Hitting two targets with one shot on pesticide genotoxicity assessment - Identifying risk while unveiling ex vivo approach as a throughput tool in gill-breathing animals.

Pesticides in the environment often compromise the ecosystem, thus requiring reliable approaches to assess their effects. Commonly used approaches, such as in vivo, come with several disadvantages, namely in the light of the 3 R's policy. Seeking for accurate and ethical approaches, this study intended to validate the ex vivo technique as an alternative, and to assess the genotoxicity of chemically-based pesticides and a biopesticide. The ex vivo approach was applied to gill cells of Procambarus clarkii for 2, 4 and 8 h. Cell viability and DNA integrity were evaluated to determine the applicability of this approach. Crayfish gill cells only showed to be suitable for exposures of 2 h. Accordingly, genotoxicity was evaluated in gill cells exposed, for 2 h, to environmentally relevant concentrations of the chemically-based pesticides dimethoate (20 µg L-1), imazalil (160 µg L-1) and penoxsulam (23 µg L-1), as well as to the bioinsecticide Turex® (25, 50, 100, 200 and 400 µg L-1). Every chemically-based pesticide demonstrated to be genotoxic, despite not inducing oxidative DNA damage. On the other hand, Turex® showed no genotoxic effects. Overall, the ex vivo approach demonstrated to be possible and practical to implement, improving the number of outcomes with a lower number of organisms. The findings from the screening test suggest that biological pesticides may pose a lower risk to non-target organisms compared to chemically-based pesticides.

Biopesticide Turex®'s cytotoxicity, genotoxicity and cell cycle arrest on HepG2 cell line.

Population growth leads to the need for more efficient techniques and compounds in agriculture, such as pesticides, to deal with the ever-growing demand. Pesticides may end up in the environment, often compromising the ecosystem affecting all organisms including humans. Therefore, the consequences of exposure to these compounds to biota and humans needs to be assessed. Bearing this in mind, the aim of this study was to examine the in vitro cytotoxicity and genotoxicity attributed to exposure to the biopesticide Turex® utilizing the liver cell line HepG2. Cells were incubated with biopesticide Turex® at 250, 500, 1000, 1500 or 2000 µg/L in both non-activated and activated forms for 24 and 48 h. Subsequent effects on cell viability were assessed using the MTT. The influence on cell cycle dynamics was determined by flow cytometry, while DNA damage was measured by the comet assay. Data demonstrated that activated Turex® induced cytotoxicity and DNA damage after 48 h in HepG2 cell line. The cell cycle progression was not markedly affected by Turex® at any concentration or duration of exposure. In conclusion, data demonstrated the potential adverse effects attributed to exposure to biopesticide Turex® in human cell line HepG2. Consequently, this type of biopesticide needs to be further investigated to determine the potential adverse in vivo effects on various non-target organisms.

Stability, dissolution, and cytotoxicity of NaYF4-upconversion nanoparticles with different coatings.

Upconversion nanoparticles (UCNPs) have attracted considerable attention owing to their unique photophysical properties. Their utilization in biomedical applications depends on the understanding of their transformations under physiological conditions and their potential toxicity. In this study, NaYF4:Yb,Er UCNPs, widely used for luminescence and photophysical studies, were modified with a set of four different coordinatively bound surface ligands, i.e., citrate, alendronate (AA), ethylendiamine tetra(methylene phosphonate) (EDTMP), and poly(maleic anhydride-alt-1-octadecene) (PMAO), as well as silica coatings with two different thicknesses. Subsequently, the aging-induced release of fluoride ions in water and cell culture media and their cytotoxic profile to human keratinocytes were assessed in parallel to the cytotoxic evaluation of the ligands, sodium fluoride and the lanthanide ions. The cytotoxicity studies of UCNPs with different surface modifications demonstrated the good biocompatibility of EDTMP-UCNPs and PMAO-UCNPs, which is in line with the low amount of fluoride ions released from these samples. An efficient prevention of UCNP dissolution and release of cytotoxic ions, as well as low cytotoxicity was also observed for UCNPs with a sufficiently thick silica shell. Overall, our results provide new insights into the understanding of the contribution of surface chemistry to the stability, dissolution behavior, and cytotoxicity of UCNPs. Altogether, the results obtained are highly important for future applications of UCNPs in the life sciences and bioimaging studies.

Boosting antibiotics performance by new formulations with deep eutectic solvents.

The critical scenario of antimicrobial resistance to antibiotics highlights the need for improved therapeutics and/or formulations. Herein, we demonstrate that deep eutectic solvents (DES) formulations are very promising to remarkably improve the solubility, stability and therapeutic efficacy of antibiotics, such as ciprofloxacin. DES aqueous solutions enhance the solubility of ciprofloxacin up to 430-fold while extending the antibiotic stability. The developed formulations can improve, by 2 to 4-fold, the susceptibility of Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria to the antibiotic. They also improve the therapeutic efficacy at concentrations where bacteria present resistance, without promoting tolerance development to ciprofloxacin. Furthermore, the incorporation of DES decreases the toxicity of ciprofloxacin towards immortalized human epidermal keratinocytes (HaCat cells). The results herein reveal the pioneering use of DES in fluoroquinolone-based formulations and their impact on the antibiotic's characteristics and on its therapeutic action.

3D Printing of Macro Porous Sol-Gel Derived Bioactive Glass Scaffolds and Assessment of Biological Response.

3D printing emerged as a potential game-changer in the field of biomedical engineering. Robocasting in particular has shown excellent capability to produce custom-sized porous scaffolds from pastes with suitable viscoelastic properties. The materials and respective processing methods developed so far still need further improvements in order to obtain completely satisfactory scaffolds capable of providing both the biological and mechanical properties required for successful and comprehensive bone tissue regeneration. This work reports on the sol-gel synthesis of an alkali-free bioactive glass and on its characterization and processing ability towards the fabrication of porous scaffolds by robocasting. A two-fold increase in milling efficiency was achieved by suitably adjusting the milling procedures. The heat treatment temperature exerted a profound effect on the surface area of mesoporous powders. Robocasting inks containing 35 vol.% solids were prepared, and their flow properties were characterized by rheological tests. A script capable of preparing customizable CAD scaffold geometries was developed. The printing process was adjusted to increase the technique's resolution. The mechanical properties of the scaffolds were assessed through compressive strength tests. The biomineralization ability and the biological performance were assessed by immersing the samples in simulated body fluid (SBF) and through MTT assays, respectively. The overall results demonstrated that scaffolds with macro porous features suitable for bone ingrowth (pore sizes of ~340 µm after sintering, and a porosity fraction of ~70%) in non-load-bearing applications could be successfully fabricated by 3D printing from the bioactive glass inks. Moreover, the scaffolds exhibited good biomineralization activity and good biocompatibility with human keratinocytes, suggesting they are safe and thus suited for the intended biomedical applications.

Cellulose Nanocrystals/Chitosan-Based Nanosystems: Synthesis, Characterization, and Cellular Uptake on Breast Cancer Cells.

Cellulose nanocrystals (CNCs) are elongated biobased nanostructures with unique characteristics that can be explored as nanosystems in cancer treatment. Herein, the synthesis, characterization, and cellular uptake on folate receptor (FR)-positive breast cancer cells of nanosystems based on CNCs and a chitosan (CS) derivative are investigated. The physical adsorption of the CS derivative, containing a targeting ligand (folic acid, FA) and an imaging agent (fluorescein isothiocyanate, FITC), on the surface of the CNCs was studied as an eco-friendly methodology to functionalize CNCs. The fluorescent CNCs/FA-CS-FITC nanosystems with a rod-like morphology showed good stability in simulated physiological and non-physiological conditions and non-cytotoxicity towards MDA-MB-231 breast cancer cells. These functionalized CNCs presented a concentration-dependent cellular internalization with a 5-fold increase in the fluorescence intensity for the nanosystem with the higher FA content. Furthermore, the exometabolic profile of the MDA-MB-231 cells exposed to the CNCs/FA-CS-FITC nanosystems disclosed a moderate impact on the cells' metabolic activity, limited to decreased choline uptake and increased acetate release, which implies an anti-proliferative effect. The overall results demonstrate that the CNCs/FA-CS-FITC nanosystems, prepared by an eco-friendly approach, have a high affinity towards FR-positive cancer cells and thus might be applied as nanocarriers with imaging properties for active targeted therapy.

Encapsulation and Enhanced Release of Resveratrol from Mesoporous Silica Nanoparticles for Melanoma Therapy.

Chemotherapy has limited success in the treatment of malignant melanoma due to fast development of drug resistance and the low bioavailability of chemotherapeutic drugs. Resveratrol (RES) is a natural polyphenol with recognized preventive and therapeutic anti-cancer properties. However, poor RES solubility hampers its bioactivity, thus creating a demand for suitable drug delivery systems to improve it. This work aimed to assess the potential of RES-loaded mesoporous silica nanoparticles (MSNs) for human melanoma treatment. RES was efficiently loaded (efficiency > 93%) onto spheroidal (size~60 nm) MSNs. The encapsulation promoted the amorphization of RES and enhanced the release in vitro compared to non-encapsulated RES. The RES release was pH-dependent and markedly faster at pH 5.2 (acid environment in some tumorous tissues) than at pH 7.4 in both encapsulated and bulk forms. The RES release from loaded MSNs was gradual with time, without a burst effect, and well-described by the Weibull model. In vitro cytotoxicity studies on human A375 and MNT-1 melanoma cellular cultures showed a decrease in the cell viability with increasing concentration of RES-loaded MSNs, indicating the potent action of the released RES in both cell lines. The amelanotic cell line A375 was more sensitive to RES concentration than the melanotic MNT-1 cells.

Sperm pre-fertilization thermal environment shapes offspring phenotype and performance.

The sperm pre-fertilization environment has recently been suggested to mediate remarkable transgenerational consequences for offspring phenotype (transgenerational plasticity, TGB), but the adaptive significance of the process has remained unclear. Here, we studied the transgenerational effects of sperm pre-fertilization thermal environment in a cold-adapted salmonid, the European whitefish (Coregonus lavaretus). We used a full-factorial breeding design where the eggs of five females were fertilized with the milt of 10 males that had been pre-incubated at two different temperatures (3.5°C and 6.5°C) for 15 h prior to fertilization. Thermal manipulation did not affect sperm motility, cell size, fertilization success or embryo mortality. However, offspring that were fertilized with 6.5°C-exposed milt were smaller and had poorer swimming performance than their full-siblings that had been fertilized with the 3.5°C-exposed milt. Furthermore, the effect of milt treatment on embryo mortality varied among different females (treatment×female interaction) and male-female combinations (treatment×female×male interaction). Together, these results indicate that sperm pre-fertilization thermal environment shapes offspring phenotype and post-hatching performance and modifies both the magnitude of female (dam) effects and the compatibility of the gametes. Generally, our results suggest that short-term changes in sperm thermal conditions may have negative impact for offspring fitness. Thus, sperm thermal environment may have an important role in determining the adaptation potential of organisms to climate change. Detailed mechanism(s) behind our findings require further attention.