**3. Results and Discussion**

### *3.1. Conjugate Nanoparticles: Preparation, Characterization and Loading Studies*

Highly hydrophilic gold nanoparticles were synthetized following a previously published procedure [9], and the UV–Vis spectrum and DLS measurements confirmed their nanodimension, as shown in Figure 1a,b. The results revealed that these AuNPs are particularly suitable for Cu(I) complexes delivery. In fact, AuNPs functionalized by 3MPS showed a high degree of stability and hydrophilicity due to the small length alkyl chains thiol with a charged terminal sulphonate group. Moreover, the 3MPS choice as a ligand, with a molar ratio of Au/S = 1 4 , guarantees a balance between stability and loading and favours transport in a watery environment. This fact increases the final bioavailability of the conjugates, especially for compounds with low water solubility. Furthermore, the plasmonic absorption peaks of the AuNPs and the absorption peaks of the copper complexes (λmax at 228 nm and 268 nm for complexes A and B, respectively, as shown in Figure 1a and in Figures S1a,b [45,50]) appeared in di fferent areas of the spectrum—in the UV spectrum for complexes and in the visible spectrum for AuNPs, allowing easy detection of the loading processes. This feature makes it possible to design a loading protocol based on the simple physical contact of AuNPs and copper complexes that can be physically adsorbed.

**Figure 1.** (**a**) UV–Vis spectrum of gold nanoparticles (AuNPs) (violet curve) and complexes A (green curve) and B (blue curve); (**b**) dynamic light scattering (DLS) measurement in water of AuNPs alone (in violet): <2RH> = 15 ± 2 nm.

On the basis of these considerations, the loading protocol for AuNPs and the two Cu(I) complexes was performed in a water solution at room temperature under gentle stirring. In Figure 2a,b, the chemical structures of the anticancer Cu(I) complexes used in this study, A and B, and a sketch of the loading protocol to obtain AuNPs-A and AuNPs-B conjugates are reported. The value of the loading efficiency η (%), reported in Figure 2c, can be calculated as follows [9,13]:

$$\mathfrak{m}\left(\%\right) = \left(\mathfrak{m}\_{\text{loaded\\_drug}}/\mathfrak{m}\_{\text{drug}}\right)100\_{\text{\textquotedblleft}}$$

where mloaded drug is the mass of the loaded drug (A or B), calculated from UV–Vis quantitative data and mdrug is the mass of the drug (A or B) used in the experimental procedure. From the absorbance value of free A or B, it is possible to obtain the amount of loaded drug, by determining the di fference.

**Figure 2.** (**a**) Chemical structures of anticancer Cu(I) complexes used in this study; (**b**) sketch of loading protocol to obtain AuNPs-A and AuNPs-B conjugates; (**c**) loading efficiency η (%) for AuNPs-A (in green, η (%) = 90 ± 4 %) and AuNPs-B (in blue, η (%) = 65 ± 10 %).

The loading studies allowed to us obtain the conjugated systems: AuNPs-A with η = 90 ± 4% and AuNPs-B with η (%) = 65 ± 10%. These systems were characterized in depth to understand the chemical interaction between AuNPs and Cu(I) complexes A and B. DLS studies were performed in a water suspension and showed a dimensional increase for AuNPs-A and AuNPs-B compared with the AuNPs alone, as reported in Figure 3a. In fact, the conjugation of the complexes involved a different degree of hydration of the particles and, as a result, the hydrodynamic diameter (<2RH>) increased.

**Figure 3.** DLS data of AuNPs in violet, AuNPs-A in green and AuNPs-B in blue: (**a**) <2RH> in water: AuNPs (15 ± 2 nm), AuNPs-A (56 ± 30 nm) and AuNPs-B (76 ± 32 nm); (**b**) Z potential in water: AuNPs (–35 ± 2 mV), AuNPs-A (–30 ± 3 mV) and AuNPs-B (–23 ± 4 mV).

Moreover, DLS allowed us to measure the electrophoretic mobility and, using the Smoluchowski equation, the Z potential [51,52]. The Z potential is the potential at this slipping plane, i.e., the surrounding electrical double layer, where the liquid moves together with particles. Therefore, the measured Z potential is not exactly the surface potential (surface charge density), but it is the potential of practical interest because it determines the inter-particle forces and enables the evaluation of the stability of the colloidal system [51,52]. The Z potential studies performed on our conjugates systems confirmed these interactions between AuNPs and complexes A and B. In fact, the Z potential was –30 ± 3 mV and −23 ± 4 mV, respectively, for AuNPs-A and AuNPs-B, instead of −35 ± 2 mV for AuNPs alone, as shown in Figure 3b. This is due to two different effects introduced by the presence of complexes A and B on the surface of the AuNPs. The first effect is the decrease of the negative charge density, due to the presence of neutral or positively charged molecules on the gold surface; this is strictly related to the Stern layer and slipping plane around the nanoparticles, which produce the Z potential. The second effect is the different aggregation grade, also observable from signal enlargement and size measurements, due to the interaction between complex molecules linked on different and vicinal AuNPs, which cause the system to be less stable in general. The balance or the prevalence of one of these two effects also explains the slight difference between the Z potential values of AuNPs-A and AuNPs-B. In fact, the conjugate AuNPs-B showed a larger size and lower Z potential with respect to AuNPs-A (see Figure 3). Indeed, complex A had a positive charge, facilitating adsorption and producing greater loading efficiency. Moreover, complex A on AuNPs decreased the interaction phenomena between the absorbed complex molecules, reducing the aggregation phenomena of the colloidal system in a solution. This justifies the DLS results regarding the smaller dimensions and more negative Z potential of AuNPs-A compared to those of AuNPs-B.

FESEM-EDX investigations performed on conjugate AuNPs-A nanoparticles showed dimensions around 10 nm with the presence of some aggregates (see Figure S2). It can be noticed that the dimensions obtained from DLS were greater than those obtained from FESEM images. Such a dimensional difference is due to the intrinsic difference between the two techniques, based on different principles. In fact, DLS estimated the particles hydrodynamic diameter (<2RH>) in the aqueous environment, with the important effect of swelling, and this dimension is the Z average value, which is the mean diameter weighted over the scattered light intensity. On the other hand, microscopy measurements were carried out under a vacuum on a dry sample deposited by casting with no hydration effects. The particles were more or less aggregated due to concentration or to fast or slow solvent evaporation occurring during the preparation of the sample. For this reason, it is difficult to directly compare the two measurements.

The Energy Dispersive X-ray Analysis (EDX) evidenced the presence of Cu and, in particular, the semiquantitative analysis showed the ratio of Au:Cu to be around 0.4:0.03 (see Figure S2).

The ATR data confirmed the effective interaction between copper complexes and AuNPs. In fact, both for AuNPs-A and AuNPs-B, typical bands were found (see Figures S3a,b). Particularly for AuNPs-A, some characteristic signals of the A complex were recognizable, such as the bending of CH2 at 1456 and 1418 cm<sup>−</sup><sup>1</sup> and the C–N stretching at 1043 and 1000 cm<sup>−</sup>1, thus confirming the successful conjugation. A shift of the C–N stretching signals was also observed, which moved from 1103 and 947 cm<sup>−</sup><sup>1</sup> in the free complex to 1043 and 1000 cm<sup>−</sup><sup>1</sup> in the conjugate, suggesting a direct involvement of these groups in the interaction with the gold nanoparticle surface. For AuNPs-B, the ATR measurements showed the typical bands at 2480 cm<sup>−</sup><sup>1</sup> due to the B–H stretching, bands at 1502 and 1403 cm<sup>−</sup><sup>1</sup> due to the C–C bond of pyrazole rings and at 1301 cm<sup>−</sup><sup>1</sup> due to C–N stretching. In this case the nitrile stretching showed a shift from 2254 cm<sup>−</sup><sup>1</sup> (free complex) to 2240 cm<sup>−</sup><sup>1</sup> (conjugate system), highlighting the involvement of these groups in the conjugation formation [33].
