Cellular and Molecular Processes Are Differently Influenced in Primary Neural Cells by Slight Changes in the Physicochemical Properties of Multicore Magnetic Nanoparticles.

Herein, we use two exemplary superparamagnetic iron oxide multicore nanoparticles (SPIONs) to illustrate the significant influence of slightly different physicochemical properties on the cellular and molecular processes that define SPION interplay with primary neural cells. Particularly, we have designed two different SPION structures, NFA (i.e., a denser multicore structure accompanied by a slightly less negative surface charge and a higher magnetic response) and NFD (i.e., a larger surface area and more negatively charged), and identified specific biological responses dependent on SPION type, concentration, exposure time, and magnetic actuation. Interestingly, NFA SPIONs display a higher cell uptake, likely driven by their less negative surface and smaller protein corona, more significantly impacting cell viability and complexity. The tight contact of both SPIONs with neural cell membranes results in the significant augmentation of phosphatidylcholine, phosphatidylserine, and sphingomyelin and the reduction of free fatty acids and triacylglycerides for both SPIONs. Nonetheless, NFD induces greater effects on lipids, especially under magnetic actuation, likely indicating a preferential membranal location and/or a tighter interaction with membrane lipids than NFA, in agreement with their lower cell uptake. From a functional perspective, these lipid changes correlate with an increase in plasma membrane fluidity, again larger for more negatively charged nanoparticles (NFD). Finally, the mRNA expression of iron-related genes such as Ireb-2 and Fth-1 remains unaltered, while TfR-1 is only detected in SPION-treated cells. Taken together, these results demonstrate the substantial impact that minor physicochemical differences of nanomaterials may exert in the specific targeting of cellular and molecular processes. A denser multicore structure generated by autoclave-based production is accompanied by a slight difference in surface charge and magnetic properties that become decisive for the biological impact of these SPIONs. Their capacity to markedly modify the lipidic cell content makes them attractive as lipid-targetable nanomedicines.

Safety assessment of new nanodiamonds@corrole hybrids addressed by the response of RAW-264.7 macrophages.

Safety assessment of carbon nanomaterials is of paramount importance since they are on the frontline for applications in sensing, bioimaging and drug delivery. The biocompatibility and safety of functionalized nanodiamonds (NDs) are here addressed through the study of the pro-inflammatory response of RAW-264.7 macrophages exposed to new nanodiamonds@corrole hybrids. The corrole unit selected is as a prototype for a hydrophobic organic molecule that can function as a NIR fluorophore reporter, an optical sensor, a photodynamic therapy agent or a photocatalyst. The new functional nanohybrids containing detonated nanodiamonds (NDs) were obtained through esterification using carboxylated NDs and glycol corroles. The success of the covalent functionalization via carbodiimide activation was confirmed through X-ray photoelectron spectroscopy (XPS), Raman and Fourier transform infrared (FTIR) spectroscopy. The UV-vis absorption and emission spectra of the hybrids are additive with respect to the corrole features. The cellular uptake, localization, cell viability and effects on immune cell activation of the new hybrids and of the precursors were carefully investigated using RAW-264.7 macrophages. Overall results showed that the ND@corrole hybrids had no pro-inflammatory effects on the RAW-264.7 macrophage cell line, making them an ideal candidate for a wide range of biomedical applications.

Macrophage inflammatory and metabolic responses to graphene-based nanomaterials differing in size and functionalization.

The preparation of graphene-based nanomaterials (GBNs) with appropriate stability and biocompatibility is crucial for their use in biomedical applications. In this work, three GBNs differing in size and/or functionalization have been synthetized and characterized, and their in vitro biological effects were compared. Pegylated graphene oxide (GO-PEG, 200-500 nm) and flavin mononucleotide-stabilized pristine graphene with two different sizes (PG-FMN, 200-400 nm and 100-200 nm) were administered to macrophages, chosen as cellular model due to their key role in the processing of foreign materials and the regulation of inflammatory responses. The results showed that cellular uptake of GBNs was mainly influenced by their lateral size, while the inflammatory potential depended also on the type of functionalization. PG-FMN nanomaterials (both sizes) triggered significantly higher nitric oxide (NO) release, together with some intracellular metabolic changes, similar to those induced by the prototypical inflammatory stimulus LPS. NMR metabolomics revealed that macrophages incubated with smaller PG-FMN displayed increased levels of succinate, itaconate, phosphocholine and phosphocreatine, together with decreased creatine content. The latter two variations were also detected in cells incubated with larger PG-FMN nanosheets. On the other hand, GO-PEG induced a decrease in the inflammatory metabolite succinate and a few other changes distinct from those seen in LPS-stimulated macrophages. Assessment of TNF-α secretion and macrophage surface markers (CD80 and CD206) further corroborated the low inflammatory potential of GO-PEG. Overall, these findings revealed distinct phenotypic and metabolic responses of macrophages to different GBNs, which inform on their immunomodulatory activity and may contribute to guide their therapeutic applications.

Metabolomic response of osteosarcoma cells to nanographene oxide-mediated hyperthermia.

Nanographene oxide (nGO)-mediated hyperthermia has been increasingly investigated as a localized, minimally invasive anticancer therapeutic approach. Near InfraRed (NIR) light irradiation for inducing hyperthermia is particularly attractive, because biological systems mostly lack chromophores that absorb in this spectral window, facilitating the selective heating and destruction of cells which have internalized the NIR absorbing-nanomaterials. However, little is known about biological effects accompanying nGO-mediated hyperthermia at cellular and molecular levels. In this work, well-characterized pegylated nGO sheets with a hydrodynamic size of 300 nm were incubated with human Saos-2 osteosarcoma cells for 24 h and their internalization verified by flow cytometry and confocal microscopy. No effect on cell viability was observed after nGO uptake by Saos-2 cells. However, a proliferation delay was observed due to the presence of nGO sheets in the cytoplasm. 1H NMR metabolomics was employed to screen for changes in the metabolic profile of cells, as this could help to improve understanding of cellular responses to nanomaterials and provide new endpoint markers of effect. Cells internalizing nGO sheets showed noticeable changes in several metabolites compared to control cells, including decreased levels of several amino acids, taurine and creatine and increased levels of phosphocholine and uridine/adenosine nucleotides. After NIR irradiation, cells showed decreases in glutamate and uridine nucleotides, together with increases in glycerophosphocholine and adenosine monophosphate. Overall, this study has shown that the cellular metabolome sensitively responded to nGO exposure and nGO-mediated hyperthermia and that NMR metabolomics is a powerful tool to investigate treatment responses.

Multifunctional pH sensitive 3D scaffolds for treatment and prevention of bone infection.

Multifunctional-therapeutic three-dimensional (3D) scaffolds have been prepared. These biomaterials are able to destroy the S. aureus bacterial biofilm and to allow bone regeneration at the same time. The present study is focused on the design of pH sensitive 3D hierarchical meso-macroporous 3D scaffolds based on MGHA nanocomposite formed by a mesostructured glassy network with embedded hydroxyapatite nanoparticles, whose mesopores have been loaded with levofloxacin (Levo) as antibacterial agent. These 3D platforms exhibit controlled and pH-dependent Levo release, sustained over time at physiological pH (7.4) and notably increased at infection pH (6.7 and 5.5), which is due to the different interaction rate between diverse Levo species and the silica matrix. These 3D systems are able to inhibit the S. aureus growth and to destroy the bacterial biofilm without cytotoxic effects on human osteoblasts and allowing an adequate colonization and differentiation of preosteoblastic cells on their surface. These findings suggest promising applications of these hierarchical MGHA nanocomposite 3D scaffolds for the treatment and prevention of bone infection. STATEMENT OF SIGNIFICANCE: Multifunctional 3D nanocomposite scaffolds with the ability for loading and sustained delivery of an antimicrobial agent, to eliminate and prevent bone infection and at the same time to contribute to bone regeneration process without cytotoxic effects on the surrounding tissue has been proposed. These 3D scaffolds exhibit a sustained levofloxacin delivery at physiological pH (pH 7.4), which increasing notably when pH decreases to characteristic values of bone infection process (pH 6.7 and pH 5.5). In vitro competitive assays between preosteoblastic and bacteria onto the 3D scaffold surface demonstrated an adequate osteoblast colonization in entire scaffold surface together with the ability to eliminate bacteria contamination.

MC3T3-E1 pre-osteoblast response and differentiation after graphene oxide nanosheet uptake.

Nano-graphene oxide (GO) and its functionalized derivatives have aroused a great interest for drug delivery, tissue engineering and photothermal cancer therapy, but their biocompatibility has not yet been fully assessed. The aim of the present study was to evaluate the proliferation and differentiation of MC3T3-E1 pre-osteoblasts after the uptake of GO nanosheets (c.a. 400nm), functionalized with poly(ethylene glycol-amine) (PEG) and labelled with fluorescein isothiocyanate (FITC). Significant proliferation decrease and apoptosis increase were observed 3days after incorporation of FITC-PEG-GO by MC3T3-E1 cells. However, alterations on healthy pre-osteoblast differentiation into cells exhibiting osteoblast phenotype were not observed, as they showed normal alkaline phosphatase levels and matrix mineralization 12days after nanosheet uptake. The results suggest that 40µg/mL concentrations of these nanosheets would not affect the differentiation of healthy pre-osteoblasts, thus these PEG-GO nanosheets have potential to be used for biomedical applications after their internalization, as the induction of local hyperthermia on bone cancer.

Mitochondrial membrane potential and reactive oxygen species content of endothelial and smooth muscle cells cultured on poly(epsilon-caprolactone) films.

A transitory but significant stimulation of mitochondrial activity, increase of reactive oxygen species (ROS) and oxidative stress were previously observed in L929 fibroblasts cultured on poly(epsilon-caprolactone) (PCL) films. ROS, mainly formed in mitochondria, play a physiological role but an excessive production can promote endothelial dysfunction, cause oxidative injury to vascular cells, oxidize lipoproteins and accelerate atherothrombogenesis. On the other hand, mitochondria have a crucial position in programmed cell death control and are responsible for ATP synthesis through the coupling of oxidative phosphorylation to respiration. This coupling requires the existence of a mitochondrial membrane potential (Deltapsi(m)). The aim of the present study was to evaluate by flow cytometry the ROS content and Deltapsi(m) of both endothelial (EC) and smooth muscle cells (SMC) cultured on PCL films as a potential substrate for vascular graft development. Cell size, internal complexity and cell cycle were also analyzed to detect the possible appearance of the subG(1) cell fraction, characteristic of apoptotic cells. The effect of treating PCL films with NaOH before culture was also studied. PCL decreases the ROS content of EC during the culture but produces an increase of these levels in SMC after 7 days. PCL also induces variations of Deltapsi(m) which show a significant parallelism with the changes observed in ROS levels proving the importance and sensitivity of these measurements as indicators of the mitochondrial function. The treatment of PCL with NaOH decreases these effects demonstrating the benefits of increasing the surface hydrophilicity before cell culture which improves cell adhesion and proliferation and reduces oxidative stress. Since no important changes have been detected in subG(1) fraction of EC and SMC cultured on either PCL or PCL-NaOH, the changes of Deltapsi(m) observed in the present study cannot be related to apoptosis. These results confirm the potential utility of PCL as a suitable scaffold in Vascular Tissue Engineering.

Alkaline-treated poly(epsilon-caprolactone) films: degradation in the presence or absence of fibroblasts.

In the first stage, we observed the study of the degradation behavior of alkaline-treated poly(epsilon-caprolactone) (PCL) in two biologically-related media: phosphate buffered saline (PBS) and Dulbecco's modified Eagle's medium (DMEM) for 18 months, finding a much accelerated degradation in the last one. As expected, the degradation in the presence of cells is much pronounced even considering that the study is limited to 6 months. The characterization of the degraded substrates by chemiluminescence (CL) allows to explain the modifications of the substrate and their relations with transitory oxidative stress phenomena described in the fibroblasts seeded onto the PCL membranes.

Effects of bleaching on osteoclast activity and their modulation by osteostatin and fibroblast growth factor 2.

HYPOTHESIS: Dental bleaching with H2O2 is a common daily practice in dentistry to correct discoloration of anterior teeth. The aim of this study has been to determine whether this treatment of human teeth affects growth, differentiation and activity of osteoclast-like cells, as well as the putative modulatory action of osteostatin and fibroblast growth factor 2 (FGF-2). EXPERIMENTS: Previously to the in vitro assays, structural, physical-chemical and morphological features of teeth after bleaching were studied. Osteoclast-like cells were cultured on human dentin disks, pre-treated or not with 38% H2O2 bleaching gel, in the presence or absence of osteostatin (100 nM) or FGF-2 (1 ng/ml). Cell proliferation and viability, intracellular content of reactive oxygen species (ROS), pro-inflammatory cytokine (IL-6 and TNFα) secretion and resorption activity were evaluated. FINDINGS: Bleaching treatment failed to affect either the structural or the chemical features of both enamel and dentin, except for slight morphological changes, increased porosity in the most superficial parts (enamel), and a moderate increase in the wettability degree. In this scenario, bleaching produced an increased osteoclast-like cell proliferation but decreased cell viability and cytokine secretion, while it augmented resorption activity on dentin. The presence of either osteostatin or FGF-2 reduced the osteoclast-like cell proliferation induced by bleaching. FGF-2 enhanced ROS content, whereas osteostatin decreased ROS but increased TNFα secretion. The bleaching effect on resorption activity was increased by osteostatin, but this effect was less evident with FGF-2. CONCLUSIONS: These findings further confirm the deleterious effects of tooth bleaching by affecting osteoclast growth and function as well as different modulatory actions of osteostatin and FGF-2.

Transitory oxidative stress in L929 fibroblasts cultured on poly(epsilon-caprolactone) films.

Poly(epsilon-caprolactone) (PCL) is considered as a potential substrate for wide medical applications. In previous studies we carried out the in vitro biocompatibility assessment of PCL films using L929 mouse fibroblasts, obtaining good cell behaviour but a transitory stimulation of mitochondrial activity and cell retraction. Reactive oxygen species (ROS), mainly formed in mitochondria, can impair the function of several cellular components and produce cell oxidative stress by changing the normal red-ox status of the major cell antioxidants as glutathione. The aim of this study was to measure intracellular ROS production and glutathione content of L929 fibroblasts cultured on PCL films. Cell size, internal complexity, cell cycle and lactate dehydrogenase release were also evaluated. The films were treated with NaOH before culture to improve the cell-polymer interaction. PCL induces a transitory but significant oxidative stress in L929 fibroblasts. The treatment of PCL films with NaOH reduces this effect. PCL also induces transitory changes on cell size and complexity. Nevertheless, after 7 days in culture, cells reach control levels for all the studied parameters. Neither cell cycle nor membrane integrity appears affected by this oxidative stress respect to control cells at any culture time. These results underline the cytocompatibility of PCL films and, therefore, its potential utility as a suitable scaffold in tissue engineering.

Endocytic mechanisms of graphene oxide nanosheets in osteoblasts, hepatocytes and macrophages.

Nano-graphene oxide (GO) has attracted great interest in nanomedicine due to its own intrinsic properties and its possible biomedical applications such as drug delivery, tissue engineering and hyperthermia cancer therapy. However, the toxicity of GO nanosheets is not yet well-known and it is necessary to understand its entry mechanisms into mammalian cells in order to avoid cell damage and human toxicity. In the present study, the cellular uptake of pegylated GO nanosheets of ca. 100 nm labeled with fluorescein isothiocyanate (FITC-PEG-GOs) has been evaluated in the presence of eight inhibitors (colchicine, wortmannin, amiloride, cytochalasin B, cytochalasin D, genistein, phenylarsine oxide and chlorpromazine) that specifically affect different endocytosis mechanisms. Three cell types were chosen for this study: human Saos-2 osteoblasts, human HepG2 hepatocytes and murine RAW-264.7 macrophages. The results show that different mechanisms take part in FITC-PEG-GOs uptake, depending on the characteristics of each cell type. However, macropinocytosis seems to be a general internalization process in the three cell lines analyzed. Besides macropinocytosis, FITC-PEG-GOs can enter through pathways dependent on microtubules in Saos-2 osteoblasts, and through clathrin-dependent mechanisms in HepG2 hepatocytes and RAW-264.7 macrophages. HepG2 cells can also phagocytize FITC-PEG-GOs. These findings help to understand the interactions at the interface of GO nanosheets and mammalian cells and must be considered in further studies focused on their use for biomedical applications.

L929 fibroblast and Saos-2 osteoblast response to hydroxyapatite-betaTCP/agarose biomaterial.

Biphasic calcium phosphate, a mixture of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP), has been successfully used as an excellent bone graft substitute because of the HA capacity for direct interaction with bone and the beta-TCP resorption properties. Agarose has been recently mixtured with ceramics as natural biodegradable binder to increase the biomaterial flexibility facilitating its placement into the bone defect. In this study, the behavior of L929 fibroblasts and Saos-2 osteoblasts cultured on hydroxyapatite-betaTCP/agarose disks has been evaluated. Both cell types adhere and proliferate on the biomaterial surface maintaining their characteristic morphology. Transitory changes on cell cycle, size, and complexity are observed. The biomaterial induces apoptosis in Saos-2 osteoblasts but not in fibroblasts. A transitory stimulation of fibroblast mitochondrial activity is observed. This effect remains in osteoblasts after 9 days of culture showing a higher sensitivity of this cell type. However, the intracellular reactive oxygen species content and the lactate dehydrogenase release of Saos-2 osteoblasts indicate that hydroxyapatite-betaTCP/agarose does not induce oxidative stress in this cell type and confirm the integrity of the osteoblast plasma membrane. These results underline the good biocompatibility of hydroxyapatite-betaTCP/agarose disks and its potential utility for bone substitution and repair.

Endothelial cells derived from circulating progenitors as an effective source to functional endothelialization of NaOH-treated poly(epsilon-caprolactone) films.

Biomaterials have been widely used to prepare synthetic vascular grafts over the past thirty years, but the inherent thrombogenicity of their surface can lead to graft failure. Endothelial progenitor cells (EPC) are circulating premature cells able to differentiate in either myocardial or endothelial cells (EC). The therapeutic potential of these cells and its easy obtaining technique are important reasons why these cells could be used to improve the performance of vascular grafts. In this study, two different stages of differentiation of EC derived from EPC (EC(1) and EC(2)) were characterized and cultured on poly(epsilon-caprolactone) (PCL) films treated with NaOH (PCL-NaOH). We investigated by immunolabeling the expression of CD31, von Willebrand factor (vWF), and endothelial nitric oxide synthase (eNOS) in these cells during the differentiation process. The proliferation, cell cycle, and mitochondrial function of EC(2) cultured on PCL-NaOH were evaluated at different times. The effect of this biomaterial on the nitric oxide (NO) content was also measured. The mature EC obtained from circulating progenitor cells (EC(2)) showed an appropriate growth and functionality on NaOH-treated films. They conserved their capacity to define vessel-like structures in culture and increased their basal NO production. These results underline the potential usefulness of these EC(2) to get a functional endothelialization of polymers with applications in vascular tissue engineering.

In vitro positive biocompatibility evaluation of glass-glass ceramic thermoseeds for hyperthermic treatment of bone tumors.

A new kind of magnetic thermoseed for bone tissue engineering has been synthesized. The materials used are specially designed to restore bone tissue after tumor extirpation, because they exhibit bioactive behavior and the ability to act as thermoseeds for cancer treatment using hyperthermia. The L929 cell line of mouse fibroblasts has been used in a wide biocompatibility study concerning cell proliferation and morphology studies, mitochondrial function determination, lactate dehydrogenase measurement, and flow cytometry studies, including cell cycle analysis, cell size and complexity, and intracellular reactive oxygen species content. The results presented in this work indicate that these bioactive magnetic materials are highly biocompatible and show greater cell response for thermoseeds with a higher magnetic phase content. There were no significant alterations detected in the cell cycle, and the interaction between fibroblasts and the different mixtures did not induce significant apoptosis.

Effect of bile acids on butyrate-sensitive and -resistant human colon adenocarcinoma cells.

A controlled balance among cell proliferation, differentiation, and apoptosis is required for the maintenance of gastrointestinal mucosa; these processes are influenced by luminal components, such as butyrate and bile acids. Using butyrate-sensitive (BCS-TC2) and butyrate-resistant (BCS-TC2.BR2) human colon carcinoma cells, we wanted to establish whether colon carcinoma cells that acquire resistance to butyrate-induced apoptosis are also resistant to the cytotoxic effect of certain bile acids, contributing, in this way, to the progression of colon carcinogenesis. The effect of bile acids on BCS-TC2 cell viability is dose and time dependent and highly stereospecific. Quantification of the relative percentage of apoptotic cells and caspase-3 activity reveals that deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) induce apoptosis in BCS-TC2 cells. BCS-TC2.BR2 cells are consistently less sensitive to their cytotoxic effects, requiring concentrations to induce 50% inhibition (IC50) in cell viability of 740 microM and >1 mM for CDCA and DCA, respectively, compared with IC50 values of 310 and 540 microM for BCS-TC2 cells. DCA-treated BCS-TC2.BR2 cells show few apoptotic signs and no caspase-3 activation. On the other hand, CDCA-treated BCS-TC2.BR2 cells show caspase-3 activation and apoptotic features, although to a lower extent than BCS-TC2 cells. Our results, in an in vitro model system, point out that acquisition of butyrate resistance is accompanied by a partial resistance to the cytotoxic effects of bile acids, which may enhance the survival of tumorigenic cells.

Action of E. coli endotoxin, IL-1beta and TNF-alpha on antioxidant status of cultured hepatocytes.

We have previously reported that endotoxin induces in vivo oxidative stress in liver and a significant increase in hepatic and plasma glutathione concentrations during the acute phase of reversible endotoxic shock in rats. In the present study we examined the in vitro effects of E. coli 0111:B4 endotoxin (lipopolysaccharide, LPS), IL-1beta and TNF-alpha on antioxidant status of cultured hepatocytes in order to differentiate between the direct and mediated endotoxin action. LPS increased total glutathione (tGSH) levels after 2 h treatment but decreased oxidized glutathione (GSSG) content which lead to a marked decrease in GSSG/tGSH index. At shorter treatment times a biphasic and dose-dependent behaviour was observed. Cytokines (IL-1beta and TNF-alpha) produced significant decreases in tGSH and GSSG after 30 min treatment. Despite its prooxidant effect, TNF-alpha significantly reduced GSSG/tGSH index. Although no significant effects were observed on glutathione reductase activity, both LPS and cytokines induced an important inhibition of glutathione peroxidase which can justify the lipid peroxidation previously observed both in liver during reversible endotoxic shock and in cultured hepatocytes after treatment with endotoxin. The inhibition of hepatic glutathione peroxidase, besides the stimulation of GSH synthesis by LPS and GSH efflux by cytokines, guarantees the export of hepatic glutathione in its reduced form for other organs, contributing to the interorgan homeostasis. On the other hand, the results presented here support a new role for GSSG/tGSH index different from a mere indicator of oxidative stress.