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Kemp et al. Page 6 (1M) do not destabilize these colloidal solutions. The high stability of these DAPHP nanoparticles is both due to electrostatic and steric factors. 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 (Figure 5A and 5B). There is an increase in absorbance at ~650 nm or broadening in the absorbance of purified Au-DAPHP on increasing the concentration of NaCl (Figure 5A). Similar trends in the broadening in surface plasmon resonance are also observed in the Ag-DAPHP nanoparticle solution (Figure 5B). 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. 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 Figure 6A. 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 (Figure 6C), corresponding to Ag (0). N1s band at ~ 399 eV corresponds to N1s atom of DAPHP molecules attached to silver nanoparticles (Figure 6D). 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 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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