Facile fabrication of carboxymethylcellulose/ZnO/g-C3N4 containing nutmeg extract with photocatalytic performance for infected wound healing

New topical antibacterial agents are required to inhibit and development of bacteria and also promoting the wound healing process. This study was evaluating the healing effect of Myristica fragrans extract coated with carboxymethyl cellulose, zinc oxide and graphite carbon nitride (CMC/ZnO/g-C3N4/MyR) by photocatalytic process on the healing process of full-thickness infectious excision wounds in mice. Nanosheets were prepared and physicochemical properties were evaluated. Safety, in vitro release, antibacterial activities under in vitro and in vivo condition, wound contraction, histopathological properties and the protein expressions of tumor necrosis factor-α (TNF-α), collagen 1A (COL1A) and CD31 were also evaluated. Physicochemical properties confirmed their successful synthesis. Nanosheets exhibited antibacterial activity under in vitro and in vivo conditions. The formulations containing CMC/ZnO/g-C3N4/MyR, significantly (P < 0.05) competed with standard ointment of mupirocin for accelerating the wound healing process due to their effects on bacterial count and the expression of TNF-α and also accelerating the proliferative phase. This structure can be used as a safe structure in combination with other agents for accelerating the wound healing process following future clinical studies.

www.nature.com/scientificreports/Wound infection is a common nosocomial infection that influences people all over the world and causes a high mortality rate 1 .Wound infection is due to bacterial pathogens entered into the body through the skin gaps such as Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii 2 .The preparation of a clean and moist wound environment that can support the physiological wound healing process is a managerial strategy for wound management 3 .Broad use of antibiotics results in drug resistance and the production of superbugs bacteria and can cause side effects on the immune system 4 .New topical antibacterial agents are required to inhibit the further spread and development of complications in fighting bacterial infections 5 .Medicinal plants have been used for accelerating infected wounds.
Nutmeg (Myristica fragrans) or MyR is a commercial source used as antithrombotic, antitumor, and antiinflammatory 5 .It is a rich source of bioactive substances with antibacterial and antioxidant activities 6 .It is known to have the wound healing activities 7,8 .There are problems in dispersion of plant derivations and also drug delivery.It is essential to coating plant derivations with safe carriers with good water dispersibility such as nanosheets 9 .In addition, a combination of nanosheets and extracts can have better antimicrobial properties 10 .
The inorganic particles such as ZnO have antibacterial activities, antidiabetic and anticancer activities 11 .ZnO are economic and safe with optical and excellent thermal and chemical stability 12 .It is a candidate for the wound healing process due to its biological properties.In addition, a progressive opted strategy is producing hybrid nanostructures of ZnO via its coupling with other fascinating semiconducting materials.This work results in the preparation of structures with maximum biological and mechanical properties 13 .Graphitic carbon nitride (g-C3N4) is a metal-free photocatalyst that has attracted many attentions for the production of heterojunctions in the entire scientific society due to properties such as chemical and thermal stability, faster charge transport, and ability absorbing light 14 .It can be utilized as a photothermal agent for photothermal therapy 15 .Zn 2+ doping could increase the photocatalytic activity of g-C3N4, and promote epithelial formation in the wound healing process 4 .Another agent which can be used for tissue engineering is carboxymethyl cellulose (CMC).The dressings based on natural polymers have been used for biomedical applications 16 .It has stability and biocompatibility properties 17 .It may have synergistic effects with g-C3N4 and Zn 2+ for mechanical and biological properties in the treatment of wound.
It was hypothesized that CMC/ZnO/g-C3N4 can coat MyR and accelerate the wound healing process in the presence of visible light and have synergistic effects for the treatment of infected wounds.This study evaluates the effects of CMC/ZnO/g-C3N4/MyR on the treatment of infected wounds by assessing physicochemical properties, antibacterial activity and the expressions of tumor necrosis factor-α (TNF-α), angiogenesis (CD31) and collagen type 1A (COL1A).

Results and discussion
g-C3N4 and metal oxides such as ZnO are the active semiconductors under visible light with a wide forbidden strip and can only be activated by ultraviolet light.When these particles absorb photons with higher energy than their banned strips, couples of electrons and cavities are created in the conduction bar and their capacity strips, respectively (Eq. 1).
High-efficiency separation in this couple will allow oxidation and reduction reactions on the particle surface.In these reactions, molecular oxygen commonly works as an electron acceptor and is converted into superoxide anion (O 2 −⋅ which is a strong oxidation agent, in fact e − in CB (conduction bar) helps to produce superoxide radicals from oxygen molecules (Eq.2).
On the other hand, the cavity with surface hydroxyl group (OH − ) with a water molecule (water in the hydrogel or wound bed) absorbed on the surface of the reaction produces a hydroxyl radical (OH°), which is a strong reducing agent.H + in VB (capacity bar) plays a key role in the production of hydroxyl radicals from H2O molecules or hydroxyl ions 18,19 (Eq. 3) Reactive oxygen species (ROS; HO ⋅ , O 2 −⋅ ) are a key component in photocatalytic process.On the other hand, the rapid recombination of the produced electron-cavitational, their photocatalytic activity has been accompanied by limitations.To improve the performance of such potential semiconductors, useful solutions have been taken to pair the two semiconductors together and place them on a suitable substrate 20,21 .

Structural characterization
To demonstrate the feasibility of this project, g-C3N4 was synthesized from melamine powder according to Fig. 2A and based on previous reports 25 .To obtain more g-C3N4 nanosheets, sonication was performed in water and the obtained g-C3N4 had several single layers based on the characteristics of electron-scanning microscopy (Fig. 2B).The g-C3N4 electron microscopy images show free standing nanosheets with a thickness size of 51 nm.Also, the distribution of C and N elements can be seen in dot mapping next to SEM.The g-C3N4 nanosheets were easily dispersible in water due to the presence of -NH2 and -NH groups which were confirmed by Fourier transform infrared spectroscopy (FTIR), it is shown in Fig. 6.Such groups -NH2 and -NH can act as an effective Lewis base for charging metal ions via chelate and have a higher binding affinity to ZnO (Fig. 3) and thus provide potential growth of nanoparticles at g-C3N4 level 26 .Accordingly, g-C3N4@ZnO a can be synthesized by a facile and simple reduction method in place at room temperature.SEM images (Fig. 3A,B) and histograms of the corresponding size distribution (Fig. 3C) showed that a medium density of dispersed ZnONP with an average size of 23 nm was precipitated on g-C3N4 surface and no accumulated ZnONP were observed.In addition, ZnONP modification on g-C3N4 surface was confirmed by energy dispersed X-ray spectroscopy (EDS) (Fig. 3D).Based on the EDS results obtained from ZnO/g-C3N4 sample, 23.23% carbon, 6.28% nitrogen, 51.26% Zn and 19.23% oxygen were found.Dot mapping results (Fig. 3E) showed a uniform dispersion of elements on the surface of nanosheets.ZnO/g-C3N4 is located on CMC substrate, and SEM images (Fig. 4A) and dot mapping (Fig. 4B) are shown that the proper and uniform distribution of elements (C, N, O, and Zn) in CMC substrate is visible.www.nature.com/scientificreports/www.nature.com/scientificreports/XRD patterns were used to identify the crystalline structure of the materials (Fig. 5).The index peak for g-C3N4 sample at 13.49 °C is related to tri-s-triazine units and intra-plate structural accumulation and is dedicated to the plate (100).The peak of the index is 27.58 °C to the plate (002) and shows that the conjugated aromatic groups of g-C3N4 layers have appeared with the JCPDS No reference card (87-1526) 27 .For example, ZnO index peaks were 32.08°, 34.5°, 36.31°,47.78°, 56.80°, 63.07° and 68.31° which correspond to crystalline plates Hexagonal structure (100), (002), ( 101), ( 102), ( 110), ( 103) and (112) and were in accordance with reference card (JCPDS No. 36-1451) 28 .For the broad peak polymer matrix in the range of 20.4, the amorphous state of CMC is well represented.In ZnO/g-C3N4 and CMC/ZnO/g-C3N4 peaks of crystalline index were visible in ZnO and g-C3N4 that confirmed the synthesis of the final product (Eq.4).www.nature.com/scientificreports/In this equation, d is the average particle size (nm), k = 0.89, 0.154060 = λ nm for Cu Ka and β full width is half the peak maximum.Using the Debye-Scherr equation, the mean crystal size of ZnO/g-C3N4 nanoparticles and CMC/ZnO/g-C3N4 were 26.14 and 77.43 nm, respectively, which was consistent with other analyses such as SEM, DLS and PSD.The FTIR spectrum of g-C3N4, CMC, ZnO and CMC/ZnO/g-C3N4 sheets is shown in Fig. 6.The pure FTIR spectrum of g-C3N4 has a wide absorption band from 3000 to 3400, which shows tensile states (NH-) and (NH2-).Peaks at 1177 cm −1 , 1428 cm −1 , 1545 cm −1 and 1654 cm −1 are related to bonds (C-N) and (C=N).In addition, the peak shown in 810 cm −1 is related to the ES-triazine ring units 29 .In the CMC peak spectrum appeared at 3600-13,400 cm −1 is associated with the stretch vibrations of aromatic ring hydroxyl groups and the peaks that appeared in 1360 cm −1 are related to the asymmetric tensile vibrations of methyl (-CH) carboxymethyl cellulose groups.The asymmetric tensile vibration of the COO group closed to 1637 cm −1 and vibration at 1093 cm −1 was assigned to C-O tensile vibration.Zn-O extended band was seen in the range of 560-430 cm −130 .It can be seen that all peaks in the first three spectrums are visible in the fourth peak, indicating the successful synthesis of the CMC/ZnO/g-C3N4 composite.
The chemical composition and oxidation state of the elements were determined by XPS.As shown in Fig. 7, the wide range of XPS, CMC/ZnO/g-C3N4 shows that the index peaks are related to C, N, O and Zn.As the XPS C1s spectrum shows in Fig. Particle size and zeta potential values for g-C3N4, ZnO/g-C3N4 and CMC/ZnO/g-C3N4 nano sheets are shown in Fig. 8 diagram (Fig. S1).Zeta potential plays an important role in the physical stability of particles 17 .Higher values of zeta potential (positive and negative) indicate its stability for nanoparticles.Zeta potential for g-C3N4 is negative (− 2.3 mV), and adding ZnO results in value positive (+ 29.1 mV).Decorating the surface of the CMC/ZnO/g-C3N4 nano sheets changed the zeta potential from positive to negative (− 23.4 mV) that is in agreement with previous studies 32,33 .
Figure 9A shows the in vitro release of free MyR and CMC/ZnO/g-C3N/MyR over 48 h.Considering that during the wound healing process, the pH of the wound area changes dynamically from pH 4 to pH 8, for growth factor secretion and activation 34,35 .The underlying wound tissue shows a neutral pH of 7.4.As a result, the release profile of CMC/ZnO/g-C3N4 containing MyR at pH 7.4 was investigated.In vitro release was significantly higher in free MyR compared to CMC/ZnO/g-C3N/MyR.During the first 4 h, 63.17% of MyR was released from MyR www.nature.com/scientificreports/solution in the buffering environment while CMC/ZnO/g-C3N4/MyR about 23.63% is released in the first 4 h.Over time up to 8 h, the released values were 91.92%, 60.37% of dialysis bags for free MyR, CMC/ZnO/g-C3N4/ MyR respectively.The possible reason is the complex polymer matrix network containing CMC/ZnO/g-C3N4, which releases it slowly.Since this matrix is also considerably resistant to degradation, the emission of the extract from the polymer matrix takes a longer time.With the slow release, probably due to the decrease in MyR concentration in healthy tissues and reduction of the toxicity of the extract, the coated form of nutmeg can have equal effects on the free drug in the treatment of infections and wounds, and polymeric coating can be used to control the release of the extract.

Cytotoxicity
Figure 9B illustrates the results for the cytotoxicity of nanosheets.The results showed that nanosheets had lower toxicity.The highest toxicity was observed in the highest concentration (85.00%) for all the nanosheets.The different concentrations did not show significant differences.The findings are in accordance with literature for the  www.nature.com/scientificreports/safety of CMC 36 , ZnO 37 , g-C3N4 38 and MyR 39 .A combination of materials could not have significant cytotoxicity.Toxicity was mostly associated with higher concentrations but not combinations of materials.

Antibacterial activity
Table 2 shows in vitro antibacterial of the nanosheets in MIC and MBC tests.The results compared the effects of nanosheets compared with commercial agents.The results showed lowest antibacterial activities in MIC and MBC tests were observed in CMC/ZnO nanosheets.The addition of g-C3N4 could increase antibacterial activity for both bacteria.The addition of MyR could improve antibacterial activity compared with CMC/ZnO/g-C3N4.
The results did not show significant differences between commercial antibiotics with CMC/ZnO/g-C3N4/MyR nanosheet and commercial antibiotics in MIC test.However, CMC/ZnO/g-C3N4/MyR nanosheet exhibited www.nature.com/scientificreports/higher antibacterial activity compared with commercial antibiotics.The results are in accordance with previous studies for the antibacterial activity of CMC and ZnO 40,41 .Antibacterial activities of CMC/ZnO could be attributed to their interactions with reactive oxygen species and also the interaction of zinc oxide with bacterial cells wall and intra-cellular content of the cell such as protein, lipid, and carbohydrates which result in disruption of nucleic acids and bacteria death 42 .The addition of g-C3N4 increased antibacterial activity in nanosheets.The results are in agreement with other studies on the antibacterial activities of g-C3N4/ZnO 4 .The mechanism of g-C3N4/ZnO is attributed to their effects on disrupting membranes and increasing protein leakage, induction of bacterial apoptosis, and decreasing ATP levels 4 .The addition of MyR increased antibacterial activities.Major compounds of MyR were including 4-Terpinenyl acetate, γ-Terpinene, isoeugenol, oleic acid, nomifensine and carvacrol which disrupt bacterial membranes 43 .The results show synergistic effects between compounds for antibacterial activities.The CMC/ZnO/g-C3N4/MyR nanosheet could compete with commercial ointments.

Wound size and tissue bacteria
Figure 9C,D shows wound healing activity of nanosheets on different days.The results did not show significant differences between groups on day 3 (P = 0.914).The highest wound healing activity were observed in the mice treated with formulations prepared from CMC/ZnO/g-C3N4/MyR on days 7 (P = 0.001) and 12 (P = 0.001) compared with other groups.The values for wound contraction were 37.16 ± 0.90%, 51.60 ± 1.30%, 41.28 ± 1.60%, 47.33 ± 1.80% and 60.50 ± 1.90% in control, Mup, CMC/ZnO, CMC/ZnO/g-C 3 N 4 and CMC/ZnO/g-C 3 N 4 / MyR, respectively on day 7.The values for wound contraction were 84.21 ± 2.50%, 97.75 ± 2.10%, 91.62 ± 2.30%, 99.25 ± 0.85% and 99.56 ± 0.2% in control, Mup, CMC/ZnO, CMC/ZnO/g-C 3 N 4 and CMC/ZnO/g-C 3 N 4 /MyR, respectively on day 12.There were no significant differences between those treated with CMC/ZnO/g-C3N4 and mupirocin on days 7 and 12 (P > 0.05); however, these exhibited lower wound contraction compared with those treated with CMC/ZnO/g-C3N4/MyR.Wound contraction was lower in those treated with CMC/ZnO compared with those treated with nanosheets (P < 0.05).The lowest wound contraction was observed in control mice on days 7 and 12.The results are in agreement with other studies for wound healing activities of CMC 44 , ZnO 45 , g-C3N4 4 and MyR 8 .The results show synergistic effects between compounds for the wound healing process.The wound healing process of nanosheets could be attributed to their effects on bacteria and also the expression of genes.The nanosheets shorten the inflammatory phase and promote the wound healing process, as will be seen.Figure 9E illustrates the photocatalytic effects of nanosheets on total bacterial count on different days.The highest total bacterial count was observed in the control mice compared with other mice (P = 0.001).The mice treated with CMC/ZnO showed lower total bacterial count compared with those in control group in all the days (P = 0.001) but the same mice showed higher total bacterial count compared with those treated with mupirocin and other nanosheets on all the days.The addition of g-C3N4 and MyR could decrease total bacterial count and the lowest total bacterial count was seen in the mice treated with CMC/ZnO/g-C3N4/MyR on day 3 and 7.The values for total bacterial count were 7.15 ± 0.10 CFU/g, 5.38 ± 0.14 CFU/g, 6.50 ± 0.12 CFU/g, 5.55 ± 0.17 CFU/g and 5.46 ± 0.15 CFU/g in control, Mup, CMC/ZnO, CMC/ZnO/g-C 3 N 4 and CMC/ZnO/g-C 3 N 4 /MyR, respectively on day 3.The values were 4.58 ± 0.16 CFU/g, 1.65 ± 0.13 CFU/g, 2.45 ± 0.14 CFU/g, 1.70 ± 0.11 CFU/g and 1.21 ± 0.10 CFU/g in control, Mup, CMC/ZnO, CMC/ZnO/g-C 3 N 4 and CMC/ZnO/g-C 3 N 4 /MyR, respectively on day 7.The values were 1.30 ± 0.08 CFU/g, 0.1 ± 0.02 CFU/g, 0.6 ± 0.04 CFU/g, 0.12 ± 0.03 CFU/g and 0.11 ± 0.04 CFU/g in control, Mup, CMC/ZnO, CMC/ZnO/g-C 3 N 4 and CMC/ZnO/g-C 3 N 4 /MyR, respectively on day 12.It shows that CMC/ZnO/g-C3N4/MyR exhibits their effects on bacteria on first days.The mechanism of action of nanosheets was previously discussed under in vitro section.There is a good agreement between antibacterial results in both sections.www.nature.com/scientificreports/

Histopathological evaluation
The results for the effects of nanosheets on histopathological parameters are illustrated in Fig. 10.The results showed that edema was significantly higher in the control mice compared with other mice on days 3-12.The addition of g-C3N4 and MyR could decrease edema and the lowest total bacterial count was seen in the mice treated with CMC/ZnO/g-C3N4/MyR on all the days.The lowest fibroblast, and re-epithelization were observed in the control mice.The treatment of mice with commercial ointment and formulations prepared from CMC/ ZnO/g-C3N4/MyR and CMC/ZnO/g-C3N4 increased fibroblast, and re-epithelization.The mechanism of action of nanosheets on pathological parameters could be attributed to their photocatalytic effects on the expression of genes, as discussed.Seemingly, nanosheets shift the wound healing process from the inflammatory phase toward proliferative phase and expedite the wound healing process.

Immunofluorescent staining
The results for the effects of synthetized nanosheets on TNF-α, CD31 and COL1A protein expression with immunofluorescence staining technique are shown in Figs.11 and 12.The results showed that protein expression of CD31 and COL1A markers were significantly (P < 0.05) higher in the mice treated with CMC/ZnO/g-C3N4/MyR nanosheet formulation.The mice in control group showed lower the protein expression for CD31 and COL1A markers compared with other groups.The mice treated with mupirocin and CMC/ZnO/g-C3N4 had higher for collagen and CD31 compared with those treated with CMC/ZnO.The mice treated with CMC/ZnO/g-C3N4 and CMC/ZnO/g-C3N4/MyR showed significantly (P < 0.05) lower expression of protein for TNF-α compared with other groups.There were no significant differences between control mice and CMC/ZnO for the expression of TNF-α.Collagen plays a major role in contracting the wound healing process.The increase in the expression of collagen is due to the effects of nanosheets on the fibroblast proliferation.Previous studies indicated on M2 macrophages increase the fibroblasts migration and production of collagen 1 and 3 46 which results in wound www.nature.com/scientificreports/closure and accelerating wound healing process.In other hand, TNF-α is associated with M1 macrophage and delays wound healing processes 47 .The decrease in the expression of TNF-α and the increase in the expression of collagen promote the wound healing process by the treatment with nanosheets.The results show that nanosheets promote the wound healing process by contracting wound and decreasing the inflammation.In addition, CD31 marker participates in production of vessels (angiogenesis) and helps to supply nutrients for the wound healing process 48 .In sum, different compounds have synergism interaction effects for decreasing the inflammation and accelerating the wound healing process via angiogenesis and mature collagen synthesis.

Conclusions
In this study, CMC/ZnO/g-C3N4/MyR quaternary biocomposites were successfully synthesized and physicochemical properties confirmed their properties.The results confirmed their safety and antibacterial activities under in vitro and in vivo conditions.This study showed that CMC/ZnO/g-C3N4/MyR nanosheets with photocatalytic performance could significantly expedite the infected wound healing process by decreasing total bacterial count, edema and the expression of TNF-α and also increasing angiogenesis, mature collagen synthesis and epithelization.CMC/ZnO/g-C3N4/MyR nanosheets can compete with standard ointment of mupirocin.It can be used in combination with other ointments for the treatment of clinical wounds following future studies.Novelty is a strength point for this study and murine study is a major limitation that cannot be used for clinical uses in humans.

The preparation of extract
The amount of 40 g of nutmeg seeds purchased from Tabriz market (Iran).To avoid the effects of climate on the results, seeds collected form field a region were used.The seeds were mixed with 200 mL of solvent (water and acetone 1-50) and soaked at 20 °C for 24 h and stirred every two hours.After the extraction, the extract was filtered using filter paper and the filtered liquid obtained from acetone solvent was concentrated with the help of rotary evaporator at 40 °C until the complete removal of the extracted solvent and then dried.All methods on plants or plant materials were carried out in accordance with relevant guidelines in the method section.

7 .
Four peaks are observed at 284.52 eV, 285.34 eV, 286.55 eV and 287.63 eV, which are attributed to C-C/C=C, C-N/C-O, C=N/C=O and O=C-O.Spectrum N 1 s was converted to four peaks at 399.33, 400.66, 401.33 and 401.87 eV, which is dedicated to Pyridine-N, pyloric-N, graphitic-N and N-Ox 31 .Typical Zn 2p peaks at 1021.6 eV and 1044.7 eV are attributed to Zn 2p1 and Zn 2p3 respectively, which are in agreement with the normal Zn binding energy in ZnO.In addition, the O 1 s spectrum is characterized by two specific peaks, 229.9 eV and 351.6 eV are related to Zn-O and N-C-O, respectively.XPS results showed that nanosheet was successfully constructed without any impurities.

Figure 9 .
Figure 9. (A) Release profile of MyR and CMC/ZnO/g-C3N4/MyR; (B) cytotoxicity of nanosheets; (C) gross wound image; (D) wound contraction percentage, and (E) total tissue bacterial count in experimental groups on different days.Data are expressed as mean ± standard deviation (n = 6).Different letters (a-d) show significant differences (P < 0.05) between groups.

Figure 10 .
Figure 10.(A) The hematoxylin and eosin staining (a) of wounded samples of experimental groups.Note: Stars show edema intensity, arrows on day 7 show fibroblast cells and on day 12 epithelial thickness.An interesting point is that in the CMC/ZnO/g-C3N4/MyR group, the stratum corneum layer, unlike the other groups, has been formed; (C,D) the determined edema, fibroblast and epithelium thickness were scored as 0: absence;1: small amount; 2: low; 3: high and 4: severe at days of 3, 7, and 12. Non-similar letters on figures (a-d) show significant differences (P < 0.05) between groups.

Figure 11 .
Figure 11.(A) Immunofluorescence staining for expression of TNF-α (green area) and CD31 (red area); and (a,b) quantitative statistics based on TNF-α and CD31 staining on day 7 at wound sites, respectively.Nonsimilar letters on figures (a-d) show significant differences (P < 0.05) between groups.

Table 1 .
GC/MS analysis of MyR extract.

Table 2 .
In vitro antibacterial of the nanosheets in MIC and MBC tests (µg/mL).