In analytical chemistry, HPLC is used to determine and identify the quantity of each component and to separate the members of a mixture. HPLC efficiency is determined by the column's ability to separate the components of a sample by the number of theoretical plates. The practical application of the HPLC method was evaluated through the comparison of standard genistin (Fig. 1b) with genistin herbal extract (Fig. 1c). As can be seen, the herbal extract is a pure substance, and it is the glycoside of genistein (Fig. 1a), which is approved by the retention time of the standard sample (genistin).
1 H NMR of nano-encapsulated genistin in DMSO has been shown in (Fig. 2) to confirm the presence of this compound in nanocapsules. As it is clear from 1H NMR, genistin, due to having an isoflavone structure, its aromatic protons have appeared in the chemical shift of 6.5 to 8.5 ppm. But usually, in these compounds, aromatic protons show themselves with a weaker intensity, which is related to the lack of good solubility of this part. Also, the vinyl proton or protons attached to the electronegative functional groups in other materials that were used in making nanocapsules (PCL, span 80, tween 80, or sunflower oil) are placed in the range of δ = 4–6 ppm. The rest of the aliphatic protons of the components of nanocapsules appear in the range of δ = 2–4 ppm 26.
By comparing the Fourier transform infrared spectrum of the genistin herbal extract in (Fig. 3a) and the nano-encapsulated sample in (Fig. 3b), we realize that the stretching vibration of the -OH functional group has been removed, which can be due to the coating, while the intensity of the vibrational peak of the -C = O group has increased. The finding is related to the presence of this functional group in the polycaprolactone layer. Still, the rest of the vibrations are facing a decrease in intensity, which is related to their coating. Also, in (Fig. 3c), the electrospinning of nanofibers with nano-encapsulated genistin shows a broad and robust band in the 3290 cm− 1 region, which is the result of a robust intermolecular hydrogen bond between the hydroxyl group of PVA and the amide group of gelatin. In contrast, the small band in the region 2917 cm− 1 is assigned to the characteristic bands of aliphatic CH stretching vibrations. The decrease in the intensity of the C = O stretching vibration band and its shift to a lower wave number in the region of 1690 cm− 1 indicates the coating of polycaprolactone with gelatin and PVA layers. The vibrational band at the wave number 1425 cm− 1 is related to -CH2 groups in the second coating of gelatin and polyvinyl alcohol. Also, the stretching vibrations of 1081 cm− 1 and 1324 cm− 1 are related to C-O and C-N functional groups, respectively 27.
The UV-Vis spectra of a) standard genistin, b) genistin herbal extract, c) nano-encapsulated genistin, and d) the electrospun nanofibers have been shown in (Fig. 4), (0.001 g sample in acetonitrile solvent). Most conjugated unsaturated organic compounds or elements with non-bonding electron pairs absorb energy to electron transfer in the range of 200 to 800 nm. The absorption wavelength of genistin is 263 nm which can be related to the π → π* transition present in all samples 28.
Scanning Electron Microscope (SEM) collected the reflected electrons from the surface of the irradiated sample to create a visible image of morphology. So, the morphology of nanocapsules containing genistin and electrospun nanofibers has been shown in (Fig. 5a, b). The diameter of nano-encapsulated genistin is 16 to 26 nm. Also, the size of electrospun nanofibers containing nano-encapsulated drugs varied from 41 to 49 nm. One of the main reasons for using acetic acid to dissolve gelatin is to reduce the surface tension, which leads to a decrease in the diameter of nanofibers 29, as confirmed by SEM images. The fibers had no beads or structural defects. Since the SEM method was not able to magnify more, therefore, the TEM method was used to visualize the nano-encapsulated drug in the electrospun nanofibers.
Transmission electron microscopes (TEM) can produce images of nanostructures with higher magnification and resolution than SEM. This technique leads to seeing details with dimensions usually below 20 nm in the samples 30. So, the TEM of electrospun nanofibers (Fig. 6) was used to show the presence of the nano-encapsulated genistin across the fibers. As can be seen in this image, nanocapsules are somehow immobilized inside the electrospun nanofibers, and this achievement guarantees our goal in this work.
One of the essential properties suggested to determine the efficiency of nanofibers is to determine the maximum tensile force that the structure can withstand along the axis. Also, one of the essential parameters evaluated in the tensile test is the amount of pressure in MPa compared to the tension, which expresses the amount of change in the size of the sample compared to its original length in percentage terms. Since PVA is a semi-crystalline structured polymer, it has good chemical resistance and thermo-stability to be a suitable host material. On the other hand, GE fiber can be easily extended into three-dimensional structures to stimulate cell proliferation.
As a result, mixing PVA with GE leads to structures with high strength due to internal bonds and joints, especially H-bonding. So, adding GE to PVA improves the elasticity and increases the elongation at the breaking point. Also, utilizing methanol to cross-link the PVA/GE mixes resulted in improved mechanical characteristics. The degree of crystallinity was increased by the alcohol treatment, and the methanol removed any remaining water from the fibers, which in turn improved the physical cross-links and mechanical characteristics 29. The elastic modulus for fibers with 40% nano-encapsulated genistin is 45.26 MPa (Table 1), and for fibers without the drug, it is 11.47 Mpa, which indicates that fibers with drug capsules have better resistance. In Fig. 7, diagram 1 shows the mechanical properties of electrospun nanofibers containing 40% nano-encapsulated genistin. Diagram 2 and diagram 3 have 10% and 20% drugs, respectively. According to the results of Table 1, raising the drug content leads to an increase in the elastic modulus. Furthermore, the strength of electrospun nanofibers with the drug (Fig. 8a) is more than electrospun nanofibers without the drug (Fig. 8b), which is by their tensile strength in (Table 2). The intersection points of the elastic and plastic range in the synthesized nanofibers containing 40% of the nano-encapsulated genistin is 0.78 MPa higher than the electrospun nanofibers without drugs. Also, the drug-free sample has upper and lower yield limits, and its deformation during elongation is non-uniform, indicating inhomogeneity in the structure's strength and undesirable for related applications 31,32.
Table 1
Relative elongation test according to stress.
Items
|
Elastic Module
|
Break Strain
(%)
|
Break Extension (mm)
|
Peak Stress (MPa)
|
Peak Force (N)
|
1
|
45.26
|
19.09
|
3.82
|
1.39
|
1.45
|
2
|
28.99
|
18.23
|
3.64
|
2.18
|
0.57
|
3
|
43.64
|
17.20
|
3.44
|
1.58
|
1.64
|
Delta
|
-------
|
1.89
|
0.38
|
0.79
|
1.07
|
Deviation
|
-------
|
0.77
|
0.15
|
0.34
|
0.47
|
Mean
|
------
|
18.17
|
3.63
|
1.72
|
1.22
|
Table 2
Tensile test according to force and extension.
Items
|
Break Stress (MPa)
|
Peak Stress (MPa)
|
A
|
1.35
|
1.28
|
B
|
0.31
|
0.50
|
Delta
|
1.05
|
0.78
|
S. Deviation
|
0.52
|
0.39
|
Mean
|
0.83
|
0.89
|
In (Fig. 9a), we will examine the calibration of genistin release in electrospun nanofibers containing nanocapsules. The HPLC device checked the sub-peaks at a wavelength of 263 and in 10 minutes. So, Drug entrapment efficiency (EE%=92.01% ±0.7) was calculated using the Eq. (1) 33.
EE = (Weight of drug in nanoparticles)/ (Weight of drug fed initially)
Figure 9b shows the drug release in environments with various pHs of 8.5, 7, and 2. Since genistin is a polar substance (glycoside of genistein). It tends to diffuse toward a polar environment. Consequently, it has a rapid delivery from PCL to PVA/GE nanofibers and then to the cellular matrix. Drug release in alkaline and neutral environments is slower than in acidic environments, and the pH of the acidic environments, due to the ability to destroy PCL and gelatin, can lead to an explosive release of the active substance. It can be concluded that pH can be effective in drug delivery. In general, a significant release was observed during the first 6 hours, and the complete release of the drug lasted for 48 hours.
The MTT assay is based on the conversion of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) into formazan crystals by living cells, which determines mitochondrial activity 34. During the MTT tests on the L929 cell line, the effectiveness of the nanofibers with drug (in different concentrations) and without drugs was compared with the control cell diagram (TCP) by examining the graphs obtained (Fig. 10), and it was well established that the cell viability exposed to electrospun nanofibers containing nano-encapsulated genistin did not show any toxicity at 24 hours (red color) and 72 hours (blue color). Two factors are effective on cell viability. The first one is the concentration of the drug, and the second one is the electrospinning time (Table 3) 35. According to the results, sample 4 after 150 minutes of electrospinning with 40% drug, has the highest cell viability. This phenomenon may be explained by the fact that when the fiber density and amount of drug rise, the likelihood of 3D cell multiplication also increases 36.
Table 3
List of examined samples for the MTT test.
Sample
|
type of nanofibers
|
Electrospinning time (min)
|
Distance
(cm)
|
Flow rate (db)
|
Voltage
(V)
|
1
|
without drugs
|
90
|
10
|
0.6
|
28
|
2
|
with 20% drug
|
90
|
10
|
0.6
|
28
|
3
|
with 20% drug
|
150
|
10
|
0.6
|
28
|
4
|
with 40% drug
|
90
|
10
|
0.6
|
28
|
5
|
with 40% drug
|
150
|
10
|
0.6
|
28
|
SEM imaging (Fig. 11) was used to show the morphology of cell adhesion and proliferation of samples 1–5 (Table 3). After 72h cell culture, samples 2–5 containing the drug have shown more impact than sample 1(without the drug). The micrograph of the nanofibers seeded with cells showed that the L929 in sample 4 with 40% nano-encapsulated genistin and 150 min electrospinning time is best attached and proliferated on the nanofibers. On one side, the flavonoid structure of genistin and its immobilization in nanofibers make it easily absorbed by L929 cells 37; on the other side, the hydrophilic two-component PVA/GE provides biochemical signals to promote cell adhesion, migration, proliferation, and differentiation 38.