Bioactive compounds: Application of albumin nanocarriers as delivery systems.

Enriched products with bioactive compounds (BCs) show the capacity to produce a wide range of possible health effects. Most BCs are essentially hydrophobic and sensitive to environmental factors; so, encapsulation becomes a strategy to solve these problems. Many globular proteins have the intrinsic ability to bind, protect, encapsulate, and introduce BCs into nutraceutical or pharmaceutical matrices. Among them, albumins as human serum albumin (HSA), bovine serum albumin (BSA), ovalbumin (OVA) and α-lactalbumin (ALA) are widely abundant, available, and applied in many industrial sectors, becoming promissory materials to encapsulate BCs. Therefore, this review focuses on researches about the main groups of natural origin BCs (namely phenolic compounds, lipids, vitamins, and carotenoids), the different types of nanostructures based on albumins to encapsulate them and the main fields of application for BCs-loaded albumin systems. In this context, phenolic compounds (catechins, quercetin, and chrysin) are the most extensively BCs studied and encapsulated in albumin-based nanocarriers. Other extensively studied subgroups are stilbenes and curcuminoids. Regarding lipids and vitamins; terpenes, carotenoids (ß-carotene), and xanthophylls (astaxanthin) are the most considered. The main application areas of BCs are related to their antitumor, anti-inflammatory, and antioxidant properties. Finally, BSA is the most used albumin to produced BCs-loaded nanocarriers.

PEGylation of genistein-loaded bovine serum albumin nanoparticles and its effect on in vitro cell viability and genotoxicity properties.

Self-assembled bovine serum albumin nanoparticles loaded with the isoflavone genistein have shown apoptosis-mediated cytotoxicity against murine mammary adenocarcinoma F3II cells. Due to their protein nature and small particle size (13-15 nm), their parenteral administration could be affected by possible immunogenic reactions and rapid clearance from the bloodstream. To avoid these problems, PEGylation of the systems was achieved in this work by using a 30 kDa methoxy-polyethylene glycol carbonyl imidazole derivative through the reaction between the carbonyl imidazole group and the amino groups of Lys residues on the protein surface, which was confirmed by a 17% reduction in the available amino groups content measured by the o-phthaldialdehyde method. PEGylated isoforms were obtained, showing an increase of particle size from 13 to 15 nm to around 260 nm, and were purified by SEC-FPLC and characterized by SDS-PAGE, DLS and AFM techniques. The effect of PEGylation on BSAnp-Gen cytotoxicity and genotoxicity against F3II cells was evaluated in vitro by MTT assay, flow cytometry analysis and micronucleus assay. From the results, PEGylation produced an improvement of the biological properties of genistein-loaded nanoparticles in terms of cytotoxicity (lower IC50), not affecting the induction of apoptosis, decreasing the genotoxicity of the systems (less induction of micronucleus formation).

Nanocomplexes based on egg white protein nanoparticles and bioactive compounds as antifungal edible coatings to extend bread shelf life.

This work is aimed to obtain nanocomplexes based on egg white protein nanoparticles (EWPn) and bioactive compounds (BC), carvacrol (CAR), thymol (THY) and trans-cinnamaldehyde (CIN), and evaluate their application as antifungal edible coatings on preservative-free breads. The nanocomplex formation was studied through stoichiometry, affinity, colloidal behavior, morphology, and encapsulation efficiency (EE, %). Rounded-shape nanocomplexes with particle sizes < 100 nm were obtained. The EE values were similar for all BC (>83%). Furthermore, the in vitro antifungal activity of the nanocomplexes was verified using the Aspergillus niger species. The nanocomplexes were applied as coatings onto the crust of preservative-free breads, which were stored for 7 days (at 25 °C). The coatings had no impact on the physicochemical properties of the bread loaves (moisture, aw, texture, and color). Finally, the coatings based on EWPn-THY and EWPn-CAR nanocomplexes showed higher antifungal efficacy, extending the bread shelf life after 7 days.

Genistein loaded in self-assembled bovine serum albumin nanovehicles and their effects on mouse mammary adenocarcinoma cells.

Antitumor activity of plant-derived flavonoids has been researched during recent decades. Among them, genistein (Gen) stands out for showing cytotoxic activity against breast cancer cells. However, its low water solubility, limited bioavailability, and fast metabolism hinder its administration in chemopreventive therapies. To overcome these obstacles, bovine serum albumin nanovehicles (BSAnp) were obtained by a heat-induced self-assembly process at 70 °C and two aqueous medium pH (9.0 and 11.0) and assayed for the Gen loading. Thus, in this work, Gen loading in BSAnp was studied by spectroscopic techniques and compared with the one obtained for its stereoisomer, chrysin (Chrys). Results revealed that Gen binds to BSAnp via fluorescence quenching mechanism forming inclusion complexes. Compared to Chrys, Gen binding to BSAnp involved more molecules, whereas the association constant was similar for both flavonoids. In general, flavonoid loading in protein systems was strongly affected by the combined effects of BSA conformational state (native vs. aggregated), nanovehicle size, and flavonoid chemical structure. To evaluate the antitumor properties freeze-dried powders were obtained, and they were assayed in vitro after reconstitution by XTT technique and Annexin V-FITC flow cytometry against mouse mammary adenocarcinoma F3II cells. Gen-loaded BSAnp produced a significant decrease in cell viability compared with unloaded BSAnp systems, being the highest cytotoxic effects found for the lowest sized Gen-loaded BSAnp. The leading cytotoxicity mechanism for Gen-loaded systems was apoptosis. Summarizing, it can be concluded that BSAnp constitute versatile nanovehicles for potential flavonoid incorporation in pharmaceutical and nutraceutical matrices.

In vitro gastrointestinal digestion and cytotoxic effect of ovalbumin-conjugated linoleic acid nanocomplexes.

The aim of this work was to examine the behavior of conjugated linoleic acid (CLA) delivery systems based on ovalbumin (OVA) and their derived nanoparticles (OVAn1 and OVAn2), under static in vitro gastrointestinal digestion model. In addition, potential cytotoxic effect of these inclusion complexes on a human colon cancer cell line (HT-29) was evaluated. OVA was resistant to gastric and intestinal digestion, while OVA nanoparticles were very susceptible to digestive enzymes hydrolysis. Particle size distribution (PDS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for OVA evidenced the presence of a protein fragment of similar size after simulated digestive process. Conversely, for nanoparticles, partial and total hydrolysis in gastric and intestinal phases, respectively, was evidenced. After in vitro gastrointestinal digestion, released CLA (RCLA) was assayed. In case of OVA, as CLA carrier, RCLA was 37%, while for OVA nanoparticles, lower RCLA values (~10-20%) were obtained. From cytotoxic assays, it was observed that CLA molecule was responsible for cell death, whereas OVA or their derived nanoparticles were not cytotoxic on HT-29 cells. On the other hand, flow cytometry analysis revealed that main death mechanism for CLA, and their inclusion complexes was apoptosis. OVA-CLA and OVAn1-CLA inclusion complexes displayed the highest potential cytotoxic activity and apoptotic index. Information derived from this work could be relevant for the design of CLA delivery systems as promising nanosupplements for production of new functional and excipient foods for both prevention and control of colon cancer.

Chrysin-loaded bovine serum albumin particles as bioactive nanosupplements.

Development of nanosupplements based on bioactive compound encapsulation and their incorporation into diet constitutes a field that is growing exponentially. Among flavonoids, chrysin (Chrys) shows high antitumor activity but its poor water solubility limits its applications. Bovine serum albumin nanoparticles (BSAnp) with sizes in the range of 13-28 nm were previously prepared by applying a heat-induced self-assembly method, and they were able to load Chrys. Hence, the aim of this study is to provide information about elaboration of Chrys nanosupplements considering the incidence of the freeze-drying process of Chrys-loaded and unloaded BSAnp systems on aqueous redispersibility and on the retention of their physicochemical properties as examined by DSC, turbidity and DLS measurements. In general, the physicochemical properties of Chrys-loaded BSAnp were retained after the freeze-drying process. The cytotoxic activity of the reconstituted powders was assayed on MCF-7 and MDA-MB-231 cell lines (as models of cancer cell lines) by using the MTT assay. Furthermore, the mechanism of cell death was researched by Annexin V-FITC flow cytometry. The apoptosis-mediated cytotoxicity was higher for Chrys-loaded BSAnp than for the BSAnp system. The greatest in vitro cytotoxic effect was observed for the smallest sized Chrys-loaded BSAnp, which was obtained at pH 11.0 and 70 °C. Finally, this system was subjected to static in vitro gastrointestinal digestion using the standardized protocol, and ∼14% Chrys was released from BSAnp after digestion. These results would provide a procedure to obtain bioactive nanosupplements in a soluble form, and they would suggest that smaller sized Chrys-loaded BSAnp could be a promising oral delivery system for potential cancer therapies.

Formation and characterization of self-assembled bovine serum albumin nanoparticles as chrysin delivery systems.

Chrysin (5,7-dihydroxyflavone) (Chrys) is a natural flavone extracted from many plants, and it has been proposed as a bioactive agent for cancer therapy. Nevertheless, its use is limited mainly due to its poor water solubility. Bovine serum albumin (BSA) is a water soluble, biocompatible and non-toxic protein with a promising application in lipophilic bioactive compound delivery. Moreover, BSA is heat sensitive, feature that could be used for producing self-assembled nanoparticle with tailor-made properties. In this contribution, we studied the formation of BSA nanoparticles (BSAnp) by thermal treatment at different conditions of temperature (70 °C/5 min and 85 °C/5 min), protein concentration (1.0-4.0%wt.) and aqueous medium pH values (9.0 and 11.0) in which it is known that BSA is found in different unfolded conformations. Binding of Chrys dissolved in dimethyl sulfoxide (DMSO) was studied by fluorescence titration experiments. Characterization of Chrys-loaded and unloaded BSAnp was performed in phosphate buffered saline (PBS) pH 7.4 by applying a set of complementary techniques: dynamic light scattering (DLS), size exclusion fast protein liquid chromatography (SEC-FPLC) and transmission electron microscopy (TEM). Different populations of BSAnp were obtained, which showed different diameters in the range of 1328 nm, ζ potentials around -10.0 mV, molecular weight in the range of 400-1000 kDa and spherical shape. Chrys encapsulation efficiency (EE. %) was also determined, and values between 44-84% were obtained, which mainly depended on the mode of Chrys binding and physicochemical BSAnp properties. Results highlight the ability of self-assembled BSAnp for Chrys vehiculization in an aqueous medium which could found potential application in antitumor therapies.

Effect of limited enzymatic hydrolysis on linoleic acid binding properties of ß-lactoglobulin.

ß-Lactoglobulin (BLG) is a member of lipocalin family, proteins with ability to bind small hydrophobic ligands, such as retinol, vitamins and fatty acids. Moreover, BLG is susceptible to protease action producing a wide range of polypeptides depending on the hydrolysis degree (HD). In the present work, the effect of limited enzymatic hydrolysis on fatty acid binding properties of BLG was studied. Linoleic acid (LA) was used as a model fatty acid. Limited enzymatic hydrolysis was performed using α-chymotrypsin immobilised on agarose microparticles. BLG hydrolysates were produced at HD: 1%, 3% and 5%. In order to determine the influence of HD on BLG molecular weight SDS-PAGE was used. BLG structural modification and LA binding properties were monitored by means of fluorescence spectroscopic techniques. The increase in HD produced: (i) a BLG degradation and a molecular weight distribution of BLG hydrolysates and (ii) an increased exposition of buried hydrophobic residues, however it was observed a decrease in surface hydrophobicity possibly due to a deterioration of hydrophobic protein domains. It was observed that enzymatic hydrolysis treatment produced a decrease in BLG ability for binding LA. It was concluded that limited enzymatic hydrolysis could deteriorate the specific site on BLG structure necessary for binding LA.