*3.6. Curcumin In Vitro Release Studies*

Release kinetic studies were performed on PGS5.0C0.5 nanoparticles by using a dialysis tubing cellulose membrane (molecular weight cut-off = 14,000 Da). Curcumin-loaded NPs were diluted in an appropriate volume of water or DMEM cell culture medium and then placed into dialysis tubes. 5 mL of sample was dialyzed against 20 mL of water or DMEM at 37 ◦C. At specific time intervals (1, 2, 3, 6, 24, 36, 48, 60 and 72 h), 1 mL of the medium was analyzed by means of UPLC (chromatographic method described below) to determine the amount of released curcumin. Consequently, the same volume of fresh medium was replaced. All the measurements were performed in triplicate. The same experiment was performed at 4 ◦C and at room temperature to determine the influence of temperature during the curcumin release. The in vitro release profile was measured by using Equation (2):

$$Release\_{curum in} \left( \% \right) = \frac{\mathbb{C}\_{Relused} \left( \begin{array}{c} (t) \\ \hline \end{array} \right)}{\mathbb{C}\_{Total}} \times 100 \tag{2}$$

where *Creleased* represents the concentration (mg/mL) of released curcumin at time point (t), and *CTotal* (mg/mL) corresponds to the total amount of curcumin loaded into PGS-NPs.

#### *3.7. UPLC Analysis*

Curcumin was quantified through a Waters ACQUITY UPLC system equipped with a quaternary solvent manager system, autosampler, thermostated column compartment and a PDA detector. The analytical separation was performed using an ACQUITY UPLC® (Waters corp., Milford, MA, USA) BEH C18 column (1.7 μm × 2.1 mm × 50 mm). Curcumin release was analyzed using a mobile phase composed of water (+0.1% of formic acid) (A) and acetonitrile (+0.1% of formic acid) (B). The flow rate was set at 0.25 mL min−<sup>1</sup> and the linear gradient elution was: 0 min, 70% A; 10 min, 30% A; 11 min, 70% A; with a re-equilibration time of 3 min before the next injection. The column temperature was maintained at 34 ◦C and the wavelength set at 425 nm, corresponding to the maximum absorption of curcumin. Before samples injections, five dilutions of a curcumin methanolic solution (0.2 mg/mL) were prepared in the range 0.001–0.2 mg/mL. Standard solutions were filtered (0.2 μm nylon filters) and injected three times into the UPLC system. Curcumin was eluted as a sharp peak at retention time of 5.5 min. In the operative concentration range the trend was linear for each compound, with no saturation effects that could bend the linearity. The area under each peak was quantified by instrumental software and plotted versus the concentration. The best fit of experimental data in the plot "Peak area vs. [curcumin]" was then used for released curcumin quantification in each sample. Chromatograms relevant to curcuminoids UPLC analysis are reported in the supplementary information.

#### *3.8. Cell Culture Assays—Cytotoxicity Study*

#### 3.8.1. Cell Culture and MTT Assay

Human cervical cancer HPV18+ HeLa cell lines and the healthy fibroblast NIH-3T3 cell line were cultured at 37 ◦C in a humidified atmosphere containing 5% CO2 in DMEM supplemented with 10% (*v*/*v*) fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 100 Units/mL penicillin, 100 μg/mL streptomycin (Gibco/BRL, Carlsbad, CA, USA) and 2 mM L-glutamine (Life Technologies, Carlsbad, CA, USA). The inhibitory effect of unloaded and curcumin-loaded PGS-NPs on cervical cancer HeLa cells and NIH-3T3 fibroblasts was analyzed by cell viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) test as previously described [37]. Briefly, HeLa cells were seeded in a 96-well plate (about 25,000 cells per well for HeLa and 5000 cells per well for NIH-3T3), incubated for attachment for 24 h at 37 ◦C and then treated with free curcumin dissolved in DMSO and with unloaded and curcumin-loaded PGS-NPs in 12 replicates at different concentrations (0, 0.0025, 0.005, 0.01, 0.015, 0.02 mg/mL of curcumin). At

different time points (24, 48 and 72 h) each well was supplemented with 10% (*v*/*v*) MTT solution and incubated for 4 h. Formazan crystals were dissolved in acidified isopropanol (isopropanol, 0.1 N HCl, 0.1% Tween-20). The optical density was measured at 570 nm by using a microplate reader (Tecan Infinite F200PRO). The 50% inhibitory concentration (IC50) of curcumin-loaded PGS-NPs and free curcumin on HeLa cells was calculated at 72 h using Graph Pad Prism software.

#### 3.8.2. RNA Extraction and Real-Time RT-PCR Analysis

Total RNA was isolated from cells (1 × 106 cells per well in 6-well plate) treated with unloaded and curcumin-loaded PGS-NPs (0.005 and 0.01 mg/mL) using Direct-ZolTM RNA MiniPrep kit (Zymo Research, Irvine, CA, USA). About 1 μg of RNA was reverse-transcribed with the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) and real-time RT-PCR was performed with the QuantiNova SYBR Green kit (Qiagen, Hilden, Germany) for gene expression in a CFX96 Real-time PCR detection system (Bio-Rad, Hercules, CA, USA). Transcript levels were normalized against the *GAPDH* gene and values were expressed as fold change over control sample treated with vehicle (CNT). Primer sequences are reported in Table 4. Data are presented as mean ± SEM.

**Table 4.** Primer sequences used for real-time RT-PCR.


fw, forward; rv, reverse.
