The amount of dextran in PLGA nanocarriers modulates protein corona and promotes cell membrane damage.

Polymeric nanocarriers (NCs) are efficient vehicles to prevent drug unspecific biodistribution and increase the drug amounts delivered to tumor tissues. However, some toxicological aspects of NCs still lack a comprehensive assessment, such as their effects on cellular processes that lead to toxicity. We evaluate the interaction of poly(lactic-co-glycolic acid) (PLGA) NCs prepared using dextran (Dex) and Pluronic®-F127 as stabilizing agents with myocardial cells (H9C2), breast adenocarcinoma cells (MCF-7) and macrophages (RAW 264.7) to address the effect of Dex in PLGA NC formulations. By an emulsion diffusion method, doxorubicin-loaded NCs were prepared with no Dex (PLGA-DOX), 1% (w/v) Dex (Dex1/PLGA-DOX) and 5% (w/v) Dex (Dex5/PLGA-DOX). Uptake analyses revealed a significant reduction in Dex5/PLGA-DOX NC uptake by H9C2 and MCF-7, as in the case of Dex1/PLGA-DOX NCs in the absence of in vitro protein corona, revealing an effect of dextran concentration on the formation of protein corona. RAW 264.7 cells presented a greater uptake of Dex5/PLGA-DOX NCs than the other NCs likely because of receptor mediated endocytosis, since C-type lectins like SIGN-R1, mannose receptors and scavenger receptor type 1 that are expressed in RAW 264.7 can mediate Dex uptake. Despite the lower uptake, Dex5/PLGA-DOX NCs promote the generation of reactive oxygen species and oxidative membrane damage in MCF-7 and H9C2 even though cellular metabolic activity assessed by MTT was comparable among all the NCs. Our results highlight the importance of an in-depth investigation of the NC-cell interaction considering additional mechanisms of damage apart from metabolic variations, as nanoparticle-induced damage is not limited to imbalance in metabolic processes, but also associated with other mechanisms, e.g., membrane and DNA damage.

Microemulsion Formulations for the Transdermal Delivery of Lapachol.

This project was carried out to investigate the feasibility of using microemulsions for transdermal delivery of lapachol. From the screening of surfactants and oils, a range of microemulsions were developed using oleic acid, a mixture of Cremophor EL and Tween 20 and water. The solubility of lapachol was determined in these ingredients and in the formulated microemulsions. The microemulsions were characterised using cross-polarising light microscopy, their electrical conductivity, pH, zeta potential and rheology were analysed, and they were also investigated using small-angle X-ray scattering and differential scanning calorimetry. Ex vivo studies were performed using porcine ear skin and Franz diffusion cells to investigate the permeation and retention of lapachol. Systems containing different concentrations of Cremophor EL (8.4-41.6%), Tween 20 (5.4-41.6%) and oleic acid (12-31.9%) are able to form microemulsions. Lapachol was delivered more effectively through the skin from all of the microemulsions tested than by the control (oleic acid). These studies indicated that microemulsions incorporating lapachol were formed successfully and that these enhanced drug delivery and retention in the skin. Microemulsion systems may, therefore, provide promising vehicles for percutaneous delivery of lapachol.

Interaction between nitroheterocyclic compounds with beta-cyclodextrins: phase solubility and HPLC studies.

Chagas disease is a serious health problem for Latin America. Nitrofurazone (NF) and Hidroxymethylnitrofurazone (NFOH) are active against Trypanosoma cruzi. The effect of beta-cyclodextrin (beta-CD) and dimethyl-beta-cyclodextrin (DM-beta-CD) complexation on the UV absorption and retention time of nitrofurazone (NF) and its hydroxymethylated analog (NFOH) were studied in solution. The retention behavior was analyzed on a reversed phase C18 column and the mobile phase used was acetonitrile-water (20/80 v/v), in which cyclodextrins (beta-CD or DM-beta-CD) were incorporated as a mobile phase additive. The decrease in the retention times of NF (or NFOH) with increasing concentration of HP-beta-CD enables the determination of the complex stability constants by HPLC. A phase-solubility study was performed, according to the method reported by Higuchi and Connors, to evaluate the changes of NF/NFOH in the complexation state, and the diagrams obtained suggested that it forms complexes with a stoichiometry of 1:1. This is an important study for the characterization of potential formulations to be used as therapeutic options for the treatment of Chagas disease.

Optimizing the medium components in bioemulsifiers production by Candida lipolytica with response surface method.

A response surface methodology was used to study bioemulsifier production by Candida lipolytica. A 2(4) full experimental design was previously carried out to investigate the effects and interactions of the concentrations of corn oil, urea, ammonium sulfate, and potassium dihydrogen orthophosphate on the emulsification activity (EA) of the bioemulsifier produced by C. lipolytica. The best EA value (3.727 units of emulsification activity (UEA)) was obtained with a medium composed of 0.4 g of urea, 1.1 g of ammonium sulfate, 2.04 g of potassium dihydrogen orthophosphate, 5 mL of corn oil, 50 mL of distilled water, and 50 mL of seawater. A curvature check was performed and revealed a lack of fit of the linear approximation. The proximity of the optimum point was evident, as was the need for quadratic model and second-order designs that incorporate the effect of the curvature. Medium constituents were then optimized for the EA using a three-factor central composite design and response surface methodology. The second-order model showed statistical significance and predictive ability. It was found that the maximum EA produced was 4.415 UEA, and the optimum levels of urea, ammonium sulfate, and potassium dihydrogen orthophosphate were, respectively, 0.544% (m/v), 2.131% (m/v), and 2.628% (m/v).

Effect of the covalent modification of horseradish peroxidase with poly(ethylene glycol) on the activity and stability upon encapsulation in polyester microspheres.

Encapsulation of proteins in polyester microspheres by coacervation methods frequently causes protein inactivation and aggregation. Furthermore, an often-substantial amount of the encapsulated proteins is released within the first 24 h from the microspheres. To overcome these problems poly(ethylene glycol) (PEG) was employed as excipient and protein-modifying agent. The model protein horseradish peroxidase (HRP) was chemically modified or co-lyophilized with PEG of differing molecular weights, namely PEG(5000), PEG(20000), and PEG(40000). The lyophilized preparations were encapsulated in poly(D,L-lactide-co-glycolic) acid (PLGA) microspheres by a coacervation method. Covalent modification of HRP with PEG increased the encapsulation efficiency (EE) from 83% to about 100% while PEG when used as an excipient reduced the EE. Encapsulation caused aggregation of ca. 5% of non-modified HRP and the residual specific activity was only 57%. Covalent modification with PEG reduced HRP aggregation to less than 1% and improved its residual activity to more than 95%. When PEG was used as excipient similar results were found with respect to a reduction in encapsulation-induced aggregation, but no more than 80% of residual activity was obtained even for the best formulation after encapsulation. It was also found that covalent modification of HRP with PEG substantially reduced the unwanted initial "burst" release observed during the initial 24 h of in vitro release from about 70% to 23%. Furthermore, HRP activity and stability were also improved during in vitro release for HRP-PEG conjugates. The data show that covalent modification of proteins with PEG might be useful to improve protein stability during coacervation encapsulation and subsequent release as well as to increase EE and reduce the burst release.

Bioemulsifier production in batch culture using glucose as carbon source by Candida lipolytica.

The yeast Candida lipolytica IA 1055 produced an inducible extracellular emulsification activity while utilizing glucose at different concentrations as carbon source during batch fermentation at 27 degrees C. In all glucose concentrations studied, maximum production of emulsification activity was detected in the stationary phase of growth, after pH reached minimal values. The bioemulsifier isolated was a complex biopolymer constituting proteins, carbohydrates, and lipids. The results obtained in this work show that the biosynthesis of a bioemulsifier is not simply a prerequisite for the degradation of extracellular hydrocarbon.

The use of babassu oil as substrate to produce bioemulsifiers by Candida lipolytica.

Candida lipolytica IA 1055 produced an extracellular emulsifier when using babassu oil as its sole carbon source during batch and fed batch fermentations at 27 degrees C. Emulsification activity was detected after 60 h of growth in all conditions studied. The bioemulsifier was isolated after 144 h of fermentation from the best condition studied. The biopolymer seems to be a polysaccharide-protein-lipid complex.