Sol–Gel Treatment of Textiles for the Entrapping of an Antioxidant/Anti-Inflammatory Molecule: Functional Coating Morphological Characterization and Drug Release Evaluation

The growing interest towards textile-based drug delivery systems is due to their potential innovative medical and well-being applications. In recent years, the technique of encapsulation or inclusion of the medicine/active principle into a polymer functional matrix has been employed in order to obtain textile materials with controlled drug release. In this study, a sol–gel-based coating was developed and used as an entrapping polymeric cross-linked network for a N-Palmitoyl-ethanolamine (PEA) derivative, 2-methyl-pentadecanoic acid (4-nitro-phenyl)-amide or N-Palmitoyl-(4-nitro-phenyl)-amine (PNPA), whose anti-inflammatory and antioxidant properties have already been shown. A wide series of chemical-physical methods have been used to characterize the silica-based functional sol and to ascertain the efficient and temporary deposit of PNPA on the sol–gel coated cotton fabrics. The medicine release system achieved was shown to ensure biocompatibility, PNPA reservoir and its subsequent releasing under the action of cutaneous stimuli, thus providing useful insights in the design of medical textiles.

and van der Waals interactions) acting between the non-polar active molecule and the alkoxysilane hosting network.
Finally, the functionally coated cotton samples were employed for in vitro diffusion studies with the aim of testing their ability to release the synthesized PEA derivative in a controlled manner compared to a standard solution of the molecule. This effectively prepared functional hybrid system, based on the non-covalently immobilized PNPA, showed good results so that it can be considered a suitable scaffold for fabrics in drug release applications, thus providing useful insights in the design and the development of medical textiles.

Materials and Methods
N-Palmitoyl-(4-nitro-phenyl)-amine (PNPA) was synthetized according to a synthetic strategy described in previous researches [28]. 3-glycidyloxypropyltriethoxysilane (GPTES) and methanol were purchased from Wacker and Aldrich, respectively, and used without further purification. Two cotton scoured and bleached 100% plain-weave textile fabrics (coded CO L and CO H ) kindly supplied by Albini S.p.A. (Albino, Italy) and Mascioni S.p.A. (Cuvio, Italy), respectively, were used for this research. The fabrics showed different mass per unit area (CO L = 119 g/m 2 and CO H = 331 g/m 2 , respectively). Cotton fabrics were washed before treatment at pH 7 and at 40 • C for 20 min in a non-ionic detergent, rinsed several times with de-ionized water and then dried. The cleaned samples were conditioned at 20 (± 1) • C and under standard atmospheric pressure at 65 (± 2)% relative humidity for at least 24 h prior to all experiments.
PNPA (25 mg) was dissolved in 40 mL of methanol through ultrasonication and left under continuous stirring. Then 2 mL of a 1 M aqueous sol-gel solution of GPTES were added drop by drop to the clear methanol solution of antioxidant molecule, thus resulting in a final GPTES sol concentration of 0.05 M (1:0.034 molar ratio with respect to PNPA). The obtained solution (G-PNPA sol) was ultrasonicated and left at room temperature under stirring for at least 90 min. The same reaction was also carried out in absence of the antioxidant molecule in order to obtain a reference GPTES sol sample. Both solutions were applied separately onto cotton textile (10 cm × 10 cm) through a two-roll laboratory padder (Werner Mathis, Zurich, Switzerland) at a nip pressure of 2 bar, then dried (80 • C for 5 min) and cured (100 • C for 1 min) in an electric laboratory oven.
PNPA, G-PNPA and G sols were fully investigated through FT-IR spectroscopy and Nuclear magnetic resonance (NMR). Untreated and treated textiles were characterized by FT-IR spectroscopy, Scanning Electron Microscopy (SEM) coupled to energy dispersive X-ray (EDS) and Atomic Force Microscopy (AFM).
The PNPA controlled release from the two prepared textiles, CO L _G-PNPA and CO H _G-PNPA, was investigated by performing in vitro diffusion studies using Franz diffusion cells and according to the experimental protocol reported in a previous work [29]. For this purpose, Strat-M ® membranes (25 mm discs, Cat. No. SKBM02560, Merck Millipore, Darmstadt, Germany) were positioned between the donor and the receptor compartments of each Franz cell and the experiments were carried out at 37 ± 0.5 • C. The two tested items, CO L _G-PNPA and CO H _G-PNPA, were placed on the Strat-M ® membrane with the GPTES layer facing towards the acceptor chamber. Then, the Franz cell compartments were fixed together and filled with 0.5 and 5.5 mL of phosphate buffer at pH 7.4 (10 −3 M), respectively. The content of the receptor chamber was withdrawn at different times, such as 1, 2, 4, 6 and 24 h, for UV-Vis analysis and replaced with phosphate buffer. The same experimental conditions were applied to a control sample consisting of a standard PNPA solution. The in vitro diffusion studies were carried out in triplicate and the obtained results were expressed as diffused amount (%).
FT-IR spectra were performed by a Thermo Avatar 370 equipped with an attenuated total reflection (ATR) accessory and using a diamond crystal as internal reflectance element. Spectra were acquired with 32 scans and in the range from 4000 to 550 cm −1 with a resolution of 4 cm −1 .
One-and two-dimensional NMR experiment were recorded in methanol-d 4 at 298.2 (±0.1) K on Bruker ARX-300, equipped with a 5 mm gradient probe and operating at 300.1 MHz for 1H nucleus. All chemical shifts are shown in parts per million (δ/ppm), downfield to tetramethylsilane (Me 4 Si) as an internal standard (δ = 0.0 ppm), or referenced to the residual protiated solvent signal such as in methanol-d 4 ( 1 H NMR: 3.30 ppm). 1 H NMR signals were assigned by means of two-dimensional homonuclear NMR gradient experiments (gCOSY, gNOESY), acquired using standard Bruker pulse sequences.
SEM morphology and SEM-EDS of the investigated samples were obtained using a FEI Quanta FEG 450 microscope. An operating voltage of 5 kV in low vacuum was used for SEM images. EDS analysis was conducted with an operating voltage of 20 kV, always in low vacuum. Samples were fixed on aluminum sample holders by means of a graphitic adhesive.
AFM characterization was performed using a stand-alone SMENA head by NTMDT, equipped with a Bruker silicon probe model NCHV working in semi-contact mode. The samples were fixed onto metallic stubs using a small piece of double-sided scotch tape and studied at RT.

Sol-Gel Synthesis and Coating Application
To obtain a controlled drug release fabric, 3-glycidoxypropyltriethoxysilane (GPTES) was employed in a sol-gel synthesis in the presence of an antioxidant/anti-inflammatory molecule, the N-Palmitoyl-(4-nitro-phenyl)-amine (PNPA) [28]. The simultaneous presence of both an epoxy group and a triethoxysilane functionalities makes this specific silica precursor able to cross-link to other GPTES molecules, to entrap the antioxidant doping molecule and still to coat the textile surface, respectively. In this regard, a GPTES-based sol was synthesized in acidic aqueous medium by addition of slight amount of HCl, as catalyst, and then added to a methanol solution containing the PNPA molecule. As reported in previous studies [15][16][17][18][19][20][21], the sol-gel synthesis leads to the formation of a hybrid polymeric 3D network through the opening of the epoxy ring of GPTES and interaction of the triethoxysilane end to form an extended polyethylene oxide network (PEO) [20], in whose holes the PNPA is physically and stably entrapped (Scheme 1).

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The so obtained G-PNPA functional sol was applied by padding on cotton fabrics and cured 173 thermally with the aim to prepare a nano-hybrid coating for controlled release application (Scheme The so obtained G-PNPA functional sol was applied by padding on cotton fabrics and cured thermally with the aim to prepare a nano-hybrid coating for controlled release application (Scheme 2).

Sol NMR Characterization
A reaction mixture relative to the G-PNPA sol as obtained in methanol for coating application, has been achieved in situ (1:0.1 = [GPTES]:[PNPA] molar ratio) in methanol-d 4 and characterized by means of 1 H one-and two-dimensional NMR spectroscopy. Figure 1 shows the 1 H NMR spectra as recorded at time zero and after 24 h (in methanol-d 4 at 298 K, 300 MHz). The comparison of the aliphatic regions of the 1 H NMR spectra of the reaction mixtures recorded at different times clearly reveal the presence of the protonic pattern expected for the diol/PEO silylated (red squares in Figure 1) derivatives and the starting GPTES (black squares in Figure 1, [20]) as shown in Scheme 1. In particular, the aliphatic regions of the 1 H NMR spectra in Figure 1 clearly show: (i) the presence of the expected protonic pattern for the GPTES open ring derivative, bringing a hydroxyl and an ether group bonded to two vicinal carbon C e and C f atoms (δ = 0.68, CH 2a ; 1.71, CH 2b ; 3.48, CH 2c + CH 2d + CH 2f ; 3.88 CH 2e , red squares in lower spectrum) [30] (ii) the proton peaks relative to the starting GPTES in a decreased concentration (δ = 0.67, CH 2a ; 1.68, CH 2b ; 2.61+2.78, CH 2f ; 3.16, CH 2e ; 3.32 + 3.76, CH 2d ; 3.49, CH 2c , black squares in upper spectrum); (iii) the presence of the upper-field methylene and the methyl proton resonances relative to free ethanol moieties, compared to those relative to the ethylic groups of GPTES (δ = 3.63, CH 2 , 1.19, CH 3 vs 3.84, CH 2 , 1.22, CH 3 ; cut signals in both spectra).   Unfortunately, the long penta-decanoic protonic chain is buried under other signals, also due to the strong molar ratio exceed of GPTES. The aromatic region shows the expected and unchanged pattern of broad signals for para-substituted phenyl ring of the PNPA molecule.
The 1 H NMR experiments clearly show that the GPTES epoxy ring opening reaction by a suitable nucleophilic group of the PNPA molecule is not occurring, as well as the formation of a stable ether covalent bond. As previously shown [20], the stable encapsulation of the PNPA molecule into the PEO silylated GPTES derivative is most likely due to the formation of weak bonds (i.e., van der Waals or electrostatic) between the polymerized GPTES (i.e., ether oxygen and hydroxyl group) and the N-Palmitoyl-(4-nitro-phenyl)-amine (i.e., nitrogen, oxygen, long alkyl chain, phenyl group).

ATR FT-IR
FT-IR spectra of untreated and treated cotton fabrics were analyzed after the normalization at 1362 cm −1 , absorption band relative to the CH bending of cellulose. In Figure 2, FT-IR of CO L untreated and treated samples with GPTES sol and G-PNPA sol (CO L _UT, CO L _GPTES and CO L _G-PNPA, respectively) are reported and compared with those relative to CO H untreated and treated fabrics with the same solutions (CO H _UT, CO H _GPTES and CO H _G-PNPA, respectively). In particular, in all spectra, it is possible to distinguish clearly the characteristic absorption bands relative to cellulose moieties, such as: the broad bands around to 3331 cm −1 and 2894 cm −1 (stretching mode of OH and CH, respectively), and the absorption bands between 1097 cm −1 and 895 cm −1 relative to the asymmetric in-plane ring stretch and to C-O stretch and asymmetric out-of-phase ring stretch (C 1 -O-C 4 ) [31]. In particular, in the inset in Figure 2A    With the aim of investigating the chemical structure and interactions between silica precursor and antioxidant molecule without the interference of the intense absorption bands of cotton fabrics, the FT-IR spectra of each sol was carried out on the pure xerogel. The latter was obtained by depositing a few drops of each solution onto optical glass slides further subjected to a thermal treatment (100 • C) to remove the solvent [26]. Indeed, by analyzing the FT-IR spectra of PNPA, it was possible to identify the characteristic peaks in order to establish its interaction with the GPTES sol. Spectra of the GPTES sol ( Figure 3, green line) clearly shows the formation of the inorganic network due to the absorption bands relative to: asymmetric (1093-1010 cm −1 ) and symmetric stretching of Si-O-Si (756 cm −1 ) and its bending mode (850 cm −1 ) [17,18,20]. Furthermore, the absorption bands at 3072-3000 cm −1 , 1255 cm −1 and 906-850 cm −1 were assigned to asymmetric and symmetric C-H stretch, ring breathing, as well as the asymmetric and symmetric ring deformation, respectively [17,18], indicating that some unopened epoxy ring remain in GPTES sol.
Appl. Sci. 2020, 10, x FOR PEER REVIEW 7 of 13 possible to identify the characteristic peaks in order to establish its interaction with the GPTES sol.

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Spectra of the GPTES sol (Figure 3, green line) clearly shows the formation of the inorganic network 233 due to the absorption bands relative to: asymmetric (1093-1010 cm ₋1 ) and symmetric stretching of Si-

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ring breathing, as well as the asymmetric and symmetric ring deformation, respectively [17,18],

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indicating that some unopened epoxy ring remain in GPTES sol.

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These peaks relative to the GPTES sol are also evidenced in the sol containing PNPA (   These peaks relative to the GPTES sol are also evidenced in the sol containing PNPA (Figure 3, black line), in which the main characteristic absorption bands of the antioxidant molecules are present. As shown in Figure 2 (red line), PNPA is featured by the asymmetric and symmetric CH 2 stretching mode of alkyl chain (2916 and 2849 cm −1 , respectively) with relative bending (1470-1458 cm −1 ), the C=O and C-N stretching of the secondary amide (1737 and 3075 cm −1 , respectively) and the C-N stretching mode (1596 cm −1 ) [32].

Morphological Characterizations
All untreated and treated cotton samples were investigated by SEM and AFM microscopy, in order to characterize the morphology of the coated textile fabric and to underline structural differences before and after the coating application. SEM analysis shows that the two analyzed tissues have two different weaves: CO H _UT samples have a larger weave than CO L _UT ones, and the fibers appear less ordered compared with CO L _UT ones and more threadbare (compare Figure 4a,b). Both samples do not show modifications after treatment and the morphology appears unchanged also at higher magnification ( Figure 5). This demonstrates that, after treatment, no changes at micrometer scale occur.
Appl. Sci. 2020, 10, x FOR PEER REVIEW 8 of 13 treatment and the morphology appears unchanged also at higher magnification ( Figure 5). This  EDS analysis shows Si peak only in samples treated with GPTES (CO H _GPTES, CO L _GPTES) and subsequently with PNPA (CO H _G-PNPA, CO L _G-PNPA) ( Figure 6, only two treated samples are reported for briefness, because the other ones exhibited a similar spectrum). The other peaks refer to carbon, present in the analyzed tissues but also in graphite adhesive used for SEM analysis, oxygen, present also in vapor-low vacuum atmosphere, and aluminum, derived from the SEM stub.
Appl. Sci. 2020, 10, x FOR PEER REVIEW 9 of 13 carbon, present in the analyzed tissues but also in graphite adhesive used for SEM analysis, oxygen, 265 present also in vapor-low vacuum atmosphere, and aluminum, derived from the SEM stub.

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The mapping presented in Figure 6   The mapping presented in Figure 6 (inset c* and d*) shows that distribution of Si element (present in GPTES) is very uniform and no phase separation occurs.

PNPA in Vitro Diffusion Studies
In vitro diffusion studies on the developed cotton-based textiles were carried out with the aim of evaluating their ability to release in a controlled manner the synthesized PEA derivative compared to a standard solution of the molecule (Scheme 3).    In the performed experiments, Strat-M ® membranes were used as a synthetic alternative, which is predictive of the diffusion process occurring through human skin. The studies involved two different textiles, such as CO L _G-PNPA and CO H _G-PNPA, prepared employing the obtained G-PNPA functional sol. The obtained results are expressed as cumulative diffused amount (%) and the diffusion profiles for the tested items are reported in Figure 8.
is predictive of the diffusion process occurring through human skin. The studies involved two  The two G-PNPA-coated cotton textiles, CO L _G-PNPA and CO H _G-PNPA, show similar results in the performed in vitro diffusion studies. In the case of the CO L _G-PNPA sample, indeed, the amount of released PNPA is equal to 5% within the first hour reaching the 48% and 66% in 6 and 24 h, respectively, while the CO H _G-PNPA sample exhibits a value of 7% after the first hour achieving the 53% and 73% at the time points of 6 and 24 h. On the other hand, the 14% of PNPA is released after the first hour from the control solution reaching the 69% and 89% at 6 and 24 h. The experimental data confirm the ability of both the developed textiles to release in a controlled way the synthesized PEA derivative. These results could be related to the rate-limiting steps in drug release reported in the literature [33,34]: the drug diffusion within the polymer matrix and the rate of polymer swelling. Depending on the presence of sweat, simulated with the buffer solution in contact with the PNPA-treated fabric, the weak interactions between the treated fabric and the antioxidant molecule slowly disappear until the drug is completely released. The presence of the sol-gel matrix increases the bonding interactions between the network and the drug, slowing down its release.

Conclusions
The N-Palmitoyl-(4-nitro-phenyl)-amine, PNPA, a PEA derivative that already showed good anti-inflammatory and antioxidant properties, was firmly entrapped in a sol-gel-based matrix obtained by polymerization reaction of the epoxy-alkoxysilane GPTES, as cross-linker compound. NMR studies run on the sol evidenced that the GPTES epoxy-ring opening and the subsequent polymerization give rise to a polyethylene oxide 3D network grafted on textile with the PNPA immobilized into it. After the textile drying and curing the xerogel was morphologically studied on the treated cotton samples by SEM and AFM microscopy, revealing that no changes in the fiber morphology occurred at the micrometer scale, and that GPTES and PNPA intimately and homogeneously wrap the fibers. Furthermore, SEM mapping revealed a uniform distribution of the silica-based coating on the cotton fibers. In vitro diffusion studies were realized on the developed functionalized cotton-based textiles in order to check their ability to release the PEA derivative in a controlled manner in comparison to a standard molecule solution. As a matter of fact, this functional textile has been shown to be a suitable system for PNPA release, thanks to the chemical binding weakening of PNPA with the sol-gel polymer matrix by mean of medium effect, thus opening the way to the design of similar functional hybrid coatings for biomedical application. Although parameters of sol particles (e.g., hydrodynamic radius and electrokinetic potential) as well as the porosity of systems were not investigated, the obtained results confirmed the potential of the nanoengineered procedure as a versatile method for preparing stable and tunable drug-releasing materials. The combination of the functionality and transparency provided by the hybrid coating with its easy processability could represent an innovative route to fabricate biomaterials for healthcare. Future work will focus on the comparison of silica sols characteristics with release properties of this delivery system.