Phytochemicals, Biodegradation, Cytocompatibility and Wound Healing Profiles of Chitosan Film Embedded Green Synthesized Antibacterial ZnO/CuO Nanocomposite

Open wound ulcer treatment remains a great challenge in wound care management especially involving multi drug resistant (MDR) pathogen. Currently, lack of effectiveness of commercially available wound dressing material is one of the primary factors delaying the wound healing. Therefore, transformation of plain Chitosan (Cs) to antibacterial polymer nanocomposite with embedment of Quercetin-ZnO/CuO biocide through green synthesis approach shed light for an efficient wound healing management. The present work studied the phytochemicals, biodegradation, storage, cytocompatibility and wound healing profiles of Cs film embedded with Quercetin-ZnO/CuO from Calotropis gigantea (C. gigantea). HPLC was used to detect Quercetin bioactive constituent. Our cytocompatibility study demonstrated ZnO/CuO-Cs-1wt.% displayed highest cell viability (168.52 ± 14.46%) at 72 h treatment. Besides, the ZnO/CuO-Cs-1wt.% is fully biodegradable. The ZnO/CuO-Cs-1wt.% also exhibited significantly enhanced cell migration (26.81 μm/h) and wound closure (62.35 ± 9.46%) at 12 h. This finding is also supported by our in vivo excisional open wound studies in Sprague-Dawley (SD) rats, which showed progressive recovery in 14 days. The controllable release of multiple metal ions from ZnO/CuO-Cs might contribute to the wound recovery. This study highlighted the promising outcomes exhibited from ZnO/CuO-Cs-1wt.% in wound healing management.


Introduction
The biological healing response of open wound ulcers is interrupted and delayed by many local and systemic factors such as medication, obesity, smoking and alcoholism, microbial infection, age of bedridden and long hospitalized patients, sex hormones, stress level, history of serious illness like diabetes and oxygenation [1,2].Presently numerous scientific studies have yielded fabrication of an ideal wound dressing materials with biodegradable properties and a detailed in vivo toxicological investigation using animal model (i.e., rats, mice and rabbits) in tackling deficiencies in chronic wound healing.The reason hybrid albino male Sprague-Dawley (SD) rats were mostly chosen by scientists because they have calm temperament, easy handling, hormonal aspect and been widely used in safety test in the biomedicine [3][4][5].
Currently, biodegradable polymer-based wound dressing material attracts countless attention in wound healing application.However, these wound dressing materials are less effective towards multi drug resistant (MDR) bacteria and structurally unstable causing an adverse impact on the patients' quality of life.Therefore, modification of plain polymer structure to antibacterial polymer nanocomposites with embedment of metal/metal oxide nanoparticles through nano-based wound dressing technology provide an efficient wound healing management (Table 1).Presently, lack of effectiveness of commercially available wound dressing material is one of the primary factors affecting the wound healing process.It was stated that, 75% of wound recovery only achieved at 24 weeks when introduced with Suprasorb X + PHNB (Lohmann and rauscher GmbH) [6,7].As such, this work focuses on the green synthesis and immobilization of Quercetin/Carbon (C) decorated ZnO/CuO nanocomposite from Calotropis gigantea (C.gigantea) using biodegradable chitosan biopolymer (Cs) and investigates the cytocompatibility effects and timeline of open wound healing progression.C. gigantea medicinal plant is rich in phytochemical constituents and plays vital role in treating various skin diseases and open ulcers [8][9][10].Surprisingly in our previous study, Quercetin/C decorated ZnO/CuO nanocomposite from C. gigantea demonstrate strong synergistic bactericidal activity with minimum bactericidal concentration (MBC) of 0.3125 mg/mL towards MDR pathogens such as Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (P.aeruginosa) [11].In contrast, the cytocompatibility effect on mouse fibroblast cell line and antibacterial properties towards MDR pathogens for other green synthesized ZnO/CuO nanocomposites were not well characterized and remain questionable (Table 2).
Microbiologically-mediated biodegradation is another important aspect in producing value-added wound dressing materials.Currently, non-biodegradable synthetic polymers create more plastic waste and it became biggest environmental threats and contaminants.This issue driving force amongst scientist in formulating new biodegradable chitosan (Cs) based biomaterials with enhanced structural properties.This set of experiment was carried out to investigate and compare wound healing performance of ZnO/CuO-Cs-1wt.%nanocomposite using an in vitro wound scratch method and in vivo excisional open wound in adult male SD rats.The expected outcome of this study to have potential biodegradable and biocompatible wound dressing materials which encourage faster wound healing; enhance cell proliferation; bring complete re-epithelialization; giving miraculous scar reduction and effective in treating open wound ulcers.

Materials
In this study, zinc nitrate hexahydrate (Acros) and copper (II) nitrate trihydrate (99.5%, Merck), from Darmstadt were used as main precursors.Silver alginate (Ag-Alg) from Oxy-Max, Malaysia was used as commercial control.Medium molecular Cs flakes (Aldrich) with degree of deacetylation 75-85% and mean molecular weight of 190,000-310,000 Da was used for Cs nanocomposite formulation.The fibroblast cells lines model, L929 used in this work was purchased from American Type Culture Collection (ATCC, USA).Fibroblast was maintained in Roswell Park Memorial Institute (RPMI) 1640 media (Gibco, Life technologies) and the media was supplemented with other nutrients.The alamarBlueTM cell viability reagent DAL1025 (Invitrogen, United Kingdom) was used for cell viability assay.The MDR pathogen (MRSA 38591) was obtained from American Type Culture Collection (ATCC).Luria-bertani (LB) nutrient medium was purchased from Sigma Aldrich (Darmstadt, Germany) and was used to culture MRSA 38591.Finally, male adult SD rats were purchased from Animal Research Centre (ARC), Advanced Medical and Dental Institute, Universiti Sains Malaysia.This pre-screening method was done using mobile phase of acetonitrile solution (A) and 0.1% v/v formic acid solution in water (B) at 30 °C with different elution gradients and run times (Method C) as recommended by previous works [39].The flow rate of the mobile phase was fixed at 0.4 mL/ min [40].Then, 2 µL of diluted extract solution volume was injected.UV peak absorbance was captured at wavelengths of 280 and 360 nm.For method C, linear gradient elution was set as: 0-12 min, 85% B; 12-22 min, 75% B; 22-30 min, 85% B.

Physical Appearance and pH Determination
The physical appearance of fabricated samples was captured through visual inspection.The pH of each type of mixing compound (Cs and ZnO/CuO-Cs-1wt.%)was determined using basic bench top pH meter (EUTECH pH 700).

Storage Analysis
The stability and bactericidal performance of freeze-thawed Cs and ZnO/CuO-Cs-1wt.%nanocomposite (1 mm×1 mm) was investigated against MRSA at different storage temperature (25 and 4 °C) and different storage time (1 and 2

HPLC Pre-screening Analysis
High-performance liquid chromatograph (HPLC) with photo diode array (PDA) Detector (SHIDMAZU HPLC, PROMINENCE LC-20AT) was used to determine the finger prints of phenolic compounds.The C. gigantea leaf extract aqueous solution was prepared using deionized water at boiling condition.The C. gigantea leaf extract solution with 1:2 dilution factor was prepared accordingly.Vaccinium arctostaphylos L. Viability (%): E. coli: 12.1 ± 0.39 and S. aureus: 14.9 ± 0.65 Nil [36] Clerodendrum inerme Zone of inhibition: E. coli: 13 ± 0.09 mm, S. aureus: 14 ± 0.01 mm, Klebsiella: cell control (L929), commercial control (Ag-Alg), and a negative control (10% (v/v) DMSO) were presented as well.The cell's morphology and migration were examined under inverted microscope after different incubation period of 24, 48 and 72 h.At last, 100 µL of 10% of Alamar blue solution was placed at respective time intervals and the cell viability absorbance was determined via microplate spectrophotometer at optical density wavelength between 570 and 600 nm.

In vitro Wound Scratch Assay Using L929 Cell Line
An in vitro wound scratch method was employed to study wound closure and cell migration rate of the ATCC mouse fibroblast cells lines model, L929 according to the established protocols [55,56].Briefly, 3 × 10

Biodegradation Study
The degradation (%) of the 6 mm diametrical disc of Cs, ZnO/CuO-Cs-1wt.%nanocomposite and commercial control (Ag-Alg) samples were monitored at regular time intervals (4, 6 and 8 days) by carefully immersing them in a small air tight glass bottle with 3 mL of phosphate buffer saline (PBS) at physiological pH of 7.4 and incubation temperature at 37 °C.The weight loss of the samples over time were evaluated by recording the initial weight before immersion and final weight after immersion.Final weight of the sample at each regular time interval was measured after fully removing the excessive water content from the sample followed by gentle dabbing using tissue paper.This protocol was performed as illustrated by Maiti et al. ( 2021) [42].The degradation (%) was determined using the formula: This set of experiment was carried out to evaluate in vitro cytocompatibility for ZnO/CuO-Cs nanocomposite using ATCC fibroblast cells lines model, L929.The early passage number of L929 cell line must be split into 2-3 passages before adding the test samples for treatment.Direct contact method (ISO 10993-5) protocol was applied and cell viability was measured quantitatively via Alamar blue assay.This is a common test method used in evaluating wound healing film [43,44].A sample is classified as cytotoxic if the cell viability is lower than 70% [45].The cell lines were seeded in to 96-well plates at a seeding density of 1 × 10 4 cells/well (passage 28) [46][47][48] and incubated for overnight in the following conditions of 5% CO 2 , 37 °C and > 90% humidity.The test samples with equal size (1 mm×1 mm) [43,49] were cut and sterilized under UV for 20-30 min [50][51][52].The old culture media was removed after formation of confluent and homogeneous cell's monolayers and then, replaced with 200 µL/well of fresh culture medium [48] along with three replicates samples for each treatment according to the direct seeding method [44,46,53,54].Two biological conditions were carried out.The following controls such as Cs control, quantify the total Zn (Zinc), Cu (Copper) and Ca (Calcium) ions release (mg/L) from ZnO/CuO-Cs-1wt.%nanocomposite.Cs sample labelled as blank control, while Ag-Alg as commercial control.A standard calibration curve was prepared by running a series of standard solutions ranging from 1 ppm, 10 ppm, 25 ppm and 100 ppm made from stock solution.Square sample (1 cm×1 cm) of each treatment group was immersed in 20 mL deionized water for 24 and 72 h at room temperature for metal ion facilitation.

Statistical Analysis
Two-way ANOVA with Tukey's multiple comparison test was employed to determine the differences among the different treatment's groups.Data was considered statistically significant if P value is less than 0.05.All the test data are expressed as the mean ± standard deviation (SD) of three replicates.

Physicochemical Properties
The SEM and TEM images of the green synthesized ZnO/ CuO nanocomposite by C. gigantea are depicted in Fig. 2a, 2b and 2c.A mixture of irregular oval and quasi-spherical morphology with average diameter of 7.515 ± 1.36 nm and length of 8.126 ± 0.66 nm are visible in ZnO/CuO nanocomposite [11].The size of ZnO/CuO was measured using ImageJ software.The ZnO/CuO nanocomposite were uniformly dispersed and well embedded throughout Cs biopolymer without any agglomeration/aggregation (Fig. 2d  and 2e).The XRD results demonstrate that the ZnO/CuO nanocomposite was well decorated with natural Quercetin/C compound [73,74].The patterns displayed by Quercetin/C showed these planes around 31.53° and 40.94° [11] (Fig. 2e  and 2f).

HPLC Pre-screening Analysis
In this pre-screening study, the bioactive constituents such as phenolic and flavonoids in C. gigantea leaf extract solution were determined.The graph captured several main peaks corresponding to retention time of 3.982 min, 6.669 and 6.769 min (Table 3).Most prominent and sharp intensity peak of Quercetin bioactive compound was detected at 6.669 min [75] (Figure S1; Supplementary material).Some minor peaks at RT of 3.982 and 6.769 min might be attributed to the presence of Vanillin and Benzoic acid, respectively [39].The FTIR and UV-vis results from previous study agree with the finding of formation of flavonoid house) with corn cob bedding.Standard laboratory diet food 4 mm pellets (Altromin) and unrestricted supply of drinking water were given [63].Daily water and food intake of all animals were closely monitored.All rats were anesthetized by intraperitoneal injection of 75.0 mg/kg ketamine and 8.0 mg/kg xylazine [63][64][65].Under anaesthesia, the dorsal hair from mid back surface had been shaved carefully using hair clipper and razor.
After that, the skin on the back area were marked on the posterior part of the scapula, and then, using scissors and a surgical blade, the entire skin was removed so that the 2 mm depth of the wound included a derma and a hypoderm, and the wounds were washed with normal saline [64].The size of the shaved area is more than 4 cm square, and the wound size is 2 cm×2 cm×0.2 cm.Each animal was wounded with one square excision wounds on dorsal individually.Analgesic (Meloxicam) 1.0-4.0mg/kg subcutaneously administrated for maximum 3 days against SD rats for post-surgery.Then the initial wound area which created on Day 0 was photographed and recorded.In next, the sterilized samples were placed over the created wounds and fixed in place using an adhesive biological membrane (Tegaderm™ Transparent Film).The wound that is not treated with bacteria served as Blank or Bare group [66].The wound that is treated with commercial hydrogel bandage (Ag-Alg) served as positive control group.For other optimized samples Cs and biodegradable porous ZnO/CuO-Cs-1wt.%nanocomposite was applied in bare wound of rats.
Wound size created on first day (Day 0) will be set as benchmark.The dressing materials were changed at Days 3, 7, 11 and 14.During the changing of dressings, photographs were taken, and the wound area were measured using a UV sterilized transparent polyethylene sheet.The sheet was kept on top of the wound and area were marked using a marker pen.The marked area was then transferred to graph sheet for getting the exact value through analysis using image J software [63,64,67].After day 14, all animals were euthanized by lab technician with method of asphyxiation using 100% of CO 2 inhalation with flow rate 10-20% of the chamber volume per minute.All major internal organs, including heart, liver, spleen, lung and kidney were harvested after euthanasia for naked-eye morphological observation (i.e., swelling, tumor growth and discoloration) [53,67].The cytotoxicity effect of nanosized ZnO/CuO in Cs biopolymer against internal organs of SD rats can be investigated by comparing treated group with normal bare control group animals [68][69][70][71][72].

Quantification of Multiple Metal Ion Release
Inductively coupled plasma-optical emission spectroscopy (ICP-OES) (Perkin Elmer OPTIMA 7300DV) was used to

Physical Appearance and pH Determination
Porous Cs and Cs biopolymer containing 1 wt% of green synthesized nanocomposite (ZnO/CuO) samples, were fabricated by freeze-thawing process appears in white and greyish white, respectively.Both samples show a strong acidic pH between 4.50 and 4.69.Incorporation of 1 wt% of green synthesized ZnO/CuO nanocomposite in Cs biopolymer slightly increase the pH.Enhanced antibacterial effect and speed wound recovery could be achieved by creating an acidic environment.This could be explained that, an acidic pH level might accelerate wound healing by fighting against wound infection, boosting bactericidal activity, improving re-epithelization and angiogenesis [78].Previous studies reported that, the transparent acidic Chitosan + 75%Honey sample with pH level of 4.34 had highest zone of inhibition towards P. aeruginosa, Staphylococcus aureus (S. aureus), Klebsiella pneumoniae (K.pneumoniae) and Streptococcus pyogenes (S. pyogenes) and it also has highest healing rate of burn wounds [79].Other than that, ZnO QDs has rapid dissolution and release of zinc ions and excellent bactericidal effect at pH below than 5.5 [80].
compound during green synthesis [11].In the presence of high content of Quercetin phytocompound in C. gigantea leaf extract solution, antibacterial properties and antioxidant activities were further improved [76].Quercetin bioactive compound belongs to flavonoid group of polyphenols with molecular formula of C 15 H 10 O 7 .Naturally, it has many promising characteristics such as anticancer, antiviral, antifungal, antimicrobial, anti-inflammatory and antioxidant.In this study, we believed that the Quercetin phytocompound play an important role in promoting synergistic antibacterial activity towards MDR and non-MDR skin pathogens [11,77].The standardization of active bioactive constituents from C. gigantea using HPLC will provide a proper guide for future quality control, purity and consistency since the plant biochemical properties varies from location to location.increases proportionally.In the present study, we found that the Cs sample was degraded partially at 3 h of immersion time and fully dissolved at 8 h of immersion time which may indicate that plain Cs without an addition of green synthesized nanocomposite has very weak polymer chain and structural properties resulting in prominent degradation rate.Based on our findings, ZnO/CuO-Cs-1wt.%nanocomposite was highly swelled on 1-2 days of immersion time and the water content of the sample cannot be fully dabbed with tissue paper where it might destroy the nanoparticulated polymer structures.However, ZnO/CuO-Cs-1wt.%nanocomposite have considerable biodegradation properties which play an important role in eliminating medical waste and create green environment without soil pollution.

In vitro Cytocompatibility Investigation at Different Incubation Time
The investigation of ZnO/CuO-Cs-1wt.%nanocomposite in vitro was studied against fibroblast cell line at different incubation time (24, 48 and 72 h) and the results are depicted in Fig. 3.These results demonstrated that, ZnO/ CuO-Cs-1wt.% nanocomposite had an improved cell viability (168.52 ± 14.46%) along with normal elongated filopodia cell morphology at 72 h treatment, indicating that this bactericidal and wound healing agent owned an excellent wound closure rate and cytocompatibility nature.This was owing to the high porosity and improved swelling characteristic of ZnO/CuO-Cs-1wt.%nanocomposite which increases absorption capacity of exudate from chronic wounds and provides passages for intake of nutrients and body fluid for wound recovery and re-epithelization [81].
Besides of that, successful control on steady and slow release of free radicals such as •O 2− and •OH − from this ZnO/CuO-Cs-1wt.%nanocomposite might present with cytocompatibility properties towards human cells and can accelerate wound healing properties [82].We believed that, this green synthesized Quercetin/C-ZnO/CuO nanocomposite might further accelerate controllable amounts of metal ions release and promote cell proliferation [67,83].In line with this study, Cs sample exhibited cell viability of 129.03 ± 4.99%, which was slightly lower to the ZnO/CuO-Cs-1wt.%nanocomposite, while the negative control does not promote the cell's augmentation.Collectively, these outcomes exhibited that ZnO/CuO-Cs-1wt.%nanocomposite would be suitable candidates for wound healing application.

In vitro Wound Healing Profile: Scratch Assay
The wound healing efficacy of an optimized ZnO/CuO-Cs-1wt.%nanocomposite was further evaluated against fibroblast cell lines at different time intervals (12 and 24 h).

Storage Analysis
The storage analysis (Table 4 and Table 5) revealed that, the different storage temperatures (25 and 4 °C) and different storage time (1 and 2 months) of the Cs and ZnO/CuO-Cs-1wt.%nanocomposite were able to tune the bactericidal performance against MDR pathogen (MRSA).Particularly, the 1 month treated-storage group (ZnO/CuO-Cs-1wt.%)at different storage temperatures exhibited a significant antibacterial effect with low optical density between 0.37 ± 0.05 and 0.48 ± 0.06, while control strain (MRSA) showed highest absorbance of 0.95 ± 0.04.The material shelf life might be deteriorated if it is kept at longer period.So far very limited research works only investigated on bactericidal performance of storage condition-dependent wound dressing material [41].

Biodegradation Study
Degradation rate (%) of the prepared biomaterials are depicted in Table 6.The degradation rate for Cs, ZnO/CuO-Cs-1wt.%and commercial control (Ag-Alg) samples were 100 ± 0.00, 100 ± 0.00 and 50.35 ± 11.20%, respectively.The degradation rate of ZnO/CuO-Cs-1wt.%sample was greater than commercial control (Ag-Alg) sample where it could be fully dissolved in 8 days, while commercial control (Ag-Alg) sample degraded partially.As per observed, the percentage of degradation of these samples were time-dependent and  The findings on wound closure (%) and cell migration rate over time was shown in Table 7 and Fig. 4. The wound closure for ZnO/CuO-Cs-1wt.%nanocomposite is equivalent to the commercial control (Ag-Alg) which is around 62.35 ± 9.46% at 12 h.Meanwhile, a slightly improved wound healing extent (45.95 ± 0.76%) were seen in the Cs group in comparison with the cell control (L929).This might be linked to the release of positively charged Ca ions (Ca 2+ ) from Cs biopolymer, which was revealed from EDAX, XRD and ICP analysis [38].These ions might help in promoting cell proliferation and wound healing [84].But it was noted that, all treated groups have complete wound closure at 24 h except negative control has shown no sign of wound healing.Rapid wound closure rate was clearly seen for the ZnO/CuO-Cs-1wt.%treated group.Moreover, ZnO/ CuO-Cs-1wt.% treated group has exhibited remarkable cell migration rate of 26.81 μm/h at 12 h.This might be due to the effectiveness of optimized ZnO/CuO-Cs-1wt.%biomaterial in stimulating wound healing and angiogenesis process via synergistic activity with presence of multiple ionic system (Cu 2+ , Zn 2+ and Ca 2+ ) [67,85,86] and hydrophilic  Moreover, the use of ZnO/CuO-Cs-1wt.%nanocomposite is advantageous to increase the material absorption on wound area through melting and slow degradation, whereas the commercial control hardly absorbed on wound surface due to the slow degradation as positively captured in biodegradation study (Figure S2; Supplementary material).In addition, all SD rats gained their body weight during 14 days of treatment period (Fig. 6a).No significant differences were captured in food and water intake for all treated and bare control group during the treatment period (Fig. 6b  and 6c).All harvested major internal organs such as spleen, kidneys, heart, lungs and liver were identically healthy and revealed normal morphological appearance in term of size, porous ZnO/CuO-Cs-1wt.%nanocomposite provide an ideal wound healing environment and superficial fibroblast proliferation.Thus, low-concentrated Cu, Zn an Ca ions play an important role in stimulating cell migration in open wound [67,83].In all treated SD rats, there was no evidence of behavioral sign of illness or pain such as guarding, vocalizing, biting, restlessness, abnormal resting position, failure to groom, lack of mobility.All experimental SD rats administrated with ZnO/CuO-Cs-1wt.%nanocomposite displayed "0" grimace scale, and there were no treatment related morbidity and mortality.Besides of that, no any abnormalities were seen on the skin, fur, eyes, mucous membrane, stool and urine for all treated group of SD rats.The detected Zn concentration was significantly higher than Cu and Ca element due to the high reactivity and solubility of ZnO in deionized water.Conclusively, these multiple metal ions derivatives release from nanocomposite could be a viable solution to combat antibiotic resistance by killing the skin pathogens.

Conclusions
Development of a synergistic biodegradable ZnO/CuO-Cs-1wt.%nanocomposite has shown an enhanced in vitro and in vivo wound healing characteristics and migration rate.The wound healing properties greatly improved with multi-ionic systems (i.e., Cu 2+ , Zn 2+ and Ca 2+ ).Besides, the degradation rate of ZnO/CuO-Cs-1wt.%nanocomposite shape and color without any tumor growth and swelling (Fig. 7).
Previously, several studies have shown that the formulated wound healing agent such as AMP-and CeON-loaded catechol-modified hydrogel [12], Chitosan-based hydrogel with MOF-loaded α-lipoic acid [21], Chitosan/poloxamerbased thermosensitive hydrogels containing zinc gluconate/ recombinant human epidermal growth factor [22] and Ag-PCL/GelMA fibrous membranes [32] dramatically cured the created initial wound size (0.8-1.5 cm in diameter) within 11 to 21 days.Though, this developed biodegradable porous ZnO/CuO-Cs-1wt.%nanocomposite in the current work were found to be best formulation than other reported wound dressing products, since it managed to heal the larger size (square shape: 2 cm×2 cm) of created wound on day 0 quickly in total 14 days.Conclusively, this developed green wound dressing has emerged a promising potential in biomaterials for open wound healing application.

Multiple Metal Ion Release
The release of Zn, Cu and natural Ca ion derivatives concentration in deionized water were quantified in line with the material degradation at Day 1 and Day 3. Concentration of metal oxide ions was a very important factor in promoting synergistic killing activity towards skin pathogens.It could be seen from Fig. 8 that the dissolution of Zn and Cu

Future Scope
This initial pilot study was investigating more on in vivo wound healing performance of novel natural Quercetin/C encapsulated ZnO/CuO in Cs biopolymer against excisional open wound using smaller-scale animal model.However, was greater than commercial control where it could be fully dissolved in 8 days, while commercial control degraded partially.Thus, technology from this medicinal plant C. gigantea especially in development of advanced transdermal ZnO/CuO-Cs wound dressing could provide efficient wound healing response.

Fig. 3
Fig. 3 Cytocompatibility investigation on mouse fibroblast cell line at different incubation time.L929 cell control (untreated group) represented 100% cell viability

Fig. 6
Fig. 6 Observed weight gain, water and food intake for all treated and bare control groups during the treatment period.(a) body weight gain, (b) water intake and (c) food intake

Fig. 8 Fig. 7
Fig. 8 Multiple metal ion release.(a) Concentration ions (mg/L) at Day 1 and (b) Concentration ions (mg/L) at Day 3. No metal ion release was detected for deionized water

Table 1
An ideal wound dressing material with biodegradable properties and in vivo animal toxicological investigation

In vivo Wound Healing Study Using SD Rat Model
The samples were kept inside air tight glass bottle during the treatment.The bactericidal effect of fabricated samples was measured by reading absorbance of treated sample in 10 6 CFU/mL bacterial broth culture (200 µL/well) after 24 h of incubation using microplate spectrophotometer at optical density wavelength of 580 nm.All treatment groups were carried out in triplicate.
[41]58]well (passage 34) were seeded into 96 well culture plates and incubated for 24 h in CO 2 incubator at 37 °C.A sterile 200 µL pipette tip was used for wound scratch.Suspension containing of Cs, ZnO/CuO-Cs-1wt.%nanocompositeandcommercialcontrol(Ag-Alg)withsize sample of 1 mm×1 mm in 200 µL RPMI culture media were prepared for treatment in 24 h.Then, the test material extraction solutions were filtered using sterile syringe filter before further use.Meanwhile, 10% (v/v) of dimethyl sulfoxide (DMSO) was used as negative control.At different time intervals (0, 12 and 24 h), the wound closure distant (µm) and cell migration over time were observed under the inverted microscope and inspected via ImageJ software.This assay was carried out with two biological replicates with n = 3. Wound closure % and cell migration rate (R M ) were calculated using recommended Eq. [1.0][56]and Eq. [2.0][57,58]: per treatment.All experimental animals were approved by USM Institutional Animal Care and Use Committee (USM IACUC) with approval number USM/IACUC/2022/ (134) (1187).Animals were placed in the individual cage (singly months)[41].

Table 4 Absorbance for storage room temperature at different storage time (1 and 2 months)
. a significant difference in mean value relative to the control strain (P < 0.05)

Table 5 Absorbance for freezing temperature at different storage time
[87]32 29.36 ± 20.82 50.35 ± 11.20nature of Cs molecules as it was proven before in contact angle analysis[87].From the above discussion, it was clear that ZnO/CuO-Cs-1wt.%nanocomposite has remarkable wound healing characteristic for biomedical applications.

vivo Wound Healing Profile: Excisional Open Wound in Animal SD Rats
± 7.83 in the bare/blank control group, 80.86 ± 2.18 in the commercial control group, 75.58 ± 2.60 in the Cs control group and 86.44 ± 6.48 in the ZnO/CuO-Cs-1wt.%treated group.Among all groups, wound treated with biodegradable