3.1 Gold nanoparticles concentration and extinction coeﬀicient.
The molar concentration and extinction coeﬀicient of gold nanoparticle solutions in Table 1, have be calculated through the following steps:
1. Calculating the average number of gold atoms per nanoparticle (N) from HRTEM images as shown in Figure 4. Assuming a spherical and fcc shaped nanoparticle, the average number of gold atoms (N) per nanoparticle was calculated using Equation 1 , where π is the the circumference of a sphere (3.14), ρ is the density for fcc gold (19.3g/cm3), NA is Avogadro’s number (the number of atoms per mole) (6.023×1023), M is the atomic weight of gold (197 g/M), and D is the average core diameter of nanoparticles that are summarized in Table 2.
Table 1: Molar concentrations a and extinction coeﬀicients (ε)b of gold nanoparticle solutions.
- molar concentrations calculated from Equation 2.
- extinction coeﬀicients calculated from the slope of regression line in Figure 3.
- extinction coeﬀicient not calculated due to aggregation of AuNP s and unstable λmax.
2. Calculating the molar concentration of the prepared solutions from initial concen- tration, using Equation 2 where C is the molar concentration of the nanoparticle solution, NTotal is the total number of gold atoms (the initial amount of gold salt, HAuCl4,used), N stands for the average number of gold atoms per nanoparticle from Equation 1, and V is the volume of the reaction solution in (L), assuming that the gold(III) reduction was complete.
3. Determining the molar extinction coeﬀicient of each sample from the slope of the regression line of absorbance vs concentration curves in Figure 3 (inset-2 in 3a, 3b, and 3d). This actually based on Lambert–Beer’s Equation 3, where A stands for absorption, ε is molar absorptivity or molar extinction coeﬀicient (the slope), b is the path length of cuvette (1cm), c is the calculated molar concentration of gold nanoparticle solutions from Equation 2.
A = εbc (3)
3.2 Gold nanoparticles color stability, and UV-Vis’s absorbance spectra in the absence and presence of medications.
The synthesized gold nanoparticle solution (3.68×10−8 M) has been characterized using UV-Vis spectrophotometer and spectrofluorometer with maximum absorption and emis- sion at λmax 520 nm. The resolution of the spectrofluorimetric is high compared to UV-Vis spectrophotometry, as the FWHM (Full Width at Half Maximum) was small (5 nm) in fluorimetry, while in UV-Vis the FWHM was broader (60 nm) as in Figure 2.
The color stability of gold nanoparticles (AuNPs) has been studied visually and by UV-Vis’s spectrophotometer. Visually, the color of AuNPs was stable for long period under tight storage conditions (≃ six months) . After addition of drugs to AuNPs, the color of AuNPs was mostly stable with alendronate, while least stable with me- mantine and tobramycin. The color change and the reactivity with tobramycin was most powerful than with memantine. Once the color changed, the process is repeated until the color completely disappeared. In the nanoscale, memantine and tobramycin made no color change. However, in the micro-scale, color change has been occurred.
Using UV-Vis, The nano-ranges of memantine and tobramycin did not led to red shift at λmax 520 nm as shown in Figure 3b and 3c or nanoparticles’ aggregations (Figure 5b) over twenty minutes. Mem. nano-ranges (33.46-334.63 nM) and Tobr. nano-ranges (4.28-128.34 nM) were stable for twenty minutes as shown in (inset-1 in Figure 3b, and inset-1 in Figure 3c), while Alen. was stable in the milli-ranges (0.11-1.48 E-02 mM) as in (inset-1 in Figure 3a). However, The micro-ranges of Mem. and Tobr. led to red shift (Figure 4a and 4b) and aggregation of gold nanoparticles (Figure 5d,5e).
3.3 Gold nanoparticles core diameter in the absence and presence of medications.
HRTEM images indicated that the core diameters of gold nanoparticles do not change when adding different medications with various concentrations even when nanoparticles aggregated. The average core diameter of AuNPs was 9.58 ± 1.27 nm. After addition of alendronate, memantine, and tobramycin to AuNPs solution, the average core diameters were relatively stable (9.68 ± 0.15 nm) as obvious in HRTEM images in Figure (5) and the calculated average core diameter in Table (2). The aggregation and color change of gold nanoparticles occur due to other factors, other than increasing in the core size of gold nanoparticles which could be due to increase in hydrodynamic diameter of nanoparticles, attraction, and repulsion forces of the outermost layer of nanoparticles, and the chemical compositions of drugs.
2: Mean core diameter, hydrodynamic diameter and Zeta Potential * of gold nanoparticles in absence and presence of alendronate, memantine, and tobramycin.
*The average core diameter, hydrodynamic diameter and Zeta Potential calculated from multiple HRTEM images and DLS measures.
3.4 Gold nanoparticles hydrodynamic diameter, and surface charge in the absence and presence of medications.
The hydrodynamic diameter of gold nanoparticles solution (52.08 ± 3.54 nm), calculated in Table 2, increase with increasing drug concentrations, except with alendronate, the hydrodynamic diameter of particles showed very small change (from 52.08 to 58.94 nm). The gradual increase in hydrodynamic diameter with increasing concentrations from nano- to micro is very large with tobramycin (from 135.3 to 332.16 nm) compared to memantine (from 64.99 to 98.41 nm) as shown in Figure 5 and Table 2. This may be attributed to the bulk structure of tobramycin and its various functional groups as shown in Figure 6 which accelerate the accumulation of more drug molecules on the surface of gold nanoparticles. While memantine structure is very small and has only one primary amine functional group available for reaction. Similarly, the average zeta potentials were measured by Zeta Potential Analyzer to analyze the surface charge of gold nanoparticles as in Table 2. The greatest decrease in negativity was with memantine (from 0.26 to 0.53 mv), while with tobramycin obviously decreased in the micro-level (from -23.63 to -1.64 mv). But with alendronate, a slight increase in negativity (from -24.56 to -30.23 mv).
3.5 Proposed gold nanoparticles’ reactivity mechanism
The stabilization of Turkevich gold nanoparticles is attributed to the electrostatic inter- actions of the negative coating layers of citrate-anions with the nanoparticle core which keep the particles suspended in the colloidal solution without precipitation . The suggested stability of Turkevich gold nanoparticles after the addition of solutions of the tested drugs (Alen., Mem., Tobr.) could be explained from the chemical structures (Fig- ure 6) as follows. It can be observed that the three drugs contain a primary amine group (−NH2) which may interact with the negative case of alendronate, there is only one primary amine group (−NH2) that could interact with the weak carboxylic acid groups (−COO−) of citrate-anions, leaving the bis-phosphonate group (−COH(PO3H2)2) freely coating the outer most layer of nanoparticles. This sec- ond negative layer of the bis-phosphonate group in alendronate could be used as another stabilizing and protective layer, which may contribute to the compatibility of gold nanoparticles with alendronate. On the other hand, memantine also contains only one primary amine group (−NH2) like alendronate, that could interact with the weak carboxylic acid group (−COO−) till complete saturation of the outer most layer of gold nanoparticles with memantine leaving this layer uncharged by the hydrocarbon chains. It was observed that memantine exhibits positive zeta potential as in Table 2 which could be explained by the presence of the hydrocarbon chains, that may form weak ionic inter- actions with matrix materials which may lead the attraction of gold nanoparticles to each other at the micro-level. On the contrary, Tobramycin that contains more than one primary amine group (−NH2), may interact with the gold nanoparticles in many directions. This may explain why tobramycin has rapid reactivity toward these nanoparticles. All these suggested mechanisms have been illustrated in the proposed diagrams (Figure 6).