Physico-Chemical Characterization and Assessment of Cytotoxic and Genotoxic Effects of Poly-Ethylene-Glycol Coated and Uncoated Gold Nanoparticles on Human Kidney (HK-2) Cells.

Although gold nanoparticles (Au-NPs) have been widely used in medicine for the diagnosis and treatment of patients due to their unique physicochemical properties, chemical stability and biocompatibility, recent reports have also highlighted their potential to induce toxicity to humans. In the present study, we investigated the toxic effects of uncoated and polyethylene glycol (PEG)-coated AuNPs on human kidney (HK-2) cells. Both forms of AuNP were synthesized and characterized using standard protocols. Dynamic Light Scattering (DLS), Zeta Sizer Nano ZS analyzer, Transmission Electron Microscopy (TEM), and Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) were used to measure their distribution, zeta potential/surface charge, morphological size, and Au concentrations, respectively. Cytotoxicity was measured by Cyto-Tox assay and trypan blue exclusion test. Oxidative stress (OS) was assessed by quantifying the levels of Glutathione (GSH), and Mitochondria Membrane Potential (MMP). Genotoxicity was assessed by single cell gel electrophoresis (Comet assay) and Chromosomal Aberration (CA) assay. Uncoated AuNPs significantly reduced cell viability, increased ROS, decreased GSH, depolarized the MMP, and induced significant DNA damage and chromosomal alterations including chromosome gaps, centric rings, breaks, deletions, and intra and inter-chromosome exchanges, in a concentration-dependent manner. PEG-coated AuNPs displayed lower cytotoxic and genotoxic effects, and did not produce any significant increase in ROS or significant decrease in GSH along with negligible polarization of the MMP. Hence, PEG-coated AuNPs are relatively less toxic than uncoated AuNPs and therefore, may have potential applications in nanomedicine.

Biochemical and Histopathological Evaluation of Graphene Oxide in Sprague-Dawley Rats.

Graphene and its derivatives are promising material for important biomedical applications due to their versatility. A detailed comprehensive study of the toxicity of these materials is required in context with the prospective use in biological setting. We investigated toxicity of Graphene Oxide (GO) in rats following exposure with respect to hepatotoxicity and oxidative stress biomarkers. Four groups of five male rats were orally administered GOs, once a day for five days, with doses of 10, 20 and 40mg/Kg GO. A control group consisted of five rats. Blood and liver were collected 24h after the last treatment following standard protocols. GO's exposure increased induction of Reactive Oxygen Species (ROS), activities of liver enzymes (Alanine ALT, Aspartate AST, Alkaline Phosphates ALP), concentration of Lipid Hydro Peroxide (LHP) and morphological alterations of liver tissue in exposed groups compared to control. The highest two doses, 20 and 40mg/kg, showed statistically significant (p<0.05) increases in the induction of ROS, activities of ALT, ALP, LHP concentration, and morphological alterations of liver tissue compared to control. However, AST activity showed no effect. The results of this study demonstrate that GO may be hepatotoxic, and its toxicity might be mediated through oxidative stress.

Solvent-dependent properties of electrospun fibrous composites for bone tissue regeneration.

Biodegradable polymer-ceramic composite scaffolds have gained importance in recent years in the field of orthopedic biomaterials and tissue engineering scaffolds for improving the rate of degradation and limited mechanical properties of bioactive ceramics. This study sought to create composites using the electrospinning process to achieve fibrous scaffolds with uniform fiber morphologies and uniform ceramic dispersions. Composites consisting of 20% hydroxyapatite/80% beta-tricalcium phosphate (20/80 HA/TCP) and poly (epsilon-caprolactone) (PCL) were fabricated. The 20/80 HA/TCP composition was chosen as the ceramic component because of previous reports of greater bone tissue formation in comparison with HA or TCP alone. For electrospinning, PCL was dissolved in either methylene chloride (Composite-MC) or a combination of methylene chloride (80%) and dimethylformamide (20%) (Composite-MC + DMF). Composite-MC mats contained a bimodal distribution of fiber diameters with nanofibers between larger, micron-sized fibers with an average pore size of 79.6 + or - 67 microm, whereas Composite-MC + DMF fibers had uniform fiber diameters with an average pore size of 7.0 + or - 4.2 microm. Elemental mapping determined that the ceramic was distributed throughout the mat and inside the fiber for both composites. However, physical characterization using differential scanning calorimetry (DSC) and mechanical testing revealed that the ceramic in the mats produced with MC + DMF were more uniformly dispersed than the ceramic in the mats produced with MC alone. Maximum tensile stress and strain were significantly higher for Composite-MC + DMF mats compared with Composite-MC mats and were comparable with the mechanical properties of mats of PCL alone. For both composites, there was molecular interaction between the PCL and the ceramic, as demonstrated by a maximum increase of approximately 10 degrees C in the glass transition values with the addition of the ceramic, as confirmed by Fourier transform infrared analysis. In addition, the crystallization behavior of the composites suggested that the ceramic was acting as a nucleating agent. Cell viability studies using human mesenchymal stem cells (MSC) showed that both composite scaffolds supported cell growth. However, cell numbers at early time points in culture were significantly higher on mats produced from MC + DMF compared with mats prepared with MC alone. Further examination revealed that cells were able to infiltrate the pores of the Composite-MC mats, but remained on the outer surface of the Composite-MC + DMF and unfilled PCL mats during the culture period. The results of this study demonstrate that the solvent or solvent combination used in preparing the electrospun composite mats plays a critical role in determining its properties, which may, in turn, affect cell behavior.

The effect of processing history on physical behavior and cellular response for tyrosine-derived polyarylates.

Polyarylates have shown promise as fully degradable polymers for drug delivery as well as for structural implant applications due to their range of physicomechanical properties. Processing history, however, could have a significant impact on their overall performance in biologically relevant environments. More specifically, structural changes at the molecular level can occur that will affect a polymer's physical properties and subsequent, cell attachment and growth. The present study was aimed at comparing cell growth on tyrosine-derived polyarylates with that of polylactic acid (PLLA) in their original state and after processing (i.e. undrawn and drawn forms). Two polyarylates having distinct molecular structures were chosen. Strictly, amorphous poly(DTE adipate), denoted as poly(DT 2,4), and poly(DTD) dodecandioate, denoted as poly(DT 12,10), having a more complex, non-crystalline organization, were compared with semi-crystalline PLLA. The degree of shrinkage, thermal characterization, air-water contact angle and surface morphology were determined for each polymer in its undrawn and drawn states. Poly(DT 2,4) and PLLA after processing resulted in greater shrinkage and a slight decrease in hydrophilicity whereas poly(DT 12,10) had minimal shrinkage and became slightly more hydrophilic in its drawn state. Surface morphology or roughness was also altered by processing. In turn, the rate of cell growth and overall cell numbers were reduced significantly on drawn forms of poly(DT 2,4) and PLLA, whereas more favorable growth rates were supported on drawn poly(DT 12,10). These findings indicate that processing effects in amorphous as well as oriented polymeric structures can significantly alter their biological performance.

Hemolysate activates P21RAS in rabbit basilar artery.

Cerebral vasospasm is the major factor of mortality and morbidity in the patients who have an aneurysmal subarachnoid hemorrhage (SAH). Erythrocyte lysate (hemolysate), oxyhemoglobin (OxyHb), and bloody cerebrospinal fluid (CSF) are the causative agents for vasospasm. However, the signal transduction pathways for the action of these spasmogens are not clear. In this study, we examined the possible effect of these spasmogens on the p21Ras protein, an important factor in the signal cascade, in rabbit basilar artery. Hemolysate enhanced p21Ras precipitation over a 7-day period. The initial increase of p21Ras precipitation occurred after the tissues were incubated for 2 days with hemolysate. The peak effect of hemolysate, which was markedly increased compared with control (P<0.05, ANOVA), was observed on day 3. OxyHb and blood CSF, in contrast, failed to produce consistent or marked changes in p21Ras precipitation. p21Ras inhibitors FTPase inhibitor 1 and manumycin abolished hemolysate-induced enhancement of p21Ras immunoprecipitation. Genistein, a tyrosine kinase inhibitor, failed to reduce the effect of hemolysate on p21Ras. We concluded that hemolysate activates p21Ras in the rabbit basilar artery.

Role of tyrosine kinase in fibroblast compaction and cerebral vasospasm.

Hemolysate, a proposed causative agent for cerebral vasospasm following subarachnoid hemorrhage, produces contraction of cerebral arteries by activation of tyrosine kinases. In addition, hemolysate accelerates fibroblast collagen compaction that could play a role in cerebral vasospasm. We studied the effect of hemolysate on tyrosine phosphorylation and fibroblast collagen compaction in cultured dog cerebral and human dermal fibroblasts using tyrosine kinase inhibitors and tyrosine antibodies (Western blot). 1) Hemolysate was found to enhance tyrosine phosphorylation of two proteins approximately 64 and 120 kDa. The effect of hemolysate was time- and concentration-dependent. 2) Two main components in hemolysate, oxyhemoglobin and adenosine triphosphate (ATP), produced similar results to that of hemolysate. 3) Tyrosine kinase inhibitor genistein and tyrphostin A51 (30 microM) markedly reduced the effect of hemolysate on tyrosine phosphorylation. 4) In another study, hemolysate increased fibroblast collagen compaction and the effect of hemolysate was reduced by genistein and tyrphostin A51. We conclude that hemolysate activates tyrosine kinase that may lead to acceleration of fibroblast compaction. This effect of hemolysate may contribute to cerebral vasospasm.

Hemolysate induces tyrosine phosphorylation and collagen-lattice compaction in cultured fibroblasts.

Hemolysate, a proposed causative agent for cerebral vasospasm after subarachnoid hemorrhage, produces contraction of cerebral arteries by activation of tyrosine kinases. In addition, hemolysate increases fibroblast-collagen compaction that could play a role in cerebral vasospasm. We studied the effect of hemolysate on tyrosine phosphorylation and fibroblast-collagen compaction in cultured canine basilar and human dermal fibroblasts using tyrosine kinase inhibitors and tyrosine antibodies. Hemolysate enhanced tyrosine phosphorylation of two proteins, 64 and 120 kDa, in cultured canine basilar artery and human dermal fibroblast cells. The effect of hemolysate was time-dependent and concentration-dependent. Oxyhemoglobin and ATP, the two major components of hemolysate, produced similar tyrosine phosphorylation, however, with a different time course. Tyrosine kinase inhibitors genistein and tyrphostin A51 abolished the effect of hemolysate in both cerebral and dermal fibroblasts. Hemolysate increased fibroblast-populated collagen-lattice compaction and tyrosine kinase inhibitors genistein and tyrphostin A51 attenuated the effect of hemolysate. We conclude that hemolysate activates tyrosine kinase that leads to the increase of fibroblast compaction. This effect of hemolysate may contribute to cerebral vasospasm.

Mitogen-activated protein kinase mediation of hemolysate-induced contraction in rabbit basilar artery.

OBJECT: Mitogen-activated protein kinase (MAPK) is an important signaling factor in vascular proliferation and contraction, which are the two features of cerebral vasospasm that follow subarachnoid hemorrhage. The authors studied the possible involvement of MAPK in hemolysate-induced signal transduction and contraction in rabbit basilar artery (BA). METHODS: Isometric tension was used to record the contractile response of rabbit BA to hemolysate, and Western blots were obtained using antibodies for MAPK. The following results are reported. 1) Hemolysate produced a concentration-dependent contraction of rabbit BA; however, preincubation of arteries with the MAPK kinase (MEK) inhibitor PD-98059 markedly reduced this contraction. The administration of PD-98059 also relaxed, in a concentration-dependent fashion, the sustained contraction induced by 10% hemolysate. 2) The Janus tyrosine kinase 2 inhibitor AG-490, preincubated with arterial rings, reduced the contractile response to hemolysate but failed to relax the sustained contraction induced by this agent. The Src-tyrosine kinase inhibitor damnacanthal and the phosphatidylinositol 3-kinase inhibitor wortmannin failed to reduce hemolysate-induced contraction. 3) Hemolysate produced a time-dependent elevation of MAPK immunoreactivity as seen on Western blots of rabbit BA. The MAPK was enhanced 1 minute after hemolysate exposure and the effect reached maximum levels at 5 minutes. The immunoreactivity of MAPK decayed slowly over time, but the level of this kinase was still higher than the basal level, even at 2 hours after exposure to hemolysate. Preincubation of arteries with the MEK inhibitor PD-98059 abolished the effect of hemolysate on MAPK immunoreactivity. CONCLUSIONS: Hemolysate produced contraction of rabbit BA, possibly by activation of MAPK, and therefore MAPK inhibitors may be useful in the treatment of cerebral vasospasm.