Pioneering wound care solutions: triaxial wet-spun fibers with bioactive agents for chronic wounds, part II (controlled release and biological activity of the active agents)

© 2025 The Author(s). Published by the Royal Society of Chemistry Mater. Adv., 2025, 6, 2029–2047 | 2029 Cite this: Mater. Adv., 2025, 6, 2029 Pioneering wound care solutions: triaxial wet-spun fibers with bioactive agents for chronic wounds, part II (controlled release and biological activity of the active agents)† Catarina S. Miranda, a A. Francisca G. Silva, b Camille Evenou, c Je ´ro ˆme Lamartine, c Berengere Fromy, c Sı ´lvia M. M. A. Pereira-Lima, b Artur Ribeiro, de Susana P. G. Costa, b Nata ´lia C. Homem f and Helena P. Felgueiras * a The incidence of bacterial infections associated with chronic wounds (CWs) has increased in recent years. Thus, a triaxial wet-spun fibrous system (containing three layers) was produced for CW healing. The triaxial fibers were loaded with cinnamon leaf oil (CLO), endowed with high antibacterial, antioxidant and anti-inflammatory features, and an antimicrobial peptide –alanine–alanine–proline–valine (AAPV) – capable of regulating the activity of human neutrophil elastase (HNE; highly expressed during inflammatory processes). To overcome the characteristic high volatility of essential oils (EOs), CLO was loaded at the system’s core and blended with polycaprolactone (PCL) which has excellent elasticity and tensile strength. The intermediate layer was composed of sodium alginate (SA) which has high hydration capacity and AAPV. Finally, the shell was made of cellulose acetate (CA), ensuring the system’s structural integrity and providing a porous network for the controlled release of AAPV and CLO. This research was divided into two parts, with the present addressing the biological characterization of the system, namely the controlled release of bioactive agents, their antibacterial, antioxidant and cytocompatibility profiles and the peptide-loaded fiber ability to inhibit HNE activity. AAPV-loaded wet-spun fibers attained a sustained release of up to 55% during 24 h of incubation in physiological-like media, also presenting effective HNE inhibition (E65%). Additionally, CLO-loaded fibers demonstrated a controlled release of up to E52% during 24 h of incubation in PBS, reaching higher antibacterial and antioxidant profiles in comparison with the unloaded fibers. Data confirmed the biological potential, safety and suitability of the proposed system for future applications in CW care. 1. Introduction Chronic wounds (CWs) cannot heal at the same pace as acute wounds. 1 CWs often stall at inflammation, conditioning the patients’ health and their quality of life, leading to chronic pain, loss of function and mobility, and ultimately raising morbidity rates. 2 Frequently, these wounds are the result of bacterial infections, triggered by Staphylococcus aureus,Staphylococcus epidermidis,Escherichia coli and Pseudomonas aeruginosa, some of the most prevalent microorganisms and a major concern for the World Health Organization. 2,3 Between 2005 and 2022, P. aeruginosa was detected in 14.5% of CWs in the United States of America (USA), while in the United Kingdom (UK) and Portugal, its presence was detected in 8.6% and 11.4% of the cases, respectively. 4 Moreover, in CWs, neutrophils release proteases (including human neutrophil elastase, HNE) in an uncontrolled manner, as soon as bacteria start colonizing the wound site. Such abnormally high concentrations of HNE a Centre for Textile Science and Technology (2C2T), University of Minho, Campus of Azure ´m, 4800-058 Guimara ˜es, Portugal. E-mail: [email protected], helena.felgu[email protected]; Fax: +351-253-510-293; Tel: +351-253-510-283 b Centre of Chemistry (CQ), University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal. E-mail: [email protected], [email protected], [email protected] c E ´quipe Inte ´grite ´fonctionnelle du tissu cutane ´(SKIN), Laboratoire de biologie tissulaire et d’inge ´nierie the ´rapeutique (LBTI), CNRS UMR5305, Universite ´Lyon I, 7 passage du Vercors, 69367 Lyon Cedex 07, France. E-mail: [email protected], [email protected], [email protected] d CEB – Centre of Biological Engineering, University of Minho, Braga, 4710-057, Portugal. E-mail: [email protected] e LABBELS – Associate Laboratory, Braga, Guimara ˜es, Portugal f Simoldes Plastics S.A., Rua Comendador Anto ´nio da Silva Rodrigues, 165, 3720193, Oliveira de Azeme ´is, Portugal. E-mail: [email protected] †Electronic supplementary information (ESI) available. See DOI: https://doi.org/ 10.1039/d4ma01104j Received 5th November 2024, Accepted 14th February 2025 DOI: 10.1039/d4ma01104j rsc.li/materials-advances Materials Advances PAPER This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal | View Issue 2030 | Mater. Adv., 2025, 6, 2029–2047 © 2025 The Author(s). Published by the Royal Society of Chemistry during inflammatory processes lead to the degradation of endogenous and supplemental growth hormones, which prevent the wounds from progressing to the following steps of the healing process. 1,5,6 The inhibitory effect of the alanine–alanine–proline–valine (AAPV) tetrapeptide against HNE has been proven to be effective. 6,7 The mechanism of action of the AAPV peptide is not yet fully understood. Still, according to Toth et al., the peptide fits the P-P1 subsites of HNE, resulting in competitive inhibition of HNE activity. 6,8 As for other small peptides, AAPV cannot be used freely in CW therapies due to its low stability in physiological media, as well as high sensitivity to changes in environmental conditions. Also, these peptides present toxicity when used at high concentrations during systemic delivery. 9,10 To prevent such events, these bioactive agents can be incorporated into polymeric structures that offer protection against environmental changes and allow their controlled and localized release, working as an alternative for the use of antibiotics. 7 Antibiotics are a common tool in the treatment of bacterial infections in CWs. 10,11 However, their excessive use has resulted in the need to think of alternate solutions, including essential oils (EOs), therapeutic agents of natural origin that are mainly composed of mixtures of volatile and lipophilic compounds extracted from several parts of plants. 9,11 EOs contain hydrophobic elements responsible for their inherent analgesic, anti-inflammatory, antioxidant and antibacterial properties. 12,13 The antibacterial effects of EOs result from their ability to disrupt processes related to ion or solute transport, due to their lipophilicity, which facilitate their penetration through the bacterial membrane and the interference with intracellular components. 9,11,14 Cinnamon leaf oil (CLO) is extracted from cinnamon barks and leaves and is mainly formed from eugenol and cinnamaldehyde, which endows the EO with antibacterial abilities. 12,15 The antioxidant and antibacterial properties of eugenol have been extensively addressed. 9,16,17 Additionally, this compound has been reported to possess anti-inflammatory properties, by suppressing the expression of the cyclooxygenase II enzyme. 18 However, similarly to small peptides, the use of EOs in therapeutic approaches comes with limitations, including cytotoxicity at high concentrations, high volatility and sensitivity to external factors, such as temperature, light and oxygen. 9,13 Still, in recent years, research has been disclosed on the loading of EOs and therapeutic peptides onto polymeric fibers that increase these bioactive agent’s physiological stability, while preserving their chemical features. 9,19 Producing polymeric fibrous structures loaded with EOs and/or peptides can be accomplished using spinning techniques, including wet-spinning. 20,21 Wet-spun fibrous constructs display a wide range of diameters and morphologies, along with high porosity. Such properties are favored in biomedicine by enabling cell penetration, adhesion and proliferation, and by resembling physiological microenvironments. 20,22 The goal of the current research was to engineer a triaxial system with bacterial and enzyme-related inhibitory features for promoting wound healing. For that, a wet-spinning approach was carried out to produce fibers with three layers, also known as a triaxial system: (1) a core (innermost layer) made of polycaprolactone (PCL), intended to offer the fibers high elasticity, loaded with CLO to inhibit the growth of both Gram-positive and Gramnegative bacteria; 7,23,24 (2) an intermediate layer made of a blend of sodium alginate (SA) and AAPV, to maintain a moist environment and to regulate the local enzymatic activity, respectively; 6,7 and (3) a shell (outermost layer) composed of cellulose acetate (CA) that ensures structural integrity of fibers and offers protection to the inner layers containing the bioactive compounds. 25 The porosity inherent to the shell allowed a somewhat controlled access to the active agents present in the intermediate layer and the core, releasing AAPV and CLO in a sustained manner. Research was divided in two parts: part I (under peer revision), chemical, physical, mechanical and thermal characterization of the triaxial system, along with a proof of concept consisting of the production of a small dressing; and part II (present manuscript), biological characterization of the triaxial system. The minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) of CLO, in its free form, were examined against S. aureus,S. epidermidis,E. coli and P. aeruginosa. HNE inhibition evaluations were performed to determine the AAPV’s maximum inhibitory concentration (IC M ), also in its free form. Additionally, bioactive agent release kinetics studies, from within the fiber systems, were carried out. The HNE inhibition capacity of the engineered fibers was evaluated, as well as their antibacterial and antioxidant properties. Finally, the safety of the triaxial fibers towards skin cell models were investigated in vitro. Recently, this technique has drawn attention as a promising tool for developing drug delivery systems for biomedical purposes. However, to the authors’ knowledge, there are no reports on the production of Eos and peptide-loaded triaxial wet-spun fibers for wound healing applications. 2. Materials and methods 2.1. Materials CA (M n 50.000), PCL (M n 80.000), SA (from brown algae, medium viscosity), 1,1-diphenyl-2-picrylhydrazyl (DPPH), ethyl cyanoglyoxylate-2-oxime (Oxyma), 1,3-diisopropylcarbodiimide (DIC), dichloromethane (DCM), chloroacetic acid, deuterium oxide (D 2 O), elastase from human leukocytes, trypsin inhibitor from soybean, N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide (N-MeO-Suc-Ala-Ala-Pro-Val-p-NA), Dulbecco’s Phosphate Buffered Saline (DPBS), trypsin–EDTA, Tryptan Blue solution and fetal bovine serum (FBS) were obtained from Sigma-Aldrich (St Louis, Missouri, USA). Antibiotic–antimycotic was obtained from Grisp (Porto, Portugal). N,N-dimethylformamide (DMF), acetic acid (AcOH), acetonitrile (ACN), piperidine, sodium hydroxide, sodium carbonate, 2,2,2-trifluoroethanol (TFE) and trifluoroacetic acid (TFA) were acquired from Merck (Darmstadt, Germany). Dimethyl sulfoxide (DMSO) and 6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were Paper Materials Advances This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online © 2025 The Author(s). Published by the Royal Society of Chemistry Mater. Adv., 2025, 6, 2029–2047 | 2031 obtained from Fisher (Maharashtra, India). Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin–streptomycin, Alamar Blue and lactate dehydrogenase (LDH) kit were purchased from Invitrogen (Massachusetts, USA). Triton was acquired from BioRad Laboratories (California, USA). Anhydrous calcium chloride was employed as coagulation/crosslinking agent during wet-spinning and was supplied by Chem-Lab (Zedelgem, Belgium). Tris–hydrochloride (Tris–HCl) was purchased from Roche (Basel, Switzerland). AAPV and WAAPV were synthesized by us as described in ref. 7. Pure cinnamon leaf oil (CLO, origin Cinnamomum zeylanicum Blume, r= 1.049) was obtained from Folha d’A ´gua Company (Santo Tirso, Portugal). 19 Sodium phosphate dibasic (SigmaAldrich), monosodium phosphate monohydrate (Merck), potassium chloride (Merck) and sodium chloride (Merck) were used in the preparation of phosphate buffer saline solution (PBS at 0.01 M: 1.44 g L 1 of Na 2 HPO 4 , 0.24 g L 1 of KH 2 PO 4 , 0.20 g L 1 KCl and 8.00 g L 1 of NaCl, adjusted to physiological pH 7.4). Gram-positive bacteria S. aureus (ATCC 6538) and S. epidermidis (ATCC 35984), along with Gram-negative bacteria E. coli (ATCC 25922) and P. aeruginosa (ATCC 25853) were supplied by American Type Culture Collection (ATCC, Virginia, USA). For bacteria growth, trypticase soy broth (TSB), trypticase soy agar (TSA), nutrient agar (NA) and nutrient broth (NB) were purchased from VWR (Alfragide, Portugal), while the Mueller Hinton broth (MHB) was obtained from CondaLab (Madrid, Spain). Human keratinocytes cell line (HaCaT) – DKFZ HaCaT adherent cell line (immortalized human keratinocytes) – was obtained from Cytion (previously known as CLS) (Eppelheim, Germany), and provided by DKFZ (Helmtoz, Germany). 26 Mouse embryonic fibroblast cell line (NIH 3T3) from Sigma Aldrich (Darmstadt, Germany). All reagents were used without further purification. 2.2. Wet-spun fiber production A PCL solution was prepared in DMF at 10% w/v and stirred for 1 h at 50 1C. CLO was then combined at 16.40 mg mL 1 (amount established in ref. 27), consisting of 4its MIC. Both compounds were left to homogenize for 1 h at 50 1C (PCL–CLO solution). 28 A 2% w/v aqueous SA solution was also prepared and stirred for 3 h at 50 1C. Afterwards, AAPV was added to the previous solution at 50 mgmL 1 , corresponding to its maximum inhibitory concentration (IC M ) (concentration established in ref. 7) (SA–AAPV solution). Finally, a 10% w/v CA solution was dissolved in DMF for 3 h at 50 1C. A wet-spinning setup was used, containing three syringe pumps (NE-300, New Era Pump Systems, Norleq, Santo Tirso, Portugal), responsible for controlling the rate and ejection volume, a triaxial spinneret (sealed needles of three layers with 21, 15 and 11 gauge, from the inner to the outer layer) and a large tray containing 500 mL of a 2% w/v CaCl 2 coagulation bath, at room temperature (RT). PCL or PCL–CLO solutions were loaded onto the syringe connected to the innermost port (core) and ejected at 0.11 mL min 1 , whereas SA or SA–AAPV solutions were loaded onto the intermediate port and ejected at 0.13 mL min 1 . Additionally, CA solution was ejected at 0.15 mL min 1 and loaded onto the outermost port (shell). Fibers containing only one layer (PCL, PCL–CLO, SA, SA–AAPV, CA) were also produced, as well as fibers without one of the layers (e.g., PCL/SA, PCL/CA, SA/CA). To each group of fibers, samples without one/ both of the active agents were also produced in order to test the influence of each compound on the properties of the system (e.g., PCL/SA–AAPV, PCL–CLO/SA/CA). The identification of the fibers used ‘‘/’’ for separating elements belonging to different layers and ‘‘–‘‘ for components within the same layer (Table 1). 2.3. WAAPV and CLO release kinetics The monitoring of AAPV release by using IV-visible absorption spectroscopy was not possible, since an overlapping of maximum wavelengths of absorption between all fiber compounds (all biodegradable polymers) was observed. Due to the absence of fluorescence from AAPV, a modified version of the peptide was synthesized, by including the fluorescent amino acid tryptophan (WAAPV), used to monitor the release by fluorimetry. Although the presence of such amino acid may have led to differences in the peptide release rates, such modification still Table 1 List of wet-spun fiber typologies analyzed in this research Fiber typology Core composition Intermediate layer composition Shell composition PCL PCL — — PCL–CLO PCL blended with CLO — — SA — SA — SA–AAPV — SA blended with AAPV — CA — — CA PCL/SA PCL SA — PCL–CLO/SA PCL blended with CLO SA — PCL/SA–AAPV PCL SA blended with AAPV — PCL–CLO/SA–AAPV PCL blended with CLO SA blended with AAPV — SA/CA — SA CA SA–AAPV/CA — SA blended with AAPV CA PCL/CA PCL — CA PCL–CLO/CA PCL blended with CLO — CA PCL/SA/CA PCL SA CA PCL–CLO/SA/CA PCL blended with CLO SA CA PCL/SA–AAPV/CA PCL SA blended with AAPV CA PCL–CLO/SA–AAPV/CA PCL blended with CLO SA blended with AAPV CA Materials Advances Paper This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online 2032 | Mater. Adv., 2025, 6, 2029–2047 © 2025 The Author(s). Published by the Royal Society of Chemistry allowed the prediction of the peptide’s behavior in physiological media, due to their similar chemical structures. The fluorescent peptide was loaded onto the fibers as described in ref. 7. A WAAPV calibration curve in PBS at concentrations ranging from 0.500 and 62.500 mgmL 1 was prepared using a spectrometer Sarspec (Vila Nova de Gaia, Portugal) and LightScan 2.0 software. WAAPV-loaded fibers were immersed in PBS, pH 7.4, for 1, 2, 4, 6 and 24 h of incubation at 37 1C and 120 rpm, and aliquots of 150 mL were collected at each time point. Fluorescence was then read using an LED lamp of 275 nm, in the range 180–900 nm, with a resolution of 1 nm and wavelength accuracy of 0.5 nm. Results were reported as intensity counts (IC) vs. wavelength, based on the WAAPV calibration curve (IC = 32.413 Concentration + 4144.7; R 2 = 0.9858). Regarding the EO, the amount of CLO released from all wetspun fibers were estimated using a UV-1800 UV-visible spectrophotometer (Shimadzu) (measurement of remaining concentration after fiber immersion). Initially, a calibration curve of CLO at different concentrations in PBS, ranging from 0.02 to 0.20 mg mL 1 , was prepared to enable reliable detection of the spectra’s region of interest. The results were plotted as absorbance vs. wavelength. The release of CLO from the wet-spun fibers was assessedinPBSafter1,2,4,6and24h.Theselectionofsuch medium was based on previous research studies from our team, in an attempt to approximate that of physiological-like media, also considering the desired future application of this system as a wound dressing. 7,10 Samples of CLO-containing fibers weighing 10 mg each were left in contact with 1 mL of PBS at 37 1Cand 120 rpm, and aliquots of 150 mL were collected at each time point. Their absorbances were then measured in the range 200–900 nm. The release of CLO was determined by the differences in absorbance between the first moment of interaction (0 h) and the subsequent measuring periods, up to the 24 h mark. 2.4. AAPV-loaded fibers’ inhibitory effect against HNE AAPV-loaded and unloaded fibers were incubated in 1 mL of PBS, pH 7.4, at 37 1C and 120 rpm for 1, 2, 4, 6 and 24 h. After each incubation period, aliquots of 150 mL were collected; these represented the testing solutions. AAPV-unloaded fibers were also analyzed, in this way subtracting the polymers’ influence from the results of AAPV-loaded fibers. HNE inhibition experiments were conducted as described in ref. 7. Briefly, a substrate solution (N-MeO-Suc-Ala-Ala-Pro-Val-p-NA) (125 mL) was mixed with 0.1 M Tris–HCl buffer (405 mL), along with the 150 mL aliquots of the testing solutions in PBS and 20 mL of HNE solution (45 mU). After 1 h of incubation at 37 1C, an inhibitor solution (500 mL) was added to stop the reaction and absorbances were read at 405 nm (EZ Read 2000 Microplate Reader, Biochrom, Cambridge, UK). Data were reported as maximum inhibitory concentration (IC M ). Experiments were conducted in triplicate, with three absorbance readings being done per replicate (mean averaging nine measurements). 2.5. Antioxidant activity Free radical-scavenging activity was determined using the 1,1diphenyl-2 picrylhydrazyl (DPPH) assay. Samples of 10 mg of each wet-spun fiber typology were immersed in 1 mL of absolute ethanol and incubated at 37 1C and 120 rpm for 1, 2, 4, 6 and 24 h. Ethanol was selected, not only because of the EO’s high solubility, but also due to its common use in DPPH assays and the low water content, which could lead to interferences in the absorbance readings. 29–31 Aliquots of 10 mL were collected at each time period and mixed, in a 96-well plate, with 140 mL of a DPPH stock solution (400 mM in absolute ethanol). DMSO and Trolox were used as negative and positive controls, respectively. The absorbances were monitored every 5 min for 1 h and measured at 515 nm, using an EZ Read 2000 Microplate Reader (Biochrom, Cambridge, UK). Experiments were conducted in triplicate and data were reported in terms of the percentage of reduced DPPH at a steady state, which was calculated as follows (eqn (1)): DPPHr%ðÞ¼ AiAr Ai 100;(1) in which A r represents the absorbance registered at a steady state and A i corresponds to the initial absorbance. 2.6. Bacteria inhibition: time-kill kinetics Time-kill kinetics tests were conducted according to standard ASTM E2149-01, adapted as reported previously. 7,10 The antibacterial profile of the triaxial wet-spun fibers containing the AAPV peptide and the CLO was evaluated against 1 10 5 CFUs per mL suspensions of S. aureus,S. epidermidis and E. coli in TSB, and a suspension of P. aeruginosa prepared in NB. Samples of 10 mg were immersed in 1 mL of each bacterium suspension and incubated at 37 1C and 120 rpm for 1, 2, 4, 6 and 24 h. At each time point, bacterial suspensions were serially diluted in PBS (10 1 to 10 4 ), plated in TSA and NA, and incubated at 37 1C for 24 h. Grown colonies were counted, and results were expressed in percentage (%) of bacteria inhibition. All measurements were performed in triplicate (three measurements were done per replicate, with data averaging nine values) and data were processed using the GraphPad Prism 8.0 software. 2.7. Biofilm formation Bacteria inoculums of S. aureus,S. epidermidis and E. coli were prepared in TSB and of P. aeruginosa in NB and left overnight to grow at 37 1C and 120 rpm. Four replicates from each fiber typology (10 mg) were weighed into 48-well plates and covered with 5 mL of each inoculum mixed with 500 mL of MHB. Control wells containing the diluted inoculums but without samples were also considered for this assay. The plates were incubated for 48 h at 37 1C to allow biofilm formation. Afterwards, fibers were removed from each well and the plate was washed 3with PBS to remove loosely bound biofilm cells. The plate was left at 37 1C for 20 min for fixating the biofilms. Then, a 0.1% w/v crystal violet (CV) aqueous solution was added to each well and the plate was left at RT for 15 min. This was followed by 3 washes with PBS to remove unbound CV. Finally, 500 mLof a 30% v/v acetic acid solution were added to each well for 15 Paper Materials Advances This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online © 2025 The Author(s). Published by the Royal Society of Chemistry Mater. Adv., 2025, 6, 2029–2047 | 2033 min and aliquots were transferred to a 96 well-plate for absorbance readings at 595 nm. 2.8. Cytocompatibility testing 2.8.1. Cell culture conditions. The cytocompatibility and cell proliferation of all fiber typologies towards mouse embryonic fibroblast cell line (NIH 3T3) and human keratinocyte cell line (HaCaT) were evaluated using a direct contact assay. Cells were thawed and sub-cultured in DMEM supplemented with 10% v/v inactivated FBS (30 min, 56 1C) and 1% v/v penicillin– streptomycin, at 37 1C in a humidified atmosphere of 5% CO 2 . Thereafter, cells were chemically detached from cell culture flasks using trypsin–EDTA solution. The number of cells was determined using a Malassez cell (Marienfeld, Germany), with cells being labelled with trypan blue (Sigma Aldrich, UK), to evaluate the proportion of dead cells, and counted using a Carl Zeiss Suzhou Co., Ltd (Suzhou, China) microscope. Cells were seeded in 96-well flat-bottom tissue plates (Greiner, Germany) and incubated for 24 h at 37 1C in a humidified atmosphere of 5% CO 2 . HaCaT and NIH 3T3 were seeded at 15 000 and 20 000 cells per well, respectively (concentrations determined in a previous investigation; unpublished data). The experiments were performed using fibroblast cell line in passages 2 to 12 and keratinocyte cell line in passages 20 to 30. Fibrous samples of 2 mg were used for each test. Experiments were conducted in triplicate. Fibers were washed and disinfected 3with deionized water (dH 2 O) and 1ethanol at 70% v/v, during 5 min each, prior to any testing. 2.8.2. Cell viability assessment. Prior to any fiber testing, the determination of the optimum cell number was carried out by seeding cells into 96-well flat black tissue plates at concentrations ranging from 5000 to 25 000 cells per well. Cells were incubated overnight at 37 1C and 5% CO 2 atmosphere. Afterwards, the medium was removed and 100 mL of a 10% v/v Alamar Blue TM (Invitrogen) were added to each well. Cells were again incubated for 1 h and the fluorescence was read (l exc = 560 and l em = 590 nm) using a Tecan fluorimeter (Tecan Spark, Lyon, France) with Tecan Spark Control 3.2 software. The metabolic activity for each cell concentration was calculated using eqn (2): Metabolic activity %ðÞ ¼Flu sample Flu negative control Flu positive control Flu negative control 100; (2) where the Flu sample corresponds to the fluorescence of each sample, Flu negative control corresponds to cells grown in 5% v/v DMSO solution, and Flu positive control represents a seeding of 50 000 cells per well, superior to the maximum tested density in the assay to assure that a very high growth of both cell lines was achieved. After determining the optimum cell number for each cell line, cells were seeded and 6 h later fibers were added to the wells. After 24 and 48 h of incubation at 37 1C and 5% CO 2 atmosphere, the metabolic activity of the cells was evaluated using the Alamar Blue assay. 2.8.3. Cell lysis assessment. The cell lysis test was performed by following the instructions from LDH kit (Invitrogen). Firstly, the optimum cell number for performing the LDH assay was determined by seeding cells at concentrations from 0 to 10 000 cells per well in 96-well flat black tissue plates (6 replicates for each cell concentration). Cells were incubated overnight at 37 1C and 5% CO 2 atmosphere. Afterwards, 10 mLof sterile water were added to the first three replicates of each cell concentration (spontaneous LDH activity), whereas 10 mLof2% v/v Triton solution in DPBS were added to the second set of replicates for each cell concentration (maximum LDH activity). Cells were again incubated for 45 min, protected from light, followed by the transfer of 50 mL of each supernatant to a new plate, to which 50 mL of a reaction mixture was added. Cells were once more incubated at room temperature (RT) during 10 min, protected from light. Then, 50 mL of a stop solution were added to all wells and the fluorescence was measured (l exc = 560 and l em = 590 nm) using Tecan fluorimeter. The maximum LDH release activity absorbances minus the spontaneous LDH release absorbances versus the tested cell numbers were plotted using the GraphPad Prism 8.0 software. Cells were then seeded at the determined optimum cell number and, 6 h later, fibers were added. After 24 h of incubation, the cell lysis test was repeated. The percentages of cell lysis were then determined using the following eqn (3): Cell lysis %ðÞ¼ Fluo sample Fluo spontaneous ctrl Fluo maximum ctrl Fluo spontaneous ctrl 100;(3) where the Fluo sample corresponds to the fluorescence of cells treated with each fibrous sample, Fluo spontaneous ctrl represents the cells treated with sterile dH 2 O (spontaneous LDH activity) and Fluo maximum ctrl corresponds to the cells treated with 2% v/v Triton (maximum LDH activity). 2.8.4. Cell morphology observations. Cells were visualized at brightfield using a light microscope (Carl Zeiss Suzhou Co., Ltd). Light microscope images were obtained at the beginning of the experiment (t= 0 h) and after 24 h of incubation in contact with the samples, to detect any possible changes in cell morphology. The images were acquired at a 10magnification objective using an Axiocam 105 color camera (Zeiss, Switzerland), using a Zeiss ZEN 3.8 software. 2.9. Cell migration 2.9.1. Cell culture. Immortalized HaCaT were cultured in DMEM, supplemented with 10% v/v FBS and 1% v/v antibiotic– antimycotic solution. Cellular subcultures were performed when confluence reached values close to 80–90%. HaCaT cells were maintained in 25 cm 2 or 75 cm 2 tissue culture flasks in an incubator at 37 1C in a humidified atmosphere with 5% CO 2 . The cell culture medium was renewed 2per week. For subcultures and plating, the adherent cells were detached with trypsin solution 0.05% v/v, and fresh medium was added in order to neutralize the trypsin. The cell suspension was centrifuged 5 min at 160 g. The supernatant was discarded, and Materials Advances Paper This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online 2034 | Mater. Adv., 2025, 6, 2029–2047 © 2025 The Author(s). Published by the Royal Society of Chemistry fresh medium was added to obtain a new cell suspension. The cell suspension was injected in a Neubaeur chamber, and the cell concentration was determined. 2.9.2. Scratch assay. The effect of PCL/SA/CA, PCL/SA– AAPV/CA, PCL–CLO/SA/CA and PCL–CLO/SA–AAPV/CA samples on the migration profiles of HaCaT cells was evaluated by microscopic visualization after the scratch assay. Fibers were washed and disinfected 3with dH 2 O and 1with ethanol at 70% v/v, during 5 min each, prior to the experiment. Cells were seeded at a density of 5.0 10 4 cells per well, on a 24-well tissue culture plate (Trasadingen, Switzerland) the day before the experiments. After confirmation of cell confluency, a scratch on the cell monolayer was performed with the help of a disposable standard 200 mL pipette tip. The wells were washed with sterile PBS and fresh medium was added to the wells prior to the visualization and recording of the scratch in a Leica DMI 3000 B inverted fluorescence microscope (Heerbrugg, Switzerland) equipped with Leica DFC450 Ccamera (Heerbrugg, Switzerland). The cells were then exposed to the samples and were further incubated at 37 1C in a humidified atmosphere of 5% CO 2 . Cells incubated only with fresh culture medium were used as control of cell migration. At the end of 24 and 48 h of contact, the cell migration was evaluated by microscopic visualization (phase contrast, 10magnification) and determined using the ImageJ s software (version 1.53, National Institutes of Health, Bethesda, Maryland, USA), respectively. The percentages of wound area were determined at 24 h and 48 h, using the following eqn (4): 32 %wound area ¼AtðÞ A0 100;(4) where A(t) corresponds to the area of each sample determined at 24 h/48 h and A 0 corresponds to the area of each sample at t= 0h. 2.10. Statistical analysis All measurements were conducted in triplicate unless otherwise mentioned in the experimental sections. Numerical data were reported as mean standard deviation (SD). Data were treated using GraphPad Prism 8.0 Software (GraphPad Software Inc, USA). Normality analysis was performed, and results were analyzed using One-way ANOVA and Tukey tests. Statistically significant differences were considered at po0.05. 3. Results and discussion The triaxial wet-spun fibers were produced via wet-spinning, as described in Section 2.2. The presence of three distinct layers on the fibers was confirmed via brightfield microscopy and scanning electron microscopy (SEM; Fig. 1). Through Fig. 1 Micrographs of triaxial wet-spun fiber morphology obtained via brightfield microscopy of (a) the complete system (1: core composed of PCL– CLO; 2: intermediate layer composed of SA–AAPV; 3: shell composed of CA); and (b) of the tri-axial fibers without additives (1: core composed of PCL; 2: intermediate layer composed of SA; 3: shell composed of CA). Visualization of the (c) surface morphology (magnification of 500) and (d) cross-section (magnification of 70) of the complete triaxial fibers obtained by SEM. Paper Materials Advances This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online © 2025 The Author(s). Published by the Royal Society of Chemistry Mater. Adv., 2025, 6, 2029–2047 | 2035 morphological, mechanical and thermal property analyses (data under peer revision), the flexibility and elasticity of the fibers was demonstrated, reaching values up to E300% of maximum elongation. Also, the engineered triaxial fibers were deemed highly stable when exposed to physiological-like media, during 28 days of incubation, losing only 23% in mass throughout that period. In the end, the triaxial wet-spun fibers were proven effective for knitting a wound dressing, in this way assuring the suitability of this system for wound healing applications. The current work focused on the biological properties of the triaxial system, including antimicrobial and antioxidant activities, the release profiles of the active agents (AAPV and CLO), the regulation of the local enzymatic activity, and the cytocompatibility of each fiber typology (safety). 3.1. WAAPV release kinetics In order to map AAPV release from peptide-loaded wet-spun fibers, a modified version of AAPV, in which a tryptophan was attached to the N-terminus, was used (WAAPV). Such modification was necessary due to an overlap of maximum absorbance bands from PCL, SA and CA polymers, centered at E200 nm, detected via UV-visible spectroscopy. As a result, WAAPV presented a maximum fluorescence peak at E275 nm, detected using fluorimetry, thus allowing for reliable detection of this compound without any interference from the remaining fiber compounds (confirmed by the analysis of the unloaded fibers). Cumulative release profiles of WAAPV were determined by comparison with the calibration curve of the analyzed peptide (Fig. 2). SA–WAAPV monolayered fibers attained the highest release profiles, since no inner or outer layers were present to protect the payload, leading to a higher exposure of the peptide to the media. In its presence, the inner layer made of PCL or PCL–CLO conditioned the peptide release from both PCL/ SA–WAAPV (difference of E3.25% compared to SA–AAPV) and PCL–CLO/SA–WAAPV coaxial fibers (difference of E7.04% compared to SA–AAPV). As observed in a previous research, 7 PCL tends to partially incorporate the SA or SA–WAAPV intermediate layers, interfering with the possible migration of the peptide towards the surrounding media. However, in this combination, the presence of visible folds and breakable sites along the SA–WAAPV shell (Fig. S1 in the ESI†) may have potentiated its degradation, accelerating peptide release. Moreover, the partial dissolution of SA while in contact with the physiological media in response to its highly hydrophilic nature and increased affinity towards water may also justify such outcome. 33,34 PCL/SA–WAAPV/CA triaxial fibers reported the lowest WAAPV release profiles, since it was conditioned by both the innermost layer of PCL and an outermost layer made of CA, which was endowed with high mechanical resistance and hydrophobic behavior, limiting the interactions of WAAPV with water molecules from PBS. 35–37 That same conclusion could be retained to explain the low release profiles registered for SA– WAAPV/CA coaxial fibers. On the other hand, PCL–CLO/SA– WAAPV/CA triaxial fibers showed a slightly superior release rate compared to PCL/SA–AAPV/CA triaxial fibers (E7.70% of increment). The presence of CLO, a compound with high affinity towards PCL, is expected to have altered the conformation of PCL polymeric chains, leading to a lower partial incorporation of the SA–WAAPV intermediate layer, therefore facilitating access to the peptide from the physiological media. 13 Overall, all WAAPV-loaded fibers displayed a prolonged and sustained release of the peptide, assuring their suitability as effective drug delivery platforms. Even though the mechanism of action of WAAPV is not yet fully understood, it is plausible to assume a similarity with AAPV, since both peptides share equal amino acids, apart from tryptophan. 3.2. CLO release profile CLO’s release kinetics was assessed after fiber contact with PBS up to 24 h of incubation (Fig. 3). Data from unloaded fibers were also collected to eliminate interference of polymers from the CLO-loaded fiber results. As expected, the PCL–CLO monolayered fibers release the most CLO in the shortest period of time, because of the easier access to the oil by the surrounding media. 22,38 Interestingly, both PCL–CLO/SA and PCL–CLO/SA– AAPV coaxial fibers also reached similar outcomes. One plausible explanation for this occurrence may rely on the feeble mechanical properties and media instability of the SA and SA– AAPV outer layers. 39 It is likely that the reduced structural integrities along with the hydrophilic character of the outer layers in both PCL–CLO/SA and PCL–CLO/SA–AAPV coaxial fibers caused the partial dissolution of SA and SA–AAPV onto the physiological media and, thus, prompted the release of CLO. 33,34 As CA displays high rigidity and structural integrity, conferring a barrier effect on the wet-spun fibers, PCL–CLO/CA and the triaxial fibers reported a slower release kinetics for CLO. 22,38,40 Generally, all fiber typologies experienced a burst release after 1 h of incubation, continuously increasing the CLO liberation from that point and until the 6 h mark. After that period oil release became more sustained, most likely because Fig. 2 Cumulative release kinetics of WAAPV-loaded wet-spun fibers. Data are reported as mean SD (n= 3). Statistical significance was determined via the Tukey test, applying multiple comparisons between the different fiber typologies (no statistical significances were detected). Materials Advances Paper This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online 2036 | Mater. Adv., 2025, 6, 2029–2047 © 2025 The Author(s). Published by the Royal Society of Chemistry access to the remaining molecules was more challenging (stronger interactions with the polymers). 3.3. HNE inhibition The inhibition of the HNE activity by AAPV-loaded wet-spun fibers was assessed by their incubation in PBS at 37 1C for a period of 24 h, simultaneously confirming the successful incorporation of the peptide (Fig. 4). Data from unloaded fibers was also collected and subtracted from the results from AAPVloaded fibers, thus removing the polymer interference. All fibers presented HNE inhibitions superior to 15% in their first hour of incubation, which was associated with an initial burst release of the peptide (Fig. 2). However, after 6 h and until the 24 h mark, all samples reported a smaller inhibitory profile, since the peptide was released at a slower pace (Fig. 3). As expected, triaxial fibers reported lower HNE inhibitory capacities in comparison with coaxial and monolayered fibers, due to the presence of the CA protective layer, which limited the access to AAPV. 22,38,40 Additionally, in the presence of PCL, SA is partially lost, becoming entrapped in the PCL polymer chains, therefore interfering with the availability of AAPV and its release to the media. 7 That same theory explains not only the superior HNE inhibition from SA–AAPV/CA coaxial fibers, but also the lower HNE inhibitory profiles observed for PCL–CLO/ SA–AAPV coaxial fibers. Since the affinity between CLO and PCL is greatly promoted by the interaction between the hydroxyl groups of eugenol (one of the main elements of CLO) and the carbonyl groups of PCL, leading to the molecular dispersion of CLO throughout the PCL polymeric matrix, the incorporation of the SA–AAPV intermediate layer was exacerbated. 13,41 However, after 6 h, such differences were less pronounced, most likely due to the characteristic high volatility of the EOs. 9,13 Despite inducing a smaller HNE inhibitory profile (in average), the triaxial fibers did not report significant differences compared to the other samples, being still classified as efficient in regulating HNE activity, with inhibitory capacities up to E65%, and thus capable of contributing to the healing process. 6 3.4. Antioxidant activity The antioxidant activities of CLO-loaded fibers were evaluated by incubating the samples for 24 h in absolute ethanol, prior to conducting the DPPH radical scavenging assay (Fig. 5 and Fig. S2 in the ESI†). Again, data from unloaded fibers were collected to remove the polymer interference in the results. CLO’s strong antioxidant properties originate in eugenol, one of its main components. 31 Brand-Williams et al. showed that the scavenging activity of eugenol is achieved by the initial formation of an antioxidant radical, followed by three types of reaction pathways, including the donation of a second hydrogen atom to the DPPH radical, the formation of a complex between one DPPH radical and one aryl radical, and the dimerization between two phenoxyl radicals that regenerate two hydroxyl groups capable of interacting with DPPH. 42 Eugenol’s ability to interfere with free radicals from hydrogen peroxide offers cell protection against oxidative stress, which, consequently, accelerates wound healing. 43 PCL–CLO monolayered fibers reached one of the highest DPPH reduction rates (varying between E57.87% at 2 h, the maximum, and E36.34% at 4 h, the minimum activity). This occurred because of the absence of an outer, protective barrier, therefore increasing the availability and exposure of CLO to the media. 22,38 PCL–CLO/SA and PCL–CLO/SA–AAPV coaxial fibers also reported high antioxidant activities, because of the SA and SA–AAPV outer layers mechanical limitations, along with their partial incorporation by PCL, which facilitated access to the oil and resulted in higher releases in comparison with CAcontaining fibers (Fig. 3). 7 As expected, PCL–CLO/CA and the triaxial fibers reported the lowest antioxidant profiles confirming the protective functions of the CA shell. 22,38,40 Both the triaxial constructs and the PCL–CLO/CA coaxial fibers Fig. 3 Cumulative release kinetics of CLO-loaded wet-spun fibers. Data are reported as mean SD (n= 3). Statistical significance was determined via the Tukey test, applying multiple comparisons between the different fiber typologies (no statistical significances were found). Fig. 4 HNE inhibition profile from AAPV-loaded wet-spun fibers. Data are reported as mean SD (n= 3). Statistical significance was determined via the Tukey test, applying multiple comparisons between the different fiber typologies (no statistical significances were found). Paper Materials Advances This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online © 2025 The Author(s). Published by the Royal Society of Chemistry Mater. Adv., 2025, 6, 2029–2047 | 2037 demonstrated antioxidant abilities overtime (in average 6.89– 56.67%, 1.51–34.30% and 1.87–32.49% for PCL–CLO/CA, PCL– CLO/SA/CA and PCL–CLO/SA–AAPV/CA fibers, respectively), making them suitable for their intended application. 3.5. Antibacterial testing S. aureus,S. epidermidis,E. coli and P. aeruginosa growth inhibition was evaluated after 24 h of exposure to the fibers at 37 1C (Fig. 6). Results showed that the antibacterial activity increased after the first hour of incubation, a clear effect of the CLO initial burst release (Fig. 2). CLO-loaded fibers achieved higher antibacterial efficacy in comparison with unloaded fibers. Such an occurrence was predicted since many reports have addressed that CLO’s strong antibacterial properties as a result of one of its main elements, eugenol. 43,44 In fact, according to the literature, the CLO mechanism of action is based on its accumulation at the bacteria cell surfaces, disrupting the cytoplasmic membrane function and integrity, consequently leading to the leakage of cell content and cell death. 9,11 PCL–CLO monolayered fibers reported superior antibacterial activities due to the larger availability and exposure of CLO to the media as was also evidenced in the release kinetics and DPPH reduction profiles (Fig. 2 and 4). PCL–CLO/SA and PCL– CLO/SA–AAPV coaxial fibers promoted one of the largest inhibitions of bacteria activity, once more due to the feeble structural integrity of the SA layer, in which polymer fragments could have been released and partially dissolved in the media. This way, the fibers’ outer layer no longer restricted the access to the EO loaded into the fibers, explaining their antibacterial activity. 45 In contrast, the antibacterial effectiveness of PCL–CLO/CA Fig. 5 DPPH reduction induced by CLO-loaded wet-spun fibers after (a) 1, (b) 2, (c) 4, (d) 6 and (e) 24 h of incubation. Data are reported as mean SD (n= 3). Statistical significance was determined via the Tukey test, applying multiple comparisons between the different fiber typologies (1 h: po0.030 significance between PCL and PCL–CLO/SA–AAPV/CA; 2 h: no statistical significances detected; 4 h: po0.030 significance between PCL–CLO/SA, PCL–CLO/SA–AAPV and PCL/SA/CA, PCL–CLO/SA/CA, PCL/SA–AAPV/CA, PCL–CLO/SA–AAPV/CA; 6 h: po0.030 significance between PCL–CLO and PCL–CLO/SA–AAPV, PCL–CLO/CA and PCL–CLO/SA/CA; 24 h: po0.002 significance between PCL–CLO, PCL–CLO/SA, PCL–CLO/SA–AAPV and PCL/SA/CA, PCL–CLO/SA/CA, PCL/SA–AAPV/CA, PCL–CLO/SA–AAPV/CA). Materials Advances Paper This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online 2044 | Mater. Adv., 2025, 6, 2029–2047 © 2025 The Author(s). Published by the Royal Society of Chemistry 9 J. C. Antunes, T. D. Tavares, M. A. Teixeira, M. O. Teixeira, N. C. Homem, M. T. P. Amorim and H. P. 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