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37 Figure 33: Cell viability assay using the CellTiter-Blue reagent with MCF-7 cells exposed to Ag NCs with serial dilutions for (A) 6 or (B) 24 hours. As read-out the OD at 570 nm is displayed. The results are represented as mean values ± standard error of the mean and were analyzed statistically using one-way ANOVA followed by Scheffé’s test using SPSS version 17.0 to establish the significance of any differences. The level of statistical significance was set at p < 0.05.
3.5.2 Effect of Ag NP on NIH/3T3 cells
The uptake and the cytotoxicity of the four different modified Ag NPs were measured by using the NIH/3T3 fibroblast cells. The difference in the uptake between not PEGylated and PEGylated NPs after 15 h of incubation is shown in figure 34 for the example of Ag-PMA and Ag-PMA-satPEG NPs.
Here both types were additionally modified with the dye DY-636 to get a fluorescent label. It is clearly seen that the uptake is reduced dramatically by saturating the surface of the particle with PEG.
Unfortunately the uptake could not be quantified. The uptake of the Ag-MUA particles is not shown but it is qualitatively in the same range like the Ag-PMA NPs. For the Ag-PMA-1PEG particles the uptake is also in the same range.
Figure 34: Fluorescence images of NIH/3T3 fibroblasts which had been exposed for 15 hours to fluorescence (DY636) labeled (A) polymer-coated Ag NPs or (B) polymer coated Ag NPs whose surface has been saturated with 10kDa PEG molecules. The images correspond to the overly images of the transmission and fluorescence channel. Scale bars correspond to 20 µm.
38 The toxicity of the Ag NPs was probed with a standard resazurin assay[98](cf. figure 34). For the positive control silver nitrate was used. The summarized results are shown in the response curves in figure 35. As expected the silver nitrate showed the highest toxicity. By comparing just the different Ag NPs, Ag-MUA NPs showed the highest toxicity with a LD50 value of 0.04 mM. The toxicity of the Ag-PMA and the Ag-PMA-1PEG is in the same range with LD50 values of 0.65 mM and 0.73 mM.
The toxicity of the Ag-PMA-satPEG showed the lowest toxicity (LD50: 1.34 mM). This low toxicity is due to the lower uptake of the Ag-PMA-satPEG NPs.
Figure 35: Reduction of resazurin to the fluorescent resofurin, which is done by living cells.
Figure 36: Resazurin-based viability test of 3T3 fibroblasts which had been incubated for 24 hours with Ag NPs. Onset of fluorescence (as quantified by the measured intensity I) is an indicator for viability of cells. The amount of Ag is quantified in a) the total amount of Ag (c(Ag)), b) the amount of Ag atoms which are present on the NP surface (csurf(Ag)), and c) the amount of Ag NPs (c(Ag NP)).
The following scaling factors were used: csurf(Ag) = 0.29⋅c(Ag) and c(Ag NP) = 4.2· 10-9 mol/mg⋅c(Ag). In case of AgNO3 as silver source only the c(Ag) concentration scale is valid, in case of all the NPs all three concentration scales are valid.
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4 Conclusion
The synthesis of defined Ag NPs is very important in the field of particle science because the effect of a particle belongs to its size, shape or not at least to its surface chemistry. Two different wet chemical syntheses were established for well defined and stable Ag NPs. First, very small (2.2 nm) and fluorescent nanoparticles were synthesized via an etching step and afterwards a ligand exchange with a hydrophilic ligand. Second, bigger (4.2 nm) and not fluorescent particles were synthesized via simple reduction and capping with a hydrophobic ligand. In both cases the particles were stable enough that several purification and/or modification steps could be done without endanger the colloidal stability of the different nanoparticles. Both types and their modifications were tested for their use in biological systems.
The first type of Ag NPs, the small Ag NCs, showed a red fluorescence, a tolerable toxicity and an unspecific uptake in MCF-7 cells. Unfortunately, the presented particles showed a limited use for labeling cells due to their low quantum yield (QY). By solving the problem of the low QY in further studies, maybe by using of other ligands, the use of this kind of particles for labeling will get more possible. One major advantage of these particles was their small size. Because of the size it could be possible that they could penetrate tissue locations which are impenetrable for bigger particles. Another important point of these particles was that they could be used, with a more detailed photophysical characterization, for increasing the further understanding on their optical properties.
The, as produced, second type of Ag NPs were not soluble in water because of the hydrophobic ligand at the surface. To transfer the particles into the aqueous phase two different methods were used. The transfer to the aqueous phase was necessary for the investigation of the uptake and the cytotoxicity in cells. The first method was an exchange of the hydrophobic ligands with hydrophilic ligand molecules.
The second method was the coating of the hydrophobic particle with an amphiphilic polymer. Both methods showed different advantages. Both methods were easy to do and also an additional modification was still possible at a later time point. An advantage of the simple ligand exchange reaction was that the ligand molecules do not need to be modified before they were used. The preparation of the amphiphilic polymer for the polymer coating, which first needed to be synthesized, cost an additional day. But this disadvantage is negligible because of the advantage that the colloidal stability of the coated Ag NPs was increased dramatically in comparison to the Ag NPs, which were stabilized by hydrophilic ligands. This increased stability was the main factor why the PMA coated particles showed a lower toxicity than the MUA stabilized particles. The polymer coated particles showed a reduced toxicity. This effect could be explained by clouding the cell membrane with the aggregated Ag-MUA particles[58].
PEGylation was another key factor for the cytotoxicity of the Ag NPs. Here not the release of Ag+ ions but the uptake of the whole particle in the cell was the most important factor. If the surface of a particle was saturated with PEG molecules and showed because of this almost no surface charge, the uptake was reduced compared to the same but “naked” particle. Already the addition of one big (10 kDa) PEG molecule to the surface reduced the cytotoxicity. So here it was clearly seen that extracellular silver was less toxic than intracellular silver.
The modification of the different Ag NPs showed no influence to the corrosion of the Ag cores. After two weeks under neutral conditions, the release of free silver cations could not been detected. Just one sample showed a release of around 0.1%. Under acidic condition (pH 3) the release of silver cations was increased. After one day release rates between 0.7 and less than 1.2% could be measured. These values increased after one week up to 0.7 – 1.4%. These small amounts of released silver ions confirm the good stability of the Ag NPs.
By comparing the cytotoxicity of the different Ag NPs to silver nitrate, the particles showed a lower toxicity referred to the absolute amount of silver. But one had to consider that only 30% of the silver of the Ag NPs was located to the surface. An additional point was that just about 1% of this surface
40 silver was released into the surrounding media under acidic conditions. With the silver salt 100% of the Ag ions were free in the media and these Ag(I) ions are not permeable for the cell membrane (trough they can be complexed by serum proteins). So there were a lot of silver ions in the extracellular medium of the cells for the silver salt but only a few ions in the intracellular medium of the cells for the Ag NPs.
Summarizing all these facts one can say that the synthesis of different Ag NPs with difference in size, optical properties, stability and surface chemistry was done in a defined way. These particles could be taken up by cells and show that intracellular Ag is more toxic than extracellular and that the Ag NPs are an efficient carrier to bring the silver inside the cells.
In addition to the silver nanoparticles commercial gold nanoparticles were used to show that the here used methods to increase the stability of a particle against salt can be used in a general way. It could be shown that the coating of particle increased the stability it than using hydrophilic ligand molecules. By replacing the weak citrate ligand molecules with strong binding thiol-PEG molecules, the stability could also be increased showing comparable results like the coated particles. For using the coating procedure, the commercial particles first were transferred to the organic phase by replacing hydrophilic ligand molecules with hydrophobic molecules in two steps. First, the weak hydrophilic ligand was preplaced by a weak phase transfer ligand, which was replaced by a strong binding thiol ligand in a second step. Afterwards the gold nanoparticles showed the same surface chemistry like the silver nanoparticles and could be used in the same manner.
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