Joana Poejo

Alginate-based hybrid aerogel microparticles for mucosal drug delivery

The application of biopolymer aerogels as drug delivery systems (DDS) has gained increased interest during the last decade since these structures have large surface area and accessible pores allowing for high drug loadings. Being biocompatible, biodegradable and presenting low toxicity, polysaccharide-based aerogels are an attractive carrier to be applied in pharmaceutical industry. Moreover, some polysaccharides (e.g. alginate and chitosan) present mucoadhesive properties, an important feature for mucosal drug delivery. This feature allows to extend the contact of DDS with biological membranes, thereby increasing the absorption of drugs through the mucosa. Alginate-based hybrid aerogels in the form of microparticles (<50μm) were investigated in this work as carriers for mucosal administration of drugs. Low methoxyl pectin and κ-carrageenan were co-gelled with alginate and further dried with supercritical CO2 (sc-CO2). Spherical mesoporous aerogel microparticles were obtained for alginate, hybrid alginate/pectin and alginate/κ-carrageenan aerogels, presenting high specific surface area (370-548m(2)g(-1)) and mucoadhesive properties. The microparticles were loaded with ketoprofen via adsorption from its solution in sc-CO2, and with quercetin via supercritical anti-solvent precipitation. Loading of ketoprofen was in the range between 17 and 22wt% whereas quercetin demonstrated loadings of 3.1-5.4wt%. Both the drugs were present in amorphous state. Loading procedure allowed the preservation of antioxidant activity of quercetin. Release of both drugs from alginate/κ-carrageenan aerogel was slightly faster compared to alginate/pectin. The results indicate that alginate-based aerogel microparticles can be viewed as promising matrices for mucosal drug delivery applications.

Preparation and characterization of soluble branched ionic β-cyclodextrins and their inclusion complexes with triclosan

This study aims to synthesize, characterize and investigate the water solubility and cytotoxicity of branched anionic/cationic β-cyclodextrins (bβCDs) obtained by reaction with epichlorohydrin and chloroacetic acid or choline chloride, respectively, by a single step polycondensation reaction. Obtained ionic bβCDs were investigated as an attempt to comparatively study anionic and cationic bβCDs. Water solubility of both ionic derivatives was similar (400 mg/mL) at neutral and basic pHs and remarkably higher than that of their neutral homologues. Additionally, a pH-dependent solubility of anionic bβCDs was observed. Cytotoxicity of ionic bβCDs was evaluated on Human colon carcinoma Caco-2 cells and high cell viability (>99%) was observed in the range of 0-100 mg/mL for anionic and cationic samples, in the same range of that of neutral and parent β-CDs. Additionally, complexes formation capacity with triclosan, a poor water soluble antimicrobial agent, was confirmed by several techniques observing a complexation limit around 4 mg/mL for both systems and higher stability constant for anionic bβCDs than cationic derivatives.

Using different natural origin carriers for development of epigallocatechin gallate (EGCG) solid formulations with improved antioxidant activity by PGSS-drying

Epigallocatechin gallate (EGCG) is the catechin with the highest antioxidant activity present in green tea. Nevertheless, due its low bioavailability, it is necessary to develop EGCG formulations capable of improving its stability resulting in increased bioavailability and thus higher biological activity of this catechin (e.g. antioxidant activity). The purpose of this work was the formulation of EGCG using three distinct natural origin carriers, namely OSA-starch, soybean lecithin and β-glucan, by particles from gas saturated solution drying (PGSS-drying). Non-cytotoxic solid formulations of EGCG in the range of micrometers and encapsulation efficiencies up to 80.5% were obtained. An improved antioxidant activity (AA) determined by the oxygen radical absorbance capacity (ORAC) method was obtained for all formulations. Furthermore, lecithin:EGCG and β-glucan:EGCG presented higher cellular antioxidant activity (Caco-2 cells) values than free EGCG at the same concentrations tested (around 1.5-fold higher values). Moreover, the solid formulations presented preservation of the AA over 4 months, and an improved storage stability in comparison with non-encapsulated EGCG over 48 h at 328 K in the absence of light (accelerated storage). These results show that PGSS-drying conditions enabled the preservation of the bioactive properties of catechin, allowing the formulation of solid particles with enhanced features.

Recovery of antioxidant and antiproliferative compounds from watercress using pressurized fluid extraction

In this work, pressurized fluid extraction was explored to recover isothiocyanates (ITCs) and phenolic compounds from watercress. Pretreatment of the raw material was studied, using different conditions of temperature (25 °C; 35 °C), pressure (Patm; 25 MPa), incubation time (0; 30; 60; 120 min) and moisture content (125; 250; 900% of water, dry basis), aiming at promoting the enzymatic hydrolysis of glucosinolates in ITCs. Extractions of ITCs were performed with supercritical CO2, and different mixtures of CO2 : ethanol (0–50, % w/w) were applied to obtain phenolic-enriched ITCs extracts. Extractions were performed at 35 °C and 25 MPa for 2 h and extracts analyzed concerning total ITCs, phenethyl isothiocyanate (PEITC) content, phenolic composition, antioxidant capacity (using ORAC, HORAC and HOSC assays) and antiproliferative effect in a human colorectal cancer cell line (HT-29). Results showed that supercritical CO2 was highly selective in isolating ITCs from watercress (up to 31.7 ± 1.6 μmol ITC per g). When mixtures of CO2 : ethanol were used, extraction of phenolics (up to 10.1 ± 0.8 mg GAE per g) and antioxidants (up to 204.4 ± 21.5 μmol TE per g concerning ORAC, 70.8 ± 10.7 μmol CAE per g for HORAC and 189.5 ± 22.9 μmol TE per g for HOSC), was promoted. PEITC was the main compound responsible for the inhibition of cancer cell growth of all extracts (EC5024 h = 27.8 ± 1.9 μM PEITC). However, extracts obtained by supercritical CO2 extraction after a 30 minute incubation period with CO2 and with CO2 : ethanol mixtures (80 : 20 and 60 : 40, % w/w) were revealed to be effective in isolating other bioactive compounds that enhanced the antiproliferative response of extracts (EC5024 h values of 23.1 ± 0.9; 20.7 ± 1.9 and 19.8 ± 0.7 μM PEITC, respectively).

Formulation of pea protein for increased satiety and improved foaming properties

Pea protein has been associated with promoting the satiety effect. One of the issues associated with the incorporation of pea protein in food products is the product homogeneity due to its solubility and dispersibility issues. Within this context, one goal of this study was to exploit the use of supercritical fluid technology to develop Solid Lipid Pea Particle (SLPP) aiming at improving dispersibility in fat-based products. PP was encapsulated by the PGSS® (Particles from Gas Saturated Solutions) technique into glyceryl dipalmitostearate (E471) and olive oil. Different process conditions, namely pressure (7.3–20.7 MPa), temperature (51–75 °C) and equilibrium time (3–37 min) were tested in order to optimize the encapsulation of pea protein via Response Surface Methodology (RSM), following a Central Composite Rotatable Design (CCRD). Results showed that pressure and the interaction between pressure and temperature had a significant impact (p < 0.05) on the protein load and thus on the encapsulation efficiency. The highest encapsulation efficiency (96%) was achieved at 14 MPa, 51 °C and 20 min. Under these conditions, SLPP presented 0.15 mg of protein per mg of particles and 84% of lipase inhibitory activity. When compared with the PP (non-encapsulated), liposoluble pea protein particles contributed to a better product homogenization. The food industry can also take advantage of the ability of pea protein for foam stabilization in aqueous food products. Therefore, PP was treated with high-pressure supercritical CO2 treatment (HPT-scCO2) that has led to improved foaming properties when compared with the non-treated PP.

Application of RPMI 2650 as a cell model to evaluate solid formulations for intranasal delivery of drugs.

During the development of intranasal drug delivery systems for local/systemic effect or brain targeting, it is necessary to assess its cytotoxicity and drug transport through nasal epithelium. In order to avoid animal experiments or the use of excised tissues, in vitro cell models, such as RPMI 2650 cells, are being preferred during recent years. Nevertheless, the deposition of solid formulations into nasal cell layers with further transepithelial transport rate of drugs has been poorly studied or reported. Thus, the purpose of this work is to further investigate RPMI 2650 cell line as an effective alternative to animal tissues for solid drug-loaded formulations cytotoxicity and drug permeation studies in order to become an option as a tool for drug discovery. Furthermore, we wanted to determine the extent to which the administration of drugs in particulate forms would differ in relation to the permeability of the same compounds applied as solutions. RPMI 2650 cells were cultured in submersed or at air-liquid interface conditions and characterized regarding transepithelial electrical resistance (TEER) and production of mucus. Pure ketoprofen (used as model compound) and five formulations loaded with same drug, namely solid lipid particles (Gelucire 43/01™), structured lipid particles (Gelucire 43/01™:Glyceryl monooleate) and aerogel microparticles (Alginate, Alginate:Pectin, Alginate:Carrageenan), were evaluated with RPMI 2650 model in terms of cytotoxicity and permeability of drug (applied as solution, dispersion or powder+buffer). RPMI 2650 cells were capable to grow in monolayer and multilayer, showing the same permeability as excised human nasal mucosa for sodium fluorescein (paracellular marker), with analogous TEER values and production of mucus, as referred by other authors. None of the powders showed cytotoxicity when applied to RPMI 2650 cells. Regarding permeation of drug through cell layers, not only the form of application of powders but also their physical and chemical properties affected the final permeation of active pharmaceutical ingredient. Aerogel microparticles administered directly to the cell layer (powder+buffer) exhibited the highest permeation-enhancing effect compared to the pure drug, which can be attributed to the mucoadhesive properties of the materials composing the carriers, proving to be an attractive formulation for nasal drug delivery. According to these results, RPMI 2650 showed to be a promising alternative to ex vivo or in vivo nasal models for cytotoxicity and evaluation of drug permeability of nasal drug-loaded formulations.

Microencapsulation of α-tocopherol with zein and β-cyclodextrin using spray drying for colour stability and shelf-life improvement of fruit beverages

α-Tocopherol (TOC) has become an important food additive due to its antioxidant effect and capacity to increase shelf-life of products. However, it is a hydrophobically active compound, which compromises its applicability in soft drinks and fruit beverages. The aim of this study was to develop sustained release forms of α-tocopherol using zein and cyclodextrin as carriers to be further applied in fruit beverage products for improved shelf-life. For this purpose, the encapsulation of α-tocopherol into zein protein was first optimized using a spray-drying technology. Different process conditions, namely temperature (110–180 °C) and solids concentration (0.01–0.04 g mL−1), were tested aiming at maximizing the encapsulation of α-tocopherol and antioxidant capacity using response surface methodology (RSM) following a Face Centred Design (FCD). Results showed that lower inlet gas temperature and lower solids concentration enable the production of α-tocopherol:zein forms with a higher antioxidant activity (oxygen radical absorbance capacity >90 μmol Trolox equivalent antioxidant capacity per g), and the highest encapsulation efficiency (42.05%) was obtained using 0.02 g mL−1 of solids concentration. β-Cyclodextrin was further added to the best system to increase the solubility capacity of α-tocopherol in fruit juice beverages. Despite the lower antioxidant activity (46.2 μmol Trolox equivalent antioxidant capacity per g), α-tocopherol:zein:cyclodextrin forms reduced colour losses when compared to the normal juice, and this effect was probably related to the suppression of anthocyanin degradation due to the formation of inclusion complexes with β-cyclodextrin. Importantly, none of these forms induced cytotoxicity in the Caco2 cell model, confirming the safety of these systems.