Stabilized Au and Ag nanoparticles were prepared using heparin derivatized with a diaminopyridine group at the reducing end synthesized through reductive amination (DAPHP). It is believed that the aldehyde on the reducing end of the residual underivatized heparin present in the DAPHP reduces AuCl4 and AgNO3, while the DAP on the derivatized DAPHP provides a strong interaction between its amino/pyridine group and the Au or Ag nanoparticles. The bioconjugates prepared in this synthesis were analyzed by UV-Vis spectroscopy. The UV-Vis spectra recorded on Au and Ag nanoparticles solutions synthesized using DAPHP are shown in . A strong resonance at approximately 533 and 400 nm, respectively, is observed for Au and Ag nanoparticles in solution, due to the excitation of surface plasmon vibrations. The surface plasmon resonance band is shifted towards a higher wavelength, compared with Au and Ag nanoparticles of the similar sizes synthesized with other conventional methods. This shift can be explained by the interaction between the DAPHP and the particles, which influences the surface plasmon resonance band. The UV-Vis spectra of Au and Ag nanoparticle solutions remained unchanged over several months when stored at 20 °C, indicating the nanoparticle size distribution in water was extremely stable. Nanoparticles are stabilized by the bound DAPHP and show no sign of aggregation.
Figure 1 UV-visible spectra of gold (solid) and silver (dashed) nanoparticles synthesized by DAPHP molecules. The plasmon peak at ~533 nm confirms the presence of AuNPs, while the plasmon peak at ~400 nm corresponds to the formation of AgNPs. Arbitrary units (a.u.). (more ...)
Au and Ag nanoparticles synthesized using DAPHP were analyzed by TEM. A drop-coated film of aqueous solution of nanoparticles was formed on carbon-coated copper grid by solvent evaporation and analyzed by TEM. TEM images of Au and Ag nanoparticle films formed are shown in and , respectively. The diffraction patterns of the particles show the face-centered cubic (FCC) of Au () and Ag () bioconjugate. The particle size distribution (PSD) of the DAPHP capped Au and Ag nanoparticles were calculated based on and , affording particle sizes 10 ± 3 nm and 7 ± 3 nm for Au-DAPHP and Ag-DAPHP nanoparticles, respectively.
TEM image of Au-DAPHP capped gold nanoparticles show uniform size distribution at (a) 100kx and (b) 160kx. (c) Diffraction pattern of AuNPs show their corresponding FCC. (d) Particle size distribution (PSD) of AuNPs is 10 nm ± 3 nm.
TEM image of Ag-DAPHP capped silver nanoparticles at (a) 160 kx and (b) 340kx. (c) Diffraction pattern reveal FCC of AgNPs. (d) Particle size distribution (PSD) of AgNPs is 7 nm ± 3 nm.
The instability of nanoparticles, particularly in the presence of electrolytes, is a major issue in colloidal chemistry. Electrolyte-induced precipitation of nanoparticles from the aqueous phase is commonly observed. Strouse et al
. has shown that colloidal gold nanoparticles can be precipitated by addition of an electrolyte and that, under certain conditions, these nanoparticles can be redispersed in water.36
The stability, of Au-DAPHP and Ag-DAPHP nanoparticle solutions in the current study, were checked as a function of NaCl (electrolyte) concentration. The UV-Vis spectra of solution of Au-DAPHP and Ag-DAPHP nanoparticle were examined after addition of sodium chloride as shown in . After addition of different amounts of NaCl, the UV-Vis spectra remained unchanged. Even high NaCl concentrations (1M) do not destabilize these colloidal solutions. The high stability of these DAPHP nanoparticles is both due to electrostatic and steric factors.
UV-visible spectra of (a) gold and (b) silver nanoparticles synthesized by DAPHP molecules as a function of increasing NaCl concentration from 0 – 1 M. Arbitrary units (a.u.).
Au-DAPHP and Ag-DAPHP particles were further purified by centrifugation and washing. The stability of these purified nanocomposites was again examined as a function of NaCl concentration, to obtain further insight into colloidal stabilization. The UV-Vis spectra of redispersed purified Au-DAPHP and Ag-DAPHP nanoparticles solution were taken after addition of 0 to 150 mM NaCl (). There is an increase in absorbance at ~650 nm or broadening in the absorbance of purified Au-DAPHP on increasing the concentration of NaCl (). Similar trends in the broadening in surface plasmon resonance are also observed in the Ag-DAPHP nanoparticle solution (). The broadening and increase in plasmon resonance at higher wavelength indicates aggregation of nanoparticles. While some aggregation takes place in 150 mM NaCl, the activity of the particles within an electrolyte solution might behave differently in vivo, since the particles are coming in contact with many other biomolecules present in biological fluids. Therefore, the tolerance limits for specific biomedical purposes would need to be further addressed depending on the application being used. This decrease in stability of purified samples, suggest that some DAPHP polysaccharide molecules bind the bioconjugate via week electrostatic interaction and that these free polysaccharides can be desorbed from the surface of bioconjugate on repeated washing.
UV-visible spectra of purified and redispersed (a) gold and (b) silver nanoparticles synthesized by DAPHP molecules as a function of NaCl concentration from 0 – 150 mM. Arbitrary units (a.u.).
Quantification of DAPHP chains bound to the surface of the purified Au-DAPHP and Ag-DAPHP nanoparticles was then determined using carbazole assay.28
This assay detects the uronic acids that are present within these polysaccharides. The theoretical calculation DAPHP attached to Au and Ag nanoparticles using heparinase digestion was calculated to be ~50 heparin chains/AuNP and ~20 heparin chains/AgNP. This method was used to determine DAPHP concentration for anticoagulant assays. To further investigate the loading of DAPHP onto AuNPs the concentration of AuNPs was estimated by two different methods and heparin was displaced by an excess amount of DTT; based on these concentrations heparin loading on the nanoparticles was ~80 heparin chains/AuNP (Supporting Information
). Heparin has the highest negative charge of any known biomolecule.21
The number of chains/particle is certainly limited due to the electrostatic repulsion of heparin, hindering a tighter packing of chains onto the particles. The heparin loading onto AgNPs is theorized to be slightly less since the binding of the amine groups is not as strong to Ag as it is to Au.
Purified DAPHP-stabilized Au and Ag nanoparticle films were drop coated on Si (111) substrates and analyzed by XPS. The general scan spectrum showed the presence of the principal C1s, N1s, Au 4f and Ag 3d core levels with no evidence of impurities. The film was sufficiently thick and therefore, no signal was measured from the substrate (Si 2p core level). The C1s, N1s, and Au 4f core levels recorded from this film are shown in . The spectra have been corrected for any background signals using the Shirley algorithm37
prior to curve resolution. The Au 4f core level could be satisfactorily fit to a two spin-orbit pair at 84 and 86 eV (4 f7/2). These values are in good agreement with published values for gold nanoparticles.38
These bands correspond to Au (0) and Au (III) state. Presence of higher binding energy components indicates the presence of Au (III) ions adsorbed on gold nanoparticles surface or as an impurity in purified solution. N1s band is observed at 399 eV, which correspond to the nitrogen atom of DAPHP molecules attached to gold nanoparticles. Similarly spin-orbit 367 (3 d5/2) can be seen in Ag-DAPHP film molecules (), corresponding to Ag (0). N1s band at ~ 399 eV corresponds to N1s atom of DAPHP molecules attached to silver nanoparticles (). Both films show C1s core level, which could be stripped into three components at 285, 286 and 287.8 eV and are assigned to the electron emission from the advantageous carbon and the carbons coordinated to hydroxyl and carboxylic groups, respectively, in heparin molecules (data not shown). Presence of C1s and N1s spectra reveals that the nanoparticles are stabilized by the DAPHP molecules. It is clear 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
Figure 6 XPS spectra of Au-DAPHP and Ag-DAPHP naoparticles film form by drop coating film on Si (111) substrate. (a) Au 4f core level spectra of from Au-DAPHP, (b) N1s core level spectra from Au-DAPHP, (c) Ag 3d core level spectra from Ag-DAPHP, (d) N1s core level (more ...)
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 (). 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.
TEM image of drop coated film of Au-HA at (a) 75 kx, (b) 160kx, (c) with broad size distribution of ~5 – 30 nm. TEM image of Ag-HA nanoparticles at (d) 75 kx, (e) 125 kx (f) and size distribution from ~6 – 26 nm.
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 (). 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 (). 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 (). 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 8 Effects of (a) Au-DAPHP (solid) and Ag-DAPHP (open) and (b) free DAPHP on aPTT in human plasma. The coagulation times for (c) Ag-HA or Ag-glucose, and (d) Au-HA or Au-glucose fell within the normal control ranges. Data represent mean ± SEM, n (more ...)
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 (). 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 (). 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 ().
Effect of Gold and Silver heparin Nanoparticles on Clot Dynamics in Human Blood using Thrombelastography
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
The effects of free DAPHP, Au-DAPHP and Ag-DAPHP on carrageenan-induced paw edema in rats as compared to Indomethacin were evaluated. Significant inhibition of carrageenan-induced paw edema was shown for Au-glucose or Ag-glucose, Au-HA or Ag-HA versus heparin (). In contrast, Au-DAPHP and Ag-DAPHP nanoparticles administered locally in the paw did not exhibit any systemic effect on aPTT as compared to locally administered heparin (). Data indicated significant inhibition of carrageenan-induced paw edema in rats by Au-glucose, Ag-glucose, Au-HA, Ag-HA, heparin, Au-DAPHP and Ag-DAPHP, without any systemic effects on hemostasis as compared to heparin suggesting the potential for local inhibition of inflammation without systemic adverse effect using Au-heparin or Ag-heparin as compare to free heparin.
Figure 9 Effects of free DAPHP, Au-DAPHP and Ag-DAPHP on carrageenan-induced paw edema in rats as compared to Indomethacin. Significant inhibition of carrageenan-induced paw edema was shown for Au- or Ag-glucose, Au-HA or Ag-HA versus heparin (A and B). In contrast, (more ...)