Test, KBS, and leg-press were found for the RTG, whilst a significant decrease in T10⁻20 was evidenced in the CG. The present results suggested that RT based on high velocity movements, low-to-moderate loads, and low volume produce positive effects on physical performance of under-20 futsal players.

Antibacterial layer-by-layer coatings to control drug release from soft contact lenses material.

In this study we investigated the possibility of using polyelectrolytes with antibacterial properties to form layer-by-layer (LbL) coatings on contact lens materials with the objective of controlling the release of ophthalmic drugs, while minimizing bacterial growth. A silicone-based hydrogel recently proposed by our group was chosen as a drug releasing soft contact lens (SCL) material and three drugs were tested: moxifloxacin hydrochloride (MXF), chlorhexidine diacetate monohydrate (CHX), and diclofenac sodium salt (DIC). Employed coatings involved combinations of sodium alginate (ALG), chitosan (CHI), sodium hyaluronate (HA) and polylysine hydrobromide (PLL), using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) as a cross-linking agent. Controlled release of DIC was achieved using two double layers of the following combinations: ALG/PLL (EDC), HA (EDC)/CHI and HA/PLL (EDC) + Drug. Furthermore, the physical properties of the coated lens material were kept, or even improved, and bacterial growth was reduced. In contrast, these coatings did not retard the release of MXF and CHX. The specificity of the barrier effect of these LbL films for DIC may be attributed to the formation of reversible interactions between DIC and the polyelectrolyte chains. A top layer of HA was needed to reduce the interaction with tear proteins. The in vivo efficacy of a contact lens coated with ALG/PLL (EDC)//HA and loaded with DIC was predicted using a simplified mathematical model to estimate the drug concentration in the tear fluid. The period of time during which the estimated concentration of DIC remained above the half-maximum inhibitory concentrations for the active enzymes in the process of inflammation, was at least double that obtained with the uncoated sample.

In situ forming chitosan hydrogels: Preliminary evaluation of the in vivo inflammatory response.

This study aims to provide important preliminary in vivo data on the general biocompatibility and degradation of in situ forming chitosan hydrogels which differ in the type of cross-linking: physical cross-linking (with glycerol phosphate disodium salt) and physico/chemical co-cross-linking (with glycerol phosphate disodium salt and genipin). Additionally, in vitro degradation studies, using lysozyme, were conducted for comparison. The subcutaneous implantation of the liquid formulations of both hydrogels in animal model (Wistar rats) was performed to assess tissue response at the end of two time periods (8 and 30days). Although the properties of both hydrogels are different (namely, pore size and mechanical strength), leading to remarkably distinct in vitro degradation behaviors, the in vivo degradation is quite similar for both types. Regarding biocompatibility, the in vivo evaluation performed by histological analysis, showed a mild to moderate chronic inflammatory reaction with no foreign body reaction, demonstrating that both hydrogels show good results for in vivo biodegradation and biocompatibility, therefore are most adequate for biomedical applications.

Influence of albumin on mineralization of PMMA-based/glass composites.

PURPOSE: The formation of a calcium phosphate layer on the surface of bone tissue engineering biomaterials is crucial for their integration in bone. Simulated biological fluids used to study the in vitro formation of those layers do not usually contain important organic components present in vivo-notably proteins. In this work the influence of bovine serum albumin on the mineralization process of poly(methylmethacrylate)-based composite was studied in vitro. METHODS: The effect of protein on calcium phosphate formation was followed by ion concentration analyses (ICP), x-ray diffraction (XRD), and scanning electron microscopy coupled with x-ray energy dispersive spectroscopy (SEM-EDS). RESULTS AND CONCLUSIONS: The results showed the precipitation of a calcium phosphate layer on the surface of composites immersed in SBF and SBFA. Further transformation and crystallization of this layer, initially amorphous, appears to be influenced by the presence of albumin.

Cotton gauze bandage: a support for protease immobilization for use in biomedical applications

The covalent immobilization of trypsin on sterilized cotton gauze bandage was carried out for future use as an anti-inflammatory agent on wound dressing. The optimal immobilization conditions were determined: the influence of pH, concentration, and volume of the trypsin solution used in the immobilization procedure was studied. The catalytic properties and kinetic parameters, as well as the storage stability of the immobilized and free enzyme were compared. Results showed that the optimal pH and temperature for the immobilized trypsin in the hydrolysis of N-benzoyl-DL-arginine p-nitroanilide were 9.5 and 55 ºC, which were higher than those of the free form (7.5 and 45 ºC). It was found, at 37 ºC and pH 7.0 (approximately, the physiological conditions), that the Km (Michaelis constant) was 3.98 µmol/mL and that Vmax (maximal reaction rate) was 0.719 µmol/(min mg) for the immobilized trypsin, compared to a Km of 2.46 mmol/mL and a Vmax of 2.89 µmol/(min mg) for the free trypsin. The amount of immobilized trypsin was 6 mg/g dried support. After 30 days, no trypsin had liberated from the support. The superior performance of the immobilized trypsin demonstrates its potential application in practice.

Neste trabalho foi efetuada a imobilização de tripsina numa gaze esterilizada de algodão. Foram determinadas as condições ótimas de imobilização: foi estudada a influência do pH, concentração e volume da solução de tripsina usada na imobilização na hidrólise da N-benzoil-DL-arginina p-nitroanilida. As propriedades catalíticas, os parâmetros cinéticos e as condições de estabilidade das enzimas livre e imobilizada foram comparadas. Os resultados mostraram que o pH e a temperatura ótimos para a tripsina imobilizada foram 9.5 e 55 ºC, respectivamente, maiores que os correspondentes da forma livre (7,5 e 45 ºC). A 37 ºC e a pH 7,0 (aproximadamente as condições fisiológicas) Km (constante de Michaelis) foi 3,98 µmol/mL e Vmax (velocidade máxima de reacção) foi 0,719 µmol/(min mg) para a tripsina imobilizada; para a tripsina livre os valores correspondentes foram 2,46 µmol/mL e 2,89 µmol/(min mg). A quantidade de enzima imobilizada foi de 6 mg/g (base seca). Após 30 dias não se verificou libertação de tripsina do suporte. O bom desempenho da tripsina imobilizada na gaze esterilizada de algodão comprova a sua potencial utilização como agente anti-inflamatório no tratamento de lesões cutâneas.

Immobilization of drugs for glaucoma treatment.

Recently there have been some developments in the preparation of controlled drug delivery systems for glaucoma. Many materials are being used in this area, namely gelatine and chitosan. Both of them present high levels of biocompatibility and biodegradability. In this paper, we wish to report the work we have been doing on the preparation and characterization of hydrogels based on gelatine and chitosan. The crosslinking agents used were 1-(3-(Dimethylamino)propyl)-3-Ethylcarbodiimide hydrocholide (CDI), 1,4-Butanodiol diglycidyl ether (epoxyde 1), Ethylene glycol diglycidyl ether (epoxyde 2) and genipin. The results obtained showed that all of the films were hydrogels. The surface and transversal cut showed a porous surface in all the films. The thermal analysis proved the modifications in the polymeric chains, with the stabilization of all of them by the crosslinking agents. The release pattern indicates that the gelatine films were the best since they release the adequate proportion of drug. Finally, the cytotoxicity showed that the gelatine films were all biocompatible, specially the ones crosslinked with one of the Epoxydes.

Synthesis and characterization of new methacrylate based hydrogels

Hydrogels have been used for several applications, including production of contact lenses, sanitary products and materials for wound dressing. The aim of this work was the development of new methacrylate based hydrogels. These materials present the advantage of being easily produced with different compositions and consequently different permeability and diffusion patterns. Therefore it becomes possible to synthesize hydrogels that can be used to immobilize a variety of compounds, such as drugs, proteins or even cells. During this work new polymers based on methacrylate monomers were prepared. Their characterization was accomplished by several techniques, e.g. Fourier Transform Infrared Spectroscopy (FTIR), swelling measurement, Differential Scanning Calorimetry (DSC) and evaluation of mechanical properties. Their subacute subcutaneous toxicity was also evaluated by using Wistar rats.

Hidrogéis têm sido utilizados para diversas aplicações, incluindo produção de lentes de contato, produtos sanitários e materiais para encerramento de ferimentos. O objetivo deste trabalho consistiu no desenvolvimento de novos hidrogéis a base de metacrilato. Estes materiais apresentam a vantagem de serem facilmente produzidos com diferentes composições e conseqüentemente com distintas permeabilidades e padrões de difusão. Por este motivo, torna-se possível sintetizar hidrogéis que possam ser usados para imobilizar uma grande variedade de compostos, tais como fármacos, proteínas ou mesmo células e tecidos. Neste trabalho foram preparados diferentes polímeros baseados em monômeros de metacrilato. A sua caracterização foi realizada através de diferentes técnicas, como Espectroscopia no Infravermelho com Transformada de Fourier (FTIR), avaliação da capacidade de intumescimento, Calorimetria Diferencial de Varredura (DSC) e avaliação das propriedades mecânicas. A sua toxicidade subaguda subcutânea foi também determinada utilizando ratos Wistar.

Exquisite regioselectivity and increased transesterification activity of an immobilized Bacillus subtilis protease.

Commercially available proteases and lipases were screened for their ability to acylate regioselectively sucrose with divinyladipate either in pyridine or dimethylformamide (DMF). The protease (EC 3.4.21.62) from Bacillus subtilis (Proleather FG-F) exhibited the highest conversion (100% in 24 h of reaction in DMF) yielding sucrose 2-O-vinyladipate as main product. The enzyme preference for a secondary hydroxyl group is a distinct feature of this biocatalyst compared to others described in the literature. Two sets of chemically distinct silica supports were used for Proleather immobilization presenting terminal amino (S(APTES)) or hydroxyl groups (S(TESPM)(-)(pHEMA)). The percentage of immobilized enzyme was smaller in S(APTES) (7-17%) than in S(TESPM)(-)(pHEMA) (52-56%), yet Proleather immobilized into S(APTES) supports presented higher total and specific hydrolytic activity. The highest total and specific activities were obtained with S(TESPM)(-)(pHEMA) and S(APTES), respectively. Silicas with large pore (bimodal distribution of pores, 130/1200 A, denoted as S(1000)) presented higher specific activities relative to those with smaller pore sizes. Furthermore, the synthetic specific activity of S(1000)S(APTES) immobilized protease was ca. 10-fold higher than that of the free enzyme. In addition to sucrose, the immobilized protease was used to acylate methyl alpha-D-glucopyranoside, trehalose, and maltose in nearly anhydrous DMF. Finally, immobilized Proleather was reasonably stable, retaining ca. 55% activity after six reaction cycles.