Application of Fish Collagen-Nanochitosan-Henna Extract Composites for the Control of Skin Pathogens and Accelerating Wound Healing

Skin is the largest protective organ that could be recurrently wounded and attacked by microorganisms. The wounded skin safeguarding and supporting were intended through natural derivatives. Fish collagen (Cg) type I, extracted from sea bream ( Spondyliosoma cantharus ), chitosan nanoparticles (NCht) from shrimp shells, and henna ( Lawsonia inermis L.) leaves extract (He) were produced and physiochemically characterized. The antimicrobial potentialities of these compounds and their composites were assessed toward skin pathogens ( Candida albicans and Staphylococcus aureus ) using various assaying methods and microimaging techniques. The infrared and electrophoretic analysis of Cg validated its characteristics, and the IR-spectroscopic analysis of the compounds/composites indicated their physiochemical attributes and interrelations. The produced NCht particles had a diameter range of 64.6-308.8nm, 104 nm mean diameter, and +31.3 mV zeta potentiality. Both NCht, He, and NCht/He composite exhibited signi ﬁ cant antimicrobial potentiality toward skin pathogens; NCht/He was the strongest with inhibitory concentrations of 20.0 and 22.5 μ g/mL and inhibition zones of 25.7 and 26.8mm against S. aureus and C. albicans , respectively. The electron micrographs veri ﬁ ed the synergistic microbicidal action of NCht/He, as they led to severe microbial lysis and deformations. The skin wounds ’ treatment with NCht/He/Cg composite promoted the fastest and complete healing of wounded rats ’ skin during 8 days of local treatment, with the absence of in ﬂ ammation and infection signs; treated with NCht/He/Cg composite, the wound area vastly reduced from 63.6mm 2 to 15.9 and 9.1mm 2 after 2 and 4 days, respectively. The natural NCht/He/Cg composites are recommended as topical applications for optimum skin disinfection and regeneration.


Introduction
Skin is the outmost and largest organ, which covers all the body; the skin's utmost role is the protection of muscles, bones, ligaments, and interior organs from external threats, including biological, physical, chemical, and mechanical agents [1]. However, wounds, illnesses, burns, or surgical incisions can dangerously affect the structure/functions performed by the skin; the main threat challenge for wounded skin caregivers is the microbial infections and inflammation emergence in injured tissues [2].
Although numerous medications were introduced for remediate and regenerate wounded skin, the accustomed antibiotic prophylaxis and anti-inflammatory drugs were not sufficient to overcome wound contamination and inflammation with skin's bioburden microorganisms [3].
Collagen (Cg) constitutes the foremost structural fraction of skins' extracellular matrix and the utmost researched biopolymer for tissue regeneration/engineering applications [4]. The Cg-based dressings were effectually employed for accelerating skins' wound healing (WH), which are attributable to the swelling capacity, high biocompatibility, biodegradability, facile adherence, and powerful protection of wounds' bed [5].
From~27 identified Cg types (classified with function, distribution, size, and composition amino acids), type I Cg is the principal for human skin care and industrial/cosmetic applications; it designates 2 alpha "α1 and α2" and 1 beta "β" peptide chain, with elevated tensile stiffness and stability [6]. The traditional Cg sources, e.g., porcine and bovine Cgs, have currently many problematic concerns for applications in biomedical and cosmetic disciplines, such as religious issues and diseases' transmission risks. Thus, fish Cg introduced splendid alternatives for applications in biomedicine, tissue engineering, skin care, and regeneration, with higher biocompatibility to human and minimum disease transmission risk [7][8][9].
From the astonishing bioactive natural biopolymers is chitosan (Cht), which is derived from chitin N-deacetylation, they both constitute the principal supporting components in crustaceans shells, most insects, and fungi cell walls [10,15]. The Cht is confirmed to possess superior bioactivities, e.g., antimicrobial action, antioxidant, WH acceleration, tissue engineering, biochelation, biosorption, antiinflammatory, anticancer, and drug-carrying capabilities [15][16][17][18][19][20]. The Cht was introduced as an effectual base for applications in skin care/regeneration, wound dressing, and tissue engineering [19,21]; the Cht main advantages depend on its combined unique characteristics such as biocompatibility, biodegradability, nontoxicity, and high adsorption capacity. The bioactivities of Cht are much augmented by its transforming into nanoforms, which provides it with greater reacted surfaces and charges to become more effective for WH, microbial inhibition, drug-carrying capabilities, and radical scavenging [22][23][24][25][26].
Accordingly, the aims/objectives of the research are the extraction of Cg from sea bream fish, Cht nanoparticles (NCht), and henna extract (He) and the assessment of produced agents and their conjugated composites as antimicrobial (against skin pathogens) and WH accelerators in wounded rats.

Fish Collagen (Cg) Extraction and Characterization.
Byproducts of sea bream (Spondyliosoma cantharus) filleting, e.g., fish skins, were obtained from "Kafrelsheikh University Fish Processing Research Plant"; collected samples were retained at -20°C until thawing and subjecting to extraction. The extraction process depended on illustrated protocol [39], with minor adjustments. The entire steps were executed at temperature of ≤4°C using DW "double-distilled water" to dissolve and wash. In brief, cut skin into~2 cm 2 parts was extensively washed; then, skin's noncollagenous proteins were deproteinized via immersion in 20 folds (w/v) of 0.1 N NaOH, stirring for 18 h, and rewashing. Cg was extracted in 25 folds from 0.5 M acetic acid for 48 h, followed by filtration and precipitation with 1.0 M NaCl treatment. Precipitated acid soluble Cg was harvested by centrifugation for 45 m at 18.500 × g, lyophilization of harvested pellets then dialysis against acetic acid (0.1 M) and DW. The Cg characterization involved SDS-PAGE "SDS-polyacrylamide gel electrophoresis" for appraising structural peptide molecular weight (MW) and FTIR spectroscopic analysis "Fourier transforms infrared-JASCO spectrometer 4100, Tokyo, Japan" for detecting biochemical structural bonds of the compound [40].

Extraction of Henna Leaves.
Fresh identified henna leaves (Lawsonia inermis L.) were attained from ARC "Agricultural Research Center, Giza, Egypt", cleansed, hot air-dried (45 ± 2°C), and mechanically powdered. Henna powder (~100 g) was soaked in 65% C 2 H 5 OH (ethanol, 1200 mL) and frequently agitated for 56 h, without heating. Plant residues were excluded by filtration through Whatman No. 1 paper, and the extraction filtrates were flash evaporated "Büchi, Flavil, Switzerland" at 44°C until dryness. The resulted He was redissolved via agitation for 24 h in DW to have 10% concentration.

Test
Organisms. Skin microbial pathogens "Candida albicans (ATCC-10231) and Staphylococcus aureus (ATCC-25923)" were employed to judge the products' antimicrobial activity. The microbial cultures were propagated and challenged aerobically at 37°C using YM for C. albicans and TS for S. aureus.
2.6. Antimicrobial Assays. The appearance of IZ "growth inhibition zones" was considered as an indicator for antimicrobial activity from NCht, He, and their composites using disc-diffusion assay [45]. Diluted solutions of produced agents (NCht, He, and NCht/He composite) were impregnated onto sterile discs (0.6 cm diameter), which were positioned onto the surfaces of inoculated agar plates with microbial cultures and incubated for 18-24 h. Plates (triplicated) were incubated at 37°C, and the IZ diameters' means were considered.
The MIC "minimal inhibitory concentration" measurement from each agent/composite toward skin pathogens was performed as indicated by Tayel et al. [16], through a microdilution method and 10-100 μg/mL concentration range of screened agents (NCht, He, and NCht/He composite) in microbial liquid media. The customary antibiotics (nystatin and vancomycin) were employed as positive controls for comparison.

Wound Healing (WH) Potentiality of Composited
Materials. Young Wistar healthy rats (average weight between 142 and 166 g) were individually housed under maintained conditions "25 ± 2°C; 12 h of light-dark cycle; 65 ± 3% relative humidity" in polyethylene clean cages. Animal handling and practical experiments were implemented following the guidelines of the "Kafrelsheikh University Ethical Committee for the care and use of laboratory animals" with the aid of two veterinarian technicians. The experiment period lasted for 14 days, with rats feeding on a customary pellet diet and free water access. After anesthesia induction via ketamine intramuscular injection (100 mg/kg body weight), wounds with semicircular areas of~60 mm 2 were made on shaved rats' thoracic area. From the wounding day (day 0), the experimented composites (containing 1.0% NCht + 1:0% He (T1 group) and 1.0% NCht + 1:0%He + 1:0% Cg (T2 group), dissolved in DW) were topically smeared every 12 h until complete epithelization. Wounds were digitally photographed daily; the reductions and manifestation of wounded areas were appraised from the captured photographs.

Statistical
Analysis. The means ± SD "standard deviations" from triplicated experimentations were computed (Microsoft Office-Excel sheets 2013), and the significance analysis between results was executed electronically (Med-Calc statistical software (V. 15.2), Belgium) at ≥95% CI.

Result and Discussion
3.1. Fish Cg Characterization. Fish Cg was effectually produced from sea bream shin, with 32.4% dry weight yield; this yield is in the average range of Cg extracted from other fish wastes, using acid solubilizing technique [39,46]. The structural attributes of produced Cg, including its FTIR spectrum and SDS-PAGE pattern, are illustrated in Figure 1. As appointed in Figure 1-(1), the main characteristic bands for Cg were observed, especially the corresponding band to amide A, amide B, and amides I, II, and III, at 3331, 2922, 1658, 1593, and 1239 cm -1 , respectively. The detected bands and their wavenumbers are closely related to formerly reported representative bands for fish Cg, in close spectral wavenumbers to current findings [46][47][48].
The characterization of Cg by SDS-PAGE (Figures 1-2) exemplified that extracted Cg type had harmonized polypeptide chains, containing two α chains (α1and α2); the α1 band had a higher density than α2 band, with respective MWs of~111.2 and 122.7 kDa, respectively. The β chain also appeared in the Cg pattern. These characteristic polypeptide chains indicate that produced Cg belonged to type I [6,36,49].

NCht
Characterization. The extracted Cht powder from P. monodon shells had a creamy color, a MW (molecular weight) of 162.3 kDa, and a 91.2% DD. The DD (higher than70%) and the estimated MW of shrimp Cht verified the chitin transformation to Cht with~low MW [15]. The synthesis of NCht from shrimp Cht was achieved, and the characterization of produced polymer nanoparticles revealed that the Ps ranges from 64.6 nm to 308.8 nm with mean Ps diameter of 104.3 nm.

International Journal of Polymer Science
The NCht particles were positively charged (zeta potential of +31.3 mV) and had homogenous, well dispersion, and spherical contours, as evidenced by their SEM imaging ( Figure 2).

FTIR Analysis.
The FTIR spectral assessment for NCht and He and the composites of NCht/He and NCht/He/Cg are presented (Figure 3). The NCht spectrum had the key characteristic bands that are designated in standard Cht (Figure 3, Cht). The main NCht characteristic peaks were distinguished at 1071 cm -1 (C-O stretch), 1375 (bridge O stretch), 1658 and 1630 (N-H bend), 2924 (C-H stretch), and 3440 (-OH stretch) [36]. In the FTIR spectra of He (Figure 3, He), the broad and strong peak observed at 3431 cm -1 is attributed to vibrated stretching of -OH groups; the two peaks at 2925 and 2830 cm -1 were assigned to vibrated stretching of -CH 2 , and the peak at 1650 cm -1 indicated the carboxylate anions (-COO-), whereas the weak peak appeared at 1467 cm -1 indicated N-H vibrated stretching in He amide [50,51].
The interaction between NCht and He was illustrated (Figure 3, Cht/He); this involved intermolecular hydrogen bonding of the hydroxyl and carbonyl group in He with the hydroxyl and amino group of NCht, respectively. The first peak had less sharpness and stretching, with a lower frequency of 3406 cm -1 of (OH) groups, and the stretching band of 1650 cm −1 was shifted to higher wavenumber of 1629 cm −1 in NCht/He composite [52,53].
After Cg addition to NCht/He composite, the He characteristic peaks were mostly overlapped with Cg. However, a novel peak was immerged at around 3697 cm -1 , the characteristic band for Cg amide B mostly disappeared, the band of Cg amide I also shifted to lesser wavelength whereas the amide II stretching was shifted to higher wavelength, and amide III showed more shrinking [6,49]. These apparent alterations in composited agents' structures indicate the biochemical interactions between most of these agents, i.e., NCht, He, and Cg [38]. Table 1 depicts the in vitro antimicrobial potentialities of NCht, He, and NCht/He composite against C. albicans and S. aureus. The inhibitory actions varied from examined agents; the most significantly powerful treatment was the challenge with NCht/He composite, as evidenced with its widest ZOIs and least MICs toward both skin pathogens ( Table 1). The He exhibited also strong antimycotic and antibacterial potentialities, and their conjoining with NCht synergistically augmented their activity. Interestingly, no significant differences were calculated between the ZOIs from NCht/He composite and nystatin (toward C. albicans) and vancomycin (toward S. aureus), which indicates the higher potentiality of composite compared to customary standard antibiotics. The He dosedependent antimicrobial activity was reported and attributed to its high contents from glycosides and flavonoids, which are well-recognized as potent antimicrobial compounds [38,54,55]. Additionally, the lawsone (as a major constituent in He) was verified to possess strong antimycotic and microbicidal actions toward numerous screened pathogens [56]; these pathogens included S. aureus and other Gram-positive/negative bacteria [57]. Regarding NCht, it has the main antimicrobial attributes of bulk Cht (e.g., positive charged   International Journal of Polymer Science particles, attachment to cells' membranes, interaction with intercellular components like DNA, and inhibition of their activities), but NCht particles have these attributes in superior manners, which make them ideal antimicrobial candidates [16,18,24,25]. The bioactivities of NCht (e.g., antimicrobial, anticancer, and preservative attributes) were illustrated to be augmented via nanopolymer incorporation with additional bioactive phytocompounds [18,26,41,58], which appointed the biocompatibility and synergy behavior of NCht when joining with other bioactive molecules.

Antimicrobial Potentiality.
3.5. Microbicidal Action. The consequences of exposure to NCht/He composite on the constructions of skin pathogens C. albicans and S. aureus after different durations are shown in Figure 4. Regarding control cells (Figure 4, C-0 and S-0), which appeared with contacted, smooth, and uniformed surfaces, the treated pathogens' cells with NCht/He composite for 5 h had obvious morphological alterations and distortions, accompanied with observable lysis signs (Figure 4, C-5 and S-5). After 10 h of exposure, the cells were mostly lysed and released their interior components, especially for C. albicans cells (Figure 4, C-10). The S. aureus cells' residues were remarkably coalesced as consequences of their membrane lysis (Figure 4, S-10). The NCht was comprehensively reported to exhibit powerful antimicrobial actions for fighting microbial pathogens; the main action resulted from NCht surface charges (positive), which enable its attachment to microbial surfaces, deform them, and interfere their permeability [24,25,59]. The nanosized NCht particles could also penetrate into microbial cells and interact with their functional components (e.g., polymerization enzymes, DNA, RNAs, proteins, and enzymes) to inactivate them and prohibit well-organized cell membrane synthesis [24,60,61]. The He contains numerous bioactive constituents that have elevated antimicrobial and cytotoxicity properties against microbial cells, e.g., 1,4-naphthoquinone analogues, lawsone, esculetin, isoplumbagin, hennadiol, laxanthone, lupeol, lacoumarin, fraxetin, betulinic acid, scopoletin, betulin, and flavone glycosides [27,55,56,62]; most of these compounds have diverse microbicidal actions affected the synthesis and development of cells' vital and structural organelles.
The skin wounds' treatment with NCht/He/Cg composite promoted the fastest healing of wounded rats' skin during 8 days of local treatment ( Figure 5). For the untreated group ( Figure 5, C), no complete healing was observed after 8 days, and the mean wound size reduced from 57.3 mm 2 to 52.8, 46.5, 38.5, and 23.4 mm 2 after 2, 4, 6, and 8 days, respectively. The healing signs appeared faster in the T1 group, treated with NCht/He composite ( Figure 5, T1); the wounded parts became mostly healed after 8 days of treatment.
In the T2 group, treated with NCht/He/Cg composite, the wound area vastly reduced from 63.6 mm 2 to 15.9 and 9.1 mm 2 after 2 and 4 days, respectively. The mostly complete healing was detected after 6 days of treatment ( Figure 5, T2).
The wound size reduction and their closure are principally resulting from the antimicrobial, reepithelialization, and anti-inflammatory actions of the composite components, i.e., NCht, He, and Cg [63,64].
The usage of natural derivatives for WH, as alternatives to chemotherapy, attained great concerns to manage skin infections and promote its regeneration [65]. Chitin and Cht were supposed to enhance healing process in wounded skins; the multiple monosubunits (N-acetyl glucosamine), in these polymers' compositions, are an imperative constituent in dermal tissue and have vital necessity for repairing scar tissues [19]. The Cht surface has numerous free amine groups, which can conjoin with blood acidic groups and enhance their coagulation [22]. Cht and NCht molecules (by their high positive charge) can effectively promote cells' growth and assist thrombosis/blood coagulation, which greatly foster damaged tissue repairing [12,19,66].
The NCht potentiality was suggested for developing dressing that has high ability to remove necrotic tissues and accelerate hemostasis activity in wounded skins, which led to rapid skin regeneration and WH [23]. Polymer-based nanomaterial, e.g., NCht, was therapeutically employed for developing WH dressings or as carriers for delivering skin care biomolecules [54]; the NCht skin curative actions were attributed to its reepithelialization, anti-inflammatory, and antimicrobial properties [24,63,64]. The bioactivities of NCht are frequently greater than native Cht because of the increment of contacted surface area (from NPs) with microbial pathogens and injured tissues [24].
Cg is normally produced by fibroblasts; it is the key responsible for triggering cell migration and contributes to tissues' regeneration via stimulating specific cells (e.g., fibroblasts and macrophages) enhancing and accelerating WH [67].
Several studies indicated the elevated potentiality of fish Cg in healing progress and wound contraction; they confirmed Cg actions for accelerating healing development and epithelization by stimulating keratinocytes' differentiation and proliferation [48,[67][68][69][70]. The Cg was proved to augment the gene expressions of TGF (transforming growth factors), FGF (fibroblast growth factor), and VEGF (vascular endothelial growth factors), which led to stimulating cutaneous tissue healing via activating/recruiting fibroblast proliferation, angiogenesis, and deriving macrophages to generate chemotactic factors [48,65,70].
The incorporation or individual utilization of He had significant positive impacts in wound's contraction and epithelialization in rats. He has treasures from bioactive compounds, which could contribute to healing processes of wounded skins; the combined synergistic actions of multiple He constituents are assumed to correlate with its curative beneficial effects [35]. The He phytoconstituents include phenolic amalgams (e.g., coumarins, naphthalenes, lignans, alkylphenones, xanthones, naphthoquinones, tannins, and flavonoids), alkaloids, terpenes, and steroids [28,29,34].  Amongst these constituents, coumarins, tannins, alkaloids, and flavonoids are the utmost involved in the WH process. Actually, owing to their edema protective and antioxidative functions, coumarins were stated to effectually ameliorate WH [70,71]. Additionally, the He flavonoids could augment skin WH via multiple actions, e.g., their antibacterial, astringent, cell necrosis prevention, cytokine expression modulation during inflammation, angiogenesis improvement, and prostaglandin synthesis inhibition [25,29,33,70]. The He tannins' contents were also documented for contributing to WH through enhancing tissue organization and regeneration; the He tannin actions mainly depend on their astringent, antioxidant, antibacterial, anti-inflammatory, and free radical biochelation activities [28,34,35,72]; these actions frequently lead to stimulate keratinocyte/fibroblast proliferation, improve WH and angiogenesis, and develop rapid crust via proteins' precipitation in damaged tissue [29,30,35]. The He-loaded nanopolymers, e.g., PLA and gelatin nanofibers, showed additional capabilities for controlling wound infections and WH via formation of hygienic dressings [37]. The rapid WH period, after treatment with NCht/He/Cg composite, and the absence of inflammation and infection signs in treated wounds indicated the synergistic powerful action of these agents to overcome wound infections and inflammation and promote their regeneration and epithelization.

Conclusion
The bioactive natural compounds, Cg, NCht, and He, were extracted from their original sources and assessed as skin protectant agents through their actions as antimicrobials and WH stimulators. The composite from NCht and He had outstanding antimicrobial potentiality against skin pathogens (C. albicans and S. aureus), whereas the Cg/NCht/He composite could effectually accelerate the WH in rats' skin, without the emergence of inflammation or infection signs, which advocate the composite topical application for optimum skin disinfection and regeneration. The study's prospects could be the examination/application of further biopolymers as carriers for WH accelerators, and investigation of their biosafety and modes of action biochemically and histologically.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare that they have no competing interests.