*2.2. PGS-NPs Fabrication*

Loaded and unloaded PGS-NPs were prepared by nanoprecipitation according to a general procedure [24], where an acetone solution of PGS and curcumin was precipitated in deionized water allowing the formation of NPs. In order to evaluate the effect of polymer concentration over nanoparticles stability and size, PGS-NPs were formulated at different polymer concentrations in the starting organic solution (before nanoprecipitation); namely, PGS0.1C0.01, [PGS]: 0.1 mg/mL; PGS0.5C0.05, [PGS]: 0.5 mg/mL; PGS1.0C0.1, [PGS]: 1.0 mg/mL; PGS5.0C0.5, [PGS]: 5.0 mg/mL by keeping constant the curcumin-to-PGS ratio (curcumin/PGS ratio equal to 10% by weight). Concentration values refer to the final suspension water volume, after nanoprecipitation and after ethanol evaporation. For unloaded PGS-NPs (reported as Blank in Table 1) the same procedures, as well as the same PGS concentrations, were used but in the absence of curcumin. All the adopted experimental conditions led to the formation of monodisperse nanosized (loaded and unloaded) PGS particles, displaying a narrow monomodal size distribution in the nanometer range, as shown in Figure 2. Table 1 reports the maxima values of the distribution curves together with the polydispersion index (PdI) derived from the cumulant analysis.


**Table 1.** Effect of polymer concentration on PGS-NPs particle diameter at t = 0, after 7 days and after 14 days from sample preparation.

PdI: Polydispersity index.

**Figure 2.** DLS size distribution (intensity) of unloaded (**a**) and curcumin-loaded PGS-NPs (**b**) formed at different polymer concentrations (0.1, 0.5, 1.0 and 5.0 mg/mL).

Results relevant to individual experiments are reported in Table 1. It was observed that the composition of the organic phase, determined by PGS and curcumin concentration, strongly affected the diameter distribution of the particles. Increasing the polymer concentration increased the PGS-NPs diameter.

On the contrary, the presence of curcuminoids seemed to not influence the formation of the NPs since only slight differences in terms of NP size were observed as a consequence of their loading. However, curcumin strongly affected particles stability, since all unloaded PGS-NPs (reported as Blank in Table 1) demonstrated good stability up to 14 days from the preparation of the samples, while an increase in size and polydispersity of the two curcumin-loaded samples (namely PGS0.1C0.01 and PGS0.5C0.05) was observed after 7 days from samples preparation (Table 1 and Figure 3). After 7 days, both formulations presented macroscopic yellow aggregates indicating the loss of their colloidal stability.

**Figure 3.** Hydrodynamic diameter of blank (**a**) and curcumin-loaded (**b**) PGS-NPs over time from sample preparation.

On the contrary, curcumin-loaded samples (PGS5.0C0.5 and PGS1.0C0.1) demonstrated better stability over 14 days, where only slight reductions in particles size distribution were observed, thus comparable to the blank formulations.

The encapsulation efficiency of the most promising formulations (PGS1.0C0.1 and PGS5.0C0.5 purified by centrifugation) were equal to 99.8% (as measured by ultra-highperformance liquid chromatography, UPLC) for both developed nanoparticles, confirming the complete encapsulation of the active compound within the polymeric matrix. These values were particularly high compared to those reported in the literature, where the encapsulation efficiency of curcumin-loaded NPs spanned from 5% to 95% [32–34]. Since almost the whole curcumin was entrapped into the NPs, no further purification of colloidal suspensions was deemed necessary. Most importantly, no additives such as Tween-80 or PEG, commonly used to maximize the loading, were employed [35,36].

Due to the high proven stability, two formulations (namely PGS1.0C0.1, PGS5.0C0.5) as well as the corresponding unloaded formulations (PGS1.0Blank and PGS5.0Blank) were selected as the most promising candidates for the in vitro release studies and biological assays.

#### *2.3. PGS-NPs Stability Analysis in Cells Culture Medium*

The colloidal stability of PGS1.0C0.1 and PGS5.0C0.5 was investigated in cells culture medium by diluting the samples in water at 37 ◦C and in Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS) at the highest concentration tested during the planned biological investigation ([PGS]:0.2 mg/mL). A reduction in particle diameter was observed for both nanosystems (Table 2), possibly due to the more diluted conditions. Indeed, the higher dilution can cause NPs to be on average more distant from one another, giving rise to a less probable interaction among particles over time in comparison to higher concentrations where collisions and interactions are maximized. After a 24 h incubation in water at 37 ◦C, a reduction in particle size distribution was observed for PGS1.0C0.10, suggesting lower stability at higher temperatures compared to PGS5.0C0.5, where no differences were observed even at 37 ◦C after 7 days of incubation. The results of particle stability tests in cell culture medium and deionized water at 37 ◦C are reported in Table 2 and the monitoring of size over time is shown in Figure 4.

**Table 2.** PGS1.0C0.1, PGS5.0C0.5 recorded mean diameters at different times from sample preparation in deionized water and DMEM.


PdI: Polydispersity index.

**Figure 4.** Hydrodynamic diameter of curcumin-loaded PGS-NPs over time from sample preparation in water (**a**) and in DMEM (**b**). DLS size distribution (intensity) of selected loaded PGS-NPs after their dilution in deionized water (**c**) and DMEM cell culture medium (**d**) at 37 ◦C.

The same experiment was conducted in cell culture medium. The background noise of cell culture medium caused an increase in the polydispersity index for all the analyzed samples. Indeed, a broad particle distribution of circa 120 nm was observed by DLS analysis of the sole DMEM. This may be due to the presence of several species in the medium such as amino acids, antibiotics and in particular of fetal bovine serum (FBS) which could strongly affect the particle size analysis. The DLS analysis of PGS1.0C0.1 NPs was heavily affected by the background noise of DMEM and did not allow for correct particle size distribution measurements. On the contrary, PGS5.0C0.5 revealed the presence of homogeneous particle size distribution with an unchanged maximum compared to the one observed in pure water, thus confirming the good colloidal stability even in the cell medium. In this context, PGS5.0C0.5 was selected as the most promising curcumin-loaded preparation to test anticancer activity. Our results evidence the importance of particle size since bigger PGS-NPs ensured better stability of the whole system, especially when encapsulating curcumin.

#### *2.4. PGS-NPs Morphological Characterization*

The selected PGS5.0C0.5 and PGS5.0Blank samples were characterized by transmission electron microscopy, confirming their spherical shape (Figure 5). The dimensions of PGS5.0C0.5 and PGS5.0Blank estimated by TEM analysis indicated an average size of 121 ± 11 nm and 124 ± 13 nm, respectively. Moreover, TEM analysis allowed confirmation of monomodal size distribution for both developed NPs. A few large aggregates were present in the micrographs as black spots of higher size compared to most of the obtained PGS-NPs, possibly formed during the particle deposition on formvar grids.

**Figure 5.** PGS5.0C0.5 (**a**) and PGS5.0Blank (**b**) TEM images and relevant size distribution graphs calculated from TEM images.

#### *2.5. Curcumin Release in Cell Culture Medium*

The experimental solubility of curcumin in water at room temperature, at 37 ◦C and in DMEM at 37 ◦C were determined by UPLC as 2 × <sup>10</sup>−<sup>4</sup> mg/mL, 7 × <sup>10</sup>−<sup>4</sup> mg/mL and <sup>1</sup> × <sup>10</sup>−<sup>3</sup> mg/mL, respectively. Then, curcumin release from developed nanostructures was investigated. Figure 6 shows the release profile of curcumin from PGS5.0C0.5 nanoparticles through the dialysis membrane and curcumin solubility limit in medium. After 24 h, 4.4% of the total loaded curcumin was released from PGS5.0C0.5 in DMEM at 37 ◦C, reaching the experimental curcumin solubility limit in the medium (black dotted line in Figure 6). On the other hand, due to the lower solubility of curcumin in water, the plateaus corresponding to 3.2% and 0.8% of release were reached after 60 h in water at 37 ◦C and room temperature, respectively. The low release of curcumin from PGS5.0C0.5 was ascribable to the low solubility of curcumin in aqueous solvents.

**Figure 6.** Release kinetics of curcumin from PGS5.0C0.5 NPs in DMEM at 37 ◦C (blank), water at 37 ◦C (blue), and water at room temperature (red).

#### *2.6. Cytotoxic Effects of Unloaded and Curcumin-Loaded PGS-NPs in HeLa Cervical Cancer Cells*

HeLa cells were treated with different concentrations of free curcumin dissolved in DMSO and of unloaded (PGS5.0Blank) and curcumin-loaded PGS-NPs (PGS5.0C0.5) for 24, 48 and 72 h (Figure 7). No significant decrease in cell viability was observed when HeLa cells were treated with PGS5.0Blank, indicating that PGS-NPs are not cytotoxic even at higher concentrations (Figure 7a). Instead, PGS5.0C0.5 showed dose-dependent cytotoxicity similar to that obtained with free curcumin dissolved in DMSO (Figure 7b), thus suggesting that the water solubility of curcumin-loaded NPs is similar to that of free curcumin. To better compare the cytotoxic efficacy of curcumin-loaded PG-NPs (PGS5.0C0.5) vs. free curcumin, the 50% inhibitory concentrations (IC50) were calculated. Interestingly, the IC50 value of curcumin-loaded PGS-NPs at 72 h (15.95 μM) was significantly lower than that of free curcumin (21.27 μM, Figure 7c,d), suggesting a higher cytotoxic effect of curcuminloaded PGS-NPs compared to free curcumin. No significant reduction in cell viability was observed when NIH-3T3 healthy fibroblast cells were treated with PGS5.0Blank and different concentrations of PGS5.0C0.5, suggesting that unloaded and curcumin-loaded PGS-NPs have no cytotoxic effect on non-cancerous cells (Figure S5).

**Figure 7.** Cytotoxicity assay in HeLa cervical cancer cells with different concentrations of PGS5.0Blank and PGS5.0C0.5 dissolved in sterile dH2O (**a**) and of free curcumin dissolved in DMSO (DMSO-C, **b**). Values are means ± SEM (n = 12). IC50 values calculated are based on cell viability of HeLa cells at 72 h after treatment with PGS5.0C0.5 (**c**) and free curcumin dissolved in DMSO (**d**).
