Carbon Nanomaterials and LED Irradiation as Antibacterial Strategies against Gram-Positive Multidrug-Resistant Pathogens.

BACKGROUND: Due to current antibiotic resistance worldwide, there is an urgent need to find new alternative antibacterial approaches capable of dealing with multidrug-resistant pathogens. Most recent studies have demonstrated the antibacterial activity and non-cytotoxicity of carbon nanomaterials such as graphene oxide (GO) and carbon nanofibers (CNFs). On the other hand, light-emitting diodes (LEDs) have shown great potential in a wide range of biomedical applications. METHODS: We investigated a nanotechnological strategy consisting of GO or CNFs combined with light-emitting diod (LED) irradiation as novel nanoweapons against two clinically relevant Gram-positive multidrug-resistant pathogens: methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE). The cytotoxicity of GO and CNFs was studied in the presence of human keratinocyte HaCaT cells. RESULTS: GO or CNFs exhibited no cytotoxicity and high antibacterial activity in direct contact with MRSE and MRSA cells. Furthermore, when GO or CNFs were illuminated with LED light, the MRSE and MRSA cells lost viability. The rate of decrease in colony forming units from 0 to 3 h, measured per mL, increased to 98.5 ± 1.6% and 95.8 ± 1.4% for GO and 99.5 ± 0.6% and 99.7 ± 0.2% for CNFs. CONCLUSIONS: This combined antimicrobial approach opens up many biomedical research opportunities and provides an enhanced strategy for the prevention and treatment of Gram-positive multidrug-resistant infections.

Effect of the lipid regulator Gemfibrozil in the Cladocera Daphnia magna at different temperatures.

In the present study, an ecotoxicological approach to the evaluation of Gemfibrozil (GEM) as an emerging organic pollutant was done. In order to assess its toxicity, tests were conducted using the cladocera Daphnia magna. Experiments were carried out at 22°C and 28°C. EC50, feeding behavior, and chronic toxicity tests (21 days) were evaluated in D. magna exposed to GEM as well as cholesterol levels at 21-day chronic exposure. D. magna GEM EC50 values (24 h) in our experimental conditions were 148.75 and 116.24 mg L-1 at 22°C and 28°C, respectively. Test concentrations of 0.1, 0.5, 1.0, 5.0 and 7.5 mg L-1 were selected for subacute and chronic experiments. Subacute short-term test (feeding study) was assessed after exposure to the toxicant. Filtration and ingestion rates of D. magna exposed animals did not show any significant difference (P > 0.05) with respect to control daphniids neither at 22°C nor at 28°C. Therefore, GEM test concentrations used in the present study did not reduce feeding behavior in D. magna. Temperature increased from 22°C to 28°C, which resulted in a decrease of the daphniids reproductive parameters such as brood size and number of young per female. Other parameters as longevity were not affected. The GEM concentrations used in the chronic test with D. magna did not affect daphniids longevity but some reproductive parameters as number of young per female or brood size were affected. Finally, a significant decreased in cholesterol levels was found in those animals exposed to the highest toxicant concentrations. More studies must be done to determine the possible implications of GEM in aquatic fauna and to derive its possible effects on the environment.

Pyriproxyfen Contamination in Daphnia magna: Identifying Early Warning Biomarkers.

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.

Multigenerational effects of the insecticide Pyriproxyfen and recovery in Daphnia magna.

In the present study, an ecotoxicological approach to the evaluation of the insecticide Pyriproxifen in the crustacean Daphnia magna was done. Acute toxicity tests (48 h), feeding behavior test (5 h) and chronic toxicity test (21 days) were carried out on a parental daphnid generation (F0). Pyriproxifen D. magna EC50 value in our experimental conditions was 336.47 µg/L. Based on this result, sublethal test concentrations were selected for the feeding study and the F0 chronic experiment. Filtration and ingestion rates of D. magna exposed animals did not show any significant difference respect to control daphnids. However, daphnids from the parental F0 generation when exposed to the insecticide during 21 days showed a decreased in all the reproductive parameters tested (mean total neonates per female, mean brood size, time to first brood, and mean number of broods per female) as well as in the population statistic growth rate (r), although survival was not affected. On the other hand, offspring from F0 females exposed to the highest Pyriproxifen concentration (14.02 µg/L) were separated in two F1 generation experiments. One group was transferred during 21 days to a medium free of toxicant (F1 generation-TC group) while the other group was exposed during 21 days to the same insecticide concentration as their mothers (14.02 µg/L) (F1 generation-TT group). Results from both experiments determined a decreased in most of the reproductive parameters which was higher in the F1-TT group, although some of them were recovered in the F1-TC group. On the other hand, the morphological analysis of the daphnids showed that the coloration pattern was altered in the daphnids exposed to the insecticide, together with a significant size decreased, and neonates from F0 progeny with the same morphological abnormality. Finally, we determined that the insecticide caused the appearance of males among the offspring generated by the F0.

Study of biological properties of gold nanoparticles: Low toxicity, no proliferative activity, no ability to induce cell gene expression and no antiviral activity.

Gold nanoparticles (AuNPs) are a fundamental building block of many applications across nanotechnology as they have excellent biosafety which make them promising for a broad range of biomedical applications. Here we explore their in vivo toxicity, cytotoxicity and proliferative capacity in human keratinocyte HaCaT cells, their ability to induce gene expression and their antiviral properties against a surrogate of SARS-CoV-2. These nanoparticles were characterized by transmission electron microscopy, dynamic light scattering and zeta potential. The results showed that these AuNPs with sizes ranging from 10 to 60 nm are non-toxic in vivo at any concentration up to 800 µg/mL. However, AuNP cytotoxicity in human HaCaT cells is time-dependent, so that concentrations of up to 300 µg/mL did not show any in vitro toxic effect at 3, 12 and 24 h, although higher concentrations were found to have some significant toxic activity, especially at 24 h. No significant proliferative activity was observed when using low AuNP concentrations (10, 20 and 40 µg/mL), while the AuNP antiviral tests indicated low or insignificant antiviral activity. Surprisingly, none of the 13 analyzed genes had their expressions modified after 24 h's exposure to AuNPs. Therefore, the results show that AuNPs are highly stable inactive materials and thus very promising for biomedical and clinical applications demanding this type of materials.

Effect of nutrition and Enteromyxum leei infection on gilthead sea bream Sparus aurata intestinal carbohydrate distribution.

The effect of a practical plant protein-based diet containing vegetable oils (VO) as the major lipid source on the mucosal carbohydrate pattern of the intestine was studied in gilthead sea bream Sparus aurata challenged with the myxosporean parasite Enteromyxum leei. Fish fed for 9 mo either a fish oil (FO) diet or a blend of VO at 66% of replacement (66VO diet) were exposed to parasite-contaminated water effluent. Samples of the anterior, middle and posterior intestine (AI, MI and PI, respectively) were obtained for parasite diagnosis and histochemistry. Fish were categorised as control (C, not exposed), early (E) or late (L) infected. Mucin and lectin histochemistry was applied to detect the different types of mucins and sialic acid in goblet cells (GC), the brush border and enterocytes. The number of GC stained with periodic acid Schiff (PAS), alcian blue (AB), aldehyde fuchsin-alcian blue (AF-AB), for the detection of neutral, acidic, sulphated and carboxylic mucins, and with the lectin Sambucus nigra agglutinin (SNA), were counted in digital images. The 66VO diet produced a significant decrease of GC with neutral and acidic mucins in the AI and MI, and also of those with carboxylic mucins and sialic acid in the MI. Sulphated mucins and sialic acid were less abundant in the AI than in the MI and PI in the C-66VO treatment. E. leei infection had a strong effect on the number of GC, as E and L infected fish had a significant decrease of GC positive for all the stains versus C fish in PI. Time and diet effects were also observed, since the lowest values were mostly registered in E-66VO fish in PI. In conclusion, though GC depletion was mainly induced by enteromyxosis, an effect of the diet was also observed. Thus, the diet can be a predisposing factor that worsens the disease course.

The prochloraz chronic exposure to Daphnia magna derived in biochemical alterations of F0 generation daphnids and malformed F1 progeny.

In the present study, D. magna individuals were exposed to several sublethal prochloraz concentrations (87, 130, 170, 230 and 380 µg/L) for 21 days according to; the previous acute toxicity results. The fungicide effects on reproduction, survival, individual size, and growth population rate were evaluated after an exposure of 21 days, and no changes were observed compared to the control group. On the other hand, F1 generation neonates were collected and their external morphology evaluated; to estimate if the fungicide concentrations used induced effects during oogenesis and; embryogenesis processes. Neonates from parents which were previously exposed to 170 µg/L and higher concentrations were malformed since 16-d of exposure onward. All animals presented the same malformation: asymmetrical shell morphology and separated valves that did not cover the complete animal body regardless of the; concentration. The biochemical parameters tested in the broodstock were cholesterol, triglycerides, glucose and LDH activity. At the end of the chronic exposure experiment, cholesterol and triglycerides remained unaltered while glucose and the LDH enzyme levels increased significantly. The results of the present work showed a direct effect of; prochloraz on D. magna individual growth, along with mobilization of some; biochemical intermediate metabolism. A daphnid stress response as a result of the fungicide presence in the medium could be an explanation for the metabolic disorders. On the other hand, the F1 malformed neonates found in the present study suggested an effect of prochloraz among different daphnid generations and more studies would be necessary in this field.

Gene expression study alerted to possible impairment in Daphnia magna individuals as a consequence of exposure to sublethal concentrations of prochloraz.

In the present study, Daphnia magna individuals were exposed for 21 days to 87, 130, 170, 230 and 380 µg/L of prochloraz. The expression of genes related to lipid metabolism (fabd), oxidative stress (cat and gst), heat shock proteins synthesis (hsp70 and hsp90), haemoglobin synthesis (hgb1 and hgb2), metallothioneins synthesis (mt-a, mt-b and mt-c), and vitellogenin synthesis (vgt1 y vgt2) were analyzed. Results showed that some gene expression in D. magna was altered as a consequence of the individual exposure to the fungicide. The genes fabd, vtg1 and vtg2, cat and gst resulted unaltered by the exposure of the daphnids to different fungicide concentrations. However, daphnid exposure to 380 µg/L of prochloraz resulted in an overexpression (p < 0.05) of hsp70 gene which indicated an alteration of the normal protein synthesis and its integrity maintenance. On the other hand, mt-b gene resulted significantly underexpressed (p < 0.05) in daphnids exposed to the lowest fungicide concentrations (87, 130 and 170 µg/L, respectively). In addition, hgb1 and hgb2 genes which are related with the haemoglobin synthesis weresignificantly overexpressed (p < 0.05). Results showed that hgb1 gene was overexpressed almost 100 times more in daphnids exposed for 21 days to 170, 230 and 380 µg/L than control values. However, many authors advocate for an association of these gene expression changes with the presence of contaminants in the medium, in fact they could be used as a good indicator of early contamination at low concentrations of toxicants.

Engineering alginate hydrogel films with poly(3-hydroxybutyrate-co-3-valerate) and graphene nanoplatelets: Enhancement of antiviral activity, cell adhesion and electroactive properties.

A new biodegradable semi-interpenetrated polymer network (semi-IPN) of two US Food and Drug Administration approved materials, poly(3-hydroxybutyrate-co-3-valerate) (PHBV) and calcium alginate (CA) was engineered to provide an alternative strategy to enhance the poor adhesion properties of CA. The synthesis procedure allows the additional incorporation of 10 % w/w of graphene nanoplatelets (GNPs), which have no cytotoxic effect on human keratinocytes. This quantity of multilayer graphene provides superior antiviral activity to the novel semi-IPN against a surrogate virus of SARS-CoV-2. Adding GNPs hardly affects the water absorption or electrical conductivity of the pure components of CA and PHBV. However, the semi-IPN's electrical conductivity increases dramatically after adding GNP due to molecular rearrangements of the intertwined polymer chains that continuously distribute the GNP nanosheets, This new hydrophilic composite biomaterial film shows great promise for skin biomedical applications, especially those that require antiviral and/or biodegradable electroconductive materials.

Pro-Myogenic Environment Promoted by the Synergistic Effect of Conductive Polymer Nanocomposites Combined with Extracellular Zinc Ions.

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.

Zinc Chloride: Time-Dependent Cytotoxicity, Proliferation and Promotion of Glycoprotein Synthesis and Antioxidant Gene Expression in Human Keratinocytes.

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 versus Silver Nanoparticles: Time-Dependent Cytotoxicity, Proliferation, and Gene Expression.

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.

Physical and biological properties of alginate/carbon nanofibers hydrogel films.

Alginates are renewable materials with excellent biocompatibility and cost-effectiveness in comparison with other biodegradable polymers. However, these hydrogels have poor mechanical properties that restrict their applications in biomedical fields such as skin tissue engineering. In this regard, the study follows an enhanced engineering route to produce alginate-based films reinforced with different amounts (0, 0.1, 0.5, 1 and 2% w/w) of carbon nanofibers (CNFs) and characterize their physical and biological properties. The results of this study showed that these composites possess similar biological properties to neat alginate hydrogels. Thus, none of the synthesized composite materials were cytotoxic and no cell adhesion was observed on the films. Water sorption at the body temperature did not suffer strong changes with the incorporation of CNFs into the alginate matrix. The dynamic mechanical and tensile/compressive properties of calcium alginate significantly improved with the addition of even a very low amount of CNFs. Thus, the tensile and compression modulus of the calcium alginate films in the dry and hydrated state increases up to three and six times, respectively, with the addition of 2% w/w CNFs. In addition, the composites reinforced with the lowest CNFs content have the advantage of possessing more transparency and lower production costs.

Study of 1D and 2D Carbon Nanomaterial in Alginate Films.

Alginate-based materials hold great promise in bioengineering applications such as skin wound healing and scaffolds for tissue engineering. Nevertheless, cell adhesion of mammalian cells on these hydrophilic materials is very poor. In cases such as polycaprolactone, poly(hydroxy-3-butyrate-co-3-valerate) and gelatin, the incorporation of hydrophobic carbon nanofibers (CNFs) and hydrophilic graphene oxide (GO) has shown significant improvement of cell adhesion and proliferation. The incorporation of these carbon nanomaterials (CNMs) into alginate films can enhance their mechanical performance, wettability, water diffusion and antibacterial properties. Herein, we report the effect of adding these CNMs into alginate films on cell adhesion for the first time. Thus, the results of this study showed that these nanocomposites are non-cytotoxic in human keratinocyte HaCaT cells. Nevertheless, contrary to what has been reported for other polymers, cell adhesion on these advanced alginate-based composites was not improved. Therefore, both types of composite films possess similar biological behavior, in terms of cell adhesion and non-cytotoxicity, and enhanced physical and antibacterial properties in comparison to neat alginate for potential biomedical and bioengineering applications.

Carbon Nanofibers in Pure Form and in Calcium Alginate Composites Films: New Cost-Effective Antibacterial Biomaterials against the Life-Threatening Multidrug-Resistant Staphylococcus epidermidis.

Due to the current global health problem of antibiotic resistant recently announced by the World Health Organization, there is an urgent necessity of looking for new alternative antibacterial materials able to treat and impede multidrug-resistant infections which are cost-effective and non-toxic for human beings. In this regard, carbon nanofibers (CNFs) possess currently much lower cost than other carbon nanomaterials, such as graphene oxide, and exhibit excellent chemical, mechanical and electric properties. Furthermore, here, the first report on the antibacterial activity of CNFs was demonstrated. Thus, these nanomaterials, in pure form or incorporated in a minuscule amount into calcium alginate composite films to reduce production costs as much as possible, showed to be new weapons against a globally spreading multidrug-resistant pathogen, the methicillin-resistant Staphylococcus epidermidis (MRSE). This Gram-positive bacterium is becoming one of the most dangerous pathogens, due to its abundance on skin. In this study, these hollow filamentous materials, in direct contact with cells and loaded in the low-cost calcium alginate composite films, showed no cytotoxicity for human keratinocyte HaCaT cells, which render them very promising for biomedical applications. The CNFs used in this work were characterized by Raman spectroscopy and observed by high-resolution transmission electron with energy-disperse X-ray spectroscopy.

Graphene oxide in zinc alginate films: Antibacterial activity, cytotoxicity, zinc release, water sorption/diffusion, wettability and opacity.

Alginate is considered an exceptional biomaterial due to its hydrophilicity, biocompatibility, biodegradability, nontoxicity and low-cost in comparison with other biopolymers. We have recently demonstrated that the incorporation of 1% graphene oxide (GO) into alginate films crosslinked with Ca2+ cations provides antibacterial activity against Staphylococcus aureus and methicillin-resistant Staphylococcus epidermidis, and no cytotoxicity for human keratinocyte HaCaT cells. However, many other reports in literature have shown controversial results about the toxicity of GO demanding further investigation. Furthermore, the synergic effect of GO with other divalent cations with intrinsic antibacterial and cytotoxic activity such as Zn2+ has not been explored yet. Thus, here, two commercially available sodium alginates were characterised and utilized in the synthesis of zinc alginate films with GO following the same chemical route reported for the calcium alginate/GO composites. The results of this study showed that zinc release, water sorption/diffusion and wettability depended significantly on the type of alginate utilized. Furthermore, Zn2+ and GO produced alginate films with increased water diffusion, wettability and opacity. However, neither the combination of GO with Zn2+ nor the use of different types of sodium alginates modified the antibacterial activity and cytotoxicity of the zinc alginates against these Gram-positive pathogens and human cells respectively.