*2.2. Naproxen Extraction*

Commercialized Proxen 500 mg tablets were ground into a fine powder using an electric grinder. The Proxen powder obtained is added to a 3M HCl solution and stirred for 24 h then left to stand for 1 h. Naproxen (NPX) or (S)-2-(6-methoxynaphthalen-2-yl) propionic acid, which is very poorly soluble in water precipitates, the additives dissolve, and the precipitant is then recovered by filtration. The extracted NPX powder is washed several times with water and then dried under a vacuum at 25 ◦C to constant weight. In order to remove the residual matter from the organic phase, the dry precipitate obtained is

dissolved in chloroform and then transferred to a separating funnel containing an equivalent amount of distilled water. The whole is then stirred until the complete dissolution of Naproxen. The two phases are finally separated by settling. This process was repeated three times to ensure the purification of the product. Pure Naproxen is then extracted from the isolated organic phase by evaporating chloroform at room temperature (25 ◦C) using a rotary. The melting point of the pure NPX white crystals obtained, measured by DSC analysis, indicates 166 ◦C, which agree with the literature [34].

### *2.3. Preparation of NPX/pHEMA and NPX/pHPMA*

NPX/pHEMA and NPX/PHPMA composites containing 2, 5, 7, and 10 wt% of NPX were prepared in situ by free radical polymerization at 25 ◦C in the presence of NPX using camphorquinone as a photoinitiator. Using known amounts distillated under reduced pressure of HEMA and HPMA monomers, camphorquinone and NPX were weighed with precision and placed in a Teflon pan traversed with a stream of nitrogen U. These mixtures are irradiated throughout the reaction time by means of UV light coming from a UV lamp with a wavelength of 380 nm and a power of 13.3 MW. A solid film deposed in the Teflon pan is obtained indicating the completion of the polymerization reaction. To remove all traces of residual monomer encrusted in the film obtained, the Teflon pan and polymer film set are placed in a vacuum oven maintained at 40 ◦C until constant mass. The aggregated NPX particles deposited or glued to the film surface are removed by washing three times with distillated water. Two series of NPX/pHEMA and NPX/pHPMA mixtures containing 2, 5, 7, and 10 wt% of NPX content are prepared by this same method, and the preparation conditions are summarized in Table 1.


**Table 1.** Preparation conditions of NPX/pHEMA and NPX/pHPMA composites.

#### *2.4. Characterization*

The FTIR spectra of NPX powder, PHEMA homopolymer, NPX/PHEMA, and NPX/ PHPMA composites films were performed in the wavenumber range 400–4000 cm−<sup>1</sup> on a Nicolet 6700 FT-IR from the company Thermo Scientific. A 30,000–200 cm−<sup>1</sup> diameter diamond-like Smart orbit crystal reflector, supplied by the same company, was used to accomplish this task. The DSC thermograms of drug, polymer, and their mixtures were performed on a Shimadzu DSC-60 (Japan) previously calibrated with indium. An amount of 8–10 mg of NPX powder or film samples were deposited in an aluminum pan and then closed, being placed in the DSC analysis cell. All samples were scanned from −40 to +240 ◦C under nitrogen gas atmosphere at a heating rate of 20 ◦C·min−1. All the thermograms taken from the second scan run revealed no traces of polymers or drug degradation. The *Tg* value of pure constituents or their mixture was taken precisely as the median point on the thermogram indicating the variation in the heat capacity versus the temperature. The *Tm* value was taken exactly at the top of the endothermic peak. The surface morphology of NPX particles, polymers, and their composites were examined by scanning electronic microscope using a JEOL JSM-6360LV SEM (Tokyo, Japan) at an accelerating voltage of 10 kV. The surface and cross-sections of samples were sputter-coated with a thin layer of gold prior and was imaged at a magnification range of 300–3000 nm. The crystalline

structures of NPX powder, polymers, and mixtures were examined by XRD analysis on an X-ray diffractometer (Rigaku D/max 2000) equipped with a Cu anode tube. The applied voltage was 40 kV and a generator current of 100 mA. All samples were examined at <sup>2</sup>*<sup>θ</sup>* = 5◦–80◦ at a scanning rate of 1.0◦·min<sup>−</sup>1.

A U-2910 spectrophotometer manufactured by Hitachi Company was used to measure ultraviolet and visible light absorbance of NPX released. Absorbance was measured using quartz cuvettes with a side length of 1 cm. The wavelength corresponding to the maximum absorbance of NPX was 230 nm. The released NPX concentration was deduced from a linear calibration curve indicating the change in absorbance versus concentration.

#### *2.5. Cell Culture and Proliferation Assessment*

The human oral cancer cell line Ca9–22 cells were cultured at 37 ◦C and 5% CO2 in RPMI-1640 medium (Thermo Fisher Scientific, Burlington, ON, Canada), supplemented with L-glutamine, 5% fetal bovine serum (FBS, Gibco) provided by the same company, and 1% penicillin/streptomycin solution (Sigma-Aldrich, Oakville, ON, Canada). Cell proliferation was bi-evaluated using two MTT and LDH tests as described by Semlali et al. 2021 [35,36] and Contant et al. 2021 [37]. For the MTT assay, 10<sup>5</sup> Ca9-22 cells per well containing the sample were seeded in 24-well plates for 24 h. After adhesion and growth of the cells, the culture medium is replaced by a new one containing a solution of MTT at 5 mg·ml−<sup>1</sup> in PBS and left for 3 h at 37 ◦C in the dark. Then, the cells were lysed with HCl, 0.05 N, in 1 mL of isopropanol. Addition of 100 μL of analysis buffer to test wells from 96-well microplates was required to measure absorbance at 550 nm by an iMark reader (Bio-Rad). The percentage of viable proliferating cells was determined using Equation (1)

$$\text{Cell}.viability(\%) = \frac{OD\_T - OD\_B}{OD\_C - OD\_B} \times 100,\tag{1}$$

where *ODT*, *ODB,* and *ODC* are the optic densities of the treated cell, blank, and control, cell respectively.

Adhesion positive control was the plate for the treated cultured tissues. The negative control for cell adhesion was the plate for untreated cultured tissue. LDH assay was realized by the LDH Cytotoxicity Detection Kit from Roche, which allows to directly quantify the cell death in culture based on the measurement of lactate dehydrogenase released into growth media. As described in our previous work [35,36], 105 cells per well were seeded in 24-well plates containing NPX/pHEMA and NPX/pHPMA with different compositions. After adhesion for 24 h, 50 μL of each supernatant was transferred in triplicate into a 96-well plate and supplemented with 50 μL reconstituted substrate mixture. Then, the plates were incubated for 30 min at room temperature in the dark until the yellow color developed, before reading at 490 nm with an xMark microplate absorbance spectrophotometer (Bio-Rad, Mississauga, ON, Canada). Triton X-100 (1%) was used as a positive control for LDH, and the negative one was obtained with untreated cells. LDH release activity was calculated using Equation (2)

$$\text{LDH.activity}(\%) = \frac{ABS\_p - ABS\_{\text{nc}}}{ABS\_{pc} - ABS\_{\text{nc}}} \times 100,\tag{2}$$

where *ABSp*, *ABSnc*, and *ABSpc* are the absorbance of drug-carrier system, positive control, and negative control, respectively.

#### *2.6. Swelling Properties*

The swelling behavior of pHEMA and pHPMA hydrogels was studied on samples of thin films of dimensions 3 cm × 3 cm and thickness varying between 2.20 and 2.56 mm. Each film sample of determined mass (*mo*) was placed in 50 mL of an aqueous solution at known pH (1 or 7) and maintained at 37 ◦C, then stirred (260 rpm) until the swelling equilibrium was reached. The mass of the medium absorbed at each time interval (*mt*) is

obtained by weighing the film after delicately wiping the droplets deposited on the two surfaces using tissue paper. The swelling degree of pHEMA and pHPMA film samples was determined from Equation (3).

$$S(wt\% ) = \frac{m\_l - m\_o}{m\_o} \times 100 \,\text{\AA} \tag{3}$$

#### *2.7. Density Measurements*

Polymer density values were determined at 25 ◦C using a pycnometer, in which cyclohexane was used as a non-solvent and Equation (4) [38]:

$$\rho = \frac{m\_p \times \rho\_{\rm chx}}{m\_p + m\_{pc} + m\_T} \,\prime \tag{4}$$

where *mp* is the mass of the polymer, *mpc* is the mass of the pycnometer with cyclohexane, and *mT* is the mass of the pycnometer with cyclohexane and polymer. *ρchx* is the density of cyclohexane (0.78 g·cm−3). Each experiment was triplicated, and the density was taken from the average arithmetic values obtained.

#### *2.8. In Vitro Release Dynamic of NPX*

The "in vitro" release dynamic of NPX from the NPX/pHEMA and NPX/pHPMA drug carrier systems was investigated at body temperature (37 ◦C) in aqueous media of pH1, 3, 5, and 7. NPX released was monitored for 72 h, in which 0.5 mL of the solution was withdrawn after each time interval, then dosed by UV analysis. The accumulative drug release percent ADR (wt %) at time *t* was calculated at chosen time intervals using the following equation:

*ADR*(*wt*%) = *mt* <sup>×</sup> <sup>100</sup> *mo* , (5)

where *mt* and *mo* are the total mass of NPX released at a certain time *t* and the initial mass of drug loaded in the polymer.

#### **3. Results and Discussions**

#### *3.1. Characterization*

#### 3.1.1. FTIR Analysis

A comparison between the FTIR spectra of the NPX/pHEMA composites with those of their components shown in Figure 1 reveals, for the composite absorption bands, that they are practically similar to those of pure pHEMA. The NPX/pHEMA spectra exhibits a shift in the broad absorption band of the hydroxyl group vibrations from 3472 to 3463 cm−<sup>1</sup> and a shift of the sharp absorption band attributed to the carbonyl group (C=O) vibration of pHEMA from 1727.62 cm−<sup>1</sup> to 1724.32 cm−1. It is well known that the position of the vibration peak of the carbonyl group suggests that most of the carboxylic acid groups are associated with the intermolecular hydrogen bonds formed between the HEMA derived moieties and the acid groups [39–41]. In addition, the wide absorption band in the spectral region 3200–3600 cm−<sup>1</sup> corresponding to the vibrations of the OH group also confirms that hydrogen bonds form in the structure of poly(2-hydroxyethyl methacrylateco-acrylic acid). The deconvolution in Lorentzian peaks of the hydroxyl absorption band between 2600 cm−<sup>1</sup> and 4000 cm−<sup>1</sup> (Figure 2) also reveals the appearance of a new band at 3274.54 cm−1, attributed to the vibration of the hydrogen bond between hydroxyl group of pHEMA and carbonyl group of NPX. On the carbonyls side, the deconvolution of the absorption band between 1600 cm−<sup>1</sup> and 2000 cm−<sup>1</sup> (Figure 3) reveals another new absorption band at 1723.5 cm−1, thus confirming this finding. Similar results are also observed for NPX/pHPMA composites in Figure 4. Indeed, the comparison between the FTIR spectra of the NPX/pHPMA composites with that of its pure polymer reveals a small shift in the absorption band of the carbonyl group of pHPMA toward the lower wave number (from 1724.12 cm<sup>−</sup>1to 1727.34 cm−1) and in the hydroxyl group (from 3400.07 cm−<sup>1</sup>

to 3395.36 cm−1). These facts are without doubt attributed to a dynamic caused by the hydrogen bond interactions leading to a miscibility of these two components.

**Figure 1.** Comparative FTIR spectra of: (**A**) pure NPX; (**B**) NPX/pHEMA2; (**C**) NPX/pHEMA5; (**D**) NPX/pHEMA7; (**E**) NPX/pHEMA10; (**F**) virgin pHEMA.

**Figure 2.** Deconvolution in Lorentzian of the FTIR spectra of pure pHEMA and NPX/pHEMA7 composite spectra between 4000 cm−<sup>1</sup> and 2600 cm<sup>−</sup>1.

**Figure 3.** Deconvolution in Lorentzian curves of the FTIR spectra of pure pHEMA and NPX/pHEMA7 composite spectra between 2000 cm−<sup>1</sup> and 1600 cm<sup>−</sup>1.

**Figure 4.** Comparative FTIR spectra of: (**A**) pure NPX; (**B**) NPX/pHPMA2; (**C**) NPX/pHPMA5; (**D**) NPX/pHPMA7; (**E**) NPX/pHPMA10; (**F**) virgin pHPMA.

#### 3.1.2. XRD Analysis

The crystalline structure of NPX in the pHEMA and pHPMA polymer matrices was investigated by X-ray diffraction, and the results obtained are gathered with their pure components in Figures 5 and 6, respectively. As shown in Figure 5, the XRD pattern of pure NPX aggregated powder shows its highly crystalline structure and nature through the distinct peaks at 6.5◦, 12.4◦, 16.6◦, 19◦, 20◦, 22.5◦, 24◦, and 28.6◦ 2*θ*, which are in good agreement with the literature [42,43]. The XRD spectra of pHEMA and pHPMA reveal an amorphous structure. The XRD motif of the NPX/pHEMA7 composite shows no new crystallinity signals or those characterizing the crystallinity of pure NPX; as for the pure polymer, this material exhibits a completely amorphous structure. Similar results are also obtained for the NPX/pHPMA systems as shown in Figure 6. This indicates that the NPX drug is uniformly distributed in its molecular level inside the polymer matrix.

**Figure 5.** X-ray diffraction spectra of NPX, pHEMA, and NPX/pHEMA systems with different NPX contents.

#### 3.1.3. DSC Analysis

The DSC thermograms of pure NPX, pHEMA, and NPX/pHEMA systems with different NPX contents are shown in Figure 7. The thermal curve of pure pHEMA shows a glass transition temperature (*Tg*) at 86 ◦C, which agrees with that of the literature [44], while the pure Naproxen shows, through its thermal plot, a sharp endothermic peak at 166 ◦C characterizing its melting temperature [34]. The NPX/pHEMA thermograms reveal a small shift in the *Tg* of pHEMA toward the low temperatures and a complete disappearance of the NPX melting peak. This reveals a uniform distribution of the NPX filler in the polymer matrix in its molecular level, in which Naproxen loses its crystallinity, thus confirming the results obtained by FTIR and XRD analysis. The decrease in the *Tg* value of the polymer in the mixture is probably due to the increase of the free volume between the polymer chains caused by the insertion of MPX molecules between them, thus promoting the chain sliding.

**Figure 6.** X-ray diffraction spectra of NPX, pHPMA, and NPX/pHPMA systems with different NPX contents.

**Figure 7.** DSC thermograms of pure NPX, pHEMA, and NPX/pHEMA7 systems.

The thermal analysis of NPX/pHPMA systems by DSC technique led to the results of Figure 8. As it can be observed on the thermogram of pure pHPMA, a transition appears at 83 ◦C characterizing the glass transition of this polymer [45]. Concerning the NPX/pHEMA system, as for the system containing PHEMA carrier, a shift was shown in the Tg of the pHPMA from 82 ◦C to 54 ◦C as the NPX content in the mixture increased. A complete disappearance of the transition characterizing the fusion of the NPX is also observed on the thermograms of NPX/pHPMA mixtures, except that containing 10 wt% of NPX content, in which a weak transition at 131 ◦C attributed to the melting point of excess of NPX aggregates.

**Figure 8.** DSC thermograms of NPX, pHPMA, and NPX/pHPMA systems with different NPX contents.

#### *3.2. Cells Adhesion and Toxicity*

As shown in Figure 9 (in blue), the NPX/pHEMA drug-carrier system with different NPX contents presents, in general, a good adhesion compared to the negative (untreated tissue culture plate) and positive (tissue culture plate treated for cell adhesion) control used in this study. However, the NPX amount incorporated in the pHEMA in drug carrier system seemed to not significantly affect the cell adhesion when the Ca9-22 cells were treated with naproxen. These results were also confirmed by the LDH assay (Figure 10 in green). In addition, NPX/pHEMA drug carrier systems, as well as the pure pHEMA, induce low cytotoxicity compared to the negative and positive controls (2% Triton). Comparable results were also observed when the pHEMA was replaced by pHPMA in the drug carrier system, regardless of the range of the composition investigated (Figure 9 in blue and Figure 10 in green).

**Figure 9.** Effect of NPX content in NPX/pHEMA and NPX/pHPMA drug carrier systems on Ca9-22 cells adhesion

**Figure 10.** Effect of NPX content in NPX/pHEMA and NPX/pHPMA drug carrier systems on Ca9-22 cells cytotoxicity.
