Beatriz Salesa

Pyriproxyfen is an insecticide currently employed in numerous countries for the management of agricultural and indoor pests. Several studies indicate that this insecticide has been detected in multiple rivers, with concentrations reaching as high as 99.59 ng/L in the Júcar River in Spain. Therefore, the determination of some biochemical and genetic effects of this insecticide on aquatic organisms could serve as an early warning mechanism to identify potential disruptions in various biomarkers. Based on this, Daphnia magna organisms were exposed to pyriproxyfen sublethal concentrations for 21 days. Some biochemical parameters, including cholesterol, triglycerides, glucose, lactate, and LDH activity, were determined. Additionally, some genetic biomarkers associated with oxidative stress, heat shock proteins, lipid metabolism, hemoglobin, metallothioneins, and vitellogenin synthesis were evaluated in daphnids exposed to the insecticide for 21 days. LDH activity increased significantly in those daphnids exposed to the highest insecticide concentration (14.02 µg/L), while cholesterol levels decreased significantly. In contrast, glucose, total proteins, and triglycerides remained unaffected in D. magna exposed to pyriproxyfen. On the other hand, exposure to the insecticide led to notable alterations in gene expression among individuals. Specifically, genes associated with lipid metabolism and reproduction exhibited a significant reduction in gene expression. Fabd expression was decreased by approximately 20% in exposed daphnids, while vtg expression was suppressed as much as 80% when compared to control values. Furthermore, it was observed that the hgb1 and hgb2 genes, associated with hemoglobin synthesis, exhibited significant overexpression. Notably, the dysfunction observed in both hemoglobin genes was linked to an increase in pigmentation in Daphnia magna during the course of the experiment. These alterations in gene expression could serve as effective indicators of early contamination even at low pesticide concentrations.

A new strategy based on the combination of electrically conductive polymer nanocomposites and extracellular Zn²⁺ ions as a myogenic factor was developed to assess its ability to synergically stimulate myogenic cell response. The conductive nanocomposite was prepared with a polymeric matrix and a small amount of graphene (G) nanosheets (0.7% wt/wt) as conductive filler to produce an electrically conductive surface. The nanocomposites’ surface electrical conductivity presented values in the range of human skeletal muscle tissue. The biological evaluation of the cell environment created by the combination of the conductive surface and extracellular Zn²⁺ ions showed no cytotoxicity and good cell adhesion (murine C2C12 myoblasts). Amazingly, the combined strategy, cell–material interface with conductive properties and Zn bioactive ions, was found to have a pronounced synergistic effect on myoblast proliferation and the early stages of differentiation. The ratio of differentiated myoblasts cultured on the conductive nanocomposites with extracellular Zn²⁺ ions added in the differentiation medium (serum-deprived medium) was enhanced by more than 170% over that of non-conductive surfaces (only the polymeric matrix), and more than 120% over both conductive substrates (without extracellular Zn²⁺ ions) and non-conductive substrates with extracellular Zn²⁺. This synergistic effect was also found to increase myotube density, myotube area and diameter, and multinucleated myotube formation. MyoD-1 gene expression was also enhanced, indicating the positive effect in the early stages of myogenic differentiation. These results demonstrate the great potential of this combined strategy, which stands outs for its simplicity and robustness, for skeletal muscle tissue engineering applications.

Multi-layer graphene (2–10 layers), also called graphene nanoplatelets (GNPs), is a carbon-based nanomaterial (CBN) type with excellent properties desirable for many biomedical applications. Despite the promising advantages reported of GNPs, nanoscale materials may also present a potential hazard to humans. Therefore, in this study, the in vivo toxicity of these nanomaterials at a wide range of concentrations from 12.5 to 500 µg/mL was evaluated in the Caenorhabditis elegans model for 24 h (acute toxicity) and 72 h (chronic toxicity). Furthermore, their in vitro toxicity (from 0 to 10 µg/mL for 12 and 24 h), proliferative activity at 72 and 96 h, and their effect on the expression of thirteen genes in human keratinocytes HaCaT cells were studied. The physico-chemical and morphological aspects of the GNPs used in this study were analyzed by Raman scattering spectroscopy, electron microscopy, zeta potential as a function of pH, and particle size measurements by dynamic light scattering. The results of this study showed that GNPs showed in vivo non-toxic concentrations of 25 and 12.5 µg/mL for 24 h, and at 12.5 µg/mL for 72 h. Moreover, GNPs present time-dependent cytotoxicity (EC₅₀ of 1.142 µg/mL and 0.760 µg/mL at 12 h and 24 h, respectively) and significant proliferative activity at the non-toxic concentrations of 0.005 and 0.01 μg/mL in the HaCaT cell line. The gene expression study showed that this multi-layer-graphene is capable of up-regulating six of the thirteen genes of human keratinocytes (SOD1, CAT, TGFB1, FN1, CDH1, and FBN), two more genes than other CBNs in their oxidized form such as multi-layer graphene oxide. Therefore, all these results reinforce the promising use of these CBNs in biomedical fields such as wound healing and skin tissue engineering.

The use of ionic metals such as zinc (Zn²⁺) is providing promising results in regenerative medicine. In this study, human keratinocytes (HaCaT cells) were treated with different concentrations of zinc chloride (ZnCl₂), ranging from 1 to 800 µg/mL, for 3, 12 and 24 h. The results showed a time–concentration dependence with three non-cytotoxic concentrations (10, 5 and 1 µg/mL) and a median effective concentration value of 13.5 µg/mL at a cell exposure to ZnCl₂ of 24 h. However, the zinc treatment with 5 or 1 µg/mL had no effect on cell proliferation in HaCaT cells in relation to the control sample at 72 h. The effects of the Zn²⁺ treatment on the expression of several genes related to glycoprotein synthesis, oxidative stress, proliferation and differentiation were assessed at the two lowest non-cytotoxic concentrations after 24 h of treatment. Out of 13 analyzed genes (superoxide dismutase 1 (SOD1), catalase (CAT), matrix metallopeptidase 1 (MMP1), transforming growth factor beta 1 (TGFB1), glutathione peroxidase 1 (GPX1), fibronectin 1 (FN1), hyaluronan synthase 2 (HAS2), laminin subunit beta 1 (LAMB1), lumican (LUM), cadherin 1 (CDH1), collagen type IV alpha (COL4A1), fibrillin (FBN) and versican (VCAN)), Zn²⁺ was able to upregulate SOD1, CAT, TGFB1, GPX1, LUM, CDH1, FBN and VCAN, with relative expression levels of at least 1.9-fold with respect to controls. We found that ZnCl₂ promoted glycoprotein synthesis and antioxidant gene expression, thus confirming its great potential in biomedicine.

Carbon nanofibers (CNFs) are one-dimensional nanomaterials with excellent physical and broad-spectrum antimicrobial properties characterized by a low risk of antimicrobial resistance. Silver nanoparticles (AgNPs) are antimicrobial metallic nanomaterials already used in a broad range of industrial applications. In the present study these two nanomaterials were characterized by Raman spectroscopy, transmission electron microscopy, zeta potential, and dynamic light scattering, and their biological properties were compared in terms of cytotoxicity, proliferation, and gene expression in human keratinocyte HaCaT cells. The results showed that both AgNPs and CNFs present similar time-dependent cytotoxicity (EC₅₀ of 608.1 µg/mL for CNFs and 581.9 µg/mL for AgNPs at 24 h) and similar proliferative HaCaT cell activity. However, both nanomaterials showed very different results in the expression of thirteen genes (superoxide dismutase 1 (SOD1), catalase (CAT), matrix metallopeptidase 1 (MMP1), transforming growth factor beta 1 (TGFB1), glutathione peroxidase 1 (GPX1), fibronectin 1 (FN1), hyaluronan synthase 2 (HAS2), laminin subunit beta 1 (LAMB1), lumican (LUM), cadherin 1 CDH1, collagen type IV alpha (COL4A1), fibrillin (FBN), and versican (VCAN)) treated with the lowest non-cytotoxic concentrations in the HaCaT cells after 24 h. The AgNPs were capable of up-regulating only two genes (SOD1 and MMP1) while the CNFs were very effective in up-regulating eight genes (FN1, MMP1, CAT, CDH1, COL4A1, FBN, GPX1, and TGFB1) involved in the defense mechanisms against oxidative stress and maintaining and repairing tissues by regulating cell adhesion, migration, proliferation, differentiation, growth, morphogenesis, and tissue development. These results demonstrate CNF nanomaterials’ unique great potential in biomedical applications such as tissue engineering and wound healing.

Few-layer graphene oxide (GO) has shown no or very weak cytotoxicity and anti-proliferative effects in a wide range of cell lines, such as glioma cells and human skin HaCaT cells at concentrations up to 100 µg/mL. However, as multi-layer GO has hardly been explored in the biomedical field, in this study, this other type of GO was examined in human keratinocyte HaCaT cells treated with different concentrations, ranging from 0.01 to 150 µg/mL, for different periods of time (3, 12, and 24 h). The results revealed a time–concentration dependence with two non-cytotoxic concentrations (0.01 and 0.05 µg/mL) and a median effective concentration value of 4.087 µg/mL at 24 h GO exposure. Contrary to what has previously been reported for few-layer GO, cell proliferation of the HaCaT cells in contact with the multi-layer GO at 0.01 μg/mL showed identical proliferative activity to an epidermal growth factor (1.6-fold greater than the control group) after 96 h. The effects of the multi-layer GO on the expression of 13 genes (SOD1, CAT, MMP1, TGFB1, GPX1, FN1, HAS2, LAMB1, LUM, CDH1, COL4A1, FBN, and VCAN) at non-cytotoxic concentrations of GO in the HaCaT cells were analyzed after 24 h. The lowest non-cytotoxic GO concentration was able to upregulate the CAT, TGFB1, FN1, and CDH1 genes, which confirms multi-layer GO’s great potential in the biomedical field.