Design a coordinated platform for coumarin-regulated delivery in line with the biological systems' growth phases

Design a coordinated platform for coumarin-regulated delivery in line with the biological systems' growth phases | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Design a coordinated platform for coumarin-regulated delivery in line with the biological systems' growth phases Rojan Akhbarati, Rahebeh Amiri Dehkharghani, Soheila Zamanlui Benisi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5122397/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Nov, 2024 Read the published version in Journal of Polymers and the Environment → Version 1 posted 13 You are reading this latest preprint version Abstract A full-control design can significantly improve drug release and cell proliferation for tissue engineering applications in medicine. The present investigation encompassed a molecular docking study which was performed to investigate the interaction of selected active ligand (coumarin) with the L929 mouse fibroblast cell line protein as the receptor. After that, the coumarin was extracted from the roots of p.ferulacea and its subsequent nanoencapsulation with polycaprolactone, employing the coacervation technique to achieve a narrow distribution of nano particle sizes. Subsequently, the electrospinning technique was utilized to apply a second coating to the nano-encapsulated coumarin. Polyvinyl alcohol and gelatin compounds were used to produce electrospun nanofibrous scaffolds for their similarity to the extracellular matrix (ECM). This coordinated nano platform aimed to assess its effectiveness in regulating drug release, evaluate its biocompatibility, and examine its impact on L929 cell proliferation according to the Lag and Log phases of their growth. In silico analyses demonstrated significant interactions and high binding energy values between the coumarin ligand and essential residues of the L929 mouse fibroblast proteins. The results of the experiments were checked using analyses of 1 H NMR, FTIR, UV, SEM, mechanical properties, DSC, HRTEM, and HPLC. The biological effects and cell proliferation were conducted employing the MTT method (up to 5 days). Notably, no cytotoxicity was detected throughout the assessment. In this way, it is feasible to create a synergistic nano delivery system by delaying the release of the drug into account the timing of distinct cell lines' development phases. Nano-encapsulation Electrospinning Nanofibrous scaffold Controlled release Coumarin L929 cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 1. Introduction The development of multifunctional drug delivery systems (DDSs) for therapeutic and ‎diagnostic purposes presents the potential for simultaneous disease diagnosis and treatment [ 1 , 2 ]. Developing an effective therapeutic delivery system targets specific cellular or tissue ‎entities in the human body to enhance the drug's therapeutic effects, minimize its side ‎effects, and enhance its physicochemical properties [ 3 , 4 ]. In recent years, a significant focus has been on investigating a range of controlled release systems, including nanoparticles, micelles, hydrogels, and electrospun membranes [ 5 – 7 ]. Also, the utilization of nanostructures such as nanofibers and nanocapsules, has demonstrated notable efficacy in the fields of tissue engineering and controlled drug release [ 8 – 10 ]. This can be attributed to their heightened surface-to-volume ratio, appropriate size, porous permeability, and convenient carriers for drug delivery [ 11 – 13 ]. Different compounds are used for the synthesis of these nanostructures which can include biodegradable polymers. Polyvinyl alcohol (PVA), gelatin, and polycaprolactone (PCL) are ‎also utilized in several biomedical applications, including wound healing, medication delivery, ‎and tissue engineering scaffolds [ 14 – 17 ]. Among the techniques that might be used for the synthesis of nanofibers and nanocapsules, the electrospinning and coacervation methods can be mentioned. Electrospinning is a robust technological process that facilitates the production of nanofibrous ‎membranes through the application of electric current across various polymer materials [ 18 – 20 ]. This process has demonstrated its efficacy in transforming a wide range of polymers into nanofibers, encompassing both natural and synthetic polymers, as well as hybrid polymer materials [ 21 – 23 ]. The coacervation technique can be used to load drugs in polymeric nanocapsules. The method involves the utilization of a coacervating compound like a desolvating agent (such as nonsolvent, pH alteration, temperature, or electrolyte) or a crosslinking agent like glutaraldehyde, to segregate the hydrocolloid from the initial solution. This technique is also employed to produce a consistent colloidal system that diminishes the average particle size (on a nanometer scale) and enhances the loading capacity with minimal coagulation and uniform distribution of nanoparticles [ 24 ]. Coumarin is a colorless and odorless crystalline substance that belongs to the benzopyrone chemical class. It is found naturally in many plants, mainly in the form of phenol. Coumarins are an important class of secondary metabolites that are widely found in several parts of plants. Ground plants such as Tonka bean and vanilla plants have high concentrations of coumarin. Among the biological and medicinal activities of coumarin and its derivatives, we can mention anticoagulant, antitumor, antibacterial, antifungal, anti-inflammatory, anti-HIV, and antioxidant [ 25 – 27 ]. Based on our continued effort with the purpose of skin tissue engineering, the present study involved the production of a coordinate scaffold composed of PVA/gelatin, and PCL nanocasules containing coumarin. To have the relative release of the active ingredient during the phase of cell matching to the culture medium (Lag phase), we employed an external polar coating for cell proliferation, taking into consideration the time needed for the Lag phase of L929 cell line. However, to ensure that the drug release would continue until the second stage of replication (Log phase), we employed an additional non-polar coating to encapsulate the coumarin. 2. Materials and Methods 2.1. Materials and apparatus Polycaprolactone (PCL, Mw of 80000 g/mol), polyvinyl alcohol (PVA, Mw of 31000–50000 g/mol, 99% hydrolyzed), gelatin (type A), and 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich, USA. Span 80, tween 80, sunflower oil, lactic acid, acetone, acetic acid, methanol, acetonitrile, glutaraldehyde solution (2.5%), and ethanol were purchased from Merck, Germany. Sodium citrate dihydrate, acid citric, sodium bicarbonate, and sodium carbonate, which were used as buffers, were also purchased from Merck, Germany. Mouse fibroblasts (L929 Cells) were obtained from Geniran, Iran. Dulbecco's Modification of Eagle's Medium (DMEM), penicillin/streptomycin, and trypsin-(EDTA) were obtained from Bioidea, Iran. Fetal bovine serum (FBS) was obtained from Gibco, USA. A KBr disk technique (Perkin Elmer Spectrum ASCII, USA) with a range of 4000 to 400 cm − 1 was used to perform the FT-IR spectra. The assessment of the product was conducted within a wavelength range of 200 to 400 nm using a UV–Vis spectrophotometer (Perkin Elmer/ Lambda 25 UV/Vis spectrophotometer, USA). The morphology of the nano-encapsulated coumarin and the electrospun nanofibrous scaffold was investigated using a Field Emission Scanning Electron Microscope (FE-SEM, MIRA3 TESCAN, Brno, Czech Republic). The morphology of the electrospun nanofibrous scaffold after cell culture was investigated using a Field Emission Scanning Electron Microscope (FE-SEM, SIGMA VP-500, ZEISS, Germany). The particle size was determined using high-resolution transmission electron microscopy (HRTEM, FEI TECNAI G2 F20 S-TWIN TEM, USA). The controlled drug release was evaluated using high-performance liquid chromatography (HPLC, SCL-10AVP, LC-20AD, DGU-20A, SPD-20A, SHIMADZU, Japan). The ELISA plate reader (Biotek Epoch Microplate Spectrophotometer, USA) was utilized to examine the plates by measuring the absorbance at a wavelength of 570 nm. The 1 HNMR spectra were performed by Bruker AVANCE AQS-300MHZ using CDCl 3 as an NMR solvent. The thermal characteristics of nano-encapsulated coumarin, and electrospun nanofibrous scaffold were investigated using a differential scanning calorimetry (DSC, NETZSCH DSC 214 Polyma, Germany). A study was carried out to create a nanofibrous scaffold through the electrospinning technique, employing a horizontal setup with a cylindrical collector wrapped in aluminum foil (Co881007NYI, ANSTCO, Iran). The mechanical characteristics were assessed utilizing a mechanical analyzer (SANTAM, STM-20, Iran). 2.2. Computational method DFT (B3LYP) optimized the coumarin ligand geometries, which was visualized using Gauss View 6 software. The 6-311 + G(d) basis set assessed the nonmetallic atoms. Frequency was calculated on the optimum geometry to confirm that it represented a global minimum, indicated by the absence of imaginary frequencies. The molecular electrostatic surface potential (MESP) and frontier molecular orbitals (FMO) were calculated based on the optimum structures. The Gaussian-09 [ 28 ] software conducted the calculations, and the Gauss View 6 created visual representations. Molecular Operating Environment (MOE) [ 29 ] software was used for coumarin into the L929 structures docking calculations that the 3D structure of it was taken from the site ( https://www.rcsb.org ). The binding pocket size for L929 was determined to be 40 × 40 × 40 points. The docking parameter “exhaustiveness” was set to 10, and a grid spacing of 0.375 Å calculated the energetic map for the docking study. The root mean square deviation (RMSD) values validated the molecular docking protocol. During the validation study, the ligand was treated as flexible while the protein remained rigid. 2.3. Preparation of coumarin herbal extract The fresh roots of p.ferulacea were collected from Yasouj, Iran, and further pulverized into powder. The extraction by maceration method was performed with 0.5 g of dried roots of p. ferulacea and 10 ml of methanol solvent. The mixture was left at room temperature for 72 hours [ 30 ]. The extracted material was then placed in an ultrasonic bath and passed through a filter. After purification, 1 H NMR analysis was carried out on the sample. 2.4. Coacervation method to prepare nano-encapsulated coumarin The coacervation technique was used to prepare nanocapsules and implemented by using polycaprolactone. An adequate quantity of PCL/acetone (0.25 g/67 mL) was combined, and the amalgam was sealed with foil before being positioned in an ultrasonic bath at a 40°C temperature. Subsequently, 0.8 mL of sunflower oil (SFO) was blended with 0.196 mL of Span 80 (water-soluble), and the mixture was introduced to the PCL/acetone from the previous step utilizing a syringe, and positioned on a magnetic stirrer. In the subsequent stage, coumarin (0.0114 g) was introduced in a nitrogen environment and covered with foil. To obtain a homogeneous mixture, it was placed under magnetic stirring for 2 hours. Lactic acid solution (coacervating solution) was incrementally added in an ultrasonic bath until the pH level dropped below the isoelectric point (4.0). The resulting colloid was placed in a water bath and swiftly cooled to 15°C to enhance the formation of nanocapsules while being stirred at 300 rpm for stabilization of the colloid. The nanocapsules were then filtered and dried utilizing a freeze-dryer for 72 hours at -70°C. The samples were then analyzed by SEM, FTIR, DSC, and UV techniques. 2.5. Preparation of nanofibrous scaffold Initially, the solutions of gelatin (9% w/v) and PVA (9% w/v) were prepared in acetic acid under gentle stirring at 35–40°C for 1h and in distilled water with continuous stirring at 90°C for 2h, respectively. Both solutions were mixed afterward with 5%, 10%, and 15% nano-encapsulated coumarin, added at independent instances. The solutions that were prepared were introduced into a 5 ml syringe and then subjected to electrospinning at ambient temperature. This process was carried out using a horizontal setup equipped with a cylindrical collector that was coated with aluminum foil (Co881007NYI, ANSTCO, Iran). The conditions of electrospinning and the amount of drug were optimized, and the samples were checked with a microscope to have the least bead density. The distance (between the tip of the needle and the collector) was set to 170 mm, the voltage was set to 27kV, the flow rate was 0.6 ml/h, and the amount of nano-encapsulated coumarin at 10% (due to the drug release control). The nanofiber scaffolds were finally examined using SEM and FT-IR, UV, DSC, mechanical test, measurement of porosity, and HR-TEM analyses. 2.6. Mechanical properties The mechanical characteristics were assessed through the utilization of a mechanical analyzer (SANTAM, STM-20, Iran). The samples consisted of rectangular discs (30×10 mm 2 ) with a scaffold thickness of 0.134 mm. These specimens underwent a consistent tensile deformation rate of 5 mm/min under dry ambient conditions at room temperature. The fracture stress and elongation at failure were subsequently quantified. 2.7. Differential scanning calorimetry (DSC) The thermal characteristics of nano-encapsulated coumarin and electrospun nanofibrous scaffold were investigated by a DSC analyzer. The samples were then subjected to a scanning velocity of 10°C /min, along with a gas stream of nitrogen at a rate of 50 mL/min, within a temperature range of 0 to 300°C. 2.8. Porosity The liquid displacement technique [ 31 ] was employed to determine the porosity of nanofiber scaffolds. The choice of ethanol as the displacing solution was made to prevent any modification in the polymeric matrix. This selection was based on ethanol's ability to enter the nanofiber scaffolds without inducing any swelling or shrinking effects. The scaffolds (dry weight, w d ) were submerged in ethanol for 30 minutes and the immersed scaffold weights were recorded as w l afterward. The filtration process was conducted after the extraction of the substances from the ethanol medium. The weight of moist scaffolds was then quantified as w w : Porosity (%) = (W w -W d )/ (W w -W l ) × 100 The values are the mean ± standard error (n = 3). 2.9. Drug release HPLC analysis was performed to evaluate the coumarin release rate from the electrospun sample containing nanocapsules. Standard solutions of coumarin were made to measure the peak of the desired drug and the inhibition time of the drug. Drug release was evaluated at different times in acidic, alkaline, and neutral environments. The HPLC device was set with a washing solvent of 60% acetonitrile and 40% water, an optical measurement was conducted, using a wavelength of 270 nm and a liquid flow rate of 1 mL/min. For the evaluation of drug release in a different environment, the electrospun sample (0.005 g) was dissolved in 50 mL of distilled water (pH = 7) under a neutral environment. Sodium citrate dihydrate (0.496 g) was dissolved in 40 ml of distilled water (40 ml). Acid citric (0.636 g) was then dissolved in distilled water (10 mL) and 5 mg of the electrospun sample at (pH = 4) in an acidic environment. Sodium bicarbonate (0.382 g) was dissolved in 40 ml of distilled water (40 ml). Next, sodium carbonate (0.047 g) was dissolved in distilled water (10 mL) and 5 mg of the electrospun sample (pH = 9.1) in an alkaline environment. Then, it was placed in a shaker incubator at 37 ℃. The release operation was performed for 72 hours. Then, the samples were injected into the HPLC device within 6 min. 2.10. MTT assay The viability of L929 cells (the mouse fibroblast cell line) was assessed on the created electrospun nanofibrous scaffold using the MTT assay. The cells were seeded onto sterilized scaffolds in a 96-well culture plate at an initial density of 5000 cells/cm 2 . The cells were subjected to treatment with 100 µl of MTT solution (500 µg/ml) on days 1, 3, and 5. This treatment was carried out for 3.5 hours following the removal of the cell culture media. Subsequently, an ELISA plate reader was utilized to examine the plates by measuring the absorbance at a wavelength of 570 nm. 2.11. Cell culture The purified L929 cells (Mouse fibroblast cell line) were grown in T25 flasks [DMEM supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin)]. The cells were treated with trypsin–EDTA solution (GIBCO) and centrifuged after being cleaned with PBS to facilitate cell transit. 2.12. Morphological analysis Scanning electron microscopy (SEM) was used to analyze the surface morphology of aligned scaffolds before and after cell culture. After PBS washing, the scaffolds were seeded with cells in 2.5% glutaraldehyde within 40 min. As the alcohol concentration increased, the scaffolds underwent dehydration. The samples were then sputter-coated with gold after being allowed to dry at room temperature. 2.13. Statistical analysis To conduct a statistical analysis of the experiment, and determine the variability in scores between samples, the one-way analysis of variance (ANOVA) was used. Data was subsequently analyzed using SPSS software. Statistics were judged significant at P < 0.05. 3. Results and Discussion 3.1. Docking results Figure 1 has been shown the optimized structure of coumarin. Several significant bond distances, including C-C and C-O, were highlited in the structure. For the global minimum identification on the potential energy surface, the frequency was calculated finding no imaginary frequencies. The stretching frequencies of the corresponding functional groups were validated against experimental data. The FMO analysis revealed that the energy gap between the LUMO and HOMO for the ligand was − 4.61 eV. Figure 2 a shows their schematic diagram. The HOMO and LUMO orbital energies are highly stabilized, suggesting that coumarin is a highly reactive ligand. The electrostatic potential mapping considering the overall electron density surface, offers a real-time depiction of the distribution of electrostatic potential (taking into account both electrons and nuclei), as well as the molecular morphology, size, and the molecule dipole moments. This visual representation can assess relative polarity. Figure 2 b displays the MESP for coumarin. The MESP surface, depicted with blue, red, green and yellow regions, highlights areas that are electron-deficient, electron-rich, neutral and slightly electron-deficient, respectively. The area surrounding the oxygen group has the most negative potential (red), whereas the area near the carbon group has a more positive charge. Additionally, light blue and green regions on the MESP surfaces of coumarin are also observed [ 32 , 33 ]. Precise anticipation of protein-ligand binding free energies through practical computational approaches is capable of revolutionizing drug identification. Using protein-ligand interaction modeling with quantum mechanical approaches, rather than relying on empirical classical mechanics approaches this objective is achieved. The 6-311 + G(d) method was explored to determine the binding free energies of coumarin as the ligand to the L929 cell line, utilizing linear-scaling density functional theory for the complex containing protein and ligand. The ligand B binding free energy to receptor protein A presents the variation between the complex mean free energy and that of its individual components., ΔGbind = 〈GAB〉 − 〈GA〉 − 〈GB〉 In the three-trajectory method, 〈GAB〉 can be determined from the bound complex simulation, while 〈GB〉 and〈GA〉 are obtained from the free ligand and unbound protein simulations, respectively. A one-trajectory approach is used, where 〈GA〉 and 〈GB〉 are computed from the complex simulation by sequentially removing the ligand and protein from the trajectory. The one-trajectory approach merely requires one bound complex simulation, allowing for the cancellation of entire intra-molecular energies, which reduces noise in the binding free energies. However, this approach does not capture the unbound protein and ligand dynamics, meaning that the entropic changes due to the restriction of conformational freedom following binding are not considered [ 34 ]. It also overlooks the potential that the unbound and bound protein and ligand can adopt various conformations. The binding affinities of the coumarin against the receptor of L929 are shown in Fig. 3 and Fig. 4 . The binding energies of the coumarin with the target protein receptor is -10.18 kcal/mole, which agrees with the experimental results. The molecular docking appraoch identified the preferable binding site and enhanced the understanding of the interplay between L929 and coumarin. In the free ligand, two significant interactions were noted (Fig. 4 ). One prominent interaction is the π-π stacking between the aromatic ring and Glu H46 (distance: 4.8 Å). Moreover, hydrophobic interactions were noted between the O2 of the ligand and the H2 of the Lys H43. Strong interactions were observed with several (four) amino acids in relation to the free ligand, leading to a binding energy of − 10.18 kcal/mole. 3.2. 1 H NMR analysis Figure 5 shows the 1 H NMR analysis of coumarin. The chemical shift of CDCl 3 was 7.21 ppm. The result shows that the chemical shifts at 7.23–7.70 ppm are related to the protons of the benzene ring (4H, Ar-H) of coumarin, and the chemical shifts at 6.36 ppm are related to the protons of the lactone ring (2H, = CH) of coumarin [ 35 , 36 ]. For the peak centers, the chemical shifts related to the middle points of the cluster and the type of coupling were performed and are as follows: A: δ7.68(d, 1H, Ar-H), B: δ 7.47(dd, 2H, Ar-H), C: δ 7.26(m, 1H, Ar-H) and D: δ 6.36 (d, 2H, =CH). According to the data obtained from the 1 HNMR spectrum, it is clear that pure coumarin was obtained from the extract of P. Ferulacea plant. 3.3. FT IR analysis Fig. 6a, 6b, and 6c show the FTIR spectrum of coumarin, nano-encapsulated coumarin, and electrospun nanofibrous scaffolds with nano-encapsulated coumarin, respectively. By comparing the FT-IR spectrum of coumarin and nano-encapsulated coumarin, the lower intensity of the OH functional group peak (3448 cm − 1 ) may be related to encapsulation. The higher intensity of the C-H aliphatic stretching vibration band (2928 cm − 1 ), and the transmittance band of C = O (1736 cm − 1 ) in Fig. 6b, can be related to the presence of the PCL carrier. By comparing the FT-IR patterns of nano-encapsulated coumarin and electrospun nanofibrous scaffold containing nano-encapsulated coumarin, it is obvious that the peak intensity of C = O (1647 cm − 1 ) has decreased due to the shell coverage of PCL with PVA and gelatin. In addition, an intermolecular hydrogen bonding interaction between the amide group of gelatins and the hydroxyl group of PVA is seen in a broad and robust peak of about 3426 cm − 1 . The stretching vibrations in the region of 1550 cm − 1 , 1250 cm − 1, and 1081 cm − 1 are also related to the N-H, C-N, and C-O groups respectively. CH 2 groups of gelatin and polyvinyl alcohol coatings can also be seen at 1448 cm − 1 [ 37 , 38 ]. The data obtained from the FT-IR spectrum exhibits the nanoencapsulation of coumarin and its recoating by the electrospinning method. 3.4. Ultraviolet (UV) analysis Fig. 7a, 7b, and 7c show the UV spectrum of coumarin, nano-encapsulated coumarin, and electrospun nanofibrous scaffold containing nano-encapsulated coumarin respectively. Chloroform was the agent used as a solvent in this process. According to Fig. 7a, The maximum absorption wavelength observed for Coumarin was in the range of 211, 214, and 216 nm, possibly associated with the π → π* transition [ 39 ]. As can be seen in Figs. 7a and 7b, it is concluded that some of the peak points at the maximum wavelength in Fig. 7b (nano-encapsulated coumarin) are the same as Fig. 7a (coumarin), which indicates the encapsulation of coumarin without any chemical interaction between them. As shown in Fig. 7c, most of the absorption peaks have disappeared, especially at higher wavelengths, which could be affiliated with the transfer of non-bonded electron pairs in the structure of coumarin and PCL, and this indicates the interactions of these electron pairs with the functional groups in the scaffold consisting of PVA and gelatin. 3.5. Scanning electron microscopy (SEM) analysis The morphology of nano-encapsulated coumarin and the electrospun nanofibrous scaffold containing 10% nano-encapsulated coumarin is shown in Fig. 8. The range of nanocapsules diameter containing coumarin (Fig. 8a) is between 13.45 and 18.54 nm. The structure of the electrospun nanofibrous scaffold (Fig. 8b-d) does not have any beads and the diameter range of them is from 181.58 to 250.57 nm. As can be seen in Fig. 8a, the coacervation method has made it possible to synthesize nanocapsules with almost the same size. Also, the use of water and acetic acid solvents to achieve homogeneity in the solution of PVA and GE mixture can facilitate the reduction of surface tension and so, promote enhanced evaporation between the needle tip and the electrospinning solution collector. Consequently, the network structure of the scaffold is well formed and the nanocapsules with drugs are regularly distributed. 3.6. High-resolution transmission electron microscopy (HRTEM) analysis HRTEM analysis, or high-resolution transmission electron microscopy, is one of the valuable tools for imaging the nanostructure of materials with the resolution of atomic distances [ 40 ]. Figure 9 illustrates the HRTEM, selected area diffraction pattern (SAED), and reduced Fast Fourier transform (FFT) patterns of the final product. As can be seen in the figures, the electrospun nanofibrous scaffold containing nano-encapsulated coumarin has a ploy crystalline structure, and the FFT spot patterns (Fig. 9 b and 9 d) confirm the crystal planes. Furthermore, SAED patterns can identify the phase mapping at the nanoscale. By analyzing the diffraction spots, we can deduce the polymeric carriers including the drug have been presented in different phases which can be related to their lack of interaction with each other, and this is one of the important factors for drug delivery. On the other hand, the symmetry observed by the reduced FFT technique and the uniform distribution of phases, lead to recognition of high homogeneity of the final product [ 41 ]. 3.7. The Porosity of electrospun nanofibrous scaffold containing 10% nano-encapsulated coumarin The porosity of the electrospun nanofibrous scaffold, which incorporated 10% nano-encapsulated coumarin, was measured to be approximately 92.8%. One significant benefit of electrospun nanofibrous scaffolds is their ability to create a network of interconnected pores, achieved through the overlapping arrangement of nanofibers. Hence, the utilization of the electrospinning approach to create a nanofibrous scaffold results in a structure that accurately mimics the intrinsic properties of the Extracellular Matrix (ECM), thereby enhancing its effectiveness as a scaffold in the field of tissue engineering. 3.8. Mechanical properties of the electrospun nanofibrous scaffold The evaluation of mechanical properties, such as tensile strength and elongation before structural breakdown, plays a significant role due to its potential application in medical interventions. The tensile strength of scaffolds can be influenced by various aspects, including porosity, polymer type, and fiber orientation. The porosity of the sample may lead to a decrease in its tensile strength [ 37 ]. Figure 10 diagram conveys the mechanical properties of an electrospun nanofibrous scaffold containing 10% drug and without drug. According to the results in Table 1 , it can be concluded that the no-drug sample has less tensile strength and elastic modulus than the drug-containing sample. So, the drug-containing sample has better resistance. Also, the percent elongation at break for the sample with the drug is less than the sample without the drug. It can be caused by poor interfacial adhesion between the PVA/GE nanofibers and PCL nanocapsules containing the drug, due to their polarity difference. Thus, the stress is not effectively transferred across the interface and leads to early failure or initiation of cracks at lower strains [ 42 , 43 ]. Table 1 Tensile test results of an electrospun nanofibrous scaffold containing 10% nano-encapsulated coumarin and without drug. Sample Tensile strength(MPa) Elongation at break(%) Elastic module as long of peak(MPa) Without drug 0.78 10.89 8.13 With drug 1.09 7.51 25.16 3.9. Differential scanning calorimetry (DSC) analysis Figure 11a-c shows the DSC thermograms of nano-encapsulated coumarin, electrospun nanofibrous scaffold without drug, and electrospun nanofibrous scaffold containing nano-encapsulated coumarin respectively. In all thermograms, the first endothermic peak has been observed at about 54°C (the dehydration temperature (T H )) due to the connection of water molecules with the hydrophilic groups of PVA and gelatin and nano-encapsulated coumarin. In Fig. 11c, two peaks were observed at 157.6 and 172°C (the degradation temperatures (T D )) [ 37 ]. This temperature range may reveal significant changes in the thermal properties of each of the polymers used. This phenomenon is potentially related to the physical interactions between PVA, gelatin, PCL, and coumarin (hydrogen bond or electrostatic attraction) [ 44 , 45 ]. On the other hand, the T D peak can also represent the thermal stability limit of the synthesized nanofibers containing nano-encapsulated coumarin. The results of the peak temperature and enthalpies of the samples are presented in Table 2 . Table 2 DSC results of a) nano-encapsulated coumarin b) electrospun nanofibrous scaffold without drug, and c) electrospun nanofibrous scaffold containing nano-encapsulated coumarin. Samples Peak Area Peak Area ---- T H (֯C) ∆H H (J/g) T D (֯C) ∆H D (J/g) a 61.8 -134.9 --- --- b 58.5 -24.62 --- --- c 43.6 -187.7 157.6 and 172 -8.124 and − 2.282 3.10. High-performance liquid chromatography (HPLC) analysis Figure 12 shows the calibration curve of pure coumarin, which was executed at a wavelength of 270 nm and a volumetric flow rate of 1 mL /min. The drug loading efficiency in the capsulation process was 87%, calculated from the following equation. EE(%) = Drug weight in nanocapsules/ Initial drug weight ×100 Figure 13 depicts the release profiles observed in electrospun nanofibrous scaffolds containing nano-encapsulated coumarin under acidic (pH = 4), neutral (pH = 7), and alkaline (pH = 9.1) conditions. The release rate is comparatively slower in a neutral environment when compared to acidic or alkaline conditions. The release operation was carried out for 72 hours. An acidic or alkaline environment can degrade polycaprolactone and gelatin and may result in an explosive drug release at the time of use. In a neutral environment, the possibility of drug release will increase approximately after 40 hours. In an alkaline environment, about 56% of coumarin is released in 30 minutes, but the drug delivery drops below 40% after 40 hours which can be related to the cross-linking between PVA in the alkaline condition. However, in all pHs, the release rate of the coumarin after 40 hours has been done with a suitable slope up to 72 hours, which is essential for the main goal of this work. Drug half-life plays a pivotal role in determining the appropriate dosing regimen and achieving the desired peak-to-trough ratio, and a half-life range of 12–48 hours is optimal for once-daily dosing. Consequently, half-life stands out as a critical parameter in the realm of research and development, and offering a pathway to enhance the efficacy of half-life optimization strategies is important [ 46 ]. Thus, our designed platform, regardless of the pHs targeting 50% drug delivery after a 40-hour timeframe (after the drug half-life and the lag phase of 24 h for L929 cells which cells adapt to the culture environment) [ 47 ], is structured to effectively achieve our goals. Putting the drug in the suitable coating (PCL) and placing it in the main framework (PVA/GE) has served our purpose. Figure 13 Release curves in acidic, neutral, and alkaline environments from electrospun nanofibrous Scaffold containing nano-encapsulated coumarin. 3.11. MTT assay The viability of L929 cells on electrospun nanofibrous scaffolds containing drug, and without drug, and the graph of cell control (TCP) at 1 day, 3 days, and 5 days is illustrated in Fig. 14 . The scaffolds produced had no harmful effects on the cells, and the cell viability is the higher in the electrospun samples containing drug, which increases the probability of cell proliferation and adhesion. In cell culture, proliferation is often described through different phases. The log phase is the main phase in which cells begin to proliferate exponentially. For L929 cells, under optimal conditions, this phase can last between 1 to 3 days [ 48 ]. So, the MTT test was carried on after complete drug release (3 days) up to 5 days. According to Table 3 , the survival and the proliferation of L929 cells on the electrospun nanofibrous scaffold containing coumarin is higher than TCP (tissue culture plate), and the sample without drug, even after the log phase (5 days) which is due to the persistence of the drug effect in the cellular environment. Table 3 The survival and the proliferation of L929 cells on the electrospun nanofibrous scaffold with drug and without drug on days 1, 3, and 5. Day 1 TCP (tissue culture plate) without drug with drug mean 2.405 2.528 2.634 cell viability 1 1.050 1.095 cell viability% 100 105 109.5 Day 3 TCP (tissue culture plate) without drug with drug mean 2.276 2.493 2.733 cell viability 1 1.095 1.200 cell viability% 100 109.5 120 Day 5 TCP (tissue culture plate) without drug with drug mean 1.083 1.245 1.410 cell viability 1 1.149 1.301 cell viability% 100 114.9 130.1 3.12. Morphological analysis of L929 cells Figure 15 shows the SEM images of L929 cells to study cell proliferation on nanofiber scaffolds with and without drugs on days 1, 3, and 5. The cell adhesion and proliferation were observed at the lowest proliferation on the first day and highest proliferation on the fifth day. Generally, the density of the L929 cell culture with DNA synthesis and mitosis is limited to 5% of the cells per day in high-density cultures [ 49 ]. As shown in Fig. 15 , L929 cell proliferation on the sample without drugs (a, c, and e) followed almost the same percentage of the reported density. However, nanofibrous scaffolds with coumarin (Figs. 15 b, 15 d, and 15 f) have been able to significantly increase cell proliferation and adhesion (more than twofold). This can be linked to the biological property of coumarin [ 25 , 50 , 51 ]. Since, cell proliferation is essential for tissue growth, and cell adhesion is necessary for the metabolic activities of cells, the designed drug delivery platform can be used for this application. 4. Conclusion In this research, the goal was to design a drug delivery with the purpose of tailored control, focusing on creating an ideal platform that can act like the body's natural ECM (extracellular matrix). Coumarin was used as an active herbal ingredient in this study, due to its strong pharmacological properties and beneficial effects on human health. Also, the strong binding interaction of it with L929 was confirmed by the molecular docking. The π-π interactions and hydrophobic interactions are an important force of binding interaction between coumarin and L929. Coumarin was simply extracted from the root of the p.ferulacea plant and the structure was characterized via 1 H NMR spectroscopy analysis. After that, biodegradable polycaprolactone polymer was used to encapsulate coumarin through the coacervation method which was confirmed by SEM analysis. The coacervation technique helped in making the spherical nanocapsules of almost equal size so that they could be easily distributed inside the scaffold fibers. A combination of polyvinyl alcohol and gelatin was used due to their hydrophilic properties to make electrospun nanofibrous scaffolds which can create suitable conditions for proliferation, adhesion, and cell function. In drug release, the release rate is relatively slower in a neutral environment compared to acidic or alkaline conditions. At all pHs, the release rate of coumarin after 40 hours (Lag phase) with a suitable slope up to 72 hours was performed, which is essential for the main purpose of this work and it is exactly according to the time required for L929 cell proliferation (Log phase). Therefore, placing the drug in a binary coating allowed for a fully controlled delivery. The drug loading efficiency was at 87%. The electrospun nanofibrous scaffolds containing the drug were then tested for porosity, tensile strength, and elongation at break, and the outcome was 92.8%, 1.09 MPa, and 7.51% respectively. Using SEM and HRTEM analysis, the nanofibrous scaffold from gelatin, and PVA with 10% nano-encapsulated coumarin was studied, and the images have shown the polycrystalline structure with high uniformity. Throughout the MTT test, no cytotoxicity was found, and the survival and the proliferation of L929 cells on the electrospun nanofibrous scaffold containing coumarin was higher than the sample without the drug, even after the 5 days, which is due to the persistence of the drug effect in the cellular environment. SEM analysis of L929 cells showed that nanofibrous scaffolds with coumarin have been able to significantly increase cell proliferation and adhesion, which could be related to the biological property of coumarin. Therefore, unlike in the past, we think it is possible to design the drug release system using a principled process that takes into account the time required for each cell line to go through two phases of adaptation and proliferation as well as the choice of appropriate two- or multi-phase platforms depending on the kind of drugs. In future studies, two or more medications might be embedded in distinct carriers to achieve sequential release, thereby preventing any interference among them. Declarations Author Contribution Rahebeh Amiri Dehkharghani made substantial contributions to the conception or design of the work and the interpretation of data and the writing the manuscript.Rojan Akhbarati has done the experimental section.Soheila Zamanlui Benisi helped in the biological part. References Pei J, Yan Y, Palanisamy CP, Jayaraman S, Natarajan PM, Umapathy VR, et al. Materials-based drug delivery approaches: Recent advances and future perspectives. 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Cite Share Download PDF Status: Published Journal Publication published 30 Nov, 2024 Read the published version in Journal of Polymers and the Environment → Version 1 posted Editorial decision: Revision requested 05 Nov, 2024 Reviews received at journal 01 Nov, 2024 Reviewers agreed at journal 25 Oct, 2024 Reviewers agreed at journal 23 Oct, 2024 Reviews received at journal 09 Oct, 2024 Reviewers agreed at journal 09 Oct, 2024 Reviewers agreed at journal 05 Oct, 2024 Reviewers agreed at journal 27 Sep, 2024 Reviewers agreed at journal 27 Sep, 2024 Reviewers invited by journal 26 Sep, 2024 Editor assigned by journal 20 Sep, 2024 Submission checks completed at journal 20 Sep, 2024 First submitted to journal 20 Sep, 2024 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. 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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-5122397","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":374406286,"identity":"c2f03544-c70a-46e1-8396-7217ec976497","order_by":0,"name":"Rojan Akhbarati","email":"","orcid":"","institution":"Islamic Azad University, Tehran","correspondingAuthor":false,"prefix":"","firstName":"Rojan","middleName":"","lastName":"Akhbarati","suffix":""},{"id":374406291,"identity":"5398d9d3-35d6-4e9b-96fc-72ce4d44dacf","order_by":1,"name":"Rahebeh Amiri Dehkharghani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYBAC+wYeBoYEBgkGNvbGBpBAAgOCxA4MDgC1fABp4TkI0mJAnBbGGSCWBFiZAT7FUC3Hzx5g5s2xyOeTfNz4mafmTx4D++EHDA/34PFLT14CM+82Ccs26cRmaZ5jBsUMPGkGDAnPcGuxY8gxAGkxYJNObJDmbTBIbGDIAfrlAG4txvxvDJj/grRIHmz+DdbC/wa/FsMZMFskGNsgtkgQsMXgxhuDw2AtPIltlnOOGRezSTwzOIBXy/kcw8e82+oM5NuPP77xpkYuj58/+eHDH3i0gACqNBuGyCgYBaNgFIwCkgEAX0ZL5aQkjYQAAAAASUVORK5CYII=","orcid":"","institution":"Islamic Azad University, Tehran","correspondingAuthor":true,"prefix":"","firstName":"Rahebeh","middleName":"Amiri","lastName":"Dehkharghani","suffix":""},{"id":374406292,"identity":"3c311881-d35f-42fe-9928-a14cb3971187","order_by":2,"name":"Soheila Zamanlui Benisi","email":"","orcid":"","institution":"Islamic Azad University, Tehran","correspondingAuthor":false,"prefix":"","firstName":"Soheila","middleName":"Zamanlui","lastName":"Benisi","suffix":""}],"badges":[],"createdAt":"2024-09-20 09:22:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5122397/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5122397/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10924-024-03458-4","type":"published","date":"2024-11-30T15:57:35+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68998326,"identity":"2a907f91-5027-4077-80cf-0e00a4762fd1","added_by":"auto","created_at":"2024-11-14 11:02:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":43113,"visible":true,"origin":"","legend":"\u003cp\u003eOptimized geometries of coumarin.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5122397/v1/6747ed00fdcf9b7ffc1ba6ea.png"},{"id":68998330,"identity":"0c5bc16d-c8d5-4839-8820-6a1f72d9e3ec","added_by":"auto","created_at":"2024-11-14 11:02:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":188917,"visible":true,"origin":"","legend":"\u003cp\u003ea) FMO analysis of coumarin and b) MESP analysis of coumarin.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5122397/v1/5c8d13ba32085f4fe0b17339.png"},{"id":68998950,"identity":"85dbfa42-4d9c-4553-b576-464c94aad1dc","added_by":"auto","created_at":"2024-11-14 11:10:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":204374,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking simulation studies of the interaction between the coumarin with the active site of the receptor of the mouse fibroblast L929. 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Hydrophobic interactions with amino acid residues are shown with dotted curves.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5122397/v1/c49867e9250d72623a3f0920.png"},{"id":68998328,"identity":"e3c9ecc0-da86-46d8-a220-d17d9ba5f526","added_by":"auto","created_at":"2024-11-14 11:02:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":79513,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH NMR analysis of coumarin\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5122397/v1/b0a53bc576a4a39e1d0f09c2.png"},{"id":68998954,"identity":"d2a28486-c073-46a3-9a77-323586881b76","added_by":"auto","created_at":"2024-11-14 11:10:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":200480,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR analysis of a) Coumarin, b) Nano-encapsulated coumarin, c) Electrospun nanofibrous scaffold containing nano-encapsulated coumarin\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5122397/v1/47c16e41e1033887b0406336.png"},{"id":68998332,"identity":"678554c9-a0d5-42ee-9d24-0189b67006c8","added_by":"auto","created_at":"2024-11-14 11:02:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":71001,"visible":true,"origin":"","legend":"\u003cp\u003eUV analysis of a) coumarin, b) nano-encapsulated coumarin, and c) electrospun nanofibrous scaffold containing nano-encapsulated coumarin.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5122397/v1/fdcbb76363484cdd3921dc58.png"},{"id":68998951,"identity":"cd2f876a-1969-42f7-a2dc-71b78092242f","added_by":"auto","created_at":"2024-11-14 11:10:49","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":649089,"visible":true,"origin":"","legend":"\u003cp\u003eSEM analysis of nano-encapsulated coumarin (a) and electrospun nanofibrous scaffold containing 10% nano-encapsulated coumarin (b, c, and d).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5122397/v1/dc9a1d0a80aea9b3716441eb.png"},{"id":68998334,"identity":"b0815ad8-e005-4077-95d9-8033557a6a96","added_by":"auto","created_at":"2024-11-14 11:02:49","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":705411,"visible":true,"origin":"","legend":"\u003cp\u003eHRTEM and SAED images of an electrospun nanofibrous scaffold containing nano-encapsulated coumarin (a and c) \u0026nbsp;and its reduced FFT (b and d).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5122397/v1/6030fb9ada09fdb48f35034c.png"},{"id":69000273,"identity":"6d9d6740-6fa8-4e1d-bd69-e4fecc92e153","added_by":"auto","created_at":"2024-11-14 11:26:50","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":93477,"visible":true,"origin":"","legend":"\u003cp\u003eThe mechanical properties of an electrospun nanofibrous scaffold containing 10% nano-encapsulated coumarin and without drug.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-5122397/v1/2ec5d3924b6f1850de4789a5.png"},{"id":68999114,"identity":"f71e795c-5d2c-493d-9cf0-2ac2ffd7c2b5","added_by":"auto","created_at":"2024-11-14 11:18:49","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":44534,"visible":true,"origin":"","legend":"\u003cp\u003eThe DSC thermograms of a)nano-encapsulated coumarin b) electrospun nanofibrous scaffold without drug and c) electrospun nanofibrous scaffold containing nano-encapsulated coumarin.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-5122397/v1/63fb4cbc102e445561e3864d.png"},{"id":68999116,"identity":"ca0fa7a1-8f0d-47c8-8f08-5dbe2f83ba93","added_by":"auto","created_at":"2024-11-14 11:18:49","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":50374,"visible":true,"origin":"","legend":"\u003cp\u003eThe calibration curve of coumarin\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-5122397/v1/9ad1620506023a44a3dad49e.png"},{"id":68998337,"identity":"d1c8db69-9cee-4c09-8a17-b9375bbb8df6","added_by":"auto","created_at":"2024-11-14 11:02:49","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":105629,"visible":true,"origin":"","legend":"\u003cp\u003eRelease curves in acidic, neutral, and alkaline environments from electrospun nanofibrous Scaffold containing nano-encapsulated coumarin.\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-5122397/v1/bb505b61199640bd5515c31c.png"},{"id":68998956,"identity":"309bfc0e-f11b-4f06-bd62-a007df67dc63","added_by":"auto","created_at":"2024-11-14 11:10:49","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":74487,"visible":true,"origin":"","legend":"\u003cp\u003eMTT test results on days 1, 3, and 5. The statistical analysis was conducted using SPSS software, employing a significance level of P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-5122397/v1/3ea947acca3a034493f3e96d.png"},{"id":68999117,"identity":"65358511-76a8-461a-9ce9-03e3f308bd1d","added_by":"auto","created_at":"2024-11-14 11:18:49","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":789617,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of L929 cells to study cell proliferation on nanofibrous scaffolds without drug ( a, c, and e) and with drug ( b, d, and f) on 1, 3, and 5 days respectively.\u003c/p\u003e","description":"","filename":"15.png","url":"https://assets-eu.researchsquare.com/files/rs-5122397/v1/610849cb3e4b14168ca89c40.png"},{"id":70382635,"identity":"29c874ea-cc6b-48d6-878f-8aabe297b642","added_by":"auto","created_at":"2024-12-02 16:28:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4311148,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5122397/v1/9737fdf4-0dbf-4cb8-982a-3a454b51551f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eDesign a coordinated platform for coumarin-regulated delivery in line with the biological systems' growth phases\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe development of multifunctional drug delivery systems (DDSs) for therapeutic and \u0026lrm;diagnostic purposes presents the potential for simultaneous disease diagnosis and treatment [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Developing an effective therapeutic delivery system targets specific cellular or tissue \u0026lrm;entities in the human body to enhance the drug's therapeutic effects, minimize its side \u0026lrm;effects, and enhance its physicochemical properties [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In recent years, a significant focus has been on investigating a range of controlled release systems, including nanoparticles, micelles, hydrogels, and electrospun membranes [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Also, the utilization of nanostructures such as nanofibers and nanocapsules, has demonstrated notable efficacy in the fields of tissue engineering and controlled drug release [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This can be attributed to their heightened surface-to-volume ratio, appropriate size, porous permeability, and convenient carriers for drug delivery [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Different compounds are used for the synthesis of these nanostructures which can include biodegradable polymers. Polyvinyl alcohol (PVA), gelatin, and polycaprolactone (PCL) are \u0026lrm;also utilized in several biomedical applications, including wound healing, medication delivery, \u0026lrm;and tissue engineering scaffolds [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Among the techniques that might be used for the synthesis of nanofibers and nanocapsules, the electrospinning and coacervation methods can be mentioned. Electrospinning is a robust technological process that facilitates the production of nanofibrous \u0026lrm;membranes through the application of electric current across various polymer materials [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This process has demonstrated its efficacy in transforming a wide range of polymers into nanofibers, encompassing both natural and synthetic polymers, as well as hybrid polymer materials [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The coacervation technique can be used to load drugs in polymeric nanocapsules. The method involves the utilization of a coacervating compound like a desolvating agent (such as nonsolvent, pH alteration, temperature, or electrolyte) or a crosslinking agent like glutaraldehyde, to segregate the hydrocolloid from the initial solution. This technique is also employed to produce a consistent colloidal system that diminishes the average particle size (on a nanometer scale) and enhances the loading capacity with minimal coagulation and uniform distribution of nanoparticles [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Coumarin is a colorless and odorless crystalline substance that belongs to the benzopyrone chemical class. It is found naturally in many plants, mainly in the form of phenol. Coumarins are an important class of secondary metabolites that are widely found in several parts of plants. Ground plants such as Tonka bean and vanilla plants have high concentrations of coumarin. Among the biological and medicinal activities of coumarin and its derivatives, we can mention anticoagulant, antitumor, antibacterial, antifungal, anti-inflammatory, anti-HIV, and antioxidant [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on our continued effort with the purpose of skin tissue engineering, the present study involved the production of a coordinate scaffold composed of PVA/gelatin, and PCL nanocasules containing coumarin. To have the relative release of the active ingredient during the phase of cell matching to the culture medium (Lag phase), we employed an external polar coating for cell proliferation, taking into consideration the time needed for the Lag phase of L929 cell line. However, to ensure that the drug release would continue until the second stage of replication (Log phase), we employed an additional non-polar coating to encapsulate the coumarin.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials and apparatus\u003c/h2\u003e \u003cp\u003ePolycaprolactone (PCL, Mw of 80000 g/mol), polyvinyl alcohol (PVA, Mw of 31000\u0026ndash;50000 g/mol, 99% hydrolyzed), gelatin (type A), and 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich, USA. Span 80, tween 80, sunflower oil, lactic acid, acetone, acetic acid, methanol, acetonitrile, glutaraldehyde solution (2.5%), and ethanol were purchased from Merck, Germany. Sodium citrate dihydrate, acid citric, sodium bicarbonate, and sodium carbonate, which were used as buffers, were also purchased from Merck, Germany. Mouse fibroblasts (L929 Cells) were obtained from Geniran, Iran. Dulbecco's Modification of Eagle's Medium (DMEM), penicillin/streptomycin, and trypsin-(EDTA) were obtained from Bioidea, Iran. Fetal bovine serum (FBS) was obtained from Gibco, USA.\u003c/p\u003e \u003cp\u003eA KBr disk technique (Perkin Elmer Spectrum ASCII, USA) with a range of 4000 to 400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was used to perform the FT-IR spectra. The assessment of the product was conducted within a wavelength range of 200 to 400 nm using a UV\u0026ndash;Vis spectrophotometer (Perkin Elmer/ Lambda 25 UV/Vis spectrophotometer, USA). The morphology of the nano-encapsulated coumarin and the electrospun nanofibrous scaffold was investigated using a Field Emission Scanning Electron Microscope (FE-SEM, MIRA3 TESCAN, Brno, Czech Republic). The morphology of the electrospun nanofibrous scaffold after cell culture was investigated using a Field Emission Scanning Electron Microscope (FE-SEM, SIGMA VP-500, ZEISS, Germany). The particle size was determined using high-resolution transmission electron microscopy (HRTEM, FEI TECNAI G2 F20 S-TWIN TEM, USA). The controlled drug release was evaluated using high-performance liquid chromatography (HPLC, SCL-10AVP, LC-20AD, DGU-20A, SPD-20A, SHIMADZU, Japan). The ELISA plate reader (Biotek Epoch Microplate Spectrophotometer, USA) was utilized to examine the plates by measuring the absorbance at a wavelength of 570 nm. The \u003csup\u003e1\u003c/sup\u003eHNMR spectra were performed by Bruker AVANCE AQS-300MHZ using CDCl\u003csub\u003e3\u003c/sub\u003e as an NMR solvent. The thermal characteristics of nano-encapsulated coumarin, and electrospun nanofibrous scaffold were investigated using a differential scanning calorimetry (DSC, NETZSCH DSC 214 Polyma, Germany). A study was carried out to create a nanofibrous scaffold through the electrospinning technique, employing a horizontal setup with a cylindrical collector wrapped in aluminum foil (Co881007NYI, ANSTCO, Iran). The mechanical characteristics were assessed utilizing a mechanical analyzer (SANTAM, STM-20, Iran).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Computational method\u003c/h2\u003e \u003cp\u003eDFT (B3LYP) optimized the coumarin ligand geometries, which was visualized using Gauss View 6 software. The 6-311\u0026thinsp;+\u0026thinsp;G(d) basis set assessed the nonmetallic atoms. Frequency was calculated on the optimum geometry to confirm that it represented a global minimum, indicated by the absence of imaginary frequencies. The molecular electrostatic surface potential (MESP) and frontier molecular orbitals (FMO) were calculated based on the optimum structures. The Gaussian-09 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] software conducted the calculations, and the Gauss View 6 created visual representations. Molecular Operating Environment (MOE) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] software was used for coumarin into the L929 structures docking calculations that the 3D structure of it was taken from the site (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The binding pocket size for L929 was determined to be 40 \u0026times; 40 \u0026times; 40 points. The docking parameter \u0026ldquo;exhaustiveness\u0026rdquo; was set to 10, and a grid spacing of 0.375 \u0026Aring; calculated the energetic map for the docking study. The root mean square deviation (RMSD) values validated the molecular docking protocol. During the validation study, the ligand was treated as flexible while the protein remained rigid.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Preparation of coumarin herbal extract\u003c/h2\u003e \u003cp\u003eThe fresh roots of p.ferulacea were collected from Yasouj, Iran, and further pulverized into powder. The extraction by maceration method was performed with 0.5 g of dried roots of p. ferulacea and 10 ml of methanol solvent. The mixture was left at room temperature for 72 hours [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The extracted material was then placed in an ultrasonic bath and passed through a filter. After purification, \u003csup\u003e1\u003c/sup\u003eH NMR analysis was carried out on the sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Coacervation method to prepare nano-encapsulated coumarin\u003c/h2\u003e \u003cp\u003eThe coacervation technique was used to prepare nanocapsules and implemented by using polycaprolactone. An adequate quantity of PCL/acetone (0.25 g/67 mL) was combined, and the amalgam was sealed with foil before being positioned in an ultrasonic bath at a 40\u0026deg;C temperature. Subsequently, 0.8 mL of sunflower oil (SFO) was blended with 0.196 mL of Span 80 (water-soluble), and the mixture was introduced to the PCL/acetone from the previous step utilizing a syringe, and positioned on a magnetic stirrer. In the subsequent stage, coumarin (0.0114 g) was introduced in a nitrogen environment and covered with foil. To obtain a homogeneous mixture, it was placed under magnetic stirring for 2 hours. Lactic acid solution (coacervating solution) was incrementally added in an ultrasonic bath until the pH level dropped below the isoelectric point (4.0). The resulting colloid was placed in a water bath and swiftly cooled to 15\u0026deg;C to enhance the formation of nanocapsules while being stirred at 300 rpm for stabilization of the colloid. The nanocapsules were then filtered and dried utilizing a freeze-dryer for 72 hours at -70\u0026deg;C. The samples were then analyzed by SEM, FTIR, DSC, and UV techniques.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Preparation of nanofibrous scaffold\u003c/h2\u003e \u003cp\u003eInitially, the solutions of gelatin (9% w/v) and PVA (9% w/v) were prepared in acetic acid under gentle stirring at 35\u0026ndash;40\u0026deg;C for 1h and in distilled water with continuous stirring at 90\u0026deg;C for 2h, respectively. Both solutions were mixed afterward with 5%, 10%, and 15% nano-encapsulated coumarin, added at independent instances. The solutions that were prepared were introduced into a 5 ml syringe and then subjected to electrospinning at ambient temperature. This process was carried out using a horizontal setup equipped with a cylindrical collector that was coated with aluminum foil (Co881007NYI, ANSTCO, Iran). The conditions of electrospinning and the amount of drug were optimized, and the samples were checked with a microscope to have the least bead density. The distance (between the tip of the needle and the collector) was set to 170 mm, the voltage was set to 27kV, the flow rate was 0.6 ml/h, and the amount of nano-encapsulated coumarin at 10% (due to the drug release control). The nanofiber scaffolds were finally examined using SEM and FT-IR, UV, DSC, mechanical test, measurement of porosity, and HR-TEM analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Mechanical properties\u003c/h2\u003e \u003cp\u003eThe mechanical characteristics were assessed through the utilization of a mechanical analyzer (SANTAM, STM-20, Iran). The samples consisted of rectangular discs (30\u0026times;10 mm\u003csup\u003e2\u003c/sup\u003e) with a scaffold thickness of 0.134 mm. These specimens underwent a consistent tensile deformation rate of 5 mm/min under dry ambient conditions at room temperature. The fracture stress and elongation at failure were subsequently quantified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Differential scanning calorimetry (DSC)\u003c/h2\u003e \u003cp\u003eThe thermal characteristics of nano-encapsulated coumarin and electrospun nanofibrous scaffold were investigated by a DSC analyzer. The samples were then subjected to a scanning velocity of 10\u0026deg;C /min, along with a gas stream of nitrogen at a rate of 50 mL/min, within a temperature range of 0 to 300\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Porosity\u003c/h2\u003e \u003cp\u003eThe liquid displacement technique [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] was employed to determine the porosity of nanofiber scaffolds. The choice of ethanol as the displacing solution was made to prevent any modification in the polymeric matrix. This selection was based on ethanol's ability to enter the nanofiber scaffolds without inducing any swelling or shrinking effects. The scaffolds (dry weight, w\u003csub\u003ed\u003c/sub\u003e) were submerged in ethanol for 30 minutes and the immersed scaffold weights were recorded as w\u003csub\u003el\u003c/sub\u003e afterward. The filtration process was conducted after the extraction of the substances from the ethanol medium. The weight of moist scaffolds was then quantified as w\u003csub\u003ew\u003c/sub\u003e:\u003c/p\u003e \u003cp\u003ePorosity (%) = (W\u003csub\u003ew\u003c/sub\u003e-W\u003csub\u003ed\u003c/sub\u003e)/ (W\u003csub\u003ew\u003c/sub\u003e-W\u003csub\u003el\u003c/sub\u003e) \u0026times; 100\u003c/p\u003e \u003cp\u003eThe values are the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Drug release\u003c/h2\u003e \u003cp\u003eHPLC analysis was performed to evaluate the coumarin release rate from the electrospun sample containing nanocapsules. Standard solutions of coumarin were made to measure the peak of the desired drug and the inhibition time of the drug. Drug release was evaluated at different times in acidic, alkaline, and neutral environments. The HPLC device was set with a washing solvent of 60% acetonitrile and 40% water, an optical measurement was conducted, using a wavelength of 270 nm and a liquid flow rate of 1 mL/min. For the evaluation of drug release in a different environment, the electrospun sample (0.005 g) was dissolved in 50 mL of distilled water (pH\u0026thinsp;=\u0026thinsp;7) under a neutral environment. Sodium citrate dihydrate (0.496 g) was dissolved in 40 ml of distilled water (40 ml). Acid citric (0.636 g) was then dissolved in distilled water (10 mL) and 5 mg of the electrospun sample at (pH\u0026thinsp;=\u0026thinsp;4) in an acidic environment. Sodium bicarbonate (0.382 g) was dissolved in 40 ml of distilled water (40 ml). Next, sodium carbonate (0.047 g) was dissolved in distilled water (10 mL) and 5 mg of the electrospun sample (pH\u0026thinsp;=\u0026thinsp;9.1) in an alkaline environment. Then, it was placed in a shaker incubator at 37 ℃. The release operation was performed for 72 hours. Then, the samples were injected into the HPLC device within 6 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. MTT assay\u003c/h2\u003e \u003cp\u003eThe viability of L929 cells (the mouse fibroblast cell line) was assessed on the created electrospun nanofibrous scaffold using the MTT assay. The cells were seeded onto sterilized scaffolds in a 96-well culture plate at an initial density of 5000 cells/cm\u003csup\u003e2\u003c/sup\u003e. The cells were subjected to treatment with 100 \u0026micro;l of MTT solution (500 \u0026micro;g/ml) on days 1, 3, and 5. This treatment was carried out for 3.5 hours following the removal of the cell culture media. Subsequently, an ELISA plate reader was utilized to examine the plates by measuring the absorbance at a wavelength of 570 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Cell culture\u003c/h2\u003e \u003cp\u003eThe purified L929 cells (Mouse fibroblast cell line) were grown in T25 flasks [DMEM supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin)]. The cells were treated with trypsin\u0026ndash;EDTA solution (GIBCO) and centrifuged after being cleaned with PBS to facilitate cell transit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Morphological analysis\u003c/h2\u003e \u003cp\u003eScanning electron microscopy (SEM) was used to analyze the surface morphology of aligned scaffolds before and after cell culture. After PBS washing, the scaffolds were seeded with cells in 2.5% glutaraldehyde within 40 min. As the alcohol concentration increased, the scaffolds underwent dehydration. The samples were then sputter-coated with gold after being allowed to dry at room temperature.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Statistical analysis\u003c/h2\u003e \u003cp\u003eTo conduct a statistical analysis of the experiment, and determine the variability in scores between samples, the one-way analysis of variance (ANOVA) was used. Data was subsequently analyzed using SPSS software. Statistics were judged significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cstrong\u003e3.1. Docking results\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e has been shown the optimized structure of coumarin. Several significant bond distances, including C-C and C-O, were highlited in the structure.\u003c/p\u003e\n \u003cp\u003eFor the global minimum identification on the potential energy surface, the frequency was calculated finding no imaginary frequencies. The stretching frequencies of the corresponding functional groups were validated against experimental data. The FMO analysis revealed that the energy gap between the LUMO and HOMO for the ligand was \u0026minus;\u0026thinsp;4.61 eV.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea shows their schematic diagram. The HOMO and LUMO orbital energies are highly stabilized, suggesting that coumarin is a highly reactive ligand. The electrostatic potential mapping considering the overall electron density surface, offers a real-time depiction of the distribution of electrostatic potential (taking into account both electrons and nuclei), as well as the molecular morphology, size, and the molecule dipole moments. This visual representation can assess relative polarity. Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb displays the MESP for coumarin. The MESP surface, depicted with blue, red, green and yellow regions, highlights areas that are electron-deficient, electron-rich, neutral and slightly electron-deficient, respectively. The area surrounding the oxygen group has the most negative potential (red), whereas the area near the carbon group has a more positive charge. Additionally, light blue and green regions on the MESP surfaces of coumarin are also observed [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003ePrecise anticipation of protein-ligand binding free energies through practical computational approaches is capable of revolutionizing drug identification. Using protein-ligand interaction modeling with quantum mechanical approaches, rather than relying on empirical classical mechanics approaches this objective is achieved. The 6-311\u0026thinsp;+\u0026thinsp;G(d) method was explored to determine the binding free energies of coumarin as the ligand to the L929 cell line, utilizing linear-scaling density functional theory for the complex containing protein and ligand. The ligand B binding free energy to receptor protein A presents the variation between the complex mean free energy and that of its individual components.,\u003c/p\u003e\n \u003cp\u003e\u0026Delta;Gbind = 〈GAB〉 \u0026minus; 〈GA〉 \u0026minus; 〈GB〉\u003c/p\u003e\n \u003cp\u003eIn the three-trajectory method, 〈GAB〉 can be determined from the bound complex simulation, while 〈GB〉 and〈GA〉 are obtained from the free ligand and unbound protein simulations, respectively. A one-trajectory approach is used, where 〈GA〉 and 〈GB〉 are computed from the complex simulation by sequentially removing the ligand and protein from the trajectory. The one-trajectory approach merely requires one bound complex simulation, allowing for the cancellation of entire intra-molecular energies, which reduces noise in the binding free energies. However, this approach does not capture the unbound protein and ligand dynamics, meaning that the entropic changes due to the restriction of conformational freedom following binding are not considered [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. It also overlooks the potential that the unbound and bound protein and ligand can adopt various conformations.\u003c/p\u003e\n \u003cp\u003eThe binding affinities of the coumarin against the receptor of L929 are shown in Fig. 3 and Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. The binding energies of the coumarin with the target protein receptor is -10.18 kcal/mole, which agrees with the experimental results.\u003c/p\u003e\n \u003cp\u003eThe molecular docking appraoch identified the preferable binding site and enhanced the understanding of the interplay between L929 and coumarin. In the free ligand, two significant interactions were noted (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). One prominent interaction is the \u0026pi;-\u0026pi; stacking between the aromatic ring and Glu H46 (distance: 4.8 \u0026Aring;). Moreover, hydrophobic interactions were noted between the O2 of the ligand and the H2 of the Lys H43. Strong interactions were observed with several (four) amino acids in relation to the free ligand, leading to a binding energy of \u0026minus;\u0026thinsp;10.18 kcal/mole.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cstrong\u003e3.2.\u003c/strong\u003e \u003csup\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eH NMR analysis\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e shows the \u003csup\u003e1\u003c/sup\u003eH NMR analysis of coumarin. The chemical shift of CDCl\u003csub\u003e3\u003c/sub\u003e was 7.21 ppm. The result shows that the chemical shifts at 7.23\u0026ndash;7.70 ppm are related to the protons of the benzene ring (4H, Ar-H) of coumarin, and the chemical shifts at 6.36 ppm are related to the protons of the lactone ring (2H, = CH) of coumarin [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]. For the peak centers, the chemical shifts related to the middle points of the cluster and the type of coupling were performed and are as follows:\u003c/p\u003e\n \u003cp\u003eA: \u0026delta;7.68(d, 1H, Ar-H), B: \u0026delta; 7.47(dd, 2H, Ar-H), C: \u0026delta; 7.26(m, 1H, Ar-H) and D: \u0026delta; 6.36 (d, 2H, =CH). According to the data obtained from the \u003csup\u003e1\u003c/sup\u003eHNMR spectrum, it is clear that pure coumarin was obtained from the extract of P. Ferulacea plant.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. FT IR analysis\u003c/h2\u003e\n \u003cp\u003eFig.\u0026nbsp;6a, 6b, and 6c show the FTIR spectrum of coumarin, nano-encapsulated coumarin, and electrospun nanofibrous scaffolds with nano-encapsulated coumarin, respectively. By comparing the FT-IR spectrum of coumarin and nano-encapsulated coumarin, the lower intensity of the OH functional group peak (3448 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) may be related to encapsulation. The higher intensity of the C-H aliphatic stretching vibration band (2928 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and the transmittance band of C\u0026thinsp;=\u0026thinsp;O (1736 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in Fig.\u0026nbsp;6b, can be related to the presence of the PCL carrier. By comparing the FT-IR patterns of nano-encapsulated coumarin and electrospun nanofibrous scaffold containing nano-encapsulated coumarin, it is obvious that the peak intensity of C\u0026thinsp;=\u0026thinsp;O (1647 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) has decreased due to the shell coverage of PCL with PVA and gelatin. In addition, an intermolecular hydrogen bonding interaction between the amide group of gelatins and the hydroxyl group of PVA is seen in a broad and robust peak of about 3426 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The stretching vibrations in the region of 1550 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1250 cm\u003csup\u003e\u0026minus;\u0026thinsp;1,\u003c/sup\u003e and 1081 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are also related to the N-H, C-N, and C-O groups respectively. CH\u003csub\u003e2\u003c/sub\u003e groups of gelatin and polyvinyl alcohol coatings can also be seen at 1448 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]. The data obtained from the FT-IR spectrum exhibits the nanoencapsulation of coumarin and its recoating by the electrospinning method.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. Ultraviolet (UV) analysis\u003c/h2\u003e\n \u003cp\u003eFig.\u0026nbsp;7a, 7b, and 7c show the UV spectrum of coumarin, nano-encapsulated coumarin, and electrospun nanofibrous scaffold containing nano-encapsulated coumarin respectively. Chloroform was the agent used as a solvent in this process. According to Fig.\u0026nbsp;7a, The maximum absorption wavelength observed for Coumarin was in the range of 211, 214, and 216 nm, possibly associated with the \u0026pi; \u0026rarr; \u0026pi;* transition [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. As can be seen in Figs. 7a and 7b, it is concluded that some of the peak points at the maximum wavelength in Fig. 7b (nano-encapsulated coumarin) are the same as Fig. 7a (coumarin), which indicates the encapsulation of coumarin without any chemical interaction between them. As shown in Fig. 7c, most of the absorption peaks have disappeared, especially at higher wavelengths, which could be affiliated with the transfer of non-bonded electron pairs in the structure of coumarin and PCL, and this indicates the interactions of these electron pairs with the functional groups in the scaffold consisting of PVA and gelatin.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5. Scanning electron microscopy (SEM) analysis\u003c/h2\u003e\n \u003cp\u003eThe morphology of nano-encapsulated coumarin and the electrospun nanofibrous scaffold containing 10% nano-encapsulated coumarin is shown in Fig. 8. The range of nanocapsules diameter containing coumarin (Fig. 8a) is between 13.45 and 18.54 nm. The structure of the electrospun nanofibrous scaffold (Fig. 8b-d) does not have any beads and the diameter range of them is from 181.58 to 250.57 nm. As can be seen in Fig. 8a, the coacervation method has made it possible to synthesize nanocapsules with almost the same size. Also, the use of water and acetic acid solvents to achieve homogeneity in the solution of PVA and GE mixture can facilitate the reduction of surface tension and so, promote enhanced evaporation between the needle tip and the electrospinning solution collector. Consequently, the network structure of the scaffold is well formed and the nanocapsules with drugs are regularly distributed.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6. High-resolution transmission electron microscopy (HRTEM) analysis\u003c/h2\u003e\n \u003cp\u003eHRTEM analysis, or high-resolution transmission electron microscopy, is one of the valuable tools for imaging the nanostructure of materials with the resolution of atomic distances [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. Figure \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e illustrates the HRTEM, selected area diffraction pattern (SAED), and reduced Fast Fourier transform (FFT) patterns of the final product.\u003c/p\u003e\n \u003cp\u003eAs can be seen in the figures, the electrospun nanofibrous scaffold containing nano-encapsulated coumarin has a ploy crystalline structure, and the FFT spot patterns (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eb and \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003ed) confirm the crystal planes. Furthermore, SAED patterns can identify the phase mapping at the nanoscale. By analyzing the diffraction spots, we can deduce the polymeric carriers including the drug have been presented in different phases which can be related to their lack of interaction with each other, and this is one of the important factors for drug delivery. On the other hand, the symmetry observed by the reduced FFT technique and the uniform distribution of phases, lead to recognition of high homogeneity of the final product [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7. The Porosity of electrospun nanofibrous scaffold containing 10% nano-encapsulated coumarin\u003c/h2\u003e\n \u003cp\u003eThe porosity of the electrospun nanofibrous scaffold, which incorporated 10% nano-encapsulated coumarin, was measured to be approximately 92.8%. One significant benefit of electrospun nanofibrous scaffolds is their ability to create a network of interconnected pores, achieved through the overlapping arrangement of nanofibers. Hence, the utilization of the electrospinning approach to create a nanofibrous scaffold results in a structure that accurately mimics the intrinsic properties of the Extracellular Matrix (ECM), thereby enhancing its effectiveness as a scaffold in the field of tissue engineering.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003e3.8. Mechanical properties of the electrospun nanofibrous scaffold\u003c/h2\u003e\n \u003cp\u003eThe evaluation of mechanical properties, such as tensile strength and elongation before structural breakdown, plays a significant role due to its potential application in medical interventions. The tensile strength of scaffolds can be influenced by various aspects, including porosity, polymer type, and fiber orientation. The porosity of the sample may lead to a decrease in its tensile strength [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. Figure \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e diagram conveys the mechanical properties of an electrospun nanofibrous scaffold containing 10% drug and without drug. According to the results in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, it can be concluded that the no-drug sample has less tensile strength and elastic modulus than the drug-containing sample. So, the drug-containing sample has better resistance. Also, the percent elongation at break for the sample with the drug is less than the sample without the drug. It can be caused by poor interfacial adhesion between the PVA/GE nanofibers and PCL nanocapsules containing the drug, due to their polarity difference. Thus, the stress is not effectively transferred across the interface and leads to early failure or initiation of cracks at lower strains [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e,\u0026nbsp;\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eTensile test results of an electrospun nanofibrous scaffold containing 10% nano-encapsulated coumarin and without drug.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTensile strength(MPa)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eElongation at break(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eElastic module as long of peak(MPa)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eWithout drug\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.78\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e10.89\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e8.13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eWith drug\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.09\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.51\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e25.16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\n \u003ch2\u003e3.9. Differential scanning calorimetry (DSC) analysis\u003c/h2\u003e\n \u003cp\u003eFigure 11a-c shows the DSC thermograms of nano-encapsulated coumarin, electrospun nanofibrous scaffold without drug, and electrospun nanofibrous scaffold containing nano-encapsulated coumarin respectively. In all thermograms, the first endothermic peak has been observed at about 54\u0026deg;C (the dehydration temperature (T\u003csub\u003eH\u003c/sub\u003e)) due to the connection of water molecules with the hydrophilic groups of PVA and gelatin and nano-encapsulated coumarin. In Fig.\u0026nbsp;11c, two peaks were observed at 157.6 and 172\u0026deg;C (the degradation temperatures (T\u003csub\u003eD\u003c/sub\u003e)) [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. This temperature range may reveal significant changes in the thermal properties of each of the polymers used. This phenomenon is potentially related to the physical interactions between PVA, gelatin, PCL, and coumarin (hydrogen bond or electrostatic attraction) [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]. On the other hand, the T\u003csub\u003eD\u003c/sub\u003e peak can also represent the thermal stability limit of the synthesized nanofibers containing nano-encapsulated coumarin. The results of the peak temperature and enthalpies of the samples are presented in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDSC results of a) nano-encapsulated coumarin b) electrospun nanofibrous scaffold without drug, and c) electrospun nanofibrous scaffold containing nano-encapsulated coumarin.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSamples\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePeak\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eArea\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePeak\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eArea\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e----\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003c/sub\u003e (֯C)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e∆H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(J/g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003c/sub\u003e (֯C)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e∆H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(J/g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e61.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-134.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e---\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e---\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e58.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-24.62\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e---\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e---\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e43.6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-187.7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e157.6 and 172\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-8.124 and \u0026minus;\u0026thinsp;2.282\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\n \u003ch2\u003e3.10. High-performance liquid chromatography (HPLC) analysis\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e shows the calibration curve of pure coumarin, which was executed at a wavelength of 270 nm and a volumetric flow rate of 1 mL /min. The drug loading efficiency in the capsulation process was 87%, calculated from the following equation.\u003c/p\u003e\n \u003cp\u003eEE(%)\u0026thinsp;=\u0026thinsp;Drug weight in nanocapsules/ Initial drug weight \u0026times;100\u003c/p\u003e\n \u003cp\u003eFigure\u0026nbsp;13 depicts the release profiles observed in electrospun nanofibrous scaffolds containing nano-encapsulated coumarin under acidic (pH\u0026thinsp;=\u0026thinsp;4), neutral (pH\u0026thinsp;=\u0026thinsp;7), and alkaline (pH\u0026thinsp;=\u0026thinsp;9.1) conditions. The release rate is comparatively slower in a neutral environment when compared to acidic or alkaline conditions. The release operation was carried out for 72 hours. An acidic or alkaline environment can degrade polycaprolactone and gelatin and may result in an explosive drug release at the time of use. In a neutral environment, the possibility of drug release will increase approximately after 40 hours. In an alkaline environment, about 56% of coumarin is released in 30 minutes, but the drug delivery drops below 40% after 40 hours which can be related to the cross-linking between PVA in the alkaline condition. However, in all pHs, the release rate of the coumarin after 40 hours has been done with a suitable slope up to 72 hours, which is essential for the main goal of this work. Drug half-life plays a pivotal role in determining the appropriate dosing regimen and achieving the desired peak-to-trough ratio, and a half-life range of 12\u0026ndash;48 hours is optimal for once-daily dosing. Consequently, half-life stands out as a critical parameter in the realm of research and development, and offering a pathway to enhance the efficacy of half-life optimization strategies is important [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]. Thus, our designed platform, regardless of the pHs targeting 50% drug delivery after a 40-hour timeframe (after the drug half-life and the lag phase of 24 h for L929 cells which cells adapt to the culture environment) [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e], is structured to effectively achieve our goals. Putting the drug in the suitable coating (PCL) and placing it in the main framework (PVA/GE) has served our purpose.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFigure\u0026nbsp;13\u003c/strong\u003e Release curves in acidic, neutral, and alkaline environments from electrospun nanofibrous Scaffold containing nano-encapsulated coumarin.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\n \u003ch2\u003e3.11. MTT assay\u003c/h2\u003e\n \u003cp\u003eThe viability of L929 cells on electrospun nanofibrous scaffolds containing drug, and without drug, and the graph of cell control (TCP) at 1 day, 3 days, and 5 days is illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003e. The scaffolds produced had no harmful effects on the cells, and the cell viability is the higher in the electrospun samples containing drug, which increases the probability of cell proliferation and adhesion. In cell culture, proliferation is often described through different phases. The log phase is the main phase in which cells begin to proliferate exponentially. For L929 cells, under optimal conditions, this phase can last between 1 to 3 days [\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e]. So, the MTT test was carried on after complete drug release (3 days) up to 5 days. According to Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, the survival and the proliferation of L929 cells on the electrospun nanofibrous scaffold containing coumarin is higher than TCP (tissue culture plate), and the sample without drug, even after the log phase (5 days) which is due to the persistence of the drug effect in the cellular environment.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe survival and the proliferation of L929 cells on the electrospun nanofibrous scaffold with drug and without drug on days 1, 3, and 5.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTCP\u003c/p\u003e\n \u003cp\u003e(tissue culture plate)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ewithout drug\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ewith drug\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003emean\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e2.405\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e2.528\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e2.634\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ecell viability\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e1.050\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e1.095\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ecell viability%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e100\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e105\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e109.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eDay 3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTCP\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(tissue culture plate)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ewithout drug\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ewith drug\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003emean\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.276\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.493\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.733\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ecell viability\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.095\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.200\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ecell viability%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e100\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e109.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e120\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eDay 5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTCP\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(tissue culture plate)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ewithout drug\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ewith drug\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003emean\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.083\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.245\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.410\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ecell viability\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.149\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.301\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ecell viability%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e100\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e114.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e130.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\n \u003ch2\u003e3.12. Morphological analysis of L929 cells\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e15\u003c/span\u003e shows the SEM images of L929 cells to study cell proliferation on nanofiber scaffolds with and without drugs on days 1, 3, and 5. The cell adhesion and proliferation were observed at the lowest proliferation on the first day and highest proliferation on the fifth day. Generally, the density of the L929 cell culture with DNA synthesis and mitosis is limited to 5% of the cells per day in high-density cultures [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e]. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e15\u003c/span\u003e, L929 cell proliferation on the sample without drugs (a, c, and e) followed almost the same percentage of the reported density. However, nanofibrous scaffolds with coumarin (Figs. \u003cspan class=\"InternalRef\"\u003e15\u003c/span\u003eb, \u003cspan class=\"InternalRef\"\u003e15\u003c/span\u003ed, and \u003cspan class=\"InternalRef\"\u003e15\u003c/span\u003ef) have been able to significantly increase cell proliferation and adhesion (more than twofold). This can be linked to the biological property of coumarin [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e]. Since, cell proliferation is essential for tissue growth, and cell adhesion is necessary for the metabolic activities of cells, the designed drug delivery platform can be used for this application.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this research, the goal was to design a drug delivery with the purpose of tailored control, focusing on creating an ideal platform that can act like the body's natural ECM (extracellular matrix). Coumarin was used as an active herbal ingredient in this study, due to its strong pharmacological properties and beneficial effects on human health. Also, the strong binding interaction of it with L929 was confirmed by the molecular docking. The π-π interactions and hydrophobic interactions are an important force of binding interaction between coumarin and L929. Coumarin was simply extracted from the root of the p.ferulacea plant and the structure was characterized via \u003csup\u003e1\u003c/sup\u003eH NMR spectroscopy analysis. After that, biodegradable polycaprolactone polymer was used to encapsulate coumarin through the coacervation method which was confirmed by SEM analysis. The coacervation technique helped in making the spherical nanocapsules of almost equal size so that they could be easily distributed inside the scaffold fibers. A combination of polyvinyl alcohol and gelatin was used due to their hydrophilic properties to make electrospun nanofibrous scaffolds which can create suitable conditions for proliferation, adhesion, and cell function. In drug release, the release rate is relatively slower in a neutral environment compared to acidic or alkaline conditions. At all pHs, the release rate of coumarin after 40 hours (Lag phase) with a suitable slope up to 72 hours was performed, which is essential for the main purpose of this work and it is exactly according to the time required for L929 cell proliferation (Log phase). Therefore, placing the drug in a binary coating allowed for a fully controlled delivery. The drug loading efficiency was at 87%. The electrospun nanofibrous scaffolds containing the drug were then tested for porosity, tensile strength, and elongation at break, and the outcome was 92.8%, 1.09 MPa, and 7.51% respectively. Using SEM and HRTEM analysis, the nanofibrous scaffold from gelatin, and PVA with 10% nano-encapsulated coumarin was studied, and the images have shown the polycrystalline structure with high uniformity. Throughout the MTT test, no cytotoxicity was found, and the survival and the proliferation of L929 cells on the electrospun nanofibrous scaffold containing coumarin was higher than the sample without the drug, even after the 5 days, which is due to the persistence of the drug effect in the cellular environment. SEM analysis of L929 cells showed that nanofibrous scaffolds with coumarin have been able to significantly increase cell proliferation and adhesion, which could be related to the biological property of coumarin. Therefore, unlike in the past, we think it is possible to design the drug release system using a principled process that takes into account the time required for each cell line to go through two phases of adaptation and proliferation as well as the choice of appropriate two- or multi-phase platforms depending on the kind of drugs. In future studies, two or more medications might be embedded in distinct carriers to achieve sequential release, thereby preventing any interference among them.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eRahebeh Amiri Dehkharghani made substantial contributions to the conception or design of the work and the interpretation of data and the writing the manuscript.Rojan Akhbarati has done the experimental section.Soheila Zamanlui Benisi helped in the biological part.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePei J, Yan Y, Palanisamy CP, Jayaraman S, Natarajan PM, Umapathy VR, et al. Materials-based drug delivery approaches: Recent advances and future perspectives. 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Materials Science and Engineering: C. 2014;34:402-9. https://doi.org/10.1016/j.msec.2013.09.043.\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":"journal-of-polymers-and-the-environment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jooe","sideBox":"Learn more about [Journal of Polymers and the Environment](https://www.springer.com/journal/10924)","snPcode":"10924","submissionUrl":"https://submission.nature.com/new-submission/10924/3","title":"Journal of Polymers and the Environment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Nano-encapsulation, Electrospinning, Nanofibrous scaffold, Controlled release, Coumarin, L929 cells","lastPublishedDoi":"10.21203/rs.3.rs-5122397/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5122397/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA full-control design can significantly improve drug release and cell proliferation for tissue engineering applications in medicine. The present investigation encompassed a molecular docking study which was performed to investigate the interaction of selected active ligand (coumarin) with the L929 mouse fibroblast cell line protein as the receptor. After that, the coumarin was extracted from the roots of p.ferulacea and its subsequent nanoencapsulation with polycaprolactone, employing the coacervation technique to achieve a narrow distribution of nano particle sizes. Subsequently, the electrospinning technique was utilized to apply a second coating to the nano-encapsulated coumarin. Polyvinyl alcohol and gelatin compounds were used to produce electrospun nanofibrous scaffolds for their similarity to the extracellular matrix (ECM). This coordinated nano platform aimed to assess its effectiveness in regulating drug release, evaluate its biocompatibility, and examine its impact on L929 cell proliferation according to the Lag and Log phases of their growth. In silico analyses demonstrated significant interactions and high binding energy values between the coumarin ligand and essential residues of the L929 mouse fibroblast proteins. The results of the experiments were checked using analyses of \u003csup\u003e1\u003c/sup\u003eH NMR, FTIR, UV, SEM, mechanical properties, DSC, HRTEM, and HPLC. The biological effects and cell proliferation were conducted employing the MTT method (up to 5 days). Notably, no cytotoxicity was detected throughout the assessment. In this way, it is feasible to create a synergistic nano delivery system by delaying the release of the drug into account the timing of distinct cell lines' development phases.\u003c/p\u003e","manuscriptTitle":"Design a coordinated platform for coumarin-regulated delivery in line with the biological systems' growth phases","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-14 11:02:44","doi":"10.21203/rs.3.rs-5122397/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-05T13:57:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-01T12:42:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"94457306912040467140304322024072315700","date":"2024-10-25T14:53:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"178752773192365482177681011671398463709","date":"2024-10-23T17:10:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-09T07:26:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"277730098335151092364470950858897639044","date":"2024-10-09T07:03:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"188859886399054506477740563774029358727","date":"2024-10-06T03:48:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"139981926253876575542282456988390532201","date":"2024-09-27T19:58:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"281918061204690355360859518502434235296","date":"2024-09-27T07:43:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-26T09:01:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-20T18:24:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-20T18:23:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Polymers and the Environment","date":"2024-09-20T09:21:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-polymers-and-the-environment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jooe","sideBox":"Learn more about [Journal of Polymers and the Environment](https://www.springer.com/journal/10924)","snPcode":"10924","submissionUrl":"https://submission.nature.com/new-submission/10924/3","title":"Journal of Polymers and the Environment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2251eafe-0b8b-424e-85a6-98626e625707","owner":[],"postedDate":"November 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-02T16:02:17+00:00","versionOfRecord":{"articleIdentity":"rs-5122397","link":"https://doi.org/10.1007/s10924-024-03458-4","journal":{"identity":"journal-of-polymers-and-the-environment","isVorOnly":false,"title":"Journal of Polymers and the Environment"},"publishedOn":"2024-11-30 15:57:35","publishedOnDateReadable":"November 30th, 2024"},"versionCreatedAt":"2024-11-14 11:02:44","video":"","vorDoi":"10.1007/s10924-024-03458-4","vorDoiUrl":"https://doi.org/10.1007/s10924-024-03458-4","workflowStages":[]},"version":"v1","identity":"rs-5122397","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5122397","identity":"rs-5122397","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}