Ronaldo N.M. Pitombo

Effects of polyethylene glycol attachment on physicochemical and biological stability of E. coli l-asparaginase

L-asparaginase obtained from E. coli strains is an important enzyme widely used in leukemia treatment. However, hypersensitivity reactions must be considered a relevant adverse effect of asparaginase therapy. One approach to reduce the hypersensitivity reactions caused by this enzyme is to change its physicochemical and biological properties by means of polyethylene glycol (PEG) conjugation, resulting in a less immunogenic enzyme with much longer half-time of plasmatic life. This work investigated the factors that could interfere in PEG-enzymes stability. The complexation did not affect the range of pH activity and stability was improved in acid medium remaining stable during 1 h at pH 3.5. The PEG-enzyme exhibited activity restoration capacity (32% after 60 min) when subjected to temperatures of 65 degrees C in physiological solution. The PEG-enzyme in vitro assays showed a very high stability in a human serum sample, keeping its activity practically unchanged during 40 min (strength to non-specific antibodies or proteases in serum). An increase of PEG-enzyme catalytic activity during the lyophilization was observed. The process of modification of L-asparaginase with PEG improved both physicochemical and biological stability.

Nuclear magnetic resonance and water activity in measuring the water mobility in Pintado (Pseudoplatystoma corruscans) fish

Abstract In this study a pulse nuclear magnetic resonance 1 H NMR technique was used to determine the relaxation time ( T 2 ) from Carr–Purcell–Meiboom–Gill (CPMG) experiments on fillets of Pintado (Pseudoplatystoma corruscans) at −70 to 60 °C and in the freeze-dried fillets, reconstituted to a moisture content of 1–32%, at 10, 25 and 40 °C. Analysis of data obtained from CPMG experiments using the MARAN WinDXP software resulted in T 2 spectra. Profiles of water fractions with different mobility have been obtained, and glass transition values have been determined. These profiles were affected by environment conditions, such as temperature, moisture and water activity. We compared the water activity method with NMR technique in measuring water molecular mobility. The NMR technique proved that it is an alternative tool to better understand water behavior in complex biological systems.

Care during freeze-drying of bovine pericardium tissue to be used as a biomaterial: A comparative study

Bovine pericardium (BP) tissue is widely used in the manufacture of bioprosthetics. The effects of freeze-drying on the BP tissue have been studied by some researchers in order to decrease their cytotoxicity due to preservation in formaldehyde solution, and to increase the lifetime of the product in storage. This study was undertaken in order to study the effect of freeze-drying in the structure of BP. To perform this study BP samples were freeze-dried in two different types of freeze-dryers available in our laboratory: a laboratory freeze-dryer, in which it was not possible to control parameters and a pilot freeze-dryer, wherein all parameters during freezing and drying were controlled. After freeze-drying processes, samples were analyzed by SEM, Raman spectroscopy, tensile strength, water uptake tests and TEM. In summary, it has been demonstrated that damages occur in collagen fibers by the loss of bulk water of collagen structure implicating in a drastic decreasing of BP mechanical properties due to its structural alterations. Moreover, it was proven that the collagen fibrils suffered breakage at some points, which can be attributed to the uncontrolled parameters during drying.

Natural and prosthetic heart valve calcification: morphology and chemical composition characterization.

Calcification is the most common cause of damage and subsequent failure of heart valves. Although it is a common phenomenon, little is known about it, and less about the inorganic phase obtained from this type of calcification. This article describes the scanning electron microscopy (SEM)/energy dispersive X-ray spectroscopy and Ca K-edge X-ray absorption near edge structure (XANES) characterization performed in natural and bioprosthetic heart valves calcified in vivo (in comparison to in vitro-calcified valves). SEM micrographs indicated the presence of deposits of similar morphology, and XANES results indicate, at a molecular level, that the calcification mechanism of both types of valves are probably similar, resulting in formation of poorly crystalline hydroxyapatite deposits, with Ca/P ratios that increase with time, depending on the maturation state. These findings may contribute to the search for long-term efficient anticalcification treatments.

Freeze-dried snake antivenoms formulated with sorbitol, sucrose or mannitol: Comparison of their stability in an accelerated test

Freeze-drying is used to improve the long term stability of pharmaceutical proteins. Sugars and polyols have been successfully used in the stabilization of proteins. However, their use in the development of freeze-dried antivenoms has not been documented. In this work, whole IgG snake antivenom, purified from equine plasma, was formulated with different concentrations of sorbitol, sucrose or mannitol. The glass transition temperatures of frozen formulations, determined by Differential Scanning Calorimetry (DSC), ranged between -13.5 °C and -41 °C. In order to evaluate the effectiveness of the different stabilizers, the freeze-dried samples were subjected to an accelerated stability test at 40 ± 2 °C and 75 ± 5% relative humidity. After six months of storage at 40 °C, all the formulations presented the same residual humidity, but significant differences were observed in turbidity, reconstitution time and electrophoretic pattern. Moreover, all formulations, except antivenoms freeze-dried with mannitol, exhibited the same potency for the neutralization of lethal effect of Bothrops asper venom. The 5% (w:v) sucrose formulation exhibited the best stability among the samples tested, while mannitol and sorbitol formulations turned brown. These results suggest that sucrose is a better stabilizer than mannitol and sorbitol in the formulation of freeze-dried antivenoms under the studied conditions.

Prospects in lyophilization of bovine pericardium.

Almost 30 years after the introduction of heart valve prostheses patients worldwide are benefiting from the implant of these devices. Among the various types of heart valves, the ones made of treated bovine pericardium have become a frequently used replacement of the hearts native valve. Lyophilization, also known as freeze-drying, is an extremely useful technique for tissue storage for surgical applications. This article gives a brief overview on the current bovine pericardium lyophilization development, including the chemical modification to improve physical-chemical characteristics and the advanced technologies used to guarantee a high-quality product. It was shown that lyophilization process can be successfully applied as a method of bovine pericardium preservation and also as a technological tool to prepare new materials obtained by chemical modification of native tissues.

Synthesis and physicochemical characterization of chemically modified chitosan by succinic anhydride

A N-succinil-quitosana e um derivado quimicamente modificado do biopolimero quitosana. A insercao de substituintes de anidrido succinico nas aminas protonadas presentes ao longo da cadeia do polimero quitosana, conferem diferentes caracteristicas fisico-quimicas a molecula de quitosana. Esta modificacao quimica, possibilitou a quitosana, solubilidade em pHs que variam do acido (2.0 a 3.0) ate alcalino (13.0 a 14.0). Estas propriedades sao atribuidas ao alongamento da cadeia alquilica, que afasta a parte hidrofilica da cadeia fechada da D-glicosamina, facilitando o acesso da agua, a qual ira estabelecer uma interacao mais forte com a molecula de quitosana. Esta propriedade nao esta presente em amostras de quitosana pura, a qual sabe-se que solubiliza-se apenas em pH abaixo de 5.5. Estas modificacoes na quitosana possibilitam novas aplicacoes na area de biotecnologia, uma vez que a solubilidade em meio neutro e levemente alcalino e importante na area biologica.

Histological Evaluation of Biocompatibility of Lyophilized Bovine Pericardium Implanted Subcutaneously in Rats

This article aims at investigating in vivo evaluation of lyophilization procedure on the biocompatibility of bovine pericardium treated with glutaraldehyde (GA). The bovine pericardium was fixed with 0.5% glutaraldehyde during 10 days and preserved in 4% formaldehyde (FA). Two groups of samples were prepared from treated membranes: Group 1, nonlyophilized samples and Group 2, lyophilized samples. Male Sprague-Dawley rats (4 weeks after birth) were anesthetized (pentobarbital sodium 25 mg/kg of body weight) and in each one were implanted subcutaneously in the dorsal region a sample from Group 1 and another from Group 2. These samples were explanted after 30 days for histological analysis. No intercurrences took place after the surgery. No differences (P > 0.05) in the calcification, granulomatous reaction, mononuclear infiltration, and granulation tissue development was observed between both groups. The implanted lyophilized samples presented a trend for a reduced inflammatory reaction. Lyophilization of the bovine pericardium does not seem to increase the above listed tissue reaction.

Evaluation of Shrinkage Temperature of Bovine Pericardium Tissue for Bioprosthetic Heart Valve Application by Differential Scanning Calorimetry and Freeze-drying Microscopy

Bovine pericardium bioprosthesis has become a commonly accepted device for heart valve replacement. Present practice relies on the measurement of shrinkage temperature, observed as a dramatic shortening of tissue length. Several reports in the last decade have utilized differential scanning calorimetry (DSC) as an alternative method to determine the shrinkage temperature, which is accompanied by the absorption of heat, giving rise to an endothermic peak over the shrinkage temperature range of biological tissues. Usually, freeze-drying microscope is used to determine collapse temperature during the lyophilization of solutions. On this experiment we used this technique to study the shrinkage event. The aim of this work was to compare the results of shrinkage temperature obtained by DSC with the results obtained by freeze-drying microscopy. The results showed that both techniques provided excellent sensitivity and reproducibility, and gave information on the thermal shrinkage transition via the thermodynamical parameters inherent of each method.

Liposome-Loaded Cell Backpacks.

Cell backpacks, or micron-scale patches of a few hundred nanometers in thickness fabricated by layer-by-layer (LbL) assembly, are potentially useful vehicles for targeted drug delivery on the cellular level. In this work, echogenic liposomes (ELIPs) containing the anticancer drug doxorubicin (DOX) are embedded into backpacks through electrostatic interactions and LbL assembly. Poly(allylamine hydrochloride)/poly(acrylic acid) (PAH/PAA)n , and poly(diallyldimethylammonium chloride)/poly(styrene sulfonate) (PDAC/SPS)n film systems show the greatest ELIP incorporation of the films studied while maintaining the structural integrity of the vesicles. The use of ELIPs for drug encapsulation into backpacks facilitates up to three times greater DOX loading compared to backpacks without ELIPs. Cytotoxicity studies reveal that monocyte backpack conjugates remain viable even after 72 h, demonstrating promise as drug delivery vehicles. Because artificial vesicles can load many different types of drugs, ELIP containing backpacks offer a unique versatility for broadening the range of possible applications for cell backpacks.