Nanofiber-coated implants: Development and safety after intravitreal application in rabbits.

Intravitreal injections are the preferred choice for drug administration to the posterior segment of the eye. However, the required frequent injections may cause complications to the patient and low adherence to the treatment. Intravitreal implants are able to maintain therapeutic levels for a long period. Biodegradable nanofibers can modulate drug release and allow the incorporation of fragile bioactive drugs. Age-related macular degeneration is one of the world major causes of blindness and irreversible vision loss. It involves the interaction between VEGF and inflammatory cells. In this work we developed nanofiber-coated intravitreal implants containing dexamethasone and bevacizumab for simultaneously delivery of these drugs. The implant was successfully prepared and the efficiency of the coating process was confirmed by scanning electron microscopy. Around 68% of dexamethasone was released in 35 days and 88% of bevacizumab in 48hs. The formulation presented activity in the reduction of vessels and was safe to the retina. It was not observed any clinical or histopathological change, neither alteration in retina function or thickness by electroretinogram and optical coherence tomography during 28 days. The nanofiber-coated implants of dexamethasone and bevacizumab may be considered as a new delivery system that can be effective for the treatment of AMD.

Production and Evaluation of Chicken Egg Yolk Immunoglobulin (IgY) against Human and Simian Rotaviruses.

Producing specific antibodies in chickens is an attractive approach for diagnosis or therapeutic applications. Besides the high immunoglobulin Y (IgY) yield transferred to the egg yolk and its suitability for large-scale production, such an approach is more bioethical for animal maintenance. The IgY technology offers new possibilities for application in human and veterinary diagnostics and therapeutics, including strategies for treating severe intestinal diseases in children, particularly in emerging countries. Herein, we describe the production and purification of polyclonal antibodies against rotavirus group A (RVA) in immunised hens aiming at its application in prophylaxis and treatment of rotavirus-induced diarrhoea. For this purpose, we inoculated Rhodia laying chickens (Gallus gallus domesticus) with two or three doses of RVA combined with adjuvants or only adjuvants (control group). As the egg-laying period began, the yolk protein purification processes yielded a high concentration of specific IgY, the highest titre resulting from the group of hens that received three doses of the immunogen. The purified IgY blocked the functional activity of RVA in MA-104 cells, thus confirming the neutralisation ability. Therefore, anti-RVA IgY could be a promising candidate for pre- and post-exposure prevention or treatment of rotavirus-induced diarrhoea.

Production and purification of Clostridium perfringens type D epsilon toxin and IgY antitoxin.

The aim of this study was to purify Clostridium perfringens type D epsilon toxin and produce and purify anti-epsilon chicken immunoglobulin Y (IgY). A single-step ion exchange chromatography resulted in a high-yield and high-purity toxin, while ion exchange chromatography followed by gel filtration resulted in the highest purity of the toxin, but at a lower yield. Purified and inactivated epsilon toxin were then administered in chickens via four inoculations and IgY was obtained at a high purity and yield, with an antibody titer of 50 IU/mL and high levels of avidity (73.2%). In summary, C. perfringens type D epsilon toxin and chicken anti-epsilon IgY were successfully produced and purified, and may be used for the diagnosis of enterotoxemia caused by the epsilon toxin, as well as in potency tests of existing and future vaccines against enterotoxemia.

Non-Enzymatic Impedimetric Sensor Based on 3-Aminophenylboronic Acid Functionalized Screen-Printed Carbon Electrode for Highly Sensitive Glucose Detection.

A highly sensitive glucose sensor was prepared by a one-step method using 3-aminophenyl boronic acid as a unit of recognition and a screen-printed carbon electrode (SPCE) as an electrochemical transducer. Scanning Electron Microscopy confirmed the success of the functionalization of the SPCE due to the presence of clusters of boronic acid distributed on the carbon surface. In agreement with the Electrochemical Impedance Spectroscopy (EIS) tests performed before and after the functionalization, Cyclic Voltammetry results indicated that the electroactivity of the electrode decreased 37.9% owing to the presence of the poly phenylboronic acid on the electrode surface. EIS revealed that the sensor was capable to selectively detect glucose at a broad range of concentrations (limit of detection of 8.53 × 10-9 M), not recognizing fructose and sucrose. The device presented a stable impedimetric response when immediately prepared but suffered the influence of the storage time and some interfering species (dopamine, NaCl and animal serum). The response time at optimized conditions was estimated to be equal to 4.0 ± 0.6 s.

Biodegradable core-shell electrospun nanofibers containing bevacizumab to treat age-related macular degeneration.

Age-related macular degeneration (AMD) is a degenerative ocular disease that affects the central retina. It is considered the main cause of blindness and loss of vision worldwide. Angiogenic factors are associated with AMD, which has led to the use of antiangiogenic drugs, such as bevacizumab, to treat the disease using frequent intravitreal injections. In the present study, biodegradable core shell nanofibers containing bevacizumab were prepared by the coaxial electrospinning technique. It is thought that the shell could control the release of the drug, while the core would protect and store the drug. Poly(caprolactone) (PCL) and gelatin were used to form the shell of the nanofibers, while poly(vinyl alcohol) (PVA) and bevacizumab comprised the core. The nanofibers were characterized using microscopy techniques, thermal analysis, and FTIR. The results showed that core-shell nanofibers were produced as designed. Bevacizumab activity was evaluated using a chicken embryo chorioallantoic membrane (CAM) assay. An enzyme-linked immunosorbent assay was used to quantify the amount of the drug released from the different nanofibers in vitro. The toxicity of the nanofibers was evaluated in human retinal pigment epithelial (ARPE) cells. The CAM results demonstrated that bevacizumab maintained its antiangiogenic activity when incorporated into the nanofibers. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tests revealed that the nanofibers showed no cellular toxicity, even in the presence of bevacizumab. The core-shell structure of the nanofibers reduced the release rate of bevacizumab compared with PVA nanofibers. The bevacizumab-loaded biodegradable nanofibers presented interesting properties that would potentially constitute an alternative therapy to intravitreal injections to treat AMD.

Antibacterial activity of different types of snake venom from the Viperidae family against Staphylococcus aureus / Atividade antibacteriana de diferentes tipos de veneno de serpentes da família Viperidae contra Staphylococcus aureus

Toxins and venoms produced by living organisms have exhibited a variety of biological activities against microorganisms. In this study, we tested seven snake venoms from the family Viperidae for antibacterial activity and the activities of reversal of antibiotic resistance and inhibition of biofilm formation against 22 clinical isolates of Staphylococcus aureus. Bothrops moojeni venom exhibited anti staphylococcal activity with the lowest mean value of minimum inhibitory concentration (MIC). Moreover, reversal of antibiotic resistance was observed for combinations of B. moojeni venom (½ x MIC) and norfloxacin or ampicillin (both ½ x MIC) for 86.4% and 50% of the isolates, respectively. B. moojeni venom alone at ½ MIC inhibited 90% of biofilm formation, whereas in combination with ciprofloxacin, both at ½ MIC, a reduction on the NorA efflux pump activity was observed. The detection of in vitro mutants colonies of S. aureus resistant to B. moojeni venom was low and they did not survive. A phospholipase A2 was purified from the venom of B. moojeni and displayed anti-staphylococcal activity when tested alone or in combination with ciprofloxacin. The results presented here will contribute to the search for new antimicrobial agents against resistant S. aureus.

Toxinas e venenos exibem uma variedade de atividades biológicas contra micro-organismos. Neste estudo, investigou-se a atividade de sete venenos de serpentes, da família Viperidae, sobre o crescimento de Staphylococcus aureus, na reversão fenotípica da resistência a antibióticos e inibição de formação de biofilme contra 22 isolados clínicos de S. aureus. O veneno de Bothrops moojeni apresentou a menor média de concentração inibitória mínima (CIM). Além disso, observou-se reversão da resistência a antibióticos para combinações do veneno de B. moojeni (½ x CIM) e norfloxacina ou ampicilina (ambos ½ x CIM) para 86,4% e 50% dos isolados, respectivamente. O veneno de B. moojeni na concentração de ½ CIM inibiu 90% de formação de biofilme, enquanto ele em combinação com ciprofloxacina, ambos na concentração de ½ CIM, diminuiu a atividade da bomba de efluxo NorA. A detecção in vitro de colônias mutantes de S. aureus resistente ao veneno de B. moojeni foi baixa e eles não sobreviveram. Uma fosfolipase A2 purificada a partir do veneno de B. moojeni exibiu atividade antibacteriana quando testada sozinha ou em combinação com ciprofloxacina. Os dados obtidos poderão contribuir para a pesquisa de novos agentes antimicrobianos contra S. aureus.

HPLC-UV method validation for the identification and quantification of bioactive amines in commercial eggs.

A quantitative and confirmatory high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method for the determination of bioactive amines in the albumen and yolk of commercial eggs was developed, optimized and validated by analyte extraction with trichloroacetic acid and pre-column derivatization with dansyl chloride. Phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine standards were used to evaluate the following performance parameters: limit of detection (LoD), limit of quantification (LoQ), selectivity, linearity, precision, recovery and ruggedness. The LoD of the method was defined from 0.2 to 0.3 mg kg(-1) for the yolk matrix and from 0.2 to 0.4 mg kg(-1) for the albumen matrix; the LoQ was from 0.7 to 1.0 mg kg(-1) for the yolk matrix and from 0.7 to 1.1 mg kg(-1) for the albumen matrix. The validated method exhibited excellent selectivity and separation of all amines with coefficients of determination higher than 0.99. The obtained recovery values were from 90.5% to 108.3%, and the relative standard deviation (RSD) was lower than 10% under repeatability conditions for the studied analytes. The performance parameters show the validated method to be adequate for the determination of bioactive amines in egg albumen and yolk.

Development of an amperometric immunosensor for detection of staphylococcal enterotoxin type A in cheese.

This paper reports a novel electrochemical immunosensor for the sensitive detection of staphylococcal enterotoxin A (SEA) based on self-assembly monolayer (SAM) and protein A immobilization on gold electrode. Three different methods of protein A immobilization were tested: physical adsorption, cross-linking using glutaraldehyde and covalent binding after activation with N-hydroxysuccinimide (NHS)/N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) on cysteamine-modified gold electrode. The EDC/NHS method for protein A immobilization was selected to lead development of the biosensor. The coating steps of the surface modification were characterized by cyclic voltammetry and the biosensor response by chronoamperometry. The advantages of the immunosensor were exposed in its high sensitivity and specificity. The proposed amperometric immunosensor was successfully used for determination of SEA in contaminated and non-contaminated cheese samples with excellent responses.