Stabilization of Graphene Oxide Dispersion in Plasma-like Isotonic Solution Containing Aggregating Concentrations of Bivalent Cations.

Graphene oxide's (GO) intravascular applications and biocompatibility are not fully explored yet, although it has been proposed as an anticancer drug transporter, antibacterial factor or component of wearable devices. Bivalent cations and the number of particles' atom layers, as well as their structural oxygen content and pH of the dispersion, all affect the GO size, shape, dispersibility and biological effects. Bovine serum albumin (BSA), an important blood plasma protein, is expected to improve GO dispersion stability in physiological concentrations of the precipitating calcium and magnesium cations to enable effective and safe tissue perfusion. METHODS: Four types of GO commercially available aqueous dispersions (with different particle structures) were diluted, sonicated and studied in the presence of BSA and physiological cation concentrations. Nanoparticle populations sizes, electrical conductivity, zeta potential (Zetasizer NanoZS), structure (TEM and CryoTEM), functional groups content (micro titration) and dispersion pH were analyzed in consecutive preparation stages. RESULTS: BSA effectively prevented the aggregation of GO in precipitating concentrations of physiological bivalent cations. The final polydispersity indexes were reduced from 0.66-0.91 to 0.36-0.43. The GO-containing isotonic dispersions were stable with the following Z-ave results: GO1 421.1 nm, GO2 382.6 nm, GO3 440.2 nm and GO4 490.1 nm. The GO behavior was structure-dependent. CONCLUSION: BSA effectively stabilized four types of GO dispersions in an isotonic dispersion containing aggregating bivalent physiological cations.

In vitro effects of cobalt and chromium nanoparticles on human platelet function.

Nanoparticles (NPs) are released from orthopedic and neurosurgical prostheses and can interact with a number of blood components once in the bloodstream. Potential toxic effects of Co and Cr NPs on blood platelets have not been thoroughly investigated. The aim of this study was to analyze the effect of Co and Cr NPs on platelet function in vitro. The ability of the tested NPs to induce platelet activation and aggregation was measured using light transmission aggregometry, flow cytometry, and quartz crystal balance with dissipation (QCM-D). This was confirmed by transmission electron microscopy (TEM), scanning electron microscopy, and optical and immunofluorescence microscopy. Perfusion of QCM-D sensor crystals with platelet-rich-plasma in the presence of Co 28 nm, CoO 50 nm, Co2O3 50 nm, Co3O4 30-50nm, Cr 35-45nm, Cr2O3 60 nm NPs (0.5-5.0 µg/mL) resulted in significant changes in frequency and dissipation, indicating that these NPs caused platelet microaggregation. Transmission electron microscopy also revealed that Cr NPs led to platelet swelling and lysis. Our study shows that both Co and Cr NPs affect platelet function in vitro with two distinct mechanisms. While Co NPs result in standard platelet aggregation, Cr NPs cause both platelet aggregation and decreased platelet membrane integrity and lysis. Based on these findings, monitoring serum NP levels and platelet-mediated hemostasis can be advised in patients with metal-on-metal Co-Cr prostheses.

Novel Barbiturate-Nitrate Compounds Inhibit the Upregulation of Matrix Metalloproteinase-9 Gene Expression in Intestinal Inflammation through a cGMP-Mediated Pathway.

Matrix metalloproteinase-9 is upregulated in inflammatory bowel disease. Barbiturate nitrate hybrid compounds have been designed to inhibit MMP secretion and enzyme activity. In this study, we investigated the mechanism of action of barbiturate-nitrate hybrid compounds and their component parts using models of intestinal inflammation in vitro. Cytokine-stimulated Caco-2 cells were used in all in vitro experiments. The NO donors SNAP and DETA-NONOate were used to study the effect of NO on MMP-9 mRNA. Mechanistic elucidation was carried out using the soluble guanylate cyclase (sGC) inhibitor, ODQ, and the cGMP analogue, 8-Bromo-cGMP. Further experiments were carried out to elucidate the role of NF-κB. NO donors exerted an inhibitory effect on MMP-9 mRNA in cytokine-stimulated cells. While the non-nitrate barbiturates had a limited effect on MMP-9 expression, the hybrid compounds inhibited MMP-9 expression through its NO-mimetic properties. No effect could be observed on mRNA for MMP-1 or MMP-2. The sGC inhibitior, ODQ, abolished the nitrate-barbiturate inhibition of MMP-9 gene expression, an effect which was reversed by 8-Br-cGMP. This study shows that the barbiturate scaffold is suitable for hybrid design as an MMP-9 inhibitor in cytokine-stimulated Caco-2 cells. The inhibition of MMP-9 levels was largely mediated through a reduction in its mRNA by a sGC/cGMP pathway mediated mechanism.

Role of matrix metalloproteinases 2 and 9, toll-like receptor 4 and platelet-leukocyte aggregate formation in sepsis-associated thrombocytopenia.

BACKGROUND: The development of thrombocytopenia in sepsis is a poor prognostic indicator associated with a significantly increased mortality risk. Mechanisms underlying this phenomenon remain to be clearly elucidated. Matrix metalloproteinases (MMPs) are enzymes that regulate the turnover of the extra-cellular matrix. MMP-2 is recognised as a platelet agonist with MMP-9 proposed as an inhibitor of platelet activation. The existence of MMP-9 in platelets is a subject of debate. There is limited evidence thus far to suggest that toll-like receptor 4 (TLR-4) and platelet-leukocyte aggregate (PLA) formation may be implicated in the development of sepsis-associated thrombocytopenia. OBJECTIVES: To investigate whether MMP -2/-9, toll-like receptor 4 (TLR-4) or platelet-leukocyte aggregate (PLA) formation are implicated in a decline in platelet numbers during septic shock. METHODS: This was an observational study which recruited healthy controls, non-thrombocytopenic septic donors and thrombocytopenic septic donors. MMP-2, MMP-9 and TLR-4 platelet surface expression as well as PLA formation was examined using flow cytometry. In addition MMP-2 and MMP-9 were examined by gelatin zymography and enzyme-linked immunosorbent assay (ELISA) using a 3 compartment model (plasma, intraplatelet and platelet membrane). RESULTS: There was no difference found in MMP-2, MMP-9 or TLR-4 levels between non-thrombocytopenic and thrombocytopenic septic donors. PLA formation was increased in thrombocytopenic patients. MMP-9 was detected in platelets using flow cytometry, gelatin zymography and ELISA techniques. CONCLUSIONS: Platelet consumption into PLAs may account for the development of thrombocytopenia in septic shock. MMP-9 is found in platelets and it is upregulated during septic shock.

The Role of Mucin in the Toxicological Impact of Polystyrene Nanoparticles.

The development of novel oral drug delivery systems is an expanding area of research and both new approaches for improving their efficacy and the investigation of their potential toxicological effect are crucial and should be performed in parallel. Polystyrene nanoparticles (NPs) have been used for the production of diagnostic and therapeutic nanosystems, are widely used in food packaging, and have also served as models for investigating NPs interactions with biological systems. The mucous gel layer that covers the epithelium of the gastrointestinal system is a complex barrier-exchange system that it is mainly constituted by mucin and it constitutes the first physical barrier encountered after ingestion. In this study, we aimed to investigate the effect of polystyrene NPs on mucin and its potential role during NP⁻cell interactions. For this purpose, we evaluated the interaction of polystyrene NPs with mucin in dispersion by dynamic light scattering and with a deposited layer of mucin using a quartz crystal microbalance with dissipation technology. Next, we measured cell viability and the apoptotic state of three enterocyte-like cell lines that differ in their ability to produce mucin, after their exposure to the NPs. Positive charged NPs showed the ability to strongly interact and aggregate mucin in our model. Positive NPs affected cell viability and induced apoptosis in all cell lines independently of their ability of produce mucin.

Platelet microaggregation in sepsis examined by quartz crystal microbalance with dissipation technology.

Antiplatelet therapies remain an area of potential interest for the treatment of sepsis; however, studies of platelet aggregation in sepsis have yielded conflicting results. We examined platelet aggregation patterns in patients with septic shock using quartz crystal microbalance with dissipation technology, a microfluidic device capable of measuring platelet microaggregate formation under flow conditions. Platelet aggregation was increased in the washed platelet samples of septic patients. Conversely, these same platelets aggregated less than healthy controls when examined in their plasma.

Differential eNOS-signalling by platelet subpopulations regulates adhesion and aggregation.

AIMS: In addition to maintaining haemostasis, circulating blood platelets are the cellular culprits that form occlusive thrombi in arteries and veins. Compared to blood leucocytes, which exist as functionally distinct subtypes, platelets are considered to be relatively simple cell fragments that form vascular system plugs without a differentially regulated cellular response. Hence, investigation into platelet subpopulations with distinct functional roles in haemostasis/thrombosis has been limited. In our present study, we investigated whether functionally distinct platelet subpopulations exist based on their ability to generate and respond to nitric oxide (NO), an endogenous platelet inhibitor. METHODS AND RESULTS: Utilizing highly sensitive and selective flow cytometry protocols, we demonstrate that human platelet subpopulations exist based on the presence and absence of endothelial nitric oxide synthase (eNOS). Platelets lacking eNOS (approximately 20% of total platelets) fail to produce NO and have a down-regulated soluble guanylate cyclase-protein kinase G (sGC-PKG)-signalling pathway. In flow chamber and aggregation experiments eNOS-negative platelets primarily initiate adhesion to collagen, more readily activate integrin αIIbß3 and secrete matrix metalloproteinase-2, and form larger aggregates than their eNOS-positive counterparts. Conversely, platelets having an intact eNOS-sGC-PKG-signalling pathway (approximately 80% of total platelets) form the bulk of an aggregate via increased thromboxane synthesis and ultimately limit its size via NO generation. CONCLUSION: These findings reveal previously unrecognized characteristics and complexity of platelets and their regulation of adhesion/aggregation. The identification of platelet subpopulations also has potentially important consequences to human health and disease as impaired platelet NO-signalling has been identified in patients with coronary artery disease.

The Nanopharmacology and Nanotoxicology of Nanomaterials: New Opportunities and Challenges.

The very dynamic growth of nanotechnology, nanomaterials (sized 1-100 nm) and their medical applications over the past 10 years has promised to add a new impetus to the diagnostics and therapeutics of a wide range of human pathologies, including cancer, cardiovascular diseases and diseases of the central nervous system. This growth in nanomedicine also fuels advances in bioengineering, regenerative medicine and the development of medical devices. However, as with all new pharmaceuticals and medical devices, new opportunities are inherently accompanied by new challenges due to the ability of nanomaterials to interact with the body on the cellular, subcellular and molecular levels. This article reviews some of the most compelling problems related to the nanopharmacology and nanotoxicology of nanomaterials. The overview focuses on opportunities emerging from the development of multifunctional nanomaterials and nanotheranostics for the diagnostics and therapy of both major and rare diseases. Challenges related to the hemocompatibility of nanomaterials are also discussed.

Pharmacokinetics and bio-distribution of novel super paramagnetic iron oxide nanoparticles (SPIONs) in the anaesthetized pig.

Manufactured nanomaterials have a variety of medical applications, including diagnosis and targeted treatment of cancer. A series of experiments were conducted to determine the pharmacokinetic, biodistribution and biocompatibility of two novel magnetic nanoparticles (MNPs) in the anaesthetized pig. Dimercaptosuccinic acid (DMSA) coated superparamagnetic iron oxide nanoparticles (MF66-labelled 12 nm, core nominal diameter and OD15 15 nm); at 0.5, or 2.0 mg/kg) were injected intravenously. Particles induced a dose-dependent decrease in blood pressure following administration which recovered to control levels several minutes after injection. Blood samples were collected for a 5-h period and stored for determination of particle concentration using particle electron paramagnetic resonance (pEPR). Organs were harvested post-mortem for magnetic resonance imaging (MRI at 1.5 T field strength) and histology. OD15 (2.0 mg/kg) MNP had a plasma half-life of approximately 15 min. Both doses of the MF66 (0.5 and 2.0 mg/kg) MNP were below detection limits. MNP accumulation was observed primarily in the liver and spleen with MRI scans which was confirmed by histology. MRI also showed that both MNPs were present in the lungs. The results show that further modifications may be required to improve the biocompatibility of these particles for use as diagnostic and therapeutic agents.

Magnetic Nanoparticles in Cancer Theranostics.

In a report from 2008, The International Agency for Research on Cancer predicted a tripled cancer incidence from 1975, projecting a possible 13-17 million cancer deaths worldwide by 2030. While new treatments are evolving and reaching approval for different cancer types, the main prevention of cancer mortality is through early diagnosis, detection and treatment of malignant cell growth. The last decades have seen a development of new imaging techniques now in widespread clinical use. The development of nano-imaging through fluorescent imaging and magnetic resonance imaging (MRI) has the potential to detect and diagnose cancer at an earlier stage than with current imaging methods. The characteristic properties of nanoparticles result in their theranostic potential allowing for simultaneous detection of and treatment of the disease. This review provides state of the art of the nanotechnological applications for cancer therapy. Furthermore, it advances a novel concept of personalized nanomedical theranostic therapy using iron oxide magnetic nanoparticles in conjunction with MRI imaging. Regulatory and industrial perspectives are also included to outline future perspectives in nanotechnological cancer research.

miR-134 in extracellular vesicles reduces triple-negative breast cancer aggression and increases drug sensitivity.

Exosomes (EVs) have relevance in cell-to-cell communication carrying pro-tumorigenic factors that participate in oncogenesis and drug resistance and are proposed to have potential as self-delivery systems. Advancing on our studies of EVs in triple-negative breast cancer, here we more comprehensively analysed isogenic cell line variants and their EV populations, tissues cell line variants and their EV populations, as well as breast tumour and normal tissues. Profiling 384 miRNAs showed EV miRNA content to be highly representative of their cells of origin. miRNAs most substantially down-regulated in aggressive cells and their EVs originated from 14q32. Analysis of miR-134, the most substantially down-regulated miRNA, supported its clinical relevance in breast tumours compared to matched normal breast tissue. Functional studies indicated that miR-134 controls STAT5B which, in turn, controls Hsp90. miR-134 delivered by direct transfection into Hs578Ts(i)8 cells (in which it was greatly down-regulated) reduced STAT5B, Hsp90, and Bcl-2 levels, reduced cellular proliferation, and enhanced cisplatin-induced apoptosis. Delivery via miR-134-enriched EVs also reduced STAT5B and Hsp90, reduced cellular migration and invasion, and enhanced sensitivity to anti-Hsp90 drugs. While the differing effects achieved by transfection or EV delivery are likely to be, at least partly, due to specific amounts of miR-134 delivered by these routes, these EV-based studies identified miRNA-134 as a potential biomarker and therapeutic for breast cancer.

Pharmacological characterization of nanoparticle-induced platelet microaggregation using quartz crystal microbalance with dissipation: comparison with light aggregometry.

BACKGROUND: Engineered nanoparticles (NPs) can induce platelet activation and aggregation, but the mechanisms underlying these interactions are not well understood. This could be due in part to use of devices that study platelet function under quasi-static conditions with low sensitivity to measure platelet microaggregation. Therefore, in this study we investigated the pharmacological pathways and regulators of NP-induced platelet microaggregation under flow conditions at nanoscale using quartz crystal microbalance with dissipation (QCM-D) and compared the data thus obtained with those generated by light aggregometry. METHODS: Blood was collected from healthy volunteers, and platelet-rich plasma was obtained. Thrombin receptor-activating peptide, a potent stimulator of platelet function, and pharmacological inhibitors were used to modulate platelet microaggregation in the presence/absence of silica (10 nm and 50 nm) and polystyrene (23 nm) NPs. Light aggregometry was used to study platelet aggregation in macroscale. Optical, immunofluorescence, and scanning electron microscopy were also used to visualize platelet aggregates. RESULTS: Platelet microaggregation was enhanced by thrombin receptor-activating peptide, whereas prostacyclin, nitric oxide donors, acetylsalicylic acid, and phenanthroline, but not adenosine diphosphate (ADP) blockers, were able to inhibit platelet microaggregation. NPs caused platelet microaggregation, an effect not detectable by light aggregometry. NP-induced microaggregation was attenuated by platelet inhibitors. CONCLUSION: NP-induced platelet microaggregation appears to involve classical proaggregatory pathways (thromboxane A2-mediated and matrix metalloproteinase-2-mediated) and can be regulated by endogenous (prostacyclin) and pharmacological (acetylsalicylic acid, phenanthroline, and nitric oxide donors) inhibitors of platelet function. Quartz crystal microbalance with dissipation, but not light aggregometry, is an appropriate method for studying NP-induced microaggregation.

Biodistribution and pharmacokinetic studies of SPION using particle electron paramagnetic resonance, MRI and ICP-MS.

AIM: Superparamagnetic iron oxide nanoparticles (SPIONs) may play an important role in nanomedicine by serving as drug carriers and imaging agents. In this study, we present the biodistribution and pharmacokinetic properties of SPIONs using a new detection method, particle electron paramagnetic resonance (pEPR). MATERIALS & METHODS: The pEPR technique is based on a low-field and low-frequency electron paramagnetic resonance. pEPR was compared with inductively coupled plasma mass spectrometry and MRI, in in vitro and in vivo. RESULTS: The pEPR, inductively coupled plasma mass spectrometry and MRI results showed a good correlation between the techniques. CONCLUSION: The results indicate that pEPR can be used to detect SPIONs in both preclinical and clinical studies.

Nanodiagnostics, nanopharmacology and nanotoxicology of platelet-vessel wall interactions.

In physiological conditions, the interactions between blood platelets and endothelial cells play a major role in vascular reactivity and hemostasis. By contrast, increased platelet activation contributes to the pathogenesis of vascular pathology such as atherosclerosis, thrombosis, diabetes mellitus, hypertension and carcinogenesis. Nanomedicine, including nanodiagnostics and nanotherapeutics is poised to be used in the management of vascular diseases. However, the inherent risk and potential toxicity resultant from the use of nanosized (<100 nm) materials need to be carefully considered. This review, basing on a systematic search of literature provides state-of-the-art and focuses on new discoveries, as well as the potential benefits and threats in the field of nanodiagnostics, nanopharmacology and nanotoxicology of platelet-vessel wall interactions.

CdTe quantum dots induce activation of human platelets: implications for nanoparticle hemocompatibility.

New nanomaterials intended for systemic administration have raised concerns regarding their biocompatibility and hemocompatibility. Quantum dots (QD) nanoparticles have been used for diagnostics, and recent work suggests their use for in vivo molecular and cellular imaging. However, the hemocompatibility of QDs and their constituent components has not been fully elucidated. In the present study, comprehensive investigation of QD-platelet interactions is presented. These interactions were shown using transmission electron microscopy. The effects of QDs on platelet function were investigated using light aggregometry, quartz crystal microbalance with dissipation, flow cytometry, and gelatin zymography. Platelet morphology was also analyzed by phase-contrast, immunofluorescence, atomic-force and transmission electron microscopy. We show that the QDs bind to platelet plasma membrane with the resultant upregulation of glycoprotein IIb/IIIa and P-selectin receptors, and release of matrix metalloproteinase-2. These findings unravel for the first time the mechanism of functional response of platelets to ultrasmall QDs in vitro.

Blood biocompatibility of surface-bound multi-walled carbon nanotubes.

Blood clots when it contacts foreign surfaces following platelet activation. This can be catastrophic in clinical settings involving extracorporeal circulation such as during heart-lung bypass where blood is circulated in polyvinyl chloride tubing. Studies have shown, however, that surface-bound carbon nanotubes may prevent platelet activation, the initiator of thrombosis. We studied the blood biocompatibility of polyvinyl chloride, surface-modified with multi-walled carbon nanotubes in vitro and in vivo. Our results show that surface-bound multi-walled carbon nanotubes cause platelet activation in vitro and devastating thrombosis in an in vivo animal model of extracorporeal circulation. The mechanism of the pro-thrombotic effect likely involves direct multi-walled carbon nanotube-platelet interaction with Ca(2+)-dependant platelet activation. These experiments provide evidence, for the first time, that modification of surfaces with nanomaterials modulates blood biocompatibility in extracorporeal circulation.

Pharmacological and toxicological effects of co-exposure of human gingival fibroblasts to silver nanoparticles and sodium fluoride.

BACKGROUND: Silver nanoparticles (AgNPs) and fluoride (F) are pharmacological agents widely used in oral medicine and dental practice due to their anti-microbial/anti-cavity properties. However, risks associated with the co-exposure of local cells and tissues to these xenobiotics are not clear. Therefore, we have evaluated the effects of AgNPs and F co-exposure on human gingival fibroblast cells. METHODS: Human gingival fibroblast cells (CRL-2014) were exposed to AgNPs and/or F at different concentrations for up to 24 hours. Cellular uptake of AgNPs was examined by transmission electron microscopy. Downstream inflammatory effects and oxidative stress were measured by real-time quantitative polymerase chain reaction (PCR) and reactive oxygen species (ROS) generation. Cytotoxicity and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and real-time quantitative PCR and flow cytometry, respectively. Finally, the involvement of mitogen-activated protein kinases (MAPK) was studied using Western blot. RESULTS: We found that AgNPs penetrated the cell membrane and localized inside the mitochondria. Co-incubation experiments resulted in increased oxidative stress, inflammation, and apoptosis. In addition, we found that co-exposure to both xenobiotics phosphorylated MAPK, particularly p42/44 MAPK. CONCLUSION: A combined exposure of human fibroblasts to AgNPs and F results in increased cellular damage. Further studies are needed in order to evaluate pharmacological and potentially toxicological effects of AgNPs and F on oral health.

Nitric oxide-matrix metaloproteinase-9 interactions: biological and pharmacological significance--NO and MMP-9 interactions.

Nitric oxide (NO) and matrix metalloproteinase 9 (MMP-9) levels are found to increase in inflammation states and in cancer, and their levels may be reciprocally modulated. Understanding interactions between NO and MMP-9 is of biological and pharmacological relevance and may prove crucial in designing new therapeutics. The reciprocal interaction between NO and MMP-9 have been studied for nearly twenty years but to our knowledge, are yet to be the subject of a review. This review provides a summary of published data regarding the complex and sometimes contradictory effects of NO on MMP-9. We also analyse molecular mechanisms modulating and mediating NO-MMP-9 interactions. Finally, a potential therapeutic relevance of these interactions is presented.

Mechanisms of platelet-stimulated colon cancer invasion: role of clusterin and thrombospondin 1 in regulation of the P38MAPK-MMP-9 pathway.

Platelets have been implicated in colon cancer metastasis and prognosis but the underlying molecular mechanisms remain unclear. We evaluated the role of the different mitogen-activated protein kinase (MAPK) pathways in platelet-stimulated matrix metalloproteinase-9 (MMP-9) generation and colon cancer invasion. In addition, proteins released during platelet-tumour cell interactions were studied. For this purpose, interactions of Caco-2 and HT29 cells with platelets were studied using scanning electron microscopy, aggregometry, flow cytometry and cell invasion chambers. Quantitative PCR and zymography were used to study MMP-9 gene expression and activity, respectively, whereas western blot was used to study p38MAPK. Finally, the origin of proteins during platelet-cancer cell interactions was investigated using stable isotope labelling by amino acids in cell culture (SILAC)-based proteomics. We found that platelets promoted p38MAPK phosphorylation and MMP-9 up-regulation in both cell lines, with the subsequent cell-invasion-promoting effects. Pharmacological inhibition of p38MAPK led to a significant down-regulation of MMP-9 and colon cancer cell invasiveness. Also, p38MAPK-small interfering RNA abolished the induction of platelet-stimulated MMP-9. SILAC experiments demonstrated that thrombospondin 1 (TSP1) was released mainly from platelets and clusterin by both platelets and cancer cells. Finally, inhibition of TSP1 and clusterin abolished p38MAPK phosphorylation, MMP-9 activity and platelet-stimulated colon cancer invasion. Our results indicate that platelet-secreted TSP1 and clusterin promote the signal regulation of MMP-9 in platelet-induced colonic cancer invasion via a P38MAPK-regulated pathway. These findings are relevant to the development of therapeutic approaches to preventing and reducing tumour cell metastasis induced by colon adenocarcinoma.