Functional Chitosan–Collagen Microaerogels Incorporating Bursera microphylla Fruit Extract with Antibacterial Properties

Functional Chitosan–Collagen Microaerogels Incorporating Bursera microphylla Fruit Extract with Antibacterial Properties | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Functional Chitosan–Collagen Microaerogels Incorporating Bursera microphylla Fruit Extract with Antibacterial Properties Víctor Alonso Reyna-Urrutia, Ramón Enrique Robles Zepeda, Miriam Estevez, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8913713/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 21 You are reading this latest preprint version Abstract Bursera microphylla A. Gray is a native medicinal plant from northwestern Mexico whose fruit extract has demonstrated relevant bioactivity. Chitosan (Cs) and collagen (Co) are naturally derived polymers widely recognized for their biodegradability, biocompatibility, and suitability for environmentally friendly biomedical applications. In this study, Cs/Co microaerogels loaded with B. microphylla fruit extract were synthesized, physicochemically characterized, and evaluated for in vitro degradation, cytotoxicity, and antibacterial activity. Three-dimensional Cs/Co hydrogels were prepared by physical crosslinking using ammonium hydroxide, followed by lyophilization and mechanical grinding to obtain microaerogels. Morphological and structural characterization was conducted using scanning electron microscopy (SEM), optical microscopy (OM), Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and moisture absorption assays. Cytotoxicity was assessed in RAW 264.7 macrophages by MTT assay, while antibacterial activity was evaluated against Staphylococcus aureus . In vitro degradation was analyzed under physiological conditions in the presence of lysozyme (Lz). The resulting microaerogels exhibited irregular morphologies with rounded protrusions and particle sizes mainly ranging from 45 to 90 µm. FTIR spectra confirmed the preservation of native functional groups without new chemical bond formation, while TGA indicated adequate thermal stability of the encapsulated extract. The microaerogels showed hydrophilic properties and a reduced lysozyme-mediated degradation rate due to the presence of collagen and the encapsulated extract. Compared to the free extract, Cs/Co microaerogels displayed lower cytotoxicity, reducing macrophage viability by only 32% at 200 µg/mL. Additionally, extract-loaded microaerogels significantly decreased S. aureus viability by 37% and 57% for the 0.5% and 1% extract formulations, respectively (p < 0.05). These results highlight the role of collagen in modulating structural stability and support the potential of Cs/Co microaerogels loaded with B. microphylla fruit extract as sustainable biopolymeric delivery systems for antibacterial applications. Bursera microphylla chitosan collagen microaerogels encapsulation antibacterial performance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Bacterial infections represent a major public health concern worldwide [ 1 ]. It is estimated that within the next 30 years, antibiotic-resistant bacterial infections could become the leading cause of mortality globally [ 2 ]. The accelerated emergence of resistance has significantly reduced the effectiveness of conventional antibiotic therapies, resulting in infections that are increasingly difficult to control. Among clinically relevant pathogens, Staphylococcus aureus remains one of the most frequently isolated microorganisms in infectious processes [ 3 ]. Consequently, the World Health Organization (WHO) has classified S. aureus as a high-priority pathogen due to its alarming resistance to antibiotics [ 4 ]. This scenario underscores the urgent need for alternative, sustainable strategies capable of addressing bacterial infections while reducing reliance on conventional antibiotics. In this context, polymer-based encapsulation systems have gained increasing attention, particularly those derived from natural and environmentally friendly materials. Chitosan (Cs) is a polysaccharide industrially obtained through the alkaline deacetylation of chitin, a renewable resource abundantly found in the exoskeletons of crustaceans such as shrimp and crabs [ 5 ]. Cs is a non-toxic, biodegradable biopolymer with well-documented biomedical properties, including hemostatic, antibacterial, biocompatible, and cytocompatible activities. Additionally, Cs is recognized by the U.S. Food and Drug Administration (FDA) as a Generally Recognized as Safe (GRAS) material, and its low cost and commercial availability further enhance its applicability [ 6 , 7 ]. Owing to its cationic nature in acidic environments, Cs enables the formation of diverse material architectures, such as nano- and microaerogels, emulsions, fibers, hydrogels, films, and membranes, making it a versatile matrix for the encapsulation of plant extracts and other bioactive compounds [ 8 – 10 ]. Collagen (Co) is another naturally derived polymer of high relevance due to its biocompatibility, biodegradability, and favorable interaction with biological tissues [ 11 ]. Co-based delivery systems have demonstrated the ability to encapsulate active compounds while enabling controlled and sustained release profiles [ 12 ]. Among the available encapsulation approaches, ionic gelation stands out as a promising technique, as it avoids the use of chemical crosslinkers and preserves the intrinsic biocompatibility of the polymeric matrix [ 5 , 6 ]. This method employs ammonium hydroxide to induce physical crosslinking, maintaining free amino groups in Cs that are essential for its functional and bioactive properties, while subsequent lyophilization ensures material stability and preservation [ 7 ]. Consequently, Cs/Co systems represent sustainable carriers for bioactive compounds derived from medicinal plants (18,19,25). Bursera microphylla A. Gray (Burseraceae), commonly known as “torote,” “copal,” or “elephant tree,” is a medicinal species native to the desert regions of northern Mexico and the southwestern United States, particularly within the Sonoran Desert [ 15 , 16 ]. In traditional Mexican medicine, the Seri people utilize the bark, fruits, and leaves to treat wounds, sore throats, and headaches, while tinctures are used for gum sores and dental abscesses, and stems and leaves are applied for anti-inflammatory and urinary discomfort treatments [ 16 , 17 ]. In vitro studies have demonstrated that B. microphylla fruit extracts inhibit inflammatory mediators such as nitric oxide (NO) and tumor necrosis factor alpha (TNF-α) in lipopolysaccharide-stimulated RAW 264.7 macrophages [ 18 , 19 ]. Although Cs-based microsystems encapsulating B. microphylla extract have shown antibacterial activity against S. aureus , the development of Cs/Co microaerogels is expected to further enhance structural stability, biocompatibility, and controlled release profile. Therefore, this study aimed to synthesize and characterize Cs/Co-based microaerogels loaded with B. microphylla fruit extract as sustainable polymeric systems designed to enhance tissue compatibility and antimicrobial performance. Materials and Methods Materials Cs (Catalog No. 448877) and type I Co (Catalog No. C9791) derived from calf skin, used for the preparation of microaerogels, were sourced from Sigma-Aldrich. Ethyl alcohol (EA, 94.9–96%, Catalog No. 493538) was utilized to extract the B. microphylla plant material. Ammonium hydroxide (AH, 28% NH₃ in H₂O, ≥ 99.99% trace metals basis, Catalog No. 338818) and glacial acetic acid (AA, ≥ 99.5% purity, Catalog No. 33209) were employed in the preparation of microaerogels. Magnesium nitrate hexahydrate (MNH, 99%, Catalog No. 237175) and sodium sulfate decahydrate (SSD, 99%, Catalog No. 403008) were applied for relative humidity absorption measurements. MTT (bromuro de 3-(4,5-dimetiltiazol-2-il) (MTT, Catalog No. 88417), and isopropanol (Catalog No. 563935) were used in the in vitro assays. Lz (Catalog No. L6876) and Phosphate Buffered Saline (PBS, Catalog No. P4417) were used for enzymatic degradation tests. All solvents and reagents were obtained from Sigma-Aldrich (Toluca, Mexico). Plant Material and Extract Elaboration Specimens of B. microphylla (B) were collected in the state of Sonora, Mexico, specifically in the “Puerto Lobos” area (30.177211, − 112.664644) of the Caborca municipality during spring (April 30, 2023). The collected fruits were weighed and immersed in ethanol (1:10 w/v) at 25°C for 10 days, with daily manual agitation. The resulting extracts were filtered and concentrated using an IKA rotary evaporator (model RV-10B-S99) set at 125 rpm and 50°C. The ethanolic extracts were then dried and stored at 4°C until further use [ 19 ]. Obtaining Microaerogels Different solutions were prepared for the microencapsulation process, with and without B. microphylla extract: (1) Cs solution 2% (w/w) was prepared by dissolving it in 0.3 M glacial acetic acid at room temperature, followed by continuous stirring until complete dissolution was achieved [ 20 ]. This solution was subsequently used for the fabrication of Cs hydrogels. (2) Separately, a 1% (w/w) Co suspension was prepared in 0.1 M acetic acid under constant stirring for 24 h at room temperature. The resulting suspension was then incorporated into a 3% (w/w) Cs solution at an 80/20 (w/w) ratio (Cs/Co) to obtain Cs/Co hydrogel-forming systems. (3) Cs/Co solutions were mixed with 0.5 or 1.0% B. microphylla extract to obtain CsCoB-0.5 (99.5/0.5) and CsCoB-1.0 (99/1) (w/w ratio), respectively. The extract was incorporated into the Cs/Co mixture and stirred for 30 min [ 7 ]. Aliquots of the polymer suspensions were made in 10 mL beakers and placed inside a hermetically closed chamber, containing 100 mL of ammonium hydroxide. Physical cross-linking (gelation) was induced by ammonia diffusion for 24 h [ 21 ]. Subsequently, the hydrogels were washed with distilled water to eliminate ammonium acetate (CH 3 COONH 4 ), and the rest of the ammonium hydroxide (NH 4 OH) remained in the hydrogels until a pH of 7 was reached. Afterward, the hydrogels were placed in a refrigerator at a temperature of − 26°C for 24 h and subsequently introduced into a freeze dryer (SCIENTZ-10N LYOPHILIZER, Kansas City, MO, USA) for 24 h, at -58°C and a pressure of 1 Pa [ 22 ]. Once the aerogels were obtained, they were mechanically crushed for 5 min to obtain the microparticulate materials. Subsequently, the microaerogels were passed through a stainless-steel screening filter (Hightop brand) with a pore size of 400 µm Physicochemical Characterization of Microaerogels Scanning Electron Microscope (SEM) and Optical Microscopy (OM) The morphology and size of the microaerogels were examined using scanning electron microscopy (SEM) and optical microscopy (OM). SEM observations were performed with a Hitachi SU8230 microscope operating at 20–30 kV. Samples were placed on aluminum tape and coated with a thin gold layer using a Desk II high-vacuum coater (LLC). For OM analysis, a LABOMED CX model microscope equipped with a 12 V, 20 W lamp (Labo America, Inc., Fullerton, CA, USA) was employed. An OEM objective micrometer was utilized to determine the apparent dimensions of the microaerogels. Fourier Transform Infrared Spectroscopy (FTIR) Fourier Transform Infrared Spectroscopy (FTIR) is commonly employed to evaluate encapsulation processes, as it enables the identification of potential chemical interactions between the encapsulating polymer and the active compounds [ 23 – 25 ]. The spectra of the microaerogels were recorded using a Perkin Elmer Spectrum Two FTIR spectrometer (Fremont, CA, USA). Measurements were performed over a wavenumber range of 4000–650 cm⁻¹, with a resolution of 4 cm⁻¹ and an average of 100 scans. Thermogravimetric Analysis (TGA) Thermogravimetric Analysis (TGA) is a useful technique to evaluate thermal decomposition and the stability of formulations under elevated temperatures [ 26 , 27 ]. The TGA of the microaerogels was performed using a Mettler Toledo TGA/DSC 2 + thermal analyzer (Greifensee, Switzerland) under a nitrogen atmosphere. The samples were heated from 25 to 600°C at a constant rate of 10°C/min. Moisture Absorption The evaluation of humidity effects on formulation prototypes provides insight into their performance under challenging environmental conditions. Moisture absorption is assessed using a gravimetric technique that tracks changes in the water content of a polymer over time [ 28 ]. These properties also indicate whether the material exhibits hydrophilic or hydrophobic characteristics [ 29 ]. For this analysis, the freeze-dried microaerogels were stored at two controlled relative humidity (RH) levels: 40 ± 1% and 80 ± 1%, employing magnesium nitrate hexahydrate (Mg (NO₃)₂·6H₂O) and sodium sulfate decahydrate (Na₂SO₄·10H₂O), respectively. The mass of each sample was recorded from the start of the experiment until equilibrium was achieved. The moisture absorption percentage (M.A.) was calculated using Eq. ( 1 ): $$\:A\left(t\right)=\left(\frac{mt-m0}{m0}\right)\times\:100$$ 1 Here, m₀ represents the initial mass of the sample at the onset of moisture exposure, while mt corresponds to the mass of the sample after a period t (in hours). For each microaerogel, the mean and standard deviation values are calculated from triplicate measurements In vitro degradation The in vitro degradation of the scaffolds was evaluated at 37°C in a Phosphate Buffered Saline (PBS, pH 7.4) solution supplemented with 1.5 mg/mL of Lz [ 30 ]. This enzyme concentration was selected because it corresponds to the level typically present in human serum [ 31 , 32 ]. To maintain enzymatic activity, the Lz solution was replaced daily [ 33 ]. Scaffolds weighing approximately 7 mg were placed into vials containing 1 mL of the enzyme solution and incubated for a total of 28 days. At predetermined time points (7, 14, 21, and 28 days), the samples were removed from the solution, rinsed with distilled water, and subsequently freeze-dried for 24 h before being weighed [ 7 ]. The degradation rate was expressed as the percentage of mass loss of the scaffolds, calculated using the following Eq. ( 2 ): $$\:Mass\:loss\:\left(\%\right)=\left(\frac{mi-m\text{f}}{mi}\right)\times\:100$$ 2 Where mi is the initial scaffold weight and mf is the weight of the scaffold after degradation. In Vitro Analysis of Microaerogels Sample Sterilization The samples were sterilized by exposure to UV radiation for 20 min using a laminar flow hood (Labconco®, Class II type A2, Kansas City, MO, USA). Cell Culture Macrophages of the RAW 264.7 cell line, originally transformed by the Abelson leukemia virus, were kindly provided by Dr. Emil A. Unanue from the Department of Pathology and Immunology at Washington University, St. Louis, MO, USA. The RAW 264.7 murine macrophages (induced by the Abelson leukemia virus) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 5% heat-inactivated fetal bovine serum and 100 U/mL penicillin, using 25 cm² culture flasks. Cells were incubated in an Isotherm incubator (Thermo Fisher Scientific, Waltham, MA, USA) under controlled conditions of 5% CO₂, 37°C, and 95% relative humidity. Cell Viability Assay Once RAW 264.7 cells reached approximately 80% confluence, they were detached using enzymatic digestion with 3 mL of trypsin and then collected in DMEM. The cell suspension was centrifuged (Beckman Coulter® Allegra X-15R, SX4750 rotor) at 1700 rpm for 7 min at 4°C. After discarding the supernatant, the resulting cell pellet was re-suspended in DMEM. A suspension of RAW 264.7 cells (500,000 cells/mL, viability > 90%) was seeded in a 96-well plate (Costar, Corning, NY, USA) and incubated for 24 h. The cells were then exposed to microaerogels (0–200 µg/mL) for 24 h using the indirect method (microaerogels were previously incubated at pH 5 buffer for 4 h, and 100 µL of the resulting supernatant was used to treat the cells). Afterward, 5 mg/mL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added and incubated for 4 h. The resulting formazan crystals were dissolved in isopropanol, and absorbance was measured at 570–630 nm using an iMARK BIO-RAD® microplate reader [ 34 ]. Antibacterial Activity The antibacterial activity of the microerogels (Cs, CsCo, CsCoB-0.5, and CsCoB-1.0) was determined following the methodology previously described [ 35 ]. Bacterial strains were cultured overnight at 37°C and subsequently adjusted to a turbidity equivalent to the 0.5 McFarland standard (≈ 1 × 10⁸ CFU/mL). An aliquot of 0.1 mL of this suspension was transferred into 9.9 mL of Mueller-Hinton broth, yielding a final inoculum concentration of 1 × 10 8 CFU/mL. Microaerogels containing B. microphylla extract were mixed with saline solution (pH 5.5) at a 1:1 ratio with the bacterial suspension and incubated at 37°C for 24 h. Following incubation, 50 µL from each treatment was serially diluted (1:10) in sterile saline (0.85% w/v), and 10 µL aliquots from each dilution were plated on agar for colony enumeration. Plates were incubated at 37°C for an additional 24 h, and antibacterial activity was quantified as CFU/mL based on colony counts [ 35 ]. Statistical Analysis The data are presented as the mean ± standard deviation. Statistical comparisons were conducted using a one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test with GraphPad PRISM® software (version 6.0c). A value of p < 0.05 was regarded as statistically significant. Results and Discussion Physicochemical Characterization of Microaerogels Microaerogels Morphology The morphological features of the microaerogels (Cs, CsCo, CsCoB-0.5, and CsCoB-1.0) are shown in Fig. 1 . Optical microscopy (OM) and scanning electron microscopy (SEM) analyses revealed that the samples consist of non-spherical microaerogels exhibiting a heterogeneous surface morphology with pronounced irregularities. In the CsCoB-0.5 (Fig. 1 C, 1 G; indicated with red arrows) and CsCoB-1.0 (Fig. 1 D, 1 H; indicated with yellow arrows), oval-shaped structures can be observed on the walls forming the three-dimensional (3D) network of the designed encapsulation systems. These findings suggest that the extract is incorporated within the walls of the 3D polymeric microsystem. This implies that the formation of these spherical bodies can be associated with the integration of the extract into the encapsulating matrix. Similar behaviors have been previously reported in studies involving Cs based microsystems loaded with B. microphylla extract, suggesting the presence of the extract within the polymeric matrix [ 35 ]. Microaerogels Size Distribution The particle size analysis of the microaerogels (Cs, CsCo, CsCoB-0.5, and CsCoB-1.0) revealed a unimodal distribution, with predominant frequencies ranging between 45 µm and 90 µm for all formulations (Fig. 2 ). Regarding histogram skewness, the particle size distributions of CsCo and CsCoB-1.0 exhibited a slight leftward skew, while Cs and CsCoB-0.5 showed a rightward skew. These findings indicate more compact and uniform distributions, with uniformity indices of 68%, 80%, 74%, and 74% for Cs, CsCo, CsCoB-0.5, and CsCoB-1.0, respectively. These values represent an improvement compared to previous research focused on chitosan-based systems, where the predominant percentages were around 50% [ 22 , 35 ]. Such enhanced uniformity could potentially influence the bioactivity of the encapsulating systems. Structure for the Fourier Transform Infrared Spectroscopy (FTIR) Figure 3 displays the FTIR spectra of the microencapsulated systems. All spectra exhibited comparable patterns, highlighting the characteristic absorption bands attributed to Cs. A broad and intense band was identified between 3500 and 3300 cm⁻¹, corresponding to the stretching vibrations of hydroxyl (O–H) and amine (N–H) groups. Additional absorption peaks appeared at 2923 and 2880 cm⁻¹, which are associated with methylene (–CH₂–) group vibrations. A prominent band at 1653 cm⁻¹ was assigned to the carbonyl (C = O) stretching of amide I, while the band at 1580 cm⁻¹ was linked to the bending vibrations of amide II groups [ 21 , 36 , 37 ]. Finally, the region between 1200 and 500 cm⁻¹ showed signals characteristic of the polysaccharide backbone [ 38 ]. The main FTIR absorption bands and their corresponding assignments are summarized in Table 1 . Table 1 Summary of FTIR absorption bands observed in Cs/Co microaerogels loaded with Bursera microphylla fruit extract. Wavenumber (cm − 1 ) Functional group Zone I 3500 − 3300 O-H stretching N-H stretching Zone II 2920 2870 C-H stretching methylene groups Zone III 1730 1630 C = O stretching amide I 1550 C = O deformation of amide II 1380 Zone IV 1190 saccharide structure 1140 1060 1017 890 The characteristic FTIR absorption band at 1580 cm⁻¹, corresponding to the amide II region of pure Cs scaffolds (as discussed previously), exhibited a bathochromic shift toward higher wavenumbers in the CsCo, CsCoB-0.5, and CsCoB-1.0 formulations. This displacement toward the Co-specific amide II band at 1545 cm⁻¹ is indicative not only of successful Co incorporation within the polymeric matrix but also of molecular-level interactions, most likely hydrogen bonding between Cs and Co, consistent with previously reported findings [ 7 , 39 , 40 ]. The absorption band observed at 1451 cm⁻¹ is primarily associated with the bending vibrations of methylene (–CH₂) and methyl (–CH₃) groups, as well as the stretching vibrations of the C–N bond, characteristic of the Co component. The presence and consistency of this band across all Co containing formulations suggest a conserved molecular environment for Co, further supporting its structural integrity and uniform incorporation within the Cs-based matrix [ 41 ]. Furthermore, the FTIR spectra of the extract-loaded microaerogels (CsCoB-0.5 and CsCoB-1.0) displayed a distinct absorption band at 1710 cm⁻¹, which can be attributed to the stretching vibrations of carbonyl (C = O) functional groups, typically associated with aldehydes and ketones present in phytochemical constituents [ 35 ]. An additional band of increased intensity at 2923 cm⁻¹ was observed, corresponding to C–H stretching vibrations of aliphatic moieties, further suggesting the incorporation of B. microphylla phytocompounds into the polymeric network [ 42 ]. Spectral comparison between extract-loaded and unloaded formulations revealed no appearance of new functional groups, suggesting the absence of covalent interactions or chemical bonding between the chemical compounds of the extract and the Cs/Co matrix. This suggests that the structure of the phytoconstituents is preserved during the encapsulation process, which is an essential factor for preserving their bioactivity. Instead, the extract appears to be physically entrapped within the three-dimensional polymeric network of Cs; this suggests that the microaerogels function as controlled-release systems. The release kinetics are likely governed by environmental pH fluctuations, mediated by the reversible protonation/deprotonation of the Cs amino groups, facilitating controlled diffusion of the extract from the microaerogels core to the external medium [ 43 ]. Thermogravimetric Analysis (TGA) Thermogravimetric analysis (Fig. 4 ) was employed to assess the thermal stability of Cs, Co type I, and their corresponding microencapsulated formulations (CsCo) loaded with B. microphylla fruit extract. The results indicate that Co exhibits lower thermal stability compared to Cs. The thermogram for Co shows three distinct weight loss events: an initial 7% loss around 100°C, a second drop of 30% at approximately 185°C (indicated by the blue arrow), and a third loss of 15% near 295°C (between the blue and green arrows), a thermal profile consistent with previous findings in the literature [ 44 ]. The thermogram of B. microphylla extract showed a significant mass loss of nearly 90% between 148–500°C, reaching its peak decomposition around 300°C. In contrast, the microaerogels (Cs, CsCo, CsCoB-0.5, and CsCoB-1.0) displayed a markedly lower mass loss, approximately 10% at 100°C, primarily attributed to water evaporation. A second weight loss event was observed around 302°C (red arrow), corresponding to the thermal decomposition of saccharide structures in the Cs backbone [ 45 – 47 ]. Similar thermal behavior was noted in the formulations containing B. microphylla extract (CsCoB-0.5% and CsCoB-1.0%), suggesting that the Cs/Co matrix enhances the thermal stability of the encapsulated extract. This increased stability likely results from the protective effect of the polymeric network, which shields the bioactive components from degradation at elevated temperatures, findings in line with recent studies [ 22 , 35 ]. Moreover, the incorporation of Co into the Cs matrix forms a semi-interpenetrating polymer network (CsCo), which offers greater thermal resistance than either of the individual components [ 39 ]. Moisture Absorption The moisture absorption (MA) analysis revealed that the microaerogels (Cs, CsCo, CsCoB-0.5, and CsCoB-1.0) reached a maximum moisture uptake of 14–18% at a RH of 40 ± 1% (Fig. 5 ). Conversely, under an RH of 80 ± 1%, all microaerogels exhibited maximum absorption values ranging from 26–37%. These findings align with previous reports indicating that Cs, as the primary polymeric matrix, can achieve an equilibrium moisture absorption of approximately 41% under high RH conditions and around 20% at low RH [ 22 , 35 ]. Another study reported that in materials where Cs is the majority polymer in the mixture only represents 10% absorption under low RH conditions; however, in this research, the value was 14% [ 48 ]. These results confirm the hydrophilic nature of the microaerogels when exposed to elevated humidity levels. This behavior is advantageous under physiological conditions, as contact with biological fluids facilitates the release of the encapsulated extracts from the polymeric matrix. The hydrophilic character of the Cs matrix is attributed to the free amino groups within the Cs polymer chain, further confirming the absence of significant chemical interactions between B. microphylla extracts and the Cs structure [ 49 ]. In vitro degradation Lysozyme (Lz), an enzyme naturally present in wound exudates and biological fluids, plays a key role in the enzymatic degradation of Cs-based materials. Evaluating the in vitro degradation of the microaerogels in the presence of Lz provides valuable insight into their structural stability and expected biodegradation behavior under physiological conditions. This allows for a better understanding of their bioactivity upon contact with wounds or biological tissues. Specifically, Lz recognizes three acetylated units in the Cs backbone to initiate its enzymatic activity, thereby promoting material breakdown and the subsequent release of the encapsulated active compound [ 2 , 30 , 50 ]. Most of the investigations have been performed under accelerated conditions, such as elevated Lz concentrations and acidic pH values (below 6.2), where the enzyme exhibits its highest catalytic activity. In contrast, the present study utilized an Lz concentration comparable to that found in human serum (1.5 µg/mL), while maintaining physiological pH (7.4) and temperature (37°C), thereby aiming to more accurately mimic human body conditions [ 30 ]. As shown in Fig. 6 , the degradation kinetics revealed that the Cs microaerogels exhibited the highest mass loss, reaching approximately 24% after incubation with Lz. This pronounced degradation can be attributed to the higher availability of glycosidic bonds susceptible to enzymatic cleavage due to the absence of Co, which provides structural reinforcement. In contrast, the CsCo microaerogels showed a slower degradation rate, with a mass loss of about 17%, suggesting that the incorporation of Co enhances the structural integrity of the polymeric matrix by forming additional hydrogen bonds with Cs chains, thereby limiting Lz accessibility. When the B. microphylla extract was incorporated, both CsCoB-0.5 and CsCoB-1.0 formulations exhibited the lowest degradation rates, around 14% (p > 0.05). This reduced degradation may be related to the partial filling of the polymeric network by the encapsulated extract, which could hinder enzyme diffusion and substrate recognition, resulting in slower matrix degradation. Overall, these findings suggest that the inclusion of Co improves the mechanical and enzymatic stability of Cs microaerogels, while the encapsulation of the extract further decreases degradation through steric and diffusional effects. Such modulation of degradation is advantageous for achieving controlled release, as the Lz-mediated breakdown of Cs N-acetylglucosamine sequences allows gradual and predictable liberation of the bioactive compound. Consequently, topical administration to wounds emerges as the most suitable route, enabling localized delivery, minimizing systemic exposure, and exploiting the Lz-rich wound environment to sustain release and enhance healing through the presence of Co within the matrix [ 30 ]. Biological Properties of Microaerogels Cell Viability As shown in Fig. 7 , the microaerogel exhibited no significant cytotoxicity toward RAW 264.7 macrophages at concentrations below 50 µg/mL, maintaining cell viability above 90% across all treatments. However, at 200 µg/mL, formulations Cs, CsCo, CsCoB-0.5, and CsCoB-1.0 resulted in a reduction in cell viability to 83%, 87%, 83%, and 68%, respectively. These results align with those previously reported by Torres-Moreno et al. (2022), where B. microphylla stem, fruit, and leaf extracts preserved approximately 90% cell viability at comparable concentrations [ 18 ]. On the other hand, the CsCoB-0.5 and CsCoB-1 microaerogels showed a greater reduction in cell viability compared to that previously reported for Cs microaerogels loaded with the fruit extract. At 200 µg/mL, CsCoB-1 reduced cell viability by 32%, whereas the previous study observed that Cs microaerogels loaded with 1% B. microphylla fruit extract decreased cell viability by 16%. These results indicate that incorporating the extract into a collagen-based matrix increases its cytotoxicity. This effect may be attributed to the three-dimensional organization of the polymer matrix, which, due to its less compact structure and greater network interconnectivity, could facilitate the rapid release of the B. microphylla extract. Although the encapsulation of B. microphylla extract in Cs based microaerogels has demonstrated favorable biocompatibility, further research is still needed to elucidate possible adverse effects, such as immunogenicity or hypersensitivity responses, to validate its safety profile for future biomedical applications [ 35 ]. Antibacterial Evaluation The antibacterial activity of the microaerogels loaded with Bursera microphylla extract is presented in Fig. 8 . These findings demonstrate a significant reduction in microbial load across all tested microaerogels, with CsCoB-0.5 and CsCoB-1.0 exhibiting the strongest antibacterial effects. Specifically, these microaerogels achieved logarithmic reductions of 37.8% and 57.7% ( p < 0.05), respectively, while CsCo and Cs showed moderate decreases of 15.6 and 9.7%, respectively, relative to the control. These results suggest that the microaerogels successfully release the encapsulated extract and its active compounds, allowing interaction with S. aureus and inducing bacterial death. The results obtained in this study suggest that the presence of Co in the encapsulating matrix produces a partial solubilization of Cs, promoting the release of the extract from the polymeric matrix [ 51 ]. It is context, it is important to highlight that S. aureus remains one of the most clinically relevant Gram-positive pathogens, frequently associated with both hospital-acquired and community-acquired infections, and is well known for its remarkable capacity to develop resistance to various antibacterial therapies [ 52 ]. Therefore, the significant antibacterial activity observed for the extract-loaded microaerogels is particularly relevant, as it addresses a pathogen of high clinical concern and reinforces the potential applicability of these systems as alternative or complementary antibacterial strategies. The antibacterial effect observed from B. microphylla extract may be related to its phytochemical composition. Previous studies have demonstrated that the fruit extract contains a variety of phenolic compounds, including flavonoids, phenolic acids, and lignans [ 18 , 19 ]. These bioactive constituents have shown potent antibacterial activity against S. aureus , primarily through mechanisms involving inhibition of bacterial enzymes and toxins, disruption of the cell wall and membrane, interference with metabolic pathways, induction of DNA fragmentation, and suppression of virulence gene expression [ 53 – 55 ]. Based on these promising results, future studies should evaluate the efficacy of these microaerogels against other bacterial strains, including antibiotic-resistant clinical isolates, as well as in vivo models to evaluate their effectiveness in infected wounds. This could open new avenues for the development of alternative antibacterial therapies based on Cs/Co polymeric matrices. Conclusions This study demonstrated the successful development of chitosan/collagen (Cs/Co) type I microaerogels loaded with B. microphylla fruit extract as a sustainable biopolymeric platform with antibacterial activity against S. aureus . The incorporation of Co as a structural component significantly influenced the physicochemical behavior of the microaerogels, particularly in terms of surface morphology, hydrophilicity, and resistance to enzymatic degradation, when compared with Cs only systems. These features are especially relevant for environmentally friendly polymeric materials intended for biomedical applications, as they contribute to enhanced stability and functionality while maintaining biodegradability. The use of naturally derived polymers and a plant-based bioactive extract highlights the environmental relevance of the proposed system, supporting its potential within the framework of green materials and sustainable polymer engineering. Moreover, the reduced cytotoxicity observed for the encapsulated extract, together with the improved antibacterial performance of Cs/Co microaerogels, underscores the advantages of polymeric encapsulation in modulating bioactivity and biological response. Although the results are promising, further investigations are required to fully elucidate the in vivo antibacterial efficacy and long-term safety of the CsCoB-0.5 and CsCoB-1.0 formulations. Comprehensive in vitro and in vivo studies will be essential to validate their pharmacological properties and to support the future development of these systems as environmentally responsible biopolymeric carriers for the treatment of bacterial infections. Abbreviations The following abbreviations are used in this manuscript: Cs Chitosan SEM Scanning electron microscopy OM Optical microscopy FTIR Fourier-transform infrared spectroscopy TGA Thermogravimetric analysis NSAIDs Non-steroidal anti-inflammatory drugs WHO World Health Organization FDA Food and Drug Administration GRAS Generally Recognized as Safe Co Collagen type I NO Nitric oxide TNF-α Tumor Necrosis Factor alpha LPS Lipopolysaccharide CsCo Chitosan/collagen CsCoB-0.5 Chitosan/collagen-0.5% B. microphylla extract CsCoB-1.0 Chitosan/collagen-1.0% B. microphylla extract 3D Three-dimensional B Bursera microphylla extract A. Gray RH Relative humidity AH Ammonium hydroxide EA Ethyl alcohol AA Glacial acetic acid AH Ammonium hydroxide MNH Magnesium nitrate hexahydrate SSD Sodium sulfate decahydrate MTT (bromuro de 3-(4,5-dimetiltiazol-2-il) Lz Lysozyme PBS Phosphate Buffered Saline DMEM Dulbecco’s Modified Eagle’s Medium CLSI Clinical and Laboratory Standards Institute MA Moisture Absorption Declarations Funding This research is funded by the Secretaría de Ciencia, Humanidades, Tecnología e Innovación (SECIHTI) with the project CBF-2023-2024-3824, Ciencia Básica y de Frontera 2023–2024, Mexico. Author Contribution Víctor Alonso Reyna-Urrutia, Julio César López-Romero and Heriberto Torres-Moreno conceptualization, investigation, resources, and supervision; Víctor Alonso Reyna-Urrutia, Ramón Enrique Robles-Zepeda, Miriam Estevez, José Luis López-Miranda, Juan Ramón Cáñez-Orozco, Karen Lillian Rodríguez-Martínez, Julio César López-Romero and Heriberto Torres-Moreno investigation, methodology, and validation; Víctor Alonso Reyna-Urrutia, Ramón Enrique Robles-Zepeda Karen Lillian Rodríguez-Martínez, Julio César López-Romero and Heriberto Torres-Moreno data curation and validation; Víctor Alonso Reyna-Urrutia and Heriberto Torres-Moreno writing of the original draft; Julio César López-Romero and Heriberto Torres-Moreno writing, review, and editing. All authors have read and agreed to the published version of the manuscript. Acknowledgments Víctor Reyna-Urrutia thanks SECIHTI for his postdoctoral fellowship. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8913713","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":601270242,"identity":"7f905a7f-baea-4639-a2cc-3ebc1e9ebd78","order_by":0,"name":"Víctor Alonso Reyna-Urrutia","email":"","orcid":"","institution":"Universidad de Sonora","correspondingAuthor":false,"prefix":"","firstName":"Víctor","middleName":"Alonso","lastName":"Reyna-Urrutia","suffix":""},{"id":601270243,"identity":"8fefa78b-d4c2-4c32-bbd4-95e463b72d66","order_by":1,"name":"Ramón Enrique Robles Zepeda","email":"","orcid":"","institution":"Universidad de Sonora","correspondingAuthor":false,"prefix":"","firstName":"Ramón","middleName":"Enrique Robles","lastName":"Zepeda","suffix":""},{"id":601270244,"identity":"1213c4dd-1324-4826-b234-fabb510a43ff","order_by":2,"name":"Miriam Estevez","email":"","orcid":"","institution":"National Autonomous University of Mexico","correspondingAuthor":false,"prefix":"","firstName":"Miriam","middleName":"","lastName":"Estevez","suffix":""},{"id":601270245,"identity":"c7975194-8410-49d2-9083-afe78ab42923","order_by":3,"name":"José Luis López-Miranda","email":"","orcid":"","institution":"National Autonomous University of Mexico","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"Luis","lastName":"López-Miranda","suffix":""},{"id":601270246,"identity":"f372f4b0-b4f3-4a36-ab21-5aeca7f84e57","order_by":4,"name":"Juan Ramón Cáñez-Orozco","email":"","orcid":"","institution":"Universidad de Sonora","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"Ramón","lastName":"Cáñez-Orozco","suffix":""},{"id":601270247,"identity":"cd9f8197-e47b-47af-b320-ba2833228b4d","order_by":5,"name":"Karen Lillian Rodríguez-Martínez","email":"","orcid":"","institution":"Universidad Estatal de Sonora","correspondingAuthor":false,"prefix":"","firstName":"Karen","middleName":"Lillian","lastName":"Rodríguez-Martínez","suffix":""},{"id":601270248,"identity":"fa9d263f-3fcd-412c-a44a-76a2393a1519","order_by":6,"name":"Julio César López-Romero","email":"","orcid":"","institution":"Universidad de Sonora","correspondingAuthor":false,"prefix":"","firstName":"Julio","middleName":"César","lastName":"López-Romero","suffix":""},{"id":601270249,"identity":"4f3e751f-d179-470d-8481-982d3b4564bf","order_by":7,"name":"Heriberto Torres-Moreno","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYBACAyA+jCLCD8QHiNEiwcDADBGRbCBCCzOKFgO86oHAnL334eGCint1/Az8Bx9X7rGxN76Re/Dgj4o7DLozErBqsew5bnB4xpliCckGZmbDM8/SErfdyEs4zHPmGYPZGez2GdxIYzjM25YgYXCAmU2y4cDhBLMbOQaHGdsOM5gdb8Cu5f4zoJZ/cC3/7Y1n5Bgc/AnSchirDqAtbEAtDXAtBxg3SOQYHODFZ8sZoMNmHEuQnNnMbGzYcCA5ccaZNwZAvxzmwemX48eYPxfUJPDzszc+fNhwwM6evz3H+OOPisNyZjewhxgCMKPxeQioHwWjYBSMglGABwAAJldiiCAVIYoAAAAASUVORK5CYII=","orcid":"","institution":"Universidad de Sonora","correspondingAuthor":true,"prefix":"","firstName":"Heriberto","middleName":"","lastName":"Torres-Moreno","suffix":""}],"badges":[],"createdAt":"2026-02-19 04:27:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8913713/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8913713/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104402572,"identity":"ef52484d-b376-4c21-bd5b-8b23b643cf4d","added_by":"auto","created_at":"2026-03-11 12:15:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1935405,"visible":true,"origin":"","legend":"\u003cp\u003eOM micrographs (\u003cstrong\u003eleft\u003c/strong\u003e column) of microaerogels and SEM micrographs (\u003cstrong\u003eright\u003c/strong\u003e column) at 1000X. Cs (\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eE\u003c/strong\u003e), CsCo \u003cstrong\u003e(B, F\u003c/strong\u003e), CsCoB-0.5 (\u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eG\u003c/strong\u003e), and CsCoB-1.0. (\u003cstrong\u003eD\u003c/strong\u003e, \u003cstrong\u003eH\u003c/strong\u003e). Cs: Cs microaerogels; CsCo: Cs/Co microaerogels; CsCoB-0.5: Cs/Co microaerogels loaded with 0.5% of \u003cem\u003eB. microphylla\u003c/em\u003e extract; and CsCoB-1.0: Cs/Co microaerogels loaded with 1.0% of \u003cem\u003eB. microphylla\u003c/em\u003e extract. Yellow and red arrows suggest the presence of \u003cem\u003eB. microphylla\u003c/em\u003eextract in the microaerogels. (\u003cstrong\u003eA\u003c/strong\u003e,\u003cstrong\u003e B, C, D\u003c/strong\u003e;\u003cstrong\u003e left\u003c/strong\u003e column): 10 µm scale on the ruler.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8913713/v1/45466934830e2cd0974f28d2.png"},{"id":104058033,"identity":"a284b08d-9f9c-44a3-8197-f4a34df5bccf","added_by":"auto","created_at":"2026-03-06 09:08:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1180294,"visible":true,"origin":"","legend":"\u003cp\u003eParticle size distribution of the of \u003cem\u003eB. microphylla \u003c/em\u003emicroaerogels. Cs: Cs microaerogels; CsCo: Cs/Co microaerogels; CsCoB-0.5: Cs/Co microaerogels loaded with 0.5% of \u003cem\u003eB. microphylla\u003c/em\u003e extract; and CsCoB-1.0: Cs/Co microaerogels loaded with 1.0% of \u003cem\u003eB. microphylla\u003c/em\u003e extract.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8913713/v1/b1805afa8960630c22a15132.png"},{"id":104058039,"identity":"f0c071f0-426e-45c3-b1a1-edd33f7baeb7","added_by":"auto","created_at":"2026-03-06 09:08:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5915570,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of \u003cem\u003eB. microphylla \u003c/em\u003emicroaerogels\u003cem\u003e. \u003c/em\u003eCo: Collagen; Cs: Cs microaerogels; CsCo: Cs/Co microaerogels; CsCoB-0.5: Cs/Co microaerogels loaded with 0.5% of \u003cem\u003eB. microphylla\u003c/em\u003e extract; and CsCoB-1.0: Cs/Co microaerogels loaded with 1.0% of \u003cem\u003eB. microphylla\u003c/em\u003e extract.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8913713/v1/9894c3069feaa937fa3553c2.png"},{"id":104058035,"identity":"784e19b0-0f6b-46ba-bb0b-0c2b88b7bd39","added_by":"auto","created_at":"2026-03-06 09:08:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1226913,"visible":true,"origin":"","legend":"\u003cp\u003eThermogravimetric analysis of the \u003cem\u003eB. microphylla\u003c/em\u003e aerogels. B: \u003cem\u003eB. microphylla \u003c/em\u003eextract; Co: Collagen; Cs: Cs microaerogels; CsCo: Cs/Co microaerogels; CsCoB-0.5: Cs/Co microaerogels loaded with 0.5% of \u003cem\u003eB. microphylla\u003c/em\u003eextract; and CsCoB-1.0: Cs/Co microaerogels loaded with 1.0% of \u003cem\u003eB. microphylla\u003c/em\u003e extract. Blue and green arrow: Maximum loss of Co; Red arrow: Maximum loss of Cs, CsCo, and CsCo with \u003cem\u003eB. microphylla\u003c/em\u003e extract.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8913713/v1/863515ef0e01c6a6ee66807e.png"},{"id":104402486,"identity":"f7af7608-0814-4fbc-bdb2-9fed33311048","added_by":"auto","created_at":"2026-03-11 12:15:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1079938,"visible":true,"origin":"","legend":"\u003cp\u003eMoisture absorption of microaerogels at a relative humidity of 40 ± 1% and 80 ± 1%. Values of \u003cem\u003en \u003c/em\u003e=\u003cem\u003e \u003c/em\u003e3 ± SD are shown. Cs: Cs microaerogels; CsCo: Cs/Co microaerogels; CsCoB-0.5: Cs/Co microaerogels loaded with 0.5% of \u003cem\u003eB. microphylla\u003c/em\u003eextract; and CsCoB-1.0: Cs/Co microaerogels loaded with 1.0% of \u003cem\u003eB. microphylla\u003c/em\u003e extract.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8913713/v1/098cdfc4793408725521b9c0.png"},{"id":104402889,"identity":"320c20f6-e9b6-4d7b-8b32-15a9ce886922","added_by":"auto","created_at":"2026-03-11 12:16:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":859976,"visible":true,"origin":"","legend":"\u003cp\u003eEnzymatic degradation of \u003cem\u003eB. microphylla\u003c/em\u003emicroaerogels. At the endpoint measurement, different letters (a–c) indicate statistically significant differences (p \u0026lt; 0.05). All values represent the mean ± standard deviation of three independent experiments (±SD, \u003cem\u003en\u003c/em\u003e = 3). Cs: Cs microaerogels; CsCo: Cs/Co microaerogels; CsCoB-0.5: Cs/Co microaerogels loaded with 0.5% of \u003cem\u003eB. microphylla\u003c/em\u003e extract; and CsCoB-1.0: Cs/Co microaerogels loaded with 1.0% of \u003cem\u003eB. microphylla\u003c/em\u003e extract\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8913713/v1/dbe8972466659ffc969f9663.png"},{"id":104058040,"identity":"a583197d-043c-4d48-875f-d4c9339edd12","added_by":"auto","created_at":"2026-03-06 09:08:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4176169,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxic effect of \u003cem\u003eB. microphylla\u003c/em\u003e microaerogels on RAW 264.7. Bars with different letters a–e\u003csup\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sup\u003eindicate statistically significant differences (\u003cem\u003ep \u003c/em\u003e\u0026lt;\u003cem\u003e \u003c/em\u003e0.05). All values represent the mean ± standard deviation of three independent experiments (±SD, \u003cem\u003en \u003c/em\u003e=\u003cem\u003e \u003c/em\u003e3). Cs: Cs microaerogels; CsCo: Cs/Co microaerogels; CsCoB-0.5: Cs/Co micraerogels loaded with 0.5% of \u003cem\u003eB. microphylla\u003c/em\u003e extract; and CsCoB-1.0: Cs/Co micraerogels loaded with 1.0% of \u003cem\u003eB. microphylla\u003c/em\u003e extract.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8913713/v1/fa49506234bc8117b581e120.png"},{"id":104058036,"identity":"88a6746b-6378-42d4-8a2e-cebb62eaaeb1","added_by":"auto","created_at":"2026-03-06 09:08:04","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":572815,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial activity of microaerogels loaded with \u003cem\u003eB. microphylla \u003c/em\u003efruit extract against \u003cem\u003eS. aureus\u003c/em\u003e. Bars with different letters a–c indicate statistical differences (\u003cem\u003ep \u003c/em\u003e\u0026lt;\u003cem\u003e \u003c/em\u003e0.05). All values represent the mean ± standard deviation of three independent experiments (±SD, \u003cem\u003en\u003c/em\u003e = 3). Cs: Cs microaerogels; CsCo: Cs/Co microaerogels; CsCoB-0.5: Cs/Co microaerogels loaded with 0.5% of \u003cem\u003eB. microphylla\u003c/em\u003e extract; and CsCoB-1.0: Cs/Co microaerogels loaded with 1.0% of \u003cem\u003eB. microphylla\u003c/em\u003e extract.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-8913713/v1/f9a8b33b49043405515b9e58.png"},{"id":104408568,"identity":"6de7d934-1ea6-41b8-b1d3-e6e1003ceda5","added_by":"auto","created_at":"2026-03-11 12:42:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16205751,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8913713/v1/3be8015c-3161-4616-8fb9-5178ede9c1a8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Functional Chitosan–Collagen Microaerogels Incorporating Bursera microphylla Fruit Extract with Antibacterial Properties","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBacterial infections represent a major public health concern worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is estimated that within the next 30 years, antibiotic-resistant bacterial infections could become the leading cause of mortality globally [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The accelerated emergence of resistance has significantly reduced the effectiveness of conventional antibiotic therapies, resulting in infections that are increasingly difficult to control. Among clinically relevant pathogens, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e remains one of the most frequently isolated microorganisms in infectious processes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Consequently, the World Health Organization (WHO) has classified \u003cem\u003eS. aureus\u003c/em\u003e as a high-priority pathogen due to its alarming resistance to antibiotics [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This scenario underscores the urgent need for alternative, sustainable strategies capable of addressing bacterial infections while reducing reliance on conventional antibiotics.\u003c/p\u003e \u003cp\u003eIn this context, polymer-based encapsulation systems have gained increasing attention, particularly those derived from natural and environmentally friendly materials. Chitosan (Cs) is a polysaccharide industrially obtained through the alkaline deacetylation of chitin, a renewable resource abundantly found in the exoskeletons of crustaceans such as shrimp and crabs [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Cs is a non-toxic, biodegradable biopolymer with well-documented biomedical properties, including hemostatic, antibacterial, biocompatible, and cytocompatible activities. Additionally, Cs is recognized by the U.S. Food and Drug Administration (FDA) as a Generally Recognized as Safe (GRAS) material, and its low cost and commercial availability further enhance its applicability [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Owing to its cationic nature in acidic environments, Cs enables the formation of diverse material architectures, such as nano- and microaerogels, emulsions, fibers, hydrogels, films, and membranes, making it a versatile matrix for the encapsulation of plant extracts and other bioactive compounds [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCollagen (Co) is another naturally derived polymer of high relevance due to its biocompatibility, biodegradability, and favorable interaction with biological tissues [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Co-based delivery systems have demonstrated the ability to encapsulate active compounds while enabling controlled and sustained release profiles [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Among the available encapsulation approaches, ionic gelation stands out as a promising technique, as it avoids the use of chemical crosslinkers and preserves the intrinsic biocompatibility of the polymeric matrix [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This method employs ammonium hydroxide to induce physical crosslinking, maintaining free amino groups in Cs that are essential for its functional and bioactive properties, while subsequent lyophilization ensures material stability and preservation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Consequently, Cs/Co systems represent sustainable carriers for bioactive compounds derived from medicinal plants (18,19,25).\u003c/p\u003e \u003cp\u003e \u003cem\u003eBursera microphylla\u003c/em\u003e A. Gray (Burseraceae), commonly known as \u0026ldquo;torote,\u0026rdquo; \u0026ldquo;copal,\u0026rdquo; or \u0026ldquo;elephant tree,\u0026rdquo; is a medicinal species native to the desert regions of northern Mexico and the southwestern United States, particularly within the Sonoran Desert [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In traditional Mexican medicine, the Seri people utilize the bark, fruits, and leaves to treat wounds, sore throats, and headaches, while tinctures are used for gum sores and dental abscesses, and stems and leaves are applied for anti-inflammatory and urinary discomfort treatments [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. \u003cem\u003eIn vitro\u003c/em\u003e studies have demonstrated that \u003cem\u003eB. microphylla\u003c/em\u003e fruit extracts inhibit inflammatory mediators such as nitric oxide (NO) and tumor necrosis factor alpha (TNF-α) in lipopolysaccharide-stimulated RAW 264.7 macrophages [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Although Cs-based microsystems encapsulating \u003cem\u003eB. microphylla\u003c/em\u003e extract have shown antibacterial activity against \u003cem\u003eS. aureus\u003c/em\u003e, the development of Cs/Co microaerogels is expected to further enhance structural stability, biocompatibility, and controlled release profile. Therefore, this study aimed to synthesize and characterize Cs/Co-based microaerogels loaded with \u003cem\u003eB. microphylla\u003c/em\u003e fruit extract as sustainable polymeric systems designed to enhance tissue compatibility and antimicrobial performance.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eCs (Catalog No. 448877) and type I Co (Catalog No. C9791) derived from calf skin, used for the preparation of microaerogels, were sourced from Sigma-Aldrich. Ethyl alcohol (EA, 94.9\u0026ndash;96%, Catalog No. 493538) was utilized to extract the \u003cem\u003eB. microphylla\u003c/em\u003e plant material. Ammonium hydroxide (AH, 28% NH₃ in H₂O, \u0026ge;\u0026thinsp;99.99% trace metals basis, Catalog No. 338818) and glacial acetic acid (AA, \u0026ge;\u0026thinsp;99.5% purity, Catalog No. 33209) were employed in the preparation of microaerogels. Magnesium nitrate hexahydrate (MNH, 99%, Catalog No. 237175) and sodium sulfate decahydrate (SSD, 99%, Catalog No. 403008) were applied for relative humidity absorption measurements. MTT (bromuro de 3-(4,5-dimetiltiazol-2-il) (MTT, Catalog No. 88417), and isopropanol (Catalog No. 563935) were used in the \u003cem\u003ein vitro\u003c/em\u003e assays. Lz (Catalog No. L6876) and Phosphate Buffered Saline (PBS, Catalog No. P4417) were used for enzymatic degradation tests. All solvents and reagents were obtained from Sigma-Aldrich (Toluca, Mexico).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlant Material and Extract Elaboration\u003c/h3\u003e\n\u003cp\u003eSpecimens of \u003cem\u003eB. microphylla\u003c/em\u003e (B) were collected in the state of Sonora, Mexico, specifically in the \u0026ldquo;Puerto Lobos\u0026rdquo; area (30.177211, \u0026minus;\u0026thinsp;112.664644) of the Caborca municipality during spring (April 30, 2023). The collected fruits were weighed and immersed in ethanol (1:10 w/v) at 25\u0026deg;C for 10 days, with daily manual agitation. The resulting extracts were filtered and concentrated using an IKA rotary evaporator (model RV-10B-S99) set at 125 rpm and 50\u0026deg;C. The ethanolic extracts were then dried and stored at 4\u0026deg;C until further use [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eObtaining Microaerogels\u003c/h3\u003e\n\u003cp\u003eDifferent solutions were prepared for the microencapsulation process, with and without \u003cem\u003eB. microphylla\u003c/em\u003e extract: (1) Cs solution 2% (w/w) was prepared by dissolving it in 0.3 M glacial acetic acid at room temperature, followed by continuous stirring until complete dissolution was achieved [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This solution was subsequently used for the fabrication of Cs hydrogels. (2) Separately, a 1% (w/w) Co suspension was prepared in 0.1 M acetic acid under constant stirring for 24 h at room temperature. The resulting suspension was then incorporated into a 3% (w/w) Cs solution at an 80/20 (w/w) ratio (Cs/Co) to obtain Cs/Co hydrogel-forming systems. (3) Cs/Co solutions were mixed with 0.5 or 1.0% \u003cem\u003eB. microphylla\u003c/em\u003e extract to obtain CsCoB-0.5 (99.5/0.5) and CsCoB-1.0 (99/1) (w/w ratio), respectively. The extract was incorporated into the Cs/Co mixture and stirred for 30 min [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAliquots of the polymer suspensions were made in 10 mL beakers and placed inside a hermetically closed chamber, containing 100 mL of ammonium hydroxide. Physical cross-linking (gelation) was induced by ammonia diffusion for 24 h [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Subsequently, the hydrogels were washed with distilled water to eliminate ammonium acetate (CH\u003csub\u003e3\u003c/sub\u003eCOONH\u003csub\u003e4\u003c/sub\u003e), and the rest of the ammonium hydroxide (NH\u003csub\u003e4\u003c/sub\u003eOH) remained in the hydrogels until a pH of 7 was reached. Afterward, the hydrogels were placed in a refrigerator at a temperature of \u0026minus;\u0026thinsp;26\u0026deg;C for 24 h and subsequently introduced into a freeze dryer (SCIENTZ-10N LYOPHILIZER, Kansas City, MO, USA) for 24 h, at -58\u0026deg;C and a pressure of 1 Pa [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Once the aerogels were obtained, they were mechanically crushed for 5 min to obtain the microparticulate materials. Subsequently, the microaerogels were passed through a stainless-steel screening filter (Hightop brand) with a pore size of 400 \u0026micro;m\u003c/p\u003e\n\u003ch3\u003ePhysicochemical Characterization of Microaerogels\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eScanning Electron Microscope (SEM) and Optical Microscopy (OM)\u003c/h2\u003e \u003cp\u003eThe morphology and size of the microaerogels were examined using scanning electron microscopy (SEM) and optical microscopy (OM). SEM observations were performed with a Hitachi SU8230 microscope operating at 20\u0026ndash;30 kV. Samples were placed on aluminum tape and coated with a thin gold layer using a Desk II high-vacuum coater (LLC). For OM analysis, a LABOMED CX model microscope equipped with a 12 V, 20 W lamp (Labo America, Inc., Fullerton, CA, USA) was employed. An OEM objective micrometer was utilized to determine the apparent dimensions of the microaerogels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFourier Transform Infrared Spectroscopy (FTIR)\u003c/h2\u003e \u003cp\u003eFourier Transform Infrared Spectroscopy (FTIR) is commonly employed to evaluate encapsulation processes, as it enables the identification of potential chemical interactions between the encapsulating polymer and the active compounds [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The spectra of the microaerogels were recorded using a Perkin Elmer Spectrum Two FTIR spectrometer (Fremont, CA, USA). Measurements were performed over a wavenumber range of 4000\u0026ndash;650 cm⁻\u0026sup1;, with a resolution of 4 cm⁻\u0026sup1; and an average of 100 scans.\u003c/p\u003e \u003cp\u003eThermogravimetric Analysis \u003cb\u003e(TGA)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThermogravimetric Analysis (TGA) is a useful technique to evaluate thermal decomposition and the stability of formulations under elevated temperatures [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The TGA of the microaerogels was performed using a Mettler Toledo TGA/DSC 2\u0026thinsp;+\u0026thinsp;thermal analyzer (Greifensee, Switzerland) under a nitrogen atmosphere. The samples were heated from 25 to 600\u0026deg;C at a constant rate of 10\u0026deg;C/min.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMoisture Absorption\u003c/h3\u003e\n\u003cp\u003eThe evaluation of humidity effects on formulation prototypes provides insight into their performance under challenging environmental conditions. Moisture absorption is assessed using a gravimetric technique that tracks changes in the water content of a polymer over time [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These properties also indicate whether the material exhibits hydrophilic or hydrophobic characteristics [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. For this analysis, the freeze-dried microaerogels were stored at two controlled relative humidity (RH) levels: 40\u0026thinsp;\u0026plusmn;\u0026thinsp;1% and 80\u0026thinsp;\u0026plusmn;\u0026thinsp;1%, employing magnesium nitrate hexahydrate (Mg (NO₃)₂\u0026middot;6H₂O) and sodium sulfate decahydrate (Na₂SO₄\u0026middot;10H₂O), respectively. The mass of each sample was recorded from the start of the experiment until equilibrium was achieved. The moisture absorption percentage (M.A.) was calculated using Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e):\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:A\\left(t\\right)=\\left(\\frac{mt-m0}{m0}\\right)\\times\\:100$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eHere, \u003cem\u003em₀\u003c/em\u003e represents the initial mass of the sample at the onset of moisture exposure, while \u003cem\u003emt\u003c/em\u003e corresponds to the mass of the sample after a period \u003cem\u003et\u003c/em\u003e (in hours). For each microaerogel, the mean and standard deviation values are calculated from triplicate measurements\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003edegradation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e degradation of the scaffolds was evaluated at 37\u0026deg;C in a Phosphate Buffered Saline (PBS, pH 7.4) solution supplemented with 1.5 mg/mL of Lz [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This enzyme concentration was selected because it corresponds to the level typically present in human serum [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. To maintain enzymatic activity, the Lz solution was replaced daily [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Scaffolds weighing approximately 7 mg were placed into vials containing 1 mL of the enzyme solution and incubated for a total of 28 days. At predetermined time points (7, 14, 21, and 28 days), the samples were removed from the solution, rinsed with distilled water, and subsequently freeze-dried for 24 h before being weighed [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The degradation rate was expressed as the percentage of mass loss of the scaffolds, calculated using the following Eq.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e):\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:Mass\\:loss\\:\\left(\\%\\right)=\\left(\\frac{mi-m\\text{f}}{mi}\\right)\\times\\:100$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere \u003cem\u003emi\u003c/em\u003e is the initial scaffold weight and \u003cem\u003emf\u003c/em\u003e is the weight of the scaffold after degradation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn Vitro\u003c/b\u003e \u003cb\u003eAnalysis of Microaerogels\u003c/b\u003e\u003c/p\u003e\n\u003ch3\u003eSample Sterilization\u003c/h3\u003e\n\u003cp\u003eThe samples were sterilized by exposure to UV radiation for 20 min using a laminar flow hood (Labconco\u0026reg;, Class II type A2, Kansas City, MO, USA).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture\u003c/h2\u003e \u003cp\u003eMacrophages of the RAW 264.7 cell line, originally transformed by the Abelson leukemia virus, were kindly provided by Dr. Emil A. Unanue from the Department of Pathology and Immunology at Washington University, St. Louis, MO, USA. The RAW 264.7 murine macrophages (induced by the Abelson leukemia virus) were maintained in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM), supplemented with 5% heat-inactivated fetal bovine serum and 100 U/mL penicillin, using 25 cm\u0026sup2; culture flasks. Cells were incubated in an Isotherm incubator (Thermo Fisher Scientific, Waltham, MA, USA) under controlled conditions of 5% CO₂, 37\u0026deg;C, and 95% relative humidity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell Viability Assay\u003c/h2\u003e \u003cp\u003eOnce RAW 264.7 cells reached approximately 80% confluence, they were detached using enzymatic digestion with 3 mL of trypsin and then collected in DMEM. The cell suspension was centrifuged (Beckman Coulter\u0026reg; Allegra X-15R, SX4750 rotor) at 1700 rpm for 7 min at 4\u0026deg;C. After discarding the supernatant, the resulting cell pellet was re-suspended in DMEM. A suspension of RAW 264.7 cells (500,000 cells/mL, viability\u0026thinsp;\u0026gt;\u0026thinsp;90%) was seeded in a 96-well plate (Costar, Corning, NY, USA) and incubated for 24 h. The cells were then exposed to microaerogels (0\u0026ndash;200 \u0026micro;g/mL) for 24 h using the indirect method (microaerogels were previously incubated at pH 5 buffer for 4 h, and 100 \u0026micro;L of the resulting supernatant was used to treat the cells). Afterward, 5 mg/mL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added and incubated for 4 h. The resulting formazan crystals were dissolved in isopropanol, and absorbance was measured at 570\u0026ndash;630 nm using an iMARK BIO-RAD\u0026reg; microplate reader [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAntibacterial Activity\u003c/h2\u003e \u003cp\u003eThe antibacterial activity of the microerogels (Cs, CsCo, CsCoB-0.5, and CsCoB-1.0) was determined following the methodology previously described [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Bacterial strains were cultured overnight at 37\u0026deg;C and subsequently adjusted to a turbidity equivalent to the 0.5 McFarland standard (\u0026asymp;\u0026thinsp;1 \u0026times; 10⁸ CFU/mL). An aliquot of 0.1 mL of this suspension was transferred into 9.9 mL of Mueller-Hinton broth, yielding a final inoculum concentration of 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/mL. Microaerogels containing \u003cem\u003eB. microphylla\u003c/em\u003e extract were mixed with saline solution (pH 5.5) at a 1:1 ratio with the bacterial suspension and incubated at 37\u0026deg;C for 24 h. Following incubation, 50 \u0026micro;L from each treatment was serially diluted (1:10) in sterile saline (0.85% w/v), and 10 \u0026micro;L aliquots from each dilution were plated on agar for colony enumeration. Plates were incubated at 37\u0026deg;C for an additional 24 h, and antibacterial activity was quantified as CFU/mL based on colony counts [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical comparisons were conducted using a one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post-hoc test with GraphPad PRISM\u0026reg; software (version 6.0c). A value of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was regarded as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePhysicochemical Characterization of Microaerogels\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003eMicroaerogels Morphology\u003c/h2\u003e \u003cp\u003eThe morphological features of the microaerogels (Cs, CsCo, CsCoB-0.5, and CsCoB-1.0) are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Optical microscopy (OM) and scanning electron microscopy (SEM) analyses revealed that the samples consist of non-spherical microaerogels exhibiting a heterogeneous surface morphology with pronounced irregularities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the CsCoB-0.5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG; indicated with red arrows) and CsCoB-1.0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH; indicated with yellow arrows), oval-shaped structures can be observed on the walls forming the three-dimensional (3D) network of the designed encapsulation systems. These findings suggest that the extract is incorporated within the walls of the 3D polymeric microsystem. This implies that the formation of these spherical bodies can be associated with the integration of the extract into the encapsulating matrix. Similar behaviors have been previously reported in studies involving Cs based microsystems loaded with \u003cem\u003eB. microphylla\u003c/em\u003e extract, suggesting the presence of the extract within the polymeric matrix [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMicroaerogels Size Distribution\u003c/h2\u003e \u003cp\u003eThe particle size analysis of the microaerogels (Cs, CsCo, CsCoB-0.5, and CsCoB-1.0) revealed a unimodal distribution, with predominant frequencies ranging between 45 \u0026micro;m and 90 \u0026micro;m for all formulations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Regarding histogram skewness, the particle size distributions of CsCo and CsCoB-1.0 exhibited a slight leftward skew, while Cs and CsCoB-0.5 showed a rightward skew. These findings indicate more compact and uniform distributions, with uniformity indices of 68%, 80%, 74%, and 74% for Cs, CsCo, CsCoB-0.5, and CsCoB-1.0, respectively. These values represent an improvement compared to previous research focused on chitosan-based systems, where the predominant percentages were around 50% [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Such enhanced uniformity could potentially influence the bioactivity of the encapsulating systems.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStructure for the Fourier Transform Infrared Spectroscopy (FTIR)\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e displays the FTIR spectra of the microencapsulated systems. All spectra exhibited comparable patterns, highlighting the characteristic absorption bands attributed to Cs. A broad and intense band was identified between 3500 and 3300 cm⁻\u0026sup1;, corresponding to the stretching vibrations of hydroxyl (O\u0026ndash;H) and amine (N\u0026ndash;H) groups. Additional absorption peaks appeared at 2923 and 2880 cm⁻\u0026sup1;, which are associated with methylene (\u0026ndash;CH₂\u0026ndash;) group vibrations. A prominent band at 1653 cm⁻\u0026sup1; was assigned to the carbonyl (C\u0026thinsp;=\u0026thinsp;O) stretching of amide I, while the band at 1580 cm⁻\u0026sup1; was linked to the bending vibrations of amide II groups [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Finally, the region between 1200 and 500 cm⁻\u0026sup1; showed signals characteristic of the polysaccharide backbone [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The main FTIR absorption bands and their corresponding assignments are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of FTIR absorption bands observed in Cs/Co microaerogels loaded with \u003cem\u003eBursera microphylla\u003c/em\u003e fruit extract.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWavenumber\u003c/p\u003e \u003cp\u003e(cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFunctional group\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZone I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3500\u0026thinsp;\u0026minus;\u0026thinsp;3300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eO-H stretching\u003c/p\u003e \u003cp\u003eN-H stretching\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZone II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2920\u003c/p\u003e \u003cp\u003e2870\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC-H stretching methylene groups\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZone III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1730\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u0026thinsp;=\u0026thinsp;O stretching amide I\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1550\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u0026thinsp;=\u0026thinsp;O deformation of amide II\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1380\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZone IV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1190\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003esaccharide structure\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1060\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e890\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe characteristic FTIR absorption band at 1580 cm⁻\u0026sup1;, corresponding to the amide II region of pure Cs scaffolds (as discussed previously), exhibited a bathochromic shift toward higher wavenumbers in the CsCo, CsCoB-0.5, and CsCoB-1.0 formulations. This displacement toward the Co-specific amide II band at 1545 cm⁻\u0026sup1; is indicative not only of successful Co incorporation within the polymeric matrix but also of molecular-level interactions, most likely hydrogen bonding between Cs and Co, consistent with previously reported findings [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The absorption band observed at 1451 cm⁻\u0026sup1; is primarily associated with the bending vibrations of methylene (\u0026ndash;CH₂) and methyl (\u0026ndash;CH₃) groups, as well as the stretching vibrations of the C\u0026ndash;N bond, characteristic of the Co component. The presence and consistency of this band across all Co containing formulations suggest a conserved molecular environment for Co, further supporting its structural integrity and uniform incorporation within the Cs-based matrix [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, the FTIR spectra of the extract-loaded microaerogels (CsCoB-0.5 and CsCoB-1.0) displayed a distinct absorption band at 1710 cm⁻\u0026sup1;, which can be attributed to the stretching vibrations of carbonyl (C\u0026thinsp;=\u0026thinsp;O) functional groups, typically associated with aldehydes and ketones present in phytochemical constituents [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. An additional band of increased intensity at 2923 cm⁻\u0026sup1; was observed, corresponding to C\u0026ndash;H stretching vibrations of aliphatic moieties, further suggesting the incorporation of \u003cem\u003eB. microphylla\u003c/em\u003e phytocompounds into the polymeric network [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSpectral comparison between extract-loaded and unloaded formulations revealed no appearance of new functional groups, suggesting the absence of covalent interactions or chemical bonding between the chemical compounds of the extract and the Cs/Co matrix. This suggests that the structure of the phytoconstituents is preserved during the encapsulation process, which is an essential factor for preserving their bioactivity. Instead, the extract appears to be physically entrapped within the three-dimensional polymeric network of Cs; this suggests that the microaerogels function as controlled-release systems. The release kinetics are likely governed by environmental pH fluctuations, mediated by the reversible protonation/deprotonation of the Cs amino groups, facilitating controlled diffusion of the extract from the microaerogels core to the external medium [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eThermogravimetric Analysis (TGA)\u003c/h2\u003e \u003cp\u003eThermogravimetric analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) was employed to assess the thermal stability of Cs, Co type I, and their corresponding microencapsulated formulations (CsCo) loaded with \u003cem\u003eB. microphylla\u003c/em\u003e fruit extract. The results indicate that Co exhibits lower thermal stability compared to Cs. The thermogram for Co shows three distinct weight loss events: an initial 7% loss around 100\u0026deg;C, a second drop of 30% at approximately 185\u0026deg;C (indicated by the blue arrow), and a third loss of 15% near 295\u0026deg;C (between the blue and green arrows), a thermal profile consistent with previous findings in the literature [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe thermogram of \u003cem\u003eB. microphylla\u003c/em\u003e extract showed a significant mass loss of nearly 90% between 148\u0026ndash;500\u0026deg;C, reaching its peak decomposition around 300\u0026deg;C. In contrast, the microaerogels (Cs, CsCo, CsCoB-0.5, and CsCoB-1.0) displayed a markedly lower mass loss, approximately 10% at 100\u0026deg;C, primarily attributed to water evaporation. A second weight loss event was observed around 302\u0026deg;C (red arrow), corresponding to the thermal decomposition of saccharide structures in the Cs backbone [\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSimilar thermal behavior was noted in the formulations containing \u003cem\u003eB. microphylla\u003c/em\u003e extract (CsCoB-0.5% and CsCoB-1.0%), suggesting that the Cs/Co matrix enhances the thermal stability of the encapsulated extract. This increased stability likely results from the protective effect of the polymeric network, which shields the bioactive components from degradation at elevated temperatures, findings in line with recent studies [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Moreover, the incorporation of Co into the Cs matrix forms a semi-interpenetrating polymer network (CsCo), which offers greater thermal resistance than either of the individual components [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMoisture Absorption\u003c/h2\u003e \u003cp\u003eThe moisture absorption (MA) analysis revealed that the microaerogels (Cs, CsCo, CsCoB-0.5, and CsCoB-1.0) reached a maximum moisture uptake of 14\u0026ndash;18% at a RH of 40\u0026thinsp;\u0026plusmn;\u0026thinsp;1% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Conversely, under an RH of 80\u0026thinsp;\u0026plusmn;\u0026thinsp;1%, all microaerogels exhibited maximum absorption values ranging from 26\u0026ndash;37%. These findings align with previous reports indicating that Cs, as the primary polymeric matrix, can achieve an equilibrium moisture absorption of approximately 41% under high RH conditions and around 20% at low RH [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Another study reported that in materials where Cs is the majority polymer in the mixture only represents 10% absorption under low RH conditions; however, in this research, the value was 14% [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. These results confirm the hydrophilic nature of the microaerogels when exposed to elevated humidity levels. This behavior is advantageous under physiological conditions, as contact with biological fluids facilitates the release of the encapsulated extracts from the polymeric matrix. The hydrophilic character of the Cs matrix is attributed to the free amino groups within the Cs polymer chain, further confirming the absence of significant chemical interactions between \u003cem\u003eB. microphylla\u003c/em\u003e extracts and the Cs structure [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003edegradation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eLysozyme (Lz), an enzyme naturally present in wound exudates and biological fluids, plays a key role in the enzymatic degradation of Cs-based materials. Evaluating the \u003cem\u003ein vitro\u003c/em\u003e degradation of the microaerogels in the presence of Lz provides valuable insight into their structural stability and expected biodegradation behavior under physiological conditions. This allows for a better understanding of their bioactivity upon contact with wounds or biological tissues. Specifically, Lz recognizes three acetylated units in the Cs backbone to initiate its enzymatic activity, thereby promoting material breakdown and the subsequent release of the encapsulated active compound [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Most of the investigations have been performed under accelerated conditions, such as elevated Lz concentrations and acidic pH values (below 6.2), where the enzyme exhibits its highest catalytic activity. In contrast, the present study utilized an Lz concentration comparable to that found in human serum (1.5 \u0026micro;g/mL), while maintaining physiological pH (7.4) and temperature (37\u0026deg;C), thereby aiming to more accurately mimic human body conditions [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the degradation kinetics revealed that the Cs microaerogels exhibited the highest mass loss, reaching approximately 24% after incubation with Lz. This pronounced degradation can be attributed to the higher availability of glycosidic bonds susceptible to enzymatic cleavage due to the absence of Co, which provides structural reinforcement. In contrast, the CsCo microaerogels showed a slower degradation rate, with a mass loss of about 17%, suggesting that the incorporation of Co enhances the structural integrity of the polymeric matrix by forming additional hydrogen bonds with Cs chains, thereby limiting Lz accessibility. When the \u003cem\u003eB. microphylla\u003c/em\u003e extract was incorporated, both CsCoB-0.5 and CsCoB-1.0 formulations exhibited the lowest degradation rates, around 14% (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). This reduced degradation may be related to the partial filling of the polymeric network by the encapsulated extract, which could hinder enzyme diffusion and substrate recognition, resulting in slower matrix degradation. Overall, these findings suggest that the inclusion of Co improves the mechanical and enzymatic stability of Cs microaerogels, while the encapsulation of the extract further decreases degradation through steric and diffusional effects. Such modulation of degradation is advantageous for achieving controlled release, as the Lz-mediated breakdown of Cs N-acetylglucosamine sequences allows gradual and predictable liberation of the bioactive compound. Consequently, topical administration to wounds emerges as the most suitable route, enabling localized delivery, minimizing systemic exposure, and exploiting the Lz-rich wound environment to sustain release and enhance healing through the presence of Co within the matrix [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eBiological Properties of Microaerogels\u003c/h2\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eCell Viability\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the microaerogel exhibited no significant cytotoxicity toward RAW 264.7 macrophages at concentrations below 50 \u0026micro;g/mL, maintaining cell viability above 90% across all treatments. However, at 200 \u0026micro;g/mL, formulations Cs, CsCo, CsCoB-0.5, and CsCoB-1.0 resulted in a reduction in cell viability to 83%, 87%, 83%, and 68%, respectively. These results align with those previously reported by Torres-Moreno et al. (2022), where \u003cem\u003eB. microphylla\u003c/em\u003e stem, fruit, and leaf extracts preserved approximately 90% cell viability at comparable concentrations [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn the other hand, the CsCoB-0.5 and CsCoB-1 microaerogels showed a greater reduction in cell viability compared to that previously reported for Cs microaerogels loaded with the fruit extract. At 200 \u0026micro;g/mL, CsCoB-1 reduced cell viability by 32%, whereas the previous study observed that Cs microaerogels loaded with 1% \u003cem\u003eB. microphylla\u003c/em\u003e fruit extract decreased cell viability by 16%. These results indicate that incorporating the extract into a collagen-based matrix increases its cytotoxicity. This effect may be attributed to the three-dimensional organization of the polymer matrix, which, due to its less compact structure and greater network interconnectivity, could facilitate the rapid release of the \u003cem\u003eB. microphylla\u003c/em\u003e extract. Although the encapsulation of \u003cem\u003eB. microphylla\u003c/em\u003e extract in Cs based microaerogels has demonstrated favorable biocompatibility, further research is still needed to elucidate possible adverse effects, such as immunogenicity or hypersensitivity responses, to validate its safety profile for future biomedical applications [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eAntibacterial Evaluation\u003c/h2\u003e \u003cp\u003eThe antibacterial activity of the microaerogels loaded with \u003cem\u003eBursera microphylla\u003c/em\u003e extract is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. These findings demonstrate a significant reduction in microbial load across all tested microaerogels, with CsCoB-0.5 and CsCoB-1.0 exhibiting the strongest antibacterial effects. Specifically, these microaerogels achieved logarithmic reductions of 37.8% and 57.7% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), respectively, while CsCo and Cs showed moderate decreases of 15.6 and 9.7%, respectively, relative to the control. These results suggest that the microaerogels successfully release the encapsulated extract and its active compounds, allowing interaction with \u003cem\u003eS. aureus\u003c/em\u003e and inducing bacterial death. The results obtained in this study suggest that the presence of Co in the encapsulating matrix produces a partial solubilization of Cs, promoting the release of the extract from the polymeric matrix [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is context, it is important to highlight that \u003cem\u003eS. aureus\u003c/em\u003e remains one of the most clinically relevant Gram-positive pathogens, frequently associated with both hospital-acquired and community-acquired infections, and is well known for its remarkable capacity to develop resistance to various antibacterial therapies [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Therefore, the significant antibacterial activity observed for the extract-loaded microaerogels is particularly relevant, as it addresses a pathogen of high clinical concern and reinforces the potential applicability of these systems as alternative or complementary antibacterial strategies.\u003c/p\u003e \u003cp\u003eThe antibacterial effect observed from \u003cem\u003eB. microphylla\u003c/em\u003e extract may be related to its phytochemical composition. Previous studies have demonstrated that the fruit extract contains a variety of phenolic compounds, including flavonoids, phenolic acids, and lignans [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These bioactive constituents have shown potent antibacterial activity against \u003cem\u003eS. aureus\u003c/em\u003e, primarily through mechanisms involving inhibition of bacterial enzymes and toxins, disruption of the cell wall and membrane, interference with metabolic pathways, induction of DNA fragmentation, and suppression of virulence gene expression [\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Based on these promising results, future studies should evaluate the efficacy of these microaerogels against other bacterial strains, including antibiotic-resistant clinical isolates, as well as \u003cem\u003ein vivo\u003c/em\u003e models to evaluate their effectiveness in infected wounds. This could open new avenues for the development of alternative antibacterial therapies based on Cs/Co polymeric matrices.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrated the successful development of chitosan/collagen (Cs/Co) type I microaerogels loaded with \u003cem\u003eB. microphylla\u003c/em\u003e fruit extract as a sustainable biopolymeric platform with antibacterial activity against \u003cem\u003eS. aureus\u003c/em\u003e. The incorporation of Co as a structural component significantly influenced the physicochemical behavior of the microaerogels, particularly in terms of surface morphology, hydrophilicity, and resistance to enzymatic degradation, when compared with Cs only systems. These features are especially relevant for environmentally friendly polymeric materials intended for biomedical applications, as they contribute to enhanced stability and functionality while maintaining biodegradability.\u003c/p\u003e \u003cp\u003eThe use of naturally derived polymers and a plant-based bioactive extract highlights the environmental relevance of the proposed system, supporting its potential within the framework of green materials and sustainable polymer engineering. Moreover, the reduced cytotoxicity observed for the encapsulated extract, together with the improved antibacterial performance of Cs/Co microaerogels, underscores the advantages of polymeric encapsulation in modulating bioactivity and biological response.\u003c/p\u003e \u003cp\u003eAlthough the results are promising, further investigations are required to fully elucidate the \u003cem\u003ein vivo\u003c/em\u003e antibacterial efficacy and long-term safety of the CsCoB-0.5 and CsCoB-1.0 formulations. Comprehensive \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies will be essential to validate their pharmacological properties and to support the future development of these systems as environmentally responsible biopolymeric carriers for the treatment of bacterial infections.\u003c/p\u003e"},{"header":"Abbreviations ","content":"\u003cp\u003eThe following abbreviations are used in this manuscript:\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"524\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eCs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eChitosan\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eSEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eScanning electron microscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eOM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eOptical microscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eFTIR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eFourier-transform infrared spectroscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eTGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eThermogravimetric analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eNSAIDs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eNon-steroidal anti-inflammatory drugs\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eWHO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eWorld Health Organization\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eFDA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eFood and Drug Administration\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eGRAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eGenerally Recognized as Safe\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eCo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eCollagen type I\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eNitric oxide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eTNF-\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eTumor Necrosis Factor alpha\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eLPS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eLipopolysaccharide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eCsCo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eChitosan/collagen\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eCsCoB-0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eChitosan/collagen-0.5% \u003cem\u003eB. microphylla\u003c/em\u003e extract\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eCsCoB-1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eChitosan/collagen-1.0% \u003cem\u003eB. microphylla\u0026nbsp;\u003c/em\u003eextract\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e3D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eThree-dimensional\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003e\u003cem\u003eBursera microphylla\u003c/em\u003e extract A. Gray\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eRH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eRelative humidity\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eAH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eAmmonium hydroxide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eEA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eEthyl alcohol\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eAA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eGlacial acetic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eAH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eAmmonium hydroxide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eMNH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eMagnesium nitrate hexahydrate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eSSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eSodium sulfate decahydrate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eMTT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003e(bromuro de 3-(4,5-dimetiltiazol-2-il)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eLz\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eLysozyme\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003ePBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003ePhosphate Buffered Saline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eDMEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eDulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eCLSI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eClinical and Laboratory Standards Institute\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eMA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eMoisture Absorption\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research is funded by the Secretar\u0026iacute;a de Ciencia, Humanidades, Tecnolog\u0026iacute;a e Innovaci\u0026oacute;n (SECIHTI) with the project CBF-2023-2024-3824, Ciencia B\u0026aacute;sica y de Frontera 2023\u0026ndash;2024, Mexico.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eV\u0026iacute;ctor Alonso Reyna-Urrutia, Julio C\u0026eacute;sar L\u0026oacute;pez-Romero and Heriberto Torres-Moreno conceptualization, investigation, resources, and supervision; V\u0026iacute;ctor Alonso Reyna-Urrutia, Ram\u0026oacute;n Enrique Robles-Zepeda, Miriam Estevez, Jos\u0026eacute; Luis L\u0026oacute;pez-Miranda, Juan Ram\u0026oacute;n C\u0026aacute;\u0026ntilde;ez-Orozco, Karen Lillian Rodr\u0026iacute;guez-Mart\u0026iacute;nez, Julio C\u0026eacute;sar L\u0026oacute;pez-Romero and Heriberto Torres-Moreno investigation, methodology, and validation; V\u0026iacute;ctor Alonso Reyna-Urrutia, Ram\u0026oacute;n Enrique Robles-Zepeda Karen Lillian Rodr\u0026iacute;guez-Mart\u0026iacute;nez, Julio C\u0026eacute;sar L\u0026oacute;pez-Romero and Heriberto Torres-Moreno data curation and validation; V\u0026iacute;ctor Alonso Reyna-Urrutia and Heriberto Torres-Moreno writing of the original draft; Julio C\u0026eacute;sar L\u0026oacute;pez-Romero and Heriberto Torres-Moreno writing, review, and editing. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eV\u0026iacute;ctor Reyna-Urrutia thanks SECIHTI for his postdoctoral fellowship. He also thanks the University of Sonora, Caborca Campus, for providing the facilities to carry out the research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCocoletzi HH, Almanza E\u0026Aacute;, Agustin OF, Nava ELV, Cassellis ER (2009) Obtaining and characterizing chitosan from shrimp exoskeletons. \u003cem\u003e22\u003c/em\u003e, 57\u0026ndash;60, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.24275/rmiq/Alim2315\u003c/span\u003e\u003cspan address=\"10.24275/rmiq/Alim2315\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang Y, Onyeri S, Siewe M, Moshfeghian A, Madihally SV (2005) In vitro characterization of chitosan-gelatin scaffolds for tissue engineering. 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Food Chem 440:138198. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.foodchem.2023.138198\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2023.138198\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-polymers-and-the-environment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jooe","sideBox":"Learn more about [Journal of Polymers and the Environment](https://www.springer.com/journal/10924)","snPcode":"10924","submissionUrl":"https://submission.nature.com/new-submission/10924/3","title":"Journal of Polymers and the Environment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bursera microphylla, chitosan, collagen, microaerogels, encapsulation, antibacterial performance","lastPublishedDoi":"10.21203/rs.3.rs-8913713/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8913713/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eBursera microphylla\u003c/em\u003e A. Gray is a native medicinal plant from northwestern Mexico whose fruit extract has demonstrated relevant bioactivity. Chitosan (Cs) and collagen (Co) are naturally derived polymers widely recognized for their biodegradability, biocompatibility, and suitability for environmentally friendly biomedical applications. In this study, Cs/Co microaerogels loaded with \u003cem\u003eB. microphylla\u003c/em\u003e fruit extract were synthesized, physicochemically characterized, and evaluated for \u003cem\u003ein vitro\u003c/em\u003e degradation, cytotoxicity, and antibacterial activity. Three-dimensional Cs/Co hydrogels were prepared by physical crosslinking using ammonium hydroxide, followed by lyophilization and mechanical grinding to obtain microaerogels. Morphological and structural characterization was conducted using scanning electron microscopy (SEM), optical microscopy (OM), Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and moisture absorption assays. Cytotoxicity was assessed in RAW 264.7 macrophages by MTT assay, while antibacterial activity was evaluated against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. \u003cem\u003eIn vitro\u003c/em\u003e degradation was analyzed under physiological conditions in the presence of lysozyme (Lz). The resulting microaerogels exhibited irregular morphologies with rounded protrusions and particle sizes mainly ranging from 45 to 90 \u0026micro;m. FTIR spectra confirmed the preservation of native functional groups without new chemical bond formation, while TGA indicated adequate thermal stability of the encapsulated extract. The microaerogels showed hydrophilic properties and a reduced lysozyme-mediated degradation rate due to the presence of collagen and the encapsulated extract. Compared to the free extract, Cs/Co microaerogels displayed lower cytotoxicity, reducing macrophage viability by only 32% at 200 \u0026micro;g/mL. Additionally, extract-loaded microaerogels significantly decreased \u003cem\u003eS. aureus\u003c/em\u003e viability by \u003cb\u003e37% and 57% for the 0.5% and 1% extract formulations, respectively\u003c/b\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results highlight the role of collagen in modulating structural stability and support the potential of Cs/Co microaerogels loaded with \u003cem\u003eB. microphylla\u003c/em\u003e fruit extract as sustainable biopolymeric delivery systems for antibacterial applications.\u003c/p\u003e","manuscriptTitle":"Functional Chitosan–Collagen Microaerogels Incorporating Bursera microphylla Fruit Extract with Antibacterial Properties","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-06 09:07:53","doi":"10.21203/rs.3.rs-8913713/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-03T08:23:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-23T11:53:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-19T09:36:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-18T17:20:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-10T11:27:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-09T12:49:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-04T20:00:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-04T15:02:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"175299113138000903103884524280747312643","date":"2026-03-04T13:10:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"39112151760275590882438487979172322404","date":"2026-03-04T12:27:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"166204406991072785807040729166214767760","date":"2026-03-04T07:38:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"113846503623984703552759437404265963266","date":"2026-03-03T21:23:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-03T16:15:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197828270677507593835666840660578006059","date":"2026-03-03T15:12:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"173068656333614910772200464393923938587","date":"2026-03-03T12:15:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"328372865987180068613419747531288595875","date":"2026-03-03T10:32:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"33311717554604597445117316437457868320","date":"2026-03-03T10:11:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-03T08:53:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-19T18:52:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-19T18:51:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Polymers and the Environment","date":"2026-02-19T04:07:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-polymers-and-the-environment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jooe","sideBox":"Learn more about [Journal of Polymers and the Environment](https://www.springer.com/journal/10924)","snPcode":"10924","submissionUrl":"https://submission.nature.com/new-submission/10924/3","title":"Journal of Polymers and the Environment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ba5abf3c-cc6d-47d2-a860-4f4c58154011","owner":[],"postedDate":"March 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T15:08:55+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-06 09:07:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8913713","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8913713","identity":"rs-8913713","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}