Ecofriendly one-pot fabrication of 7-methoxy coumarin encapsulated zeolitic imidazole nanocomposite for pH-responsive wound healing application and its biosafety evaluations

doi:10.21203/rs.3.rs-2862242/v1

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Research Article

Ecofriendly one-pot fabrication of 7-methoxy coumarin encapsulated zeolitic imidazole nanocomposite for pH-responsive wound healing application and its biosafety evaluations

https://doi.org/10.21203/rs.3.rs-2862242/v1

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Biocompatible 7-methoxy coumarin encapsulated 7-MC@ZIF-L nanocomposite was fabricated to develop efficient wound healing. The stimuli-responsive drug release mechanism was examined at pH 5 and 7.4. The release of coumarin was increased within three hours by burst release under acidic conditions. The 7-MC@ZIF-L nanocomposite shows significant bacterial growth control against human infectious pathogens S. aureus, E. coli, P. aeruginosa, and S. epidermis. Moreover, the MTT and AO/EB assays revealed that nanocomposite had high cell viability towards L929 fibroblast cells. The present study enlightens a novel one-pot fabrication of 7-MC@ZIF-L nanocomposite as a drug delivery system and its stimuli-responsive drug release ability. The current methodology is an effective approach than any existing post-synthesis methods. The synthesized 7-MC@ZIF-L nanocomposite demonstrates ultrahigh thermal and chemical stability. The anti-biofilm results confirmed that the nanocomposite effectively inhibits biofilm formation and damages the biofilm architectural matrix based on the dose-dependent manner. In vitro wound scratch assay revealed that the sustainable and slow release of 7-methoxy coumarin and zinc ions promotes the cell proliferation rate. Bio-compatibility study results confirmed that the 7-MC@ZIF-L nanocomposite is safer and non-toxic for human use. In vivo artemia, salina lethality assay concluded that the nanocomposite is highly suitable for wound healing and other biomedical applications. The overall results signify that the 7-MC-ZIF-L nanocomposite is an efficient material for stimuli-responsive wound healing treatment in the future for wound care therapy.

Anti-biofilm

Artemia salina

Cytotoxicity

7-methoxy coumarin

Nanocomposite

wound healing

Most antibiotics suffer from non-specific delivery leads to lethal effects in normal cells and tissues. So unwanted harmful effects of antibiotics on healthy cells are common trouble, and programmed drug delivery is essential for biomedicine [1–3]. To overcome the side effects of uncontrolled drug delivery, we need a specific and efficient vehicle to deliver the targeted molecules to targeted sites. A perfect drug delivery system (DDS) must have high drug loading capacity, biocompatibility, control over the release, and fewer adverse effects [4–10]. Several DDS has developed in past decades, including organic and inorganic materials. Among these materials, metal-organic frameworks (MOFs) are prominent in biomedical applications. Recent works show that MOFs play a vital role in targeted drug delivery techniques [11–15]. In this context, zeolitic imidazole frameworks (ZIFs) are members of the MOF family, composed of zinc and imidazole ligands. Due to the unique physicochemical properties such as microporosity, large surface area, controlled drug release, hydrothermal stability, stimuli responsibility, and biocompatibility, ZIFs greatly impact biomedical applications [16–18]. ZIFs-based drug delivery systems provide synergistic effects in medical techniques, such as chemotherapy, phototherapy, chemo-photothermal therapy, and immunotherapy. Zinc-based MOFs are potential carriers for various stimuli-responsive approaches such as pH, enzyme, and temperature [19–22]. As stated in existing reports, all ZIFs have been popularly used in stimuli-responsive drug delivery techniques. The mechanism of pH-responsive intracellular corrosion of ZIF particles and its biological effects are given more advantages in biomedicine. Previously, many synthetic antibiotics and chemotherapeutics such as doxorubicin, cisplatin, 5-fluorouracil, camptothecin, vincristine, and ciprofloxacin combined with ZIFs and utilized in various biomedical applications [23–26]. Some studies suggested that encapsulating plant-derived compounds with nano drug delivery systems plays a major role in anticancer, antimicrobial, and tissue engineering techniques. Much biomedical research focused on separating, characterization, and utilization of phytocompounds as natural anticancer, antioxidants, antiviral, anti-tubercular, and antibiotics agents. Plant compounds and MOFs exhibit a synergistic effect on several biological processes in biomedicine [27–31]. In this case, coumarin is a phytocompound derived from edible and medicinal plants, which exhibits many pharmacological activities. Coumarin derivatives are used to discover a variety of new antibiotics, anticoagulants, bio-enzymes, chemotherapeutics, and bioluminescence. Coumarin derivatives have a good complexing ability with metal ions [32–36]. The formation of metal coumarin complexes plays an ideal role in biological activities. Recently, it has been reported that metal and hydroxycoumarin complexes might be applicable as anticoagulants, luminescent for DNA conformation, chemical sensors, and molecular devices [37–40]. N and S donor ligands greatly enhanced the coordination bonding with metal ions. Nowadays, antibiotic resistance of bacteria is a considerable health threat worldwide due to its continuous usage [41–47]. In the wound care process, controlling bacterial infections in the wound site is challenging for physicians. The need for effective antibiotics and wound-healing agents urged us to find new materials for wound care [48–51]. This research work mainly focuses on fabricating 7-methoxy coumarin encapsulated ZIF-L nanocomposite to control wound-infecting bacteria and safe drug delivery in wound sites. This 7-MC@ZIF-L nanocomposite will be an efficient, low-cost, biocompatible drug delivery system for wound healing applications.

2.1. Chemicals

7-methoxy coumarin, 2-methyl imidazole, Zinc nitrate, Acridine orange, ethidium bromide, and MTT was bought from Sigma Aldrich, USA. Nutrient broth, nutrient agar, Ciprofloxacin, Muller Hinton agar, crystal violet, and ethanol were obtained from Himedia Chemicals, India. All the chemicals and reagents were used directly without further purification. Milli-Q water was used in all the experiments.

2.2. Characterization techniques

The Shimadzu UV2450 spectrometer identified the absorption spectra of the 7-MC@ZIF-L nanocomposite in the 200–800 nm wavelength range. The crystalline nature of the nanocomposite was confirmed by using a Panalytical Xpert powder diffractometer with Cu Kα (λ = 1.540598Å) radiation. Fourier transform infrared (FTIR) spectroscopy (Bruker Optik Vector 33-MIR FTIR spectrometer) identified functional groups of synthesized nanocomposites ranging from 400 to 4000 cm^− 1. The surface morphologies were characterized via field emission scanning electron microscopy (FE-SEM, Zeiss Merlin, operating at 5 kV). The thermal stability of the nanocomposite was quantified using TGA, Perkin Elmer Diamond TG under a nitrogen atmosphere. The optical density (OD) was quantified by (Biotek Synergy Neo2) microplate reader. DLS and HR-TEM (JEOL JEM2100) analysis examined average particle size and ultra-fine nanoparticle structures.

2.3. Synthesis of 7-MC@ZIF-L nanocomposite

7-methoxy coumarin encapsulated ZIF-L nanocomposite (7-MC@ZIF-L) was synthesized according to Prabhu et al. (2022) with some modifications [28]. In brief, Zinc nitrate hexahydrate (200 mg) was dissolved in 90 ml of Milli-Q deionized water. 2-methylimidazole (600 mg) and 7-methoxy coumarin (20 mg) was dissolved in 10 ml of methanol. The two separate solutions were mixed for 30 min under magnetic agitation at room temperature. After, the coumarin and 2-methyl imidazole mixture were slowly added to the zinc nitrate solution. The color of the reaction mixture changed from transparent to milky white colloid, which indicates the formation of the 7-MC@ZIF-L nanocomposite. Then the nano colloid was separated by centrifugation at 8000 rpm for 10 min and washed with Milli-Q water thrice. The unreacted metal and organic compounds were removed, and the absorbance of the supernatant solution was detected using a UV-vis spectrophotometer. Finally, the hydrated 7-MC@ZIF-L nanocomposite was dried in a hot air oven at 60 ºC for 12 h.

2.4. In vitro drug release study

The pH-responsive drug releasing the potential of 7-MC@ZIF-L nanocomposite was quantified by in vitro drug release kinetics [29]. The 50 mg of the 7-MC@ZIF-L nanocomposite was immersed in 50 mL phosphate buffer saline medium at pH 5 and 7.4 with gentle magnetic stirring (150 rpm) at room temperature. After a specific time, 2 mL of mother solution intervals were centrifuged and replaced with 2 ml of fresh PBS medium. The content of 7-methoxy coumarin in the supernatant was detected by atomic absorption. The absorption at 275 nm to determine coumarin release was measured by UV–the vis absorption spectrum. The total amount of drug released has been calculated using the standard calibration curve.

2.5. Cytotoxicity Assay

Human fibroblast L929 cells (Density 6×103 per well) were inoculated in 96-well plates at 37 ᵒC for 24 hours [29]. After the incubation culture medium was replaced and added with different concentrations (20–100µg/ml) of the 7-MC@ZIF-L nanocomposite and coumarin to continue incubation for 24 h. The cell group was incubated without adding nanocomposite, which was considered a negative control. After incubation, the culture medium was discarded, and 100 µL of Cell Counting Kit-8 (CCK-8) solution was added. The absorbance value of treated wells was quantified at 450 nm using a multi-plate reader at the end of 30 mins incubation. To evaluate the in vitro cytocompatibility of nanocomposite, compare it to the negative control. The percentage of cell viability was calculated by using the formula:

$$CT \% =\frac{\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e} \text{o}\text{f} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}-\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e} \text{o}\text{f} \text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d} }{\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e} \text{o}\text{f} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\text{X} 100$$

2.6. Biocompatible nature of 7-MC@ZIF-L nanocomposite

The Biosafety nature of fabricated 7-MC@ZIF-L nanocomposite was evaluated based on in vitro cytotoxicity mechanism against human peripheral blood cells using MTT (3–4,5-dimethylthiazol diphenyltetrazolium bromide) assay [31]. PBMC cells were obtained from National Centre for Cell Science (Pune, India). The PBMC cells were seeded into DMEM with antibiotic penicillin (20 µg/mL) and incubated overnight at 37 ºC in a CO₂ incubator. After incubation cells were treated with different concentrations of 7-MC@ZIF-L nanocomposite (100–500 µg/ml) for 24 h at 37 ºC under CO₂ flow. Hydrogen peroxide (250 µM) was used as a positive control to compare cytotoxicity. After 24 h, cell viability was measured by a multi-plate reader (Molecular Device Spectramax M3) at 570 nm.

2.7. In vitro wound scratch assay

The wound healing potential of 7-MC@ZIF-L nanocomposite by in vitro cell scratch assay [48]. Human fibroblast L929 cells were obtained from the National Center for Cell Science (Pune, India). The cells were cultured in Dulbecco's modified eagle medium with 20% fetal bovine serum and 1% penicillin. The cell proliferation rate was quantified by using a Click-iTEdU Imaging Kit. The healthy L929 cells were treated with a 100 µg/ml concentration of 7-MC@ZIF-L nanocomposite. After the nanocomposite treatment, cells were allowed overnight culture at 37°C in a 90% oxygenated tissue culture hood. The L929 cells were scratched with a sterile 200µl micro tip. The initial wounding area and migration of scratched cells after 12, 24, 48, and 72 h were photographically monitored using an inverted fluorescence microscope. The percentage of wound healing was calculated by using Image J software. Measurements were performed in triplicate values. The percentage of the wound healing potential of nanocomposite is calculated to be the following formula:

$$Wound closure \% =\frac{\text{A}0-\text{A}\text{t} }{\text{A}\text{t}}\text{X} 100$$

A0 -is the area of wound scratch at the time of scratching. At -is the area of the wound after the scratches are performed.

2.8. Assessment of antibacterial activity

The antibacterial efficacy of the 7-MC@ZIF-L nanocomposite was evaluated by the good diffusion method [30]. The four human infectious bacteria (E. coli, S. aureus, S. epidermis, and P. aeruginosa) were overnight cultured in nutrient broth. The 20 ml of nutrient agar was poured into sterile Petri dishes. The thickness of the agar medium in all Petri dishes was maintained in equal quantity. After the bacterial strains were uniformly streaked on the surface of nutrient agar plates. The zone of inhibitions illustrates the inhibitory activity of the nanocomposite on bacterial growth. The average diameter was calculated for each sample. In each of these Petri plates, 5 mm in diameter sized wells were made using a sterile Cork borer No. 4. Alternatively, wells were filled with different concentrations (20–100 µg/ml) of the 7-MC@ZIF-L nanocomposite. The nanocomposite-treated bacterial plates were incubated for 12 hours at 37 ºC. Streptomycin was used as a positive control. The diameters of the bacterial inhibition zones were measured using a zone scale. The average zone of inhibition in all bacteria calculates from triplicate values.

2.9. Assessment of anti-biofilm potential

The biofilm inhibition efficiency of 7-MC@ZIF-L nanocomposite against ATCC MRSA 33591 and N 7 clinical strains were analyzed following the protocol [27] of Prabhu et al. (2020). MRSA and N7 bacterial cultures (108 CFU/ml) were diluted using TSB broth with 0.5% glucose. Following, bacterial strains were treated with 25–100 µg/ml of different concentrations of 7-MC@ZIF-L nanocomposite for 48 hours at 37 ºC under anaerobic conditions. The excess of free-floating bacteria was removed with PBS solution by centrifugation. Nanocomposite-treated bacteria stained by crystal violet (0.4%) and slides were subjected to distilled water to remove excess stain. Finally, the bacterial inhibition was quantified from the absorbance of color intensity at 570 nm using a multi-plate reader. Morphological changes and structural deformities of 7-MC@ZIF-L nanocomposite-treated bacteria groups were observed, and images were captured by inverted phase contrast light microscopy.

2.10. Live-dead Cell Staining and wound scratch

The survival of 7-MC@ZIF-L nanocomposite-treated cells was measured using a live-dead assay [29]. L929 fibroblast cells were inoculated in 12-well culture plates and cultured for 24 hours at 37 ºC under CO₂ flow. After one day of cell culture, fresh media containing 7-MC@ZIF-L nanocomposite was replaced with a concentration of IC₅₀ in each cell group. Cells are treated with blank media, considered a negative control. Cell scratches were made by 200 µl sterile micro tips. After the nanocomposite treatment, cells were washed with PBS solution. The excess number of media and residues removed from cells. The AO/EB dual stains 0.5 mL was added into control and treated cell groups for 30 min. Images were taken under an inverted fluorescence microscope at different time intervals.

2.11. Brine shrimp lethality assay

Biocompatibility of fabricated 7-MC@ZIF-L nanocomposite was examined by using brine shrimp lethality bioassay. Artemia salina cysts are maintained in laboratory conditions and were used for cytotoxicity assay [26], according to Prabhu et al. (2021). Briefly, Artemia salina cysts of 0.5 gm were aerated in a 1-liter capacity glass jar containing seawater near the light source. The cysts containing seawater were aerated continuously for 48 h at a temperature 25–35°C range. After hatching, active nauplii were collected from bright illumination and transferred to a sterile 12-well plate. The different doses of 7-MC@ZIF-L nanocomposite (25–175 µg/ml) were added into nauplii-containing wells. The plates were allowed to incubate for 48 h in dark conditions, and several dead nauplii were counted after incubation. Percentages of nauplii mortality and lethal dose (LD₅₀) for 7-MC@ZIF-L nanocomposite were quantified by using probit analysis. A nanocomposite-loaded well is considered a negative control. The dead nauplii in each well were counted by magnification lenses every 12 h. The percentage of mortality was calculated by using the standard formula:

$$\text{L}\text{e}\text{t}\text{h}\text{a}\text{l}\text{i}\text{t}\text{y} \% =\frac{\text{N}\text{t}-\text{N}\text{c} }{\text{N}\text{t}}\text{X} 100$$

Where the number of surviving Artemia salina nauplii in the treated group (Nt) and control group (Nc).

2.12. Statistics

Data are expressed as mean ± SD. Statistical evaluation was performed by two-tailed Student’s t-test or one-way ANOVA. ns represents no statistical significance, * is p < 0.05, ** is p < 0.01, and *** is p < 0.001.

3.1. FTIR and Powder XRD analysis

FTIR spectroscopy studies examined the active functional groups of ZIF-L and 7-MC@ZIF-L nanocomposite. The results are shown in the figure. 1A&B. The spectra of the 7-MC@ZIF-L nanocomposite were found to be more similar to bare ZIF-L frameworks with some additional peaks. The sharp and intensive peaks at 562 cm-¹ and 658 cm^− 1 correspond to the Zn-N stretching vibration of zinc and imidazole binder co-ordinations. The sharp maximum absorption peaks at around 763 and 1148 cm^− 1 correspond to C–N stretching vibrations of imidazole linkers. The long peak observed at 1484 and 1624 cm^− 1 were attributed to C-O stretching and C = C stretching vibrations of amide and carboxyl groups from methyl imidazole. The 2347 and 2932 cm^− 1 peaks represent aliphatic C–H stretching vibrations of imidazole binding units. The broad peak at around 3456 cm^− 1 indicates the presence of a hydroxyl group in ZIF-L frameworks. The additional peaks and shifting of characteristic peaks confirmed the incorporation of 7-methoxy coumarin within ZIF-L frameworks.

The crystal structure and patterns of the fabricated 7-MC@ZIF-L nanocomposite and bare ZIF-L frameworks were examined through the powdered XRD technique (Figure.1C&D). Powder XRD spectra of ZIF-L and 7-MC@ZIF-L nanocomposite showed distinct diffraction peaks at 13.82°, 15. 38°,17.46°, 21.43°, 26.54°, 27.83°, 29.76°, 32.52°, 36.28°, 42.64°, and 43.72° respectively, codifying the Bragg’s reflection planes (100), (002), (101), (102), (110), (103), (102), (104), (203), (114) and (104), confirming the hexagonal crystalline structure according to JCPDS: 01-1136. The powder XRD pattern of ZIF-L frameworks matched well with the previous report [29] by Prabhu et al. (2021). In this context, 7-MC@ZIF-L nanocomposite showed the same diffraction patterns as ZIF-L frameworks indicating good crystallinity and stable metal-organic framework formation. However, some additional X-ray diffraction peaks correspond to the presence of 7-methoxy coumarin inside the ZIF-L framework without alteration in the crystal structure of ZIF-L frameworks.

3.2. UV-vis spectroscopy and TGA analysis

The formation of ZIF-L frameworks and 7-methoxy coumarin encapsulation was primarily confirmed by the UV-visible spectroscopy technique (Figure.2A). The UV-visible spectra of the alone ZIF-L frameworks showed sharp absorbance at 305 nm due to the absorbance of zinc ions. 7-methoxy coumarin exhibits absorbance at 326 and 425 nm, respectively. In this context, coumarin encapsulated 7-MC@ZIF-L nanocomposite shows sharp absorbance in the coumarin range 327nm, which indicates the uniform encapsulation of 7-methoxy coumarin within ZIF-L frameworks. The encapsulation of coumarin overcomes the absorbance of zinc ions from the ZIF-L frameworks. The thermal stability of nanomaterials plays an important role in biomedical applications. The thermal heat tolerance and decomposition range of fabricated 7-MC@ZIF-L nanocomposite and ZIF-L was investigated by the TGA technique, and the results are shown in the figure. 2B&C. TGA spectra of bare ZIF-L frameworks (Figure.2C) show high stability until 550°C with a minimum reduction in the total mass of 4.32% between 50–550°C range, confirming the high stability. When the temperature increases to 550–750°C, a sharp weight loss event of 46.28% was observed. The maximum weight loss region above 550°C indicates the decomposition of the ZIF-L due to the denaturing of organic linkers. In this case, the 7-MC@ZIF-L nanocomposite showed three different weight loss events (Figure.2B). At a temperature range of 80–190°C, the first weight loss of 4.32% indicates the removal of water molecules from frameworks. The second weight loss was 11.39% from 250 to 550°C, corresponding to the decomposition of 7-methoxy coumarin from ZIF-L frameworks. The major total mass 39. 21% reduction from 570 to 800°C confirmed the decomposition of ZIF-L frameworks.

3.3. Morphology and particle size analysis of 7-MC@ZIF-L nanocomposite

Morphology and particle size of nanomaterials play a prestigious role in biomedicine. Crystalline structure and particle size distribution are determining factors in cellular uptake and internalization across biological systems. The crystal morphology of nanocomposite in FE-SEM and TEM analysis (Figure.3). Microscopic images of 7-MC@ZIF-L nanocomposite exhibited a typical sphere shape, but they are not stable in the crystal structure. It also formed agglomerated polygon particles. FE-SEM images (Figure.3A&B) showed two-dimensional flaky particles with agglomeration. TEM images of nanocomposite figure. 3C&D show crystalline particles well of 70–100 nm in size, and no other crystal morphology were observed. The less than 100 nm size particles can easily travel across the cell wall and intracellular organelles. The FE-SEM and TEM images concluded that the fabricated 7-MC@ZIF-L nanocomposite is highly suitable for wound healing applications. In this study, the figure shows the average particle size of the fabricated 7-MC@ZIF-L nanocomposite (Figure. 4A) was observed to be 120 nm. It is a suitable size for potential cellular penetration and endocytosis of living cells.

3.4. pH responsible drug release kinetics of 7-MC@ZIF-L nanocomposite

Stimuli-responsive drug release potential of 7-MC@ZIF-L nanocomposite was evaluated at different pH (5 and 7.4) in a time-dependent way. The line graph figure.4B showed a constant increase in 7-methoxy coumarin released under acidic pH-5, which reached a stationary phase at 38 hours, and no further changes in drug release up to 72 hours. However, the physiological pH-7.4 show a very minimum amount of coumarin release was observed in the same period. At physiological pH-7.4, the 7-MC@ZIF-L nanocomposite exhibits a stable with a drug release efficiency of 20% at the end of the first 16 hours, followed by 32% at 72 h. In this context, when the pH decreased to 5, the drug release was observed to be 62% at 16 h, which constantly increased to 85% at 72 hours. The slow and sustainable release of 7-methoxy coumarin at the physiological pH of 7.4 confirmed the hydrophobic nature of ZIF-L frameworks. On the contrary, in the acidic pH-5, the threefold increase in coumarin release under acidic conditions due to the disintegration and breaking of organic linker 2-methyl imidazole from ZIF-L frameworks. The protonation of imidazole linkers leads to the cleavage of bonds between zinc and imidazolate binder and causes drug release. The in vitro drug release kinetics illustrated that 7-MC@ZIF-L nanocomposite could be a more suitable material for stimuli-responsive wound care applications.

3.5. Cytotoxic nature of 7-MC@ZIF-L nanocomposite against L929 cells

The unique physicochemical and biological characteristics of nanomaterials have great interest in biomedicine. Nanomaterials are used as agents for drug delivery or imaging and gene delivery, and biological devices. Due to toxicity, the chemical composition and morphological properties become more complex for human use. If nanomaterials accumulate or interact with biological molecules, humans face many health risks. Recently, very limited information has been available about nanomaterials' biological properties and effects. Therefore, the biosafety assessment of fabricated 7-MC@ZIF-L nanocomposite is necessary for implementing its wound healing application. In vitro cytotoxic effects of the nanocomposite were evaluated against human peripheral mononuclear cells (PBMC) as a model system. PBMC cytotoxicity study has been proven as preliminary toxicity effects of fabricated nanocomposite against human cells. In vitro cytotoxic effects of the 7-MC@ZIF-L nanocomposite were examined based on the cell membrane damage and stability via trypan blue exclusion assay. The 7-methoxy coumarin encapsulated ZIF-L showed no cytotoxic effects and cell viability reduction until 500µg/ml concentration. The result was more similar to the negative control (Figure. 5A). The positive control hydrogen peroxide (H2O2) treated cells showed 84.6 ± 0.13% cell toxicity at 250 mM concentration. The results revealed that the 7-MC@ZIF-L nanocomposite is more biocompatible with human cells and suitable antibiotic material for wound healing applications.

3.6. The anti-biofilm potential of 7-MC@ZIF-L nanocomposite

MRSA is a dreadful pathogen affecting human skin and causing wound infection-related life-threatening diseases. It is a major causative bacterium in hospital-associated infections. Biofilm constructing potential of MRSA incorporated with resistance to antibiotics. The advantages of nanomaterials in biomedicine have revolved great interest among biomedical researchers. The antibiofilm efficacy of the 7-MC@ZIF-L nanocomposite was assessed by a simple crystal violet staining method. The figure. 5B illustrated the percentage of bacterial inhibition in nanocomposite-treated bacterial pathogens at different concentrations. The 7-MC@ZIF-L treated group showed 91% of bacterial inhibition at 82 ± 1.24 µg/ml and IC50 value of 41.13 ± 0.94 µg/ml concentrations. The cell adherence and growth inhibition of ATCC MRSA 33591 and clinical strain N7 were shown in the figure. 6. This illustrates that the 7-MC@ZIF-L nanocomposite damages the biofilm colonies of MRSA and N7 pathogens in a dose-dependent manner. Encapsulation of 7-methoxy coumarin within ZIF-L frameworks increased the antibiofilm effect by attenuating the adhesion of bacterial cells. The presence of zinc ions and phytochemicals of 7-methoxy coumarin is the leading ingredient of efficient anti-biofilm effects of the 7-MC@ZIF-L nanocomposite.

3.7. Antibacterial activity of 7-MC@ZIF-L nanocomposite

Antibacterial efficiency of fabricated 7-MC@ZIF-L nanocomposite was evaluated on four human pathogenic bacteria Escherichia coli, Staphylococcus epidermis, Pseudomonas aeruginosa, and Staphylococcus aureus. The antibacterial effects of the 7-MC@ZIF-L nanocomposite in well diffusion assay (Figure. 7) with different concentrations were shown in figure.7. Results revealed that the nanocomposite potentially inhibits the multiplication of bacterial strains in dose depending on the manner with the highest activity at 100 µg/ml. Comparative assessment of the anti-bacterial activity of 7-MC@ZIF-L nanocomposite and Ciprofloxacin revealed that 7-MC@ZIF-L nanocomposite exhibited the highest bacterial inhibition S. aureus and P. aeruginosa with an inhibition zone of 18 mm and 22 mm respectively. Compared to commercial antibiotic ciprofloxacin fabricated, the 7-MC@ZIF-L nanocomposite is similar in bacterial growth inhibition. It is seen in the figure. 7 that extraordinary zone of inhibition appeared in all the bacteria S. aureus, S. epidermis, P. aeruginosa, and E. coli displaying excellent antibacterial activities. The inhibition zones induced by 7-MC@ZIF-L nanocomposite reach 18, 20, 22, and 17 mm, respectively. Biomechanism of antibacterial effect of 7-MC@ZIF-L nanocomposite due to electrostatic interaction between Zn²⁺ions in ZIF-L frameworks and lipopolysaccharide of the bacterial cell wall [30, 49]. The attachment of 7-MC@ZIF-L nanoparticles alters the integrity of the phospholipid bilayer and leads to penetration into the bacterial intracellular organelles. The hydrolysis-mediated releasing of Zn²⁺ ions and methoxy coumarin molecules promotes intracellular ROS generation inducing the apoptosis mechanism. In wounds, controlling bacterial infection is the main process for effective wound care. Antimicrobial agents are applied to control bacterial infections, and they are spreading [48, 50]. In this case, 7-MC@ZIF-L nanocomposite significantly controls the wound pathogens' multiplication in minimum dose. It will be a potential material for wound infection control and the wound care process.

3.8. The wound healing potential of 7-MC@ZIF-L nanocomposite

Zeolitic imidazole frameworks are promising for wound healing and skin tissue regeneration applications. Due to their large surface area, hydrothermal stability, biocompatibility, and antimicrobial properties to promote efficient wound healing. In the present study, the wound-healing ability of the 7-MC@ZIF-L nanocomposite was evaluated against L929 fibroblast cells. The in vitro wound scratch assay revealed that fabricated nanocomposite promotes the cell migration and proliferation mechanism within 24 h (Figure.8). The biocompatible nature of ZIF-L frameworks allows the surface to nutrient transport and cell proliferation. The presence of 7-methoxy coumarin activates the enzymatic activities of L929 cells. The release of zinc ions acts as a micronutrient for cell growth and an immune booster in cell multiplications [51]. Morphological analysis of the 7-MC@ZIF-L nanocomposite-treated L929 cells showed a healthy shape and internal structures. The porous and interconnected nanostructure of ZIF-L frameworks provides more advantages to cell growth and transport of bioactive coumarin molecules. The microscopic fluorescence images of wound scratch and live-dead assay exhibit a high cell proliferation and migration rate within 24 h of exposure (Figure.9). The bright green fluorescence intensity confirmed the more live cells in the scratched groups. The 7-MC@ZIF-L treated cells showed threefold increased cell regeneration, granulation, and wound closure at 72 h compared to the control group. This in vitro wound scratch study concluded that the loading of 7-methoxy coumarin is superior to improving wound healing by fast wound closure. Inflammation of wound sites significantly decreases the healing process in many cases. In this context, the antibacterial proficiency of the 7-MC@ZIF-L nanocomposite will control bacterial infection at wound sites and enhance the healing mechanism. Undoubtedly, the 7-MC@ZIF-L nanocomposite might be a new biocompatible and low-cost material for wound healing applications.

3.9. Biocompatibility of 7-MC@ZIF-L nanocomposite against Artemia salina

The in vivo toxicological effects of the synthesized nanocomposite were evaluated against the Artemia salina model system. Lethality assay of Artemia salina is an alternative method to existing methods due to its low cost and reliability for results of experimental toxicity studies. The LC₅₀ value of the 7-MC@ZIF-L nanocomposite was calculated to be 148.13 ± 2.82 µg/mL using Anova data analysis software from triplicates values. The morphological changes of nanocomposite-treated Artemia salina nauplii were observed under an inverted phase contrast microscopy (Figure.10). The images exhibit no significant malformations and growth inhibition up to 150 µg/mL concentration. However, the microscopic images of nanocomposite-treated Artemia salina show very mild toxicity, and growth reductions such as size and mortality have appeared at the highest concentration of 175 µg/mL. The results concluded that the 7-MC@ZIF-L nanocomposite exposed negligible cytotoxic effects against nauplii at a maximum of 175 µg/mL dose. And no other behaviour changes in swimming and structural longevity were observed until 48 hours of treatment. The fabricated 7-MC@ZIF-L nanocomposite is non-toxic at the lower dose and only mildly toxic at higher concentrations. In vitro and in vivo toxicity studies concluded that 7-MC@ZIF-L is highly suitable and biocompatible for wound healing applications.

In conclusion, we constructed a pH-responsive 7-methoxy coumarin-loaded ZIF-L nano-delivery system to improve the wound healing effect of 7-methoxy coumarin on wound care treatment. The fabrication of 7-MC@ZIF-L nanocomposite was simple and under eco-friendly conditions, making it possible for mass production. This zeolitic imidazole frameworks-based drug delivery system has the potential release of 7-methoxy coumarin and zinc ions in the acidic environment. The biocompatible and antimicrobial properties of the prepared nanocomposite showed excellent bacterial inhibition and biofilm control against human pathogens. The 7-MC@ZIF-L nanocomposite showed a 91% of reduction in biofilm production of MRSA and N7 clinical strains. In the antibacterial study, the nanocomposite exposed high levels of inhibition zones against four wound infectious pathogens, S. aureus, P. aeruginosa, E. coli, and S. epidermis, at lower concentrations. The antimicrobial mechanism of the 7-MC@ZIF-L nanocomposite exhibits the synergistic effects of zinc ions and 7-methoxy coumarin release mediated bacterial cell death. In vitro wound scratch assay confirmed that the nanocomposite could potentially wound cell regeneration quickly. Further investigation of in vitro and in vivo biocompatibility studies concluded that there are no cytotoxic effects against fibroblast cells and model organism Artemia salina. The overall experimental results strongly concluded that 7-MC@ZIF-L nanocomposite is a promising biomaterial for antimicrobial activity-mediated wound healing application. Nanocomposite could be a suitable and low-cost material for wound care that will facilitate effective wound healing; it can also reduce the cost of treatment.

Competing interests

The authors declared that they have no conflict of interest in this research.

Availability of data and materials

All the data generated or analysed during the study are included in this published article.

Funding

R. Prabhu Thanks to BioMe Live analytical centre, Karaikudi for financial and experimental support.

Authors' contributions

Prabhu Raju: Writing & editing the original draft. Mangaiyarkarasi Rajkumar: Investigation. Jegatheeswaran Sona Muthu: Formal analysis, Methodology.

Ethical Approval

There is no human and animal trials were carried out in this research work.

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No competing interests reported.

Scheme.1.jpg
Scheme. 1- The graphical abstract of 7-MC@ZIF-L mediated wound healing mechanism.

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Ecofriendly one-pot fabrication of 7-methoxy coumarin encapsulated zeolitic imidazole nanocomposite for pH-responsive wound healing application and its biosafety evaluations

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Chemicals

2.2. Characterization techniques

2.3. Synthesis of 7-MC@ZIF-L nanocomposite

2.4. In vitro drug release study

2.5. Cytotoxicity Assay

2.6. Biocompatible nature of 7-MC@ZIF-L nanocomposite

2.7. In vitro wound scratch assay

2.8. Assessment of antibacterial activity

2.9. Assessment of anti-biofilm potential

2.10. Live-dead Cell Staining and wound scratch

2.11. Brine shrimp lethality assay

2.12. Statistics

3. Results and discussions

3.1. FTIR and Powder XRD analysis

3.2. UV-vis spectroscopy and TGA analysis

3.3. Morphology and particle size analysis of 7-MC@ZIF-L nanocomposite

3.4. pH responsible drug release kinetics of 7-MC@ZIF-L nanocomposite

3.5. Cytotoxic nature of 7-MC@ZIF-L nanocomposite against L929 cells

3.6. The anti-biofilm potential of 7-MC@ZIF-L nanocomposite

3.7. Antibacterial activity of 7-MC@ZIF-L nanocomposite

3.8. The wound healing potential of 7-MC@ZIF-L nanocomposite

3.9. Biocompatibility of 7-MC@ZIF-L nanocomposite against Artemia salina

4. Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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