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Back to Journal »International Journal of Nanomedicine» Volume 16

Novel self-assembled nanofibers loaded with Acaciin as an anti-BCV MPro inhibitor as an alternative model for SARS-CoV-2

Author Mohamad SA, Zahran EM, Abdel Fadeel MR, Albohy A, Safwat MA 

Published on March 2, 2021, Volume 2021: 16 pages, 1789-1804 pages

DOI https://doi.org/10.2147/IJN.S298900

Single anonymous peer review

Editor approved for publication: Dr. Thomas Webster

Soad A Mohamad,1,* Eman Maher Zahran,2,* Maha Raafat Abdel Fadeel,3 Amgad Albohy,4 Mohamed A Safwat5 1 Department of Pharmacy, Deraya University School of Pharmacy, University District, New Minya City, 61111, Egypt; 2 Egyptian New Rice Department of Pharmacognosy, Faculty of Pharmacy, University of De Laia, University District, Nya City, 61111; 3 Veterinary Serum and Vaccine Research Institute (VSVRI), Cairo, Egypt; 4 Department of Medicinal Chemistry, Faculty of Pharmacy, British University (BUE), Egypt, El-Sherouk, 1837 , Egypt; 5 Department of Pharmacy, School of Pharmacy, South Valley University, Qena, 83523, Egypt *These authors have contributed equally to this work. SARS-COVID-2 has recently become one of the most life-threatening problems, and new treatments are urgently needed Antiviral drugs, especially those derived from herbal medicine. Purpose: This study aims to load acaciin (ACA) into new self-assembled nanofibers (NFs), and then study their possible antiviral effects on bovine coronavirus (BCV) as an alternative model for SARS-COV-2. Methods: ACA used 1H-NMR and DEPT-Q 13C-NMR spectra for identification, Autodock 4 was used for molecular docking research, and the traditional solvent injection method was improved to synthesize biodegradable NF. Different characterization techniques are used to examine the formation of NF, followed by antiviral studies for BCV and MTT assay using MDBK cells. Results: Formed core/shell NFs, ranging from 80-330 nm, with tiny spiny branches, enhanced encapsulation efficiency (97.5 ± 0.53%, P <0.05) and dual controlled release (burst release 65%) ) 1 hour, sustained release up to> 24 hours). The antiviral studies on the formed NF showed that the inhibition rate on BCV cells was 98.88 ± 0.16% (P <0.05), and the IC50 was 12.6 μM. Conclusion: The result introduces a new, time/cost-saving strategy for the synthesis of biodegradable NF without the need for electrical current or dangerous cross-linking agents. In addition, it provides an innovative way to discover herbal-derived drugs to combat SARS-CoV-2 infection. Key words: acaciin, molecular docking, nanofiber, core-shell, BCV/SARS-COV-2

Acaciin, a known compound isolated from Ocimum menthiifolium in the Lamiaceae family, 1,2 is a biologically active flavonoid compound, reported to have antioxidant and hepatoprotective activities. 3 As several plant-derived flavonoids, such as apigenin, quercetin, and kaempferol, have proven to have multiple antiviral properties, 4 acaciin was proposed to follow the same behavior 5, and then SARS-CoV-2 was investigated . Recently, severe SARS-CoV-2 virus infection has caused a global outbreak of Coronavirus Disease (SARS-COV-2)6, which requires the development of diagnostic and detection methods. 7,8 It is worth noting that recent rapid detection and monitoring strategies have been reported as DNA sensors based on DNA-modifying enzymes, the diagnosis of pathogenic SARS-Cov-2 viruses and diseases caused by viruses. 9 Coronaviruses are divided into 4 subfamilies: α, β, γ, and δ coronaviruses, of which SARS-CoV-2 belongs to the beta category. 10 The beta coronavirus maintains its integrity through four proteins: E (envelope), M (membrane), N (nucleocapsid), and S (spike) proteins. 11 The latter and its binding site help the virus to attach to the host through cell receptors (ACE2) (a key receptor for coronavirus entry) and episialic acid receptors on the respiratory epithelium. 12 Infect cells, thereby promoting their aggregation and Onset. The second step involves adapting the virus to its host through genomic coding, which facilitates the expression of genes encoding necessary auxiliary proteins. Fortunately, a high similarity has been observed between SARS-COV-2 and bovine coronavirus (BCV), both of which belong to the beta coronavirus. Analysis of the supporting phylogenetic development of its genome 13 shows that the difference lies in their host, among which BCOV causes gastrointestinal and respiratory diseases in cattle14 and COVID-19 causes the same diseases in humans, both of which target the respiratory system. 12 They obtained the same sequence of the backbone and binding site of the main protease (Mpro) target. 13 is considered a key protein in its pathogenesis and plays a key role in its replication and maturation. Therefore, structural similarity and common Mpro support the use of BCV as an alternative model for SARS-CoV-2,15,16, especially in the presence of government and ethical issues. 17 Our research started with computer-aided drug design to hypothesize the antiviral potential of ACA targeting Mpro, and then nanoformulations to improve the bioavailability of lipophilic ACA. According to the electrostatic interaction between the negatively charged lecithin and the positively charged chitosan polysaccharide, NFs are manufactured by improving the solvent injection method, which is the first report here. The main principles they chose were due to their biocompatibility, biodegradability and non-toxicity. 18 This process uses a self-assembly method that allows lecithin and chitosan to assemble spontaneously through electrostatic interactions, without the use of electric current, harmful organic solvents, or crossover. 19 Finally, ACA-loaded NF was tested against BCV to reveal their effects on viruses The developmental impact depends on the mechanism of the virus entering the cell and the inhibition of RNA replication.

Therefore, the purpose of our research is to study the anti-SARS-CoV-2 potential of the isolated flavonoid ACA loaded on NFs by modifying some parameters to enhance the morphological and biological characteristics (Scheme 1). The obtained NFs have a large surface area to volume ratio, sustained drug release, high yield, high safety and low cost. Scheme 1 shows the diagram, (A) separation of compounds, NMR analysis, ADME and docking studies, (B) using solvent injection and SEM analysis to manufacture NF, (C) studying the antiviral activity of NF.

Scheme 1 shows the diagram, (A) separation of compounds, NMR analysis, ADME and docking studies, (B) using solvent injection and SEM analysis to manufacture NF, (C) studying the antiviral activity of NF.

Brüker Avance 400 MHz NMR spectrometer (Germany) was used for NMR analysis. For NF characterization, microscope with Leica camera, model: DM1000 (microscope marketed in the US), with (LEICA EC3 digital camera), UV spectrophotometer (Jasco, Japan), Nano Zeta Sizer (ZS) (Malvern Instruments, Malvern, UK) ) A particle size analyzer (Zeta sizer 3000 HAS; Malvern Instruments Ltd, Worcestershire, UK), a rotational viscometer (Brookfield-DVBT) and a DSC 131 evo (SETARAM Inc., France) were used. For biological research, a confocal laser scanning microscope (CLSM) equipped with an environmental chamber (SP2 type; Leica, Mannheim, Germany) is used.

The above-ground part of O. menthiifolium was collected from the Jizan National Garden in Jizan, Saudi Arabia. The air-dried aerial part (500 g) was extracted by immersion in 95% ethanol, and then concentrated under pressure to a syrup-like consistency (70 g). It was then suspended in water and extracted with petroleum ether, dichloromethane, ethyl acetate and butanol in sequence. The butanol fraction (6 g) was chromatographed on silica gel using ethyl acetate: methanol (70:30), and then sephadex was performed using methanol to obtain 4 fractions, from which the compound was precipitated, purified, and analyzed.

The compound was analyzed by H1 and DEPT-Q-C13 NMR (Figure S1 a, b), and the data obtained was compared with the previously reported data, 20 and the results of previous metabolomics studies. 2

Based on the typical SMILES of the selected ligand obtained from PubChem, the online SwissADME program is used to calculate the ADME properties of the compound under study. 21 The software calculates the physicochemical and pharmacokinetic properties and drug-like properties of compounds to detect their bioavailability through Lipinski's five rules. 22 The observed attribute values ​​are shown in Figure S2.

The 6LU7 PDB code was used for ACA docking at the active site of SARS-CoV-2 Mpro. 23 The 25*25*25Å3 grid frame used is centered on the eutectic ligand with a detail level of 16.0, and the 3D image is generated using PyMOL. 24 The structure of ACA was downloaded from PubChem and used 1000 steps for energy minimization according to the steepest descent method, and then the 1000-step conjugate gradient algorithm was performed on the Avogadro software. The water molecules and non-protein residues in 24 use PyMOL to remove each enzyme by adding hydrogen, and then use the Make Macromolecule command on PyRx.25,26 to prepare proteins. RMSD values ​​are reported by the DockRMSD server, 27 3D images are generated using PyMOL, and 2D interaction diagrams are generated using LigPlot Plus.28

Lecithin was used to test PVA, CMC, PPA, and chitosan, and the resulting nanoformulation was studied using an optical microscope (Figure S3). NFs are only formed by the combination of lecithin/chitosan, in which 9 different formulations with different L/ACA concentrations were prepared according to the method described in 29, and some parameter modifications were made. Lecithin 25, 50 and 70% (w/v) were dissolved in 1:20 DMSO/water solution, in which ACA (5%, 10% and 15%) was dissolved to obtain different weight ratios. The preparation method of the chitosan aqueous solution is to dilute 20% chitosan (w/v) in acetic acid (0.1% v/v) standard solution, and then use a metal needle to slowly inject 2 mL of the 8 mL ACA/lecithin solution into the tube . The inner diameter of the syringe is close to 0.1 mm and the distance from the collector is about 1-2 mm. Under mechanical stirring at 1500 rpm for 15 minutes, the injection rate is adjusted to 1.8 mL h-1. The resulting suspension was filtered through a filter membrane (0.8 μm), in which α-tocopherol was added to the resulting filtrate (Table 1). Table 1 Composition of Acaciin NFs loaded nanofibers with different formulations

Table 1 Composition of Acaciin NFs loaded nanofibers with different formulations

LEM is used for preliminary inspection of NF materials during the manufacturing process.30 Polymer samples can be monitored even in the expanded state, which is the same as they appear in in vitro and in vivo experiments.

The UV absorption spectra of ACA/NFs and free ACA were obtained with a spectrophotometer in the range of 200-700 nm at 1.0 nm intervals. The sample is measured three times in a rectangular quartz cuvette with a path length of 1 cm at 25 °C.

Zeta potential uses Nano Zeta Sizer to measure the charge of particles in nanocolloids through electrophoretic light scattering of nanoparticles in aqueous solutions. The Smoluchowski equation is used to calculate the Ʒ potential at 25°C, collecting 15° scattered light for each sample every 10 times. 31 Ʒ The potential value is expressed as the average value and standard deviation of nine measurements obtained with nine different formulations. Dynamic Light Scattering (DLS) is used at 25°C using a 90° fixed angle particle size analyzer and 633 nm He-Ne Laser to determine the particle size of NF.

The viscosity of the polymer solution was measured by a rotational viscometer at room temperature.

The NF solution was centrifuged at 4500 rpm for 60 minutes, and then an ultraviolet spectrophotometer (Jasco, Japan) was used to detect the amount of drug embedded in the supernatant at a wavelength of 240 nm. The EE and DL of ACA/NF are calculated according to formulas 1 and 2: (1) (2)

Wherein W initial drug represents the initial added amount of the drug, and W wrapped drug represents the amount of drug wrapped in NF. WNP represents the total weight of all components in NF. 32

Compared with ACA-NFs (1% w/w), ACA's Differential Scanning Calorimetry (DSC) analysis was performed, using standard calibration instruments: mercury, indium, tin, lead, zinc, and aluminum, and nitrogen And helium as the purge gas. The procedure of the test (samples 1-7) is a heating zone of 25°C to 500°C and a heating rate of 10°C/min (the heating zone of samples 8-10 is -50 to 200°C). Use CALISTO data Processing software v.149.32 processing thermogram results

The dialysis membrane method is used to release ACA from the corresponding NF in vitro. 32 Transfer 1 ml of NF suspension to a dialysis bag with a molecular mass cut-off value of 12,000 Da. Then the bag was suspended in 40 mL ethanol: phosphate buffered saline at 37°C ± 0.5°C at a ratio of 3:7 (v/v), running at 50 rpm at pH 6.8 ± 0.2 (JSSB-series water bath) . At intervals of 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 hours after administration, 1 mL of receptor solution was taken out, and 1 mL of blank medium at the same temperature was added to the receptor compartment. Compare the results with the results obtained with the corresponding ACA in a DMSO/water (1:10) suspension. 33 Determine the release mechanism of ACA according to the kinetic model:

Zero order R=K0t24,34

Baker-Lonsdale Model: 27,37

Hixson–Crowell cube root law: 28,38

R, Q and Mt/M∞ refer to the fraction of the drug released at time t, K or KH is the rate constant associated with each model, UR represents the unreleased fraction of the drug, and n is the diffusion index that characterizes the release during dissolution Mechanism.

Use high-resolution SEM to study NF morphology, which allows structures at different distances from the scan level to have a high depth of clarity. 39 Dilute the sample in ultrapure water (1:10), then apply 20 µL of the slurry on an amorphous polycarbonate grid and dry it at room temperature. They use CO2 for additional drying, sputter gold plating in a metalizer, and then inspect under an SEM with an accelerating voltage of 200 kV. 32

Madin Darby bovine kidney cells (MDBK) obtained from the VSVRI Institute were grown in Dulbecco's modified Eagle medium (Gibco, Bethesda, Md.) supplemented with 10% fetal bovine serum (Gibco) at 37°C and 5% CO2 . The stock of BCV (Mebus strain) comes from the VSVRI Institute. BCV, propagated in MDBK cells, 40 supplemented with 10% fetal bovine serum (FBS) according to standard tissue culture procedures. The cells were cultured with 5% CO2 in a humidified incubator at 37°C, and then subcultured at 80-90% confluence.

MDBK cells were grown in a Petri dish (Martek, Ashland, Massachusetts) with a glass slide bottom for 2 days for confocal time-lapse microscopy on the coverslip. Petri dishes, coverslips and sapphire discs are covered with 8-10 nm thick carbon, which is evaporated under high vacuum to promote cell growth. Then, the cells were infected with BCV at a multiplicity of infection (MOI) of 1, 5, and 10, and kept at 4°C for 1 hour to allow adsorption, and then incubated at 37°C for up to 48 hours.

The time-lapse microscope was performed using a confocal laser scanning microscope (CLSM) equipped with a 37°C and 5% CO2 environmental chamber. For nuclear analysis, cells were fixed with 4% paraformaldehyde and stained with DAPI in phosphate buffered saline (1 μg/mL). The sample was embedded in a fluorescent mounting medium (DakoCytomation, Glostrup, Denmark) and analyzed by CLSM. Use Huygens Essential program suite (SVI, Hilversum, Netherlands) to deconvolve the image by blind deconvolution algorithm. 42

Conventional microtiter cell culture assays used to titrate viral cells are performed on MDBK cells. First, the cells are seeded in 96 wells, and then cultured at 37°C in a humidified atmosphere containing 5% CO2 for 24-48 hours. Then, prepare a 10-fold serial dilution of the virus stock solution and infect the cells with 100 μL of the virus. After 2 hours, remove excess virus, and then record the cytopathic effect every day during the incubation period for 5-6 days, and calculate IC50, CC50 and SI.

The MTT assay of ACA-NF using the MDBK cell line is essential to determine the cytotoxicity of ACA and ACA-NF before testing its antiviral potential. 43 The test started with chemical reduction of tetrazole, using mitochondrial enzymes to form formazan crystals, and then adding acidified alcohol to dissolve them. Then, the MDBK cell line was prepared by trypsinization and suspended in minimal medium using trypan blue as a repelling dye. After staining and cell preparation, inoculate them in a tissue culture plate (96-well) and adjust the cell concentration to 5 × 105 cells/well. The wells were incubated overnight at 37°C in a humidified incubator with 5% CO2; then different concentrations of ACA and ACA/NFs were added to the wells and incubated under the same conditions for 8-10 hours. After the incubation, add 10 μL (5 mg mL-1) of MTT reagent to each well, complete the incubation for 4 hours, then add acidified isopropanol (100 μL, 0.1 N) to each well, and keep it at room temperature for 30 minutes in the dark. Minutes, then place on the shaker for another 1 minute.

The antiviral potential was determined by adding 25 μL of MDBK cell virus suspension (106 TCD50 mL-1) to 25 μL of different concentrations of ACA-NF in a PCR tube and incubating at 37°C for 1 minute. 44 . The virus suspension (25 μL 106 TCD50 mL-1) was added to different tissue culture well plates containing MDBK cells at a concentration of 5×105 cells mL-1, and then the wells were shaken and placed in a CO2 incubator for 1 hour. Add different concentrations of ACA-NF to the wells and incubate for a further 72 hours at 37°C. Two controls were kept in parallel to incubate with the treated wells: a triple control containing cell and virus suspension without NF and a triple control containing only cells without NFS or virus suspension. Then, the detection of antiviral activity was determined by checking the tissue culture for virus cytopathicity every day and applying MTT assay after 72 hours. The values ​​of CC50 (cytotoxic concentration of 50% cells) and IC50 (inhibitory concentration of 50% infected cells) were estimated from the concentration-effect curve after linear regression analysis. Then calculate the average of three independent experiments and the selectivity index (SI=CC50/IC50). 45

All results are expressed as mean ± standard deviation and analyzed using one-way analysis of variance, and then a multi-parameter Tukey post-hoc test was performed in Graph Prism® (GraphPad Software Inc., San Diego, CA), and a statistical display was established at p Significance <0.05.

This compound was isolated from O. menthiifolium for the first time, and was identified as acaciin (linamarin, acacetin-7-O-rutinoside) 20 (Figure 1), and is considered to be one of the common classification markers of the Ocimum genus in the Lamiaceae. 46 Acaciin : Off-white, solid; 1HNMR (DMSO-d6) ppm: δH 12.9 (1H, br. s, OH-5), 8.04 (2H, d, J = 9.2 Hz, H-2ʹ, H-6ʹ), 7.17 (2H , d, J = 9.2 Hz, H-3ʹ, H-5ʹ), 6.95 (1H, s, H-3), 6.8 (1H, s, H-8), 6.46 (1H, s, H-6), 5.07 (1H, d), J = 9.2 Hz, H-1ʹ'), 4.53 (1H, s, H-1ʹ''), 3.89 (3H, s, OMe-4ʹ), 3.65 (1H, d, J = 8.5 Hz, H-6ʹ'), 1.08 (3H, d, J = 6.3 Hz, H-6ʹ''); 13C-NMR (DMSO-d6) ppm: δC 182.5 (C-4), 164.4 (C-7 ), 161.6 (C-9), 157.5 (C-5), 130.7 (C-6), 128.9 (C- 2ʹ,6ʹ), 123.1 (C-1ʹ), 115.2 (C-3ʹ,5ʹ), 105.9 ( C-10), 104.3 (C-3), 100.4 (C-1ʹ'), 100.1 (C-1ʹ'') ), 95.3 (C-8), 76.1 (C-3ʹ', C-5ʹ'), 73.5 (C-2ʹ'), 72.5 (C-4ʹ''), 70.8 (C-2ʹ''), 70.1 (C-4ʹ'), 68.8 (C-5ʹ''), 66.5 (C-6ʹ') , 56.1 (OMe-4ʹ'), 18.2 (C-6ʹ''). Figure 1 The chemical structure of acaciin.

Figure 1 The chemical structure of acaciin.

Lipinski's five rules stipulate that for any compound selected as a potential drug, the following conditions shall not be violated more than once: (a) Molecular weight <500 Daltons (b) High lipophilicity (expressed as Log P<5) (C) Less than 5 hydrogen bond donors (d) Less than 10 hydrogen bond acceptors. If the compound of interest has three or more of the above criteria, the compound is likely to be a potential candidate for drug development. 22 Therefore, ACA has a relatively low solubility, cannot cross the blood-brain barrier and has no effect on cytochrome P450. It is worth noting that the aglycon itself does not violate Lipinski's five rules, but the inclusion of the disaccharide moiety will cause some related violations (Figure S2).

ACA docks with the four main targets of SARS-CoV-2, and the docking scores with nsp16/10, ACE2-PD and RBD-S-protein are -9.0, -6.7, -7.1 Kcal/mol, and -9.1 Kcal, respectively /mol uses Mpro. When docked with nsp16/10 and Mpro, the internal ligands reached -8.2 and -7.8 Kcal/mol, respectively. The docking procedure provides good accuracy, and the RMSD value between docking and co-crystallization ligand is 1.839, calculated by the DockRMSD server (Figure 2). ACA overlaps with the co-crystalline ligand, follows the same behavior and occupies the same binding pocket, has the same interaction (Figure 2), but has a better docking score. It forms an H bond with Ser 144, which is reported to be one of the key residues of Mpro, through C=O, and forms an H bond with His163, Leu141, and Thr26 through 5-OH, C=O, and 4ʹ-OMe. Part of it forms H bonds with His164, Thr190, Tyr54 and Asp187 (Figure 2). It has been reported that co-crystal ligands interact with most of these residues. 49 Figure 2 (A) The overlap of the internal ligand docked in the active site of Mpro (blue) and the co-crystallized ligand (green), (B) the Acaciin docked in the active site of Mpro overlaps the co-crystal ligand, (C ) Acaciin 3D interaction with amino acid residues in the active site of Mpro, showing H bond (yellow dotted line) and double interaction.

Figure 2 (A) The overlap of the internal ligand (blue) and the eutectic ligand (green) docked in the active site of Mpro, (B) the docking of Acaciin in the active site of Mpro overlaps the eutectic ligand, (C) Acaciin 3D interaction with amino acid residues in the active site of Mpro, showing H bond (yellow dashed line) and double interaction.

Apply the solvent injection method and modify some parameters that are expected to affect the shape of the nanoformulation formed (Figure 3). It was observed that when PVA, CMC, PPA and lecithin were tested and microscopically examined, only lecithin formed nanofibers, while other polymers formed nanoparticles (Figure S3). The principle of our new NF formation technology is to use the electrostatic interaction between positively charged chitosan and negatively charged lecithin. 50 This leads to a decrease in surface tension, which leads to nanoparticle elongation and chain entanglement to form short nanofibers. Figure 3 A cartoon representation of the improved solvent injection method.

Figure 3 A cartoon representation of the improved solvent injection method.

According to reports, the key parameter for manufacturing NF (Figure 3) utilizes the electrostatic interaction between chitosan and lecithin. 51 This is why other tested polymers (PVA, CMC, and PPA) provide NP instead of NF ( Figure S3). Therefore, this interaction generates electrostatic force to overcome the surface tension of the polymer solution (Figure 4). In addition to using metal nozzles to generate more electrostatic friction, all of them undergo self-assembly and phase separation, 52 leading to the formation of core/shell Fiber (Figure 4). Another parameter that controls fiber morphology and diameter is the molecular weight of the polymer, because it affects the rheological properties of the solution and the higher fluidity organizes the polymer into a core-shell rather than a continuous structure. Imagine a droplet on the tip of a nozzle. The inner polymer is covered by a continuous phase of another polymer. When the power is strong enough, the continuous phase captures the droplet and sucks it into the core-shell jet. It is worth noting that the core-shell NF is considered to be one of the most important breakthroughs in the formation of NF. It significantly affects the dual drug release profile and minimizes the direct contact of the bioactive agent in the core aqueous solution with potentially hazardous solvents. Shell, leaving only the core-shell interface. 53 In addition, viscosity is also a parameter; it supports the formation of core/shell, where the more viscous phase (lecithin that dissolves acaciin) forms the core, while the less viscous (chitosan) wraps the core that forms the shell. This is confirmed by the high Ʒ potential of the surface. 54 In addition, increasing the L/Cs ratio increases the high viscosity, which then generates additional charges, which overcomes surface tension and promotes chain entanglement 54 (Figure 4). In addition, adjusting the minimum flow rate will reduce the solution passing through the gap between the needle tip and the collector, which allows for longer stretch times of the sprayed droplets, more solvent evaporation and spray formation. Therefore, the stable cone-shaped jet alternately forms with the unstable receding jet, resulting in a change in the NF diameter (Figure 4). 55 In addition, shortening the distance between the needle tip and the collector will prevent the fiber from over-stretching and shorten it slightly. 56 It is worth noting that the diameter of the orifice directly determines the flow of the solution and the initial diameter of the fiber. When it is narrow enough, it will cause the fiber form. 57 Finally, it is reported that reducing the rotation speed can eliminate 55. In short, imagine a spherical droplet in a vacuum state at the tip of the needle, while adjusting the previous parameters, can actually produce a self-assembled, stable and bead-free short core/shell NF. Figure 4 The cartoon representation of NF formation, (A) the electrostatic interaction step of fiber formation, (B) the arrangement of polymer and core/shell formation, (C) the effect of high viscosity on chain entanglement, (D) cone The alternating and receding jets cause the fiber diameter to change.

Figure 4 The cartoon representation of NF formation, (A) the electrostatic interaction step of fiber formation, (B) polymer arrangement and core/shell formation, (C) the effect of high viscosity on chain entanglement, (D) cone The alternating and receding jets cause the fiber diameter to change.

The LEM survey of nine formulations (Table 1) revealed the formation of short NF, but formulation 3 obtained the best characterization results and was further selected for SEM analysis (Table S1). Significantly high Ʒ potential> 50 mV (Figure 5), which is proportional to the increased L/Cs ratio and high drug content, indicates that the formed NFs have high stability. It has been reported that size and polydispersity index (PDI) affect the loading, release and stability of drugs in NFs. The smaller the particle size, the lower the drug concentration, the lower the PDI (≤0.4), and the larger the exposed surface area, resulting in faster release of the encapsulated drug and lower aggregation. 18 Such a low PDI value (0.2±0.02, P <0.05) causes mechanical effects through electrical stability, 58 and biological inhibition through the interaction between the positively charged NFs and the negatively charged surfaces of microbial cells. 51 Considering the particle size, its range is 80-330 nm, which is inversely proportional to increasing the L/Cs ratio and reducing the drug content. This is very valuable in antibacterial evaluation. The smaller particle size will cause the microbial cells to affect the carrier. The absorption increases. 51 DSC thermogram (Figure 5) detects any changes in the crystalline properties of the loaded aca. In NFs, it is known that when the drug-polymer interaction is appropriate, the presence of the drug will not have a significant impact on the Tg temperature of the polymer. 59 Therefore, the DSC thermograms of co-compared pure ACA and ACA-loaded NF show relatively similarity. The exothermic zone fluctuates around the glass transition temperature (Tg) of 120°, and it is confirmed that there is no chemical and physical due to preparation. Variety. When the drug release profile was studied (Figure 5), it showed dual controlled release, which is a typical feature of the core/shell structure. The initial burst release rate after 60 1 hour was 65%, and the subsequent gradual release was extended to 24 hr. Excellent release curve. This is expected to achieve a high therapeutic index and a long-lasting biological effect. In addition, the observed high encapsulation efficiency (EE) (> 97.3 ± 0.5%, p <0.05) (Figure 5) indicates that most of the ACA is preferably inserted into the lipid core of NFs, and only a small part of the water is lost during the preparation process. In phase. 18 Figure 5 (A) UV scan of acaciin (200-400 nm) at pH=6.8, (B) standard calibration curve of acaciin at pH 6.8 at λmax=240nm, (C) Ʒ-formed The potential of NFs (> 50 mV), the particle size distribution of (D, E) NFs, (F) the DSC mode of acaciin and formed NFs, (G) the dual drug release profile of acaciin and formed NFs, (H) NFs The relationship between different formulations and viscosity and EE.

Figure 5 (A) UV scan of acaciin (200-400 nm) at pH=6.8, (B) standard calibration curve of acaciin at pH 6.8 at λmax=240nm, (C) Ʒ-formed NFs (> 50 mV) , (D, E) particle size distribution of NFs, (F) DSC mode of acaciin and formed NFs, (G) dual drug release curve of acaciin and formed NFs, (H) different formulations of NFs are related to viscosity and EE.

Finally, the SEM examination revealed hairless, bead-free, spindle-shaped, core/shell NF, with a diameter range of 250 nm–3 µm and a length range of 5–20 µm. The morphology of ordinary NF (Figure 6) and ACA-NF (Figure 6) are roughly similar, with the latter being slightly wider due to the intrusion of ACA into the core. In addition, it has been reported that the previously discussed difference between the steady jet and the receding jet causes some diameter changes,61 as shown in Figure 6. In addition, the core/shell structure shows a shell thickness range of 300-500 nm and a core diameter range of 500 nm-3 µm (Figure 7). Figure 6 (A) Free ACA and (B) ACA-NFs, ce) nanofibers expressing NF diameter changes Figure 7 (A, B) Different core/shell NFs, spiny branches of (CF) NFs (red arrow) , (G) Immature spiny branches (yellow arrows).

Figure 6 (A) free ACA and (B) ACA-NFs, ce) nanofibers expressing NF diameter changes

Figure 7 (A, B) Different core/shell NFs, spiny branches of (CF) NFs (red arrow), (G) immature spiny branches (yellow arrow).

Surprisingly, thorn-like branches were obtained on the outer surface of NFs, with a width ranging from 200-400 nm, a length ranging from 800 nm-2 µm (Figure 7), and some immature branches of about 200-300 nm. Width and length up to 500 nm (Figure 7). It is reported that such branches are produced by the temperature treatment 62 or continuous ion layer adsorption and reaction after electrospinning. 63 They are often used for support purposes to provide a higher surface/volume ratio, higher mechanical buildup64, and due to improved substrate adhesion, the contact area with the tested microorganisms in biocidal research is large and sufficient. 63 Obtaining such a branch can be initially explained by relatively high temperature and humidity (the operating room temperature is 43°C and the humidity is 60%).

Through MTT cell viability determination (the gold standard of cytotoxicity 65) and microscopic examination, the possible toxic effects of nine ACA-NFs preparations on MBDK cells were evaluated. 66 The antiviral activity of ACA-NFs is based on the ability of ACA to prevent the cytopathic effect (CPE) of the virus on MBDK cell culture, and the results are recorded (Table S2). The antiviral activity was significantly increased to 78.14%, 87.16% and 98.88% in a dose-dependent manner, in which the ACA-NF concentration was 1, 3 and 6 ppm, with slight cytotoxicity of 6.30 ± 4.31%, 5.00 ± 0.023% and 8.3 . They were ± 0.16% (P<0.05) (Figure 8). Increasing the concentration to 10 ppm has no effect on the activity, the cytotoxicity slightly increases to 9.21 ± 0.01%, P <0.05, all of which are compared with the free ACA (12.5 µg/mL) with 75.40% activity, and are significantly less toxic 10.8 ± 0.1% (P<0.05). Figure 8 Biaxial graph of the effect of different NF concentrations on antiviral activity and cytotoxicity.

Figure 8 Biaxial graph of the effect of different NF concentrations on antiviral activity and cytotoxicity.

CTEM (Scanning Transmission Electron Microscope) (Figure 9) shows the plaque changes between normal cells (a), negative control (b), and 6 μL/mL ACA-NFs (C) and free ACA (D) treated cells . ACA-NFs reduced cell damage and reduced plaque formation and cell fusion, which indicated significant viral suppression and cell protection for up to 48 hours. According to reports, if SI ≥ 4,45 acaciin has an SI of 4.2 (IC50 = 12.6 µM, CC50 = 52.8 µM), then promising antiviral activity can be achieved, which indicates a good antiviral potential. Figure 9 CTEM micrographs of MDBK cells: (A) normal cells, (B) cells infected with BCV and forming plaques (C) cells incubated with ACA-NFs for 48 hours (D) cells incubated with free ACA for 48 hours .

Figure 9 CTEM micrographs of MDBK cells: (A) normal cells, (B) cells infected with BCV and forming plaques (C) cells incubated with ACA-NFs for 48 hours (D) cells incubated with free ACA for 48 hours .

The possible antiviral effect is expected to be initiated by the electrostatic interaction between the positively charged NFs and the negatively charged lipid bilayer on the microbial envelope (Figure 10). Biological effects may be carried out in two ways: the first is the binding affinity of ACA (predicted by computer studies) to the Mpro target, which inhibits the translation of viral RNA polyproteins and interferes with the early stages of viral replication. The second is the mechanical action of ACA-loaded NF, which inhibits virus receptor binding through electrostatic interaction and prevents the virus from entering the host cell. Figure 10 A cartoon summary of two predicted pathways to inhibit SARS-COV-2, 1) electrostatic binding of NFs to the microbial envelope, 2) release of acaciin from NFs and binding to Mpro, inhibiting polyprotein from viral RNA and stopping viral replication .

Figure 10 A cartoon summary of two predicted pathways to inhibit SARS-COV-2, 1) electrostatic binding of NFs to the microbial envelope, 2) release of acaciin from NFs and binding to Mpro, inhibiting polyprotein from viral RNA and stopping viral replication .

The current research has stimulated the significant antiviral potential of acacetin as a flavonoid isolated from herbal sources for the first time. This research produced short core/shell nanofibers with excellent physicochemical properties and no current or toxic crosslinking agents. As recognized biodegradable candidates (lecithin and chitosan) are used in the synthesis of NF, our new technology is safer and more advantageous than the usual methods. In addition, the application of the self-assembly method protects the encapsulated drug from severe stress due to the current used in common methods such as electrospinning. The new nanofibers have obtained a branched surface with good mechanical properties and a core/shell structure that achieves dual controlled release, which is considered to be one of the most important breakthroughs in the formation of nanofibers. Our procedure successfully produces uniform NF with perfect surface characteristics and can save time and money spent in other technologies with additional dangerous steps. In addition, from a biological point of view, the obtained NFs can mechanically inhibit the virus from entering the host cell, combined with the Mpro inhibitory activity of ACA itself, all of which may hinder the progress of BCV. BCV is usually used as SARS-COV-2. Alternative model. Our breakthrough discovery provides deep insights for the exploration and development of natural-based antiviral drugs, especially the combination of nano-formulations with improved mechanical and functional properties to support the global fight against the current SARS-CoVID-2 disease.

The author thanks Dr. Fahad AbdelAziz, Professor of Botany and Head of Jazan Garden, who certified, approved and allowed the plant to work. I would also like to thank the VSVRI Institute for providing cell line cells and virus strains for antiviral testing, and thanks to D. Usama Ramadan Abdelmohsen, Associate Professor of Student Pharmacy, Miniyatraya University, Egypt for his help.

The authors report no conflicts of interest in this work.

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