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The effect of chrysin–curcumin-loaded nanofibres on the wound-healing process in male rats

Abstract Aim The aim of the present study was to produce chrysin–curcumin-loaded PCL-PEG nanofibres by an electrospinning technique and to evaluate the biological activity of the chrysin–curcumin-loaded PCL-PEG fibres for wound healing and its related genes using in vivo methods. Materials and methods The electrospinning method was carried out for the preparation of the chrysin, curcumin and chrysin–curcumin-loaded PCL-PEG nanofibres with different concentrations. FTIR and SEM were performed to characterize the chemical structures and morphology of the nanofibres. In vitro drug release, as well as in vivo wound-healing studies were investigated in male rats. The expressions of genes related to the wound-healing process were also evaluated by real-time PCR. Results Our study showed that the chrysin–curcumin-loaded nanofibres have anti-inflammatory properties in several stages of the wound-healing process by affecting the IL-6, MMP-2, TIMP-1, TIMP-2 and iNOS gene expression. Our results demonstrated that the effect of the chrysin-loaded nanofibre, the curcumin-loaded nanofibre and the chrysin–curcumin-loaded nanofibre in the wound-healing process is dose dependent and in accordance with the obtained results in that it might affect the inflammation phase more than the other stages of the wound-healing process. Conclusion We have introduced chrysin–curcumin-loaded PCL-PEG nanofibres as a novel compound for shortening the duration of the wound-healing process.


Introduction
Chronic wound healing can result in infection and eventually cancer. One of the aims of modern medicine is shortening wound-healing time, increasing bioavailability and reducing the side effects of drugs [1][2][3]. Among the many proposed wound-healing protocols, physical methods such as infrared waves [4], high-voltage electric waves [5], and drug delivery involving biomolecules such as endostatin [6], vitamin A [7], ascorbic acid [8] and growth factors [9] can be considered. A myriad of studies has also introduced natural compounds and "therapeutic agents" for decreasing wound "or ulcer" healing time [10][11][12][13] for decreasing wound-healing time. However, none of these approaches has been widely accepted to be effective enough since wound healing is a very complicated biological process that involves intracellular and extracellular pathways [14]. Different types of immune cells, endothelial cells, keratinocytes and fibroblasts play a pivotal role in wound healing. Moreover, the proliferation, differentiation and cellular migration of the aforementioned cells can play a vital role in the wound healing process [15][16][17].
Using a high dose of antibiotics also appears to not have a strong effect on the wound-healing process since sometimes a low level of bacteria may aid in the wound-healing process through the recruitment of immune cells [18]. Thus, a balance of the normal flora should be maintained in the wound area [19]. In this regard, several studies showed that natural compounds such as herbal and mushroom extracts possess biomedical potential [20][21][22][23][24]. It has been demonstrated that honey can remove harmful bacteria such as E.coli, salmonella and Staphylococcus aureus [25,26] and also plays a role in maintaining wound moisture which is necessary for healing [27,28]. It has also been declared that honey can promote angiogenesis in the wound area and epithelial tissue regeneration [27].
Chrysin (5,7-dihydroxyflavone) is a natural and biologically active flavonoid in many extracts, such as plants gum, honey, and propolis. Previous studies have reported that Chrysin possesses potent anti-inflammatory and anti-oxidant properties [29,30]. On the other hand, curcumin is a naturally occurring polyphenolic compound with a broad range of favourable biological functions, including anti-cancer, antioxidant and anti-inflammatory activities [31]. The low bioavailability and in vivo stability of curcumin, however, require the development of suitable carrier vehicles to deliver the molecule in a sustained manner at therapeutic levels.
Despite the aforementioned functions, the poor water solubility of chrysin and curcumin hinders its application as a proper drug for wound healing [32][33][34]. This limitation calls for the creation of an appropriate conveyor to increase the in vivo stability of the bioactive compound, bioavailability and eliminate their poor water solubility.
The wound-healing process involves a series of genes such as IL-6 [35,36], tissue inhibitor of metalloproteinase (TIMPs) [37,38], inducible nitric oxide synthase (iNOS) [39,40] and matrix metalloproteinases (MMPs) [41,42]. There are several studies that suggest the role of IL-6 in the early phase of wound healing [35,43]. MMPs are involved in extracellular matrix (ECM) degradation which makes for a poor wound healing process. On the other hand, TIMPs are essential for wound healing in smooth muscle cells, keratinocytes, fibroblasts and also endothelial cells by targeting MMPs [44]. Furthermore, iNOS plays a significant role in wound healing when free radical levels are elevated [45]. In recent years, the effect of reactive oxygen species (ROS) is under focus in different tissues because of its great influence on healing pathways [46][47][48][49].
The aim of the present study is to fabricate chrysin-curcumin-loaded PCL-PEG nanofibres using the electrospinning technique [50,51] and then evaluate the biological activity of the chrysin-curcumin-loaded PCL-PEG fibres on the wound healing process and its related genes using an in vivo rat method.

Materials
Polyethylene glycol (PEG) with a molecular weight (Mw) of 4000, dichloromethane, e-caprolactone (e-CL), dimethyl sulfoxide (DMSO), diethyl ether and methanol were purchased from Merck (Darmstadt, Germany). Tween20, chrysin and curcumin were provided by Sigma-Aldrich (St Louis, MO, USA) and Stannous octoate (Sn(Oct) 2 ) was supplied by Alfa Aesar. Healthy male rats (2 and 3 weeks of age) were purchased from Pasteur Institute of Iran. TRIzol reagent, the QuantiTect reverse transcription kit, and the QuantiTect SYBR Green realtime PCR (RT-PCR) kit were purchased from Roche (Mannheim, Germany). A cDNA synthesis kit and a Gene JET RNA purification kit were purchased from Thermo Fisher Scientific (Waltham, USA).

Synthesis of PCL/PEG/PCL triblock copolymer
The most methods that follow goes on, described before in our previous study [1]. As we showed in our previous study, PCL/PEG/PCL copolymers were prepared by the ring-opening polymerization (ROP) of e-CL in the presence of PEG, using stannous octoate as a catalyst. In summary, 1 g of polyethylene glycol (PEG4000), 10 ml of e-CL, and Sn(Oct) 2 (0.5 wt.% of monomers) were added to a 25-ml round-bottom flask supported by a nitrogen atmosphere [1]. The solution was heated to 130 C for 6 h while stirring. dichloromethane was used for dissolving the resulting polymer and precipitated in cold diethyl ether three times and after that dried at room temperature for 72 h.
Finally, a PCL-PEG-PCL triblock copolymer with a ratio of 10% (w/v) was added to the above-described solutions and stirred for 24 h at room temperature. A 5-ml plastic syringe with a blunt-ended metal needle tip (gauge 16) was used for each solution. The spinning solution was delivered at a controlled feeding rate of 2 ml/h, and the voltage between the ground collector on the electrospinning device and the needle tip (Fanavaran Nano-Meghyas, Tehran, Iran) was adjusted to a range of 28-30 kV. The distance between the nozzle and the collector and the rotating speed of the drum were fixed at 10 cm and 200 rpm, respectively. The metal collector was covered with an aluminium foil. All electrospinning experiments were carried out at room temperature [52,53]. To remove the excess DMSO after electrospinning, all nanofibres were washed with deionized water and dried in vacuum at 40 C.

Fourier transform infrared spectroscopy (FTIR)
The chemical structure of the PCL-PEG nanofibres, as well as the chrysin-and curcumin-loaded PCL-PEG nanofibres were performed by FTIR. The FTIR spectra of the nanofibres were obtained by using a Shimadzu 8400 s infrared spectrophotometer (Kyoto, Japan).

In vitro chrysin and curcumin release assay
To investigate the release kinetics of chrysin and curcumin from the nanofibres mats, a piece of fibre mat (20-30 mg, 25Â25Â0.1 mm 3 ) was placed into a 20 ml vial of phosphatebuffered saline (PBS, pH 7.4) containing Tween 20 (0.5%) and DMSO (5%) following by incubation at 37 C with a shaking rate of 150 rpm for 15 days. In specific intervals, 1 ml of the media solution was taken to evaluate the absorbance of the chrysin and curcumin nanofibres by a UV spectrophotometer at a wavelength of 425 nm, and then, 1 ml fresh buffer was replaced in the solution. The buffer solution was used for the medium to determine the calibration curve. The release percentages of the chrysin and curcumin nanofibres were calculated according to the following equation: In vivo wound-healing studies In the Faculty of Advanced Sciences at Tabriz University of Medical Sciences this experiment was conducted. We purchased ninety male rats (weight: 250-300 grams) from the Tabriz University of Medical Sciences central animal house, and they caged in pathogen-free and in addition to food and water ad libitum, facilities on a 12-h12-h lightdark cycle. University of Tabriz animal ethics committee have approved all experiments. The rats were allocated to 10 groups and each group included nine rats. The most steps of wound creation and the other manners method is similar to our previous study [1]. After anaesthesia, we created an open excision-type wound (2 Â 2 cm 2 ) on the thoracic-lumbar region of the rats to the depth of the panniculus carnosus region of rats. The wound was not dressed or covered. After recovery of the animals from anaesthesia, appropriate disinfected individual cages were prepared for caging them. All nanofibres were UV sterilized before use and each of them was placed on the wound site and slightly around the wound. We considered 10 groups; The first group was control group which received the PCL-PEG nanofibres for treatment while the 2nd, 3th and 4th groups were treated with 5% (w/w), 10% (w/w) and 15% (w/w) chrysin-loaded nanofibres and the 5th, 6th and 7th groups received 5% (w/w), 10% (w/w) and 15% (w/w) curcumin-loaded nanofibres, respectively. The 8th, 9th and 10th groups were treated with the chrysin 5%-curcumin 10% (w/w), chrysin 10%-curcumin 5% (w/w) and chrysin 7.5%-curcumin 7.5% (w/w) loaded nanofibres, respectively. Differences in the wound area were measured at 5, 10 and 15 days after treatment with nanofibres. The extent of wound healing was reported as the percentage of wound area that remained exposed. Each group, as well as the control, were tested on three rats and the mean value was recorded.
Wound size % ð Þ ¼ W 5: 10 and 15 W 0 Â 100 where W 5, W 10 and W 15 are the wound areas after 5, 10 and 15 days treatment, and W 0 is the initial wound area. Also wound sampling was done at 5, 10 and 15 days after treatment. At the end of each treatment time point, full-thickness sections of the wound were dissected and stored in À70 C until RNA extraction.

RNA extraction and cDNA synthetic
As described before [1], the sections which included full thickness of the wound were stored in À70 C until RNA extraction. For RNA recovery from tissue biopsies by disruption, 500 ml TRIzol solution (Invitrogen, Carlsbad, CA, USA) was used, and then, for homogenization, we used a pestle mixer (Scientific Specialties, Randallstown, MD, USA), and then, RNA was extracted in 50 ml chloroform that prepared form Sigma-Aldrich. After that, isopropanol (Sigma, Castle Hill, NSW, Australia) used for precipitating RNA from the aqueous phase, and ice-cold 75% (w/w) ethanol used for washing the pellet. Then, in 100 ll Milli-Q water the pellet was dried and resuspended. 1 ll of Oligo (dT) primers, 1 lg of total RNA and 11 ll of diethylpyrocarbonate (DEPC) treated water were mixed in dry ice followed by incubation at 65 C for 5 min and then stored on dry ice for 1 min to synthesize the complementary DNA (cDNA). 4 ll of 5X reaction buffer, 1 ll of RiboLockTM RNase Inhibitor (20 U/ll) and 2 ll of (10 mM) dNTP Mix, mixed as the reaction mixture of the components, was added before incubation at 25 C for 5 min. 2 ll of M-MuL V reverse transcriptase (20 U/ll) was used for RNA reverse-transcription and then incubated at 25 C for 5 min and further incubated at 42 C for 60 min. The reaction was terminated at 70 C for 5 min.

Real-time quantitative PCR
Based on the same previous method [1], for all tissue samples, reverse transcription was performed in a single batch and in the same PCR run were analysed for a given primer Table 1. The primer sequences used in the present study.
Primer Sequences set. Each reaction volume (20 ml total) in the PCR contained 3 ll of cDNA and a SYBR Green PCR Master Mix that purchased from Applied Biosystems was used as the amplification reagent supplement. The program for real-time PCR included the following: initial denaturation at 95 C for 10 s, followed by cycles of denaturation at 94 C for 15 s, annealing at 58 C for 30 s, and 72 C for 30 s. After that, the melting curve analysis of 70 C to 95 C used for assessing the amplicons. Table 1 reports the primers sequences for the RT-PCR. The 2 ÀDDCT method was used to calculate DCT values in relation to b-actin CT values, in which DCT used for representing the difference between the CT value of the target genes and the CT value of b-actin.

Morphology of the nanofibrous scaffolds
To investigate the physicochemical characterization of the nanofibres, the scaffolds were characterized by SEM. The average diameter of the PCL-PEG nanofibres and chrysinloaded PCL-PEG nanofibres, curcumin-loaded PCL-PEG nanofibres and the chrysin-curcumin-loaded PCL-PEG nanofibres were in the range of 50-300 nm. Figure 1(A) shows the drugfree nanofibre of PCL-PEG with a nearly smooth surface and net-like structure. Likewise, the chrysin-and curcumin-loaded PCL-PEG nanofibre at 5% (w/w) showed a net-like structure with beads (Figures 1(B,E)). Increasing the chrysin concentration to 10% (w/w) significantly changed the size of beads and granular beads (Figure 1(C)). According to Figure 1(D), although the chrysin-loaded PCL-PEG nanofibres 15% (w/w) were almost the same as the 10% (w/w), the granular beads were more populated. As shown in Figure 1(F), the diameter of the nanofibres increased in the curcumin-loaded PCL-PEG nanofibre 10% (w/w) and according to Figure 1(G) the diameter increased even more for the curcumin-loaded PCL-PEG nanofibre 15% (w/w). Figure 1(H) reveals that the diameter of nanofibres increased for the 5% chrysin 10% curcumin (w/w). According to the Figure 1(I,J) which is related to 7.5% chrysin-7.5% curcumin (w/w) and 10% chrysin-5% curcumin (w/w), the beads transformed to the granular surface and their diameter increased. Due to the precipitation of curcumin at higher concentrations, the maximum concentration of curcumin that could be loaded in the PCL-PEG nanofibres was 15% (w/w).  PCL-PEG nanofibre, the chrysin PCL-PEG nanofibre and the curcumin-chrysin PCL-PEG nanofibre, respectively.

FTIR results
In vitro release study Figure 3 shows the release behaviour of the chrysin and curcumin in PBS from the nanofibre mat (A: chrysin and B: curcumin). The maximum amount of chrysin and curcumin were released from the nanofibres for day 1 (Figure 3(A,B)). As shown, the maximum release of chrysin and curcumin from the nanofibres is related to the amount of chrysin and curcumin. For example, the maximum release of chrysin from the nanofibre mat with 5, 10 and 15% chrysin was about 59, 68 and 81.5%, respectively. Based on the Figure 3(A), the in vitro release of chrysin from the nanofibre mat was greater and faster than the curcumin.
In vivo wound closure study Figure 4 shows the picture of one of the rats treated with curcumin nanofibres (using chrysin 7.5%-curcumin 7.5% nanofibre, 5 days after treatment). The wound closure percentage in all rats was measured on 5, 10, and 15 days post-injury and the effect of chrysin, curcumin and chrysin-curcumin-loaded nanofibres on wound closure is shown in Figure 5(A-C), respectively. According to these graphs, in all groups, the percentage of wound closure was   higher than controls. In higher doses, the chrysin-loaded nanofibre significantly promoted the wound-healing process, in a way that the percentage of wound closure on day 15 was 78, 82 and 88%, for 5, 10 and the 15% chrysin-loaded nanofibres, respectively ( Figure 5(A)). On day 15, the percentage of wound closure for 5, 10, and 15% curcumin nanofibre was around 85, 87, and 92%, respectively (Figure 4(B)). In general, although the wound closure efficacy of the nanofibres containing curcumin was higher than that of chrysin, there was no significant difference between the different percentage of curcumin compared to chrysin. Figure 5 also demonstrates that there is no synergistic effect between chrysin and curcumin for wound closure.

The effect of chrysin and curcumin nanofibres on IL-6 expression
On day 5, the results revealed that the chrysin-loaded PCL-PEG nanofibre 15% (w/w) significantly elevated the expression of IL-6 compared to 5, 10% (w/w) and the curcumin-loaded PCL-PEG 10% (w/w) more significantly elevated the expression of IL-6 than 5, 15% (w/w) in comparison with the control group (p values <.05). Likewise, the chrysin-curcumin-loaded PCL-PEG 7.5% (w/w) more significantly elevated IL-6 expression than the other chrysin-curcumin-loaded PCL-PEG nanofibres compared to the control group (p values <.05) (Figure 6(A)).
The effect of chrysin and curcumin nanofibres on TIMP-1 expression On day 5, in comparison with the control group, the chrysinloaded PCL-PEG 15% (w/w) significantly elevated the expression of TIMP-1 compared to the nanofibres 5, 10% (w/w); and the same trend was observed in the effect of curcuminloaded PCL-PEG 15% (w/w) on the expression of TIMP-1 than the nanofibres 5, 10% (w/w) (p values <.05). Likewise, the chrysin 5%-curcumin 10% remarkably elevated the expression of TIMP-1 more than the chrysin 10%-curcumin 5% loaded PCL-PEG and the chrysin-curcumin-loaded PCL-PEG 7.5% (w/w) (p values <.05) (Figure 8(A)). On day 10, the chrysinloaded PCL-PEG 10% (w/w) promoted the expression of TIMP-1 more than the nanofibres 5, 15% (w/w) and the control group; and a similar trend was noticed for the curcumin-loaded PCL-PEG 15% (w/w) in the expression of TIMP-1 compared to the nanofibres 5, 10% (w/w) and the control group, but these levels were not significantly different. In comparison with control group, the chrysin 5%-curcumin 10% elevated the expression of TIMP-1 more than the chrysin  10%-curcumin5% loaded PCL-PEG and the chrysin-curcuminloaded PCL-PEG 7.5% (w/w), but these levels were not significant (Figure 8(B)).

Discussion
The purpose of using the nanofibres in this study was to solve the problem associated with the insolubility of chrysin and curcumin-based on a simple principle that increasing the surface area of the drug and carrier can enhance the dissolution rate of the drug and provide a helpful pathway for the delivery of water-insoluble drugs. Therefore, the chrysin-curcumin-loaded nanofibre can be a more effective and promising candidate in wound healing applications [54].
This interesting phenomenon for the chrysin-curcuminloaded nanofibre has not been reported elsewhere. In this study, chrysin and curcumin presented a positive effect on an open wound-healing model in rats and also its effect on the expression of the different type of genes that are related to wound healing such as IL-6, TIMPs, iNOS and MMPs were systematically investigated.
IL-6 plays a critical role in inflammation and is produced by inflammatory cells [43,55]. Based on previous studies, IL-6 has a regulatory role in leukocyte recruitment in inflammatory conditions [56,57]. Also, it has been shown that IL-6 deficient mice have a low level of macrophages and neutrophils [36]. As the results show, chrysin and curcumin nanofibres elevated IL-6 gene expression during the wound-healing process after 10 days, and on day 10, the maximum expression was elevated by chrysin 7.5%-curcumin 7.5% loaded PCL-PEG. Based on these results, high doses of curcumin play a crucial role in inducing IL-6 expression.
Previous studies have been shown that MMP-2 gene is expressed at wound sites during the healing process and also MMPs have a crucial role in keratinocyte detachment from the basement membrane and the other healing process [58][59][60][61][62]. Based on these results, MMP-2 gene expression was variable between groups for day 5 and day 10, so that in chrysin and curcumin groups, the 15% concentration elevated more MMP-2 expression and chrysin 10%-curcumin 5% loaded PCL-PEG reduced MMP-2 expression. For day 10, MMP-2 gene expression reduced in comparison with day 5 and also the curcumin group, especially curcumin 5% more greatly reduced MMP-2 expression.
TIMP genes are related to the cell proliferation stage in the process of wound healing [23], and any disturbance in the expression of these genes may lead to a delayed wound healing [36]. As our results showed, the expression of TIMP-1 reduced from day 1 to day 10. Also, it has been shown that for day 5, a higher level of TIMP-1 expression was related to the chrysin 5%-curcumin 10% loaded PCL-PEG, and for day 10, it was related to the chrysin 10% loaded PCL-PEG. On day 5, TIMP-2 expression higher level was related to chrysin 5%-curcumin 10% loaded PCL-PEG and for day 10, it was related to the chrysin 10%-curcumin 5% loaded PCL-PEG, but generally, TIMP-2 gene expression levels for day 10 were lower than day 5.
Wound-healing processes have an interconnected relationship with many components and one of these components is iNOS which plays a key role in wound healing and closure [63]. As the present results showed, the down-regulation of iNOS gene for day 5 was related to the chrysin 5%-curcumin 10% loaded PCL-PEG and for day 10, the chrysin group in comparison with curcumin group more greatly reduced iNOS gene expression especially chrysin 15%. On the other hand, chrysin 7.5%-curcumin 7.5% loaded PCL-PEG reduced more iNOS gene expression than all of the other groups.

Conclusion
In conclusion, we have introduced here chrysin-curcuminloaded PCL-PEG nanofibres as a novel compound to shorten the duration of the wound-healing process. The electrospinning of a PCL-PEG-chrysin-curcumin solution was performed and ended by the production of a yellow colour electrospun mat without any significant accumulation of chrysin on the surface. Our results showed that the chrysin-curcumin-loaded nanofibres have anti-inflammatory properties in several stages of the wound-healing process by affecting the IL-6, MMP-2, TIMP-1, TIMP-2 and iNOS gene expression. These results demonstrated that the effect of the chrysin-loaded nanofibre, the curcumin-loaded nanofibre and the chrysin-curcumin-loaded nanofibre in the wound-healing process is somewhat dose dependent and in accordance with the obtained results, it might affect the inflammation phase more than the other stages of wound-healing process. In this study, we focused on gene expression related to the inflammation phase and the evaluation of the expression of genes related to other phases, the proliferation and wound closure properties need to be investigated further.