29
30 The results of the measurements of the different Au NPs showed the expected results. The original and the Au-Phosphine NPs showed the smallest hydrodynamic diameter. Also the size of the Au-PEG-SH particles was in the expected range. Here a wrapping of the PEG molecules around the particle was not expected because of the gold-thiol bond. This bond is so stable[94] that a “wrapped polymer” will be removed from other molecules to enter the gold-thiol bond. The results of the Au-PMA NPs were in the same range like the Ag-PMA NPs. One possible explanation for the fact that the hydrodynamic diameter of the Au-PMA-satPEG was bigger than the one of the Ag-PMA-satPEG NPs was that smaller PEG molecules were used to saturate the surface (Au: 2 kDa PEG; Ag: 10 kDa PEG) and because of this more molecules could be linked to the surface.
Sample dh [nm] ζ [mV]
Au-citrate 7 ± 2 -19.0 ± 2.5 Au-Phosphine 6 ± 2 -51.0 ± 4.8 Au-PEG-SH 22 ± 6 -44.5 ± 7.7 Au-PMA 10 ± 3 -33.0 ± 6.4 Au-PMA-satPEG 19 ± 5 -25.3 ± 2.4
Table 2: Hydrodynamic diameter dh and zeta-potential ζ of commercial Au NPs with different surface coating, with a core diameter of dc = 5 nm.
3.4.2 Dissolving behavior of the Ag NPs
Because of the cytotoxic effect of the Ag NPs their dissolving behavior, releasing silver cations, under different pH values was a critical factor. The released Ag+ ions were measured via ICP-MS at different times and under two different pH values. First, a pH value of 7 (pure water) was chosen and second a value of pH 3, which is even 2 pH units less than in the late endosome[95,96]. This was done by separating the solid Ag NPs from the released Ag+ ions by ultrafiltration, using 3 kDa MWCO filters with a speed of 9000 rpm for 15 minutes. The detection limit of the ICP-MS was measured and calculated to 0.0015% of the used Ag+ ions. For this purpose the attainable dilution limit of a standard solution, which gives a signal 10 times the standard deviation of a blank sample, was measured.
At the beginning the total amount of silver in the different samples were measured (ctot(Ag)). Then an aliquot of each sample was taken and the dissolved silver was separated from the Ag NPs by ultrafiltration, described above. The dissolved silver cations were quantified (c(Ag+)) via ICP-MS and the amount of released silver was calculated by calculating c(Ag+)/ctot(Ag). This was done at day 0, day 7 and day 14 for neutral conditions and at day 0 and day 7 for acidic conditions. Under neutral conditions even after 14 days the Ag particles were stable. A release of silver cations could just been measured in one sample. Even this value was very low, around 0.1%. This showed again the good stability of the different Ag NPs. Under acidic conditions (pH 3) the release of Ag+ ions should be increased and could been measured. After one day all three samples released silver cations. The measured values were between 0.7 and less than 1.2%. These values increased just a little bit after 7 days under the acidic conditions up to 0.73 und less than 1.4%. Even under these extreme conditions the Ag NPs show a good stability.
Sample released Ag (water): c(Ag+)/ctot(Ag) [%]
day 0 day 7 day 14
Ag-MUA < 0.0015 < 0.0015 < 0.0015 Ag-PMA < 0.0015 < 0.0015 0.146 ± 0.001 Ag-PMA-satPEG < 0.0015 < 0.0015 < 0.0015
31 Sample released Ag (pH 3): c(Ag+)/ctot(Ag) [%]
day 1 day 7
Ag-MUA 0.781 ± 0.002 1.316 ± 0.002 Ag-PMA 1.120 ± 0.004 1.390 ± 0.003 Ag-PMA-satPEG 0.701 ± 0.001 0.735 ± 0.001
Table 3: The amount of released Ag+ ions c(Ag+)/ctot(Ag), in dependence on their exposure to water or acidic solution. At day 0 all residual Ag+ had been removed by ultrafiltration.
3.4.3 Stability of Ag NPs under different sodium chloride concentrations
For using the Ag NPs in cells it is important that the particles keep the colloidal stability with high salt concentrations over a longer time period. Therefore the hydrodynamic diameters of the different modified particles were measured first directly after adding the salt and a second time after 24 h. The results are shown in figures 25 and 26.
The Ag-MUA particles differ in the behavior to the other three modified particles. Only the Ag-MUA NPs show an increasing hydrodynamic diameter with an increasing salt concentration. Additionally these particles aggregated and precipitated after 24 h with a NaCl concentration higher than 160 mM.
The hydrodynamic diameters of the other three samples kept constant for 24 h even at very high salt concentrations up to 2.5 M.
Figure 25: Selected results of DLS measurements for stability test of Ag-MUA (left) and Ag-PMA (right) NPs vs. NaCl from 0 mM slat to 2.5 M salt concentration. The results directly after mixing is shown in straight lines and the results after 24 h in dashed lines. The Ag-MUA NPs kept constant up to 160 mM of salt and aggregated at higher concentrations. The Ag-PMA NPs kept constant up to a concentration of salt of 2.5 M.
32 Figure 26: Selected results of DLS measurements for stability test of PMA-1PEG (left) and Ag-PMA-satPEG (right) NPs vs. NaCl from 0 mM salt to 2.5 M salt concentration. The results directly after mixing is shown in straight lines and the results after 24 h in dashed lines. Both types of Ag NPs kept constant up to a concentration of 2.5 M of salt.
The hydrodynamic diameters of the four Ag NP samples with different salt concentrations directly after addition of the salt are shown in figure 27. Some concrete values including the standard deviations of the above measurements are shown in table 4. It is clearly shown that the hydrodynamic diameter of the Ag-MUA NPs increases to more than 1000 nm by salt concentrations higher than 625 mM.
Figure 27: Sketches of the four different modified particles and summary of the hydrodynamic diameter of the different modified Ag NPs vs. NaCl directly after addition of the salt. Only the Ag-MUA particles show an increasing diameter with higher salt concentrations. The other three samples keep the same diameter up to 2.5 M.
33 t = 0 h
c(NaCl) 0 mM 20 mM 160 mM 2.5 M
Ag-MUA 11.23±4.4nm 13.54±4.2nm 93.48±63.9nm 1180±361nm Ag-PMA 12.04±2.7nm 9.97±2.4nm 10.60±2.4nm 13.77±3.3nm Ag-PMA-1PEG 13.22±3.8nm 12.36±3.8nm 10.87±3.5nm 12.88±3.9nm Ag-PMA-satPEG 12.16±3.0nm 12.68±3.2nm 13.49±3.6nm 20.84±5.9nm t = 24 h
c(NaCl) 0 mM 20 mM 160 mM 2.5 M
Ag-MUA 11.91±5.2nm 11.46±3.9nm 122.3±42.1nm →∞
Ag-PMA 11.27±2.8nm 10.52±2.5nm 11.19±2.6nm 14.82±4.1nm Ag-PMA-1PEG 11.87±3.7nm 11.87±3.5nm 12.19±3.6nm 13.43±3.9nm Ag-PMA-satPEG 12.09±3.2nm 12.67±3.4nm 14.26±3.9nm 25.64±7.6nm
Table 4: Summary of mean hydrodynamic diameters dh and their corresponding standard deviation, as derived from the data presented in Figures 23 + 24.
3.4.4 Commercial gold nanoparticles vs. NaCl
The stability of commercial Au NPs against NaCl was also measured with different modifications. The results directly after the addition of the salt and after 24 h are shown in figure 28 - 30. The Au NPs without modification (Au-citrate) showed the worst stability. By increasing the salt concentration above 40 mM, the hydrodynamic diameter started to increase up to almost 900 nm with a concentration of 2.5 M salt. After 24 hours all the particles with a concentration higher than 80 mM were aggregated.
The Au-Phosphine particles behaved similar. Here the hydrodynamic diameter also started to increase by increasing the salt concentration to more than 160 mM up to 700 nm with a concentration of 2.5 M.
The particles with concentrations higher than 625 mM were aggregated after 24 h.
The Au NPs stabilized by PEG-SH or PMA coating and additional PEG modification did not show any increase of the hydrodynamic diameter up to a salt concentration of 2.5 M and were also stable for at least 24 hours.
Figure 28: Selected results of DLS measurements for stability test of Au-citrate NPs vs. NaCl from 0 mM slat to 2.5 M salt concentration. The results directly after mixing is shown in straight lines and the results after 24 h in dashed lines. All the samples containing salt showed an increased diameter after 24 h and samples with more than 80 mM salt were aggregated and precipitated.
34 Figure 29: Selected results of DLS measurements for stability test of Au-Phosphine (left) and Au-PEG-SH (right) NPs vs. NaCl from 0 mM slat to 2.5 M salt concentration. The results directly after mixing is shown in straight lines and the results after 24 h in dashed lines. The Au-Phosphine samples kept constant for 24 h up to a concentration of 625 mM of salt. With higher concentrations the samples aggregated and precipitated. The Au-PEG-SH NPs kept constant up to a concentration of 2.5 M of salt.
Figure 30: Selected results of DLS measurements for stability test of Au-PMA (left) and Au-PMA-satPEG (right) NPs vs. NaCl from 0 mM slat to 2.5 M salt concentration. The results directly after mixing is shown in straight lines and the results after 24 h in dashed lines. Both types of Au NPs kept constant up to a concentration of 2.5 M of salt.
The results of the measurements directly after addition were summarized in figure 31. A summary of the measured values shown in above figures is presented in table 5.
35 Figure 31: Summary of the hydrodynamic diameter of the Au NPs vs. NaCl direct after addition and sketches of the different modified Au NPs.
t = 0 h
c(NaCl) 0 mM 160 mM 625 mM 2.5 M
Au-citrate 7.10±1.9nm 149.8±49.4nm 833±324nm 898±315nm Au-Phosphine 6.30±1.8nm 6.49±1.8nm 539±248nm 693±297nm Au-PEG-SH 21.47±5.8nm 23.08±5.7nm 22.16±5.9nm 26.51±7.4nm Au-PMA 9.48±2.9nm 10.00±2.6nm 10.78±3.0nm 12.34±3.1nm Au-PMA-satPEG 18.64±5.1nm 17.75±5.0nm 21.06±5.7nm 22.50±5.9nm t = 24 h
c(NaCl) 0 mM 160 mM 625 mM 2.5 M
Au-citrate 7.26±2.0nm →∞ →∞ →∞
Au-Phosphine 6.68±1.8nm 7.40±1.9nm →∞ →∞
Au-PEG-SH 21.31±5.8nm 22.47±4.9nm 21.80±5.9nm 25.08±7.2nm Au-PMA 9.10±2.7nm 10.43±2.9nm 13.39±4.7nm 15.39±4.4nm Au-PMA-satPEG 18.57±5.1nm 18.65±5.2nm 18.65±5.2nm 22.63±6.1nm
Table 5: Summary of mean hydrodynamic diameters dh and their corresponding standard deviation, as derived from the data presented in Figures 27 - 29.
36