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Article

Development of κ-Carrageenan Films Reinforced with Magnesium Oxide Nanoparticles for the Potential Treatment of Chronic Wounds: In Vitro and In Vivo Insights

by
Lesly Rodríguez-Vicens
1,
Jorge L. Mejía-Méndez
2,
Edgar R. López-Mena
3,* and
Sergio A. Bernal-Chávez
1,*
1
Departamento de Ciencias Químico Biológicas, Universidad de las Américas Puebla, San Andrés Cholula, Puebla 72810, Mexico
2
Programa de Edafología, Colegio de Postgraduados, Campus Montecillo, Carr. México Texcoco km 36.4, Montecillo 56264, Mexico
3
Tecnologico de Monterrey, Escuela de Ingeniería y Ciencias, Av. Gral. Ramón Corona No 2514, Colonia Nuevo México, Zapopan 45121, Jalisco, Mexico
*
Authors to whom correspondence should be addressed.
Polysaccharides 2025, 6(2), 45; https://doi.org/10.3390/polysaccharides6020045 (registering DOI)
Submission received: 27 February 2025 / Revised: 5 May 2025 / Accepted: 28 May 2025 / Published: 30 May 2025
(This article belongs to the Special Issue Seaweed Polysaccharides: Innovations in Isolation, Characterization, Chemical Modification and Processing)
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Abstract

:
In this work, κ-carrageenan (κ-C) and polyethylene oxide (PEO) were utilized to synthesize polymeric films (κ-C-PEO). A 2k experimental design was employed to optimize the synthesis of κ-C-PEO systems by considering the content of κ-carrageenan, PEO, and glycerin and their influence on the mechanical features of the resultant films. The κ-C-PEO systems were robustly characterized by FTIR spectroscopy, thermogravimetric analyses, and scanning electron microscopy (SEM). Magnesium oxide nanoparticles (MgO-NPs) were utilized to load κ-C-PEO films as an efficient approach to enhance their biological performance. The activity of κ-C-PEO films was studied against Gram-negative bacteria through the Kirby–Bauer assay. Artemia salina nauplii were cultured to assess the possible toxicity of κ-C-PEO films. The results demonstrated that κ-C-PEO films were elongated with the heterogeneous distribution of MgO-NPs. The tensile strength, thickness, and swelling capacity of κ-C-PEO films were 129 kPa, 0.19 mm, and 52.01%, respectively. TGA and DTA analyses revealed that κ-C-PEO films are thermally stable structures presenting significant mass loss patterns at >200 °C. Treatment with κ-C-PEO films did not inhibit the growth of Escherichia coli nor Pseudomonas aeruginosa. Against A. salina nauplii, κ-C-PEO films did not decrease the survival rate nor compromise the morphology of the tested in vivo model. The retrieved data from this study expand the knowledge about integrating inorganic nanomaterials with polysaccharide-based structures and their possible application in treating chronic wounds. Even though this work provides innovative insights into the optimal design of bioactive structures, further approaches are required to improve the biological performance of the synthesized κ-C-PEO films.

1. Introduction

Wound healing, a critical physiological process that is documented across all ages, from children to the elderly [1], ensures the restoration of tissue integrity and functionality after injury. Mechanistically, the wound-healing process is divided into four stages: hemostasis, inflammation, proliferation, and remodeling [2]. The hemostasis stage is the immediate response to injury and involves vasoconstriction, platelet adhesion, and fibrin clot formation phenomena. In the inflammatory and proliferation stages, the expansion of blood vessels leads to the infiltration of neutrophils and macrophages, the release of pro-inflammatory cytokines (e.g., IL-1 and IL-6), and the formation of fibrin matrix by fibroblasts [3]. In the remodeling stage, the collagen fibers are replaced and reorganized to improve the tensile strength of the wound [4]. In the same stage, there is an extracellular matrix adjustment, vascular regression, and increased fibroblast activity, resulting in improved wound tensile strength, scar formation, and tissue integrity.
Epidemiologically, the development of wounds by traumas, surgical procedures, burns, chronic conditions (e.g., obesity, diabetes, and autoimmune diseases) [5,6,7], and infections were associated with 2.5% of the total population of the United States of America (USA) and correlated with the investment of more than 96.8 million USD in management strategies and treatment schemes [8]. Current treatment regimens to promote wound healing encompass the use of topical agents (e.g., antiseptics, growth factors, or insulin sensitizers) [9,10,11], negative pressure wound therapy, hyperbaric oxygen therapy [12], and electrical stimulation delivery [13]. In clinical practices, the consideration of wound-healing therapies has occurred with significant efficacy [14]; however, various factors can still hamper their performance, for example, inflammatory events, microbial contamination, or constant exposure to oxidative stress conditions [15,16].
Microorganisms in wounds can cause aberrations in the wound-healing mechanism by promoting biofilm formation, host immunity evasion, and reducing nutrient availability [17]. Common microorganisms involved in wound contamination include Escherichia coli [18], Pseudomonas aeruginosa [19], Proteus mirabilis [20], Staphylococcus aureus [21], and Pseudomonas aeruginosa [22]. They are characterized by their capability to cause tissue damage and loss of elasticity through the production of enzymes (e.g., ureases and collagenases), mild to severe skin infections through the production of toxins (e.g., lipopolysaccharides and hemolysins), abscess formation, and exacerbate inflammation by upregulating the production of pro-inflammatory cytokines (e.g., IL-6 and TNF-α). The infection of wounds comprises the same stages mentioned in previous paragraphs; however, in the presence of pathogenic bacteria, it is initiated by contamination upon injury with foreign objects, and it is followed by colonization, which confers protection against antibiotics and immunological responses [23]. In contrast to healthy wound-healing events, infected wounds also include the exacerbated release of pro-inflammatory cytokines, which can be acute or chronic and lead to tissue damage, impaired healing, and disrupted proliferation and remodeling stages [24]. The treatment of infected wounds is based on the administration of antibiotics such as amikacin [25], vancomycin [26], and ceftriaxone [27]; however, its efficacy is challenging, predominantly due to antibacterial resistance mechanisms [28].
Nanobiotechnology is devoted to designing, manipulating, and applying materials with nanometric architecture. For biomedical applications, nanomaterials (NMs) such as liposomes, nanotubes, and nanoparticles (NPs) are frequently utilized in drug delivery systems, tissue engineering, magnetic resonance imaging, and biosensing [29]. According to their chemical composition, NMs are categorized into organic and inorganic. Polymers are biocompatible, biodegradable, and low-toxic materials with significant flexibility, tensile strength, and uniformity to synthesize nanostructures such as micelles, dendrimers, and capsules [30]. In contrast to other natural polysaccharides, κ-carrageenan is a sulfated polysaccharide derived from red algae that has gained considerable attention in research and clinical pipelines. Chemically, κ-carrageenan is constituted by alternating units of 1,3-linked β-D-galactose and 1,4-linked α-D-galactose units. For nanobiotechnological applications, κ-carrageenan has been utilized to fabricate biocompatible scaffolds for tissue engineering [31], spheres for cosmeceutical development [32], and films for antimicrobial and wound-healing applications [33,34].
For wound-healing applications, κ-carrageenan fibers are convenient materials since they are biocompatible structures exhibiting bioadhesive and occlusive properties while preventing inflammation [35]. The synthesis of fibers with biopolymers is often performed through cross-linking and electrospinning [36]. Cross-linking is a convenient route to obtain films with suitable barrier properties, surface area, and biodegradability for wound healing [37]. In this approach, cross-linking agents such as polyethylene oxide (PEO), formaldehyde [38], polyethylene glycol [39], and sodium tripolyphosphate can promote the formation of κ-carrageenan films [40]. In contrast to other cross-linking agents, PEO and its branch derivatives are advantageous polymers for manufacturing polymeric films for wound healing due to their non-toxicity, biocompatibility, and versatility when combined with other materials.
Biopolymeric films can be reinforced with NPs to enhance their properties and ensure their functionality together with mechanical strength, durability, and thermal stability [41]. Magnesium oxide (MgO) is a white inorganic compound that, due to its role in enzyme function, nutritional importance, and suitability to develop bioactive structures, is exploited to synthesize NMs with applications in cancer therapy [42], bone regeneration [43], and wound healing [44]. In wound healing, MgO-based NMs have been reported to be beneficial as their porosity and high surface area can promote cell attachment, infiltration, and proliferation. Together with this, they can dissolve in physiological media, release Mg2+ ions, modulate signaling pathways involved in cell migration and differentiation, and provide optimal alkaline environments for enzyme activity and growth factors release. Considering this, MgO-based NMs have attracted special attention for potential applications in developing wound-healing systems. The evidence regarding using MgO-NPs to reinforce κ-carrageenan films intended for wound healing is scarce since only a limited number of studies have focused on their modification with other NMs like Ag-NPs to disrupt the growth of pathogenic bacteria such as S. aureus [45], a Gram-positive pathogen involved in wound-healing infections.
Given the need to develop novel alternatives to treat wound healing through nanobiotechnological approaches, this work sought to optimize the synthesis of κ-carrageenan films reinforced with MgO-NPs by a 2k factor experimental design. The morphology of the fabricated materials was analyzed by scanning electron microscopy (SEM). In contrast, their thermal stability and chemical composition were investigated through thermogravimetric analysis (TGA), differential thermal analysis (DTA), and Fourier transform infrared (FTIR) spectroscopy. The tensile strength, swelling capacity, and thickness of DS and DS-MgO-NPs were also studied. The antibacterial activity of the developed materials was evaluated against Gram-negative (E. coli and P. aeruginosa) bacteria. The in vivo toxicity of the obtained materials was determined against Artemia salina nauplii.

2. Materials and Methods

2.1. Reagents

κ-carrageenan (1.007 × 106 Da molecular weight, >99.0% purity, 10 Pa-s viscosity) was purchased from Chemico (Jalisco, Mexico). Polyethylene oxide with an approximate molecular weight of 200,000 (PolyOX™ WSR N80) and 7,000,000 (PolyOX™ WSR 303) was obtained from DuPont™ (Midland, MI, USA). Glycerin USP was from AzuMex® (Mexico City, Mexico). All systems were prepared using water from a Milli-Q® (MQ) filtration system Millipore (Billerica, MA, USA). MgO-NPs were obtained as published [46].

2.2. 2k Experimental Design and Development of κ-C-PEO Films

A 2k experimental design was conducted using Minitab® V-21.1.0 software. Table 1 outlines the factors and levels proposed for the study.
To develop κ-C-PEO films, two systems were prepared in separate containers containing 50% of the total water. PEO was added to the first container, and κ-C with glycerin at 70% of the polymer weight was incorporated into the second. Both solutions were maintained for 30 min under agitation at 300 rpm and 70 ± 5 °C. Subsequently, the two systems were mixed for 30 min at the same stirring speed and temperature. Then, 6.5 mL of the resulting mixture was poured into a glass Petri dish with dimensions of and dried in an oven at 45 ± 5 °C for 5 h. After this period, the obtained κ-C-PEO was refrigerated at 8 °C for 1 day. The same procedure was repeated to reinforce κ-C-PEO films with 1.0% w/w of MgO-NPs previously dispersed into the PEO system. The percentage of raw materials was considered based on the design matrix. Scheme 1 represents the synthesis process of κ-C-PEO films.

2.3. Analysis of the Tensile Strength, Thickness, Swelling Capacity, and Water Solubility of κ-C-PEO Films

The tensile strength of κ-C-PEO films was measured using a ZP-500N tensiometer (Shenzhen Ailigu Instrument Co., Ltd., Shenzhen, China). Briefly, empty or reinforced κ-C-PEO films were cut into a dumbbell shape (6.57 ± 0.55 cm in length and 6.57 ± 0.55 cm in width) and uniaxially stretched until they broke at a controlled speed. Then, Equation (1) was considered to determine their tensile strength (TS); in this equation, Fo is the loading force, whereas w and t are the original width and thickness of the sample, respectively. Moreover, the TC of κ-C-PEO films was carried out with a digital micrometer. For the SC evaluation, a plastic tube filled with 2 mL of aqueous solution at pH 7.5 was placed in a dry bath (Benchmark, BSH300, Sayreville, NJ, USA) until it reached 33 ± 0.5 °C. After this, 0.2 g of κ-C-PEO films was placed into the tube and swelled for 2 min. The swelling capacity (SC) of κ-C-PEO films was determined using Equation (2). The SC of sample 4 was evaluated for 0, 5, 15, and 30 min, and 4 and 24 h; this was according to optimization results. The water solubility of the same sample was analyzed as reported [47]. Briefly, sample 4 (2 × 2 cm) was placed into Erlenmeyer flasks containing 30 mL of deionized water. The mixture was maintained in contact with water at 25 °C for 24 h. After this, the resultant solution was filtered, and the filter paper was allowed to air dry for another day.
T S   ( k P a )   =   F o W   ×   t ′
S C % = W f   −   W i W f   × 100

2.4. Characterization of κ-C-PEO Films

Considering these results and the data listed in Table 2, sample 4 was utilized to incorporate MgO-NPs and further utilized for characterization and biological activity analysis. Samples were designated as DS (dressing system; unloaded κ-C-PEO film) and DS-MgO-NPs (dressing system/MgO-NPs). The developed κ-C-PEO films were characterized through FTIR spectroscopy, TGA and DTA analyses, and SEM. The chemical composition of κ-C-PEO films was investigated using a Cary 630 FTIR spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) within the 4000–500 cm−1 range. The measurements were performed utilizing the attenuated total reflectance mode and 4 cm−1 resolution with a one-minute collection time. For TGA and DTA evaluation, 10 mg of raw materials and κ-C-PEO films were placed in alumina crucibles and analyzed with an STA 2500 Regulus TGA/DTA thermal analyzer (Netzsch Japan, Ltd., Yokohama, Japan). Measurements were conducted under nitrogen flow within the temperature range of 20 °C to 600 °C at a heating rate of 10 °C/min. The structural features of empty or reinforced κ-C-PEO films, previously coated with gold in a vacuum ion sputter coater (SEC, MCM-100), were investigated using a Tescan MAIA3 (Brno-Kohoutovice, Czech Republic) at an accelerating voltage of 6.5 kV.

2.5. Strains Culture and Antibacterial Activity Analysis of κ-C-PEO Films

The antibacterial activity of DS and DS-MgO-NPs was studied against E. coli (ATCC 25922) and P. aeruginosa (ATCC 14210) through the Kirby–Bauer method. Briefly, strains were cultured in Muller–Hinton (MH) broth (Becton & Dickinson), maintained under orbital shaking for 24 h, and adjusted to 0.05 at 600 nm in a Cary 60 spectrophotometer (Agilent Technologies, Santa Clara, CA, USA), with 100 mL of the final suspension were dispensed into Petri dishes containing MH agar. Then, DS or DS-MgO-NPs were carefully cut and placed into Petri dishes, which were further incubated at 37 °C for 24 h. Discs impregnated with amikacin were considered the positive control for E. coli and P. aeruginosa. Discs without treatment were considered the negative control. The antibacterial activity of samples was determined by measuring the zone of inhibition (ZI) around the films. All experiments were performed in triplicate.

2.6. Treatment of A. salina with κ-C-PEO Films

A. salina dried cysts were mixed into 1 L of distilled water that contained 35 g of sea salt and kept at 32 °C for 48 h. The toxicity assay on A. salina nauplii was carried out by carefully placing 250 µL of specimens into a 96-well plate, along with empty or reinforced κ-C-PEO films at concentrations of 60, 120, 180, 240, and 300 μg/mL, respectively. The survival rate of nauplii was observed after 24 h of treatment using a Leica DMi1 inverted microscope (Leica, Wetzlar, Germany) with a FLEXACAM C1 camera. Images were captured using Leica software version 3.3.0 (Leica Microsystems, Wetzlar, Germany). Treatment with K2Cr2O7 at the same concentrations as the synthesized materials was appraised as the positive control.

2.7. Statistical Analysis

A one-way analysis of variance (ANOVA) followed by Tukey’s mean separation test utilizing OriginPro 2023 data processing software (OriginLab, Northampton, MA, USA) was utilized to determine significant statistical differences between the retrieved data from the antibacterial and antioxidant activity of empty or reinforced κ-C-PEO films.

3. Results

3.1. Analysis of Tensile Strength, Swelling Capacity, and Thickness

The tensile strength, swelling capacity, and thickness of the developed formulations are listed in Table 2. According to the optimization results and the determined tensile strength, thickness, and swelling capacity, only sample 4 was utilized for further characterization and biological activity analyses. In an independent experiment, its content was maintained, but 1% of MgO-NPs was added. In this context, sample 4 was designated as DS, and when reinforced with MgO-NPs, it was referred to as DS-MgO-NPs.

3.2. Effect of Outcome Variables on κ-C-PEO Films

Figure 1 illustrates the effect of κ-carrageenan, PEO, and PEO MW content on the thickness, tensile strength, and swelling capacity of κ-C-PEO films. The swelling capacity of sample 4 (2.5% κ-carrageenan and 0.50% PEO N303) after 0, 5, 15, and 30 min and 4 and 24 h is represented in Figure S1 and listed in Table S1. The calculated water solubility of sample 4 was 14.67 ± 7.74% after 24 h.

3.3. Characterization of DS and DS-MgO-NPs

3.3.1. SEM Analysis

DS exhibited a continuous polymeric matrix constituted by elongated films (see Figure 2A–C). The surface of such structures presented a rough texture and visible heterogeneous distribution of MgO-NPs (see Figure 2D–F).

3.3.2. TGA and DTA Analysis

As observed in Figure 3A, κ-carrageenan presented thermal stability between 26.07–218.57 °C, presenting mass loss patterns from 99.28 to 79.81%. The primary decomposition of κ-carrageenan was recorded from 221.07 to 578.57 °C, causing 79.37–30.28% mass loss. The TGA of PEO-303 revealed that from 18.56–258.56, it exhibited 99.87–67.18% mass loss, whereas at 266.06–348.56, it lost 59.39–0.69% mass (see Figure 3B). Meanwhile, it was noted that glycerin did not exhibit significant mass loss patterns (100–80.21%) as the temperature increased (25.35–185.38 °C) until it reached 240.35 °C, causing 4.49% mass loss (see Figure 3C). In Figure 3D, it can be noted that MgO-NPs remained stable within the range of 26.97–69.38°C; however, it presented mass loss patterns (78.31–50.06%) from 71.97–334.49 °C. According to Figure 3E, the significant mass loss of the developed DS was recorded until 247.53–577.53 °C, which caused 47.69–21.14% mass loss. Similarly, DS-MgO-NPs did not exhibit significant mass loss changes until 220.91–588.41°C (60.92–23.43%). DTA analyses are also illustrated in Figure 3 and indicate the phase transitions and decomposition of DS and DS-MgO-NPs between 0 and 600 °C. The same phenomena can be associated with the raw materials utilized for their development.

3.3.3. FTIR Analysis

The chemical composition of DS and DS-MgO-NPs together with the raw materials utilized for their development is represented in Figure 4. For DS, the 3400 cm−1 band can be related to the stretching vibration of hydroxyl groups, whereas the bands at 2800 and 1200 cm−1 can be associated with the stretching of methyl groups. The peaks presented at 1250 cm−1 can correspond to sulfate esters, which are representative of κ-carrageenan. Similarly, the FTIR spectrum of DS-MgO-NPs demonstrates the stretching of hydroxyl and methyl groups at 3400 and 2800 cm−1, respectively. However, it also contained broad peaks related to the stretching (800–600 cm−1) and bending (500–400 cm−1) vibrations of Mg–oxygen bonds. Comparably, the FTIR spectra of PEO 303 and glycerin demonstrated the stretching of hydroxyl, methyl, and carbonyl groups. Regarding the analysis of DS, it was noted that the signal intensity of the peaks decreased to 648.55–775.28 cm−1, which may be associated with the carbonyl stretching and glycoside bond interaction between κ-carrageenan and PEO 303. Similar findings have been reported in other studies involving the use of these polymers for hydrogel membrane development [48].

4. Biological Activities

4.1. Antibacterial Activity

As illustrated in Figure 5, treatment with DS did not inhibit the growth of E. coli and P. aeruginosa. Similar results were obtained after bacteria cultures were exposed to DS-MgO-NPs. The center of the plates represents the zone of inhibition of amikacin, the positive control utilized in this study toward E. coli and P. aeruginosa. For the former, treatment with amikacin inhibited 23 mm of its growth, whereas for the latter, it resulted in a 21 mm inhibition zone.

4.2. Treatment of A. salina with DS and DS-MgO-NPs

As represented in Figure 6, A. salina nauplii exposed to DS and DS-MgO-NPs did not present significant anatomical aberrations. In addition, treatment with the developed films did not decrease their viability.

5. Discussion

Biopolymers such as polysaccharides, proteins, and nucleic acids constitute attractive sources for designing structures for biomedical applications. Polysaccharides are complex carbohydrates composed of long chains of monosaccharide units linked together by glycosidic bonds [49]. Biologically, polysaccharides and their derivatives are recognized because of their capacity to participate in energy storage, structural support, cell recognition, the modulation of cell signaling pathways, cell adhesion, development, and the upregulation of the growth of beneficial gut microbiota events [50,51,52]. Biomedically, the intrinsic therapeutic features of polysaccharides are leveraged to rationally design structures for drug delivery [53], wound healing, vaccine development [54], skin, bone, and cartilage engineering [55], and next-generation medical devices [56].
Optimization is a systematic process by which the properties and performance of NMs are assessed for specific applications. Current approaches to optimize the development of NMs, such as bimetallic NPs [57], carbon nanotubes [58], and smart hybrid composites [59], encompass factorial experimental design, response surface methodology, molecular dynamics simulation, and machine learning. In contrast to other statistical methods, the 2k factorial experimental design evaluates the effect factors on outcome variables categorizing it into low and high.
Here, the evaluated factors consisted of κ-carrageenan, PEO, and PEO MW content, and the outcome variables included tensile strength, swelling capacity, and thickness. According to the factorial design analysis performed in Minitab® (V-21.1.0) software, it was evidenced that the κ-carrageenan and PEO content highly influence the thickness of films (see Figure 1). In the same context, it was observed that the content of κ-carrageenan and PEO MW affects the tensile strength and swelling capacity of the developed films (see Figure 1). The cross-linking of κ-carrageenan and PEO is based on the hydrogen bonding between the hydroxyl groups of both polymers, which leads to the formation of a network-type structure. The results illustrated in Figure 1 can be associated with the fact that higher concentrations of κ-carrageenan and PEO MW can occur in thicker films due to an extensive network formation. The κ-carrageenan and PEO MW content can also increase the developed films’ tensile strength since it can enhance the entanglement of the polymer chains, number of intermolecular forces, and resilience under tensile stress [60].
According to the optimization process, one formulation of κ-carrageenan films was selected, designated as sample 4 in Table 2, and utilized to incorporate MgO-NPs. Notably, the amount of MgO-NPs was not considered during the 2k factorial experimental design since this study envisioned optimizing the development of films based on their κ-carrageenan and PEO MW content. Together with this, the amount of MgO-NPs was selected based on other reports where it has been demonstrated that adding 1% of metal-based NPs (e.g., ZnO- and FexOy-NPs) can occur in films with improved physicochemical and mechanical features and antimicrobial and antioxidant performance [60,61].
The developed films were analyzed for their tensile strength, swelling capacity, and thickness. Tensile strength is appraised as the maximum tensile stress a material can withstand before breaking, deforming, or fracturing. For polymer-based films designed for biomedical applications such as wound healing, evaluating their tensile strength is required to ensure their integrity over time, functional barrier capability, and biological performance (e.g., antimicrobial, antioxidant, or anti-inflammatory) during their application. For instance, it was noted that samples containing 2.5% κ-carrageenan and 0.5% PEO N303 resulted in 129 kPa, whereas films synthesized with 0.75% PEO N303 occurred in 87 kPa. On the other hand, it was determined that the tensile strength of films containing 0.5% of κ-carrageenan and 0.5% of PEO N80 occurred at 92 kPa. In contrast, samples of 1.5% of κ-carrageenan and 0.75% of PEO N80 exhibited 46 kPa. When films were developed with 2.5% κ-carrageenan and 0.5% PEO N80, the calculated tensile strength of the obtained material was 214 kPa.
Swelling capacity is the ability of diverse materials to absorb moisture and expand without dissolving. For developing wound-healing dressing systems from polymeric films, evaluating their swelling capacity is necessary to correlate their possible capacity to facilitate cell migration and tissue regeneration, absorb excess exudate, enhance the release of bioactive compounds, and confer protection against external irritants. Regarding their swelling capacity, it can be observed in Table 2 that films with 0.5 and 1.5% κ-carrageenan tended to disintegrate completely, while those with 2.5% of the same polymer exhibited 3.76–52.01% swelling capacity. It was noted that the swelling capacity of sample 4 increased in a time-dependent manner (0–30 min and 4–24 h). Even though no statistical differences were determined between the retrieved values (61.8 ± 1.86–64.10 ± 3.87%), it was observed that the κ-carrageen film softened and became fragile in the medium due to manipulation. Still, when dried, the sample maintained its original appearance. This is presented in Figure S1 and Table S1 and can be associated with the fact that the content of MgO-NPs influenced the capability of the film to absorb water. The differences between the swelling capacity and content of κ-carrageenan can be associated with the presence of PEO and its derivatives since it was recorded that samples with 0.5% of PEO N303 or PEO N80 presented 52.01% and 0% swelling capacity. In contrast, films containing 0.75% of the same PEO MW occurred in 34.52 and 15.89% swelling capacity, respectively. Interestingly, it was assessed that 1.0% of PEO N303 affected its swelling capacity, resulting in 3.76%, whereas it benefited the swelling capacity of films containing 1.0% of PEO N80, occurring in 24.88%. The water solubility was only determined for sample 4 based on the optimization results. In this regard, it was recorded that sample 4 presented 14.64 ± 74% mass loss in solution after 24 h, indicating its suitability to be applied for wound-healing applications by being slowly solubilized and controllably releasing the entrapped MgO-NPs under the physiological conditions of the wound site.
Thickness is related to measuring how wide a film or material is, and it can be expressed in micrometers or millimeters. For wound-healing systems, the thickness of films is of great significance since it influences their ability to act as competent barriers against potential pathogenic microorganisms or contaminants, moisture retention or evaporation management, durability under physiological stress, comfort and wearability, and adhesion efficiency. When evaluated for their thickness, it was recorded that the content of κ-carrageenan and PEO derivatives also alters the thickness of the obtained materials. For example, it was seen that samples containing 2.5, 1.5, or 0.5% of κ-carrageenan and varying proportions of PEO N303 or PN80 occurred in films with thicknesses ranging from 0.05 to 0.29, meaning that films with the highest thickness contained 2.5% of κ-carrageenan and 1.0% of PEO N80. In the same context, it was noted that films with lower thickness contained 2.5% of κ-carrageenan and 0.5–0.75% of PEO N80.
The variabilities between the swelling capacity of the developed κ-carrageenan films can be correlated to the molecular weight of the PEO used since it has been documented that higher-molecular-weight PEO derivatives lead to greater water retention due to their larger chain structure. Comparably, the differences among samples can be associated with thickness and its influence on restricting or enhancing water diffusion patterns. The variabilities of tensile strength between films can be associated with the fact that PEO 303 has a higher molecular weight. Still, parameters such as type, molecular weight, degree of crystallinity, and processing conditions can also influence the tensile strength of the developed materials.
Infectious diseases can be caused by pathogenic microorganisms such as bacteria, fungi, viruses, and parasites. In contrast to other disorders, infections caused by bacteria are challenging to treat due to their multiple drug-resistance mechanisms. E. coli is a Gram-negative bacterium that, in the clinical pipeline, can cause diarrhea, vomiting, sepsis, or neonatal meningitis [62]. During wound healing, E. coli is considered an opportunistic pathogen that can lead to localized pain, redness, or severe disorders such as peritonitis. P. aeruginosa is another opportunistic Gram-negative pathogen related to respiratory, urinary, and wound infections [63]. The persistence of P. aeruginosa in contaminated surfaces such as medical equipment is associated with delayed epithelialization, inflammation, and increased pain [64]. Here, it was recorded that treatment did not inhibit the growth of E. coli nor P. aeruginosa when slices of DS or DS-MgO-NPs were placed into Petri dishes.
In contrast to these results, recent scientific evidence suggests that κ-carrageenan-based films can inhibit the growth of bacteria strains (e.g., Staphylococcus aureus, Bacillus cereus, and Salmonella enterica) when incorporated into melanin pigment [65], polyoxometalates [66], or orange essential oil [67]. Even though there are no studies where κ-carrageenan-based films have been incorporated into MgO-NPs for potential wound treatment, it has been reported that, when incorporated with nanostructures such as copper NPs, κ-carrageenan-based films can decrease the viability of E. coli and S. aureus together with near-infrared irradiation [68]. Similarly, it has been reported that κ-carrageenan-based films reinforced with zinc oxide (ZnO-NPs) can decrease the growth of Listeria monocytogenes [69]. Comparable results have been documented in another study utilizing iron oxide NPs [70]. The variabilities between the retrieved results in this work and other studies can be associated with experimental differences during the development of films, the NPs synthesis route, and the implemented antibacterial assay. For instance, it has been reported that NPs with small sizes (<50 nm) exhibit greater antibacterial activity due to their high surface area-to-volume ratio, enhancing their interaction with bacterial components. The larger size (102.60 ± 23.34 nm) of the synthesized MgO-NPs in work by a previous research group might have limited their activity toward the cultured strains under the proposed conditions [46]. In the same context, it has been documented that green synthesis routes are preferred to synthesize antibacterial NPs since they can act in synergy with extracts from natural sources such as Falcaria vulgaris to inhibit the growth of pathogenic bacteria involved in wound infections [71]. Similar effects have been unveiled when additional elements such as cobalt, copper, and zinc have been used as dopants during the synthesis of MgO-NPs [72]. Regarding film features, it has been reported that films with high porosity tend to exhibit enhanced bacterial interaction [73]. In contrast, films with high tensile strength can exert prolonged contact with bacteria, leading to their death.
A. salina, or brine shrimp, belongs to the family Artemiidae. They are an elongated and translucent species frequently found in hypersaline environments (e.g., salt lakes, salt flats, and coastal lagoons). Compared to other species, A. salina poses significant ecological importance as a food source for invertebrates and as a research model to investigate genetic diversity, microbial interactions, and toxicity of organic and inorganic compounds. The use of A. salina nauplii to evaluate the biocompatibility of polymeric films is limited; however, it can provide important data about their capacity to cause growth and development aberrations, modify behavior, and have a potential environmental impact. Here, it was determined that DS and DS-MgO-NPs did not decrease the viability of A. salina nauplii nor cause anatomical aberrations. In both cases, the retrieved results suggest the compatibility of the developed κ-carrageenan based films. The evidence about the evaluation of the in vivo toxicity of κ-carrageenan-based films is limited; however, it has been reported that, when incorporated with ZnO-NPs, they can decrease the survival rate and deposit in the gut regions of A. salina at 25–500 mg/mL [74]. Table 3 presents a comparative overview of the characterization features of κ-carrageenan-based films developed in other studies.

6. Conclusions

This study reported the synthesis of κ-carrageenan films through a 2k factorial experimental design, which evidenced that the content of κ-carrageenan and PEO highly influences the tensile strength, swelling capacity, and thickness of the developed films. Considering optimization results, samples utilized in this work were termed DS and DS-MgO-NPs. Through SEM analyses, it was evidenced that DS-MgO-NPs exhibited an elongated structure and heterogeneous distribution of MgO-NPs. TGA and DTA studies revealed the thermal stability of DS and DS-MgO-NPs as they exhibited mass loss patterns at >200 °C. FTIR spectroscopy evaluation confirmed the chemical composition of films, indicating the presence of characteristic functional groups such as hydroxyl, methyl, and sulfate esters. The antibacterial activity of DS and DS-MgO-NPs was poor since they did not inhibit the growth of E. coli and P. aeruginosa. In the same context, the developed films did not affect the survival rate or morphology of A. salina nauplii, suggesting their biocompatibility. This study reports, for the first time, the development and optimization of κ-carrageenan films incorporated with MgO-NPs for the potential treatment of chronic wounds and validates their biological performance through in vitro and in vivo models. Further approaches are required to improve the activity of the optimized formulation by modifying the synthesis route and the amount of added MgO-NPs, altering the surface chemistry of the developed films by inserting positively charged groups that can disrupt bacterial membranes, and revising the cross-link of the synthesized matrix to ensure the sustained release and activity of the added substance. Considering the retrieved data, integrating additional spectroscopy approaches, such as energy-dispersive X-ray spectroscopy, is required to confirm the uniform distribution of the incorporated NPs and correlate it with the recorded biological activities. In addition to film characterization, other variables that must be considered are its wettability, contact angle measurement, roughness, and porosity, correlating them with their biological performance.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/polysaccharides6020045/s1, Figure S1: Representative images of the swelling capacity of sample 4 after 0, 5, 15, and 30 min and 4 and 24 h; Table S1: Swelling capacity (%) of sample 4 after 0, 5, 15, and 30 min and 4 and 24 h.

Author Contributions

Conceptualization, J.L.M.-M. and S.A.B.-C.; validation, J.L.M.-M., E.R.L.-M., and S.A.B.-C.; investigation, L.R.-V., J.L.M.-M., E.R.L.-M. and S.A.B.-C.; writing—original draft preparation, L.R.-V., J.L.M.-M. and S.A.B.-C.; writing—review and editing, E.R.L.-M. and S.A.B.-C.; visualization, L.R.-V. and E.R.L.-M.; supervision, S.A.B.-C.; funding acquisition; J.L.M.-M., E.R.L.-M. and S.A.B.-C.; project administration, J.L.M.-M. and S.A.B.-C. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Acknowledgments

COECYTJAL partially funded this work through the FODECIJAL 2023 Program and Tecnologico de Monterrey through the Nanotechnology and Devices Design Research Group.

Conflicts of Interest

The authors declare no conflicts of interest.

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Polysaccharides 06 00045 sch001
Scheme 1. Development process of κ-C-PEO-MgO-NPs films.
Scheme 1. Development process of κ-C-PEO-MgO-NPs films.
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Polysaccharides 06 00045 g001
Figure 1. Effect of the outcome variables κ-carrageenan (κ-C) and polyethylene oxide (PEO) on the (a) thickness, (b) tensile strength, and (c) swelling capacity of κ-C-PEO films.
Figure 1. Effect of the outcome variables κ-carrageenan (κ-C) and polyethylene oxide (PEO) on the (a) thickness, (b) tensile strength, and (c) swelling capacity of κ-C-PEO films.
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Polysaccharides 06 00045 g002
Figure 2. SEM analysis of (A–C) DS and (D–F) DS-MgO-NPs. For this evaluation, only sample 4 was studied considering optimization analyses.
Figure 2. SEM analysis of (A–C) DS and (D–F) DS-MgO-NPs. For this evaluation, only sample 4 was studied considering optimization analyses.
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Polysaccharides 06 00045 g003
Figure 3. (A–F) TGA and DTA analyses of DS and DS-MgO-NPs utilized for their preparation. The analysis of raw materials utilized for their development is also depicted in this figure: κ-C; κ-carrageenan; PEO-303; polyethylene oxide 303; MgO-NPs, magnesium oxide nanoparticles. The analyzed samples (DS and DS-MgO-NPs) correspond to sample 4, which was selected according to optimization evaluation.
Figure 3. (A–F) TGA and DTA analyses of DS and DS-MgO-NPs utilized for their preparation. The analysis of raw materials utilized for their development is also depicted in this figure: κ-C; κ-carrageenan; PEO-303; polyethylene oxide 303; MgO-NPs, magnesium oxide nanoparticles. The analyzed samples (DS and DS-MgO-NPs) correspond to sample 4, which was selected according to optimization evaluation.
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Figure 4. FTIR analysis of the raw materials utilized to develop DS and DS-MgO-NPs. The composition of DS and DS-MgO-NPs is associated with sample 4, which was selected based on optimization analyses.
Figure 4. FTIR analysis of the raw materials utilized to develop DS and DS-MgO-NPs. The composition of DS and DS-MgO-NPs is associated with sample 4, which was selected based on optimization analyses.
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Polysaccharides 06 00045 g005
Figure 5. Antibacterial activity DS and DS-MgO-NPs against E. coli and P. aeruginosa. The evaluated samples (DS and DS-MgO-NPs) correspond to sample 4, which was selected based on optimization analyses.
Figure 5. Antibacterial activity DS and DS-MgO-NPs against E. coli and P. aeruginosa. The evaluated samples (DS and DS-MgO-NPs) correspond to sample 4, which was selected based on optimization analyses.
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Figure 6. Microscopy images of A. salina treated with DS and DS-MgO-NPs. The formulation of the tested samples corresponds to sample 4, which was selected based on optimization results.
Figure 6. Microscopy images of A. salina treated with DS and DS-MgO-NPs. The formulation of the tested samples corresponds to sample 4, which was selected based on optimization results.
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Table 1. Outcome variables considered for the development of κ-C-PEO films.
Table 1. Outcome variables considered for the development of κ-C-PEO films.
FactorsLevelsOutcome Variables
LowHigh
κ-carrageenan concentration0.52.5Tensile strength
PEO concentration0.51.0Thickness
PEO MWN80N303Swelling capacity
Abbreviations: PEO, polyethylene oxide; MW, molecular weight.
Table 2. Tensile strength, swelling capacity, and thickness of κ-carrageenan and PEO films.
Table 2. Tensile strength, swelling capacity, and thickness of κ-carrageenan and PEO films.
Sampleκ-CarrageenanPEO (%)PEO
MW		Tensile Strength (kPa)Swelling
Capacity (%)		Thickness (mm)
12.50.50N8021444.420.05
22.50.75N3038734.520.10
42.50.50N30312952.010.19
52.51.00N303923.760.13
72.51.00N808724.880.29
81.50.75N804615.890.06
90.50.50N80920.000.06
Table 3. Comparative overview of characterization features and antibacterial outcome of κ-carrageenan-based films.
Table 3. Comparative overview of characterization features and antibacterial outcome of κ-carrageenan-based films.
SampleLoaded
Substance		Characterization
Features		Reference
κ-carrageenan and glycerolPA
  • Thermal analysis revealed the thermal decomposition of κ-carrageenan and PA at 250 °C.
  • The incorporation of PA caused a rough surface in films and decreased moisture absorption.
  • FTIR evaluation indicated the interaction of κ-carrageenan with water and the successful incorporation of PA.
[33]
κ-carrageenan and HPMCAS CUR
  • SEM analysis revealed the formation of pores in the internal structure and the amorphous state of films.
  • Mass loss patterns from films containing CUR were noted at 375 °C.
  • DSC demonstrated glass transition and melting at 56.30 and 145 °C, respectively.
[34]
κ-carrageenan Ag-NPs
  • The WCA of the developed films was ≥90°, indicating their hydrophobic behavior.
  • The tensile strength of films varied from 10 to 15 kPa.
[45]
κ-carrageenan and OHSSPH-NPs
  • The incorporation of SPH-NPs increased the tensile strength of films (13.61 MPa), WCA (87.85°), and improved 46.15% the elongation at break.
  • According to SEM analysis, adding NPs contributed to surface uniformity and density.
[75]
κ-carrageenanBITC
  • The addition of BITC increased 33.70% the tensile strength and the maximum thermal degradation temperature by 48.80%.
  • Contrarily, incorporating BITC decreased the elongation at break, water vapor, and oxygen permeability by 33.80, 60.10, and 42%, respectively.
[76]
κ-carrageenan and glycerolGreen synthesized Cu-NPs with Argemone mexicana extract
  • Varying concentrations of Cu-NPs enhanced the thickness (42.80–47.60 mm), tensile strength (37.38–39.72 MPa), elastic modulus (5.66–8.26 GPa), and elongation at break (7.82–12.23%) of films.
  • TGA analysis demonstrated the degradation of glycerol and κ-carrageenan at 300 °C. The incorporation of Cu-NPs increased the thermal degradation temperature to 241.66 °C.
  • The incorporation of Cu-NPs decreased the WVP (1.08 × 10−9 g m/m2) and MC (10.35%) of films.
[77]
κ-carrageenan and gelatinMekwiya date palm seeds extract
  • Thermal degradation analysis revealed that films presented a residual weight of 27.99% when the extract was incorporated.
  • The addition of extract also decreased the film’s tensile strength from 24.19 to 8.24 MPa but increased its elongation at the breaking point to 46.17%.
[78]
Abbreviations: PA, proanthocyanin; HPMCAS, hydroxypropylmethylcellulose succinate acetate; DSC, differential scanning calorimetry; OHS, potato oxidized hydroxypropyl starch; WCA, water contact angle; SPH-NPs, Broccoli leaf polyphenol/soybean protein isolate/hydroxypropyl methylcellulose composite nanoparticles; BITC, benzyl isothiocyanate; Ag-NPs, silver nanoparticles; Cu-NPs, copper nanoparticles; WVP, water vapor permeability; MC, moisture content.
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Rodríguez-Vicens, L.; Mejía-Méndez, J.L.; López-Mena, E.R.; Bernal-Chávez, S.A. Development of κ-Carrageenan Films Reinforced with Magnesium Oxide Nanoparticles for the Potential Treatment of Chronic Wounds: In Vitro and In Vivo Insights. Polysaccharides 2025, 6, 45. https://doi.org/10.3390/polysaccharides6020045

AMA Style

Rodríguez-Vicens L, Mejía-Méndez JL, López-Mena ER, Bernal-Chávez SA. Development of κ-Carrageenan Films Reinforced with Magnesium Oxide Nanoparticles for the Potential Treatment of Chronic Wounds: In Vitro and In Vivo Insights. Polysaccharides. 2025; 6(2):45. https://doi.org/10.3390/polysaccharides6020045

Chicago/Turabian Style

Rodríguez-Vicens, Lesly, Jorge L. Mejía-Méndez, Edgar R. López-Mena, and Sergio A. Bernal-Chávez. 2025. "Development of κ-Carrageenan Films Reinforced with Magnesium Oxide Nanoparticles for the Potential Treatment of Chronic Wounds: In Vitro and In Vivo Insights" Polysaccharides 6, no. 2: 45. https://doi.org/10.3390/polysaccharides6020045

APA Style

Rodríguez-Vicens, L., Mejía-Méndez, J. L., López-Mena, E. R., & Bernal-Chávez, S. A. (2025). Development of κ-Carrageenan Films Reinforced with Magnesium Oxide Nanoparticles for the Potential Treatment of Chronic Wounds: In Vitro and In Vivo Insights. Polysaccharides, 6(2), 45. https://doi.org/10.3390/polysaccharides6020045

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