Assessing the Influence of Betaine-Based Natural Deep Eutectic Systems on Horseradish Peroxidase.

To validate the use of horseradish peroxidase (HRP) in natural deep eutectic systems (NADES), five different betaine-based NADES were characterized in terms of water content, water activity, density, and viscosity experimentally and by thermodynamic modeling. The results show that the NADES under study have a water activity of about 0.4 at 37 °C for water contents between 14 and 22 wt %. The densities of the studied NADES had values between 1.2 and 1.3 g.cm-3 at 20 °C. The density was modeled with a state-of-the-art equation of state; an excellent agreement with the experimental density data was achieved, allowing reasonable predictions for water activities. The system betaine:glycerol (1:2) was found to be the most viscous with a dynamic viscosity of ∼600 mPa.s at 40 °C, while all the other systems had viscosities <350 mPa.s at 40 °C. The impact of the NADES on the enzymatic activity, as well as on, conformational and thermal stability was assessed. The system betaine/sorbitol:water (1:1:3) showed the highest benefit for enzymatic activity, increasing it by two-folds. Moreover, upon NADES addition, thermal stability was increased followed by an increment in a-helix secondary structure content.

Subcritical Water as a Pre-Treatment of Mixed Microbial Biomass for the Extraction of Polyhydroxyalkanoates.

Polyhydroxyalkanoate (PHA) recovery from microbial cells relies on either solvent extraction (usually using halogenated solvents) and/or digestion of the non-PHA cell mass (NPCM) by the action of chemicals (e.g., hypochlorite) that raise environmental and health hazards. A greener alternative for PHA recovery, subcritical water (SBW), was evaluated as a method for the dissolution of the NPCM of a mixed microbial culture (MMC) biomass. A temperature of 150 °C was found as a compromise to reach NPCM solubilization while mostly preventing the degradation of the biopolymer during the procedure. Such conditions yielded a polymer with a purity of 77%. PHA purity was further improved by combining the SBW treatment with hypochlorite digestion, in which a significantly lower hypochlorite concentration (0.1%, v/v) was sufficient to achieve an overall polymer purity of 80%. During the procedure, the biopolymer suffered some depolymerization, as evidenced by the lower molecular weight (Mw) and higher polydispersity of the extracted samples. Although such changes in the biopolymer's molecular mass distribution impact its mechanical properties, impairing its utilization in most conventional plastic uses, the obtained PHA can find use in several applications, for example as additives or for the preparation of graft or block co-polymers, in which low-Mw oligomers are sought.

Assessment of deep eutectic solvents toxicity in zebrafish (Danio rerio).

Deep Eutectic Systems (DES) have emerged as a "green alternative" to organic solvents and have been coined as biocompatible and biodegradable. However, the number of studies concerning the real biodegradability and biocompatibility are scarce. Thus, to study the toxicity of certain DES, two different approaches were used: i) zebrafish exposure via water, where the system (DES) was tested at potentially realistic environmental concentrations and ii) via intraperitoneal injection, where the system was tested in different concentrations, relevant to the pharmaceutical industry. These studies were performed using zebrafish, a standardized animal model often used in biomedicine and toxicological assays. The results show low toxicity according to tested concentrations (up to 73.47 µM), when the system CA:T:W, with a 2:1:3 molar ratio, was tested through exposure via water and also in the intraperitoneal injection tests with concentrations up to 6000 µM. The activity of different enzymes involved in antioxidant pathways (glutathione S-transferase, catalase, glutathione peroxidase), the total antioxidant capacity (TAC) and lipoperoxidation (MDA content) were determined suggesting low toxicity of the tested system (DES). The promising results herein presented show that DES present the potential to be used as the new class of green solvents, not only for use in the pharmaceutical industry, but also in cosmetic and chemical engineering processes without causing negative impact on living organisms.

Supercritical CO2 Assisted Impregnation of Ibuprofen on Medium-Chain-Length Polyhydroxyalkanoates (mcl-PHA).

In this work, we propose the utilization of scCO2 to impregnate ibuprofen into the mcl-PHA matrix produced by Pseudomonas chlororaphis subs. aurantiaca (DSM 19603). The biopolymer has adhesive properties, is biocompatible and has a melting temperature of 45 °C. Several conditions, namely, pressure (15 and 20 MPa) and impregnation time (30 min, 1 h and 3 h) were tested. The highest ibuprofen content (90.8 ± 6.5 mg of ibuprofen/gPHA) was obtained at 20 MPa and 40 °C, for 1 h, with an impregnation rate of 89 mg/(g·h). The processed mcl-PHA samples suffered a plasticization, as shown by the decrease of 6.5 °C in the Tg, at 20 MPa. The polymer's crystallinity was also affected concomitantly with the matrices' ibuprofen content. For all the impregnation conditions tested the release of ibuprofen from the biopolymer followed a type II release profile. This study has demonstrated that the mcl-PHA produced by P. chlororaphis has a great potential for the development of novel topical drug delivery systems.

Improved storage of influenza HA-VLPs using a trehalose-glycerol natural deep eutectic solvent system.

Vaccines are typically stored under refrigerated conditions (2-8 °C) to limit potency and efficacy loss. Maintaining the cold chain is costly and prone to vaccine wastage due to unexpected changes in the storage conditions. The development of formulations conferring enhanced stability can extend vaccine shelf-life and facilitate storage under non-refrigerated conditions, thus simplifying the distribution process and reducing vaccine wastage. In this study, the suitability of a natural deep eutectic system (NADES) consisting of trehalose and glycerol (TGly) for storage of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs) was successfully demonstrated. TGly was efficient in maintaining the activity and physical integrity of HA-VLPs for up to 4 h at 50 °C (accelerated stability study), with half-life values ≈ 20-34 h for the best TGly formulations. Importantly, improved storage is achieved at increasingly higher TGly concentrations, hence confirming the importance of TGly content in its protective capacity. In addition, HA-VLPs were stable in TGly for over one month at room temperature (short-term stability study), with no impact on HA titer or particle size distribution. This work highlights the potential of NADES to improve stability of VLP-based vaccines, showing promising protective capacity under non-refrigerated conditions and short-term thermal stress, and thus having a notable impact on vaccine's cold chain.

Natural deep eutectic systems, an emerging class of cryoprotectant agents.

This work aimed at evaluating the potential of using natural deep eutectic systems (NADES) as cryoprotectant agents (CPAs). Several combinations between natural primary metabolites that have been identified in animals that live in extreme cold climates were prepared. All systems showed very little cytoxicity towards L929 cells at concentrations high as 1-2 M. Moreover, this cell line was highly tolerant to 10% (w/v) of NADES when compared to Me2SO. To test NADES as CPAs, two cell lines were used, L929 and HacaT cells. After freeze/thawing cycle, it was possible to observe that for L929 cells, NADES presented similar behaviour to Me2SO. For Hacat cell line a significant improvement on post-thawing recovery was observed. Moreover, the results presented herein showed that NADES do not need to be removed from the freezing media after thawing the cells, which is a great advantage of these materials. Additionally, we have shown that NADES can act as CPA when cells are frozen at -20 °C. In overall, the results demonstrate the high potential of NADES to be used in cryobiology as alternative CPAs.

Pseudomonas chlororaphis as a multiproduct platform: Conversion of glycerol into high-value biopolymers and phenazines.

Pseudomonas chlororaphis subsp. aurantiaca DSM 19603 was cultivated using glycerol as the sole carbon source for the simultaneous production of medium-chain length polyhydroxyalkanoates (mcl-PHA), extracellular polysaccharide (EPS) and phenazines. A maximum cell dry mass of 11.79 g/L was achieved with a mcl-PHA content of 19 wt%, corresponding to a polymer concentration of 2.23 g/L. A considerably higher EPS production, 6.10 g/L, was attained. Phenazines synthesis was evidenced by the bright orange coloration developed by the culture during the cell growth phase. The mcl-PHA produced by P. chlororaphis was composed mainly of 3-hydroxydecanoate (50 wt%) with lower amounts of 3-hydroxyoctanoate (17 wt%), 3-hydroxytetradecanoate (17 wt%), 3-hydroxydodecanoate (13 wt%) and 3-hydroxyhexanoate (3 wt%). This PHA showed unique thermal features being highly amorphous, with a degree of crystallinity of 27% and a low melting temperature (45.0 °C). The secreted EPS was mostly composed of glucose, glucosamine, rhamnose and mannose, with smaller amounts of three other unidentified monomers. Although the bioprocess can be improved further to define the optimal conditions to produce each bioproduct (mcl-PHA, EPS or phenazines), this study has demonstrated for the first time the ability of P. chlororaphis to simultaneously produce three high-value products from a single substrate.

Preparation of Binary and Ternary Deep Eutectic Systems.

The preparation of deep eutectic systems (DES) is a priori a simple procedure. By definition, two or more components are mixed together at a given molar ratio to form a DES. However, from our experience in the laboratory, there is a need to standardize the procedure to prepare, characterize and report the methodologies followed by different researchers, so that the results published can be reproduced. In this work, we test different approaches reported in the literature to prepare eutectic systems and evaluated the importance of water in the successful preparation of liquid systems at room temperature. These published eutectic systems were composed of citric acid, glucose, sucrose, malic acid, ß-alanine, L-tartaric acid and betaine and not all of preparation methods described could be reproduced. However, in some cases, it was possible to reproduce the systems described, with the inclusion of water as a third component of the eutectic mixture.