In Vitro Evaluation of Bioactive PCL/Alginate Hierarchical Fibers with Controlled Liposomal Silymarin Release for Enhanced Tissue Engineering: Breaking Barriers in MSC Transplantation

In Vitro Evaluation of Bioactive PCL/Alginate Hierarchical Fibers with Controlled Liposomal Silymarin Release for Enhanced Tissue Engineering: Breaking Barriers in MSC Transplantation | 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 Article In Vitro Evaluation of Bioactive PCL/Alginate Hierarchical Fibers with Controlled Liposomal Silymarin Release for Enhanced Tissue Engineering: Breaking Barriers in MSC Transplantation Payam Moharreri, Amir Mahdi Molavi, Arman Abroumand Gholami, Tahere Mokhtari, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6279660/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Purpose : The clinical application of mesenchymal stem cells (MSCs) in tissue engineering is hindered by critical challenges, including low cell survival rates, poor retention at injury sites, and the lack of bioactive scaffolds that mimic the native tissue microenvironment. To address these limitations, this study developed a multifunctional platform using liposomal silymarin (Lip-Sil)-enriched polycaprolactone/alginate (PCL/Alg) hierarchical fibers to enhance the delivery, adhesion, and functionality of adipose-derived MSCs (AMSCs) for tissue regeneration. Methods : Lip-Sil was synthesized using the remote loading method and characterized for particle size, zeta potential, encapsulation efficiency, and dissolution behavior. PCL/Alg hierarchical fibers were fabricated via electrospinning and evaluated for mechanical properties, morphology, hydrophilicity, degradation rate, , and surface chemistry using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The biological performance of the scaffolds was assessed through in vitro studies, including cell viability, adhesion, and proliferation of AMSCs using MTT assay, DAPI staining, and FE-SEM imaging. Results : The Lip-Sil formulation exhibited a particle size of 94.7 nm, a zeta potential of -29 mV, and an encapsulation efficiency of 73%. The cumulative dissolution profile showed a sustained release, reaching 65% after 2 weeks. The PCL/Alg fibers demonstrated a significant reduction in diameter (157.7 ± 42.8 nm) compared to pure PCL fibers (323.3 ± 122.8 nm). Mechanical testing revealed that the PCL and PCL/Alg scaffolds had a tensile strength of 10 ± 1.3 and 2.7 ± 0.17 MPa and a strain at break of 67.4 ± 2.41% and 55.1 ± 2.9%, respectively. The addition of alginate improved hydrophilicity (water contact angle: 31.8 ± 4.1° vs. 126.9 ± 9.6° for PCL) and degradation rate. The water uptake rate of PCL/Alg scaffolds reached 80.7 ± 5.3% within 18 hours, significantly higher than that of PCL scaffolds (18.6 ± 0.88%) and these ratios for both samples remained constant until 28 hours. AMSCs cultured on PCL/Alg/Lip-Sil scaffolds showed an excellent increase in cell proliferation compared to control groups (p<0.01) after 7 days of incubation. DAPI staining revealed a mean cell adhesion index of 1.6 ± 0.1 for the composite scaffold. FE-SEM imaging confirmed enhanced cell spreading and expansion on the composite scaffolds. Conclusion : The developed PCL/Alg/Lip-Sil scaffold represents a promising platform for tissue engineering, offering controlled drug release, improved cell adhesion, and enhanced AMSC proliferation. This multifunctional system addresses key challenges in stem cell delivery and tissue regeneration, providing a robust foundation for future clinical applications. Biological sciences/Biotechnology Biological sciences/Cell biology Biological sciences/Molecular biology Adipose Tissue Derived Mesenchymal Stem Cells Biomedical applications Drug Carrier Electrospinning Guided Tissue Regeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Tissue regeneration remains a significant challenge in modern medicine, particularly in cases of extensive tissue damage where the body's natural repair mechanisms are insufficient. When tissue injury exceeds a critical threshold, the disruption of cellular signaling pathways and the loss of structural integrity impede the regeneration process, often leading to incomplete or dysfunctional tissue repair (1,2). Traditional approaches, such as autografts and allografts, are limited by donor site morbidity, immune rejection, and the risk of disease transmission. As a result, there is a growing need for innovative strategies to promote effective tissue regeneration (3). Cell therapy, particularly the use of mesenchymal stem cells (MSCs), has emerged as a promising solution for tissue repair and regeneration (4). MSCs are multipotent stromal cells capable of differentiating into various cell types, including osteoblasts, chondrocytes, and adipocytes (5). Beyond their differentiation potential, MSCs exert paracrine effects by secreting growth factors, cytokines, and extracellular vesicles that modulate the local microenvironment, reduce inflammation, and promote angiogenesis (6). These properties make MSCs a powerful tool for enhancing tissue regeneration and functional recovery (7). Among the various sources of MSCs, adipose-derived MSCs (AMSCs) have gained significant attention due to their abundance, ease of isolation, and minimal ethical concerns (8). Adipose tissue represents a readily accessible and rich source of MSCs, with a higher yield compared to other sources such as bone marrow (9). AMSCs also exhibit robust proliferative capacity, multilineage differentiation potential, and immunomodulatory properties, making them an attractive candidate for cell-based therapies (6,10). Furthermore, their autologous nature eliminates the risk of immune rejection, addressing a major limitation of allogeneic cell transplantation (4,11). Despite their therapeutic potential, the clinical application of AMSCs faces several challenges. One major limitation is the low survival rate and poor retention of transplanted cells at the injury site (4). Additionally, the lack of a supportive scaffold to guide cell behavior and provide structural stability can hinder the integration of AMSCs into the host tissue. To overcome these challenges, there is a critical need for advanced biomaterial platforms that can enhance cell survival, promote adhesion, and provide a conducive microenvironment for tissue regeneration (5,9,10). In biomedical engineering, various methods are employed to fabricate scaffolds, each offering distinct advantages and limitations. Among these, electrospinning stands out as a highly effective and versatile technique, primarily due to its ability to closely mimic the natural extracellular matrix (ECM) (9). Electrospun fibers provide a high surface area-to-volume ratio, interconnected porous structures, and tunable mechanical properties, making them ideal for supporting cell adhesion and proliferation (5). The topography of polymer fibers, especially random fibers, promotes cell adhesion, proliferation, and exhibit paracrine functions (12). Additionally, random fibers exhibit uniform mechanical properties in all directions, which is advantageous for tissue regeneration (11). However, the success of this method heavily depends on the choice of materials used for scaffold fabrication (13). In recent times, there has been a growing interest in certain biomaterials that possess unique characteristics such as flexibility, ductility, gel formation ability, and promotion of cellular behavior (7). Gel frameworks, resembling the ECM, have gained attention due to their 3D structure creation, high water absorption capacity, and flexibility (10). Natural biopolymers, such as chitosan, alginate (Alg), and collagen, are widely used in tissue engineering due to their biocompatibility, biodegradability, and ability to mimic the ECM (11). In particular, Alg is known for its hydrophilic nature, gel-forming ability, and low immunogenicity, making it suitable for creating a cell-friendly microenvironment (5). However, its application in long-term implantable devices is often limited by issues such as uncontrolled enzymatic degradation, insufficient mechanical strength, and rapid dissolution in physiological environments (2). On the other hand, synthetic polymers like polycaprolactone (PCL) have gained widespread use in tissue engineering due to their design flexibility, ease of fabrication, and excellent mechanical properties (9). PCL is a biodegradable polyester with a slow degradation rate, making it suitable for applications requiring long-term structural support (7). However, PCL is not without drawbacks. Its hydrophobic nature, lack of cell recognition sites, and suboptimal bioactivity often result in poor cell adhesion and limited integration with host tissues (10,14). Composite scaffolds combining PCL and Alg have been widely explored in tissue engineering due to their synergistic properties (7,14). For instance, Habibizadeh et al. (2021) demonstrated the potential of PCL/Alg scaffolds in skin regeneration, while Shirehjini et al. (2022) utilized these scaffolds for cartilage repair (2,6). However, these studies often lack bioactive signals to enhance cell survival and functionality, which limits their clinical applicability. Despite these advancements, a critical challenge remains: the lack of bioactive signals within the scaffold to further enhance cell survival, proliferation, and differentiation (1). While composite scaffolds like PCL/Alg improve mechanical and biological properties, they often fail to provide the necessary biochemical cues to fully mimic the native tissue microenvironment. This limitation can be addressed by incorporating small molecules or bioactive agents into the scaffold to modulate cellular behavior and promote tissue regeneration (15). Polyphenols, a class of naturally occurring compounds with diverse biological activities, have garnered significant attention due to their antioxidant, anti-inflammatory, and pro-regenerative properties (16,17). Silymarin (Sil), a well-studied polyphenolic flavonoid derived from milk thistle, is particularly promising for tissue engineering applications (15). It has been shown to enhance cell viability, reduce oxidative stress, and promote tissue repair through its ability to upregulate endogenous antioxidant enzymes and improve mitochondrial function (18–21). However, the therapeutic potential of Sil is often limited by its poor bioavailability, low water solubility, and rapid metabolism (logP value of 1.4), which hinder its effective delivery to target tissues (17). Furthermore, loading directly onto fibrous scaffolds without encapsulation often results in rapid release, uneven distribution, and reduced therapeutic efficacy (1,19). To overcome these challenges, advanced drug delivery systems, such as liposomes, have been explored as carriers for polyphenols like Sil (17). Liposomes are spherical nanoparticles composed of lipid bilayers that can encapsulate both hydrophilic and hydrophobic compounds, providing controlled release, improved stability, and enhanced cellular uptake (18,22). This is because lipid molecules are regarded as safe, biocompatible, and capable of breaking down naturally within the body (16,23). They can enhance the transport of substances through cells by temporarily disrupting the cell's lipid-based membranes and improve drug permeability by modulating tight junctions between cells (24). Liposomal encapsulation not only enhances the solubility and stability of polyphenols but also enables controlled and sustained release, ensuring prolonged exposure of cells to their therapeutic effects (25,26). Moreover, liposomes play a critical role in improving the interaction between cells and the scaffold. By coating the scaffold with liposomes, the surface properties of the scaffold are modified, leading to enhanced hydrophilicity and improved cell adhesion (1,24). This is particularly important for tissue engineering, where strong cell-scaffold interactions are essential for promoting cell survival, proliferation, and differentiation. We hypothesize that the integration of liposomal Sil (Lip-Sil) into a PCL/Alg scaffold will create a multifunctional platform capable of enhancing AMCS adhesion, proliferation, and metabolic activity while providing a controlled release of therapeutic agents to promote tissue regeneration. By combining the mechanical strength of PCL, the bioactivity of alginate, and the therapeutic potential of silymarin, this platform not only delivers AMSCs to the target site but also enhances their survival, functionality, and regenerative potential, thereby addressing critical challenges in tissue engineering. This study aims to develop and evaluate a hierarchical PCL/Alg scaffold enriched with lip-Sil as a multifunctional platform for the delivery and support of AMSCs in tissue engineering applications. 2. Material and methods 2.1. Synthesis of Lip-Sil Liposomes were prepared by the remote loading method, which had been previously described with some modifications (1). A stock solution of lipids containing HSPC-DPPC:mPEG2000-DSPE (Avanti Polar Lipids, USA), and cholesterol (Sigma-Aldrich, USA) in molar ratios of 55:5:40 was prepared using chloroform (Merck, Germany). The stock containers were then placed under argon gas to remove oxygen. The lipid film was sterilized by washing it multiple times with methanol and chloroform, and then heated in the oven at 200°C for 90 min. To remove the chloroform, the test tubes were placed in a rotary device (Heidolph, Germany) at 55°C and 15 rpm for 2 hours and then subjected to freeze-drying (Christ Alpha 1-4 LDplus, Germany) for 90 min. The lipid film was hydrated by adding the required amount of mannitol acetic acid buffer (pH=7.5). Then the solution was vortexed for 10 min to dissolve the lipid film and refrigerated at 5°C for 24 hours. Next, the liposomes underwent sonication (Bandelin Sonopuls, Germany) for 15 min at 55°C and were then passed through 100 and 200 µm filters (Whatman, UK) 11 times, as well as 15 times through a 50 µm filter using an extruder at 55°C. Sil extract with a concentration of 10 mg/ml was added to 1 ml of the lipid formulation. The resulting liposomes were gently shaken at 60°C for 150 min. To remove any free Sil molecules and the external buffer of the liposomes, a dialysis buffer (10% sucrose and 10 mM phosphate, pH=7.4) was utilized. Finally, the liposomes were sterilized using a 0.2 µm syringe filter (Millipore, USA). 2.3. Fabrication of scaffolds To create a 20% (w/v) solution, PCL granules (Sigma-Aldrich, USA) were dissolved in a mixture of tetrahydrofuran (Merck, Germany) and dimethylformamide (Merck, Germany) with constant stirring at room temperature (in a ratio of 1:1). A homogeneous solution of polyvinyl alcohol (PVA) at 8% concentration was prepared by dissolving proper amount of PVA distilled water at 90 °C. Also, homogeneous solution of Alg (Sigma-Aldrich, USA) with 5% (w/v) was prepared by dissolving in deionized water and stirred overnight at room temperature. The PVA:Alg (1:2) blend solution was created by combining the two solutions in appropriate volumes and briefly stirring them at room temperature for 12 hours. Before electrospinning (Fanavaran Nano-Meghyas Co, Iran), the freshly prepared solutions were sonicated. The polymer solution was loaded into a syringe and pumped at a flow rate of 1 ml/h. The distance between the needle tip and the collector was maintained at 15 cm. The voltage was gradually increased until a Taylor cone was formed and then fixed at 25 kV. The nanofibers were produced at room temperature (25-28 °C) and a relative humidity of 45-50%, without any beads. Finally, the developed scaffolds were dried in a vacuum oven for 24 hours and stored in desiccators for future use. 2.4. Characterization of liposomes 2.4.1. Dynamic Light Scattering Dynamic Light Scattering (DLS) instrument in a Zetasizer (Nano ZS90, Malvern Instruments, Worcester, UK) was used to measure the particle size, polydispersity index (PDI), and zeta potential. 2.4.2. Encapsulation efficiency The quantity of silymarin contained in the prepared liposome was determined using a UV/vis spectrophotometer (UV-2600, Shimadzu, Japan). The standard silymarin graph was generated at a wavelength of 288 nm using methanol as the solvent. To assess the encapsulation efficiency (EE) percentage, the liposome formulation was mixed with methanol at a v/v ratio of 1:20, causing the liposomes to break down. The mixture was incubated at 37 °C for 15 minutes and then measured at a wavelength of 288 nm. The EE percentage of silymarin in the formulation was then calculated using the following formula: 2.4.3 Dissolution study The method of dialysis was utilized to determine the release of the drug. The fabricated samples were subjected to dissolution study in a shaking incubator (120 rpm; IKA, Germany) using a PBS solution at a pH of 7.4 and a temperature of 37 °C to evaluate the release rate of Sil from the formulation. The test involved placing 10 ml of Lip-Sil into a dialysis bag with a molecular weight cutoff of 12-14 kDa, which was then immersed in 75 mL of buffer. The release of Sil was monitored over a period of 2 weeks. At specific time intervals (0, 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72, 96, 120, 144, 168, 240, and 336 h), 1 mL of the solution was withdrawn and replaced with an equal amount of fresh medium. The release of Sil from the liposomes in the dialysis bag served as a control group. The quantity of Sil in the withdrawn samples was determined by comparing the experimental results to a calibration curve using a UV/Vis spectrophotometer (UV-2600, Shimadzu, Japan) at a 288 nm wavelength. 2.5. Characterization of scaffolds 2.5.1. Morphology of scaffolds To analyze the microstructure of the scaffolds, a field emission scanning electron microscope (FE-SEM) with an accelerating voltage of 10 kV was utilized (Tescan Mira3 LMU, Czech Republic). Prior to observation, all samples were placed on metal stubs using conductive double-sided tape, and a thin layer of gold was sputter deposited on each one. The ImageJ software was used to determine the average diameter of the fibers in the FE-SEM images, with at least 30 fibers being measured. 2.5.2. Chemical surface composition analysis To distinguish the unique vibrational frequencies of scaffolds, attenuated total reflection fourier transform infrared (ATR-FTIR) spectroscopy was utilized with a Thermo Nicolet Avatar 370 instrument (USA). The samples were scanned with a resolution of 4 cm-1 in the range of 400-4000 cm -1 . 2.5.3. Mechanical properties Rectangular samples, measuring 5 cm × 1 cm were taken from the PCL and PCL/Alg scaffolds (n = 5). One centimeter from both ends of the samples was secured within the holders, and the initial distance between the gauges was established as 3 cm. The micro-tensile testing was carried out with the help of equipment that employed a load cell of 100 N and had a crosshead speed of 10 mm/min (TA Plus model, USA). The test was terminated once the samples had fractured. 2.5.4. Wettability To evaluate the hydrophilicity of the scaffolds, the water contact angle (WCA) was measured. This involved placing 1 ml of deionized water onto the PCL, PCL/Alg, and PCL/Alg/Lip-Sil scaffolds and capturing images using an optical contact angle apparatus (JC2000A, Powereach Co. Ltd., China). The angle between the liquid-scaffold interfaces was then determined using ImageJ software. The average of five measurements taken at different positions on the scaffold surface was considered the final contact angle value. 2.5.5. Water uptake measurement To measure the amount of water uptake, scaffolds were immersed in PBS solution. After six time points (1, 3, 6, 18, 24, and 28h) of immersion in PBS solution, the dry weight and the wet weight of the scaffolds were assessed to evaluate the water uptake of the scaffolds. After each time point, the samples were taken out and their weight was measured following a careful removal of surface water using a sampler. The following equation was used to get the percentage water uptake: S 0 is the weight of dried samples before soaking and S 2 is the weight of soaked samples. 2. 5.6. Degradation rate In order to determine the rate of degradation of the PCL and PCL/Alg scaffolds, an in vitro biodegradation study was conducted using a PBS solution. The scaffolds were incubated in a shaking chamber at a temperature of 37 °C. This procedure continued until the scaffold containing Alg was completely destroyed. To account for changes in concentration during the degradation process, the solution was replaced every three days. The weight of the samples was measured weekly to measure biodegradation. The results were reported in the form of the percentage of the remaining scaffolds of the initial weight of the scaffolds. 2.6. Biological assessments 2.6.1. Cell viability The viability and growth of human AMSCs (Sinacell Knowledge-Based Production and Research Company, Iran) were assessed using the MTT assay. Cells were seeded on various scaffolds (PCL, PCL/Alg, and PCL/Alg/Lip-Sil) as well as on tissue culture grade polystyrene (Control; Corning, USA). The cells were seeded at a density of 1 × 10 4 cells per scaffold and incubated in a humidified environment at 37 °C with 5% CO 2 . After 2, 4, and 6 days of cell culture, 20 µL of MTT reagent (Sigma-Aldrich, USA) was added to each well containing the scaffolds, and the plates were incubated at 37 °C for 4 h. Subsequently, DMSO (Sigma-Aldrich, USA) was added to each well as a solvent, and the plates were placed on a shaker for 30 min to enhance dissolution. The absorbance was then measured at a wavelength of 570 nm using a microplate reader spectrophotometer (SPECTROstar Nano, BMG LABTECH, Germany). Details of the pilot study, including the methodology for optimal dose selection of Lip-Sil, dose-response curves, and cytotoxicity analysis, are provided in the Supplementary Materials. The study protocol was approved by the Ethics Committee of Mashhad University of Medical Sciences (MUMS) under the ethical approval code: IR.MUMS.MEDICAL.REC.1399.546. All procedures involving AMSCs were conducted in accordance with the ethical guidelines and regulations. 2.6.2. Cell adhesion To evaluate the visualization of cells attached to the scaffolds in comparison to the control sample (tissue culture plate), DAPI staining (Sigma-Aldrich, USA) was conducted on the 6th day of cell culture. The cells attached to the scaffolds were fixed by removing the culture medium, washing with PBS, and fixing with 4% PFA at 4°C for 30 min. Subsequently, the fixed cells were permeabilized using a buffer solution (0.2% Triton X-100 in PBS) for 5 min, and the cell nuclei were stained with DAPI for 10 min. The cells were then observed under a fluorescence microscope (Olympus, Japan). Quantification of cell adhesion was performed using ImageJ software (NIH, USA). Fluorescence images were analyzed to count the number of DAPI-stained nuclei, and the results were expressed as fold change relative to the control group. 2.6.3. Cell morphology The morphology of AMSCs cultured on the samples was examined using FE-SEM images taken 6 days after culture. The samples were fixed with 2.5% glutaraldehyde at 4°C for three hours. After fixation, the excess glutaraldehyde (Sigma-Aldrich, USA) was removed by washing the samples with PBS. The samples were then dehydrated using ethanol at gradually increasing concentrations (30-100%) for 10 min each, which removes water from the samples and prepares them for imaging. Following dehydration, the samples were placed in a desiccator, coated with gold, and analyzed using FE-SEM imaging. 2.7. Statistical The data were presented as mean ± SD, and statistical significance was considered at p<0.05. Data analysis was conducted using GraphPad Prism 9.0.0 software. Normality of the data was confirmed using the Shapiro-Wilk test, and parametric tests (e.g., t-test and ANOVA) were applied accordingly. Statistical tests, including Unpaired t-test with Welch's correction for fiber diameter test, two-way ANOVA for MTT assay, and one-way ANOVA for contact angle and optimal dose tests with the Tukey post hoc test, were performed to determine differences between parameters. Pearson’s correlation coefficient (r) was used to evaluate the relationship between drug concentration and cell viability. Potential outliers were identified using the ROUT method (Q = 1%) in GraphPad Prism. All experiments were performed with at least five independent replicates (n = 5) unless otherwise stated. 3. Results and discussion 3.1. Physicochemical characteristics of Lip-Sil Liposomal formulations are regarded as promising drug delivery systems for a variety of substances, including small hydrophilic and lipophilic drugs. In this study, we prepared Lip-Sil using a lipid composition comprising HSPC, cholesterol, and mPEG2000-DSPE, which is similar to FDA-approved liposomal formulations such as Doxil® and AmbiSome® (26). The particle size of Lip-Sil was determined to be 94.7 nm, with a size distribution ranging from 70 nm to 170 nm (Fig. 1a). The PDI of the liposomes was found to be between 0.15 and 0.3, indicating a highly uniform population of vesicles. A PDI value below 0.3 is generally considered acceptable for liposomal formulations, as it reflects a narrow size distribution and homogeneity (22). The zeta potential of Lip-Sil was measured to be -29 mV (Fig. 1b), which is close to the threshold value of -30 mV required for colloidal stability (27). This suggests that the Lip-Sil formulation possesses good physical stability, minimizing the risk of aggregation during storage and application. The negative zeta potential can be attributed to the presence of anionic lipids in the formulation, which also contributes to the electrostatic repulsion between liposomes, further enhancing their stability. This feature may also facilitate interactions with positively charged cell membranes, potentially enhancing cellular uptake. The Lip-Sil achieved approximately 70% encapsulation efficiency in this study. The achieved EE of 73% is considered acceptable for Lip-Sil, especially when compared to other studies where larger liposomes had EE in the range of 65-70% for Sil (25). It has been suggested that if the cholesterol-to-lipid ratio exceeds a certain threshold, it can disrupt the normal structure of the liposomal membrane, leading to a decrease in drug EE. Therefore, the increase in Sil encapsulation efficiency observed in this study is likely due to the suitable cholesterol ratio. The higher molar ratio of cholesterol in the liposomes affects the arrangement and interactions of liposomal membrane components with Sil, resulting in a decrease in EE (23). Figure 1c illustrates an initial burst release of Sil within the first few hours. The release profile of Lip-Sil exhibited biphasic behavior, with a faster release rate observed during the initial phase compared to the steady release phase. Within the first 24 hours, approximately 37% of Sil was released from the liposomes, while after 2 weeks, only 65% was released. Evidence shows that pegylated liposomes cause slow release of Sil due to the fast-moving hydrophilic chains of PEG. These findings imply that the drug would remain stable in the bloodstream and be released specifically at the targeted site, indicating that this liposome formulation meets the criteria for effective drug delivery systems (16). Additionally, the inclusion of cholesterol in a high molar ratio decreases the formation of transient hydrophilic holes that facilitate Sil release through liposomal layers. This reduction in membrane fluidity contributes to the stability of the liposomes and slows down the release of the Sil (18). Morphology of scaffolds Electrospun fibers made from naturally derived polymers and incorporating drug or bioactive molecule release capabilities are appealing for biomedical uses because of their advantageous characteristics such as a high surface area to volume ratio, non-toxicity, and biocompatibility (28). In this study, electrospun PCL/Alg fibers containing liposomal silymarin were fabricated. The scaffolds were successfully electrospun, and their morphologies were analyzed using FE-SEM (Fig 2a-d). The images depict smooth nanofibers that are randomly oriented and free of beads. Fibers containing Alginate exhibited a narrow distribution with an average diameter of 157.7 ± 42.8 nm (Fig 2e). On the other hand, PCL fibers showed a broader range with an average diameter of 323.3 ± 122.8 nm. The reduction in fiber diameter upon the addition of alginate can be attributed to the increased charge density and repulsive forces within the electrospinning solution, which promotes the formation of thinner fibers (29,30). In elucidating the reduction in the base diameter of the composites, Bok Kim and Hyung Kim proposed that the incorporation of dispersed alginate within the composite facilitates the dissipation of stored elastic deformation energy and impedes the elastic recovery of the deformed PCL/alginate mixture within the nozzle (31). Furthermore, to preserve the structure of the alginate nanofibers, a crosslinking process is required. However, when PCL/Alg nanofibers were immersed in PBS, they maintained their fibrous structure without the need for crosslinking (Fig 6). The diameter of the fibers, which is directly influenced by electrospinning parameters, plays a critical role in determining cellular behavior. In this study, the electrospinning parameters were carefully selected based on previous research to produce uniform, bead-free fibers with optimal morphological and mechanical properties (2,6,14). It has been widely documented that these parameters not only affect fiber morphology but also significantly influence cell-scaffold interactions. For instance, studies have shown that a voltage range of 20–25 kV and a flow rate of 1–2 mL/h yield PCL composite fibers with diameters ranging from 100 to 500 nm, which are ideal for promoting cell adhesion and proliferation (2,14). Similarly, Shirehjini et al. demonstrated that a needle-to-collector distance of 15 cm produces fibers with a high surface-to-volume ratio, enhancing cell-scaffold interactions by more effectively mimicking the ECM (6). This can be attributed to the presence of PCL, which acted as a supportive backbone for the nanofiber structure (29). Figure 2f illustrates the application of liposomes on the scaffold surface, demonstrating a consistent and thin layer of the liposome solution on the fibers, free from any aggregation. Surface chemical of scaffolds Common bond absorptions observed in PCL are the asymmetric vibration of -CH2 at 2940 cm -1 , the symmetric vibration of -CH2 at 2864 cm -1 , a strong peak at 1723 cm −1 attributing to C=O stretching, the vibration of C-O at 1293 cm -1 , the C-C vibration at 1240 cm -1 , and the symmetric vibration of C-O-C at 1159 cm -1 (14). In the PCL/Alg spectra, the carboxyl peak near 1597 cm −1 characterizes symmetric COO − stretching vibrations, asymmetric COO − stretching vibrations were presented at 1529 cm −1 , and -OH peak at 3300 cm − 1 (32). It was observed that the intensity of the C-H bands became more robust as they moved from PCL to PCL/Alg scaffold. This indicates that the CH peak from the alkyl group of PVA was positioned on the CH peaks from PCL (33). In order to enhance the bioactive characteristics of synthetic polymers, it is typically desirable to incorporate dispersed bioactive materials to the greatest extent possible. The detection of a broad -OH peak within the range of 3050-3700 cm −1 provides evidence for the existence of liposomal silymarin on the surface of the PCL/Alg/Lip-Sil scaffold (Figure 3). Mechanical properties The physical characteristics of scaffolds are crucial for maintaining their shape while new tissues regenerate. The mechanical properties of engineered materials should closely resemble those of the human tissues they are intended to replace. These characteristics also impact different cellular processes, including cell growth and the skeletal structure of cells (34). Figure 4 displays stress-strain curves, indicating that the PCL scaffold had a maximum tensile strength of 10 ± 1.3 MPa, while the PCL/Alg scaffold had a maximum tensile strength of 2.7 ± 0.17 MPa. Additionally, the percentage strain at break for the PCL and PCL/Alg scaffolds were 67.4 ± 2.41% and 55.1 ± 2.9%, respectively. The disparity in maximum strength and strain at break between PCL and PCL/Alg nanofibers was found to be statistically significant (p<0.001 and p<0.0001, respectively). The inclusion of the alginate component led to a notable decrease in both the maximum strength and strain of the scaffold compared to PCL. This can be attributed to the fact that when external stress was applied to the entire PCL/Alg scaffold, it was primarily concentrated in the alginate content (32). From the findings, it can be inferred that the mechanical characteristics of the PCL scaffolds can be readily adjusted by incorporating Alg into them. Several studies have utilized PCL as a reinforcing agent in Alg hydrogels to enhance the mechanical properties of the resulting fibers (35–37). While our findings align with these studies, our primary objective was distinct: we aimed to improve the properties of PCL fibers themselves, rather than focusing on enhancing Alg. The ability to modulate PCL flexibility by incorporating alginate represents a significant finding. Alginate may alter the viscoelastic behavior of the fibers, increasing energy absorption and reducing overall stress during strain application (38,39). However, this also introduces a limitation, as these modifications confine its applicability mainly to soft tissue engineering applications, such as native human skin and cartilage, which typically exhibit tensile strengths in the range of 2–3 MPa (40). Wettability The wettability of electrospun fibers was assessed, as it is a crucial factor affecting the potential cellular response to the surface. Based on the findings in Fig 5, the addition of Alg showed a significant decrease in the average WCA (p<0.0001), which can be attributed to Alg's chemical structure containing carboxylate and hydroxyl groups that can interact with water molecules. In contrast to the hydrophobic nature of PCL (126.9 ± 9.6°), the composite Alg fibers exhibited high hydrophilicity (31.8 ± 4.1°). Moreover, the incorporation of Lip-Sil onto the scaffold further reduced the WCA compared to the PCL and alginate composite fibers (p<0.0001 and p<0.01, respectively). In a similar research, Mohammadi et al demonstrated that the addition of HSPC/cholesterol/m-PEG2000-DSPE liposome to the poly-L-lactic acid scaffold resulted in decreased WCA measurements from a hydrophobic state to a fully hydrophilic state (24). The favorable wettability observed in fibers is believed to promote cell adhesion (41). Consequently, the cellular behavior advantage of these fibers is expected to surpass that of other types of fibers. Water uptake The capacity of scaffolds to absorb water is an important characteristic in tissue engineering applications as it indicates their ability to swell. However, excessive water uptake can result in structural damage and reduced mechanical strength (42). Conversely, low water uptake hinders cell attachment and penetration into the scaffold. Water uptake experiment was conducted to examine how Alg affects the swelling characteristics of the PCL scaffold. According to Figure 6, the PCL and PCL containing Alg both reached their maximum swelling levels at around 18.6 ± 0.88% and 80.7 ± 5.3% respectively within 18 hours. The water absorption ratios for both samples remained constant as the incubation time increased. The lower swelling ratio of PCL nanofibers compared to PCL/Alg can be attributed to the hydrophobic properties of the PCL fibers. However, our findings demonstrate a moderate increase in water uptake by the composite scaffold. Once the scaffolds undergo a moderate level of swelling, they can transform into 3D structures resembling the ECM. These structures are capable of providing the necessary nutrients for cell growth and eliminating waste generated from cellular metabolism (42). Degradation behavior In this research, PCL, an FDA-approved substance, was selected as the primary scaffold due to its lack of negative effects caused by degradation, such as the release of acidic by-products. The degradation and removal of the scaffold from the implantation area are crucial when it is used as a tissue-engineered platform. Nevertheless, minimal decomposition of the PCL nanofibers in PBS solution was observed during the in vitro hydrolysis process. This can be attributed to the hydrophobic nature of the PCL nanofibers, which undergo slow hydrolysis and have low solubility in the PBS solution and biological environment (43). Figure 7 illustrates that only a 10% weight loss was observed in the PCL samples after being submerged in the PBS solution for 4 weeks. Previous studies have also demonstrated that PCL completely degrades in vivo within about 2 years (44,45). Although the use of PCL eliminates concerns regarding the removal of implants from the patient's body, as opposed to non-degradable materials used in clinical settings (46), this degradation rate can pose challenges depending on the target tissue. The choice of the implant's in-vivo degradation rate depends on the rate at which the target tissue regenerates. The faster the target tissue regenerates, the faster the in-vivo degradation rate of the implant is required (2). Our results revealed that incorporating Alg into the scaffold can regulate the hydrolysis of PCL and cause the complete destruction of the scaffold. PCL scaffold containing Alg degradation occurs faster than PCL scaffolds because of its hydrophilicity (2). Therefore, to optimize the lifespan of the implant, the PCL scaffolds can be customized by adjusting the percentage ratio of Alg according to the specific native target tissue. Cell adhesion and proliferation The most crucial need for a scaffolding material is its ability to support cell growth and maintain metabolic functions (47). Both natural and synthetic biomaterials have demonstrated impressive benefits as matrices for supporting cells (3). Nevertheless, when cells are seeded on engineered scaffolds, the substrate can result in the generation of oxidative stress due to the mechanical and physical pressure exerted on cells. This stress can cause alterations in the structure and function of the cells (48). In this research, a biomaterial for tissue engineering was developed by combining the natural polymer alginate with the synthetic polymer PCL and covering them with a proliferation agent with controlled and sustained release. The aim was to use the advantages of synthetic and natural polymers in one material to enhance the scaffold's properties resembling the ECM. The adhesion and viability of AMSCs on different types of scaffolds, including PCL, PCL/Alg, PCL/Alg/Lip-Sil, and a control group were compared by MTT assay and DAPI staining (Fig 8). The MTT assay showed that AMSCs had a higher rate of proliferation in the PCL/Alg and PCL/Alg/Lip-Sil scaffolds compared to the PCL and control groups (Fig 8a). This means that trapping AMSCs in the scaffolds incorporating with Alg not only did not harm the cells but also increased their rate of growth compared to the PCL and control groups. The findings can be attributed to the porous structures, extensive surface area, and three-dimensional design of the PCL/Alg scaffold. This scaffold closely resembles the natural microenvironment of the ECM and offers improved cell nutrition, viability, and proliferation (2). Additionally, there were no harmful effects observed in any of the scaffolds for active AMSCs, as none of them displayed a significant decrease in absorption compared to the control group (Figure 8a). Leena et al's research findings indicated that Sil enhances the proliferation of mesenchymal stem cells on Alg composite scaffolds. They attributed these positive outcomes to the ability of silymarin to stimulate cell growth and increase cellular metabolism (49). The way the nanofiber network in the ECM is arranged and structured influences how cells grow and communicate (50). Our study reveals that Lip-Sil significantly enhances the surface of the scaffold, helping to maintain the structure of the ECM. A previous study indicated that Sil stimulates cell proliferation in polycaprolactone fibers (51), providing support for our findings. Lavi Arab et al. demonstrated that Sil nanocarriers stimulate the proliferation of AMSCs at low concentrations (52). This effect is mediated by the upregulation of endogenous mitochondrial enzyme levels and activities induced by silymarin. Additionally, evidence suggests that while high concentrations of Sil can suppress lymphocyte proliferation and induce cell death in cancerous and toxic tissues, lower doses exhibit cytoprotective properties. At these lower concentrations, Sil promotes MSCs proliferation and inhibits apoptosis, highlighting its dose-dependent dual role in cellular behavior (53,54), as noted in our supplementary evidence. The results of the quantitative analysis of DAPI images revealed that the composite scaffolds significantly enhanced cell adhesion compared to the control group (p < 0.05). Furthermore, the incorporation of Lip-Sil into the composite scaffold resulted in a remarkable increase in cell attachment, demonstrating superior performance compared to all other groups (p < 0.01). These findings align with the MTT assay results, confirming the favorable impact of the composite scaffold and Lip-Sil on cell adhesion and viability. The scaffold that holds liposomes loaded with polyphenol compounds has the potential to be a highly effective and innovative method for delivering drugs. It can be used to transport drugs accurately and continuously to specific areas, reducing burst releases, and also serve as a scaffold for tissue engineering, promoting tissue regeneration (55,56). The PCL/Alg scaffold exhibited lower cell adhesion compared to the PCL/Alg/Lip-Sil scaffold, resulting in a lower number of viable cells. The limited cell adhesion can be attributed to the inherent hydrophobic nature of the polymers, which hampers the interaction between the cells and the scaffold (57). This could be attributed to the restricted space available for cell growth and a weaker interaction between the cells and the scaffold (58). The optimal mechanism for enhancing cell growth and adhesion in this platform can be attributed to several factors. Firstly, the functional groups in alginate facilitate the adsorption of ECM proteins such as fibronectin and collagen (59), which subsequently enhance cell adhesion through cellular signaling pathways. Additionally, MSCs exhibit a strong tendency for high adhesion in the stiff PCL environment due to durotaxis (60). Furthermore, as demonstrated, the scaffold-liposome interaction enhances the hydrophilicity of the scaffold surface (1,24), making it more interactive and conducive to cell attachment. Silymarin released from liposomes improves cellular metabolism by upregulating endogenous enzymes (e.g., superoxide dismutase and catalase) and enhancing mitochondrial function (52), thereby promoting cell survival and proliferation. These distinct biochemical interactions collectively create a favorable microenvironment that supports AMSC adhesion, proliferation, and metabolic activity. Consistent with our findings, Dadashpour et al. developed a platform where silybin-loaded nanoparticles, an active compound of Sil, were incorporated into hybrid fibers made of natural and synthetic polymers (19). Their results demonstrated sustained adhesion and proliferation of AMSC on this scaffold, supporting the potential of such systems for long-term cell culture applications. Morphology of cells FE-SEM images were captured to examine the morphology of cells and assess how well AMSCs adhered to the PCL/Alg nanofiber (Figure 9). The PCL scaffold had minimal cell adhesion and density. However, the nanofibers containing Alg showed good compatibility with the AMSCs. At lower magnification, it was observed that the AMSCs adhered tightly to the nanofibers, regardless of whether Lip-Sil was present. However, there were no notable differences in the cell appearances on the PCL/Alg and PCL/Alg/Lip-Sil nanofibers. This is likely due to the presence of more pores in the nanofiber than anticipated, which helps alleviate compression pressure. This is significant because it allows for easy provision of media and nutrients, which is crucial for the use of biomaterials in tissue engineering as cells need to remain alive within the implanted scaffold (61). Both scaffolds showed potential in terms of the nanofiber's topography inducing cell expansion. However, at higher magnification, it was observed that the cells exhibited more expansion on the PCL/Alg/Lip-Sil fibers, while in the other samples, the cells were clustered together. The PCL/Alg/Lip-Sil scaffold demonstrated excellent cell expansion and high production of ECM on both the surface of the scaffold and within its pores. The composite nanofibers created a favorable environment for the growth of AMSCs. Additionally, these nanofibers not only exhibited a strong affinity for stem cells but also preserved the normal characteristics of the native tissue and promoted ECM secretion. According to Wu et al., Sil enhances cellular metabolic activity at concentrations as low as 50 μM, mediated by the improvement of ECM homeostasis through the modulation of mRNA expression and protein levels of catabolic and anabolic cytokines (20). Polyphenols not only mitigate the damage caused by oxidative stress to normal tissues but also exhibit a specific affinity for functional molecules like receptors, enzymes, transcription factors, and transduction factors (62). Consequently, polyphenols can facilitate the recovery process of damaged tissues and improve the ECM to create a favorable environment for cell growth. Through their ability to regulate the tissue microenvironment and participate in cellular events, polyphenols demonstrate growth-promoting properties (48). Conclusion We have successfully developed Lip-Sil loaded PCL/Alg nanofiber with AMSCs seeding for tissue engineering. Based on the findings from drug dissolution, efficient encapsulation, zeta potential, and DLS, the liposome formulated using the thin layer hydration method has favorable characteristics for delivering the agents to the injured tissue. These features include controlled release of silymarin, high drug loading efficiency, stability, and a small particle size of the formulation. Characterization and in vitro assessments were conducted to measure the modifications in scaffolds containing alginate in comparison to PCL fibers. The outcomes of the composite scaffold indicated an enlargement in water uptake and mass loss, as well as a reduction in fiber diameter and WCA compared to PCL fibers. The application of Lip-Sil covering on the PCL/Alg enhanced the hydrophilicity, which is justified by the presence of OH groups on the fiber surface. The implantation of AMSCs onto the scaffolds demonstrated that the PCL/Alg/Lip-Sil scaffold exhibited a high potential for cell adhesion, proliferation, and expansion, as indicated by the results of DAPI, MTT, and FE-SEM analyses. We reported a new platform for injured tissue treatment, with an enhanced cell adhesion index and improved proliferation rate of adipose mesenchymal stem cells. The requirements of tissue engineering included targeted, prolonged, and controlled release of healing agents, a suitable substrate for promoting cell growth and connection, and self-renewal cells, which can be achieved through our novel platform. This study has several limitations that should be acknowledged. First, the in vitro nature of our experiments may not fully replicate the complex biological environment of in vivo systems. Second, while we demonstrated the scaffold's ability to enhance cell viability and adhesion, we did not investigate its effects on inflammation or immune response, which are critical for tissue regeneration. Future studies should address these limitations by incorporating in vivo models, cytokine profiling, and long-term biocompatibility assessments to further validate the scaffold's potential for clinical applications. In summary, this research proposes an innovative and improved platform for tissue engineering applications. The study utilized the small molecule (silymarin) as a model of healing agents in liposomal formulation for the ability of sustained drug release, AMSCs for supporting tissue regeneration and cell replacement, and PCL/Alg fibers were employed as a substrate and co-delivery system for cells and agents. Declarations Data availability The data analyzed during the current study available from the corresponding author on reasonable request. Declaration of interest The authors declare no conflicts of interest. Acknowledgments This work was supported by grant number 98126378 from the Research Council of the Faculty of Medicine, Mashhad University of Medical Sciences (MUMS, Iran) with ethic number IR.MUMS.MEDICAL.REC.1399.546. No human participants were directly involved in this study. However, we used commercially available human adipose-derived mesenchymal stem cells purchased from Sinacell Knowledge-Based Production and Research Company (Iran). These cells were obtained as an established cell line, and no additional human samples were collected or used for this research. All ethical and legal requirements for the use of human-derived biological materials were followed in accordance with the supplier's guidelines and relevant institutional regulations. References Abroumand Gholami A, Gheybi F, Molavi AM, Tahmasebi F, Papi A, Babaloo H. 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Supplementary Files supplementary.docx GraphicalAbstract.png Cite Share Download PDF Status: Published Journal Publication published 13 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 15 Apr, 2025 Reviews received at journal 15 Apr, 2025 Reviews received at journal 14 Apr, 2025 Reviewers agreed at journal 02 Apr, 2025 Reviewers agreed at journal 02 Apr, 2025 Reviewers invited by journal 02 Apr, 2025 Editor assigned by journal 01 Apr, 2025 Editor invited by journal 01 Apr, 2025 Submission checks completed at journal 01 Apr, 2025 First submitted to journal 21 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-6279660","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":443216770,"identity":"57975a04-7e1c-4e42-8340-638488830f09","order_by":0,"name":"Payam Moharreri","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Payam","middleName":"","lastName":"Moharreri","suffix":""},{"id":443216771,"identity":"7b3903f3-b7a4-40a0-8121-7a54b8e7992c","order_by":1,"name":"Amir Mahdi Molavi","email":"","orcid":"","institution":"Ferdowsi University of Mashhad, Khorasan Razavi Branch","correspondingAuthor":false,"prefix":"","firstName":"Amir","middleName":"Mahdi","lastName":"Molavi","suffix":""},{"id":443216772,"identity":"c7104090-6483-4ac0-8b1a-68ee6c87c943","order_by":2,"name":"Arman Abroumand Gholami","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Arman","middleName":"Abroumand","lastName":"Gholami","suffix":""},{"id":443216773,"identity":"d6b9e47e-5e64-4790-9704-f939cbaf9d91","order_by":3,"name":"Tahere Mokhtari","email":"","orcid":"","institution":"University of Pittsburgh","correspondingAuthor":false,"prefix":"","firstName":"Tahere","middleName":"","lastName":"Mokhtari","suffix":""},{"id":443216779,"identity":"84341a16-8cc4-4724-ae57-a3d4cd8ab9fe","order_by":4,"name":"Fatemeh Gheybi","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Fatemeh","middleName":"","lastName":"Gheybi","suffix":""},{"id":443216781,"identity":"755d850f-f8bc-4e65-8804-58c37709826c","order_by":5,"name":"Hossein Haghir","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hossein","middleName":"","lastName":"Haghir","suffix":""},{"id":443216783,"identity":"c76ced76-213b-4f25-bab6-3686d7649841","order_by":6,"name":"Reza Kazemi Oskuee","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Reza","middleName":"Kazemi","lastName":"Oskuee","suffix":""},{"id":443216784,"identity":"85959a4a-02ef-4643-aca7-c6e30bddde09","order_by":7,"name":"Hamideh Babaloo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYBACewYexgMMDEDEzGDA8AEowsZOQIthAw8DXAvjDJAWZgJaDA7AtADZzDwgipAWw/azBw58qLgjr9vOvPGxza9t8nzMDIwfPubg8QtPXsLBGWeeGW47zFZsnNt327CNmYFZcuY2fH7JMTjM23aYcdthHjPp3J7bjEAtbMy8eLQYnH8D1mIP1GL+27Lntj1hLTcgtiSCbGFm+HE7kaAWwxnvQH45nAzyi2Rvw+3kNmbGZrx+sefPPfjgQ8Vh223nD2/88OPPbdv57c0HP3zEowUVMLaByQZi1YPAH1IUj4JRMApGwUgBAJYhWnSJIxh2AAAAAElFTkSuQmCC","orcid":"","institution":"International Campus, Shahid Sadoughi University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Hamideh","middleName":"","lastName":"Babaloo","suffix":""}],"badges":[],"createdAt":"2025-03-21 18:08:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6279660/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6279660/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-19799-6","type":"published","date":"2025-10-13T15:56:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81931785,"identity":"87fa2fe7-9d90-4855-9d9c-e454d359654e","added_by":"auto","created_at":"2025-05-05 05:31:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":407090,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The percentage frequency distribution of Lip-Sil diameter. (b) The intensity and fitting curve of the zeta potential of Lip-Sil. (c) The cumulative dissolution profile of silymarin versus time.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6279660/v1/65ebf39011a583b9a2cf29ff.png"},{"id":81933447,"identity":"da4e8236-f824-402f-be07-74b190b24fb2","added_by":"auto","created_at":"2025-05-05 05:39:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":864385,"visible":true,"origin":"","legend":"\u003cp\u003e(a) FE-SEM micrographs of (a and b) PCL and (c and d) PCL/Alg. (e) Scatter plot indicate average fiber diameters of scaffolds (****p\u0026lt;0.0001). (f) Micrograph showing the Lip-Sil on the PCL/Alg scaffold. The presence of liposomes on the scaffold is indicated by black arrows.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6279660/v1/cfc89e510f5ae59e3af6e2dc.png"},{"id":81933445,"identity":"53b15f9e-bdf0-494a-bfcf-135e87f4e7a4","added_by":"auto","created_at":"2025-05-05 05:39:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":133822,"visible":true,"origin":"","legend":"\u003cp\u003eATR-FTIR spectra for PCL, PCL/Alg, and PCL/Alg/Lip-Sil scaffolds. *, #, +, $, @, \u0026amp; and % represent O-H, CH, C=O, C-O, C-C, and C-O-C, respectively.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6279660/v1/6ea77b81d23bdc5a9171a868.png"},{"id":81933446,"identity":"538d2798-1c78-43ac-87f0-c531286128b1","added_by":"auto","created_at":"2025-05-05 05:39:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":76318,"visible":true,"origin":"","legend":"\u003cp\u003eStress (σ)-strain (Ԑ) curves for PCL and PCL/Alg. Data represent mean and SD (n=5).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6279660/v1/3bdcbcdd86ea2117254430c5.png"},{"id":81931792,"identity":"2c95f916-c220-46ce-9236-7bcf2caeb1f3","added_by":"auto","created_at":"2025-05-05 05:31:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":275092,"visible":true,"origin":"","legend":"\u003cp\u003eThe wettability angle of water drops on PCL, PCL/Alg, and PCL/Alg/Lip-Sil scaffolds (n=3). Data represent mean and SD. * and + indicate significance differences versus PCL and PCL/Alg respectively (****p\u0026lt;0.0001 and ++p\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6279660/v1/cb3f47d6c74ea1dff15fe79d.png"},{"id":81931782,"identity":"3dabf4fe-78bf-4caa-8649-0bf282a6dba0","added_by":"auto","created_at":"2025-05-05 05:31:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":97239,"visible":true,"origin":"","legend":"\u003cp\u003eThe water uptake of the PCL and PCL/Alg scaffolds after 28 hours incubation in PBS (n=5).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6279660/v1/619bc8323d243b3f2b0cee6c.png"},{"id":81933448,"identity":"b3aa7399-5ff9-4d35-83e0-864d14abc874","added_by":"auto","created_at":"2025-05-05 05:39:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":82689,"visible":true,"origin":"","legend":"\u003cp\u003eThe amount of weight loss in scaffolds after being incubated for 8 weeks in PBS solution at a temperature of 37 °C, expressed as a percentage, was measured in a sample size of 5.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-6279660/v1/59e62fb45ff46c07b93f5ccc.png"},{"id":81931790,"identity":"03c7790c-9936-4473-9ca1-1bb75f64c603","added_by":"auto","created_at":"2025-05-05 05:31:15","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":204459,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Comparison of AMSCs proliferation on the Ctrl sample, PCL, PCL/Alg, and PCL/Alg/Lip-Sil scaffolds as determined by an MTT assay during 2, 4, and 6 days (n=5). (b) Fluorescence microscope images of the DAPI staining assays respectively after 6 days of AMSCs cultured on Ctrl group (c), PCL (d), PCL/Alg (e), and PCL/Alg/Lip-Sil (f) scaffolds (n=3). The scale bar represents 500 µm for images. Data represent mean and SD (*p \u0026lt; 0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, and ****p\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-6279660/v1/782b070bcb1b1117dfed163a.png"},{"id":81933449,"identity":"76dc0737-6ab6-42bc-8ccb-779a32310a8c","added_by":"auto","created_at":"2025-05-05 05:39:15","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":402071,"visible":true,"origin":"","legend":"\u003cp\u003eFE-SEM images of AMSCs cultured on (a) PCL, (b) PCL/Alg, and (c) PCL/Alg/Lip-Sil scaffolds after 6 days. The scale bar represents 200 µm for images.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-6279660/v1/8b44183c52beac1acd824817.png"},{"id":93955814,"identity":"45807942-7bbe-49ef-9a79-19cabf58bd59","added_by":"auto","created_at":"2025-10-20 16:03:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3387308,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6279660/v1/a1f60531-a6a3-4b17-82de-edcb442cb5a1.pdf"},{"id":81931788,"identity":"dbb7ffd8-8970-4acf-a4b7-96d98a59bca0","added_by":"auto","created_at":"2025-05-05 05:31:15","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":260125,"visible":true,"origin":"","legend":"","description":"","filename":"supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-6279660/v1/d7bdf3082c40fbd393eff016.docx"},{"id":81931789,"identity":"5d0e4875-2f96-4d2a-a957-3fff19e1cf99","added_by":"auto","created_at":"2025-05-05 05:31:15","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":303492,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-6279660/v1/635161de403ee4cb1033f06e.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"In Vitro Evaluation of Bioactive PCL/Alginate Hierarchical Fibers with Controlled Liposomal Silymarin Release for Enhanced Tissue Engineering: Breaking Barriers in MSC Transplantation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTissue regeneration remains a significant challenge in modern medicine, particularly in cases of extensive tissue damage where the body\u0026apos;s natural repair mechanisms are insufficient. When tissue injury exceeds a critical threshold, the disruption of cellular signaling pathways and the loss of structural integrity impede the regeneration process, often leading to incomplete or dysfunctional tissue repair (1,2). Traditional approaches, such as autografts and allografts, are limited by donor site morbidity, immune rejection, and the risk of disease transmission. As a result, there is a growing need for innovative strategies to promote effective tissue regeneration (3).\u003c/p\u003e\n\u003cp\u003eCell therapy, particularly the use of mesenchymal stem cells (MSCs), has emerged as a promising solution for tissue repair and regeneration\u0026nbsp;(4). MSCs are multipotent stromal cells capable of differentiating into various cell types, including osteoblasts, chondrocytes, and adipocytes\u0026nbsp;(5). Beyond their differentiation potential, MSCs exert paracrine effects by secreting growth factors, cytokines, and extracellular vesicles that modulate the local microenvironment, reduce inflammation, and promote angiogenesis\u0026nbsp;(6). These properties make MSCs a powerful tool for enhancing tissue regeneration and functional recovery\u0026nbsp;(7). Among the various sources of MSCs, adipose-derived MSCs (AMSCs) have gained significant attention due to their abundance, ease of isolation, and minimal ethical concerns\u0026nbsp;(8). Adipose tissue represents a readily accessible and rich source of MSCs, with a higher yield compared to other sources such as bone marrow\u0026nbsp;(9). AMSCs also exhibit robust proliferative capacity, multilineage differentiation potential, and immunomodulatory properties, making them an attractive candidate for cell-based therapies\u0026nbsp;(6,10). Furthermore, their autologous nature eliminates the risk of immune rejection, addressing a major limitation of allogeneic cell transplantation\u0026nbsp;(4,11).\u003c/p\u003e\n\u003cp\u003eDespite their therapeutic potential, the clinical application of AMSCs faces several challenges. One major limitation is the low survival rate and poor retention of transplanted cells at the injury site\u0026nbsp;(4). Additionally, the lack of a supportive scaffold to guide cell behavior and provide structural stability can hinder the integration of AMSCs into the host tissue. To overcome these challenges, there is a critical need for advanced biomaterial platforms that can enhance cell survival, promote adhesion, and provide a conducive microenvironment for tissue regeneration\u0026nbsp;(5,9,10).\u003c/p\u003e\n\u003cp\u003eIn biomedical engineering, various methods are employed to fabricate scaffolds, each offering distinct advantages and limitations. Among these, electrospinning stands out as a highly effective and versatile technique, primarily due to its ability to closely mimic the natural extracellular matrix (ECM)\u0026nbsp;(9). Electrospun fibers provide a high surface area-to-volume ratio, interconnected porous structures, and tunable mechanical properties, making them ideal for supporting cell adhesion and proliferation\u0026nbsp;(5). The topography of polymer fibers, especially random fibers, promotes cell adhesion, proliferation, and exhibit paracrine functions\u0026nbsp;(12). Additionally, random fibers exhibit uniform mechanical properties in all directions, which is advantageous for tissue regeneration\u0026nbsp;(11). However, the success of this method heavily depends on the choice of materials used for scaffold fabrication\u0026nbsp;(13).\u003c/p\u003e\n\u003cp\u003eIn recent times, there has been a growing interest in certain biomaterials that possess unique characteristics such as flexibility, ductility, gel formation ability, and promotion of cellular behavior\u0026nbsp;(7). Gel frameworks, resembling the ECM, have gained attention due to their 3D structure creation, high water absorption capacity, and flexibility\u0026nbsp;(10).\u0026nbsp;Natural biopolymers, such as chitosan, alginate (Alg), and collagen, are widely used in tissue engineering due to their biocompatibility, biodegradability, and ability to mimic the ECM\u0026nbsp;(11). In particular, Alg is known for its hydrophilic nature, gel-forming ability, and low immunogenicity, making it suitable for creating a cell-friendly microenvironment\u0026nbsp;(5). However, its application in long-term implantable devices is often limited by issues such as uncontrolled enzymatic degradation, insufficient mechanical strength, and rapid dissolution in physiological environments\u0026nbsp;(2). On the other hand, synthetic polymers like polycaprolactone (PCL) have gained widespread use in tissue engineering due to their design flexibility, ease of fabrication, and excellent mechanical properties\u0026nbsp;(9). PCL is a biodegradable polyester with a slow degradation rate, making it suitable for applications requiring long-term structural support\u0026nbsp;(7). However, PCL is not without drawbacks. Its hydrophobic nature, lack of cell recognition sites, and suboptimal bioactivity often result in poor cell adhesion and limited integration with host tissues\u0026nbsp;(10,14).\u003c/p\u003e\n\u003cp\u003eComposite scaffolds combining PCL and Alg have been widely explored in tissue engineering due to their synergistic properties\u0026nbsp;(7,14). For instance, Habibizadeh et al. (2021) demonstrated the potential of PCL/Alg scaffolds in skin regeneration, while Shirehjini et al. (2022) utilized these scaffolds for cartilage repair\u0026nbsp;(2,6). However, these studies often lack bioactive signals to enhance cell survival and functionality, which limits their clinical applicability. Despite these advancements, a critical challenge remains: the lack of bioactive signals within the scaffold to further enhance cell survival, proliferation, and differentiation\u0026nbsp;(1). While composite scaffolds like PCL/Alg improve mechanical and biological properties, they often fail to provide the necessary biochemical cues to fully mimic the native tissue microenvironment. This limitation can be addressed by incorporating small molecules or bioactive agents into the scaffold to modulate cellular behavior and promote tissue regeneration\u0026nbsp;(15).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePolyphenols, a class of naturally occurring compounds with diverse biological activities, have garnered significant attention due to their antioxidant, anti-inflammatory, and pro-regenerative properties\u0026nbsp;(16,17). Silymarin (Sil), a well-studied polyphenolic flavonoid derived from milk thistle, is particularly promising for tissue engineering applications\u0026nbsp;(15). It has been shown to enhance cell viability, reduce oxidative stress, and promote tissue repair through its ability to upregulate endogenous antioxidant enzymes and improve mitochondrial function\u0026nbsp;(18\u0026ndash;21). However, the therapeutic potential of Sil is often limited by its poor bioavailability, low water solubility, and rapid metabolism (logP value of 1.4), which hinder its effective delivery to target tissues\u0026nbsp;(17).\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eFurthermore, loading directly onto fibrous scaffolds without encapsulation often results in rapid release, uneven distribution, and reduced therapeutic efficacy\u0026nbsp;(1,19).\u003c/p\u003e\n\u003cp\u003eTo overcome these challenges, advanced drug delivery systems, such as liposomes, have been explored as carriers for polyphenols like Sil\u0026nbsp;(17). Liposomes are spherical nanoparticles composed of lipid bilayers that can encapsulate both hydrophilic and hydrophobic compounds, providing controlled release, improved stability, and enhanced cellular uptake\u0026nbsp;(18,22). This is because lipid molecules are regarded as safe, biocompatible, and capable of breaking down naturally within the body\u0026nbsp;(16,23). They can enhance the transport of substances through cells by temporarily disrupting the cell\u0026apos;s lipid-based membranes and improve drug permeability by modulating tight junctions between cells\u0026nbsp;(24). Liposomal encapsulation not only enhances the solubility and stability of polyphenols but also enables controlled and sustained release, ensuring prolonged exposure of cells to their therapeutic effects\u0026nbsp;(25,26). Moreover, liposomes play a critical role in improving the interaction between cells and the scaffold. By coating the scaffold with liposomes, the surface properties of the scaffold are modified, leading to enhanced hydrophilicity and improved cell adhesion\u0026nbsp;(1,24). This is particularly important for tissue engineering, where strong cell-scaffold interactions are essential for promoting cell survival, proliferation, and differentiation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe hypothesize that the integration of liposomal Sil (Lip-Sil) into a PCL/Alg scaffold will create a multifunctional platform capable of enhancing AMCS adhesion, proliferation, and metabolic activity while providing a controlled release of therapeutic agents to promote tissue regeneration. By combining the mechanical strength of PCL, the bioactivity of alginate, and the therapeutic potential of silymarin, this platform not only delivers AMSCs to the target site but also enhances their survival, functionality, and regenerative potential, thereby addressing critical challenges in tissue engineering. This study aims to develop and evaluate a hierarchical PCL/Alg scaffold enriched with lip-Sil as a multifunctional platform for the delivery and support of AMSCs in tissue engineering applications.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cp\u003e\u003cstrong\u003e2.1. Synthesis of Lip-Sil\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiposomes were prepared by the remote loading method, which had been previously described with some modifications (1). A stock solution of lipids containing HSPC-DPPC:mPEG2000-DSPE (Avanti Polar Lipids, USA), and cholesterol (Sigma-Aldrich, USA) in molar ratios of 55:5:40 was prepared using chloroform (Merck, Germany). The stock containers were then placed under argon gas to remove oxygen. The lipid film was sterilized by washing it multiple times with methanol and chloroform, and then heated in the oven at 200\u0026deg;C for 90 min. To remove the chloroform, the test tubes were placed in a rotary device (Heidolph, Germany) at 55\u0026deg;C and 15 rpm for 2 hours and then subjected to freeze-drying (Christ Alpha 1-4 LDplus, Germany) for 90 min. The lipid film was hydrated by adding the required amount of mannitol acetic acid buffer (pH=7.5). Then the solution was vortexed for 10 min to dissolve the lipid film and refrigerated at 5\u0026deg;C for 24 hours. Next, the liposomes underwent sonication (Bandelin Sonopuls, Germany) for 15 min at 55\u0026deg;C and were then passed through 100 and 200 \u0026micro;m filters (Whatman, UK) 11 times, as well as 15 times through a 50 \u0026micro;m filter using an extruder at 55\u0026deg;C. Sil extract with a concentration of 10 mg/ml was added to 1 ml of the lipid formulation. The resulting liposomes were gently shaken at 60\u0026deg;C for 150 min. To remove any free Sil molecules and the external buffer of the liposomes, a dialysis buffer (10% sucrose and 10 mM phosphate, pH=7.4) was utilized. Finally, the liposomes were sterilized using a 0.2 \u0026micro;m syringe filter (Millipore, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Fabrication of scaffolds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo create a 20% (w/v) solution, PCL granules (Sigma-Aldrich, USA) were dissolved in a mixture of tetrahydrofuran (Merck, Germany) and dimethylformamide (Merck, Germany) with constant stirring at room temperature (in a ratio of 1:1). A homogeneous solution of polyvinyl alcohol (PVA) at 8% concentration was prepared by dissolving proper amount of PVA distilled water at 90 \u0026deg;C. Also, homogeneous solution of Alg (Sigma-Aldrich, USA) with 5% (w/v) was prepared by dissolving in deionized water and stirred overnight at room temperature. The PVA:Alg (1:2) blend solution was created by combining the two solutions in appropriate volumes and briefly stirring them at room temperature for 12 hours. Before electrospinning (Fanavaran Nano-Meghyas Co, Iran), the freshly prepared solutions were sonicated. The polymer solution was loaded into a syringe and pumped at a flow rate of 1 ml/h. The distance between the needle tip and the collector was maintained at 15 cm. The voltage was gradually increased until a Taylor cone was formed and then fixed at 25 kV. The nanofibers were produced at room temperature (25-28 \u0026deg;C) and a relative humidity of 45-50%, without any beads. Finally, the developed scaffolds were dried in a vacuum oven for 24 hours and stored in desiccators for future use.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Characterization of liposomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.1. Dynamic Light Scattering\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDynamic Light Scattering (DLS) instrument in a\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eZetasizer (Nano ZS90, Malvern Instruments, Worcester, UK) was used to measure the particle size, polydispersity index (PDI), and zeta potential.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.2. Encapsulation efficiency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe quantity of silymarin contained in the prepared liposome was determined using a UV/vis spectrophotometer (UV-2600, Shimadzu, Japan). The standard silymarin graph was generated at a wavelength of 288 nm using methanol as the solvent. To assess the encapsulation efficiency (EE) percentage, the liposome formulation was mixed with methanol at a v/v ratio of 1:20, causing the liposomes to break down. The mixture was incubated at 37 \u0026deg;C for 15 minutes and then measured at a wavelength of 288 nm. The EE percentage of silymarin in the formulation was then calculated using the following formula:\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" height=\"63\" width=\"511\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.3 Dissolution study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe method of dialysis was utilized to determine the release of the drug. The fabricated samples were subjected to dissolution study in a shaking incubator (120 rpm; IKA, Germany) using a PBS solution at a pH of 7.4 and a temperature of 37 \u0026deg;C to evaluate the release rate of Sil from the formulation. The test involved placing 10 ml of Lip-Sil into a dialysis bag with a molecular weight cutoff of 12-14 kDa, which was then immersed in 75 mL of buffer. The release of Sil was monitored over a period of 2 weeks. At specific time intervals (0, 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72, 96, 120, 144, 168, 240, and 336 h), 1 mL of the solution was withdrawn and replaced with an equal amount of fresh medium. The release of Sil from the liposomes in the dialysis bag served as a control group. The quantity of Sil in the withdrawn samples was determined by comparing the experimental results to a calibration curve using a UV/Vis spectrophotometer (UV-2600, Shimadzu, Japan) at a 288 nm wavelength.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5. Characterization of scaffolds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.1. Morphology of scaffolds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo analyze the microstructure of the scaffolds, a field emission scanning electron microscope (FE-SEM) with an accelerating voltage of 10 kV was utilized (Tescan Mira3 LMU, Czech Republic). Prior to observation, all samples were placed on metal stubs using conductive double-sided tape, and a thin layer of gold was sputter deposited on each one. The ImageJ software was used to determine the average diameter of the fibers in the FE-SEM images, with at least 30 fibers being measured.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.2. Chemical surface composition analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo distinguish the unique vibrational frequencies of scaffolds, attenuated total reflection fourier transform infrared (ATR-FTIR) spectroscopy was utilized with a Thermo Nicolet Avatar 370 instrument (USA). The samples were scanned with a resolution of 4 cm-1 in the range of 400-4000 cm\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.3. Mechanical properties\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRectangular samples, measuring 5 cm \u0026times; 1 cm were taken from the PCL and PCL/Alg scaffolds (n = 5). One centimeter from both ends of the samples was secured within the holders, and the initial distance between the gauges was established as 3 cm. The micro-tensile testing was carried out with the help of equipment that employed a load cell of 100 N and had a crosshead speed of 10 mm/min (TA Plus model, USA). The test was terminated once the samples had fractured.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.4. Wettability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the hydrophilicity of the scaffolds, the water contact angle (WCA) was measured. This involved placing 1 ml of deionized water onto the PCL, PCL/Alg, and PCL/Alg/Lip-Sil scaffolds and capturing images using an optical contact angle apparatus (JC2000A, Powereach Co. Ltd., China). The angle between the liquid-scaffold interfaces was then determined using ImageJ software. The average of five measurements taken at different positions on the scaffold surface was considered the final contact angle value.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.5. Water uptake measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo measure the amount of water uptake, scaffolds were immersed in PBS solution. After six time points (1, 3, 6, 18, 24, and 28h) of immersion in PBS solution, the dry weight and the wet weight of the scaffolds were assessed to evaluate the water uptake of the scaffolds. After each time point, the samples were taken out and their weight was measured following a careful removal of surface water using a sampler. The following equation was used to get the percentage water uptake:\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cimg src=\"data:image/png;base64,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\" width=\"495\" height=\"57\"\u003e\u003c/p\u003e\n\u003cp\u003eS\u003csub\u003e0\u003c/sub\u003e is the weight of dried samples before soaking and S\u003csub\u003e2\u003c/sub\u003e is the weight of soaked samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. 5.6. Degradation rate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to determine the rate of degradation of the PCL and PCL/Alg scaffolds, an in vitro biodegradation study was conducted using a PBS solution. The scaffolds were incubated in a shaking chamber at a temperature of 37 \u0026deg;C. This procedure continued until the scaffold containing Alg was completely destroyed. To account for changes in concentration during the degradation process, the solution was replaced every three days. The weight of the samples was measured weekly to measure biodegradation. The results were reported in the form of the percentage of the remaining scaffolds of the initial weight of the scaffolds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6. Biological assessments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6.1. Cell viability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe viability and growth of human AMSCs (Sinacell Knowledge-Based Production and Research Company, Iran) were assessed using the MTT assay. Cells were seeded on various scaffolds (PCL, PCL/Alg, and PCL/Alg/Lip-Sil) as well as on tissue culture grade polystyrene (Control; Corning, USA). The cells were seeded at a density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per scaffold and incubated in a humidified environment at 37 \u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. After 2, 4, and 6 days of cell culture, 20 \u0026micro;L of MTT reagent (Sigma-Aldrich, USA) was added to each well containing the scaffolds, and the plates were incubated at 37 \u0026deg;C for 4 h. Subsequently, DMSO (Sigma-Aldrich, USA) was added to each well as a solvent, and the plates were placed on a shaker for 30 min to enhance dissolution. The absorbance was then measured at a wavelength of 570 nm using a microplate reader spectrophotometer (SPECTROstar Nano, BMG LABTECH, Germany).\u003cbr\u003eDetails of the pilot study, including the methodology for optimal dose selection of Lip-Sil, dose-response curves, and cytotoxicity analysis, are provided in the Supplementary Materials. The study protocol was approved by the Ethics Committee of Mashhad University of Medical Sciences (MUMS) under the ethical approval code: IR.MUMS.MEDICAL.REC.1399.546. All procedures involving AMSCs were conducted in accordance with the ethical guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6.2. Cell adhesion\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the visualization of cells attached to the scaffolds in comparison to the control sample (tissue culture plate), DAPI staining (Sigma-Aldrich, USA) was conducted on the 6th day of cell culture. The cells attached to the scaffolds were fixed by removing the culture medium, washing with PBS, and fixing with 4% PFA at 4\u0026deg;C for 30 min. Subsequently, the fixed cells were permeabilized using a buffer solution (0.2% Triton X-100 in PBS) for 5 min, and the cell nuclei were stained with DAPI for 10 min. The cells were then observed under a fluorescence microscope (Olympus, Japan). Quantification of cell adhesion was performed using ImageJ software (NIH, USA). Fluorescence images were analyzed to count the number of DAPI-stained nuclei, and the results were expressed as fold change relative to the control group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6.3. Cell morphology\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe morphology of AMSCs cultured on the samples was examined using FE-SEM images taken 6 days after culture. The samples were fixed with 2.5% glutaraldehyde at 4\u0026deg;C for three hours. After fixation, the excess glutaraldehyde (Sigma-Aldrich, USA) was removed by washing the samples with PBS. The samples were then dehydrated using ethanol at gradually increasing concentrations (30-100%) for 10 min each, which removes water from the samples and prepares them for imaging. Following dehydration, the samples were placed in a desiccator, coated with gold, and analyzed using FE-SEM imaging.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7. Statistical\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data were presented as mean \u0026plusmn; SD, and statistical significance was considered at p\u0026lt;0.05. Data analysis was conducted using GraphPad Prism 9.0.0 software. Normality of the data was confirmed using the Shapiro-Wilk test, and parametric tests (e.g., t-test and ANOVA) were applied accordingly. Statistical tests, including Unpaired t-test with Welch\u0026apos;s correction for fiber diameter test, two-way ANOVA for MTT assay, and one-way ANOVA for contact angle and optimal dose tests with the Tukey post hoc test, were performed to determine differences between parameters. Pearson\u0026rsquo;s correlation coefficient (r) was used to evaluate the relationship between drug concentration and cell viability. Potential outliers were identified using the ROUT method (Q = 1%) in GraphPad Prism. All experiments were performed with at least five independent replicates (n = 5) unless otherwise stated.\u003c/p\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003e\u003cstrong\u003e3.1. Physicochemical characteristics of Lip-Sil\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiposomal formulations are regarded as promising drug delivery systems for a variety of substances, including small hydrophilic and lipophilic drugs. In this study, we prepared Lip-Sil using a lipid composition comprising HSPC, cholesterol, and mPEG2000-DSPE, which is similar to FDA-approved liposomal formulations such as Doxil\u0026reg; and AmbiSome\u0026reg; (26). The particle size of Lip-Sil was determined to be 94.7 nm, with a size distribution ranging from 70 nm to 170 nm (Fig. 1a). The PDI of the liposomes was found to be between 0.15 and 0.3, indicating a highly uniform population of vesicles. A PDI value below 0.3 is generally considered acceptable for liposomal formulations, as it reflects a narrow size distribution and homogeneity (22). The zeta potential of Lip-Sil was measured to be -29 mV (Fig. 1b), which is close to the threshold value of -30 mV required for colloidal stability (27). This suggests that the Lip-Sil formulation possesses good physical stability, minimizing the risk of aggregation during storage and application. The negative zeta potential can be attributed to the presence of anionic lipids in the formulation, which also contributes to the electrostatic repulsion between liposomes, further enhancing their stability. This feature may also facilitate interactions with positively charged cell membranes, potentially enhancing cellular uptake.\u003c/p\u003e\n\u003cp\u003eThe Lip-Sil achieved approximately 70% encapsulation efficiency in this study. The achieved EE of 73% is considered acceptable for Lip-Sil, especially when compared to other studies where larger liposomes had EE in the range of 65-70% for Sil (25). It has been suggested that if the cholesterol-to-lipid ratio exceeds a certain threshold, it can disrupt the normal structure of the liposomal membrane, leading to a decrease in drug EE. Therefore, the increase in Sil encapsulation efficiency observed in this study is likely due to the suitable cholesterol ratio. The higher molar ratio of cholesterol in the liposomes affects the arrangement and interactions of liposomal membrane components with Sil, resulting in a decrease in EE (23).\u003c/p\u003e\n\u003cp\u003eFigure 1c illustrates an initial burst release of Sil within the first few hours. The release profile of Lip-Sil exhibited biphasic behavior, with a faster release rate observed during the initial phase compared to the steady release phase. Within the first 24 hours, approximately 37% of Sil was released from the liposomes, while after 2 weeks, only 65% was released. Evidence shows that pegylated liposomes cause slow release of Sil due to the fast-moving hydrophilic chains of PEG. These findings imply that the drug would remain stable in the bloodstream and be released specifically at the targeted site, indicating that this liposome formulation meets the criteria for effective drug delivery systems (16). Additionally, the inclusion of cholesterol in a high molar ratio decreases the formation of transient hydrophilic holes that facilitate Sil release through liposomal layers. This reduction in membrane fluidity contributes to the stability of the liposomes and slows down the release of the Sil (18).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphology of scaffolds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eElectrospun fibers made from naturally derived polymers and incorporating drug or bioactive molecule release capabilities are appealing for biomedical uses because of their advantageous characteristics such as a high surface area to volume ratio, non-toxicity, and biocompatibility (28). In this study, electrospun PCL/Alg fibers containing liposomal silymarin were fabricated. The scaffolds were successfully electrospun, and their morphologies were analyzed using FE-SEM (Fig 2a-d). The images depict smooth nanofibers that are randomly oriented and free of beads. Fibers containing Alginate exhibited a narrow distribution with an average diameter of 157.7 \u0026plusmn; 42.8 nm (Fig 2e). On the other hand, PCL fibers showed a broader range with an average diameter of 323.3 \u0026plusmn; 122.8 nm. The reduction in fiber diameter upon the addition of alginate can be attributed to the increased charge density and repulsive forces within the electrospinning solution, which promotes the formation of thinner fibers (29,30). In elucidating the reduction in the base diameter of the composites, Bok Kim and Hyung Kim proposed that the incorporation of dispersed alginate within the composite facilitates the dissipation of stored elastic deformation energy and impedes the elastic recovery of the deformed PCL/alginate mixture within the nozzle (31). Furthermore, to preserve the structure of the alginate nanofibers, a crosslinking process is required. However, when PCL/Alg nanofibers were immersed in PBS, they maintained their fibrous structure without the need for crosslinking (Fig 6). The diameter of the fibers, which is directly influenced by electrospinning parameters, plays a critical role in determining cellular behavior. In this study, the electrospinning parameters were carefully selected based on previous research to produce uniform, bead-free fibers with optimal morphological and mechanical properties\u0026nbsp;(2,6,14). It has been widely documented that these parameters not only affect fiber morphology but also significantly influence cell-scaffold interactions. For instance, studies have shown that a voltage range of 20\u0026ndash;25 kV and a flow rate of 1\u0026ndash;2 mL/h yield PCL composite fibers with diameters ranging from 100 to 500 nm, which are ideal for promoting cell adhesion and proliferation\u0026nbsp;(2,14). Similarly, Shirehjini et al. demonstrated that a needle-to-collector distance of 15 cm produces fibers with a high surface-to-volume ratio, enhancing cell-scaffold interactions by more effectively mimicking the ECM\u0026nbsp;(6). \u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eThis can be attributed to the presence of PCL, which acted as a supportive backbone for the nanofiber structure\u0026nbsp;(29). Figure 2f illustrates the application of liposomes on the scaffold surface, demonstrating a consistent and thin layer of the liposome solution on the fibers, free from any aggregation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurface chemical of scaffolds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCommon bond absorptions observed in PCL are the asymmetric vibration of -CH2 at 2940 cm\u003csup\u003e-1\u003c/sup\u003e, the symmetric vibration of -CH2 at 2864 cm\u003csup\u003e-1\u003c/sup\u003e, a strong peak at 1723 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e attributing to C=O stretching, the vibration of C-O at 1293 cm\u003csup\u003e-1\u003c/sup\u003e, the C-C vibration at 1240 cm\u003csup\u003e-1\u003c/sup\u003e, and the symmetric vibration of C-O-C at 1159 cm\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003e(14). In the PCL/Alg spectra, the carboxyl peak near 1597 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e characterizes symmetric COO\u003csup\u003e\u0026minus;\u003c/sup\u003e stretching vibrations, asymmetric COO\u003csup\u003e\u0026minus;\u003c/sup\u003e stretching vibrations were presented at 1529 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e, and -OH peak at 3300 cm\u003csup\u003e\u0026minus; 1\u0026nbsp;\u003c/sup\u003e(32). It was observed that the intensity of the C-H bands became more robust as they moved from PCL to PCL/Alg scaffold. This indicates that the CH peak from the alkyl group of \u0026nbsp;PVA was positioned on the CH peaks from PCL (33). In order to enhance the bioactive characteristics of synthetic polymers, it is typically desirable to incorporate dispersed bioactive materials to the greatest extent possible. The detection of a broad -OH peak within the range of 3050-3700 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e provides evidence for the existence of liposomal silymarin on the surface of the PCL/Alg/Lip-Sil scaffold (Figure 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMechanical properties\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe physical characteristics of scaffolds are crucial for maintaining their shape while new tissues regenerate. The mechanical properties of engineered materials should closely resemble those of the human tissues they are intended to replace.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eThese characteristics also impact different cellular processes, including cell growth and the skeletal structure of cells (34). Figure 4 displays stress-strain curves, indicating that the PCL scaffold had a maximum tensile strength of 10 \u0026plusmn; 1.3 MPa, while the PCL/Alg scaffold had a maximum tensile strength of 2.7 \u0026plusmn; 0.17 MPa. Additionally, the percentage strain at break for the PCL and PCL/Alg scaffolds were 67.4 \u0026plusmn; 2.41% and 55.1 \u0026plusmn; 2.9%, respectively. The disparity in maximum strength and strain at break between PCL and PCL/Alg nanofibers was found to be statistically significant (p\u0026lt;0.001 and p\u0026lt;0.0001, respectively). The inclusion of the alginate component led to a notable decrease in both the maximum strength and strain of the scaffold compared to PCL. This can be attributed to the fact that when external stress was applied to the entire PCL/Alg scaffold, it was primarily concentrated in the alginate content (32). From the findings, it can be inferred that the mechanical characteristics of the PCL scaffolds can be readily adjusted by incorporating Alg into them. Several studies have utilized PCL as a reinforcing agent in Alg hydrogels to enhance the mechanical properties of the resulting fibers\u0026nbsp;(35\u0026ndash;37). While our findings align with these studies, our primary objective was distinct: we aimed to improve the properties of PCL fibers themselves, rather than focusing on enhancing Alg. The ability to modulate PCL flexibility by incorporating alginate represents a significant finding. Alginate may alter the viscoelastic behavior of the fibers, increasing energy absorption and reducing overall stress during strain application\u0026nbsp;(38,39). However, this also introduces a limitation, as these modifications confine its applicability mainly to soft tissue engineering applications, such as native human skin and cartilage, which typically exhibit tensile strengths in the range of 2\u0026ndash;3 MPa\u0026nbsp;(40).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWettability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe wettability of electrospun fibers was assessed, as it is a crucial factor affecting the potential cellular response to the surface. Based on the findings in Fig 5, the addition of Alg showed a significant decrease in the average WCA (p\u0026lt;0.0001), which can be attributed to Alg\u0026apos;s chemical structure containing carboxylate and hydroxyl groups that can interact with water molecules. In contrast to the hydrophobic nature of PCL (126.9 \u0026plusmn; 9.6\u0026deg;), the composite Alg fibers exhibited high hydrophilicity (31.8 \u0026plusmn; 4.1\u0026deg;). Moreover, the incorporation of Lip-Sil onto the scaffold further reduced the WCA compared to the PCL and alginate composite fibers (p\u0026lt;0.0001 and p\u0026lt;0.01, respectively). In a similar research, Mohammadi et al demonstrated that the addition of HSPC/cholesterol/m-PEG2000-DSPE liposome to the poly-L-lactic acid scaffold resulted in decreased WCA measurements from a hydrophobic state to a fully hydrophilic state (24). The favorable wettability observed in fibers is believed to promote cell adhesion (41). Consequently, the cellular behavior advantage of these fibers is expected to surpass that of other types of fibers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWater uptake\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe capacity of scaffolds to absorb water is an important characteristic in tissue engineering applications as it indicates their ability to swell. However, excessive water uptake can result in structural damage and reduced mechanical strength (42). Conversely, low water uptake hinders cell attachment and penetration into the scaffold. Water uptake experiment was conducted to examine how Alg affects the swelling characteristics of the PCL scaffold. According to Figure 6, the PCL and PCL containing Alg both reached their maximum swelling levels at around 18.6 \u0026plusmn; 0.88% and 80.7 \u0026plusmn; 5.3% respectively within 18 hours. The water absorption ratios for both samples remained constant as the incubation time increased. The lower swelling ratio of PCL nanofibers compared to PCL/Alg can be attributed to the hydrophobic properties of the PCL fibers. However, our findings demonstrate a moderate increase in water uptake by the composite scaffold. Once the scaffolds undergo a moderate level of swelling, they can transform into 3D structures resembling the ECM. These structures are capable of providing the necessary nutrients for cell growth and eliminating waste generated from cellular metabolism (42).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDegradation behavior\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this research, PCL, an FDA-approved substance, was selected as the primary scaffold due to its lack of negative effects caused by degradation, such as the release of acidic by-products. The degradation and removal of the scaffold from the implantation area are crucial when it is used as a tissue-engineered platform. Nevertheless, minimal decomposition of the PCL nanofibers in PBS solution was observed during the in vitro hydrolysis process. This can be attributed to the hydrophobic nature of the PCL nanofibers, which undergo slow hydrolysis and have low solubility in the PBS solution and biological environment (43). Figure 7 illustrates that only a 10% weight loss was observed in the PCL samples after being submerged in the PBS solution for 4 weeks. Previous studies have also demonstrated that PCL completely degrades in vivo within about 2 years (44,45). Although the use of PCL eliminates concerns regarding the removal of implants from the patient\u0026apos;s body, as opposed to non-degradable materials used in clinical settings (46), this degradation rate can pose challenges depending on the target tissue. The choice of the implant\u0026apos;s in-vivo degradation rate depends on the rate at which the target tissue regenerates. The faster the target tissue regenerates, the faster the in-vivo degradation rate of the implant is required (2). Our results revealed that incorporating Alg into the scaffold can regulate the hydrolysis of PCL and cause the complete destruction of the scaffold. PCL scaffold containing Alg\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003edegradation occurs faster than PCL scaffolds because of its hydrophilicity (2). Therefore, to optimize the lifespan of the implant, the PCL scaffolds can be customized by adjusting the percentage ratio of Alg according to the specific native target tissue.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell adhesion and proliferation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe most crucial need for a scaffolding material is its ability to support cell growth and maintain metabolic functions (47). Both natural and synthetic biomaterials have demonstrated impressive benefits as matrices for supporting cells (3). Nevertheless, when cells are seeded on engineered scaffolds, the substrate can result in the generation of oxidative stress due to the mechanical and physical pressure exerted on cells. This stress can cause alterations in the structure and function of the cells (48). In this research, a biomaterial for tissue engineering was developed by combining the natural polymer alginate with the synthetic polymer PCL and covering them with a proliferation agent with controlled and sustained release. The aim was to use the advantages of synthetic and natural polymers in one material to enhance the scaffold\u0026apos;s properties resembling the ECM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe adhesion and viability of AMSCs on different types of scaffolds, including PCL, PCL/Alg, PCL/Alg/Lip-Sil, and a control group were compared by MTT assay and DAPI staining (Fig 8). The MTT assay showed that AMSCs had a higher rate of proliferation in the PCL/Alg and PCL/Alg/Lip-Sil scaffolds compared to the PCL and control groups (Fig 8a). This means that trapping AMSCs in the scaffolds incorporating with Alg not only did not harm the cells but also increased their rate of growth compared to the PCL and control groups. The findings can be attributed to the porous structures, extensive surface area, and three-dimensional design of the PCL/Alg scaffold. This scaffold closely resembles the natural microenvironment of the ECM and offers improved cell nutrition, viability, and proliferation (2). Additionally, there were no harmful effects observed in any of the scaffolds for active AMSCs, as none of them displayed a significant decrease in absorption compared to the control group (Figure 8a). Leena et al\u0026apos;s research findings indicated that Sil enhances the proliferation of mesenchymal stem cells on Alg composite scaffolds. They attributed these positive outcomes to the ability of silymarin to stimulate cell growth and increase cellular metabolism (49). The way the nanofiber network in the ECM is arranged and structured influences how cells grow and communicate (50). Our study reveals that Lip-Sil significantly enhances the surface of the scaffold, helping to maintain the structure of the ECM. A previous study indicated that Sil stimulates cell proliferation in polycaprolactone fibers (51), providing support for our findings. Lavi Arab et al. demonstrated that Sil nanocarriers stimulate the proliferation of AMSCs at low concentrations\u0026nbsp;(52). This effect is mediated by the upregulation of endogenous mitochondrial enzyme levels and activities induced by silymarin. Additionally, evidence suggests that while high concentrations of Sil can suppress lymphocyte proliferation and induce cell death in cancerous and toxic tissues, lower doses exhibit cytoprotective properties. At these lower concentrations, Sil promotes MSCs proliferation and inhibits apoptosis, highlighting its dose-dependent dual role in cellular behavior\u0026nbsp;(53,54), as noted in our supplementary evidence.\u003c/p\u003e\n\u003cp\u003eThe results of the quantitative analysis of DAPI images revealed that the composite scaffolds significantly enhanced cell adhesion compared to the control group (p \u0026lt; 0.05). Furthermore, the incorporation of Lip-Sil into the composite scaffold resulted in a remarkable increase in cell attachment, demonstrating superior performance compared to all other groups (p \u0026lt; 0.01). These findings align with the MTT assay results, confirming the favorable impact of the composite scaffold and Lip-Sil on cell adhesion and viability. The scaffold that holds liposomes loaded with polyphenol compounds has the potential to be a highly effective and innovative method for delivering drugs. It can be used to transport drugs accurately and continuously to specific areas, reducing burst releases, and also serve as a scaffold for tissue engineering, promoting tissue regeneration (55,56). The PCL/Alg scaffold exhibited lower cell adhesion compared to the PCL/Alg/Lip-Sil scaffold, resulting in a lower number of viable cells. The limited cell adhesion can be attributed to the inherent hydrophobic nature of the polymers, which hampers the interaction between the cells and the scaffold (57). This could be attributed to the restricted space available for cell growth and a weaker interaction between the cells and the scaffold (58). The optimal mechanism for enhancing cell growth and adhesion in this platform can be attributed to several factors. Firstly, the functional groups in alginate facilitate the adsorption of ECM proteins such as fibronectin and collagen\u0026nbsp;(59), which subsequently enhance cell adhesion through cellular signaling pathways. Additionally, MSCs exhibit a strong tendency for high adhesion in the stiff PCL environment due to durotaxis\u0026nbsp;(60). Furthermore, as demonstrated, the scaffold-liposome interaction enhances the hydrophilicity of the scaffold surface\u0026nbsp;(1,24), making it more interactive and conducive to cell attachment. Silymarin released from liposomes improves cellular metabolism by upregulating endogenous enzymes (e.g., superoxide dismutase and catalase) and enhancing mitochondrial function\u0026nbsp;(52), thereby promoting cell survival and proliferation. These distinct biochemical interactions collectively create a favorable microenvironment that supports AMSC adhesion, proliferation, and metabolic activity. Consistent with our findings, Dadashpour et al. developed a platform where silybin-loaded nanoparticles, an active compound of Sil, were incorporated into hybrid fibers made of natural and synthetic polymers\u0026nbsp;(19). Their results demonstrated sustained adhesion and proliferation of AMSC on this scaffold, supporting the potential of such systems for long-term cell culture applications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphology of cells\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFE-SEM images were captured to examine the morphology of cells and assess how well AMSCs adhered to the PCL/Alg nanofiber (Figure 9). The PCL scaffold had minimal cell adhesion and density. However, the nanofibers containing Alg showed good compatibility with the AMSCs. At lower magnification, it was observed that the AMSCs adhered tightly to the nanofibers, regardless of whether Lip-Sil was present. However, there were no notable differences in the cell appearances on the PCL/Alg and PCL/Alg/Lip-Sil nanofibers. This is likely due to the presence of more pores in the nanofiber than anticipated, which helps alleviate compression pressure. This is significant because it allows for easy provision of media and nutrients, which is crucial for the use of biomaterials in tissue engineering as cells need to remain alive within the implanted scaffold (61). Both scaffolds showed potential in terms of the nanofiber\u0026apos;s topography inducing cell expansion. However, at higher magnification, it was observed that the cells exhibited more expansion on the PCL/Alg/Lip-Sil fibers, while in the other samples, the cells were clustered together. The PCL/Alg/Lip-Sil scaffold demonstrated excellent cell expansion and high production of ECM on both the surface of the scaffold and within its pores. The composite nanofibers created a favorable environment for the growth of AMSCs. Additionally, these nanofibers not only exhibited a strong affinity for stem cells but also preserved the normal characteristics of the native tissue and promoted ECM secretion. According to Wu et al., Sil enhances cellular metabolic activity at concentrations as low as 50 \u0026mu;M, mediated by the improvement of ECM homeostasis through the modulation of mRNA expression and protein levels of catabolic and anabolic cytokines (20). Polyphenols not only mitigate the damage caused by oxidative stress to normal tissues but also exhibit a specific affinity for functional molecules like receptors, enzymes, transcription factors, and transduction factors (62). Consequently, polyphenols can facilitate the recovery process of damaged tissues and improve the ECM to create a favorable environment for cell growth. Through their ability to regulate the tissue microenvironment and participate in cellular events, polyphenols demonstrate growth-promoting properties (48).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe have successfully developed Lip-Sil loaded PCL/Alg nanofiber with AMSCs seeding for tissue engineering. Based on the findings from drug dissolution, efficient encapsulation, zeta potential, and DLS, the liposome formulated using the thin layer hydration method has favorable characteristics for delivering the agents to the injured tissue. These features include controlled release of silymarin, high drug loading efficiency, stability, and a small particle size of the formulation. Characterization and in vitro assessments were conducted to measure the modifications in scaffolds containing alginate in comparison to PCL fibers. The outcomes of the composite scaffold indicated an enlargement in water uptake and mass loss, as well as a reduction in fiber diameter and WCA compared to PCL fibers. The application of Lip-Sil covering on the PCL/Alg enhanced the hydrophilicity, which is justified by the presence of OH groups on the fiber surface. The implantation of AMSCs onto the scaffolds demonstrated that the PCL/Alg/Lip-Sil scaffold exhibited a high potential for cell adhesion, proliferation, and expansion, as indicated by the results of DAPI, MTT, and FE-SEM analyses. We reported a new platform for injured tissue treatment, with an enhanced cell adhesion index and improved proliferation rate of adipose mesenchymal stem cells. The requirements of tissue engineering included targeted, prolonged, and controlled release of healing agents, a suitable substrate for promoting cell growth and connection, and self-renewal cells, which can be achieved through our novel platform.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study has several limitations that should be acknowledged. First, the in vitro nature of our experiments may not fully replicate the complex biological environment of in vivo systems. Second, while we demonstrated the scaffold\u0026apos;s ability to enhance cell viability and adhesion, we did not investigate its effects on inflammation or immune response, which are critical for tissue regeneration. Future studies should address these limitations by incorporating in vivo models, cytokine profiling, and long-term biocompatibility assessments to further validate the scaffold\u0026apos;s potential for clinical applications.\u003c/p\u003e\n\u003cp\u003eIn summary, this research proposes an innovative and improved platform for tissue engineering applications. The study utilized the small molecule (silymarin) as a model of healing agents in liposomal formulation for the ability of sustained drug release, AMSCs for supporting tissue regeneration and cell replacement, and PCL/Alg fibers were employed as a substrate and co-delivery system for cells and agents. \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grant number 98126378 from the Research Council of the Faculty of Medicine, Mashhad University of Medical Sciences (MUMS, Iran) with ethic number IR.MUMS.MEDICAL.REC.1399.546. No human participants were directly involved in this study. However, we used commercially available human adipose-derived mesenchymal stem cells purchased from Sinacell Knowledge-Based Production and Research Company (Iran). These cells were obtained as an established cell line, and no additional human samples were collected or used for this research. All ethical and legal requirements for the use of human-derived biological materials were followed in accordance with the supplier\u0026apos;s guidelines and relevant institutional regulations.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbroumand Gholami A, Gheybi F, Molavi AM, Tahmasebi F, Papi A, Babaloo H. Effect of polycaprolactone/carbon nanotube scaffold implantation along with liposomal ellagic acid in hippocampal synaptogenesis after spinal cord injury. Nanomedicine J [Internet]. 2023;10(3):197\u0026ndash;209. 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J Physiol Sci. 2022;72(1):1\u0026ndash;24. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Adipose Tissue Derived Mesenchymal Stem Cells, Biomedical applications, Drug Carrier, Electrospinning, Guided Tissue Regeneration","lastPublishedDoi":"10.21203/rs.3.rs-6279660/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6279660/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e: The clinical application of mesenchymal stem cells (MSCs) in tissue engineering is hindered by critical challenges, including low cell survival rates, poor retention at injury sites, and the lack of bioactive scaffolds that mimic the native tissue microenvironment. To address these limitations, this study developed a multifunctional platform using liposomal silymarin (Lip-Sil)-enriched polycaprolactone/alginate (PCL/Alg) hierarchical fibers to enhance the delivery, adhesion, and functionality of adipose-derived MSCs (AMSCs) for tissue regeneration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Lip-Sil was synthesized using the remote loading method and characterized for particle size, zeta potential, encapsulation efficiency, and dissolution behavior. PCL/Alg hierarchical fibers were fabricated via electrospinning and evaluated for mechanical properties, morphology, hydrophilicity, degradation rate, , and surface chemistry using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The biological performance of the scaffolds was assessed through in vitro studies, including cell viability, adhesion, and proliferation of AMSCs using MTT assay, DAPI staining, and FE-SEM imaging.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: The Lip-Sil formulation exhibited a particle size of 94.7 nm, a zeta potential of -29 mV, and an encapsulation efficiency of 73%. The cumulative dissolution profile showed a sustained release, reaching 65% after 2 weeks. The PCL/Alg fibers demonstrated a significant reduction in diameter (157.7 ± 42.8 nm) compared to pure PCL fibers (323.3 ± 122.8 nm). Mechanical testing revealed that the PCL and PCL/Alg scaffolds had a tensile strength of 10 ± 1.3 and 2.7 ± 0.17 MPa and a strain at break of 67.4 ± 2.41% and 55.1 ± 2.9%, respectively. The addition of alginate improved hydrophilicity (water contact angle: 31.8 ± 4.1° vs. 126.9 ± 9.6° for PCL) and degradation rate. The water uptake rate of PCL/Alg scaffolds reached 80.7 ± 5.3% within 18 hours, significantly higher than that of PCL scaffolds (18.6 ± 0.88%) and these ratios for both samples remained constant until 28 hours. AMSCs cultured on PCL/Alg/Lip-Sil scaffolds showed an excellent increase in cell proliferation compared to control groups (p\u0026lt;0.01) after 7 days of incubation. DAPI staining revealed a mean cell adhesion index of 1.6 ± 0.1 for the composite scaffold. FE-SEM imaging confirmed enhanced cell spreading and expansion on the composite scaffolds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: The developed PCL/Alg/Lip-Sil scaffold represents a promising platform for tissue engineering, offering controlled drug release, improved cell adhesion, and enhanced AMSC proliferation. This multifunctional system addresses key challenges in stem cell delivery and tissue regeneration, providing a robust foundation for future clinical applications.\u003c/p\u003e","manuscriptTitle":"In Vitro Evaluation of Bioactive PCL/Alginate Hierarchical Fibers with Controlled Liposomal Silymarin Release for Enhanced Tissue Engineering: Breaking Barriers in MSC Transplantation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-05 05:31:11","doi":"10.21203/rs.3.rs-6279660/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-15T10:47:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-15T08:17:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-14T22:18:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"132430008385046326948283874851856379294","date":"2025-04-02T17:21:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"92422520495067934514646557573432941914","date":"2025-04-02T16:42:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-02T15:09:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-02T03:30:30+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-02T03:19:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-01T06:41:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-21T17:55:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7049eacd-f538-4054-88bf-a2a90b8faf74","owner":[],"postedDate":"May 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47187058,"name":"Biological sciences/Biotechnology"},{"id":47187059,"name":"Biological sciences/Cell biology"},{"id":47187060,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-10-20T15:59:11+00:00","versionOfRecord":{"articleIdentity":"rs-6279660","link":"https://doi.org/10.1038/s41598-025-19799-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-10-13 15:56:54","publishedOnDateReadable":"October 13th, 2025"},"versionCreatedAt":"2025-05-05 05:31:11","video":"","vorDoi":"10.1038/s41598-025-19799-6","vorDoiUrl":"https://doi.org/10.1038/s41598-025-19799-6","workflowStages":[]},"version":"v1","identity":"rs-6279660","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6279660","identity":"rs-6279660","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}