Leonardo Pereira Franchi

Characterization and in vitro evaluation of bacterial cellulose membranes functionalized with osteogenic growth peptide for bone tissue engineering.

The aim of this study was to characterize the physicochemical properties of bacterial cellulose (BC) membranes functionalized with osteogenic growth peptide (OGP) and its C-terminal pentapeptide OGP[10–14], and to evaluate in vitro osteoinductive potential in early osteogenesis, besides, to evaluate cytotoxic, genotoxic and/or mutagenic effects. Peptide incorporation into the BC membranes did not change the morphology of BC nanofibers and BC crystallinity pattern. The characterization was complemented by Raman scattering, swelling ratio and mechanical tests. In vitro assays demonstrated no cytotoxic, genotoxic or mutagenic effects for any of the studied BC membranes. Culture with osteogenic cells revealed no difference in cell morphology among all the membranes tested. Cell viability/proliferation, total protein content, alkaline phosphatase activity and mineralization assays indicated that BC-OGP membranes enabled the highest development of the osteoblastic phenotype in vitro. In conclusion, the negative results of cytotoxicity, genotoxicity and mutagenicity indicated that all the membranes can be employed for medical supplies, mainly in bone tissue engineering/regeneration, due to their osteoinductive properties.

Cytotoxicity and genotoxicity of silver nanoparticles of different sizes in CHO-K1 and CHO-XRS5 cell lines.

Nanoparticles (NPs) have been used in a range of products due to their unique properties. Nevertheless, these NPs can cause adverse biological effects and because of that, there is a great concern about the health and environmental risks related to their use. Recently, silver nanoparticles (Ag NPs) have been used in a variety of cytotoxicity and genotoxicity studies, but there are still controversies regarding the association between the size and the toxicity of these particles. Therefore, in this study, we aimed to evaluate the cytotoxicity and genotoxicity of Ag NPs (10 and 100 nm) in two different cell lines, CHO-K1 and CHO-XRS5, by performing cell viability assay (XTT), clonogenic assay, micronucleus test, comet assay, as well as by investigating the cell cycle kinetics using the flow cytometry. Cell cultures were exposed to different concentrations of AgNPs (0.025-5.0 μg/ml) for 24 h. Our results indicated that cytotoxicity and genotoxicity induced by the 100 nm-Ag NPs were greater than those induced by the 10 nm-Ag NPs for both cell lines, which suggests that the exposure to greater size particles (100 nm) can cause more adverse biological effects than the exposure to the smaller ones (10 nm).

Evaluation of carbon nanotubes network toxicity in zebrafish (danio rerio) model

This is a detailed in vivo study of the biological response to carbon nanotubes network as probed by the zebrafish model. First, we prepared pristine carbon nanotubes (CNTs) by methanol chemical vapor deposition in the presence of Mn and Co as catalysts, followed by purification in acid, which furnished curved tubes with diameters lying between 10 and 130 nm. The CNT network consisted of pristine CNTs dispersed in water in the presence of a surfactant. The CNT network pellets corresponded to agglomerated multi-walled CNTs with an average diameter of about 500 nm. Although the same pristine CNTs had been previously found to exert genotoxic effects in vitro, here we verified that the CNT network was not genotoxic in vivo. Indeed, Raman spectroscopy and microscopy conducted in the intestine of the zebrafish revealed complete clearance of the CNT network as well as minimal disturbances, such as aneurysms, hyperemia, and reversible inflammatory focus in the zebrafish gills.

Cytotoxicity and genotoxicity of coronaridine from Tabernaemontana catharinensis A.DC in a human laryngeal epithelial carcinoma cell line (Hep-2)

Cancer has become a major public health problem worldwide and the number of deaths due to this disease is increasing almost exponentially. In the constant search for new treatments, natural products of plant origin have provided a variety of new compounds to be explored as antitumor agents. Tabernaemontana catharinensis is a medicinal plant that produces alkaloids with expressive antitumor activity, such as heyneanine, coronaridine and voacangine. The aim of present study was firstly to screen the cytotoxic activity of the indole alkaloids heyneanine, coronaridine and voacangine against HeLa (human cervix tumor), 3T3 (normal mouse embryo fibroblasts), Hep-2 (human laryngeal epithelial carcinoma) and B-16 (murine skin) cell lines by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); and secondly to analyze the apoptotic activity, cell membrane damage and genotoxicity of the compound that showed the best cytotoxic activity against the tumor cell lines tested. Coronaridine was the one that exhibited greater cytotoxic activity in the laryngeal carcinoma cell line Hep-2 (IC50 = 54.47 μg/mL) than the other alkaloids tested (voacangine IC50 = 159.33 g/mL, and heyneanine IC50 = 689.45 μg/mL). Coronaridine induced apoptosis in cell lines 3T3 and Hep-2, even at high concentrations. The evaluation of genotoxicity by comet assay showed further that coronaridine caused minimal DNA damage in the Hep-2 tumor cell line, and the LDH test showed that it did not affect the plasma membrane. These results suggest that further investigation of coronaridine as an antitumor agent has merit.

Citotoxicidade e genotoxicidade de nanotubos de carbono

There are many controversies regarding the cyto- and genotoxicity of carbon nanotubes (CNTs). In this work, we discuss that many of the incongruous arguments are probably associated with the poor physical-chemical characterization of the CNT samples used in many publications. This manuscript presents examples of carbon nanostructures observed under high resolution electron microscopy that can be found in typical CNT samples, and shows which roles in cyto- and genotoxicity need to be better investigated. Issues concerning chemical treatment are addressed and examples of misunderstandings that can occur during the studies of cyto- and genotoxicity of CNT samples are given.

Light source is critical to induce glioblastoma cell death by photodynamic therapy using chloro-aluminiumphtalocyanine albumin-based nanoparticles.

Selection of an efficient light source is fundamental in the development of photodynamic therapy (PDT) protocols. However, few studies provide a comparison of different light sources with regard to phototoxic effects. Here, we compared the cell death induced by photoactivation of chloro-aluminiumphtalocyanine (AlClPc)-loaded human serum albumin nanoparticles under irradiation with different light sources: continuous laser (CL), pulsed laser (PL), and light-emitting diode (LED). Cells were exposed to three different AlClPc concentrations (1, 3, and 5μM) and three different light doses (200, 500, and 700mJ/cm2) for each light source. Cell death and differentiation of apoptosis and necrosis pathway were measured by flow cytometry. CL was the best light source for improving the photodynamic action of AlClPc-loaded albumin nanoparticles in glioblastoma cells and avoiding undesirable side effects, especially at low photosensitizer doses (200mJ/cm2). In addition, apoptosis was the main cell death pathway in all evaluated cases (70% for CL, and greater than 50% for PL and LED). In conclusion, the search for optimal light sources and light/photosensitizer doses is a crucial step in improving PDT outcomes and enhancing the clinical translation of PDT.

Regenerated cellulose scaffolds: Preparation, characterization and toxicological evaluation.

Regenerated cellulose scaffolds (RCS) may be used as alloplastic materials for tissue repair. In this work, the RCS were obtained by viscose process and characterized by scanning electron microscopy (SEM), wide angle X-ray diffraction (WAXD), Fourier transform infrared spectroscopy (FTIR) and thermogravimetry analysis (TG). In vitro enzymatic degradation assay and toxicological assays were also evaluated. The physicochemical characterizations revealed the formation of a porous material with distinct thermal profile and crystallinity compared to pristine cellulose pulp. Enzymatic degradation assay revealed that lysozyme showed a mildest catalytic action when compared to cellulase, Tricoderma reesei (Tr). Nevertheless, both enzymes were efficient for degrading the RCS. RCS did not show cytotoxicity, mutagenic or genotoxic effects. The systematically characterization of this work suggests that RCS presented distinct features that make it a viable material for future studies related to the development of scaffolds for biological applications.

Carbon Nanotubes: From Synthesis to Genotoxicity

Massive industrial production of carbon nanotubes (CNTs) is increasing year after year, and it is urgent to address their safety-related issues. Due to their morphological similarities with asbestos fibers, which are classical carcinogenic materials, these CNTs have been considered as hazardous manufactured products by regulatory agencies. In this context, genotoxic effects of CNTs and the mechanisms proposed in current literature are reviewed and discussed in this chapter. Relevant aspects of preparation and physicochemical characterization of CNTs in toxicological context as well as the recent perspectives involving cytotoxicity assessment are also highlighted. Finally, this chapter aims to contribute to point out to a proactive discussion towards a responsible and sustainable development of nanotechnology lined up with environmental, health, and safety (EH&S) requirements.

Nanocellulose-collagen-apatite composite associated with osteogenic growth peptide for bone regeneration

Despite advances in the field of biomaterials for bone repair/regeneration, some challenges for developing an ideal bone substitute need to be overcome. Herein, this study synthesized and evaluated in vitro a nanocomposite based on bacterial cellulose (BC), collagen (COL), apatite (Ap) and osteogenic growth peptide (OGP) or its C-terminal pentapeptide [OGP(10-14)] for bone regeneration purposes. The BC-COL nanocomposites were successfully obtained by carbodiimide-mediated coupling as demonstrated by spectroscopy analysis. SEM, FTIR and 31P NMR analyses revealed that in situ synthesis to apatite was an effective route for obtaining of bone-like apatite. The OGP-containing (BC-COL)-Ap stimulated the early development of the osteoblastic phenotype. Additionally, the association among collagen, apatite, and OGP peptides enhanced cell growth compared with OGP-containing BC-Ap. Furthermore, none of the nanocomposites showed cytotoxic, genotoxic or mutagenic effects. These promising results suggest that the (BC-COL)-Ap associated with OGP peptides might be considered a potential candidate for bone tissue engineering applications.

DNA polymeric films as a support for cell growth as a new material for regenerative medicine: Compatibility and applicability.

Abstract DNA polymeric films (DNA‐PFs) are a promising drug delivery system (DDS) in modern medicine. In this study, we evaluated the growth behavior of oral squamous cell carcinoma (OSCC) cells on DNA‐PFs. The morphological, biochemical, and cytometric features of OSCC cell adhesion on DNA‐PFs were also assessed. An initial, temporary alteration in cell morphology was observed at early time points owing to the inhibition of cell attachment to the film, which then returned to a normal morphological state at later time points. MTT and resazurin assays showed a moderate reduction in cell viability related to increased DNA concentration in the DNA‐PFs. Flow cytometry studies showed low cytotoxicity of DNA‐PFs, with cell viabilities higher than 90% in all the DNA‐PFs tested. Flow cytometric cell cycle analysis also showed average cell cycle phase distributions at later time points, indicating that OSCC cell growth is maintained in the presence of DNA‐PFs. These results show high biocompatibility of DNA‐PFs and suggest their use in designing “dressing material,” where the DNA film acts as a support for cell growth, or with incorporation of active or photoactive compounds, which can induce tissue regeneration and are useful to treat many diseases, especially oral cancer. HighlightsOral squamous cell carcinoma cells were successfully grown on DNA polymeric films.DNA polymeric films were not toxic to the cells at low concentrations of DNA.DNA polymeric films are proposed to act as a support for cell growth.Results are relevant for designing drug delivery systems for photodynamic therapy.