*3.2. HR-XPS Studies*

Molecular and electronic structure of AuNPs-Cu(I) complexes assemblies were probed by means of synchrotron radiation-induced photoemission spectroscopy (HR-XPS); for comparison, the pristine Cu(I) complexes A and B ([Cu(PTA)4]+ [BF4]− and [HB(pz)3Cu(PCN)], respectively) were also investigated. Signals were acquired at C1s, P2p, N1s, B1s, Cu2p and Au4f core levels, and the obtained spectra were analyzed by following a peak fitting procedure that evidenced spectral components arising from atoms in di fferent electronic environments.

C1s and P2p spectra data analysis allowed us to assess the Cu(I) complex stability; as reported in Table S1 in the Supporting Information, P2p and C1s components positions, i.e., BE values, which reflect the molecular composition of the organic ligands, were not a ffected by the AuNP-Cu(I) complexes interaction. Indeed, the P2p3/2 spin-orbit component of the phosphorous signal was always found around 131.50 eV BE (complex A: 131.03 eV; AuNP-A: 131.11 eV; complex B: 131.81 eV; AuNP-B 131.86 eV), as expected from the literature for organic molecules containing P atoms [56]. The observed P2p BE stability allowed us to completely dismiss the occurrence of any degradation e ffect due to molecule oxidation, which would result in phosphane oxide formation with a noticeable shift in the P2p signal BE towards higher values [56]). P2p spectra are reported in Figure S4 in the Supporting Information. Au4f spectra appeared composed, showing a spin-orbit peak of high intensity due to metallic gold atoms at the nanoparticle cores (Au4f7/2 BE = 83.9 eV), and a second signal of low intensity at higher BE values (Au4f7/2 BE = 85.1 eV) was associated with partially positively charged gold atoms at the NP surface, as expected from the literature on analogous systems [4]. Copper Cu2p core signals were also acquired; for all samples, a single spin-orbit pair was observed, compatible with Cu(I) ions (Cu2p3/2 = 931.5 eV BE) [56]. The Au4f spectrum of AuNP-A and the Cu2p spectra of both AuNP-A and complex A were reported as examples in the Supporting Information (Figure S5).

The most interesting signal that shed light into the Cu(I) complex/AuNP interaction was the N1s core level. As reported in Table 1, both Cu(I) compounds showed N1s signals at the BE, as expected in the literature for the proposed molecular structure (tertiary amines N1s are expected at about 400 eV BE, and was found at 399.99 eV for complex A; N ≡ C − R like nitrogen N1s signal is expected at 399.6 eV BE [57], and was found at 399.7 eV BE in complex B [56]). In Figure 4 all N1s spectra are collected. Contributions related to pristine nitrogen atoms in Cu(I) complexes are represented in red.


**Table 1.** N1s BE, FWHM values and assignments for pristine Cu(I) complexes and AuNPs carriers.

**Figure 4.** High-resolution x-ray photoelectron spectroscopy (HR-XPS) N1s spectra of: (**a**) AuNPs-A; (**b**) complex A; (**c**) AuNPs-B; (**d**) complex B.

As evidenced in Table 1 and clearly observable in Figure 4, when Cu(I) complexes (Figure 4b,d) interacted with the gold nanoparticles, the N1s signal shape was modified (Figure 4a,c). For AuNP-A, a shoulder appeared at high BE (Figure 4a), suggesting a new spectral component at about 401 eV BE (in blue in the figure), which is usually assigned to positively charged N atoms in quaternary ammonium salts. On the other hand, the N1s spectrum of AuNP-B was larger at low BE, suggesting that a second spectral component appeared at a lower BE than the pristine N ≡ C − R-like N atom (Figure 4c); this behaviour indicates a partial electron transfer from the AuNPs to the nitrogen atom of the N ≡ C − R functional group.

### *3.3. Conjugate Nanoparticles: Stability and Release Studies*

It must also be highlighted that the main advantage of drug delivery with AuNPs is the possibility of studying a targeted and controlled release in terms of target site and time, as reported in many recent papers [8–10]. In this context, it is important to verify the stability of conjugates, also in view of a future formulation that makes them easily storable and at the same time quickly ready for use. The lyophilization appears to fulfil to this requirement, and was therefore performed by studying the effects on AuNPs alone and conjugates, both in terms of dimensional and structural stability. Comparing the fresh samples and the lyophilized samples by DLS measurements, aggregation phenomena were observed with important dimensional variations, as shown in Figure 5a–c. These phenomena involved both AuNPs alone and conjugated systems that remained however with dimensions under 300 nm. Regarding the structural stability of the systems, they showed unaltered structural conformation, confirmed by FTIR measurements, and good results in terms of <2RH> reproducibility when they were re-suspended in water at room temperature up to 10 days, as reported in Figure 5d.

**Figure 5.** DLS stability study in water of AuNPs in violet, AuNPs-A in green and AuNPs-B in blue: (**a**) AuNPs <2RH> before (dashed line) 15 ± 2 nm and after samples lyophilization (solid line) 25 ± 5 nm; (**b**) AuNPs-A <2RH> before (dashed line) 56 ± 30 nm and after lyophilization (solid line) 256 ± 30 nm; (**c**) AuNPs-B <2RH> before (dashed line) 76 ± 32 nm and after lyophilization (solid line) 276 ± 32 nm; (**d**) <2RH> of lyophilized AuNPs-A and AuNPs-B re-suspended in water at different days, up to 10 days.

Therefore, the study on the release was performed. The conjugate was re-suspended in water at 37 ◦C with gentle stirring and the UV–Vis analysis of the aqueous solution at defined times allowed the quantification of the released complex. This study showed strong and di fferent interactions between Cu(I) complexes A and B and AuNPs, as already evidenced by the HR-XPS studies. In particular, for AuNPs-A, the interaction involved nitrogen that partially transferred electrons to the surface of the metal, creating an interaction that caused a slow release. In Figure 6 the slow release profile of AuNPs-A, less than 10% up to 4 days, is shown. On the other hand, the AuNPs-B system involved the N ≡ C − R moiety, which strongly interacted with the gold surface, making the release not appreciable in a few days (up to 4). Taking these results into account, surely the AuNPs-A conjugate is more promising, not only for the better loading e fficiency but, above all, for the evident slow release, unlike the AuNPs-B conjugate.

**Figure 6.** Released profile of AuNPs-A with inset details showing the release in time in the range of 0–24 h.

AuNPs-A showed an excellent and promising result, because with a single administration it could be possible to achieve a slow drug delivery release in a biological site up over 4 days and more. Moreover, a main advantage of delivering a water-soluble drug with AuNPs is the accumulation of AuNPs in cancer cells, which guarantees the drug's targeting. Further, the slow release is an excellent opportunity to study the synergistic e ffects of AuNPs and copper complexes, e ffects that could occur for a long time (days and weeks), as in the case of slow-release anti-inflammatory drugs reported in the literature [9]. This fact opens new scenarios for investigations related to the action mechanisms as well as for synergistic action with AuNPs-A.
