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Kemp et al. Page 7 from the UV-visble, TEM and XPS studies that nanoparticles form on reduction of gold chloride by heparin and are stable at high electrolyte concentration. The reaction mechanism is not clear. However, it has been shown that other monosaccharide sugars reduce gold chloride by the reducing end of the residue.39 HA is commonly found in the extracellular matrix and provides a protective barrier around cells. Since this GAG has no anticoagulant activity, we extended our study to synthesize Au and Ag nanoparticles using hyaluronan. HA also acted as a reducing agent and was capable of synthesizing and stabilizing the nanoparticles. HA nanoparticle composites were analyzed by TEM (Figure 7). The Au-HA and Ag-HA nanocomposites were polydisperse with a size histogram ranging from of 5 to 30 nm. This polydispersity is greater than that observed for Au- DAPHP and Ag-DAPHP, because, the DAPHP contains a diaminopyridine moiety that binds tightly to the surface particles formed controlling their size and polydispersity. Hyaluronan binds Au and Ag weaker than DAPHP, resulting in a greater dispersity in nanocomposite size and shape. The biological activity of heparin is most commonly determined by coagulation-based assays such as aPTT, which measures inhibition of the blood coagulation cascade.29 The anticoagulant activity of DAPHP capped gold nanoparticles was compared with free DAPHP molecules (Figure 8). Either Au-DAPHP or Ag-DAPHP resulted in a concentration-dependent prolongation of clotting time or aPTT that is comparable to that obtained with DAPHP or heparin (Figure 8A and B). In contrast, Au- or Ag-glucose, Au-HA or Ag-HA did not exhibit any effect on aPTT as compared to Au-DAPHP or Ag-DAPHP at the same molar concentration (Figure 8C and D). These in vitro data clearly indicate the sole contribution of DAPHP linked to Au or Ag in the inhibition of coagulation, without any significant contributions by Au, Ag or HA (Figure 8C and D). Furthermore, the effects of free DAPHP, Au-DAPHP and Ag-DAPHP on platelet/fibrin clot dynamics in human blood was evaluated using thrombelastography.30–34 The time to clot initiation (R in minutes) or clot strength (MA in mm) for Au- or Ag-glucose, Au-HA or Ag- HA fell within the normal control ranges (Table 1). In contrast, Au-DAPHP or Ag-DAPHP at the same DAPHP molar concentration resulted in maximal inhibition of clot strength and without clot initiation for the duration of the study (Table 1). These in vitro data again clearly confirm the main contribution of DAPHP linked to Au or Ag in affecting coagulation, without any significant contributions by Au, Ag or HA (Table 1). A rat carrageenan model of paw edema was utilized to determine the potential anti- inflammatory efficacy of Au or Ag nanocomposites. In that regards, it has been previously demonstrated that the mixture of Cu, Au and Ag significantly decreased inflammatory disorders induced by adjuvant arthritis in the rat. The treatment with the mixture at low levels but not with the individual metals had a significant preventive effect. The results indicate an enhanced effect of the metal mixture in the model studied.40 Additionally, carrageenan-induced paw edema of mice and rats were suppressed by intravenous injection of heparin, corresponding well to the increase in plasma endothelial superoxide dismutase activity release by heparin in rats or mice. A dose response curve that was biphasic in nature was also observed for the ischemic paw model of mice. Electron microscopy confirmed that injections of heparin prevented the ischemia-induced mitochondrial swelling of the paw muscle.41 Similarly, the antioxidant activity of HA in a rat model of collagen-induced arthritis has been studied. Treatment with HA starting at the onset of arthritis for 10 days, limited the erosive action of the disease in the articular joints of knee and paw, reduced lipid peroxidation, restored the endogenous antioxidants reduced glutathione and superoxide dismutase, decreased plasma TNF-alpha levels, and limited synovial neutrophil infiltration.42 Biomacromolecules. Author manuscript; available in PMC 2010 March 9. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptPDF Image | Gold and Silver Nanoparticles Stabilized Glycosaminoglycans
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