*2.9. Statistical Analysis*

In all the experiments, each sample was tested in three independent analyses, each carried out in triplicate. The results are presented as the mean ± SD values obtained.

#### **3. Results and Discussion**

#### *3.1. Loading of Indole Compounds in Gelatin and Release Kinetics*

In the initial experiments, porcine skin gelatin type A was dissolved at 10% *w*/*v* concentration in PBS at pH 7.4 at 37 ◦C (HGel-A) and DHICA or MeDHICA previously dissolved in the minimal amount of DMSO, were added under stirring to a 10% *w*/*w* concentration with respect to gelatin (10% *w*/*w* DHICA or MeDHICA/HGel-A). The solutions were set for gelation for 12 h at 4 ◦C and then washed with PBS to remove not incorporated indole compounds (HGel-A, Figure 1) [32].

**Figure 1.** Preparation of gelatin and loading of indole compounds.

UV–Vis spectrophotometric analysis of the indoles (λmax 320 nm) in the washings allowed to estimate an extent of incorporation into the gelatins of 62 ± 1.7% in the case of DHICA and even higher, up to 80 ± 1.3%, for MeDHICA. Using DHICA at 1 or 5% *w*/*w* concentration with respect to gelatin, the extent of incorporation proved to be 48 and 59%, respectively.

The kinetics of release of the indoles at physiological pH and 25 ◦C was then evaluated over 72 h, by refreshing the medium every hour in the first 6 h and then every 24 h. For either indoles, the release

was smooth over the observation period reaching values around 30% of the incorporated indole for the 10% *w*/*w* DHICA or MeDHICa/HGel-A (Figure 2, panel A). In the case of DHICA, the release was comparable for the 5% *w*/*w* DHICA/HGel-A and up to 60% after 72 h for the 1% *w*/*w* DHICA/HGel-A (Figure 2, panel B).

**Figure 2.** Release kinetics of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) or the methyl ester of DHICA (MeDHICA) at 10% *w*/*w* (panel **A**), and DHICA 1, 5, 10% *w*/*w* (panel **B**) from pristine porcine skin type A gelatin (HGel-A) in PBS at pH 7.4 with refreshing of the medium over 72 h. Reported are the mean ± SD values of three experiments and the polynomial regression that fits the data.

The release of the indoles at 10% *w*/*w* in HGel-A was also monitored at pH 5.5, a pH value of relevance for topical delivery. A sustained release was observed for DHICA reaching 40% of the incorporated indole at 72 h, whereas this was much lower for MeDHICA (Figure 3).

**Figure 3.** Kinetics of release of DHICA or MeDHICA at 10% *w*/*w* from HGel-A in PBS at pH 5.5 with refreshing of the medium over 72 h. Reported are the mean ± SD values of three experiments and the polynomial regression that fits the data.

The satisfactorily high kinetics of release for DHICA also at lower pH highlights the potential use of DHICA loaded gelatin hydrogels for epidermal drug delivery in topical uses, e.g., in wound healing to ameliorate the associated inflammatory state. It is worth noting that low pHs are also found in tumor environments. Given the observed gelatin releasing ability, the proposed system may have potential as pH-dependent targeted drug delivery system in anti-cancer therapy. On the contrary, MeDHICA release is negatively affected by low pH. A possible explanation can be found in the higher solubility of the compound at pH 7.4, due to the partially ionized phenol groups. In the case of DHICA this would in part be counterbalanced by the hydrophilic character of the carboxyl group that at pH 5.5 is also ionized to a significant extent based on the pKa value of 4.25 reported for DHICA carboxyl group [41].

However, the relatively low melting temperature of gelatin hydrogels would pose severe limitations to other possible applications suggested by the biological activity of DHICA, but implying exposure to physiological temperatures. Based on this consideration, in further experiments the possibility to ge<sup>t</sup> a tougher material that could remain unaltered even after prolonged exposure to physiological temperature or higher was explored by two di fferent strategies: (i) chemical cross-linking; (ii) chitosan-gelatin blends.

#### *3.2. Preparation of Cross-linked Gelatins and Gelatin-Chitosan Blends*

DMTMM was used as the coupling agen<sup>t</sup> for gelatin cross-linking [42]. DMTMM is a zero-length coupling agen<sup>t</sup> promoting the activation of carboxyl groups for subsequent amide or ester formation. Like the *N*-(3-dimethylaminopropyl)- *N*--ethylcarbodiimide hydrochloride and *N*-hydroxysuccinimide (EDC/NHS) system for amide formation representing the standard method for zero-length cross-linking between amino and carboxyl group ligation, DMTMM is water soluble and active towards the desired reaction in a water environment. Recently, it was demonstrated that DMTMM provides better yields than the EDC/NHS system, even in the absence of pH control, that is otherwise fundamental for EDC/NHS conjugation. [42] Thus, we envisaged DMTMM as a suitable coupling agen<sup>t</sup> both for pristine gelatin and for the preparation of the hydrogel composed of gelatin and chitosan, since chitosan requires acidic pH to be solubilized.

For preparation of HGel-B, the coupling agent/gelatin ratio had firstly to be optimised, starting from a 10% *w*/*v* gelatin solution in PBS. DMTMM was used in a 5, 10, 20% molar ratio with respect to total gelatin free carboxyl groups (78–80 mmol of free carboxyl groups/g of protein). The 20% DMTMM proved the only condition a ffording thermically stable hydrogels at 37 ◦C.

In particular, HGel-B with a 1:10 or 1:5 cross-linker agen<sup>t</sup> to gelatin to molar ratios were prepared and tested for DHICA/MeDHICA incorporation and release.

HGel-C was obtained by reaction of gelatin with chitosan (≥ 75% deacetylation) in the presence of DMTMM coupling agent. 5:1 and 8:1 gelatin/chitosan *w*/*w* ratio were tested, using 10, 20, or 30% DMTMM molar ratio based on gelatin free carboxyl groups. Best conditions a ffording a stable hydrogel was the 8:1 gelatin/chitosan ratio with 10% of DMTMM.

Both HGel-B and HGel-C proved stable at 37 ◦C, but also at higher temperatures up to 100 ◦C.

#### *3.3. Characterization of the HGel-B and HGel-C Gelatins*

For both materials the swelling profile was determined in water at 25 ◦C. The swelling behavior expressed as swelling degree shown in Figure 4 appears rather di fferent for the two gelatins. In the case of HGel-B the uptake of water is very rapid with respect to what observed for HGel-C. For this latter the swelling degree was found to be 800% after 6 h, whereas that of HGel-B reached 400% at 1 h.

The surface morphology of HGel-B and HGel-C was investigated using scanning electron microscopy (SEM, Figure 5). The two hydrogels showed quite di fferent features. In particular, HGel-B had a rough wrinkled surface with some holes (Figure 5A), while the HGel-C exhibited a smooth membranous phase consisting of dome shaped orifices, microfibrils, and crystallite.

**Figure 4.** (**A**) Swelling degree over the time and (**B**) images (before and after swelling in water) of HGel-B (i) and HGel-C (ii). Reported are the mean ± SD values of three experiments and the polynomial regression that fits the data.

**Figure 5.** SEM images of HGel-B (**A**, scale bar 500 μM) and HGel-C (**B**, scale bar 400 μM).

.

FT-IR spectra taken in the ATR mode for HGel-B and HGel-C are shown in Figure 6. In either cases the characteristic bands of gelatin that is the amide I and amide II stretching at 1637 and 1540 cm-1, respectively, and in the case of HGel-B the NH stretching band (band A) is well apparent [43]. The spectrum of HGel-C is dominated by the intense OH stretching band at around 3300 cm-1 in accord with the higher content of water of this sample while the well defined band at 1087 cm<sup>−</sup><sup>1</sup> (bridge C–O–C stretch) would be evidence for the presence of chitosan [44].

**Figure 6.** Attenuated Total Reflectance (ATR)-IR spectra of HGel-B (**A**) and HGel-C (**B**).

#### *3.4. Loading and Release of DHICA and MeDHICA from HGel-B and HGel-C*

The uptake of DHICA and MeDHICA for HGel-B is shown in Figure 7 panel A. For the HGel-B loaded at 10% *w*/*w* DHICA the uptake into the hydrogel scaffold was around 40% in the first 30 min with an increase up to 90% incorporation at equilibrium (4 h). The loading of MeDHICA 10% *w*/*w* was faster reaching 90% values in 2 h. On the other hand, the HGel-B in the presence of 5% *w*/*w* DHICA reaches 50% incorporation as the maximum value, while no appreciable loading could be detected using lower DHICA or MeDHICA to gelatin ratios (data not shown).

**Figure 7.** Loading of the indole compounds in the HGel-B (panel **A**) or in HGel-C (panel **B**) over time. Reported are the mean ± SD values of three experiments and the polynomial regression that fits the data.

DHICA loading into HGel-C starting from a 10% *w*/*w* DHICA gelatin ratio appears very fast reaching a 50% incorporation in the first 30 min and up to 80% at equilibrium (4 h); on the contrary, MeDHICA incorporation was less effective, featuring only a 50% loading after 4 h (Figure 7, panel B). Again, these different behaviors may be ascribed to the free carboxy group of DHICA, that may give acid-base interactions with chitosan rich in free amino groups. These favorable interactions are not allowed with the esterified carboxy group in MeDHICA.

The release kinetics of indole derivatives was determined in PBS at 37 ◦C (Figure 8, panel A). In the case of 10% *w*/*w* HGel-B, the observed release of DHICA was very smooth with 15% values at 1 h up to 30% at 6 h with medium refreshing every 1 h, and no significant changes for longer times up to 24 h. The release of DHICA is even lower (23% at 6 h) with 5% *w*/*w* HGel-B loaded with the indole, (data not shown). MeDHICA in 10% *w*/*w* HGel-B is released rapidly (57% after 1 h), up to 90% after 4 h with repeated medium refreshing. Much slower release kinetics was observed for DHICA in the case of HGel-C with an initial value of 8% after 1 h which increases up to 54% after 6 h with medium refreshing. On the other hand, MeDHICA is released to 35% after 1 h with a steep increase up to 90% in the first 4 h after repeated medium refreshing (Figure 8, panel B).

In summary (Table 1), loading and release values seem to be dictated mainly by the interactions between the gel and the indoles carboxy groups, and to a lesser extent by the acidic phenolic functionalities. The lower incorporation of DHICA in HGel-A is likely due to the unfavorable interactions of the carboxylate group of DHICA with the carboxylate groups of the acidic aminoacids of gelatin. Such an effect would be decreased with HGel-B and HGel-C, where gelatin carboxy groups are capped by the cross-linking reaction and by the increase of basic amino groups from chitosan, respectively. On the contrary, MeDHICA, missing the ionizable and strongly hydrophilic carboxy group does not experience unfavorable interactions with pristine gelatin (HGel-A) and HGel-B as indicated by the high loading extent, but has a lower affinity for the hydrophilic HGel-C, as indicated also by the higher extent of release with respect to DHICA.

**Figure 8.** Release kinetics of the incorporated indoles from HGel-B (**A**) and HGel-C (**B**) in PBS with refreshing of the medium at 37 ◦C over 6 h. Reported are the mean ± SD values of three experiments and the polynomial regression that fits the data.

**Table 1.** Overview of the percentage values of loading and release of the indole compounds from the gelatin hydrogels investigated.


a Release evaluated in PBS 1× pH 7.4 at room temperature; b Release evaluated in PBS 1× pH 7.4 at 37 ◦C.

#### *3.5. Assessment of the Stability of DHICA in the HGel-B and -C Gelatins*

One of the advantages that should be offered by incorporation of melanin related indoles into a biopolymer like gelatin is the increase of the stability to aerial oxidation in aqueous neutral media of physiological relevance. This issue is more critical in the case of DHICA with respect to MeDHICA given its higher proness to oxidation as shown in our previous studies [36].

Therefore, in subsequent experiments the kinetics of decay of free DHICA in PBS at 37 ◦C was evaluated by HPLC analysis over the time period used for monitoring the release from cross-linked gelatins. HGel-B loaded with DHICA at 10% was immersed in PBS at 37 ◦C and the release was again monitored over 6 h without medium refreshing leading to a release of 10% of the incorporated compound. The decay of free DHICA in the PBS solution at the same concentration estimated based on the incorporation shown in Figure 7, panel A, was monitored by HPLC analysis. Figure 9 (panel A) shows that under these latter conditions DHICA was consumed to 70% over 6 h being oxidized to dark melanin, whereas the indole entrapped into the gelatin and hence released slowly into solution was preserved from oxidation to a remarkable extent (Figure 9, panel B).

#### *3.6. Evaluation of the Antioxidant Properties*

Considering the remarkable antioxidant activity of the compounds under investigation, the antioxidant properties of the DHICA/MeDHICA released in PBS at 37 ◦C from the 10% HGel-B and HGel-C were also evaluated by the DPPH and FRAP assays. Briefly, aliquots of the medium were withdrawn over time and the DPPH decay after 10 min was evaluated spectrophotometrically (Figure 10, panels A and B). A similar procedure was followed to evaluate the ferric reducing antioxidant power of the medium containing the 10% HGel-B and HGel-C (Figure 10, panels C and D). As expected, the reducing potency increased over time as a result of the progressive release of the indoles from the hydrogels further confirming the observed stability of the indole compounds incorporated into the hydrogels.

**Figure 9.** Panel **A**: Decay of free DHICA in the PBS solution monitored by HPLC analysis. Panel **B**: Appearance of the free DHICA solution in PBS (left) vs. the 10% *w*/*w* DHICA/HGel-B containing medium (right) over 6 h. Reported are the mean ± SD values of three experiments.

**Figure 10.** 2,2-diphenyl-1-picrilhydrazyl (DPPH) reduction properties of DHICA or MeDHICA 10% HGel-B (panel **A**) and 10% HGel-C (panel **B**) over time; increase of the absorbance at 593 nm due to the Fe2<sup>+</sup> TPTZ complex induced by DHICA or MeDHICA 10% HGel-B (panel **C**) and 10% HGel-C (panel **D**). Reported are the mean ± SD values of three experiments and the polynomial regression that fits the data.

The antioxidant potency of HGel-C appeared higher than that of HGel-B in either assays. This result can be interpreted considering the higher release of the indoles in terms of rate and extent from HGel-C with respect to HGel-B. Previous studies on the antioxidant effect of the two indoles have shown an around 2.5-fold higher activity for DHICA with respect to MeDHICA in the DPPH assay [36]. This result allows to rationalize the effect on DPPH consumption observed for DHICA and MeDHICA incorporated into HGel-B (28 and 32%, respectively), in the light of the concentration of the indoles actually released in incubation medium after 6 h, that is 9 μM and 19 μM for DHICA and MeDHICA, respectively. This

holds also for HGel-C with a 40 and 55% DPPH consumption for DHICA and MeDHICA, respectively, considering that DHICA and MeDHICA are present in solution at concentrations of 13 μM and 31 μM, respectively. Given the temperature conditions of these experiments HGel-A could not be tested.

The antioxidant potency of DHICA or MeDHICA incorporated into the gelatin hydrogels persisted even after prolonged storage of the material. This issue was demonstrated by DPPH assay applied to indole loaded HGel-B and HGel-C that have been exposed to air over one week. The reducing ability of the materials immersed in the 200 μM DPPH solution at a 0.04 *w*/*v* ratio at 25 ◦C increased over time reaching an almost complete consumption of the reagen<sup>t</sup> after 7 days (Figure 11). The extent of incorporation a ffected the antioxidant e ffects. In agreemen<sup>t</sup> with the higher incorporation of DHICA in HGel-C the antioxidant activity of the loaded gelatin appeared slightly higher than that of MeDHICA/HGel-C, even if the higher solubility of MeDHICA in the assay medium (methanol) would have favored it compared to DHICA. Control experiments proved the stability of the DPPH reagen<sup>t</sup> over the period of the assay. Moreover, the DPPH consumption profile of the loaded gelatins that had not been subjected to storage proved closely similar to that observed for the gelatins exposed to air over one week. For instance, in the case of HGel-B loaded with DHICA, a DPPH consumption of 86% is observed after 7 days. A DPPH consumption of 90% after seven days was obtained also for HGel-A that could be tested under these experimental conditions.

**Figure 11.** Reduction of DPPH by HGel-B ( **A**) or HGel-C (**B**) loaded with DHICA or MeDHICA at 10%. Reported are the mean ± SD values of three experiments.
