Vibrational spectroscopy of muscular tissue intoxicated by snake venom and exposed to photobiomodulation therapy.

The pathogenesis of myonecrosis caused by myotoxins from bothropic venom is associated with local extracellular matrix (ECM) disintegration, hemorrhage, and inflammation. Search for alternative methods associated with serum therapy is mandatory to neutralize the fast development of local damage following snakebites. The experimental use of photobiomodulation therapy (PBMT) in murine models has shown promising results relative to structural and functional recovery from bothropic snakebite-induced myonecrosis. This study pioneered in using Raman and Fourier transform infrared (FTIR) spectroscopies to characterize biochemical alterations in the gastrocnemius that had been injected with Bothrops jararacussu venom and exposed to local PBMT. Results show that vibrational spectra from lyophilized and diluted venom (1307 cm -1) was also found in the envenomed gastrocnemius indicating venom presence in the unirradiated muscle 48 h post-injection; but any longer visible after PBMT at this time exposure or 72 h post-injection regardless irradiated or not. Raman and FTIR analyses indicated that the bands with higher area and intensity were 1657 and 1547 cm-1 and 1667 and 1452 cm-1, respectively; all are assignments for proteins, especially collagen, and are higher in the PBMT-exposed gastrocnemius. The infrared spectra suggest that laser treatment was able to change protein in tissue and that such change indicates collagen as the main target. We hypothesize that the findings reflect remodeling of ECM with key participation of collagen and faster tissue recovery for an anabolic condition.

Sciatic nerve repair using poly(ε-caprolactone) tubular prosthesis associated with nanoparticles of carbon and graphene.

INTRODUCTION: Injuries to peripheral nerves generate disconnection between spinal neurons and the target organ. Due to retraction of the nerve stumps, end-to-end neurorrhaphy is usually unfeasible. In such cases, autologous grafts are widely used, nonetheless with some disadvantages, such as mismatching of donor nerve dimensions and formation of painful neuromas at the donor area. Tubulization, using bioresorbable polymers, can potentially replace nerve grafting, although improvements are still necessary. Among promising bioresorbable synthetic polymers, poly(l-lactic acid) (PLLA) and poly(ε-caprolactone) (PCL) are the most studied. Carbon nanotubes and graphene sheets have been proposed, however, as adjuvants to improve mechanical and regenerative properties of tubular prostheses. Thus, the present work evaluated nerve tubulization repair following association of PCL with nanoparticles of carbon (NPC) and graphene (NPG). METHODS: For that, adult Lewis rats were subjected to unilateral sciatic nerve tubulization and allowed to survive for up to 8 and 12 weeks postsurgery. RESULTS: Nanocomposites mechanical/chemical evaluation showed that nanoparticles do not alter PCL crystallinity, yet providing reinforcement of polymer matrix. Thus, there was a decrease in the enthalpy of melting when the mixture of PCL + NPC + NPG was used. Nanocomposites displayed positive changes in molecular mobility in the amorphous phase of the polymer. Also, the loss modulus (E") and the glass transition exhibited highest values for PCL + NPC + NPG. Scanning electron microscopy analysis revealed that PCL + NPC + NPG prostheses showed improved cell adhesion as compared to PCL alone. Surgical procedures with PCL + NPC + NPG were facilitated due to improved flexibility of the prosthesis, resulting in better stump positioning accuracy. In turn, a twofold increased number of myelinated axons was found in such repaired nerves. Consistent with that, target muscle atrophy protection has been observed. CONCLUSION: Overall, the present data show that nanocomposite PCL tubes facilitate nerve repair and result in a better regenerative outcome, what may, in turn, represent a new alternative to pure PCL or PLLA prostheses.

PEGylation of Reduced Graphene Oxide Induces Toxicity in Cells of the Blood-Brain Barrier: An in Vitro and in Vivo Study.

Polyethylene glycol (PEG) coating has been frequently used to improve the pharmacokinetic behavior of nanoparticles. Studies that contribute to better unravel the effects of PEGylation on the toxicity of nanoparticle formulation are therefore highly relevant. In the present study, reduced graphene oxide (rGO) was functionalized with PEG, and its effects on key components of the blood-brain barrier, such as astrocytes and endothelial cells, were analyzed in culture and in an in vivo rat model. The in vitro studies demonstrated concentration-dependent toxicity. The highest concentration (100 µg/mL) of non-PEGylated rGO had a lower toxic influence on cell viability in primary cultures of astrocytes and rat brain endothelial cells, while PEGylated rGO induced deleterious effects and cell death. We assessed hippocampal BBB integrity in vivo by evaluating astrocyte activation and the expression of the endothelial tight and adherens junctions proteins. From 1 h to 7 days post-rGO-PEG systemic injection, a notable and progressive down-regulation of protein markers of astrocytes (GFAP, connexin-43), the endothelial tight (occludin), and adherens (ß-catenin) junctions and basal lamina (laminin) were observed. The formation of intracellular reactive oxygen species demonstrated by increases in the enzymatic antioxidant system in the PEGylated rGO samples was indicative of oxidative stress-mediated damage. Under the experimental conditions and design of the present study the PEGylation of rGO did not improve interaction with components of the blood-brain barrier. In contrast, the attachment of PEG to rGO induced deleterious effects in comparison with the effects caused by non-PEGylated rGO.

Reduced graphene oxide: nanotoxicological profile in rats.

BACKGROUND: We have previously demonstrated that reduced graphene oxide (rGO) administered intravenously in rats was detected inside the hippocampus after downregulation of the tight and adherens junction proteins of the blood-brain barrier. While down-regulators of junctional proteins could be useful tools for drug delivery through the paracellular pathway, concerns over toxicity must be investigated before clinical application. Herein, our purpose was to trace whether the rGO inside the hippocampus triggered toxic alterations in this brain region and in target organs (blood, liver and kidney) of rats at various time points (15 min, 1, 3 h and 7 days). RESULTS: The assessed rGO-treated rats (7 mg/kg) were clinically indistinguishable from controls at all the time points. Hematological, histopathological (neurons and astrocytes markers), biochemical (nephrotoxicity and hepatotoxicity assessment) and genotoxicological based tests showed that systemic rGO single injection seemed to produce minimal toxicological effects at the time points assessed. Relative to control, the only change was a decrease in the blood urea nitrogen level 3 h post-treatment and increases in superoxide dismutase activity 1 h and 7 days post-treatment. While no alteration in leukocyte parameters was detected between control and rGO-treated animals, time-dependent leukocytosis (rGO-1 h versus rGO-3 h) and leukopenia (rGO-3 h versus rGO-7 days) was observed intra-treated groups. Nevertheless, no inflammatory response was induced in serum and hippocampus at any time. CONCLUSIONS: The toxic effects seemed to be peripheral and transitory in the short-term analysis after systemic administration of rGO. The effects were self-limited and non-significant even at 7 days post-rGO administration.

Reduced graphene oxide induces transient blood-brain barrier opening: an in vivo study.

BACKGROUND: The blood-brain barrier (BBB) is a complex physical and functional barrier protecting the central nervous system from physical and chemical insults. Nevertheless, it also constitutes a barrier against therapeutics for treating neurological disorders. In this context, nanomaterial-based therapy provides a potential alternative for overcoming this problem. Graphene family has attracted significant interest in nanomedicine because their unique physicochemical properties make them amenable to applications in drug/gene delivery and neural interface. RESULTS: In this study, reduced graphene oxide (rGO) systemically-injected was found mainly located in the thalamus and hippocampus of rats. The entry of rGO involved a transitory decrease in the BBB paracellular tightness, as demonstrated at anatomical (Evans blue dye infusion), subcellular (transmission electron microscopy) and molecular (junctional protein expression) levels. Additionally, we examined the usefulness of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) as a new imaging method for detecting the temporal distribution of nanomaterials throughout the brain. CONCLUSIONS: rGO was able to be detected and monitored in the brain over time provided by a novel application for MALDI-MSI and could be a useful tool for treating a variety of brain disorders that are normally unresponsive to conventional treatment because of BBB impermeability.

Haemophilus influenzae porine ompP2 gene transfer mediated by graphene oxide nanoparticles with effects on transformation process and virulence bacterial capacity.

BACKGROUND: H. influenzae is a natural competent bacterium that can uptake DNA from the environment and recombine into bacterial genome. The outbreaks of Brazilian purpuric fever, heavily polluted areas of a different H. influenzae biogroup - aegyptius - as well as gene transference between Neisseria meningitis make the transformation process an important evolutionary factor. This work studied the horizontal transference of the ompP2 gene from a multiresistant strain of H. influenzae 07 (NTHi), under the influence of graphene oxide nanoparticles in order to mimic an atmosphere rich in suspended particles and this way verify if the CFU transformants number was increased. MATERIAL AND METHODS: In this article the gene ompP2 was transformed into different strains of H. influenzae mediated or not by graphene oxide nanoparticles in suspension, followed by the adhesion tests in Hec-1B (human endometrium adenocarcinoma) and A549 (pulmonary epithelial carcinoma) cells lines. The transformation frequency and the adhesion capacity were determined in all the mutants to which the ompP2 gene was transferred and compared to their wild type strains. RESULTS: The nanoparticles increased the transformation ratio of one particular strain isolated from a pneumonia case. The adhesion patterns to A549 and Hec1b cell lines of these mutated bacteria has their capacity increased when compared to the wild type. CONCLUSIONS: Graphene oxide nanoparticles aid the transformation process, helping to increase the number of CFUs, and the mutants generated with the ompP2 gene from a H. influenzae resistant strain not only present a chloramphenicol resistance but also have an increased adherence patterns in A549 and Hec1B cell lines.

Absence of mutagenic and recombinagenic activity of multi-walled carbon nanotubes in the Drosophila wing-spot test and Allium cepa test.

In order to assess the safety of the carbon nanotubes to human health and the environment, we investigated the potential toxicity and ability of multi-walled carbon nanotubes (NT), to induce DNA damage by employing the Allium cepa genotoxicity/mutagenicity test and the Somatic Mutation and Recombination Test (SMART) in the fruitfly, Drosophila melanogaster. The results demonstrated that NT did not significantly induce genotoxic or mutagenic effects in the Allium cepa test. All concentrations evaluated in the SMART assay showed survival rates higher than 90percent, indicating the absence of chronic toxicity for NT. Furthermore, the various treatments showed no significant increase in the NT mutation and recombination frequencies in mwh/flr(3) genotype compared to respective negative controls, demonstrating the absence of DNA damage caused by NT.

Fast preparation of nano-hydroxyapatite/superhydrophilic reduced graphene oxide composites for bioactive applications.

A method for the direct electrodeposition of globular nano-hydroxyapatite (nHAp) onto reduced graphene oxide (RGO) is presented and a model for the specific growth preference is discussed. Results show that the carboxyl (carboxylic acid)/carboxylate functional groups attached directly to the RGO after oxygen plasma treatment were essential to accelerate the OH- formation and the deposition of globular nHAp crystals. High resolution scanning electron microscopy, energy dispersive X-ray and X-ray diffraction showed that homogeneous, highly crystalline, stoichiometric nHAp crystals, with preferential growth in the (002) plane direction, were formed without any thermal treatment. The nHAp/RGO composites were shown to be an appropriate surface for mesenchymal stem cell adhesion with active formation of membrane projections.