Enhancing the efficacy of Arrabidaea chica extract release from polysaccharide-based films The impact of cashew gum and cross-linking process - Cosmetologia (2024)

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Journal of Drug Delivery Science and Technology 93 (2024) 105421Available online 1 February 20241773-2247/© 2024 Elsevier B.V. All rights reserved.Enhancing the efficacy of Arrabidaea chica extract release from polysaccharide-based films: The impact of cashew gum and cross-linking process Gabriel Assis de Azevedo a, Ilza Maria de Oliveira Sousa b, Ailane de Souza Freitas b, Mary Ann Foglio b, Ângela Maria Moraes a,* a Department of Engineering of Materials and Bioprocesses, School of Chemical Engineering, University of Campinas, Av. Albert Einstein, 500, SP, Campinas, CEP 13083- 852, Brazil b Faculty of Pharmaceutical Sciences, University of Campinas, R. Cândido Portinari, 200, Campinas, SP, CEP 13083-871, Brazil A R T I C L E I N F O Keywords: Polysaccharide films Cashew gum Cross-linking Sustained-release Arrabidaea chica verlot extract Fridericia chica L A B S T R A C T Polysaccharide films containing plant-derived bioactive agents have emerged as alternatives for the treatment of mucosal lesions. Herein, polysaccharide films composed of chitosan (CH), alginate (AL), and cashew gum (CG, an exudate of Anacardium occidentale L.) containing the standardized Arrabidaea chica Verlot (synonym Fridericia chica L) (Ac) plant extract, as a bioactive agent, were produced. The properties of the films, such as morphology, thickness, mechanical properties, swelling, contact angle, and the plant extract release kinetics were evaluated. Moreover, the influence of the cross-linking process with calcium ions on these properties was also analyzed. Transparent, reddish films with lamellar cross-sections and thickness values varying from 63.5 to 123.7 μm were produced, with Ac incorporation efficiency varying from 79 to 93 %, being higher for CH-AL films. The addition of Ac to CH-AL films reduced their tensile strength, but when Ac was added to the CH-AL-CG formulation, no significant variation was observed in this property, showing that CG and Ac may interact in a positive manner towards the tensile strength. The presence of CG increased the structural stability of the CH-AL films in simulated saliva, from 300 min to more than 500 min. Besides that, the addition of CG changed the impact of Ac extract diffusion and matrix erosion on the Ac release mechanism, but did not lead to significant changes in the me-chanical properties of the films. Although the least cross-linked film exhibited only about half the swelling degree (around 5.4 g/g) when compared to the other formulations, it displayed the highest extract release ratio (45 mg of Ac extract per gram of film), showing an anomalous diffusion mechanism, which may indicate that a balance between erosion and diffusion phenomena is fundamental for efficient Ac extract release. 1. Introduction Oral lesions are usually treated locally with emulsions and gels containing the appropriate drug. However, the efficiency of this type of treatment can be affected by mouth movements and leaching of the active agent due to contact with saliva. Therefore, treatment approaches more suitable for this region of the body are necessary, such as mucoadhesive films [1,2]. These films are commonly composed of either synthetic or natural polymers. Synthetic polymers may present toxicity whenever unreacted monomers and oligomers are present in the final formulation [3]. This problem usually does not occur with polymers of natural origin, such as chitosan (CH) and alginate (AL). Furthermore, polysaccharides consist of sustainable resources, since they may be obtained from seaweed, animals, microorganisms, and plants [4]. The adhesion of the surface of a material to mucosal tissue can occur through different mechanisms, such as by receptor-specific interactions, and covalent or noncovalent bonds [5]. An initial material surface wetting stage is followed by a consolidation stage, in which adhesive interactions of different intensities are established, e.g. van der Waals bonds, hydrogen bonds, ionic interactions, and covalent bonds [6], stabilizing the interpenetration and entanglement of swollen polymer chains with mucosal tissues. Charge interactions, for instance, due to the presence of amino groups in chitosan, can improve the mucoadhesion of * Corresponding author. E-mail address: ammoraes@unicamp.br (Â.M. Moraes). Contents lists available at ScienceDirect Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst https://doi.org/10.1016/j.jddst.2024.105421 Received 20 October 2023; Received in revised form 18 January 2024; Accepted 24 January 2024 Journal of Drug Delivery Science and Technology 93 (2024) 1054212biomaterials through electrostatic interaction with sialic acid molecules from mucosal tissues [7]. Alginate-containing biomaterials, on the other hand, may exhibit mucoadhesive properties due to their ability to form hydrogen bonds with mucin-type glycoproteins through interactions between AL carboxyl and mucin hydroxyl groups [8]. Once the mucoadhesive film attaches to the oral mucosa, local release of the bioactive agent begins. However, the release rate should be controlled at adequate levels to maintain satisfactory local concentration of the bioactive agent for appropriate periods [9]. Therefore, frequently a mixture of components is used in film formulation to promote sustained release of the bioactive agent. Cashew gum (CG), for example, an exudate from Anacardium occidentale L, is a polysaccharide that can effectively exert this function, due to the compound’s chemical structure and composition. This carbohydrate has been reported as a biomaterial component that promotes sustained release and changes the release mechanism of different types of bioactive agents incorporated in tablets, beads, nanoparticles, films, scaffolds, and gels [10]. CG also has the potential to bind to mucin-type molecules present in mucosal tissues through, for instance, hydrogen bonds due to its bulky molecular structure with many hydroxyl and carboxyl groups. Due to the relevant properties of CG, its use mixed with distinct compounds and for different purposes is being considered lately, e.g. exploring biopolymer nano-composite films by blending CG with polyvinyl alcohol and incorpo-rating nanochitosan for the development of materials for organic electronics and charge storage devices [11]. In addition to film matrix components, bioactive agents also origi-nated from natural materials, such as the Arrabidaea chica Verlot (Ac) extract used in this work, are attractive. This extract is composed mainly of three anthocyanins: 6,7,3′,4′-tetrahydroxy-5-metoxy-flavilium, 6,7,4′- trihydroxy5-metoxy-flavilium (known as carajurone), and, 6,7-dihy-droxy-5,4′-dimetoxy-flavilium (denominated as carajurin). Carajurin, one of the main pigments of Ac, can serve as a chemical and biological marker of the whole extract [12,13]. The Ac extract has anti-inflammatory and healing properties, and it also shows excellent potential to be employed in the treatment of oral lesions, as demonstrated in a clinical study in which a mucoadhesive herbal gel containing 2.5 % of standardized A. chica extract was suc-cessfully tested for the therapy of mucositis oral lesions [14]. The se-lection of materials based on compounds originating from natural sources has then very positive impacts not only regarding their inherent biological activity but also from the point of view of sustainability, green chemistry, and economic circularity, which could bring additional ad-vantages and attractiveness to the product developed. Besides composition, cross-linking procedures are also relevant regarding the production of polymeric films for the controlled release of bioactive agents. Cross-linking affects matrix mass transport and sta-bility when in prolonged contact with physiological media. Moreover, the cross-linking steps may also affect the mechanicalproperties of the films [15,16]. Calcium is one of the most commonly used cross-linking agents in alginate-containing films, due to its effectiveness and biocompatibility. However, its use in high concentrations can turn the films too rigid, making more difficult the adaptation of the biomaterial to tissue con-tours, while its addition in low concentration may impact the integrity of the films exposed to physiological fluids for long periods. For instance, Ac extract was previously incorporated into CH and AL films [17], and despite the high incorporation efficiency that was achieved, these matrices showed low extract release, probably because of the high cross-linking agent concentration, which decreased the diffusivity of the extract components through the matrix. Therefore, considering the use of multiple sequential crosslinking steps may be an adequate strategy to control the reticulation degree and produce materials with tailored properties. In this context, this study aimed to answer the following question: can cashew gum and cross-linking agent concentration affect the release of Arrabidaea chica Verlot extract from polysaccharide-based films? 2. Materials and methods The Brazilian Agricultural Research Corporation (EMBRAPA-CE, Brazil) provided the CG in natura. Medium molar mass CH (75 % deacetylation degree, 208 cP viscosity in 1 % acetic acid solution at 1 %), and medium viscosity AL (2994 cP viscosity in water at 2 % and 25 ◦C) were provided by Sigma-Aldrich (Gillingham, United Kingdom). Arrabidaea chica Verlot. (Bignoniaceae) leaves were obtained from the Chemical, Biological and Agricultural Pluridisciplinary Research Center of the University of Campinas (Brazil) experimental field (voucher de-posit 1348 at CPQBA-Herbarium – Germoplasm bank). The use of plant genetic material was registered at the Brazilian System for the Man-agement of Genetic Heritage and Associated Traditional Knowledge (SisGen), under the codes A080AEE, A466Ae6 and A855C71. Other re-agents used were of, at least, analytical grade. 2.1. Production of Ac extract The standardized Ac extract was produced at the Laboratório de Fitoquímica, Farmacologia e Experimentação Animal (LAFTEX) of the Faculty of Pharmaceutical Sciences at the University of Campinas (UNICAMP, Campinas, SP, Brazil), according to the method developed by Paula et al. and Sousa et al. [18–20]. One kilogram of dried ground leaves was exposed to 5 L of 70 % hydroethanol solution acidified with 0.3 % citric acid for 1.5 h, at room temperature, under mechanical stirring. Following, the liquid phase was separated from the solid leaves residue by filtration, and the solvent was concentrated using a vacuum rotary evaporator. The residual water was removed using a mini spray dryer (Büchi Labortechnik AG, Büchi B-290) operated with nitrogen (414 L/h), at an inlet temperature of 100 ◦C and outlet temperature of 60 ◦C ± 2 ◦C. The dried powder was collected and stored in a glass vial protected from light at − 8 ◦C until further use. 2.2. CG purification The methodology used for CG purification was adapted from de Sá Pinto et al. [21] Initially, 7 g of CG in natura were dissolved in 100 mL of distilled water, thereafter the solution was kept under gentle stirring for 24 h, and the pH was adjusted to 7 with a 1 mol/L NaOH solution. Af-terward, gum cations replacement with Na+ was achieved by adding 7 g of NaCl to the solution. Subsequently, the solution was centrifuged at 1300 g for 15 min (model CT-5000R, Cientec), to remove insoluble particles by filtering the supernatant in a sintered disc funnel of porosity 1. The filtered supernatant was mixed with ethanol (95.54 ◦GL) in a ratio of 4:1 v/v (ethanol to CG solution) to precipitate CG. The mixture was further centrifuged at 1300 g for 15 min and the precipitate was collected, dried and macerated. To reduce the final proportion of salt in the CG, the purification process described above was repeated with the dried powder, but the pH neutralization and NaCl addition steps were not performed. The solid phase collected after precipitation with ethanol and centrifugation was then dried in an oven with air circulation (410D, Nova Ética) for 24 h, at 37 ◦C. 2.3. Film production and Ac extract incorporation The films were produced following a methodology adapted from Pires et al. [17] Initially, precursor solutions were prepared separately, the first consisting of 1 % CH in 1 % (v/v) acetic acid, and the second, of 0.5 % AL or a mixture of 0.25 % AL and 0.25 % CG in water. In a stirred vessel, 100 mL of the CH solution was added to 200 mL of the second solution at a flow rate of 200 mL/h, using a peristaltic pump (Minipuls 3, Gilson). After all the CH solution was transferred, the stir-ring rate was increased from 500 to 1000 rpm for 10 min. In the case of CH-AL films, the mixture pH was corrected to 7 by the addition of 2 mol/L NaOH aqueous solution. Thereafter, 4 mL of CaCl2 (1 % m/v) were added to the resulting solution to promote primary G. Assis de Azevedo et al. Journal of Drug Delivery Science and Technology 93 (2024) 1054213cross-linking of carboxyl groups from AL not bound to amino groups from CH. The resulting mixture was deaerated in a vacuum pump (Q- 355B2, Quimis) for 120 min. The resulting mass was divided equally into two 15 cm diameter polystyrene Petri dishes and taken to an oven with air circulation (410D, Nova Ética) to dry for 20 h at 37 ◦C. After drying, a secondary cross-linking procedure was carried out by immersing the films in an aqueous CaCl2 (1 % m/v) solution for 15 min to reticulate the remaining free carboxyl groups. Two washing steps of 30 min each were then performed by immersing the films in deionized water. Finally, the material was dried at room temperature for approx-imately 20 h. For the formulations containing CG, the addition of NaOH promoted phase separation. Therefore, in this case, the neutralization step was performed before the secondary cross-linking procedure, by immersing the dried film in 150 mL of 2.38 g/L HEPES buffer (pH 7.4) for 30 min. Alternatively, for CG-containing formulations, the secondary cross- linking step was not performed to analyze the effect of this procedure on the characteristics of the films. In both cases, after neutralizing with HEPES, films were washed twice with deionized water for 30 min and then dried at room temperature. The Ac extract was added to the formulations in a proportion of 10 % in weight regarding the polysaccharide mass (formulations named CH- AL-Ac and CH-AL-CG-Ac). For that, 8 mL of an ethanol solution with Ac at a concentration of 25 mg/mL were used. In all cases, the addition of the Ac extract was performed after the primary cross-linking step with CaCl2. The formulations tested and the procedures used are summarized in Table 1. 2.4. Film morphology The morphology of the films was evaluated by direct visual inspec-tion (registered using a model A10, Samsung mobile phone digital camera), and by scanning electron microscopy (SEM, model Leo 440i, Leica) operating at 15 kV voltage, 100 pA current, and 80◦ tilting. Before the SEM analysis, the samples were kept in a desiccator with silica for 24 h and coated with an ultra-thin layer of gold (200 Å) with a sputter coater (model K450, Emitech). 2.5. Thickness The thickness of the films was determined using a digital micrometer (model MDC-25S, Mitutoyo) and reported as the average of 10 inde-pendent measurements at central and peripheral points. 2.6. Mechanical properties Film samples of 2.5 cm × 8 cm, previously stored for 48 h in a desiccator with silica at room temperature, had their tensile strength and elongation at breakdetermined based on ASTM D-882 (ASTM, 2012), using a texturometer (model TA. XT2, Stable Micro System) [22]. The samples were fixed to two pneumatic clamps initially spaced 5 cm apart, thereafter the crosshead was moved away at a speed equal to 0.1 cm/s. The cell load was equal to 5.098 kgf. The tensile strength (TS) and the elongation at break (EL) of the films were calculated using Equations (1) and (2), respectively: TS =Fm / Across (1) EB =(d / di ) ∗ 100 (2) where Fm is the maximum breaking force, Across is the cross-sectional area, and di and d are the distances between the clamps at the initial moment and the time of rupture, respectively. Young’s modulus was obtained by calculating the slope of the initial linear region of the stress versus strain curve. 2.7. Swelling degree The swelling tests followed a methodology based on Kipcak et al. [23]. Initially, 2 cm × 2 cm samples, previously dried in a desiccator with silica for 12 h, were weighed and then immersed in 10 mL of simulated saliva solution (8 g/L of sodium chloride, 0.19 g/L of mono-basic potassium phosphate and 2.38 g/L of dibasic sodium phosphate, with a pH of 6.8 at 37 ◦C). Thereafter, the excess solution present on the surface of the film was blotted with absorbing paper, and the masses of the samples were determined. This process was repeated in specific time intervals and the mass values were used to calculate the swelling degree (SD) as shown in Equation (3): SD=(MW – MD) / MD (3) where MW is the mass of the wet film, and MD is the initial mass of the dried film. 2.8. Contact angle Approximately 10 μL of simulated saliva were dispensed onto the surface of the film sample and the mean apparent static contact angle was determined (CAM-MICRO contact angle meter, Tantec), considering five independent analyses for each formulation. 2.9. Efficiency of incorporation of Ac extract The efficiency of Ac extract incorporation was assessed indirectly, by determining the amount of extract transferred to the spent solutions of the secondary cross-linking and washing steps carried out during the production of the films. This evaluation was performed using a UV–vi-sible spectrophotometer (DU640, Beckman) at a wavelength of 470 nm and a calibration curve previously prepared with known amounts of standardized Ac extract. Carajurin, one of the main red components of the extract, absorbs light at the above-mentioned wavelength and was used as a reference compound to determine extract content. This pro-cedure was already used successfully to monitor the Ac extract compo-nents [17]. Considering the initial quantity of extract added to the formulations and the Ac extract lost during the above-mentioned processing steps, the incorporation efficiency (ε) was calculated according to Equation (4): ε=(M0 – MSC+W ) ∗ 100 / M0 (4) where M0 is the initial mass of Ac extract added and MSC + W is the mass of Ac determined in the total volume of the solutions used during the secondary cross-linking and washing procedures. 2.10. Kinetics of Ac extract release The Ac extract release kinetics was determined using 2 cm × 2 cm film samples immersed in 10 mL of simulated saliva solution at 37 ◦C gently mixed at 100 rpm. After specified periods, the film sample was removed and transferred to the same volume of fresh simulated saliva Table 1 Description of the different film formulations tested in this work and occurrence of the secondary cross-linking. CH: chitosan, AL: alginate, CG: cashew gum, Ac: Arrabidaea chica extract, NSC: No secondary cross-linking. Formulation Mass of each component in 300 mL of solution (g) Secondary cross- linking CH AL CG A. chica extract CH-AL 1 1 – – Yes CH-AL-CG 1 0.5 0.5 – Yes CH-AL-Ac 1 1 – 0.1 Yes CH-AL-CG-Ac 1 0.5 0.5 0.1 Yes CH-AL-CG-Ac-NSC 1 0.5 0.5 0.1 No G. Assis de Azevedo et al. Journal of Drug Delivery Science and Technology 93 (2024) 1054214solution at 37 ◦C, and this process was repeated for 48 h. The amount of extract released was determined at 470 nm by light absorption spec-trophotometry as described previously. The Korsmeyer-Peppas model (Equation (5)) [24,25] was used to assess the release mechanism of each formulation: MtM∞= ktn (5) where Mt is the amount of drug released at time t, M∞ is the amount of plant extract released at an infinitely large time, representing the Fig. 1. Morphological aspect of the different films produced (formulations identified on the left). Images in the left column represent membrane specimens of approximately 10 cm in diameter. Images at the remaining columns refer to SEM analysis (surface and cross-section at the center and right columns, respectively, performed correspondingly using magnifications of 500 X and 1500 X). G. Assis de Azevedo et al. Journal of Drug Delivery Science and Technology 93 (2024) 1054215maximum amount that could be released at equilibrium conditions (thus, the ratio Mt/M∞ represents the fraction of extract released), k is the release constant, and n is the release exponent. For thin films, values of the release exponent n lower than 0.5 describe quasi-Fickian diffusion, in which drug diffusion is the pre-dominant phenomenon, with a portion occurring within the swollen matrix and another portion through pores filled with the liquid medium, while values equal to 0.5 represent solely Fickian diffusion. Values be-tween 0.5 and 1.0 describe anomalous diffusion, and in this case, besides diffusion, other phenomena such as erosion of the polymer matrix may influence the release process. Values of n equal to 1 represent case II or zero-order transport, and values of n higher than 1 are related to supercase II mass transport, in which drug release is influenced by phenomena such as the detachment of polymeric chains, degradation, and erosion of the matrix. 2.11. Statistical analysis Data statistical analysis was performed with the aid of the Past® software, applying Tukey’s comparative test with a significance level of 5 %. Quantitative data were expressed in terms of mean values and their standard deviation. Unless otherwise specified, all experiments were repeated at least three times. 3. Results and discussion 3.1. Visual aspect, morphology and thickness of the films The formulations were transparent, and this fact could contribute to a more effective application of the films over mucosal lesions (Fig. 1A, 1D, 1G, 1J, and 1M). CH-AL films presented smooth and homogeneous surfaces (Fig. 1A and 1B), similar to what was reported previously work [17]. However, the addition of CG increased the apparent roughness of the film surface (Fig. 1D and 1E), which may indicate a possible pref-erence of CH to bind to AL, pushing the CG to the surface and increasing film roughness. The Ac extract provided a reddish aspect to the films (Fig. 1G, 1J, and 1M), with no visible impact on their roughness (Fig. 1H and 1K). However, the absence of secondary cross-linking in the CH-AL-CG- Ac-NSC formulation led to a rougher and more heterogeneous surface (Fig. 1N), which may be caused by the reduced concentration of Ca2+, the cross-linking agent, which is responsible for approximating the alginate chains closer to each other, forming a smoother and more organized structure [26]. In most cases, the cross-section of the films could be considered lamellar (Fig. 1C, 1I, 1L, and 1O), except the CH-AL-CG formulation (Fig. 1F), which showed a less organized structure. None of the films showed poreson their surfaces or cross-sections. However, fiber ag-gregates and entangled structures are visible, possibly resulting from the formation of polysaccharide polyelectrolyte complexes. According to the literature [17,26–28], the thickness of CH-AL films ranges from 25 to 72 μm, similar to what was found in this work (Fig. 2). The addition of CG to the CH-AL film increased the thickness size, which may be a result of the CG displacement towards the film’s surface, as discussed previously, or could be attributed to the larger volume occu-pied by the highly branched CG molecules. Although the addition of the Ac extract did not influence the thick-ness of CH-AL films, which indicates effective dispersion of the extract in the polysaccharide matrix, when mixed with the CG-containing formu-lations, it promoted significant disturbance of the film structure (as observed in Fig. 1L and 1O), increasing film thickness. The absence of secondary cross-linking in the CH-AL-CG-Ac-NSC formulation further increased the film mean thickness, which reached 123.7 ± 13.7d μm, the highest value observed among all the formula-tions. The lower thickness value for the more cross-linked formulation may be a consequence of greater organization and closer packing of alginate chains mediated by Ca2+ ions, which corroborates what was observed in the morphology analysis results. 3.2. Efficiency of Ac extract incorporation Ac extract incorporation in the films varied between 78.6 ± 3.4 and 93.2 ± 0.9 % (Fig. 3), which is similar to what was found previously for films containing CH and AL [17]. The CH-AL-CG-Ac formulation showed lower Ac incorporation percentage than CH-AL-Ac. An explanation for this difference may lie in the film production methodology since the CH-AL-CG-Ac formulation underwent a neutralization step with HEPES not necessary for CH-AL-Ac given that the latter was neutralized by the addition of NaOH before primary cross-linking. Thus, part of the Ac may have been leached during the HEPES neutralization step, reducing extract retention, and consequently, its final incorporation efficiency in CH-AL-CG-Ac films. Similarly, CH-AL-CG-Ac films went through the secondary cross- linking procedure, in which part of the previously incorporated Ac was leached away by the solution, leading to a lower percentage of Ac incorporated and effectively retained by this formulation when compared to the counterpart produced without the secondary cross- linking step. 3.3. Mechanical properties The results obtained in the analysis of the mechanical properties of the films are summarized in Table 2. The data achieved for the CH-AL formulation fit well among the results reported in the literature for Fig. 2. Thickness of different film formulations. The same letter indicates no significant difference between average values (Tukey test, confidence interval of 95 %). G. Assis de Azevedo et al. Journal of Drug Delivery Science and Technology 93 (2024) 1054216films prepared with approximately the same compositions and following equivalent procedures (from 0.45 to 22.60 MPa for Young’s modulus, from 4.49 to 31.14 MPa for tensile strength, and 1.72–8.42 % for elongation at break) [17,28–32]. The addition of CG to the CH-AL formulation had no statistically significant impact on any of the me-chanical properties of the film. The presence of Ac in the CH-AL film significantly reduced the values of tensile strength and Young’s modulus of the film, being probably a consequence of the interaction of molecules of the plant extract with polymers’ hydroxyl groups, reducing the number of possible hydrogen bonds formed between groups of the polysaccharide chains. Carajurin and carajurone, two important components of the Ac extract [17], for instance, show, respectively, two and three hydrogen bond donor groups and four hydrogen bond acceptor groups each [33,34]. Similar behavior was observed by Zhu et al. [35], in which CH-AL scaffolds also showed a reduction in their rupture tension after the addition of flurbiprofen, a drug with analgesic and anti-inflammatory action showing a relatively bulky structure, with three hydrogen bond acceptor groups, and one donor group [36]. There was no significant difference in elongation at break and Young’s modulus resulting from the different cross-linking procedures for CH-AL-CG-Ac and CH-AL-CG-Ac-NSC formulations. However, the formulation that underwent secondary cross-linking showed higher tensile strength, probably because of the additional bonds formed be-tween neighboring alginate chains due to Ca2+ cross-linking. 3.4. Swelling degree of the films in simulated saliva solution The analysis of swelling degree showed that CH-AL, CH-AL-Ac, CH- AL-CG, and CH-AL-CG-Ac films absorbed increasing amounts of simu-lated saliva solution in the first 100 min of the test period (Fig. 4A and B), reaching similar swelling degrees. Film fragmentation was observed by visual monitoring and confirmed by the drop in the swelling degree for formulations CH-AL and CH-AL-Ac exposed to simulated saliva so-lution for significantly longer periods (Fig. 4A). However, the presence of CG on the formulation provided these films with more resistance to degradation (Fig. 4B). The absence of the secondary cross-linking procedure reduced remarkably the swelling index of the CH-AL-CG-Ac-NSC film. This result was not expected because the higher the cross-linking agent concen-tration, the lower the number of available charged carboxyl groups that could interact with the aqueous media, and consequently, the lower the swelling [37,38]. Also, given that CG is a branched, bulky molecule, not as linear as AL or CH, it was expected that the aqueous fluid absorption would be higher in formulations containing CG since contrarily, higher concentrations of Ca2+ ions contribute to increase the size of chain segments between cross-link points, facilitating water penetration [37]. Another observation was that all film formulations showed rapid swelling during the first minutes of exposure to the solution, which probably has a relationship to the swelling of the external part of the matrix, while the center of the matrix was not fully hydrated yet. Thereafter, the polymeric chains of the solvated matrix may have started to expand, allowing the swelling of the inner part, which would have continued until the osmotic and elastic forces are balanced and the swelling degree tends to become constant [38]. After around 300 min, however, for formulations CH-AL- Ac and CH-AL-Ac, matrix destabili-zation starts to occur, leading to mass loss and an apparent decrease in the swelling degree (Fig. 4A). 3.5. Contact angle The addition of CG resulted in a significant increase of the simulated saliva contact angle with the CH-AL film (Fig. 5). As previously reported, pure CG films have a contact angle of approximately 32◦ [39]. There-fore, it is not reasonable to associate the increase in this parameter with CG alone, but rather to attribute it to its interactions with AL and CH, possibly making available less hydrogen bond-forming groups to interact with the simulated saliva. A similar trend was observed with the addition of Ac extract, that possibly also has interacted with the polysaccharide matrix by hydrogen bonds, reducing the potential of the aqueous solution interaction with the films, and increasing the contact angle. Similar behavior was re-ported by Zhu et al. (2019) [35], in which flurbiprofen was added to CH-AL films and caused an increase in the contact angle. The absence of secondary cross-linking showed no influence on the contact angle. Fig. 3. Incorporation efficiency of Ac extract in the various film formulationsproduced using different cross-linking approaches. Table 2 Mechanical properties of the films produced. Formulation Tensile strength (MPa) Elongation at break (%) Young’s modulus (MPa) CH-AL 17.18 ± 4.88a 1.44 ± 0.67a 1.71 ± 0.40a CH-AL-CG 17.73 ± 4.33a 2.58 ± 0.34a 1.21 ± 0.16a CH-AL-Ac 5.98 ± 1.90b 1.71 ± 0.74a 0.47 ± 0.15b CH-AL-CG-Ac 11.41 ± 2.01a 1.44 ± 0.07a 0.76 ± 0.10b CH-AL-CG-Ac-NSC 8.49 ± 2.86b 1.63 ± 0.66a 0.76 ± 0.20b The same letter in the same column indicates no significant difference between average values (Tukey test, confidence interval of 95 %). G. Assis de Azevedo et al. Journal of Drug Delivery Science and Technology 93 (2024) 10542173.6. Release of Ac extract Considering the mass of Ac released per mass of film, the values for CH-AL-Ac and CH-AL-CG-Ac films were statistically similar, implying that CG practically did not affect the amount of Ac extract released per gram of dried film (Fig. 6). Considering the periods of 6 and 48 h of release, in terms of release percentage, the CH-AL-CG-Ac-NSC film dis-played the highest value (54.0 ± 16.9) among the formulations (Table 3). However, in terms of concentration, the CH-AL-Ac and CH-AL- CG-Ac-NSC films released the same amount of Ac extract, because the 2 × 2 cm CH-AL-Ac specimens showed higher Ac incorporation efficiency (Fig. 3), being heavier than the CH-AL-CG-Ac-NSC ones (29.7 ± 0.6 mg and 18.9 ± 2.9 mg respectively). An inverse relationship between swelling and release was noticed, which may indicate that extract diffusion along the film plays an important role in the release, since the more swollen the film, the greater the diffusional path to be crossed by the extract molecules. Wider diffusional pathways pose a greater challenge for the release of bioactive Fig. 4. Swelling degree of the polysaccharide films containing and not containing CG in simulated saliva solution at pH 6.8 and 37 ◦C. (A) Formulations not containing G, with primary and secondary cross-linking. (B) Formulations containing CG treated with different cross-linking procedures. G. Assis de Azevedo et al. Journal of Drug Delivery Science and Technology 93 (2024) 1054218agents, especially when they are bulky or exhibit any degree of inter-action with the matrix [40]. Upon comparing the CH-AL-Ac formulation presented in this study with a corresponding formulation produced by Pires et al. [17], our study reports a substantially higher extract release percentage (30.6 ±3.3 %) than that documented in the prior publication, where the values did not exceed 10 %. This result was expected, since in the present work different procedures were used for film crosslinking. The crosslinking agent concentration and the exposure time of the films to the cross-linking solution were lower than the ones used by Pires et al. [17] Therefore, the size of the pores of the CH-AL-Ac matrix in the formu-lation developed herein is probably larger, allowing greater release of Ac in the simulated saliva. Another parameter that was possibly affected by differences in the crosslinking procedure was the time to reach the equilibrium plateau, which for the three formulations developed herein was around 6 h, lower than those reported by Pires et al. [17], of around 8 h. Data fitting using the Korsmeyers-Peppas equation showed that the release mechanism of all formulations was anomalous diffusion, also known as non-Fickian release, which comprises a combination of diffusion of the bioactive agent incorporated and erosion of the matrix (Table 4) [24,25]. In addition to the examination of the Korsmeyer-Peppas kinetic model, alternative models, including zero order, first order, and Higuchi models, were employed to analyze the Fig. 5. The contact angle of simulated saliva solution with the different polysaccharide films. The same letter indicates no significant difference between average values (Tukey test, confidence interval of 95 %). Fig. 6. Amount of Ac extract released per unit mass of polysaccharides in various film formulations exposed to simulated saliva formulation at 37 ◦C. Table 3 Percentages of Ac extract released by the polysaccharide films after 6 and 48 h in simulated saliva concentration at 37 ◦C and corresponding Ac concentrations reached. Formulation Ac released (%) Ac released (μg/mL) After 6 h After 48 h After 6 h After 48 h CH-AL-Ac 30.6 ± 3.3a 33.2 ± 3.4a 77.1 ± 9.8a 83.8 ± 10.2a CH-AL-CG-Ac 31.2 ± 5.6a 38.6 ± 5.4a 35.6 ± 5.3b 44.1 ± 5.1b CH-AL-CG-Ac-NSC 54.0 ± 16.9b 60.3 ± 17.1b 75.3 ± 19.6a 84.2 ± 19.8a The same letter in the same column indicates no significant difference between average values (Tukey test, confidence interval of 95 %). G. Assis de Azevedo et al. Journal of Drug Delivery Science and Technology 93 (2024) 1054219release data (results not shown). Nevertheless, the R2 values associated with these model fittings did not reach a sufficiently high level to assure further consideration. Data fitting using the Korsmeyers-Peppas equation showed that the values of the exponential parameter n were different for each formula-tion, which indicates that the impact of the phenomena (diffusion and erosion) on the release was different. For CH-AL films, for example, the addition of CG to the formulation decreased the n value from 0.6948 ±0.1273a to 0.5704 ± 0.3135b. This suggests that the presence of the gum favored the diffusion mechanism. Potentially this is due to the increase in the molecular weight between cross-links attributable both to the substitution of half of the mass of AL by CG in the formulation (providing less alginate molecules available to bind to calcium ions) and to the fact that CG is bulkier than AG, pushing away adjacent alginate molecules. The opposite was observed for the absence of secondary cross-linking: the n value rose from 0.5704 ± 0.3135b to 0.7608 ± 0.0744a. This Table 4 Korsmeyer-Peppas parameters and the release mechanisms indicated by the mathematical model for each film formulation. Modeling parameters, coefficient of determination and release mechanism Formulation CH-AL-Ac CH-AL-CG-Ac CH-AL-CG-Ac-NSC k (min− 1) 0.0348 ± 0.0140a 0.0639 ± 0.0469b 0.0256 ± 0.0095c n 0.6948 ± 0.1273a 0.5704 ± 0.3135b 0.7608 ± 0.0744a R2 0.9789 ± 0.009a 0.9832 ± 0.0130b 0.9911 ± 0.0057c Predominant transport mechanism Anomalous diffusion Anomalous diffusion Anomalous diffusion The same letter in the same line indicates no significant difference between average values (Tukey test, confidence interval of 95 %). Fig. 7. Visual aspect of the films before and after the release test. G. Assis de Azevedo et al. Journal of Drug Delivery Science and Technology 93 (2024) 10542110behavior was expected, as the least cross-linked formulation has fewer cross-links between AL chains and the reticulating agent (Ca2+), leading to more intense matrix erosion. Moreover, the CH-AL-Ac films showed altered morphology after the release test, with fragmentation and parts of the film detaching from the original samples (Fig. 7) after 48 h in contact with the simulatedsaliva solution, which confirms the erosion of the matrix indicated by the ki-netic parameters. Although the CH-AL-CG-Ac-NSC formulation showed no apparent degradation, the film extremities bent towards the center after 48 h, probably due to the low cross-linking concentration. The CH-AL-CG-Ac films remained apparently intact after the release kinetics analysis, which corroborates with the n value determined, being the closest to 0.5 (Fickian-diffusion), suggesting that this film release was the least influenced by matrix erosion. Regarding therapeutic concentration, in the range from 0.25 to 30 μg/mL, Ac extract can stimulate fibroblast growth [41,42] and at 12.5 μg/mL, antimicrobial activity is also detected [43]. However, at a con-centration of 189 μg/mL, a reduction of 50 % decrease on mouse peri-toneal macrophage cell viability is observed [44]. Hence, a reasonable conclusion is that all three film formulations yielded appropriate con-centrations of Ac extract (ranging from 35 to 77 μg/mL after 6 h). 4. Conclusions The absence of secondary cross-linking in the films containing CG resulted in an increase in the amount of Ac released from 27.5 ± 3.9 to 45 ± 12.7 mg/g, which stands as the highest release value among all formulations. Considering the mechanism indicated by the Korsmeyer- Peppas model for the CH-AL-CG-Ac-NSC formulation, both diffusion and erosion appear to have affected Ac release. Furthermore, after approximately 300 min in simulated saliva solu-tion, the films with no CG presented a decrease in the swelling degree, associated with significant matrix erosion. However, with the addition of CG, their resistance to erosion increased, and the films remained stable in the simulated saliva solution for more than 500 min, corrob-orating the results attained through the use of Korsmeyer-Peppas model that showed that the CH-AL-Ac film exhibited a release profile more strongly influenced by erosion than the CH-AL-CG-Ac formulation. These findings showed that an adequate combination of cross-linking procedures and CG addition may enhance important properties of CH- AL films, such as release kinetic and resistance to erosion, resulting in suitable drug delivery systems for mucosal lesion treatment. CRediT authorship contribution statement Gabriel Assis de Azevedo: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. Ilza Maria de Oliveira Sousa: Investigation, Methodology, Visualization, Writing – review & editing. Ailane de Souza Freitas: Investigation, Methodology, Visualization, Writing – review & editing. Mary Ann Foglio: Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Visualization, Writing – original draft, Writing – review & editing. Ângela Maria Moraes: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Valida-tion, Writing – original draft, Writing – review & editing. Declaration of generative AI and AI-assisted technologies in the writing process During the preparation of this work, the author G. A. Azevedo used the tool ChatGPT-3.5 exclusively to improve language and readability. After using this tool/service, the authors G. A. Azevedo, M. A. Foglio, and Â. M. Moraes reviewed and edited the content as needed and take full responsibility for the content of the publication. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. Acknowledgements The authors would like to acknowledge the support for this research from the National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq, Brazil – grants # 314724/2021–4, 130574/2020–1, 301269/2017–3, and 301724/2022–9) and the Coordination for the Improvement of Higher Educational Personnel (Coordenação de Aperfeiçoamento de Pes-soal de Nível Superior – CAPES, Brazil – finance code 001). References [1] L.G. Camargo, P. de Freitas Rosa Remiro, G.S. Rezende, S. Di Carla Santos, M. Franz-Montan, Â.M. Moraes, Development of bioadhesive polysaccharide-based films for topical release of the immunomodulatory agent imiquimod on oral mucosa lesions, Eur. Polym. J. 151 (2021) 110422, https://doi.org/10.1016/j. eurpolymj.2021.110422. [2] A.P de S.P.A. Magalhães, F.A. do Carmo, C.R.E. 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