Synthesis of Polystyrene-Based Cationic Nanomaterials with Pro-Oxidant Cytotoxic Activity on Etoposide-Resistant Neuroblastoma Cells

Drug resistance is a multifactorial phenomenon that limits the action of antibiotics and chemotherapeutics. Therefore, it is essential to develop new therapeutic strategies capable of inducing cytotoxic effects circumventing chemoresistance. In this regard, the employment of natural and synthetic cationic peptides and polymers has given satisfactory results both in microbiology, as antibacterial agents, but also in the oncological field, resulting in effective treatment against several tumors, including neuroblastoma (NB). To this end, two polystyrene-based copolymers (P5, P7), containing primary ammonium groups, were herein synthetized and tested on etoposide-sensitive (HTLA-230) and etoposide-resistant (HTLA-ER) NB cells. Both copolymers were water-soluble and showed a positive surface charge due to nitrogen atoms, which resulted in protonation in the whole physiological pH range. Furthermore, P5 and P7 exhibited stability in solution, excellent buffer capacity, and nanosized particles, and they were able to reduce NB cell viability in a concentration-dependent way. Interestingly, a significant increase in reactive oxygen species (ROS) production was observed in both NB cell populations treated with P5 or P7, establishing for both copolymers an unequivocal correlation between cytotoxicity and ROS generation. Therefore, P5 and P7 could be promising template macromolecules for the development of new chemotherapeutic agents able to fight NB chemoresistance.

disappearance of the metallic magnesium (90'), stirring was continued at r.t. for 1 h. Then, the suspension was decanted to obtain a clear solution of Grignard's reagent (1), which will be used as it is in the subsequent reaction. To know the exact title of reagent 1, an aliquot of the obtained solution (1 mL) was transferred in a flask containing 0.1014 N HCl in excess (10 mL) and back titrated with 0.1021 N NaOH (5.20 mL) in the presence of phenolphthalein as indicator. Reagent 1 resulted 0.580 N (85 % yield). S1.2. 4-(4-Bromobuthyl)styrene (2) A mixture of the 1,4-dibromobuthane (24.72 g, 114.5 mmol), dry THF (50 mL) and a solution of LiCuBr2 in dry THF (3.4 mL) was cooled to 0 °C, treated dropwise with 0.580 N 4-vinylphenyl magnesium chloride (47 mL, 27.3 mmol) in THF and stirred at room temperature for 18 h. The reaction mixture was then treated with an iced aqueous solution of NaCN (1.81 g) and NH4Cl (11.34 g) dissolved in water (70 mL) and extracted with peroxide-free ethyl ether (3x60 mL). The extracts were dried over anhydrous MgSO4 overnight and the solvent was removed by evaporation at reduced pressure obtaining a pale yellow oil (23.72 g). The unreacted 1,4-dibromobuthane was removed by distillation at reduced pressure (0.05 torr) and the oily yellow residue was furtherly purified by chromatographic column (petroleum ether 40-60°C/acetone = 85/15) to provide 2 as colorless oil (5.01 g, 21.0 mmoli, 77 % yield).
The experimental data of the copolymerization have been reported in Table S1.

S2.1. Copolymerization Procedure
In a 25 mL tailed test tube equipped with a magnetic stirrer and carefully flamed under nitrogen, monomer M5 (5) DMAA, AIBN as radical initiator, and the freshly distilled anhydrous solvent were introduced in the ratios reported A solution of P5 in just enough MeOH was filtered and transferred in a three-necks round-bottomed flask equipped with a mechanic stirrer and a funnel. It was thermostated at 25 ° C and the clear solution (S1) was slowly added with Et2O until an oily precipitate (OP5-1) was obtained. OP5-1 was decanted and separated from the supernatant (S2).
S2 was treated as the starting solution (S1) obtaining a second oily precipitate (OP5-2). OP5-1 and OP5-2 were then dissolved in MeOH and precipitated in an excess of Et2O obtaining the corresponding copolymers, namely P5-High and P5-Low.
The unreacted M5 was recovered from the mixture of the combined solvents by evaporation at reduced pressure.  (Table S2) were used to obtain a linear regression curve according to the Ordinary Least Squares (OLS) method, whose equation was Eq. (1) and extrapolating it to concentration c = 0, Kcal was determined. Kcal was found to be 501.

S2.2.2. Measurements
Solutions of P5 in MeOH were prepared at three different concentrations c (g/Kg) (Table S2) and were analyzed by VPO method at 45 °C in MeOH. The ratios between the measurement values (MV) and concentrations (c) (kg/g) were plotted vs concentrations (c) finding a regression curve whose extrapolation to concentration c = 0, worked directly by the instrument, provided the Kmeas (kg/g) for P5. The molecular mass of P5 was determined accordingly to equation Eq. (2) and was reported in Table S2 ( ) =

S2.3. Determination of NH2 equivalents contented in P5
The NH2 content of P5, in the form of hydrochloride, was obtained by volumetric titrations with a solution of HClO4 in acetic acid (AcOH), using quinaldine red as indicator [3]. Briefly, acetic anhydride (3 mL) was added to a solution of HClO4 70% (1.4 mL) in AcOH (80 mL), obtaining a colorless solution which was left under stirring at room temperature overnight. The clear yellow solution was made up to 100 mL with AcOH and standardized with potassium acid phthalate. The title of solution was found to be 0.1612 N. A sample of P5 (300.5 mg) was dissolved in AcOH (5 mL), treated with 2 mL of a solution of mercury acetate (1.5 g) in AcOH (25 mL), added with a few drops of a solution of quinaldine red (100 mg) in AcOH (25 mL) and titrated with the standardized solution of HClO4 in AcOH, using a calibrated burette with needle valve (0.02 mL). The very sharp end points were detected by observing the disappearance of the red color. Standardization and titrations were made in triplicate and the result was reported as means ± SD and expressed both as µ equiv. NH2/µ mol P5 and µ equiv. NH2/g P5 (Table S3).

S2.4. Dynamic Light Scattering (DLS) analysis
The hydrodynamic size (diameter) (Z-AVE, nm) and PDI of P5 particles were determined using Dynamic Light Scattering (DLS) analysis. Z-Ave and PDI measurements were performed in water mQ as medium at max concentration of P5 of 3 mg/mL (pH = 7.4), in batch mode using a low volume quartz cuvette (pathlength, 10 mm). The analysis was performed by a photon correlation spectroscopy (PCS) assembly, equipped with a 50 mW He-Ne laser (532 nm) and thermo-regulated at the physiological temperature of 37 °C. The scattering angle was fixed at 90°. Results were the combination of three 10-min runs for a total accumulation correlation function (ACF) time of 30 min. The hydrodynamic particle size result was volume-weighted and reported as the mean of three measurements ± SD (Table S3). PDI value was reported as the mean of three measurements ± SD made by the instrument on the sample. The Z-potential (ζ-p) was measured at 37° C in mQ water as a medium, and an applied voltage of 100 V was used. The P5 sample was loaded into pre-rinsed folded capillary cells, and twelve measurements were performed.