Marta M. D. C. Vila

Structural and functional stabilization of protein entities: state-of-the-art

Within the context of biomedicine and pharmaceutical sciences, the issue of (therapeutic) protein stabilization assumes particular relevance. Stabilization of protein and protein-like molecules translates into preservation of both structure and functionality during storage and/or targeting, and such stabilization is mostly attained through establishment of a thermodynamic equilibrium with the (micro)environment. The basic thermodynamic principles that govern protein structural transitions and the interactions of the protein molecule with its (micro)environment are, therefore, tackled in a systematic fashion. Highlights are given to the major classes of (bio)therapeutic molecules, viz. enzymes, recombinant proteins, (macro)peptides, (monoclonal) antibodies and bacteriophages. Modification of the microenvironment of the biomolecule via multipoint covalent attachment onto a solid surface followed by hydrophilic polymer co-immobilization, or physical containment within nanocarriers, are some of the (latest) strategies discussed aiming at full structural and functional stabilization of said biomolecules.

Nanoencapsulation of bovine lactoferrin for food and biopharmaceutical applications

Lactoferrin has for long captured the interest of many researchers as a natural compound with a wide variety of uses. Lactoferrin is a monomeric, iron-binding 80 kDa glycoprotein, and appears to be the subfraction of whey with the best documented antiviral, antimicrobial, anticancer and immune modulating/enhancing effects. It belongs to the family of transferrin proteins, and serves to control iron levels in body fluids by sequestering and solubilizing ferric iron. In the present research effort, production of lactoferrin derivatives (starting from a purified commercial extract), encompassing full stabilization of its three-dimensional structure, has been attempted via nanoencapsulation within lipid nanovesicles, integrating a multiple water-in-oil-in-water emulsion. Long-term storage of the multiple nanoemulsions produced did not lead to leaching of protein, thus proving the effectiveness of the encapsulation procedure. Furthermore, lactoferrin nanovesicle derivatives prepared under optimal conditions were successfully employed at lab-scale antimicrobial trials.

A simple device for quantitative colorimetric diffuse reflectance measurements

Abstract A very simple and low cost reflectometer, for colorimetric diffuse reflectance measurements is described. The proposed instrument uses a light dependent resistor (LDR), as a sensor, and a light emitter diode (LED), as light source. The performance of the device was compared with that of a commercial instrument, measuring the reflectance of the Pantone® Process Black Uncoated Palette. The quantitative analysis of nickel, in a catalyst, using dimethylglyoxime as colorimetric reagent, was also used to test the device. The observed precision was about 6% (R.S.D.). These results were compared with those obtained by the gravimetric method using the same reaction, through the statistical Student’s t-test procedure. A complete agreement was observed. The good quality of all the obtained results allows the recommendation of the instrument for diffuse reflectance measurements including quantitative analytical procedures.

Alternatives to overcoming bacterial resistances: State-of-the-art.

Worldwide, bacterial resistance to chemical antibiotics has reached such a high level that endangers public health. Presently, the adoption of alternative strategies that promote the elimination of resistant microbial strains from the environment is of utmost importance. This review discusses and analyses several (potential) alternative strategies to current chemical antibiotics. Bacteriophage (or phage) therapy, although not new, makes use of strictly lytic phage particles as an alternative, or a complement, in the antimicrobial treatment of bacterial infections. It is being rediscovered as a safe method, because these biological entities devoid of any metabolic machinery do not possess any affinity whatsoever to eukaryotic cells. Lysin therapy is also recognized as an innovative antimicrobial therapeutic option, since the topical administration of preparations containing purified recombinant lysins with amounts in the order of nanograms, in infections caused by Gram-positive bacteria, demonstrated a high therapeutic potential by causing immediate lysis of the target bacterial cells. Additionally, this therapy exhibits the potential to act synergistically when combined with certain chemical antibiotics already available on the market. Another potential alternative antimicrobial therapy is based on the use of antimicrobial peptides (AMPs), amphiphilic polypeptides that cause disruption of the bacterial membrane and can be used in the treatment of bacterial, fungal and viral infections, in the prevention of biofilm formation, and as antitumoral agents. Interestingly, bacteriocins are a common strategy of bacterial defense against other bacterial agents, eliminating the potential opponents of the former and increasing the number of available nutrients in the environment for their own growth. They can be applied in the food industry as biopreservatives and as probiotics, and also in fighting multi-resistant bacterial strains. The use of antibacterial antibodies promises to be extremely safe and effective. Additionally, vaccination emerges as one of the most promising preventive strategies. All these will be tackled in detail in this review paper.

Analytical methods for vancomycin determination in biological fluids and in pharmaceuticals

Vancomycin is a glycopeptide antibiotic employed in the treatment of infections caused by certain methicillin-resistant staphylococci. It is indicated also for patients allergic to penicillin or when there is no response to penicillins or cephalosporins. The adequate vancomycin concentration levels in blood serum lies between 5 and 10 mg/L. Higher values are toxic, causing mainly nephrotoxicity and ototoxicity. Various analytical methods are described in the literature: spectrophotometric, immunologic, biologic and chromatographic methods. This paper reviews the main analytical methods for vancomycin determination in biological fluids and in pharmaceutical preparations.

Biomimetic aqueous-core lipid nanoballoons integrating a multiple emulsion formulation: a suitable housing system for viable lytic bacteriophages.

The emergence of antibiotic-resistant bacterial strains and the weak penetration of antibiotics into bacterial biofilms put an emphasis in the need for safe and effective alternatives for antimicrobial treatments. The application of strictly lytic bacteriophages (or phages) has been proposed as an alternative (or complement) to conventional antibiotics, allowing release of the natural predators of bacteria directly to the site of infection. In the present research effort, production of bacteriophage derivatives (starting from lytic phage particle isolates), encompassing full stabilization of their three-dimensional structure, has been attempted via housing said bacteriophage particles within lipid nanovesicles integrating a multiple water-in-oil-in-water (W/O/W) emulsion. As a proof-of-concept for the aforementioned strategy, bacteriophage particles with broad lytic spectrum were entrapped within the aqueous core of lipid nanoballoons integrating a W/O/W multiple emulsion. Long-term storage of the multiple emulsions produced did not lead to leaching of phage particles, thus proving the effectiveness of the encapsulation procedure.

Development and Characterization of a Hydrogel Containing Silver Sulfadiazine for Antimicrobial Topical Applications

Development and optimization of a hydrogel with impregnated silver sulfadiazine was pursued, for antimicrobial topical applications. The selected hydrogel exhibited a homogeneous appearance, with whitish colloration and devoid of any fractures or cracks. The content in impregnated silver sulfadiazine was within established limits (1%, w/w) with a standard deviation of up to 1.28%. The hydrogel presented a good characteristic in relation to release of the active antimicrobial principle, verified through swelling tests and antimicrobial activity. The swelling tests indicated a higher increase in weight during the first 6 h of contact with a moist environment, with a maximum value of 266.00 ± 0.81, and with maintenance of the original shape of the hydrogel. The impregnated silver sulfadiazine presented antimicrobial activity, as expected, indicating a prolonged release of the drug. The infrared spectra of the hydrogel with impregnated silver sulfadiazine indicated that the drug did not engage in any bonds with the polymeric matrix, which otherwise could have reduced its antimicrobial activity. The mechanical resistance tests produced good results, indicating that the hydrogels may be utilized in different locations of the human body with skin lesions.

Structural and functional stabilization of phage particles in carbohydrate matrices for bacterial biosensing

Infections associated with health care services are nowadays widespread and, associated to the progressive emergence of microorganisms resistant to conventional chemical antibiotics, are major causes of morbidity and mortality. One of the most representative microorganisms in this scenario is the bacterium Pseudomonas aeruginosa, which alone is responsible for ca. 13-15% of all nosocomial infections. Bacteriophages have been reported as a potentially useful tool in the diagnosis of bacterial diseases, since they specifically recognize and lyse bacterial isolates thus confirming the presence of viable cells. In the present research effort, immobilization of these biological (although metabolically inert) entities was achieved via entrapment within (optimized) porous (bio)polymeric matrices of alginate and agar, aiming at their full structural and functional stabilization. Such phage-impregnated polymeric matrices are intended for future use as chromogenic hydrogels sensitive to color changes evolving from reaction with (released) intracytoplasmatic moieties, as a detection kit for P. aeruginosa cells.

Green Spectrophotometric Method for the Quantitative Analysis of Vancomycin in Pharmaceuticals and Comparison with HPLC

Abstract A spectrophotometric method for the determination of vancomycin base, (VCM), and vancomycin hydrochloride, (HVCM), based on the reaction with copper (II) ions, is presented. The obtained detection limit is about 4.5×10−5 mol L−1. The working analytical range falls between 1.0×10−3 mol L−1 and 1.0×10−2 mol L−1. Recovery studies in presence of excipients were performed. The recovery results were compared with HPLC. For HVCM the proposed method presented similar recovery to that of HPLC, 100.4% vs. 100.2%, but better precision, 1.9% vs. 6.1%. In the VCM case the recovery is quite better, 100.5% vs. 89.6%, with a little smaller precision, 2.1% vs. 1.3%.

Comparative study of two spectrophotometric reagents for catechol analysis in guaraná seeds powder

Neste trabalho sao descritos e comparados dois metodos espectrofotometricos para analise de catecol (o-hidroxifenol) em Paullinea cupana var. sorbilis, popularmente conhecido como guarana. As amostras de sementes trituradas foram extraidas com etanol 97% v/v e as solucoes tratadas com p-aminofenol em meio etanolico alcalino ou m-aminofenol e metaperiodato de sodio em solucao tampao (pH 3.0). A absorbância foi medida a 586 nm e 520 nm respectivamente. Quatro produtos comerciais diferentes foram analisados e os resultados foram comparados com aqueles obtidos com m-aminofenol. Apesar da boa qualidade das curvas de calibracao, nos dois metodos houve interferencia da matriz, o que foi contornado usando-se o metodo da adicao de padrao. A comparacao entre os resultados obtidos pelos dois metodos, usando o teste estatistico t de Student, mostrou que nao ha diferencas significantes para um nivel de confianca de 95%. In this article are described and compared two spectrophotometric methods for the analysis of catechol (o-hydroxyphenol) in Paullinea cupana var. sorbilis, popularly known as guarana. The pounded seed samples were extracted with ethanol 97% v/v and the solutions were treated with p-aminophenol in alkaline ethanolic medium or m-aminophenol and sodium metaperiodate in buffered aqueous solution (pH 3.0). The absorbance was measured at 586 nm and 520 nm respectively. Four different commercial products were analyzed and the results were compared with those obtained with the m-aminophenol method. Despite the good quality of the calibration curves, in the two methods the interference of the matrix was observed. This problem was settled using the standard addition method. Comparison between the results obtained by the two methods, using the statistical Student’s t-test, showed that there is no significant difference at the 95% confidence level.