3.1. Physicochemical characteristics
Morphological properties and surface features of the nanofibers were observed through scanning electron microscopy (SEM) images. SEM images with approximate scaffold size of poly lactic acid / chitosan nanofibers containing 30% and 15% cod liver oil displayed in Fig. 1a and 1b respectively. As can be seen from the SEM images of the nanoscaffold structures are uniform and without any cracking along the formation. The nanoscaffold structures encapsulated different concentration of cod liver oil without any systemic defects and nanofibers containing 30% cod liver oil show less diameter than 15% cod liver oil which this may be due to the higher solubility of the polymer phase at higher concentrations of oil phase. According to SEM images average size of the diameter scaffold tube estimated between about 50–150 nm. To investigation of the three-dimensional (3D) images of the fibers we did transmission electron microscopy (TEM). From the TEM images it can be seen that cod liver oil trapped uniformly in micelles during in poly lactic acid / chitosan scaffolds. The oil phase ranges in the polymer phase are well defined. The TEM image of 30% w/w cod liver oil cod liver oil distributed in poly lactic acid / chitosan nanoscaffolds is shown in Fig. 1c.
Here Fig. 1a, b and c.
Energy dispersive spectroscopy (EDAX) as a suitable technique used for the semi-quantitative analysis of elements. This method is mainly used to obtain point chemical composition and quantitative investigation of the poly lactic acid / chitosan nanoscaffolds containing cod liver oil. About 37.17 percent of the total atomic weight of the final products is related to C atoms whish this could be related to carbon atoms in poly lactic acid, chitosan, omega3 in cod liver oil and Tween structures. The existence of the O, Na and N atoms to the amount about 31.63, 5.31 and 15.36 percent could be related to the existence of these atoms in poly lactic acid, chitosan, cod liver oil, tween, NaOH and dimethylformamide structures. The small amounts of the Ti, Ca atoms could be related to the unpredictable impurities in the final products. The EDAX analysis of the poly lactic acid / chitosan nanoscaffolds containing 30% cod liver oil is shown in Fig. 2a. The size distribution obtained of the nanoscaffolds is as a plot of the relative intensity of light scattered by nanoscaffolds in various size classes and introduced as an intensity size distribution. Results related to size distribution of the nanoscaffolds obtained from dynamic light scattering analysis show well match with SEM images and estimates the size of the poly lactic acid / chitosan nanoscaffolds containing 30% cod liver after 15 min ultrasonic irradiation at 60 W fibers about 50–150 nm in Fig. 2b.
Here Fig. 2a and 2b.
UV-Vis as a general qualitative technique can be used to identify and confirm functional groups in a compound by matching the absorbance spectrum. Absorption in UV-Vis spectroscopy follows Beer's Law:
A = ε × b × C Eq. 1
where ε is the molar attenuation coefficient, b is path length, and C is concentration. UV–Vis absorption spectra of poly lactic acid / chitosan nanoscaffolds containing cod liver oil shows that with increasing concentration from 15% w/w to 30% w/w absorbent peaks become noticeably more intense, however cod liver oil not absorbed alone and it can be concluded that more loading of cod liver oil take place in poly lactic acid / chitosan nanoscaffolds. Figure 3a demonstrated UV–vis absorption spectra of poly lactic acid / chitosan nanofibers containing cod liver oil 30% w/w, 15% w/w compared with cod liver oil(32). Fourier Transform infrared spectroscopy (FT-IR) as an analytical technique used to identify functional groups in materials. Figure 3b and 3c show FT-IR spectrum of the prepared poly lactic acid / chitosan nanoscaffolds containing 30% w/w and 15% w/w cod liver oil in the region 400–4000 cm− 1 respectively. The absorption peaks at 3454 cm− 1 and 1630 cm− 1 region are attributed to the stretching and bending vibration of O–H groups from chitosan and omega3 structure in cod liver oil. The obtained peaks at 2884 cm− 1, 1650 cm− 1, 1600 cm− 1 that expressed existence of stretching mode C-H, CH2 groups, C = O, C = C and C-N region in chitosan omega3 in cod liver oil and poly lactic acid structures. The reflectance at 3093 cm− 1 shows N-H band in chitosan. In general, it can be said that cod liver oil structures with forming chemical bonds are located in nanobio polymeric nanoscaffold structures.
Here Fig. 3a, 3b and 3c
3.2. In vivo Study
Diabetic rats were evaluated in two ways; a) blood glucose measurement by glucometer, rats with blood glucose above 200 mg / dl were considered diabetic and selected for further study, b) the selected rats were examined for the appearance of diabetic symptoms including polyuria, overeating and thirst and were assured of their diabetic status. The wound healing process in diabetic rats in the four study groups was evaluated on days 0, 3, 7 and 14 by measuring the wound area and were presented in Table 2. As shown in the results and tables, in the group using nanofibers mixed with cod liver oil, the wound healing process (assessed by measuring the wound area) was investigated. Significantly, it has shown better results than the group that used nanofiber alone or cod liver oil alone. It also appears that 30% cod liver oil supplementation with polyelactic acid / chitosan nanoscaffolds heals 94.5% wound healing on day 14, whereas 15% cod liver oil can heal wounds about 86% on day 14 which were presented in Table 3. Macroscopic changes of the wound were evaluated for treatment progress on days 0, 3, 7 and 14 after treatment and recorded with a photograph. The percentage of wound healing was calculated using the following formula:
Table 2
Wound healing process (Wound Area in mm2) studied on days 0, 3, 7 and 14.
Day | Zero | Three | Seven | Fourteenth |
Control group | 152.67 | 133.88 | 116.85 | 87.05 |
Nanofiber + cod liver oil 30% | 154.15 | 96.60 | 54.31 | 8.43 |
Nanofiber + cod liver oil 15% | 154.15 | 102.83 | 71.61 | 20.07 |
Poly Lactic Acid / Chitosan Nanofibers | 154.15 | 133.88 | 100.81 | 61.53 |
Drug soluble group | 154.15 | 110.30 | 93.18 | 61.81 |
Table 3
Evaluation of wound healing rate in study groups studied on days 7 and 14.
Percentage of recovery per day | Seven | Fourteenth |
Control group | 23.5 | 42.0 |
Nanofiber + cod liver oil 30% | 64.8 | 94.5 |
Nanofiber + cod liver oil 15% | 53.5 | 86.0 |
Poly Lactic Acid / Chitosan Nanofibers | 34.1 | 60.0 |
Drug soluble group | 39.5 | 59.9 |
Percentage of recovery= (Surface wound on the first day- Surface wound in day X) / (Surface wound on the first day) × 100 Eq. 2
Figure 4 demonstrated a 14-day course examination of the wound surface and a photograph of the wound healing process. At day zero, third, seventh, and fourteenth, the rate of wound healing in the nanoscaffold saline-treated mice with 30% cod oil was significantly different from that of the untreated control group and also from the other groups. This prospect which poly lactic acid / chitosan nanoscaffolds as biocompatible and biodegradable polymers able to interact with skin cells and their environment and accelerate the healing process. Due to the high porosity of the nanoscaffold coating and also due to the hydrogel like properties of the polymers used, the coating swells after absorption of moisture and creates a very small gap between the coating and the wound surface. On the 14th day, these characteristics are well observed and the nanoscaffold porosity property permits oxygen to pass through the wound while keeping its surface moist.
Here Fig. 4
Wound area in five groups of animals studied on days 0, 3, 7 and 14 (Mean ± sd; N = 3) is shown in Fig. 5. As can be seen from the diagram, the area of the wound in the group with 30% cod oil was significantly less than the other groups, this indicates a greater improvement in this group than in the other groups. The presence of biocompatible nanofibers not only induces immune and allergic responses but also induces the body to resemble the original tissue located in the wound. As a result, biochemical signals are needed to accelerate recovery and eventually wound healing will occur faster.
Here Fig. 5