Preparation, characterization and evaluation of the anti-inflammatory activity of epichlorohydrin-β-cyclodextrin/curcumin binary systems embedded in a pluronic® /hyaluronate hydrogel

International Journal of Molecular Sciences Article Preparation, Characterization and Evaluation of the Anti-Inflammatory Activity of Epichlorohydrin-β-Cyclodextrin/Curcumin Binary Systems Embedded in a Pluronic®/Hyaluronate Hydrogel Ana-María Fernández-Romero 1, Francesca Maestrelli 2, Sara García-Gil 3, Elena Talero 3, Paola Mura 2, Antonio M. Rabasco 1and María Luisa González-Rodríguez 1,*   Citation: Fernández-Romero, A.-M.; Maestrelli, F.; García-Gil, S.; Talero, E.; Mura, P.; Rabasco, A.M.; González-Rodríguez, M.L. Preparation, Characterization and Evaluation of the Anti-Inflammatory Activity of Epichlorohydrin-β- Cyclodextrin/Curcumin Binary Systems Embedded in a Pluronic®/Hyaluronate Hydrogel. Int. J. Mol. Sci. 2021,22, 13566. https://doi.org/10.3390/ ijms222413566 Academic Editors: Lajos Szente, Antonino Mazzaglia and Francesco Trotta Received: 17 October 2021 Accepted: 14 December 2021 Published: 17 December 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, Universidad de Sevilla, C/Prof. García González 2, 41012 Seville, Spain; anaferr[email protected] (A.-M.F.-R.); [email protected] (A.M.R.) 2Department of Chemistry “Ugo Schiff” (DICUS), University of Florence, via Schiff 6, Sesto Fiorentino, 50019 Florence, Italy; francesca.maestr[email protected] (F.M.); [email protected] (P.M.) 3Department of Pharmacology, Faculty of Pharmacy, Universidad de Sevilla, C/Prof. García González 2, 41012 Seville, Spain; [email protected] (S.G.-G.); etaler[email protected] (E.T.) *Correspondence: malugor[email protected]; Tel.: +34-95-455-6397 Abstract: Curcumin (Cur) is an anti-inflammatory polyphenol that can be complexed with polymeric cyclodextrin (CD) to improve solubility and bioavailability. The aim of the present work was to prepare a CurCD hydrogel to treat inflammatory skin conditions. Epichlorohydrinβ -CD (Epi β CD) was used as polymeric CD. To characterize the binary system, solid-state and in-solution studies were performed. Afterwards, an experimental design was performed to optimize the hydrogel system. Finally, the CurEpi β CD hydrogel system was tested for anti-inflammatory activity using a HaCat psoriasis cell model. Co-grinded Cur/Epi β CD binary system showed a strong interaction and Curcumin solubility was much improved. Its combination with Pluronic ® F-127/hyaluronate hydrogel demonstrated an improvement in release rate and Curcumin permeation. After testing its anti-inflammatory activity, the system showed a significant reduction in IL-6 levels. Hydrogelcontaining CurEpiβCD complex is a great alternative to treat topical inflammatory diseases. Keywords: curcumin; cyclodextrin; epichlorohydrinβ -cyclodextrin; pluronic; hydrogel; inflammatory disease; psoriasis 1. Introduction Curcumin (Cur) is the main active ingredient of turmeric (Curcuma longa). This spice has been used in some Asian countries as traditional medicine for multiple purposes, such as worm treatment, swelling, improving digestion, etc. It is generally considered safe and efficient as treatment [1]. Curcumin has many therapeutic applications due to its anti-inflammatory and antioxidant properties. Its anti-inflammatory activity derives from Curcumin capability to modulate TNF- α , IL-1 β and IL-6 expressions and inhibit COX-2, NF- κ B and MAPK pathways [ 2 ]. Its antioxidant activity derives from its structure. Curcumin is an heptadienone with highly activated central CH 2 prone to reaction. In addition, the phenolic OH is also susceptible to reaction, losing a proton. In both cases, when an oxidant agent reacts with Curcumin, the molecule loses a proton, creating a radical that is stabilized by delocalization [2,3]. Nonetheless, Curcumin has an extremely low solubility and bioavailability that hinders its therapeutic use. Many approaches to overcome this issue have been reported, such as micelles [4], nanoemulsions [5], nanoparticles [6] and cyclodextrins [7]. Cyclodextrins (CDs) are cyclic oligosaccharides consisting of α-1,4 linked glucopyranose units listed as GRAS (Generally Recognized as Safe) excipients by the Food and Drug Int. J. Mol. Sci. 2021,22, 13566. https://doi.org/10.3390/ijms222413566 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021,22, 13566 2 of 26 Administration (FDA) and usually added to food and pharmaceutical products [ 8 ]. CDs are useful for improving low aqueous solubility of lipophilic drugs since they have the ability to form inclusion complexes with hydrophobic drugs in aqueous solutions, hosting them into their hydrophobic cavity. The main driving forces of the complex formation are van der Waals forces and hydrophobic interactions without any covalent bonding, and the solubilizing effect is given by the hydrophilic outer surface of the CD molecules. Natural CDs contain 6, 7 and 8 glucose units and are named α , β and γ cyclodextrin, respectively. β -cyclodextrin ( β CD) is able to form complexes 1:2 mol/mol with Curcumin, thus, improving its solubility and its pharmacological effect [ 9 – 11 ]. On the other hand, since the low solubility and low complexing ability of natural CDs, several derivatives have been synthesized over the years. Tønnesen et al., (2002) in a comparative study with various, CDs showed that the complexes formed were of type 1:1 mol/mol and that randomly methylated β CD and hydroxypropylγ -CD had the highest complexing ability toward Curcumin [ 12 ]. Among the derivatives, the soluble epichlorohydrin polymer derivative of β -CD (Epi β CD) showed a great ability on improving drug solubility [ 13 , 14 ] and oral bioavailability [ 15 , 16 ] after its incorporation in drug formulation. A recent study showed the enhanced antioxidant and antiproliferative activity of curcumin obtained by complexation with a purposely synthesized Epi β CD [ 17 ]. On the other hand, the preparation method of binary systems Cur/CD also has a certain importance in determining the performance of the final product [ 18 ]. With these premises, Epi β CD has been proposed as a great candidate to form complex with Curcumin in treatment of multiple diseases. The aim of the present work was to design a topical formulation to serve as treatment for skin inflammatory diseases, such as psoriasis. Several recent studies have been reported about the combination of cyclodextrins complexes and hydrogel [ 19 – 21 ] also for curcumin administration [ 22 ]. In this case, a gel formulation based on the combined use of Pluronic ® F127 and Epi β CD was developed and used to exploit benefits connected with their inherent properties, namely, beneficial Curcumin stabilization in this polymer and the complexing power and transdermal penetration enhancer of Epi β CD [ 23 ], in order to obtain an optimal local delivery of Curcumin. Finally, to demonstrate the anti-inflammatory activity of the proposed system, a HaCat cell psoriatic model was employed, which was selected due to previous experience in our research laboratory [24]. 2. Results 2.1. Phase Solubility Studies In order to evaluate the complexing and solubilizing abilities of Epi β CD towards Curcumin, phase solubility studies were performed in phosphate buffer pH 5. This pH was selected as a compromise between Curcumin stability and skin pH [ 25 ]. Curcumin solubility increased linearly with increasing Epi β CD concentrations (Figure 1a), indicating the formation of a soluble inclusion complex. The stability constant, calculated by taking the CD repeating unit as its molecular weight, was 4.5 times higher than with the parent CD. This value (6.186 M −1 ) is higher than those obtained by Tønnesen et al., (2002) with other derivatives [ 12 ]. The superior complexing and solubilizing efficiency of polymeric CD derivatives can be attributed to their higher aqueous solubility, but also to their polymeric structure, which allowed an efficient cooperation of adjacent CD cavities for interaction with the drug [ 26 ]. Since no deviations from linearity were observed in phase solubility diagrams, the formation of higher order complexes in the CD concentration range studied can be excluded. Taking into account these results, binary systems were prepared at 1:10 w/wCur:EpiβCD ratio. 2.2. Solid-State Studies In order to evaluate the effect of the preparation techniques, different binary systems of Curcumin with Epi β CD were prepared and submitted to the DSC analysis. As shown in Figure 1b, Curcumin presents a melting peak at 179.72 ◦ C with a melting enthalpy of 95.42 J/g, which is in accordance with bibliography [ 27 ], while Epi β CD showed an Int. J. Mol. Sci. 2021,22, 13566 3 of 26 amorphous profile with a broad endothermic band from 40 to 120 ◦ C associated with water loss. Curcumin melting peak was almost unchanged in the physical mixture (PM) with Epi β CD, indicating the absence of solid-state interactions between the components. On the contrary, the drug melting peak intensity gradually decreased as a function of the technique used, with the trend PM = kneading (KN) < colyophilizated (COL) < coevaporated (COE), and it completely disappeared in the co-grinding (GR). This indicated that the co-grinding procedure was the most successful to induce effective solid-state interactions between the components, resulting in complete Curcumin amorphization and/or complexation. Solid-state interactions between the components were further investigated by XRD and FTIR analysis. The XRD spectra of pure drug and Epi β CD, the PM and different binary systems samples are presented in Figure 1c. Pure Curcumin showed sharp diffraction peaks at 2 θ between 9 and 29 [ 27 ] indicating its crystalline nature, while the pattern of Epi β CD was typical of a completely amorphous substance. Diffraction patterns of the PM, KN and COL products almost corresponded to the superimposition of those of the plain components, where the peaks at 9, 15 and 18 ◦ were still present, confirming a limited interaction between the components. COE showed, instead, a marked reduction of the drug diffraction peaks intensity and GR sample presented a completely amorphous profile. FT- IR analysis further confirmed the above results (Figure 1d). In fact, the peaks of Curcumin at 1627–1602 cm −1 attributed (more detail, see Figure S1) to the stretching of the benzene ring of Curcumin [ 28 ], covered all the binary systems by the broad band of the CD, and appeared shifted at 1583 cm−1 in the GR, thus, confirming the deeper interaction obtained with this preparation technique. Figure 1. Cont. Int. J. Mol. Sci. 2021,22, 13566 4 of 26 Figure 1. Phase solubility study and solid-state characterization of Cur/Epi β CD binary systems prepared with different methods. ( a ) Phase solubility study of Curcumin and Epi β CD. Thermograms by Differential Scanning Calorimetry, ( b ) X—ray diffraction spectra, ( c ) and FT—IR spectra, ( d ) Abbreviations: GR, co—grinding; COE, co—evaporation; KN, kneading; COL, co—lyophilization. 2.3. Dissolution Rate Studies The higher effectiveness of GR in inducing drug–CD interactions was further corroborated by results of dissolution studies. The dissolution profiles are reported in Figure 2, where it is visible that all binary systems with Epi β CD exhibited improved dissolution properties with respect to drug alone (Curcumin), but a clear influence of the preparation method on the performance of the final product can be noticed. In particular, it is evident that GR system exhibited the highest dissolution rate (85 mg/L of dissolved Curcumin after 5 min) and the highest solubility (100 mg/L) followed by COE product (20 mg/L of dissolved Curcumin after 10 min) and then by KN and COL products. The overall results Int. J. Mol. Sci. 2021,22, 13566 5 of 26 indicated that the GR was the most efficacious preparation technique to obtain complete drug amorphization and/or complex formation, and maximum improvement of drug dissolution rate. Moreover, GR is a fast, highly efficient, convenient, versatile, sustainable and eco-friendly solvent-free method for obtainment of drug–cyclodextrin binary systems [ 29 ], and thus the co-ground system was chosen as the most effective product to introduce in the final formulation. Figure 2. Dissolution rate of Cur/Epi β CD binary systems (GR ; COE ; KN ; COL ; PM ) compared with plain Cur . 2.4. Optimization of Gel Formulation The aim of the present work was to design a topical formulation to serve as treatment for skin inflammatory diseases, such as psoriasis. In this case, a hydrogel was selected as hydrophilic vehicle. To formulate it, four polymers were chosen: Pluronic ® F-127, Carbopol ® 940, chitosan and sodium hyaluronate. Pluronic ® F127 is a poloxamer able form rigid gels at different concentrations and temperatures, being the temperature dependent of the concentration of the polymer. In order for it to reticulate at 32 ◦ C (skin temperature), Pluronic ® F-127 concentration should be at least of 15% w/v[ 30 ]. Contrary, this concentration is too high for Carbopol ® 940, chitosan and sodium hyaluronate. For this reason, Pluronic®F-127 was kept constant throughout the experimental design. An orthogonal Taguchi L9 array was performed and the effects of Curcumin concentration (added as GR with Epi β CD), type of polymer, polymer concentration and ratio of Pluronic ® /polymer over % of drug released at 6 h, % of permeated drug at 6 h, pH and apparent viscosity of the gel, were analyzed. Results of each experiment and their experimental conditions are shown in Table 1. In this case, permeation varied from 0.0491 to 0.3394%, drug release from 0.6183 to 3.2814%, gel viscosity from 0.0972 to 3.8998 Pa·s, and pH from 4 to 5. After analyzing all results, statistical significance of these factors on the evaluated responses was clarified with ANOM (Analysis of Means) of the main factors on each response (data not shown). Moreover, results were analyzed by ANOVA (Analysis of Variance) (Table 2) and contributions of each response were described in a Pareto chart (Figure 3). This type of chart facilitates information about how much each parameter contributes to the response and the sign of each contribution. In this case, all responses showed significant results. Int. J. Mol. Sci. 2021,22, 13566 6 of 26 Table 1. Experimental matrix for the Taguchi L9 array and results obtained. Results show the amount of Curcumin permeated (Permeation, %) and released (Release, %), gel apparent viscosity (Viscosity, Pa · s) and pH. [Pol] represents the concentration of different polymers expressed in % w/v; Ratio represents the proportion in weight between Pluronic ® and the other polymer; [Cur] represents the Curcumin concentration expressed in mM; Tp represents the type of polymer as a non-numerical factor, choosing between chitosan (Chito), Carbopol®(Carb) or sodium hyaluronate (Hyal). Run [Pol] Ratio [Cur] Tp Permeation ±SD Release ±SD Viscosity ±SD pH ±SD 1 1 20:80 0.2 Chito 0.0963 ±0.0044 1.25 ±0.12 0.0698 ±0.0017 4 ±0 2 1 80:20 0.3 Carb 0.2070 ±0.0089 3.28 ±0.56 2.473 ±0.045 4.2 ±0.3 3 1 50:50 0.4 Hyal 0.075 ±0.014 0.930 ±0.098 0.114 ±0.011 5 ±0 4 2 20:80 0.3 Hyal 0.0678 ±0.0044 0.9957 ±0.0084 0.12 ±0.11 4.7 ±0.3 5 2 80:20 0.4 Chito 0.212 ±0.011 2.452 ±0.067 0.562 ±0.077 4 ±0 6 2 50:50 0.2 Carb 0.140 ±0.019 2.25 ±0.12 3.90 ±0.17 4.5 ±0 7 3 20:80 0.4 Carb 0.0491 ±0.0071 0.618 ±0.043 4.98 ±0.12 4.2 ±0.3 8 3 80:20 0.2 Hyal 0.339 ±0.062 5.45 ±0.23 0.0972 ±0.0095 5 ±0 9 3 50:50 0.3 Chito 0.1099 ±0.00004 1.44 ±0.13 0.64 ±0.043 4.5 ±0 Table 2. Summarized results of analysis of variance (ANOVA) of experimental results for apparent gel viscosity (Y 1 ), pH (Y 2 ), % Curcumin permeation at 6 h (Y 3 ), % Curcumin release at 6h (Y 4 ). X1: ([Pol] −1 vs. +1) ; X2: Ratio ( − 1 vs. +1); X3: [Cur] ( − 1 vs. +1); X4: Tp ( − 1 vs. +1); X5: [Pol] (+1, −1 vs. 0). X6: Tp (−1, +1 vs. 0); X7: Ratio (−1, +1 vs. 0); X8: [Cur] (−1, +1 vs. 0). Factors Viscosity (Y1) pH (Y2) Permeation (Y3) Release (Y4) F-Value p-Value F-Value p-Value F-Value p-Value F-Value p-Value X1 417.9 <0.001 * 2 0.191 9.1 0.015 * 28.9 <0.001 * X2 12.1 0.007 * 8 0.020 * 7.991 0.020 * 21.28 0.001 * X3 112.9 <0.001 * 0.5 0.497 36.47 <0.001 * 169.7 <0.001 * X4 39.11 <0.001 * 40.5 <0.001 * 2.641 0.139 34.36 <0.001 * X5 9.371 0.014 * 0.667 0.435 0.2954 0.600 5.714 0.041 * X6 6612 <0.001 * 4.167 0.072 2.443 0.152 0.1107 0.747 X7 188.8 <0.001 * 0.6667 0.435 202.1 <0.001 * 509.8 <0.001 * X8 157.1 <0.001 * 0.1667 0.693 4.303 0.068 5.326 0.46 * * indicates which values are of statistical significance Apparent viscosity of the system was affected by all factors studied (Figure 3a). The polymer concentration had a positive effect on viscosity, indicating that at higher concentration, viscosity will also be higher. The type of polymer had an interesting effect on this parameter. The effect was negative when chitosan was compared with hyaluronate but it was positive when compared to Carbopol ® , which indicated that Carbopol ® increased the viscosity more than the other two polymers. As expected, the ratio also affected, in all cases in a positive way, indicating that the more polymer was added, the higher the viscosity. Curcumin concentration also had a curious behavior, as it was negative when compared with the medium level (0.3 mM of Curcumin) but positive when extreme levels were compared. Regarding pH, only two factors contributed significantly to its behavior: the proportion of Pluronic ® /polymer and the type of polymer used to create the hydrogel (Figure 3b). Interestingly, only the ratios 20:80 and 50:50 affected the pH, increasing it with polymer diminution. Probably, at 80:20 ratio, the amount of polymer compared to the amount of Pluronic ® is not enough to change the pH. Type of polymer also affects the pH, which is consistent with previous knowledge. Pluronic ® solution has a pH of 6–7, while sodium Int. J. Mol. Sci. 2021,22, 13566 7 of 26 hyaluronate and chitosan had a pH of 7 and below 6, respectively. Logically, the addition of chitosan will lower the pH while sodium hyaluronate will increase it. Carbopol ® , on the other hand, needs to be neutralized to reticulate, leading to a pH of 7 that will not affect significantly Pluronic®pH. Figure 3. Pareto charts of half effects corresponding to: ( a ) gel viscosity; ( b ) pH; ( c ) in vitro Curcumin permeation; ( d ) in vitro Curcumin release. X1: [Pol] ( − 1 vs. +1); X2: Ratio ( − 1 vs. +1); X3: [Cur] ( − 1 vs. +1); X4: Tp ( − 1 vs. +1); X5: [Pol] (+1, −1 vs. 0). X6: Tp (−1, +1 vs. 0); X7: Ratio (−1, +1 vs. 0); X8: [Cur] (−1, +1 vs. 0). In the case of in vitro permeation (Figure 3c), three actors affected the result: the polymer concentration, the Curcumin concentration and the ratio of Pluronic ® /polymer. Curcumin concentration affected negatively the amount of Curcumin able to permeate skin, when low and high levels were compared; however, when both of them were compared with the middle level (0.3 mM), the effect was neutral, so, the permeation was favorable until this drug concentration. As Figure 3c shows, the proportion of Pluronic ® /polymer affects in a positive way, indicating that the higher the Pluronic ® content, more Curcumin can permeate through the membrane. The same behavior was obtained with the polymer concentration (the amount of Curcumin permeated increased as the polymer concentration increased). The in vitro release behavior of Curcumin was similar to that of permeation except for the contribution of the type of polymer. In this case, the use of sodium hyaluronate Int. J. Mol. Sci. 2021,22, 13566 8 of 26 provided more Curcumin released than the addition of chitosan. Carbopol ® did not affect the release (Figure 3d). After analyzing ANOM and ANOVA data, optimized formulation was proposed. The main objective of this array was to maximize permeation, release and viscosity, while keeping pH close to 5.5. However, as can be seen in the Pareto charts (Figure 3), some factors affecting Curcumin release and permeation are in opposition to those affecting viscosity. This indicates that in order to maximize viscosity, permeation and release will be hindered. For that reason, permeation and release were chosen as more important and favorable responses than viscosity. As a result, optimized gel components were as follows: Curcumin concentration was kept at 0.2 mM, sodium hyaluronate 3% w/v was chosen as polymer and concentration and the ratio of Pluronic ® /polymer was set at 80:20. 2.5. Characterization Studies Once the optimized composition of the gel was obtained, physicochemical characterization was carried out as was mentioned in Section 4.9. 2.5.1. Apparent Viscosity, Density and pH Apparentviscositywasmeasured onlyto the hydrogelformulationcontaining CurEpi β CD GR. The result was 0.135 Pa · s at 62.65 s. Additionally, representation of shear rate and viscosity was also considered. On it, viscosity diminished with shear rate, indicating that this hydrogel is a non-Newtonian fluid. Regarding pH and density, they were 6 and 0.979 g/mL, respectively. 2.5.2. Gelation Temperature and Storage and Loss Moduli Pluronic ® F-127 is a poloxamer appreciated for its ability to jellify with temperature. However, the addition of other components to its solution can modify the gelling temperature. For this reason, different solutions of Pluronic ® were tested: Pluronic ® 17% w/vwater solution (PluW), Pluronic 17% w/vwater/ethanol solution (PluWE), PluWE containing Epi β CD (PluWE-Epi), optimized gel containing only Epi β CD (Epi-Gel) and optimized gel loaded with CurEpi β CD GR binary system (CurEpi β CD-Gel). Additionally, sodium hyaluronate (Hyal) was also analyzed for comparation purposes. To study changes in the gelation temperature, two tests were taken: inversion tube and the intersection between storage (G 0 ) and loss (G”) moduli. To obtain the gelling temperature via G 0 and G” crossover, both moduli should be equal. If G 0 = G”, loss tangent (tan δ ) equals 1. Consequently, the range in which this tangent is 1 or near 1 was selected as the gelling temperature. Table 3shows the results of both assays. As can be seen, both tests resulted in different gelatin temperatures. For PluW, inversion test resulted in gelation above 35 ◦ C and returned to solution state after 44 ◦ C. The range obtained by tan ( δ ) calculation indicated a narrower range (38.05–41.06 ◦ C). In both cases, the range is in accordance with bibliography [ 31 ]. Comparing PluWE and PluW, gelling temperature was lower (15.37 ◦ C) and the system returned to a liquid state at a lower temperature (23.09 ◦ C). The addition of Epi β CD to PluWE resulted in an increase in the gelling temperature in comparison with PluWE, a narrower range was obtained. Optimized gel with and without Curcumin (CurEpi β CD-gel and Epi-Gel, respectively) showed a similar temperature range to that of PluWE, indicating that the addition of sodium hyaluronate can increase gelling temperature. As can be seen in Figure S2, after 5 min at 20 ◦ C, CurEpi β CD-gel and Epi-Gel did not test positive for inversion test, indicating that even after a change in the structure occurs, the optimized hydrogel is not able to form structures as rigid as PluW. Sodium hyaluronate was also analyzed as a control sample. This component does not have a gelling temperature; however, changes in viscosity with temperature are well known. For this reason, it was also analyzed. Interestingly, an increase in tan ( δ ) until 1 was detected, followed by a plateau (range mentioned in Table 3), and, finally, a decrease in the parameter. Int. J. Mol. Sci. 2021,22, 13566 9 of 26 Table 3. Gelling temperatures obtained by inversion test and loss tangent (tan δ ) calculation. Samples are: Pluronic ® 17% w/vwater solution (PluW), Pluronic 17% w/vwater/ethanol solution (PluWE), PluWE containing Epi β CD (Plu-Epi), optimized gel containing only Epi β CD (Epi-Gel) and optimized gel loaded with CurEpi β CD GR binary system (CurEpi β CD-Gel). Inversion test was considered positive when gel remained at the bottom of the tube for 30 s after heating. Sample Inversion Test (◦C) Tan (δ) 25 28 32 35 38 41 44 47 50 Range (◦C) Hyal + + + + + + + + + 51.36–57.35 PluW - - - - + + + - - 38.05–41.06 PluWE - - - - - - - - - 15.34–23.09 PluWE-Epi - - - - - - - - - 24 Epi-Gel - - - - - - - - - 15.34–23.09 CurEpiβCD-Gel - - - - - - - - - 15.34–27.77 Regarding loss and storage moduli, the same samples were analyzed. Results are showed in Figure 4. PluW exhibited similar G 0 and G 00 that increased with temperature until 40 ◦ C, when G 00 rapidly decreased while G 0 decreased slowly. Both moduli increased again until 65 ◦ C but G 0 was higher than G 00 . The addition of ethanol resulted in a slower increase of G 0 and G 00 , with an initial plateau at 15–20 ◦ C were G 00 was higher that G 0 . At gelation temperature, G 0 surpassed G 00 . A similar behavior was noted in PluWE-Epi sample, Epi-Gel and CurEpiβCD-Gel (Figure 4c–e) performed similarly to PluWE. Sodium hyaluronate presented a different behavior than Pluronic ® . In this case, G 0 decreased while G 00 was almost constant and reach a plateau between 50 and 58 ◦ C, after which G0abruptly increased. G00 also increased, but not as abruptly as G0. 2.5.3. Curcumin Content Once the optimized hydrogel was prepared, the amount of Curcumin extracted was quantified. The result showed that a 95.54% of Curcumin can be extracted from the hydrogel. 2.5.4. In Vitro Release and Curcumin Permeation The optimized hydrogel was then assessed in terms of in vitro Curcumin release and permeation. As comparison, a solution of GR CurEpi β CD was prepared following the same procedure as described in Section 4.7. As previous results showed (Table 2), the percentage of Curcumin released and permeated after 6 h from the hydrogels in the optimization step, indicating slow processes. For that reason, in this section, both studies were performed over the course of 72 h. As can be seen in Figure 5a, Curcumin release from the solution was completely opposed to that from the gel. At 8 h, the amount of Curcumin released from the CurEpi β CD solution was about 30%, while the hydrogel released only a very low drug amount. Interestingly, later in the experiment, the hydrogel released its content slowly but regularly, while the solution exhibited a degradation process after 48 h. During the in vitro permeation studies, Curcumin behavior was completely different (Figure 5b). In this case, Curcumin permeated from the CurEpiBCD solution was only about 20% after 48 h and remained almost unchanged at 72 h. In the case of the hydrogel, the amount of Curcumin permeated was similar to that of the solution until 8 h of experiment. Afterwards, the optimized system allowed a strong increase of the amount of permeated Curcumin until 48 h (almost 60%); however, after this point, degradation overcame permeation and the amount of Curcumin permeated at 72 h, decreased (40%). Int. J. Mol. Sci. 2021,22, 13566 16 of 26 In vitro permeation of Curcumin was also sustained over 72 h. In this case, the CurEpi β CD solution and hydrogel started with slow-rate permeation. Nonetheless, the solution reached its maximum at 24 h and, subsequently, started degrading, although not as markedly as in the release assay. Epi β CD is known for its ability to enhance drug permeation, as Gidwani et al., (2017) stated. In their studies, when tretinoin was combined with this polymeric CD and lipid nanostructures, the amount of drug permeated almost doubled [ 13 ]. In some permeation assays, raw Curcumin have demonstrated an extremely low permeation [ 5 , 52 ]. In comparison, this complex represents a huge improvement in Curcumin permeation. Regarding the hydrogel, an unexpected degradation occurred at 72 h, in which 15% of Curcumin was lost. This phenomenon will be further studied as Curcumin is more stable at acidic pH [ 50 ] and the experiment was performed in the absence of light. It should be noted that, even though the system degraded over time, Curcumin permeation was greater in the hydrogel than in the solution. Two main reasons behind this are the capability of Pluronic ® to form supramolecular structures with CD (more about this topic will be addressed later) and to the occlusive nature of the hydrogel. The stability of the system was also tested. Three parameters were considered: pH, Curcumin antioxidant activity and Curcumin content. pH only decreased one unit after 21 days of experiment in both hydrogel systems (CurEpi β CD-Gel and Em-Gel), indicating a change in the structure of the gel. Considering Cur content, results showed that CurEpi β CD-Sol had better stability than the hydrogel. This can be attributed to gel composition, as it is the sole difference between both samples. pH could also contribute to Curcumin degradation, as it was higher in the hydrogel until day 21. However, degradation rate did not reduce when pH changed from 6 to 5, rendering this hypothesis as weak. Another hypothesis was that Pluronic ® or sodium hyaluronate are destabilizing the system. The poloxamer used in this work is a co-block polymer able to create supramolecular structures with CD. These structures were first introduced by Harada [ 53 ] and, since then, they have been well studied. Multiple of these studies include a variety of β CD derivatives [54–57] , including polymeric CD such as Epi β CD [ 58 ], as well as Epi α CD [ 59 ]. In all cases, these supramolecular complexes formed stable systems with different drugs, enhancing their release and/or permeation profiles with the exception of DIMEB ((2,6-di-O-methyl)-βCD) , which is capable of rupturing Pluronic ® micelles, a key part of this supramolecular structures [ 57 ]. On the other hand, sodium hyaluronate is a polysaccharide with high water binding capacity that can be useful in skin diseases treatment [ 60 ]. It has also been used in combination with CD, either in gel or to functionalize the CD [ 44 , 61 – 63 ], including combination with a poloxamer [ 44 ]. Again, there are no reports of sodium hyaluronate having a negative interaction with CD, including when it was combined with poloxamer. From this investigation, we can deduce that neither Pluronic ® nor sodium hyaluronate destabilize CD system, either alone or in combination. In addition to this information, some types of Pluronic ® are able to stabilize Curcumin by entrapping the polyphenol in its lipophilic core. The conclusion that can be extracted is that Curcumin might not be suitable for these supramolecular structures in the conditions proposed, although they definitely benefit from it in terms of release and permeation. Another positive effect of the CD was its capability to retain Curcumin antioxidant capability. On the one hand, the solution showed a consistent EC 50 over the course of the experiment, with minimal changes. On the other hand, Curcumin in the hydrogel increased its antioxidant capability at days 15 and 21, to reduce it again. Analyzing Curcumin content and antioxidant capability simultaneously, it is clear that, in both systems, the antioxidant effect cannot be attributed entirely to Curcumin. Many studies have identified Curcumin degradation products as ferulic acid, vanillin, ferulic aldehyde and feruloyl methane [ 3 , 63 , 64 ]. These products have in common the hydroxyl methoxyphenyl groups, that are key to the antioxidant activity of Curcumin [ 64 ]. Moreover, some authors even stated that the pharmacological effect of Curcumin can be attributed to its degradation products [ 65 ]. This would explain why, even when Curcumin Int. J. Mol. Sci. 2021,22, 13566 17 of 26 presence in the system was diminishing, EC 50 was kept unaltered (solution) or even increased (hydrogel). Psoriasis is a chronic inflammatory skin disorder characterized by hyperproliferation of keratinocytes. Currently, treatments for this disease include topical and systemic agents, phototherapy and biologics. Regarding topical therapies, they are applied for mild forms of psoriasis, skin irritation being the most frequent adverse effect [ 24 ]. Therefore, new topical approaches for mild psoriasis with few side effects are required. In this regard, the use of natural products with anti-inflammatory properties is gaining considerable attention for psoriasis treatment. Previous in vitro and in vivo studies have reported the anti-psoriatic properties of Curcumin via suppression of pro-inflammatory cytokines, down-regulation of NF-kB and MAPK signaling pathways and upregulation of involucrin and filaggrin, proteins that regulate skin barrier function [ 66 – 68 ]. In addition, several formulations aimed to enhance bioavailability, solubility and physicochemical properties of Curcumin have been evaluated in different animal models of psoriasis, demonstrating an improvement of its anti-psoriatic effect [ 69 – 71 ]. In the present study, we demonstrated for the first time the anti-inflammatory properties of CurEpi β CD-Gel in an in vitro model of psoriasis, established using HaCaT keratinocytes treated with TNF- α . This cytokine is released by keratinocytes and plays a main role in the pathogenesis of psoriasis since it stimulates the production of other pro-inflammatory cytokines such as IL-6. This factor is a multifunctional cytokine that promotes epidermal keratinocyte hyperplasia and stimulates the differentiation of IL-17-producing T cells [ 72 ]. High plasma levels of this cytokine and its receptor have been detected in patients with psoriasis, demonstrating an important role in the pathogenesis of this disease [ 73 ]. For this reason, IL-6 inhibitors could be a promising strategy for inhibiting the inflammatory response in psoriasis. Our data evidenced that pre-treatment with CurEpi β CD-Gel significantly reduced the production of the pro-inflammatory cytokine IL-6 in TNF- α -stimulated HaCaT keratinocytes. These results are in agreement with previous studies that reported the capacity of Curcumin to reduce IL-6 levels in both HaCaT cells and psoriatic animals [ 65 , 67 , 73 ]. All these findings suggest that Curcumin, administered in a well-tolerated topical formulation, could be a novel and safe approach for the treatment of patients with mild psoriasis. 4. Materials and Methods 4.1. Materials Soluble β -cyclodextrin-epichlorohydrin polymer (Epi β CD) MW 4500 cyclodextrin content (estimated 50–70%) residual β -cyclodextrin content max 1% was purchased from Cyclolab R&D Ltd. (Budapest, Hungary). Pluronic ® F-127 and high molecular weight chitosan were purchased from Sigma-Aldrich Co. (Barcelona, Spain). Sodium hyaluronate was purchased from Fagron Iberica (Barcelona, Spain). Carbopol ® 940 was purchased from Escuder (Barcelona, Spain). Solvents used for chromatographic analysis were HPLC quality. All other chemicals were of analytical degree. 4.2. Phase Solubility An excess amount of Curcumin (10 mg) was added to 10 mL of phosphate buffer ( pH 5 ) containing increasing amounts of Epi β CD. The concentration range for Epi β CD was 0–13.33 mM. The prepared samples in sealed glass containers protected from light were first sonicated 60 min in ultrasonic bath (Eurosonic 44 ultrasonic bath, Wilten Woltil de Meern, Utrecht, The Netherlands) and then magnetically stirred at constant temperature ( 25.0 ±0.5 ◦C ) until equilibrium (72 h). Aliquots were filtered (0.45 µ m Millipore membrane filter, Darmstadt, Germany) and drug concentration was spectrophotometrically determined at 425 nm (Shimadzu 1601 UV-VIS spectrophotometer, Milan, Italy S.R.L). Preliminary studies showed that the presence of Epi β CD did not interfere with Curcumin absorbance at this wavelength. Each experiment was repeated at least 3 times (coefficient of variation, CV < 2%). The apparent stability constants of the complexes (Ks) were calculated Int. J. Mol. Sci. 2021,22, 13566 18 of 26 from the straight portion of the phase-solubility diagrams using the following equation according to Higuchi and Connors [74]: Ks =slope S0×(1−slope)(1) where S0is the aqueous solubility of Curcumin (mM) in the absence of CD. 4.3. Preparation of Interaction Products Cur/Epi β CD 1:10 w/wphysical mixture (PM) was prepared by adding accurately weighed components in a mortar and then mixing manually with a spatula for 13 min. Solid drug/CD interaction products were prepared by using different techniques: coevaporated product (COE) was prepared by dissolving Curcumin in ethanol and adding a water solution of Epi β CD, then evaporating the solvent in a rotary evaporator at 70 ◦C and drying the resulting products in a vacuum desiccator with silica gel for 24 h to remove traces of solvents; for co-grinding (GR) product, the physical mixture was placed in a high-energy vibration micromill (MM200 Retsch GmbH, Haan, Germany) for 30 min at 24 Hz; kneading (KN) product was obtained pounding in a mortar the physical mixture, adding a minimum amount of a mixture water/ethanol 1:1 v/v, kneading thoroughly with a pestle to obtain a homogeneous slurry and continuing until the solvent was completely removed, then drying the products in a vacuum desiccator with silica gel for 24 h to remove traces of solvent; co-lyophilized product (COL) was prepared by dissolving Curcumin in the minimum amount of ethanol and adding a water solution of Epi β CD, then the solution was freeze-dried at − 50 ◦ C and 1.3 · 10 −2 mm Hg. The 75–150 µ m sieve granulometric fraction was then used for the following studies. 4.4. Differential Scanning Calorimetry (DSC) Thermograms of the individual components or drug/Epi β CD products were obtained with a Mettler TA4000 calorimeter equipped with a DSC25 cell (Columbus, OH, USA). The samples, weighed with a Mettler M3 Microbalance (5–10 mg), were scanned in Al pans pierced with a perforated lid at 10 ◦C/min, from 30 to 150 ◦C, under static air. 4.5. Powder X-ray Diffractometry (PXRD) A Bruker D8-advance (Billerica, MA, USA) X-ray diffractometer was employed for the diffraction patterns of drug and drug/Epi β CD binary systems. The parameters were: Cu K α radiation, voltage 40 kV, current 55 mA and 2 θ over a 5–45 ◦ range at a scan rate of 0.05 ◦ /s. A Sol-X ® solid-state Si (Li) was used as a detector, and C/Ni Goebel-Spiegel mirrors in the incident beam were used as a monochromator; 1.0 mm divergence, 0.2 scatter, and 0.1 for the receiving slits were used. All samples were examined at room temperature. 4.6. Fourier Transform Infrared Spectrometry (FT-IR) FT-IR spectra were performed by a Shimadzu IRPrestige21 apparatus (Shimadzu Corporation, Kyoto, Japan) on KBr tablets. Spectra were recorded in the 400–4000 cm −1 range (10 scans, resolution 4 cm−1). Samples were examined after dispersion in Nujol. 4.7. Dissolution Rate Studies In vitro dissolution studies of the prepared solid products (PM, GR, COE, COL, KN) and Curcumin were performed using a modified dispersed amount method [ 14 ]. Briefly, a sample amount corresponding to 10 mg of Curcumin was added to 20 mL of phosphate buffer (pH 5) at 37 ◦ C and gently stirred (50 rpm) by a magnetic stirrer. At predetermined time intervals, aliquots of 3 mL were withdrawn with a syringe-filter (0.45 µ m Millipore membrane filter) and immediately replaced with the same volume of fresh dissolution medium, thermostated at the same temperature. The drug amount in the samples was spectrophotometrically assayed as described previously. A correction was applied for Int. J. Mol. Sci. 2021,22, 13566 19 of 26 the cumulative dilution. All experiments were repeated three times for each sample (CV < 2.5%). 4.8. Optimization of Gel Formulation The main objective of the present work was to obtain a topical formulation of CurEpi β CD complex. For this, an experimental design–based screening study was realized in order to select the best composition of formulation. Four different polymers were evaluated: poloxamer in the form of Pluronic ® F-127, high molecular weight chitosan, carbomer in the form of Carbopol ® 940 and sodium hyaluronate. Due to its ability to jellify with temperature, Pluronic®was used as the base for all gel formulations. In order to optimize gel characteristics, an experimental design was performed using DOEpack 2000 as software (Dayton, OH, USA). The designated factors were: Curcumin concentration, type of polymer, Pluronic ® /polymer ratio and polymer concentration. These factors and their levels are collected in Table 4. Taguchi L9 array was selected as design model. Responses analyzed were: percentage of drug released after 6 h, percentage of drug permeated at 6 h, apparent viscosity and pH of the formulation. Table 4. Factors (F1, F2, F3 and F4) and their levels selected for the screening study. Experiments were realized by using a Taguchi L9 array. (−1): low level; (0): middle level; (+1): high level. Name Factor Level −1 0 +1 F1 Cur concentration 0.2 mM 0.3 mM 0.4 mM F2 Polymer type Chitosan Carbopol®Hyaluronate F3 Pluronic®/polymer ratio 20:80 80:20 50:50 F4 Polymer concentration 1% p/v 2% p/v 3% p/v Gel preparation was as follows: Cur/Epi β CD binary system was dissolved in 3.6 mL of Pluronic ® 23.6% w/v. After 30 min, 1.4 mL of absolute ethanol was added, ensuring complete dissolution. Finally, when dissolution was completed, secondary polymer was added in the amount required by the array. The mixture was stirred and left to rest at 4 ◦ C for 24 h. Afterwards, different responses were analyzed. 4.9. Characterization Studies 4.9.1. Apparent Viscosity Apparent viscosity was obtained with a rheometer (Discovery HR-3 hybrid rheometer, TA Instruments, New Castle, DE, USA). All rheological measurements were conducted by a parallel plate geometry (40 mm diameter) at 32 ◦ C. Samples were analyzed monitoring the viscosity ( η , Pa · s) as a function of the shear rate in the range of 10–1000 s −1 . The gap of the assay was 400 µm. Apparent viscosity was selected at 62.65 s. 4.9.2. Gelation Temperature In order to comprehend the interaction between the components of the gel, the gelation temperature of the system was studied by inversion method and storage–loss moduli crossover. For the inversion test, briefly, 1 mL of sample was placed in a glass tube and heated in thermostatic bath. Samples were heated at a rate of 1 ◦ C/min from 25 to 50 ◦ C. Every minute, the tube was turned for 30 s. The test was positive if gel sample stayed at the bottom of the tube. For ease of readability, data was displayed only at 25, 28, 32, 35, 41, 44, 47 and 50 ◦ C, and assay was performed in duplicates. Formulation studied were: water solution of Pluronic ® F-127 17% w/v(PlusW), Pluronic ® F-127 17% w/vwater/ethanol solution (PluWE), Epi β CD dissolved in Pluronic ® F-127 water/ethanol solution (Epi-Plu), Int. J. Mol. Sci. 2021,22, 13566 20 of 26 optimized gel containing only Epi β CD (Epi-Gel) and Cur-Epi β CD-loaded optimized gel (CurEpiβCD-Gel). Samples were prepared following the procedure in Section 4.8. For the storage–loss moduli crossover, moduli were analyzed following conditions in Section 4.9.3. Afterwards, loss tangent (tan ( δ )) was calculated and the temperature or range of temperatures were tan ( δ ) was between 0.8 and 1.2 were selected as gelling temperature . 4.9.3. Storage and Loss Moduli To further analyze the optimized formula and the interaction between gel components, storage and loss moduli were calculated. For this matter, a rheometer (Discovery HR-3 hybrid rheometer, TA Instruments, New Castle, DE, USA) was employed. Samples were analyzed by monitoring storage (G 0 ) and loss (G”) moduli as a function of temperature in the range of 15–65 ◦ C. Frequency was kept at 1 Hz and oscillatory strain at 0.01%. Temperature was increased at 2 ◦C/min. Samples were analyzed in duplicates. 4.9.4. In Vitro Release Studies The release behavior of Curcumin from the hydrogel was evaluated by dialysis, as other studies have already performed [ 75 ]. Briefly, 1 g of each formulation was placed inside a dialysis bag (Spectra ® /Por 12–14 kD MWCO membranes), previously rinsed with PBS pH 7.4/ethanol/Tween ® 80 mixture in ratio 74.5:25:0.5 (% v/v). The system was placed in 100 mL of the same medium under gentle stirring at room temperature. Samples ( 1 mL ) were taken at scheduled times (0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 24, 48 and 72 h) and the same amount of fresh medium was added. Curcumin content was quantified using HPLC technique described in [33]. Each sample was analyzed in duplicate. 4.9.5. In Vitro Permeation of Curcumin To evaluate the amount of Curcumin able to penetrate skin, an in vitro permeation study was performed. In this case, a skin model was used instead of animal skin following Mura et al.’s (2014) protocol with slight modifications [ 76 ]. Cellulose membranes with a pore width of 0.2 µ m was soaked in lauryl alcohol for 15 min at 58 ◦ C. Afterwards, the excess of lauryl alcohol was dried with filter paper and weighed to ensure an increase in weight of 90–110%. Once dried, membranes were placed in Franz-type diffusion cells filled with PBS pH 5.5/ethanol/Tween ® 80 74.5:25:0.5 (% v/v) ensuring no air bubbles were present, mimicking skin conditions. Temperature was kept at 32 ◦ C. Samples ( 0.5 mL ) were withdrawn at scheduled times (0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 24, 48 and 72 h) and the same amount of fresh medium was added. Curcumin content was analyzed by HPLC as described in [33]. Duplicates of each sample were made. 4.9.6. pH This parameter was analyzed by dipping pH strips in the samples for 30 s. Results were read as by supplier instructions. 4.9.7. Density Hydrogel density was measured with a pycnometer, which was zeroed in a precision balance at 25 ◦ C. Afterwards, gel formulation was added to the top avoiding air bubble formation and the system was closed with its corresponding cap, letting the excess gel to overflow. Finally, the pycnometer was cleaned and its weight measured. To obtain density, the following formula was applied: D=Formulation weight −Pycnometer weight Pycnometer volume (2) 4.9.8. Curcumin Extraction from Hydrogel The amount of Curcumin that can be extracted from the optimized hydrogel was analyzed. In brief, an amount of hydrogel was accurately weighed, and a sufficient amount Int. J. Mol. Sci. 2021,22, 13566 21 of 26 of chloroform was added. The mixture was vigorously stirred with the aid of a vortex mixer and the liquid was separated from the matrix. The sequence was repeated until chloroform was colorless. Afterwards, the extracted chloroform sample was centrifuged at 1000 rpm for 10 min at 4 ◦ C to separate chloroform from any hydrogel residue and the organic phase was transferred to a round bottom flask to evaporate. Once all chloroform was evaporated, the Curcumin residue was dissolved in ethanol and analyzed by UV/Vis spectrophotometry using an Agilent 8453 UV-visible spectrophotometer (Agilent Technologies, Budapest, Hungary). A volume of 100 µ L sample was diluted up to 5 mL with an acetonitrile/acetic acid 2% 1:1 v/vmixture, and absorbance was measured at 425 nm. 4.10. Stability Studies Chemical stability of the optimized hydrogel and Curcumin embedded in gel was tested over the course of 84 days. pH, Curcumin content and Curcumin oxidation were analyzed. For comparison purposes, solution of CurEpiβCD complex and hydrogel without complex (Em-gel) were also considered. Analyses were made in duplicate. pH was measured as indicated in Section 4.9.6. 4.10.1. Oxidation Study Curcumin is an antioxidant agent capable of scavenging free radicals from both lipidic and aqueous phases [ 77 ]. In this study, the antioxidant capability of Curcumin was determined by using ABTS (2,2 0 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as oxidative agent, following the methodology previously reported by Pisoschi and Negulescu [ 78 ]. To create the oxidizing radical (ABTS •+ ), 2.98 mM of ABTS and 0.98 mM of K 2 S 2 O 8 (potassium persulfate) were dissolved in purified water, forcing the loss of an electron from the nitrogen atom of ABTS. This dark blue solution is reduced by an antioxidant, losing its color in the process as the positively charged nitrogen is neutralized by a hydrogen from the antioxidant [ 77 ]. The decrease in the absorbance value was monitored, and Trolox (1.05 mg/mL in ethanol absolute) was chosen as standard antioxidant. After obtaining the corresponding working solutions, serial dilutions were prepared into 96-well plates, adding 100 µ L of working solutions and 100 µ L of fresh ABTS, leaving to react for 6 min. In the case of Trolox, further dilution was needed, adding only 10 µ L on working sample and 90 µ L prior to fresh ABTS. Finally, absorbance at 734 nm was measured with a plate reader (Synergy HT Plate Reader, Winooski, VT, USA). The antioxidant activity was defined as EC 50 , which indicated the equivalent concentration of antioxidant needed to reduce the initial concentration of ABTS to 50%. Antioxidant capability was expressed as Trolox equivalent (EC50 compound/EC50Trolox) [33]. 4.10.2. Curcumin Content In order to analyze Curcumin content, 100 µ L samples were taken directly from the hydrogel and analyzed following the procedure described in Section 4.9.8. 4.11. Cell Studies 4.11.1. Cell Viability Assay Resazurin assay was used for determining the viability of HaCaT cells upon exposure to the optimized hydrogel. In this colorimetric assay, viable cells can reduce resazurin into the resorufin product, which is pink [ 79 ]. For this reason, not only the optimized gel was analyzed, but also each one of its components. In all cases, samples followed the same procedure described in Section 4.7, substituting hydrogel by water when needed. Therefore, formulations studied were: solution of CurEpi β CD complex in water (CurEpi β CD-Sol), optimized gel formulation containing only Epi β CD (Epi β CD-Gel), optimized gel formulation containing only Curcumin (Cur-Gel), unloaded optimized gel (Em-Gel) and CurEpi β CD- loaded optimized hydrogel (CurEpiβCD-Gel). Firstly, HaCaT cells were seeded into 96-well plates in growth medium at 10 4 cells/well for 24 h to ensure the adherence. Then, cells were incubated in a humidified atmosphere Int. J. Mol. Sci. 2021,22, 13566 22 of 26 of 5% CO 2 at 37 ◦ C. After that, cells were treated with different formulations at final concentration range of 0.625–20 µ M in ethanol 0.2% v/vand the cytotoxicity was measured after 48 h of incubation. Thereafter, cells were washed once with PBS pH 7.4, and 150 µL resazurin (20 µ g/mL in medium) were added to each well. Plates were incubated for 4 h, and, finally, the absorbance was determined at 540 nm and 620 nm in a microplate spectrophotometer (Sinergy HT, Biotek ® , Bad Friedrichshall, Germany). Cell viability was expressed as a percentage respect to the untreated cells. 4.11.2. Determination of IL-6 Production HaCaT cells were seeded in 6-well plates (2.5 × 10 5 cells/well). After 24 h, cells were washed twice (PBS pH 7.4, 4 ◦ C) and medium containing CurEpi β CD-Gel (2.5, 5 and 10 µM ) was added for 24 h. Then, cells were stimulated with TNF- α (10 ng/mL) except for control group, for another 24 h. Thereafter, supernatant fluids were collected and stored at − 80 ◦ C until measurements. Commercial enzyme-linked immunosorbent assay (ELISA) kit (Diaclone GEN-PROBE, Besançon, France) was used to quantify IL-6 according to the manufacturer’s protocol. 4.11.3. Statistical Analysis All data in the figures are exhibited as arithmetic means with their standard error of means. Statistical analysis was carried out using GraphPad Prism version 5.00 software (GraphPad Software, Inc., San Diego, CA, USA). The Shapiro–Wilk test was used to verify the normality of the data. Student’s t-test was used to compare between the two control groups (control vs. TNF- α ). Statistical differences between multiple groups were compared by one-way ANOVA followed by Bonferroni’s post hoc test. A p-value less than 0.05 was considered as statistically significant. 5. Conclusions Epi β CD is a relatively new polymeric CD that is not yet widely studied. It is known for its capability to enhance solubility and permeation of poorly soluble molecules. For this reason, it was selected to improve Curcumin characteristics. In this study, this binary system was embedded in a hydrogel and its anti-inflammatory activity was tested. CurEpi β CD binary system was characterized and the most suitable complexing method was determined. The results showed a high improved affinity between Curcumin and Epi β CD compared with other β CD and an impressive increase in dissolution rate with the GR method. Afterwards, a hydrogel system was optimized and characterized. Although the system demonstrated a reduced stability over time, permeation and release of Curcumin were highly improved. In addition, the optimized gel demonstrated anti-inflammatory activity. With these findings, we can conclude that CurEpi β CD binary system embedded in a Pluronic ® /hyaluronate hydrogel is a promising treatment for skin inflammatory diseases. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/ijms222413566/s1. Author Contributions: Conceptualization: A.-M.F.-R., F.M. and M.L.G.-R.; methodology: A.-M.F.-R., F.M., S.G.-G., E.T. and M.L.G.-R.; validation: A.-M.F.-R., F.M. and M.L.G.-R.; formal analysis: F.M., E.T. and M.L.G.-R.; investigation: A.-M.F.-R., F.M., E.T., S.G.-G., P.M., A.M.R. and M.L.G.-R.; resources: F.M., E.T., P.M., A.M.R. and M.L.G.-R.; writing—original draft preparation: A.-M.F.-R., F.M., S.G.-G., E.T. and M.L.G.-R.; writing—review and editing: F.M., E.T., P.M., A.M.R. and M.L.G.-R.; supervision: F.M., E.T., P.M., A.M.R. and M.L.G.-R.; project administration: M.L.G.-R.; funding acquisition: E.T., A.M.R. and M.L.G.-R. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Junta de Andalucía (Grant numbers CTS214 and CTS658), CITIUS Services, the VI Plan Propio of the Universidad de Sevilla. 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