Abs Adsorbed Abs
4.3. Adsorption of gold nanoparticles to hydroxyapatite
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
400 500 600 700 800 900 1000
Wave length (nm)
Absorbance
Figure 7: Effect of bovine serum (10 % v/v) on the UV-Vis absorption spectra of mPEG-AlkSH, BP-PEG-AlkSH and citrate coated gold nanoparticles (from top to bottom)
4.3. Adsorption of gold nanoparticles to hydroxyapatite
The adsorption of bisphosphonate modified GNPs (GNPs-BP) to hydroxyapatite was studied to evaluate the affinity of surface-modified gold nanoparticles to bone mineral. For this study endobone was used as a porous hydroxyapatite ceramic produced by sintering of bovine bone and it consequently can be used as model of bone, because it is the major constituent of the natural bone besides the protein [42]. The hydroxyapatite binding of GNPs modified with different amounts of bisphosphonate in the polymer layer was compared with the binding of GNPs coated with 100% of mPEG-AlkSH (0% bisphosphonate). The results showed that increasing the amount of binding ligand for the hydroxyapatite surface (bisphosphonate) led to increasing amounts of GNPs adsorbed to hydroxyapatite as depicted in Figure 8.
0 20 40 60 80 100 120
0% 25% 50% 75% 100%
Bisphosphonate conc. (%)
% Binding to HA
1 day 2 days 3 days 4 days
Figure 8: Hydroxyapatite binding of GNPs modified with different amounts of bisphosphonate (e.g, 75% means that GNPs are coated with 75% BP-PEG-AlkSH and 25% mPEG-AlkSH)
GNPs modified with 0% Bp (100% mPEG-AlkSH) showed no affinity to hydroxyapatite, while about 25% of the initially added GNPs modified with 25% BP (75% mPEG-AlkSH) adsorbed to hydroxyapatite within 24 hours. This adsorbed amount increased to about 40% after 48 hours with no further increase in the adsorbed amount by shaking for longer time (up to 4 days). The bound amounts of particles modified with 50% bisphosphonate were about 45% after one day and reached 64% after two days. The adsorbed amounts of GNPs modified with 75% and 100% bisphosphonate were 58% and 37% after one day, and 76% and 81% after two days and reached 86% and 100% after four days respectively. The steady increase in the adsorbed amounts with increasing concentration of bisphosphonate in the particle surface can be explained by the higher hydroxyapatite affinity of GNPs containing more bisphosphonate in their surface. The relatively long time (about two days) to establish the equilibrium between the adsorbed and free particles can be attributed to the necessary diffusion of the particles within the large volume of
155
the dispersion medium and the necessary penetration into the macro and micro pores of endobone granules on the bottom of the flasks to reach the adsorption sites. Similar results are obtained by many other authors who studied the binding of nanoparticles to hydroxyapatite [55,60].
Since it is well known that bisphosphonate strongly binds to divalent ions, such as calcium [61], the binding assay of bisphosphonate-modified GNPs was repeated in presence of calcium chloride to explore effect of calcium ions on the affinity of the nanoparticles. The used calcium concentration was 2.5 mM, which is the physiological concentration present in blood serum. The binding affinity of 100% bisphosphonate-modified GNPs to hydroxyapatite in the presence and absence of calcium chloride is depicted in Figure 9. It could be observed that there is no difference in the binding of nanoparticles either in presence or absence of calcium. This indicates that the binding affinity of particles to hydroxyapatite is not affected by the presence of calcium ions in the colloidal solution.
0 20 40 60 80 100 120
24 48
Time (hours)
% Binding to HA
GNP-BP GNP-BP + CaCl2
Figure 9: Effect of calcium chloride on the hydroxyapatite binding of bisphosphonate-modified GNPs
Furthermore, the in-vitro binding of bisphosphonate-modified GNPs was carried out in presence of BSA (200 µg/ml) and serum (10% v/v) to determine, if the interaction of bisphosphonate with these proteins compromises the binding to hydroxyapatite. The results of these experiments are presented in Figure 10, which shows that the binding affinity of nanoparticles to hydroxyapatite is not affected by the presence of BSA or serum. The obtained data indicate that the bisphosphonate-modified GNPs are stable and the observed interaction of particles and proteins, especially BSA (Figure 5), does not influence their bone mineral affinity.
0 20 40 60 80 100 120
GNPs-BP GNPs-BP + BSA GNPs-BP + Serum
% Binding to HA
2 days 4 days
Figure 10: Effect of BSA (200 µg/ml) and bovine serum (10% v/v) on the binding of bisphosphonate-modified GNPs to hydroxyapatite
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5. Conclusions
In conclusion, it could be shown that the synthesized polymers, methoxy poly(ethylene glycol)-undecyl mercaptane and the bisphosphonate derivative, are suitable to functionalize gold nanoparticles prepared by citrate reduction. The obtained particles were characterized with respect to size, surface charges and dispersion stability. The polymer modified GNPs showed an enhanced stability against aggregation in different solutions such as high ionic strength NaCl solutions, as well as solutions containing BSA and serum.
Furthermore, the modification of the particles with bisphosphonate modified polymers allowed the preparation of particles with high affinity and strong binding to hydroxyapatite. The binding affinity to bone mineral could be adjusted by increasing the amount of bisphosphonate (targeting ligand) on the particle surfaces. Furthermore, it was demonstrated that the binding is not affected by present calcium ions as well as in presence of proteins.
Based on the conducted in vitro studies, bisphosphonate-functionalized GNPs seem to be a promising model for the investigation of the in vivo bone targeting of nanoparticles. An adjustment of the binding affinity can be achieved by changing the amounts of immobilized bisphosphonate modified polymers. An exchange for other suitable ligands allows using the nanoparticle system also for other targets and the possible exploration for other applications than bone.
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