pharmaceutics Article Polymeric Nanoparticles and Chitosan Gel Loading Ketorolac Tromethamine to Alleviate Pain Associated with Condyloma Acuminata during the Pre- and Post-Ablation Salima El Moussaoui 1,† , Ismael Abo-Horan 1,† , Lyda Halbaut 1 , Cristina Alonso 2 , Lluïsa Coderch 2 , María Luisa Garduño-Ramírez 3 , Beatriz Clares 4,5, * , José Luis Soriano 4 , Ana Cristina Calpena 1,5 , Francisco Fernández-Campos 6,‡ and Mireia Mallandrich 1,5,‡ 1 Department of Pharmacy, Pharmaceutical Technology and Physical-Chemistry, Faculty of Pharmacy and Food Sciences, University of Barcelona, Av. Joan XXIII 27-31, 08028 Barcelona, Spain; selmouel9@alumnes.ub.edu (S.E.M.); iabohora7@alumnes.ub.edu (I.A.-H.); halbaut@ub.edu (L.H.); anacalpena@ub.edu (A.C.C.); mireia.mallandrich@ub.edu (M.M.) 2 Institute of Advanced Chemistry of Catalonia-CSIC (IQAC-CSIC), 18-26 Jordi Girona St., 08034 Barcelona, Spain; cristina.alonso@iqac.csic.es (C.A.); luisa.coderch@iqac.csic.es (L.C.) 3 Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Cuernavaca 62209, Mexico; lgarduno@uaem.mx 4  Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Granada,  18071 Granada, Spain; jlsoriano@correo.ugr.es 5 Institut de Nanociència i Nanotecnologia IN2UB, Universitat de Barcelona, 08028 Barcelona, Spain Citation: El Moussaoui, S.; 6 Reig-Jofre Laboratories, Av. de les Flors s/n, 08970 Sant Joan Despí, Spain; ffernandez@reigjofre.com Abo-Horan, I.; Halbaut, L.; Alonso, * Correspondence: beatrizclares@ugr.es C.; Coderch, L.; Garduño-Ramírez, † These authors contribute equally to this work. M.L.; Clares, B.; Soriano, J.L.; Calpena, ‡ These authors contribute equally to this work. A.C.; Fernández-Campos, F.; et al. Polymeric Nanoparticles and Abstract: This study describes the preparation and evaluation of two formulations, a hydrogel and a Chitosan Gel Loading Ketorolac nanostructured system, containing ketorolac tromethamine as an anti-inflammatory agent for the Tromethamine to Alleviate Pain local therapy against the inflammatory process derived from the surgical excision of Condyloma Associated with Condyloma acuminata. Both formulations were physicochemically characterized. In vitro release profiles show Acuminata during the Pre- and Post-Ablation. Pharmaceutics 2021, 13, that the nanoparticles release 92% ± 2.3 of the total ketorolac tromethamine encapsulated, while the 1784. https://doi.org/10.3390/ chitosan gel releases 18.6% ± 0.2. The ex vivo permeation and distribution through human skin were pharmaceutics13111784 also assayed and was observed how the main amount of ketorolac tromethamine is retained in the epidermis. In vivo studies were accomplished to evaluate the anti-inflammatory efficacy in mice Academic Editor: Jesus Perez-Gil which also involved the histological analysis to confirm the in vivo results. The nanoparticles present a significantly higher anti-inflammatory efficacy than chitosan gel. The tolerability of developed Received: 31 August 2021 formulations was assessed by monitoring the biomechanical properties of the skin before and after Accepted: 20 October 2021 application of both formulations. No statistical differences in trans-epidermal water loss and skin Published: 25 October 2021 hydration with respect to the basal values were observed and the formulations exhibited higher anti-inflammatory activity compared to a reference ketotorlac tromethamine solution. Therefore, it Publisher’s Note: MDPI stays neutral can be concluded that both formulations can be proposed as outstanding candidates for offering a with regard to jurisdictional claims in local anti-inflammatory therapeutical tool with potential clinical application. published maps and institutional affil- iations. Keywords: PLGA nanoparticles; chitosan gel; Condyloma acuminata; ketorolac tromethamine; anti-inflammatory; topical delivery Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article distributed under the terms and Sexually transmitted infections (STIs) represent nowadays a significant public health conditions of the Creative Commons problem [1]. One of the most common STIs is the human papillomavirus (HPV) infection [2], Attribution (CC BY) license (https:// being Condyloma acuminata (CA) its clinical manifestation [3]. To date, more than 200 HPV creativecommons.org/licenses/by/ strains have been sequenced, of which 40 are known to cause genital infections [2]. There 4.0/). are two types of HPV in terms of the oncogenic risk, of which the virus subtypes 6 and 11, Pharmaceutics 2021, 13, 1784. https://doi.org/10.3390/pharmaceutics13111784 https://www.mdpi.com/journal/pharmaceutics Pharmaceutics 2021, 13, 1784 2 of 19 are classified as of low oncogenic risk, and are those which are mainly responsible 90% of cases. It is noteworthy that subtypes 16 and 18 in contrast are deemed as high-risk strains, due to their oncogenic potential [4]. Patients aged 20 to 39 account for more than 70% of CA infections [5]. Children [6] and pregnant women can also be affected [7]. Even though asymptomatic Condyloma acuminata is common, certain cases might be accompanied by itching, pain, and bleeding [8]. The treatment strategy could be ablative or by using topical procedures based on the localization, size and morphology of the lesions [2]. In some cases, such as intra-anal involvement, the ablative strategy might be the only option [5]. However, Condyloma acuminate is known for its recurrence, particularly if HPV 11 has been diagnosed [9]. In previous studies, our research group investigated the use of topical ketorolac tromethamine (KT) as a good alternative for managing the inflammatory process following the excision of anogenital warts with promising results by loading the drug in alginate-based hydrogel [4]. KT is a non-steroidal anti-inflammatory drug (NSAID) belonging to the Aryl acetic acid derivatives. It is used for the relief of postoperative pain and the management of inflammation [10]. Stemming from the nanoparticles (NPs) having the property of sustained drug release, the drug can reach the skin over a prolonged time period and can be retained in the skin at the desirable concentration [11]. In this context poly (D, L-lactic-co-glycolic acid) (PLGA) has demonstrated immense potential among the matrixes used for NPs owing to its biocompatible and biodegradable properties, among others [12]. Chitosan (CTS), a natural polymer, is a promising candidate for controlled drug deliv- ery systems and wound healing [13]. It being a strong candidate is due to their properties such as biocompatibility, biodegradability, antimicrobial activity, low immunogenicity, mucoadhesivity, hydrophilicity and ease modification [14]. Additionally, CTS can also provide antifungal properties and hemostatic properties as well as the anti-inflammatory effect [13,14]. All these properties are of great interest for the management and care of the areas treated post-surgically, thus positively influencing the reduction of complications such as bleeding and infections and this favors the wound healing process [15]. Skin is the largest human organ which accounts for roughly 10% of an average total body mass. The main function of skin is to act as a barrier preventing moisture and the loss of nutrients and protecting against ultraviolet (UV) radiation action, chemicals, allergens and microorganisms [16]. Transdermal Drug Delivery (TDD) offers various benefits over other traditional drug delivery routes such as it is painless and non-invasive with fewer side effects, and more patient compliance among other benefits [17]. Stratum corneum (SC) is the outer layer of the epidermis with a crucial barrier function. Many strategies have been investigated to overcome this barrier and facilitate the drug permeation [18,19]. However, overcoming the SC to reach the deeper layers of skin or the systemic circulation is not always appropriate. In certain cases, such as Condyloma acuminata, where the infection occurs in the epidermis, taking advantage of the drug accumulation in this skin layer would be a great strategy for local treatment. Taking all the above into account, in this work, we formulated KT into a PLGA nanostructured system as a vehicle for preventing the inflammatory process during the surgical excision of warts. The KT-NPs may be easily sprayable onto warts without the need to contact them. At the same time, we formulated KT into a CTS-based hydrogel as a vehicle for managing the local inflammatory process stemming from the post-operative stage in the surgical excision of warts taking advantage of the mucoadhesive and regenerative properties of CTS. Both formulations were characterized and evaluated in vitro, ex vivo and in vivo. 2. Materials and Methods 2.1. Materials KT, polyvinyl alcohol (PVA) with 90% hydrolyzation, ethyl acetate (EA), lactic acid so- lution 85%, phosphate buffer solution (PBS) pH 7.6, gentamicin sulphate and bovine serum albumin were purchased from Sigma-Aldrich (Barcelona, Spain). CTS was obtained from Pharmaceutics 2021, 13, 1784 3 of 19 FagronIberica (Terrassa, Spain). PLGA (Resomer® RG 503) was acquired from Boehringer Ingelheim (Ingelheim, Germany). Adhesive tapes were obtained from D-squame, (Cuderm Co., Dallas, TX, USA). Double distiller water was obtained from a Milli-Q1 Gradinet A10 system apparatus (Millipore Iberica S.A.U., Madrid, Spain). All the other chemicals and reagents used in the study were all of analytical grade (Panreac, Castellar del Vallès, Spain). 2.2. Preparation of Formulations 2.2.1. Preparation of CTS Gel CTS gel was prepared weighting an accurate amount of CTS and dispersing the polymer in 1% lactic acid solution under continuous stirring so as to obtain a final con- centration of 3% (w/v) until dissolution. Then, the mixture was allowed to swell in the fridge overnight. Appropriate amount of KT (2%; w/v) was added once the gel was swollen and gently homogenized. Table S1 in Supplementary Materials depicts the critical process parameters and Table S2 shows the Quality Target Product Profiles. 2.2.2. Preparation of NPs NPs were prepared using the double emulsion-solvent evaporation method as de- scribed previously with modification [20,21]. The NPs were optimized by a two-level full factorial design for 3 factors in standard order, which led to 8 candidate formulations (Table S3). In brief, 250 µL of the inner aqueous phase pH 2.0 (W1 ) consisting of a PVA 2% (w/v) and KT (80 mg/mL) solution were added to 1 mL of the oily phase (O), which con- tained 90 mg of PLGA in ethyl acetate. A probe Vibra-Cell® sonicator (Sonics and Materials, Inc., Newtown, CT, USA) was used to homogenize this mixture for 20 s in two 10 s cycles at 50 watts and 30% amplitude, resulting in a primary emulsion (W1 /O), in which 2 mL of external aqueous phase (W2 ), PVA 2% (w/v), was added drop-wise and subjected to a second sonication process for 1.5 min, 10 s cycles (50 watts and 30% amplitude) to produce the double emulsion (W1 /O/W2 ). Finally, a volume of 5 mL of PVA 0.3% (w/v) was added to the double emulsion before the organic solvent was evaporated under reduced pressure (Bücchi B-480, Flawil, Switzerland) for about 15 min at 36 ◦ C and 74 mbar vacuum. For comparison studies, blank NPs were also fabricated. Table S1 shows the critical process parameters and Table S2 shows the Quality Target Product Profiles. 2.3. Physical Characterization of Formulations 2.3.1. Characterization of CTS Gel pH Study The pH was recorded in triplicate by pH meter micropH 2000 (Crison Instruments SA, Alella, Spain) at room temperature. Swelling and Degradation Tests The swelling ratio (SR) was assayed based on a gravimetric method. For this task, known amounts of dried hydrogel were immersed in PBS (pH = 5.5) for 3 h at 32 ◦ C. Samples were removed and weighed (Wt ) after blotting the surface water at pre-established times. The PBS uptake was measured in triplicate. The swelling ratio was determined using the following equation: Ws − Wd SR = (1) Wd where Ws is the weight of the swollen hydrogel at various times and Wd is the weight of dried hydrogel. The gel degradation rate was evaluated in terms of percentage of weight loss (WL). Briefly, known amounts of dried hydrogel were immersed in PBS (pH = 5.5) at 32 ◦ C for Pharmaceutics 2021, 13, 1784 4 of 19 3.75 h. Samples (n = 3) were then dried and weighed at pre-established time intervals. The following equation was used to calculate the WL: Wi − Wd WL(%) = 100% (2) Wi where Wi denotes the initial weight of the hydrogel and Wd denotes the weight of the hydrogel at various times. Both, the swelling and the degradation were fitted to mathematical models. The best fit was chosen based on the determination coefficient value (R2 ). Morphological Studies Scanning Electron Microscopy (SEM) was used to visualize the internal structure of developed hydrogel in a JEOL J-7100F (JEOL Inc., Peabody, MA, USA). Samples were carbon-coated with an Emitech K950X coater (Quorum Technologies Ltd., Kent, UK). Rheological Study The rheological properties of CTS gel were analyzed in a rotational HaakeRheo Stress 1 rheometer (Thermo Fisher Scientific, Karlsruhe, Germany) with cone-plate geometry (Haake C60/2◦ Ti, 60 mm diameter, 0.105 mm cone-plate gap). The shear profile was evaluated following a ramp-up stage (0–100 s−1 for 3 min), a constant shear rate stage (100 s−1 for 1 min), and a ramp-down stage (100–0 s−1 for 3 min). All measurements were taken in triplicate at room temperature and the steady-state viscosity was recorded at 100 s−1 (constant shear stretch). Experimental results were then fitted to different mathematical models by regression analysis [4]. 2.3.2. Characterization of the NPs Physicochemical Characterization Both, the mean particle size (Zave), the polydispersity index (PI) and the zeta potential (ZP) of the KT-NPs were determined by dynamic light scattering (DLS). The characteri- zation was carried outby diluting the samples 1/20 in Milli-Q water and measuring by a Zetasizer Nano Zs (Malvern Instruments, Malvern, UK) at 25 ◦ C with disposable cuvettes. The pH of the resulting colloidal suspension of NPs was measured by pH metermicrop H 2000 (Crison Instruments SA, Alella, Spain). Transmission electron microscopy (TEM) technology was used to evaluate the struc- ture of KT-NPs with a JEOL 1010 (JEOL Inc., Tokyo, Japan) at 80 Kv. Uranyl acetate (1% w/v) was used as a staining solution to visualize the NPs. The image processing program Image J (1.53 e) was utilized to measure the particle size starting from the produced TEM image. Encapsulation Efficiency An indirect method was used to determine the KT encapsulated in the NPs based on the measuring of the non-entrapped KT in the dispersion medium. For this purpose, fresh KT-NPs were centrifuged for 15 min at 14,000 rpm (Sigma 301K, Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany). Subsequently, free KT was separated using Ultracel YM-100 filters (Millipore, Bedford, MA, USA) and collected in the filtered solution. The determination of the KT was performed using a high-performance liquid chro- matography (HPLC) methodology which had been previously validated. The encapsula- tion efficiency (EE) was calculated using Equation (3). Total amount of KT − free KT EE% = (3) Total amount of KT Pharmaceutics 2021, 13, 1784 5 of 19 2.4. Stability Studies The physical stability of KT-NPs stored at 4 ◦ C was studied for 3 months by monitoring potential changes in Zave, PI and EE. The stability of the KT-CTS gel was visually evaluated. The formulation was stored at room temperature for 3 months. 2.5. In Vitro Drug Release Study To investigate the KT release from NPs and the CTS gel, vertical Franz diffusion cells (Vidrafoc, Barcelona, Spain) with a diffusion area of 2.54 cm2 were used. A dialysis membrane (MWCO 12,000–14,000 Da., Medicell International Ltd., London, UK) was placed between the donor and receptor chambers in the case of NPs and nitrocellulose 0.45 µm pore size (Millipore Merck, Madrid, Spain) for CTS gel. The receptor medium was PBS solution pH 7.4. The system was kept at 32 ± 0.5 ◦ C under continuous stirring, maintaining sink conditions throughout the course of the experiment. 200 µL of KT-NPs (n = 6) and 200 mg of CTS gel (n = 6) were put into the donor compartment. Aliquots of 300 µL were removed at predetermined intervals of up to 9 h and replaced with an equivalent volume of tempered receptor medium. The experimental data were fitted to first order, using the Prism software, v. 5 (GraphPad Software, Inc., San Diego, CA, USA). 2.6. Ex Vivo Permeation Studies The penetration and permeation of KT in and through skin was evaluated ex vivo using human skin. The skin was obtained from abdominoplasties (protocol code N◦ 001 approved on 20/01/2016 by Bioethics Committee of the Barcelona-SCIAS Hospital); which was frozen and kept at −20 ± 5 ◦ C until the date of the permeation test. Then, specimens were dermatomed at 500 µm pieces. The human skin pieces were allowed to thaw at room temperature and then were placed between the donor and receptor chambers of Franz static diffusion cells (Lara-Spiral, Courtenon, France). The Franz cells consisted of a 3 mL receptor volume and 1.86 cm2 diffusion area. The experimental system used PBS (pH 7.6), gentamicin sulphate 0.04% (w/v) to prevent skin degradation [22] and bovine serum albumin 1% (w/v) as receptor fluid at 32 ◦ C continuously stirred at 500 rpm by a magnetic bar allowing sink conditions throughout the test. 10 µL samples (either NPs or CTS gel) were placed on the donor chamber. At the end of the experiment (24 h), skin membranes were demounted, and the remaining formulation amounts were recovered by a swab. Equally, the receptor fluid was collected to measure the amount of KT distributed at each skin layer [9]. KT was extracted from the skin samples by sonication in a water:methanol (1:1, v/v) medium for 20 min. KT was determined by HPLC. The extracted amount of KT is expressed as µg/cm2 and percentage (%) of the applied dose. Control cells were also used to evaluate potential interferences in the sample analysis of the receptor fluid or skin samples using unloaded gel and blank NPs. 2.7. HPLC Analysis KT in samples was determined using HPLC methodology validated in terms of linearity, accuracy, and precision according to ICH Q2 (R1) validation guidelines. The HPLC system consisted of a Waters 717 plus Autosampler with a detector Waters 2487 (Waters, Milford, MA, USA); a C18 column YMC-Pack Pro, 250 × 4.6 mm, 5 µm (YMC Co., LTD, Kyoto, Japan) was used with a flow rate of 1 mL/min at isocratic conditions. The mobile phase was carried out with acidified water (glacial acetic acid 1.65%) and acetonitrile with 0.065% of triethylamine at the elution conditions of 1:1 v/v. The volume of injection was 10 µL. The wavelength was set at 314 nm. Pharmaceutics 2021, 13, 1784 6 of 19 2.8. In Vivo Anti-Inflammatory Efficacy Evaluation 2.8.1. TPA-Induced Ear Oedema in Mice Male Swiss CD-1 mice (20–25 g) purchased from Circulo AND, S.A. de C.V. (Coyacan D.F., Mexico) were kept under standard animal housing conditions in a 12 h dark-light cycle with access to food and water ad libitum. This study was approved by the animal research ethical committee of the Vivarium at University of Morelos (protocol code BIO-UAEM 03:2019 date of approval February 2019). The protocol was carried out as previously reported by Domínguez-Villegas et al. [23]. A 12- O-detradecanoylphorbol-13-acetate (TPA) solution of 50 mg/mL in ethanol was used to induce mouse ear oedema inflammation. Animals were divided into three groups of three specimens each one (n = 3). 5 µL of TPA solution was applied to both sides of the right ear simultaneously with 100 mg of formulation (KT-CTS gel to group 1 and KT-NPs to group 2) as well as 5 µL of ethanol to both sides of the left ear. The control group was treated with an equal amount of KT solubilized in acetone applied to both sides of the right ear and 5 µL of acetone to both sides of the left ear. The animals were then sacrificed by cervical dislocation 4 h after treatment and 7 mm circular sections were cut from the left and right ears. These were weighted accurately. The anti-inflammatory activity expressed as a percentage of inhibition of the inflammatory process was calculated according to the Equation (4): Weight control ear − Weight treated ear   Inhibition(%) = × 100 (4) Weight control ear 2.8.2. Histological Analysis Histological analysis of the circular sections was conducted after TPA-induced oedema in mice to evaluate the anti-inflammatory efficacy by observing whether immunologic cells were present in the tissues. Hence, the circular ear sections were fixed in 4% formaldehyde solution and then embedded in paraffin to obtain histological cuts which were stained with hematoxylin-eosin and observed by light microscopy. 2.9. In Vivo Tolerance Study The study was carried out with 10 healthy-skinned female participants ranging in age from 21 to 64 years old. The study was authorized by the University of Barcelona’s Ethics Committee (IRB00003099, approved on 20 March 2018), which followed the Declaration of Helsinki’s guidelines. All the participants provided written informed consent forms. Volunteers were asked to not use cosmetic or another skin care products on the test areas during two days before the study. The subjects stayed in the test room for at least 30 min before taking the measurements. Several readings were collected from the flexor side of the left forearm before applying the formulation (baseline readings). Then, readings were collected newly just after application of a uniform layer (0.1 mL/cm2 ) of formulation at1 h after application and at 2 h. The total quantity of water lost (TEWL) through the skin was measured using a Tewameter® TM 300. (Courage-Khazaka electronic GmbH, Cologne, Germany). The hydration of the stratum corneum (SCH) was determined using a Corneometer® CM 825. (Courage-Khazaka electronic GmbH, Cologne, Germany). All measurements were taken in accordance with published procedures. 2.10. Statistical Analysis All statistical analyses were performed using the Prism® software, v. 5 (GraphPad Software Inc., San Diego, CA, USA). T-student tests were performed on the in vitro release and the ex vivo permeation studies to compare both formulations. Furthermore, analysis of variance (ANOVA) was conducted on the in vivo anti-inflammatory efficacy study to compare the formulations, as well as in the in vivo tolerance study to compare the biomechanical properties if the skin over time to the basal values. The level of statistical significance was set at p < 0.05. 3.1. Characterization of Formulations 3.1.1. Characterization of CTS Gel Pharmaceutics 2021, 13, 1784 7 of 19 The resulting CTS gel was transparent and extremely vis The pH of the CTS gel was 4.4 ± 0.1 and 5.5 ± 0.1 for KT-CTS g 3. Results Figure 1 shows the swelling and degradation profiles of 3.1. Characterization of Formulations gel. 3.1.1. Similar profiles Characterization of CTS Gelwere observed for both gels, indicating th gel The doesresulting not CTS gel affect was transparent the gel and extremelyNevertheless, structure. viscous with poor flowability. significant The pH of the CTS gel was 4.4 ± 0.1 and 5.5 ± 0.1 for KT-CTS gel. found between Figure the KT-CTS 1 shows the swelling gel profiles and degradation and blankof KT-CTSCTSgel andgel inCTSterms blank gel. Similar profiles were observed for both gels, indicating that the addition of KT in the swelling study. gel does not affect the gelItstructure. can beNevertheless, seen that the swelling significant is a linear statistical differences were and zero-order found between the kinetic KT-CTS gel with a lag-time and blank CTS gel inof 10ofmin. terms This the initial weightformulation on the swelling study. It can be seen that the swelling is a linear and steady process following a to 4.5-fold zero-order kineticits weight. with a lag-time of 10 min. This formulation was able to absorb PBS up to 4.5-fold its weight. Figure 1. Swelling and degradation processes of KT-CTS gel. Modelling graphic expressed as Figure 1. Swelling and degradation processes of KT-CTS gel. Modell mean ± SD (n = 3). Panel (a) shows the KT-CTS gel; and panel (b) the blank gel without KT (CTS gel). ± SD (n =3). Panel (a) shows the KT-CTS gel; and panel (b) the blank After 3.75 h ± 0.22 h, the CTS gel had utterly degraded in the medium (PBS). The weight loss fitted to a one-phase exponential decay. Table 1 shows the fitted parameters After 3.75 h ± 0.22 h, the CTS gel had utterly degraded values for both processes. weight loss Table 1. Best-fit fitted values to a and of the swelling one-phase exponential degradation processes of CTS gel withdecay. and withoutTable KT. 1s values for both processes. Parameter Swelling Degradation Y0 (g) 1 3.681 ± 0.050 * 2.952 ± 0.168 KT-CTS gel K2 0.006 ± 0.001 0.005 ± 0.002 Table 1. Best-fit values of the swelling and degradation processes of R2 0.9033 0.9322 Y0 (g) 1 3.526 ± 0.062 3.164 ± 0.183 CTS gel Parameter Swelling K2 0.008 ± 0.001 0.006 ± 0.002 (blank) R2 Y0 (g) 1 0.9085 0.9341 3.681 ± 0.050 * 1 Y = initial weight; 2 K = degradation constant (g/min) or swelling constant (min−1 ); * statistical differences 0 KT-CTS between gelgel and blank CTS gel (significanceKlevel set at p < 0.05). the KT-CTS 2 0.006 ± 0.001 R2 0.9033 CTS gel Y0 (g) 1 3.526 ± 0.062 The surface of the dried KT-CTS gel was evaluated by SEM. The gel compact and dense appearance, with smooth surface which pre (blank) K2 0.008 ± 0.001 0.006 ± 0.002 cro-irregularities where the R2 water and KT may0.9085 be interposed within 0.9341the po ture (Figure 2). 1 Y0 = initial weight; 2 K = degradation constant (g/min) or swelling constant (min−1); * statistical Pharmaceutics 2021, 13, 1784 8 of 19 differences between the KT-CTS gel and blank CTS gel (significance level set at p < 0.05). The surface of the dried KT-CTS gel was evaluated by SEM. The gel presented compact and ofdense The surface appearance, the dried withevaluated KT-CTS gel was smoothby surface SEM. Thewhich presented gel presented a m cro-irregularities compact where the water and dense appearance, and KT with smooth maywhich surface be interposed within the polymer struc presented micro-irregularities where the water ture (Figure 2).and KT may be interposed within the polymer structure (Figure 2). Figure 2. SEM images of the dried discs of KT-CTS gel. (a) Magnification 1000×; and ( tion 5000×; the scale bar is 20 µm length. Figure2.2.SEM Figure SEM images images of the of the drieddried discsdiscs of KT-CTS of KT-CTS gel. (a)gel. (a) Magnification Magnification 1000×; 1000×; and and (b) magnifica (b) magnifica- tionTo tion 5000predict 5000×; ×; the the thescale scale ispotential barbar is 20 20 µmµm topical usage of KT-CTS gel, the rheological stu length. length. ried out. The mathematical model that best fitted experimental flow data was To Topredict thethe potential topical usageusage of KT-CTS gel, the rheological study was carried model 2predict (rmathematical potential = 0.999), model confirming topical of KT-CTS a shear-thinning gel, the rheological behavior of study was car KT-CTS gel. In out. ried The that best fitted experimental flow data was out. The mathematical model that best fitted experimental flow data was the Casson model the Casso 2 depicted model (rthe (r = 0.999),2 = rheological confirming profile a shear-thinning 0.999), confirming which behaviorrevealed a shear-thinning of KT-CTS that behaviorgel. the of gel has In Figure KT-CTS a pseudoplas 3 is depicted gel. In Figure 3 i the with rheological a viscosity profile of which 1.431 revealed Pas that ±0.0004. the gel has a pseudoplastic behavior with a depicted the rheological profile which revealed that the gel has a pseudoplastic behavio viscosity of 1.431 Pas ± 0.0004. with a viscosity of 1.431 Pas ±0.0004. Figure 3. Rheological profile of KT-CTS gel. Blue curve represents the formulation viscosity. Re curve represents the shear stress of the formulation. Figure 3. Rheological profile of KT-CTS gel. Blue curve represents the formulation viscosity. Red Figure 3. Rheological curve represents the shear profile of formulation. stress of the KT-CTS gel. Blue curve represents the formulation v curve represents the shear stress of the formulation. Pharmaceutics 2021, 13, x FOR PEER REVIEW Pharmaceutics 2021, 13, 1784 9 of 19 3.1.2. Characterization of NPs The resulting 3.1.2. Characterization KT-NPs of NPs showed a high EE, concretely 93.91%. The Za 2.3The nmresulting with KT-NPs a PI value showedofa high 0.061EE,indicative of narrow concretely 93.91%. distribution, The Zave was and a Z 108.9 ± 2.3 nm Thea PI with pH of the value external of 0.061 aqueous indicative of narrowphase W2 resulted distribution, and a ZP ofin 5.2.mV. The pH of −6.20 the external aqueous phase W2 resulted in 5.2. As depicted in Figure 4, the TEM image shows round-shaped struct As depicted in Figure 4, the TEM image shows round-shaped structure of KT-NPs. The The particle particle size associated size associated was 51.9 ±was14.5 51.9 ± 14.5 nm (26.4 nm wasnmthe (26.4 nm was minimum the minimum size, whereas the the maximum maximum was was 92.7 92.7 nm). nm). Figure Figure 4. TEM 4. TEM imageimage of KT-NPs. of KT-NPs. 3.2. Stability Studies 3.2.The Stability stability Studies of the formulations was evaluated at 3 months, stored at 25 ◦ C and 4 ◦ C for the KT-CTS gel and the The stability of KT-NPs, respectively. After the formulations was3evaluated months of storage of KT-CTS gel at 3 months, stored at ◦ at room temperature (25 C), no visual changes in appearance were evidenced. forThe theKT-NPs KT-CTSstoredgel at and the 4 ◦ C for KT-NPs, 3 months respectively. neither After showed statistically 3 months significant of storage changes inatthe room Zave,temperature the PI, the ZP nor (25the°C), no visual EE (Table 2). Thechanges statistical in appearance significance were level was set eviden at p < 0.05. The KT-NPs stored at 4 °C for 3 months neither showed statistic changes Table in theofZave, 2. Comparison thesize the particle PI,(Zave), the polydispersity ZP nor theindexEE(PI), (Table 2). The zeta potential statistical (ZP), pH of sig was the set aqueous external at p < 0.05. phase and encapsulation efficiency (EE) from the KT-NPs at time 0 and time 90 days (n = 3) stored at 4 ◦ C. Results are expressed as mean ± standard deviation. Table 2. Parameter Comparison of the particle KT-NPs size Day 0(Zave),KT-NPs polydispersity Day 90 index (PI), zeta pote p-Value the external Zave aqueous (nm) phase and108.9 encapsulation ± 2.3 efficiency 111.2 ± 3.6 (EE) from the KT-NPs at p = 0.4039 90 days (n =3) PI stored at 4 ºC. Results are expressed 0.061 ± 0.013 0.064as ± mean 0.018 ± standard deviation. p = 0.8265 ZP −6.20 ± 0.48 −6.28 ± 0.39 p = 0.8337 pH aqueous Parameter external phase (W2 ) ± 0.1 5.2 KT-NPs 5.2 Day 0 ± 0.2 KT-NPsp Day = 1.00090 EE% 93.9 ± 2.83 92.4 ± 2.45 p = 0.5258 Zave (nm) 108.9 ± 2.3 111.2 ± 3.6 PI Study 3.3. In Vitro Drug Release 0.061 ± 0.013 0.064 ± 0.018 The release profiles ZP of both formulations were±investigated −6.20 0.48 by the in vitro −6.28 diffusion ± 0.39 cells technique. The NPs allowed a higher degree of release of KT than the CTS gel pH aqueous external phase (Figure 5). 5.2 ± 0.1 5.2 ± 0.2 (W2) EE% 93.9 ± 2.83 92.4 ± 2.45 3.3. In Vitro Drug Release Study rmaceutics 2021, 13, x FOR PEER REVIEW Pharmaceutics 2021, 13, 1784 10 of 19 Figure 5. In vitro release profile of the formulations, KT-NPs and KT-CTS gel (p < 0.001). Each value Figure 5. In vitro release profile of the formulations, KT-NPs and KT-CTS gel represents mean ± SD (n = 6). represents mean ± SD (n = 6). According to the drug release study, the best-fitted model to the experimental data was the first-order kinetic equation with a K value of 0.901 h−1 for KT-CTS gel and 1.841 hAccording to (Table −1 for the KT-NPs the drug release 3). Significant study, statistical the best-fitted differences model were found for all the to the parameters was evaluated. the first-order kinetic equation with a K value of 0.901 h−1 for KT −1 for the KT-NPs (Table 3). Significant statistical differences were fo hTable 3. Parameters of KT release from KT-CTS gel and KT-NPs according to first-order kinetics. rameters Values evaluated. are expressed as the Mean ± standard deviation (n = 6). Parameter KT-CTS Gel KT-NPs p-Value Table%R∞ 3. Parameters (%) 1 of 18.6 KT±release 0.3 from KT-CTS 92.0 ± 2.3 gel and KT-NPs p < 0.0001 according t K (h −1 ) 2 0.9 ± 0.2 1.8 ± 0.1 p < 0.0001 Values are expressed as the Mean ± standard deviation (n =6). Half-time (h) 0.8 0.4 p = 0.0014 R2 0.9422 0.9287 - 1 Parameter KT-CTS Gel KT-NPs %R∞ represents the maximum percentage of KT released; 2 K represents the release rate. %R∞ (%) 1 18.6 ± 0.3 92.0 ± 2.3 3.4. Ex Vivo Permeation of KT through Human Skin K (h−1) 2 0.9 ± 0.2 1.8 ± 0.1 Ex vivo permeation studies were carried out to examine the permeability of KT throughHalf-time human skin(h) 0.8 and investigate the biodistribution 0.4 A large of KT in the skin layers. percentage of KT R2 was found on the skin surface 0.9422in the residual formulation, for both, the 0.9287 NPs and the CTS gel as depicted in Table 4. NPs provided greater absorption of KT than 1%R∞ the CTSrepresents the gel. In the case of maximum percentage NPs, the KT was of KTinreleased; mainly retained 2 K represents the the epidermis whereas it was in the receptor fluid in the case of the CTS gel. The CTS gel led to higher percutaneous absorption of KT than the NPs. However, no significant statistical differences were found 3.4. in theEx KTVivo amount Permeation retained or inof theKT through epidermis or inHuman Skin the amount percutaneously absorbed. Statistical differences were only observed in the amount diffused into the receptor fluid Ex vivo permeation studies were carried out to examine the p and the amount penetrated in the stratum corneum. through human skin and investigate the biodistribution of KT in the s percentage of KT was found on the skin surface in the residual formu NPs and the CTS gel as depicted in Table 4. NPs provided greater abs the CTS gel. In the case of NPs, the KT was mainly retained in the ep was in the receptor fluid in the case of the CTS gel. The CTS gel led neous absorption of KT than the NPs. However, no significant statistic found in the KT amount retained or in the epidermis or in the amou absorbed. Statistical differences were only observed in the amount d ceptor fluid and the amount penetrated in the stratum corneum. Table 4. Skin distribution of KT (expressed as µg/cm ) contained in the formulati vitro test after an exposure time of 24 h. Values are expressed as mean ± SD (n =6). KT-CTS Gel Biodistribution KT-NPs (µg/cm2) p Pharmaceutics 2021, 13, 1784 (µg/cm2) 11 of 19 Total applied 14.61 26.91 Skin surface 12.14 ± 1.31 26.51 ± 0.84 Table 4. Skin distribution of KT (expressed as µg/cm2 ) contained in the formulations with an in vitro Stratum test after corneum an exposure 0.08 ±as0.02 time of 24 h. Values are expressed mean ± SD (n = 6).0.04 ± 0.01 Epidermis 0.71 ± 0.32 0.32 ± 0.17 Biodistribution KT-NPs (µg/cm2 ) KT-CTS Gel (µg/cm2 ) p-Value Dermis Total applied 14.61 0.001 ± 0.001 26.91 0.002 ± 0.001 - Receptor Skin surface Fluid 12.14 ± 1.31 0.02 ± 0.0126.51 ± 0.84 0.49 ± 0.09 - Stratum Total recovery 0.08 ± 0.02 corneum 12.95 ± 0.84 0.04 ± 0.01 27.36 ± 0.85 0.03 * Epidermis 0.71 ± 0.32 0.32 ± 0.17 0.13 Percutaneous 0.001 ± 0.001 Dermis 0.002 ± 0.001 0.57 Receptor Fluid 0.02 ± 0.01 0.73 ± 0.32 0.49 ± 0.09 0.81 ± 0.19 <0.01 * Absorption Total recovery 12.95 ± 0.84 27.36 ± 0.85 - * Statistical differences between formulations (p < 0.05). Percutaneous 0.73 ± 0.32 0.81 ± 0.19 0.71 Absorption 3.5. In Vivo * Statistical Anti-Inflammatory differences between formulations (pEfficacy < 0.05). Evaluation 3.5.1. 3.5. In TPA-Induced EarEfficacy Vivo Anti-Inflammatory Oedema in Mice Evaluation 3.5.1. The acute ear TPA-Induced Ear skin oedema Oedema in Mice model created by topical administration of T The acute to assess theear skin oedema model formulations created by topical administration anti-inflammatory activity. Asof aTPA was used of infl reference to assess the formulations anti-inflammatory activity. As a reference of inflammatory hibition, inhibition, aaKT KTsolution solution in acetone in acetone was was used. used. Figure Figure 6 shows 6 showsof the the percentage percentage inhibition for eachformulation. for each formulation. Figure 6. Biomechanical properties in human volunteers before the formulations applications (Basal) Figure 1 h and 26. Biomechanical h post-application: properties TEWL in humanand for both formulations; volunteers the evolutionbefore the formulations of the stratum corneum ap sal) 1 h and 2 h post-application: TEWL for both formulations; and the evolution hydration for both formulations. corneum hydration for both formulations. The formulation of the NPs was the one that exhibited the highest reduction in inflammation, and both formulations showed greater anti-inflammatory efficacy than the TheKTformulation reference of the solution in acetone. All NPs was were differences the statistically one that significant. exhibited the highest re flammation, and both formulations showed greater anti-inflammatory effi reference KT solution in acetone. All differences were statistically significan Pharmaceutics 2021, 13, 1784 12 of 19 Pharmaceutics 2021, 13, x FOR PEER REVIEW 3.5.2. Histological Analysis After trols, the TPA-induced respectively, asinflammation, well as fora histological the treated evaluation tissuesof with the mice ears was either KT-NPs conducted Figure 7a,b shows the histological images for the negative and positive controls, KT-CTS gel respectively, (Figure as well 7d). as for the No tissues treated immune system with either cells(Figure KT-NPs were7c)found in the or KT-CTS gel negat (Figure (Figure 7d).7a). On the No immune contrary, system the cells were positive found control in the negative shows control immune ear (Figure 7a). Onsystem the contrary, the positive control shows immune system cells vessels (Figure 7b). The ear treated with KT-NPs did not present in blood vessels (Figure 7b).immun The ear treated with KT-NPs did not present immune system cells (Figure 7c). A limited (Figure number 7c). A limited of immune number cells systems of immune were observed cells in the blood systems vessels were of the ear observed treated with in sels ofgel KT-CTS the ear treated (Figure 7d). with KT-CTS gel (Figure 7d). Figure Figure 7. Histological 7. Histological images ofimages the miceofearthe aftermice ear after ear the TPA-induced theoedema TPA-induced ear oedema in in mice. (a) negative tive control ear; (b) positive control ear; (c) ear treated with KT-NPs, gel. control ear; (b) positive control ear; (c) ear treated with KT-NPs, and (d) ear treated with KT-CTS and (d) e The scale bar is 50 µm in length. The small purple cells corresponding to the KT-CTS gel. The scale bar is 50 µm in length. The small purple cells corresponding immune system cells. system 3.6. cells. In Vivo Tolerance Study Figure 8 shows the evolution of biomechanical parameters as tracked before and after 3.6. the formulations In Vivo wereStudy Tolerance applied for up to 2 h. This was to assess the impact of the formulations on the skin hydration and its integrity. TEWL is measured in grams per hour perFigure square 8 shows meter (g/h·the evolution m2 ) and the stratum ofcorneum biomechanical parameters hydration is given as track in arbitrary after the formulations were applied for up to 2 h. This was to assess the units (AU). A slight decrease was observed in the stratum corneum after the application of the formulations whereas no changes in the TEWL values were observed. Neither were formulations on the skin hydration and its integrity. TEWL is measured anystatistical differences found for TEWL and nor were they found in the process of stratum hour per corneum square hydration at 1 meter (g/h·m2)toand and 2 h compared the values. the basal stratum corneum hydration is giv units (AU). A slight decrease was observed in the stratum corneum after the the formulations whereas no changes in the TEWL values were observed anystatistical differences found for TEWL and nor were they found in stratum corneum hydration at 1 and 2 h compared to the basal values. Pharmaceutics Pharmaceutics2021, 2021,13, 13,x1784 FOR PEER REVIEW 1313ofof19 19 Figure 8. Anti-inflammatory efficacy of studied formulations expressed as a percentage reduction in in-flammation Figure when compared 8. Anti-inflammatory efficacytoof a control. studied The results areexpressed formulations presentedasasa the mean ±reduction percentage SD of three inmeasurements. in-flammation*when standscompared for statistical differences to a control. Thebetween thepresented results are formulations as theandmean the positive ± SD of control three and the KT solution, ** stands for statistical differences between KT-NPs and KT-CTS measurements. * stands for statistical differences between the formulations and the positive gel (p