Synthesized zinc peroxide nanoparticles (ZnO₂-NPs): a novel antimicrobial, anti-elastase, anti-keratinase, and anti-inflammatory approach toward polymicrobial burn wounds

Introduction

Increasing multidrug resistance (MDR) has generated scientific interest to synthesize novel antimicrobial agents with broad spectrum activities to complement and/or replace conventional antibiotics.¹ There are two main categories of antimicrobial agents, organic and inorganic. Over the past decade, inorganic materials, such as metal and metal oxides, have attracted more attention as they have been found to be safe for human beings and animals and also had the efficiency to withstand harsh conditions.² To date, several types of synthetic metal oxide nanoparticles (NPs), such as titanium, silver, zinc, ferric, silicon, copper, and magnesium oxide have profound antimicrobial activities. The inorganic zinc oxide nanoparticles (ZnO-NPs) are of particular interest because of their unique properties, such as antibacterial, antifungal, wound healing, good chemical stability, biocompatibility, and low toxicity.^3–6 ZnO-NPs may be considered as bi-functional materials because of their ability to work as safe antimicrobial agents for dermatological and orthopedic applications in addition to the role of zinc as a vital trace element for building, stabilizing, and functioning of proteins and enzymes.^7,8 Moreover, most of the elements constituting the metal oxides are considered as trace elements that are vital to the human body.⁹ Therefore, there is a great interest in incorporating antimicrobial metal oxide NPs within orthopedic, dental, and skin biomaterials.^10,11 Synthesis of metal oxide NPs using wet chemical methods is an interesting nanotechnology field because wet chemical methods are economic and simple.¹² The antimicrobial activity of metal oxides has been strongly associated with their morphology, particle size, and surface area.¹³ For example, metal oxide particles of smaller size, no greater than 100 nm, and larger surface area have exhibited stronger antimicrobial activity due to their capability to interact and damage the microbial membranes.^14,15

Burn wounds are considered a huge public health issue all over the world, especially in the developing countries. Microbial infections in burn wound patients are common and are difficult to control. In Egypt, nosocomial infection requires more attention than it currently receives. It constitutes a major problem due to morbidity and mortality among hospitalized patients. In addition to the lack of universal approach, unavailability of records, statistics as well as information, makes the problem difficult to control.¹⁶ Approximately, 50%–75% of morbidity in hospitalized burn patients is due to microbial infections.¹⁷ Microbial infection is one of the most serious complications that happen in the acute period following burn.¹⁸ Polymicrobial communities (biofilms) are consortia of bacteria and fungi that colonize the damaged tissues and secrete polysaccharides that provide an optimal environment for polymicrobial survival. Polysaccharide biofilm is an important barrier to microbial consortia, enabling their escape from host immunity and confers increased tolerance to antimicrobials when compared with monomicrobial infections.^19,20 Moreover, the increase in MDR is due to the strong antibiotic selective pressure from the clinical and environmental origin. The most common etiological bacteria attributed to chronic burn wound infections are Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus spp., Klebsiella spp., Enterococcus spp., and Escherichia coli. The most prevalent fungi are Aspergillus niger, Candida spp. in addition to the genera of Aureobasidium, Malassezia, Curvularia, Ulocladium, and Trichophyton.^21,22 Limited reports have documented the bacterial and fungal co-interactions, which are often found together in different environments.²³ The contribution of fungal pathogens to polymicrobial biofilms is more than 50% in the majority of the burn wound infections.²⁴ Recently, the bacterial-fungal co-infection caused by S. aureus and Candida albicans revealed a unique architecture when S. aureus was associated with fungal hyphae. Interestingly, a number of virulence factors in S. aureus were enhanced by C. albicans.²⁵ Burns caused by both P. aeruginosa and S. aureus have resulted in a chronic non-healing wound in which such co-infection have shown that the P. aeruginosa limits S. aureus growth.²⁶ The majority of previous studies on polymicrobial burn infections have documented the pathogen-infection interaction, but often ignored the host microbiota. Grice et al²⁷ reported that the presence of host microbiota-associated pathogens in burns could lower the efficiency of burn healing.

The microbial enzyme is a virulence weapon, acts as a part of pathogen lifestyle in the infection establishment. Elastases and keratinases secreted by bacteria and fungi were investigated to help in skin invasion during burns.²⁸ Elastase (small serine protease) is a protein-degrading enzyme, including elastin substrate. It is an endopeptidase, which catalyzes the hydrolysis of internal peptide bonds within the protein. Elastin is an insoluble and highly cross-linked protein of connective tissues.²⁹ Elastase as one member of the chymotrypsin family of proteases has a tissue-damaging activity, which is primarily responsible for the breakdown of elastin and capable of degrading various plasma proteins during infections as in P. aeruginosa.^26,30 Furthermore, keratinases are an arsenal of proteases secreted by some bacterial and fungal pathogens,^31,32 that disseminate through the stratum corneum and subsequently digest the keratin network into absorbable amino acids. Dermatophytosis is a dermatophyte superficial infection in keratinized skin, caused by secretion of multiple serine-subtilisins and fungalysins (keratinases). The role of these enzymes is to reduce disulfide bonds in keratins of skin, which facilitate the penetration into host cellular system.³³ Indeed, keratinases are the major causal agents of tinea diseases. Our literature survey indicates that no available reports documented the activity of microbial elastases and/or keratinases in burn-causing infections, or reducing their activities by using metal oxide NPs. Therefore, this is the first study to evaluate the efficiency of synthesized ZnO₂-NPs as anti-elastase and anti-keratinase.

Inflammation reaction is the response of living tissues to injury. During this process, lysosomal enzymes produce a variety of disorders, by which macromolecules damaging and membrane lipid peroxidation are assumed to be responsible for certain pathological conditions.³⁴ Therefore, stabilization of lysosomal membrane is important in limitation of the inflammatory response. The mechanism of anti-inflammation depends on inhibiting the release of lysosomal constituents of activated neutrophil such as proteases, bactericidal, and fungicidal enzymes, which cause further damage and tissue inflammation upon lysosomal membrane stabilization or by extracellular releasing.³⁴ Anti-inflammatory activity of ZnO-NPs has been previously reported.³⁵ However, to the best of the authors’ knowledge, to date, there have been no reports to discuss the anti-inflammatory activity of zinc peroxide nanoparticles (ZnO₂-NPs) for further in vivo application as a novel ointment in the treatment of burn wounds.

Difficulties in the management of burn infection beside much attention toward other safe biologically active compounds have become an interesting research field and integral part of modern medical applications, and also encourage researchers to develop inorganic antimicrobial agents based on metal oxide NPs.^36,37 Therefore, the aim of the present study is to design and characterize ZnO₂-NPs as a novel antimicrobial, anti-elastase, anti-keratinase, and anti-inflammatory approach combating polymicrobial MDR pathogens of burn wound infections.

Material and methods

Synthesis of ZnO₂-NPs

ZnO₂-NPs were essentially synthesized using the following analytical grade chemicals without further purification: zinc acetate dihydrate (Zn[CH₃COO]₂·2H₂O), ammonium hydroxide (NH₄OH), hydrogen peroxide (H₂O₂; sol 40%), glycerol, ethanol, and acetone (Adwic, El-nasr Chemical Co., Cairo, Egypt). An aqueous suspension of ZnO₂-NPs was synthesized in a typical procedure by using the following co-precipitation route,

(1)

(2)

In a typical procedure, NH₄OH (10 mL) was mixed with 20 mL of 0.1 mol of Zn(CH₃COO)₂·2H₂O under magnetic stirring. Immediately, 70 mL of acetone and 3 g glycerol were rapidly added to the suspension. Then 40 mL of H₂O₂ was added to the suspension with stirring at room temperature for 30 min. The suspended precipitate was centrifuged and washed 3 times with distilled water.^38–40

Characterization of ZnO₂-NPs

ZnO₂-NPs were characterized by various conventional methods. The crystalline structure ZnO₂-NPs were characterized by X-ray diffraction (XRD) using a Philips PW 1840 diffractometer (Mahwah, NJ, USA). CuKα radiation of 1.5406Å wavelength at 40 kV and 30 mA was used to expose the samples through a Ni filter. The 2θ values were set in the range of 10°–65°. Peaks on the X-ray patterns recorded for the sample were compared with standard XRD pattern of ZnO₂ (Joint Committee on Powder Diffraction Standards [JCPDS] card No 13-0311). The mean crystallite size was calculated using the Scherer equation. Fourier transformed infrared (FT-IR) spectrum was obtained with Perkin-Elmer-1430 (Perkin Elmer, Waltham, MA, USA) using KBr pellet technique for the range 4.000 and 400 cm⁻¹. The nanostructures and size distribution of the synthetic particles were examined by transmission electron microscopy (TEM) using a JEM 100SX (JEOL, Co., Akishima, Japan) operating at an acceleration voltage of 80 kV. The specimens for TEM investigations were prepared by placing a drop of ZnO₂ suspension on a carbon-coated copper grid (400 mesh; Electron Microscopy Sciences, Hatfield, PA, USA) and allowing this to dry in air under ambient conditions. Thermal behavior of synthetic ZnO₂ was analyzed using differential scanning calorimetry (DSC, Shimadzu DSC-60A) and thermo-gravimetric analyzer (TGA, a Shimadzu TGA-50) at a heating rate of 10°C/min under a nitrogen atmosphere. The ultraviolet-visible (UV-vis) spectrum of the ZnO₂ suspension was measured by using UV-vis recording spectrophotometer (Unico-2800s).⁴¹

Isolation, identification, and selection of dominant polymicrobial pathogens

A total of 47 swabs of burn wounds were kindly provided by the Clinical Laboratories of Burn Units at Tanta University Hospitals and El-Menshawy Hospitals. All burn wound patients provided written informed consent, and the study protocol was approved by the review board of Tanta University Hospitals and El-Menshawy Hospitals for the collection of swabs from the Laboratories of Burn Units. The clinicians followed the guidelines and the standard protocols that are compatible with the requirements of the Declaration of Helsinki. The swabs were transferred to the Lab of Microbiology at Faculty of Science, Tanta University, Egypt, and immediately inoculated on selective and non-selective culture media. The identified isolates were sent to the hospital labs for diagnosis. For isolation of bacteria, the swabs were cultured on blood agar, cetrimide agar, chocolate agar, MacConkey agar, and nutrient agar (Sigma, St Louis, MO, USA). The swabs were inoculated on various mycological media (Sabouraud’s dextrose agar “Pharmacopoeia, New York, USA” with and without gentamicin and chloramphenicol and also on blood agar) for isolation of fungi. All plates were incubated under aerobic conditions for up to 72 h. Monomicrobial and polymicrobial cultures were recorded. Moderate or heavy growth was regarded as a positive culture, whereas sparse growth was neglected. Phenotypic identification and conventional biochemical tests for bacteria and fungi were performed.^42–44 Moreover, isolated yeasts were identified according to the protocols.^45,46 In order to confirm the phenotypic observation of P. aeruginosa and A. niger from polymicrobial cultures, P. aeruginosa were re-inoculated on cetrimide agar supplemented with 15 μg/mL nalidixic acid and checked by using VITEK^® 2 automated systems (BioMérieux, Marcy-L’E’toile, France). The strains of A. niger were grown on potato dextrose agar medium at 30°C for 7 days. The color and mycelial growth were observed daily. In addition, the spores-producing A. niger was identified up to the genus and species level based on macroscopic and microscopic criteria⁴⁴ as shown in supplementary additional files (Figure S1).

Antimicrobial susceptibility test for selecting MDR strains

Antibiotic susceptibility test was performed by disc diffusion method.⁴⁷ Agar plates of Muller-Hinton (Sigma) and Sabouraud’s dextrose were prepared for bacteria and fungi, respectively. For inoculum preparation, P. aeruginosa was cultured on nutrient agar and incubated for overnight. Then, it was suspended in sterile normal saline solution. The bacterial concentration was adjusted to 1×10⁷ colony-forming units (CFUs)/mL. A. niger was incubated for 7 days at 30°C on Sabouraud’s dextrose agar. The conidial suspensions were prepared according to the basic fungicidal activity test EN 1275.⁴⁸ The density of conidia in the spore suspension was optically adjusted to a concentration of (1×10⁷ CFU/mL). The prepared bacterial and fungal inocula were swabbed on the top surface of Muller-Hinton agar and Sabouraud’s dextrose agar media, respectively, and then allowed to dry for 10 min. The antibacterial and antifungal discs (Table 1) were placed aseptically on the surface of the inoculated agar plates and left for 15 min at room temperature for diffusion. All plates were then incubated for 18 h at 37°C for bacteria and 30°C for fungi for up to 3 days. The sensitivity was determined by measuring the zone of growth inhibition (mm) around the discs. The results were interpreted according to the Clinical and Laboratory Standards Institute standards.⁴⁹ MDR was defined as resistance to at least 3 or more antimicrobial categories.

Table 1 Antimicrobial activity of ZnO2-NPs against MDR Pseudomonas aeruginosa and Aspergillus niger strains Notes: Values are the mean of three replicates ± SD. Means with the same letters in the same column show the insignificant difference (P≤0.05). Abbreviations: AMB, amphotericin; AMP, antimicrobial profile; AN, Aspergillus niger; ANOVA, analysis of variance; ATM, aztreonam; AX, amoxicillin; C, chloramphenicol; CAZ, ceftazidime; CIP, ciprofloxacin; CLT, clotrimazole; CN, gentamicin; CRO, ceftriaxone; CT, colistin sulfate; CTX, cefotaxime; FEP, cefepime; FLC, fluconazole; IPM, imipenem; ITC, itraconazole; K, kanamycin; MBC, minimum bactericidal concentration; MCFG, micfungin; MDR, multidrug resistance; MFC, minimum fungicidal concentration; MIC, minimum inhibitory concentration; MIZ, miconazole; NPs, nanoparticles; ND, not detected; NYT, nystatin; PA, Pseudomonas aeruginosa; SXT, cotrimoxazole; TE, tetracycline; TOB, tobramycin; TRB, terbinafine.

Antimicrobial activity of ZnO₂-NPs

In vitro antimicrobial activity of synthesized ZnO₂-NPs was carried out by disc diffusion method as mentioned above. Different concentrations (200, 100, and 50 μg/mL) of ZnO₂-NPs-impregnated filter paper discs (6 mm) were tested against MDR P. aeruginosa and A. niger strains. Minimum inhibitory concentration (MIC) was performed by a serial dilution technique⁵⁰ using 96-well microtiter plates. Various concentrations (200, 100, 50, 25, 12.5, 6.25, and 3.12 μg/mL) of ZnO₂-NPs were prepared, and 2 μL of the prepared inoculum solution was added to every well. The plates were incubated at the optimal conditions for bacteria and fungi. MIC was the lowest concentration of ZnO₂-NPs at which no visible growth was observed. For determining minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC), a volume of the aliquots (10 μL) from the tubes, which was used in MIC assays and showed no turbidity, was sub-cultured on the surface of the nutrient agar and Sabouraud’s dextrose agar for bacteria and fungi, respectively. MBC was defined as the lowest concentration of the ZnO₂-NPs at which there was no colony formation of P. aeruginosa after 18 h incubation period, while MFC was regarded as the lowest concentration of ZnO₂-NPs that did not yield A. niger growth after days of the incubation period.

Elastase activity

The selected bacterial and fungal strains were cultured in Luria-Bertani agar and Sabouraud’s dextrose agar plates, respectively. The freshly prepared cultures were then inoculated onto agar plates containing 0.15% elastin (Sigma). The plates were incubated at 37°C for 24 h for bacteria and at 30°C for 72 h for fungi. The formation of proteolytic halo clear zone around colony is a positive result of elastase production.⁵¹ Elastase-producing strains were selected for quantitative assay as determined by diazocoupling method,⁵² using succinyl-l-alanyl-l-alanyl-l-alanyl-p-nitroanilide (STANA) (Sigma) as a substrate and measuring p-nitroanilide (p-NA) released from STANA. One unit of elastase is defined as the amount of p-NA released from STANA at 1 min.

Elastase inhibition by ZnO₂-NPs was determined spectrophotometrically using STANA as the substrate by monitoring the release of p-NA at 550 nm. Briefly, elastase crude enzyme was incubated together with different concentrations of ZnO₂-NPs. After 15 min of pre-incubation at 37°C, 20 μL of 50 mM substrate STANA was added to 0.9 mL of 50 mM Tris/HCl buffer, pH 7.5. Absorbance was measured for 60 min after addition of STANA. The inhibition activity was calculated after stopping the reaction using 1 mL of 10% trichloroacetic acid (TCA). To the reaction mixture, 0.2 mL of 0.1% sodium nitrate, 0.5% ammonium sulfamate, and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride were added and the color developed was determined spectrophotometrically at 550 nm.

Keratinase activity

The keratin-degrading capability was investigated as described by Wawrzkiewicz et al⁵³ with some modifications using keratin powder (Sigma) instead of chicken feathers, which was considered as a keratin source. The agar plates of feather meal medium (0.01% Mg₂SO₄, 0.14% K₂ HPO₄, 0.07% KH₂PO₄, 0.05% NaCl, 1.0% ground keratin, 1.5% Bacto agar) were inoculated with freshly prepared inocula of tested bacterial and fungal strains. The keratolytic activity of the strains was observed as a halo clear zone surrounding the colony. Keratinase activity of keratinase-producing strains was quantitated spectrophotometrically according to the method of Joshi et al⁵⁴ with some modifications, using azokeratin as the substrate. Briefly, an aliquot (800 μL) of the substrate azokeratin (5 mg dissolved in 50 mM phosphate buffer pH 7.5) was added to a 1.5 mL centrifuge tube. To this reaction mixture, 200 μL of the appropriately diluted crude supernatant enzyme was mixed. After 60 min of incubation at 50°C in water bath shaker, the reaction was stopped by adding 200 μL of 15% (w/v) TCA. The reaction mixture was centrifuged at 10,000 g for 15 min. The optical absorption of the supernatant was measured at 450 nm against the blank (without TCA). The increase in absorbance (A450) by 0.01 was considered as one unit (1 U) of keratinase activity when compared with the control. This formula (total activity [U/mL] = Δ OD/0.01) was used for calculation of keratinase activity. Keratinase inhibition was determined spectrophotometrically using the substrate azokeratin after incubation of the keratinase crude enzyme together with different concentrations of ZnO₂-NPs as described.⁵⁴

Anti-inflammatory activity

Membrane stabilization

The anti-inflammatory activity of ZnO₂-NPs was determined by human red blood cells membrane stabilization (HRBCsMS) method with some modifications.^55,56 Briefly, blood samples were collected from healthy volunteers who provided written informed consent, and the collection of blood samples from the healthy volunteers was approved by the review board of Tanta University Hospitals and El-Menshawy Hospitals. The blood was centrifuged at 3,000 rpm and packed cells were washed twice with normal saline solution (0.85%, pH 7.0) and 10% v/v suspension was prepared with normal saline. The assay reaction mixture contained 1 mL of ZnO₂-NPs at different concentrations and 1 mL of freshly prepared 10% human red blood cells (HRBCs) suspension. Normal saline solution (2 mL) was used as a control. Aspirin (Bayer, Leverkusen, Germany) was used as the reference drug. The assay mixture was incubated at 56°C for 30 min and centrifuged at 2,500 rpm for 5 min. The supernatant solution, which contained hemoglobin, was estimated spectrophotometrically at 560 nm. The percentage of hemolysis was calculated by assuming that 100% hemolysis resulted in the presence of normal saline solution. The percentage of HRBCsMS was calculated according to the following formula:

Albumin denaturation

To assay albumin denaturation, different concentrations of ZnO₂-NPs were mixed with 1% aqueous solution of bovine albumin (Merck, Darmstadt, Germany) fraction. The mixtures were incubated at 37°C for 20 min, then heated at 51°C for 20 min. The turbidity was measured spectrophotometrically at 660 nm. Aspirin was used as the reference drug.^57,58 The protection of protein denaturation (%) was calculated according to the following formula.^59,60 One hundred percent is the denaturation that resulted in the presence of distilled water.

Proteinase inhibitor activity

To assay trypsin inhibition, 0.06 mg trypsin (dissolved in 1 mL of 20 mM Tris-HCl buffer, pH 7.4) was mixed together with varying concentrations of ZnO₂-NPs to a final volume of 2 mL. After incubation for 5 min at 37°C, 1 mL of 0.8% azocasein (Sigma) in 20 mM NaHCO₃, pH 8.1 was added to the reaction mixture at 37°C for 20 min. The reaction was stopped by adding 2 mL of 10% (w/v) TCA solution. The assay mixture was centrifuged at 12,000 g for 10 min, then 2 mL of 1.0 M NaOH was mixed to the supernatant and absorbance was measured at 440 nm against the buffer as a blank.⁶¹ The percentage of proteinase inhibitory activity was calculated as mentioned above.

Skin burn wounds

New Zealand white rabbits were investigated to evaluate ZnO₂-NPs in the healing of their dermal burn wounds in this study. The animal experiment protocol was reviewed and approved by the Institutional Animal Ethics Committee (IAEC), Faculty of Science, Tanta University, Egypt (code; Rec-Sci-Tu-020) and the procedures in the present study were performed in accordance with the IAEC guidelines. The experimental design, ointment formulation, and the protocol of anesthesia and sacrifice were explained in detail in our previous paper.⁶² The back of rabbits was shaved, injected intramuscularly with ketamine (Vetoquinol, Lavaletrie, Canada) (15 mg/kg) and xylazine (1.1 mg/kg) to induce sedation before burning. The burn was produced by applying a hot square spatula of 1 cm² surface area, which had been heated on the flame, to the shaved backs of the rabbits for 30 s. The selected strains P. aeruginosa (PA6) and A. niger (AN4), which isolated from the patients of infected burn wounds, were used to induce infection in the burned areas of the experimental animal models. The freshly prepared cultures were used to prepare the inoculum in normal saline solution, and the microbial concentration was adjusted spectrophotometrically. The same volume of a bacterial and fungal suspension of 1×10⁷ CFU/mL was applied to the inflamed areas. The rabbits were randomly divided into 4 groups (n=6). Group 1 (negative control) in which rabbits were burnt, did not get infected or receive any treatment. Group 2 (positive control) in which rabbits were burnt, infected, but did not receive any treatment. Group 3 (ZnO₂-NPs-receiving group) in which rabbits were burnt, microbial suspensions were applied, and the rabbits received treatment with ZnO₂-NPs ointment. Group 4 (silver sulfadiazine-receiving group) in which rabbits were burnt, microbial suspensions were applied, and received treatment with silver sulfadiazine ointment (Dermazin; Sandoz, Princeton, NJ, USA). The formulation of ZnO₂-NPs ointment was prepared as described.⁶³ Skin biopsies were taken on seventh, fourteenth, and twenty-first days and clinical evaluation, including the degree of healing and wound area size, was performed macroscopically and histopathologically. The biopsy samples (0.5×1.5 cm²) were fixed in a buffered formaldehyde solution (10%). The paraffin block sections were prepared and stained with hematoxylin for histopathological examinations.

Statistical analysis

Results are presented as the mean ± standard deviation of 3 replicates. The statistical analyses were carried out using SPSS-20. The obtained data were analyzed statistically to determine the degree of significance using one-way analysis of variance and Student’s t-test at a probability level P≤0.05.

Results and discussion

Characterization of ZnO₂-NPs

XRD pattern has been used to confirm that synthetic ZnO₂-NPs have the same crystal structure of standard ZnO₂ particles. Figure 1 shows XRD pattern of the synthetic ZnO₂-NPs produced by co-precipitation method. XRD pattern of ZnO₂-NPs did not detect any impurity peaks and revealed a pure single phase structure that coincided with that of standard cubic ZnO₂ crystal structure (JCPDS card No 13-0311). XRD pattern reveals strong XRD reflections at 2θ=37.2° and 62.2° for plans (200), and (311), respectively suggesting the formation of ZnO₂-NPs with good crystal quality. The broadening of the XRD reflections indicated that the synthesized ZnO₂ particles are in the nanometer range where the average crystallite size of synthetic ZnO₂-NPs was about 13 nm as calculated by the Scherer’s equation: D = k.λ/(β.cosθ), where k=0.9; λ is the wavelength of monochromatic X-ray radiation (λ=1.5406 Å), θ is the Bragg, angle and β is the full width of X-ray pattern line at half peak.

Figure 1 X-ray diffraction pattern of the synthetic ZnO2-NPs.

The FT-IR spectrum of synthetic ZnO₂-NPs is shown in Figure 2. The spectrum reveals characteristic infrared absorption bands at 3,450, 2,370, 1,645, 1,390, and 1,050 cm⁻¹ that attributed to the vibrations of Zn-O. The absorbed H₂O on the surface of ZnO₂-NPs revealed the presence of two infrared (IR) bands: one at 3,450 cm⁻¹, belonging the stretching vibration of the O-H bond, and the another at 1,645 cm⁻¹ belonging the bending vibration of H-O-H.³⁸ FT-IR spectrum does not show any of absorption bands of −COO or −CH₃ groups of zinc acetate, suggesting the purity of synthetic ZnO₂-NPs.⁴¹ Figure 3 shows TEM images of synthetic ZnO₂-NPs. It demonstrates the possibility of precise determination of spherical ZnO₂-NPs; the particles are well-defined and well dispersed non-agglomerated NPs having spherical structures with average particle sizes in the range of 15–25 nm. The results indicate that synthetic ZnO₂-NPs have well-dispersed ZnO₂-NPs. The average particle sizes determined by TEM images were very close to the crystallite size calculated from XRD results.

Figure 2 Fourier transformed infrared spectrum of the synthetic ZnO2-NPs.

Figure 3 Transmission electron microscopy image of the synthetic ZnO2-NPs.

The TGA and DSC curves, Figure 4, showed the thermal behavior of synthetic ZnO₂-NPs from room temperature, 25°C up to 500°C. The TGA curve shows weight losses in temperatures ranging from 25°C to 500°C; a weight loss of about 4.2% below 100°C, solid arrows in Figure 4, owing to adsorbed water on the surface of ZnO₂ particles, a weight loss of about 8.7% up to 16°C owing to removal of adsorbed and chemisorbed water, and an abrupt weight loss of about 14.9%, from 92.3% to 77.4%, in the temperature range from 165°C to 245°C and then remains almost constant up to 500°C. This drop confirms the transformation of ZnO₂ to ZnO.

Figure 4 Thermogravimetric analysis and differential scanning calorimetric curves of the synthetic ZnO2-NPs.

The DSC curve revealed an exothermic peak at 211°C that is in agreement with abrupt weight loss in TGA curve, which is attributed to oxygen release due to decomposition of ZnO₂ molecules to ZnO and oxygen, according to the following chemical reaction equation,

(3)

After decomposition temperature, 211°C, up to 500°C, the formed ZnO has a stable hexagonal phase.⁴¹ Figure 5 demonstrates the UV-vis spectrum of synthetic ZnO₂-NPs suspension. The synthetic ZnO₂-NPs have an optical UV absorption in the region (200–300 nm), with maximum absorption occurrring at 239 nm.

Figure 5 Ultraviolet spectra of the synthetic ZnO2-NPs.

Incidence of monomicrobial and polymicrobial infections

Out of 47 clinical swabs recovered from skin burn wounds, polymicrobial infections were recorded in 28 patients (59.6%), while the remaining cases yielded monomicrobial infection. Out of 28 patients who had polymicrobial cultures, mixed bacteria alone were recorded in 5 (17.8%) patients, mixed fungi alone were isolated from 6 patients (21.4%), and mixed bacteria and fungi were recorded in 17 patients (60.7%). The predominant bacteria and fungi were P. aeruginosa and A. niger, respectively, and observed in 11 (64.7%) patients, while other mixed bacteria and fungi were recorded in the remaining 6 patients (33.3%). Mousa⁶⁴ reported that polymicrobial bacterial and fungal burn wound infections were observed in 57.5%. P. aeruginosa was the most common isolate (19.1%) and the predominant fungi were Aspergillus sp. Our results are in coincidence with results from previous studies.^65–69 Martin et al²⁰ found that the presence of one microorganism generates an appropriate environment for other pathogenic microorganisms, which are able to colonize the respective niche, so 2 or more pathogenic microorganisms could synergically interact to cause disease.

Multidrug resistance and antimicrobial profile (AMP)

All strains of P. aeruginosa and A. niger were tested against 15 antibacterial agents and 8 antifungals. The AMP of each strain tested is presented in Table 1. From these results, we concluded that all strains were MDR. Among the β-lactams used against P. aeruginosa, all strains were found to be resistant to amoxicillin and ceftazidime. Moreover, the incidence of resistance against cefotaxime, ceftriaxone, and aztreonam were 81.8%, while 63.6% of these bacterial strains exhibited resistance to cefepime. In addition to β-lactam antibiotics, the P. aeruginosa strains also exhibited a higher incidence of resistance to non-β-lactams. Maximum resistance was recorded to co-trimoxazole (100%), followed by kanamycin (90.9%), colistin sulphate (81.8%), tetracycline (72.7%), ciprofloxacin (72.7%), tobramycin (63.6%), gentamicin (36.4%), and imipenem (18.2%). Indeed, all A. niger strains were resistant to itraconazole, fluconazole, and terbinafine. Maximum resistance was found to clotrimazole (72.7%), miconazole (54.5%), micafungin (54.5%), nystatin (54.5%), and amphotericin (45.4%). Data of AMP revealed that all of the tested P. aeruginosa and A. niger strains demonstrated phenotypic resistance to at least 3 different classes of antibacterial and antifungal agents, and were classified as MDR P. aeruginosa and A. niger. Recently, the accumulated misuse and overuse of anti-infectious drugs have led to the development of MDR-resistant strains. Pathogens developed, by the acquisition of resistance genes or genetic mutations, defense mechanisms that made them difficult to treat, resulting in prolonged illness with a greater risk of death. At the moment, nanotechnology-based drug delivery systems emerged as a promising approach to circumvent the limitations of conventional drugs to assess the safety, quality, and efficacy.⁷⁰ Therefore, in the near future, nanotechnology could play a key role against MDR clinical pathogens.

Antimicrobial activity of ZnO₂-NPs against MDR strains

There are several reports, which have investigated ZnO-NPs due to their unique properties, such as antibacterial, antifungal, and wound healing.⁷¹ The present study is might be the first to investigate ZnO₂-NPs against polymicrobial MDR pathogens isolated from burn wound infections. Antimicrobial activities of synthesized ZnO₂-NPs were tested against MDR P. aeruginosa and A. niger (Table 1). The ZnO₂-NPs show significant inhibitory activity against strains tested with distinct differences in the susceptibility to ZnO₂-NPs in a dose-dependent manner. The mean zones of inhibition ranged from 7.0±0.0 to 40.0±1.0 mm. P. aeruginosa (PA6) and A. niger (AN4) strains were found to be more susceptible strains to ZnO₂-NPs. The highest mean zones of inhibition ranged from 12.0±2.0 to 21±1.0 mm and from 30.0±3.0 to 40±1.0 mm against PA6 and AN4, respectively. Five strains (PA2, PA5, PA10, AN3, and AN11) showed no sensitivity against ZnO₂-NPs even at its high concentration (200 μg/mL). MIC, MBC, and MFC are shown in Table 1. The values of MIC ranged between 6.25 and 50 μg/mL. MBC values ranged between 12.5 and 100 μg/mL, while MFC from 12.5 to 50 μg/mL. The antimicrobial activities of ZnO₂-NPs against MDR bacterial and fungal clinical pathogens depend on particle size, the concentration of the powder, morphology, and specific surface area. The increase in ZnO₂-NPs concentration (50, 100, and 200 μg/mL) correlated with increasing antimicrobial activities. This may be due to the increased H₂O₂ concentration from the surface of ZnO₂. By this way, the generated H₂O₂ can penetrate the cell membranes of P. aeruginosa and A. niger, and kill them. Sangeetha and Kumaraguru⁷² explained the mechanism of antimicrobial activities of metal oxide NPs. The small particle size of metal oxide NPs is associated with a larger band gap; consequently, these unfavorable conditions can prevent the recombination of excitons. Therefore, more available excitons will result in the formation of a higher concentration of reactive oxygen species, and consequently enhance the antimicrobial activities of metal oxide NPs.⁷²

ZnO₂-NPs as anti- elastase and anti-keratinase

A number of virulence factors are secreted by P. aeruginosa and can contribute to its pathogenicity. Current antifungal therapy has been threatened by the development of MDR strains, in addition to host toxicity of available polyenes as well as the fungistatic mode of azoles action.⁷³ Targeting pathogenicity and virulence are now considered valuable approaches against clinical pathogens.⁷⁴ Therefore, discovering novel drugs with enhanced efficacy, safety, and/or alternative mode of combating infections is needed. Establishment of microbial pathogenesis depends on the host-cell interaction with virulence factors secretion, mainly proteinases, including elastase (LasB protease), keratinases, lipases, gelatinases, and phospholipases. These proteinases virulence factors are extracellular enzymes, probably essential for establishing bacterial and fungal infections to degrade structural barrier, in addition to obtaining nutrients.⁷⁵ PA6 and AN4 strains were examined for qualitative production of elastase and keratinase on agar plates, and they were able to produce extracellular elastase and keratinase enzymes. The enzyme activity was assessed based on the observation of a clear zone.

Recent trends in the screening of alternative antimicrobial agents against MDR pathogens prompted us to evaluate the efficacy of ZnO₂-NPs as an anti-virulent active agent against MDR P. aeruginosa and A. niger. Elastases and keratinases production is reported to aid in the pathogenesis of P. aeruginosa and Aspergillus sp. ^75,76 and, therefore, proteinase inhibitors are being explored as potential antibacterial and antifungal agents. PA6 and AN4 strains were assessed quantitatively for the production of elastase and keratinase enzymes (Figure 6). PA6 showed relatively higher production of elastase (30 U/mL) and keratinase (28 U/mL) on the third day and second day of growth, respectively. In addition, the highest production of elastase and keratinase was observed on the fifth day of growth, and recorded at 35 and 41 U/mL, respectively, for the AN4 strain. From these results, ZnO₂-NPs were tested for their anti-elastase and anti-keratinase activity against both PA6 and AN4 strains (Table 2). A significant decrease in elastase and keratinase activities was recorded with increased concentrations of ZnO₂-NPs until 200 μg/mL, at which enzyme activities completely stopped. Proteinases are believed to contribute to microbial virulence by digesting immunologically important proteins and destroying host tissues.⁷⁷ As well, the inhibition of these enzyme activities may reduce the microbial pathogenesis. Therefore, the broad spectrum anti-proteinase activities of ZnO₂-NPs in addition to growth inhibition of PA6 and AN4 are indicative of the efficacy of ZnO₂-NPs as potential antibacterial and antifungal drugs. To the best of the authors’ knowledge, to date, there have been no reports to evaluate the efficacy of ZnO₂-NPs as anti-elastase and anti-keratinase activity against mixed bacterial and fungal pathogens isolated from burn wound infections.

Figure 6 Time course of elastase (A) and keratinase (B) production by PA6 strain (blue line) and AN4 strain (red line). Abbreviations: AN, Aspergillus niger; PA, Pseudomonas aeruginosa.

Table 2 Anti-elastase and anti-keratinase activities of ZnO2-NPs Notes: Values are the mean of three replicates ± SD. Means with the same letters in the same column showed the insignificant difference (P≤0.05). Abbreviations: AN, Aspergillus niger; ANOVA, analysis of variance; NPs, nanoparticles; PA, Pseudomonas aeruginosa.

In vitro anti-inflammatory activity of ZnO₂-NPs

Anti-inflammatory activity of ZnO₂-NPs was performed to explore its efficiency. The inhibitory activity of ZnO₂-NPs (100–2,000 μg/mL) was evaluated through membrane stabilization, albumin denaturation, and proteinase inhibitory activity compared with commercially available aspirin (Table 3). Also, ZnO₂-NPs showed significant stabilization toward human red blood cells membrane (HRBCsM). Moreover, the percentage protection of ZnO₂-NPs at concentration 2,000 μg/mL was highly similar to that of a standard anti-inflammatory drug (aspirin) at the same concentration with maximum inhibition 81% and 82.1% caused by ZnO₂-NPs and aspirin, respectively (Table 3). Other studies reported the membrane stabilization method for studying in vitro anti-inflammatory activity due to the similarity between erythrocyte membrane and lysosomal membrane.^78,79 The stabilization implies that the ZnO₂-NPs may well stabilize lysosomal membranes.

Table 3 Anti-inflammatory effect of ZnO2-NPs compared to aspirin Notes: Values are the mean of three replicates ± SD. Means with the same letters in the same column showed the insignificant difference (P≤0.05). Abbreviations: ANOVA, analysis of variance; NPs, nanoparticles.

Albumin denaturation is another mechanism to explore the efficiency of ZnO₂-NPs as an anti-inflammatory active agent. Protein denaturation is a process through which proteins lose their secondary and tertiary structure as well as their biological function by application of external stress. In addition, protein denaturation is a well-documented cause of inflammation. A significant increase in inhibiting albumin denaturation was observed by increasing the concentrations of ZnO₂-NPs and aspirin until 2,000 μg/mL. However, ZnO₂-NPs were effective in inhibiting albumin denaturation with maximum inhibition of 90% when compared with aspirin, which showed 85.3% maximum inhibition (Table 3).

Anti-inflammatory activity of ZnO₂-NPs was also conducted by proteinase inhibitory mechanism. Lysosomic neutrophils are a rich source of serine proteinase. Das and Chatterjee⁸⁰ reported that leukocytes proteinase plays a key role in the development of tissue damage during inflammation as well as a significant role of protection provided by proteinase inhibitors. In the present study, ZnO₂-NPs exhibited a significant anti-proteinase activity. Also, ZnO₂-NPs were similar to aspirin in the inhibition of proteinase. The maximum inhibition for both was reached 89% at 2,000 μg/mL (Table 3). It might be the first study to discuss the in vitro anti-inflammatory activity of ZnO₂-NPs. Therefore, we suggest it as a lead compound for designing an alternative drug with a potent anti-inflammatory activity in the treatment of skin burn wounds. Hence, new in vivo treatment was conducted in order to evaluate the feasibility of ZnO₂-NPs in curing the skin wound infections, which can avoid the problem of increasing MDR bacterial and fungal pathogens if treated by conventional antibiotics.

In vivo histopathological evaluation of ZnO₂-NPs

At the beginning (first day) of the in vivo evaluation, the average diameter of the rabbit’s skin burns was 1.5±0.3 cm. All model groups were controlled during the 3 weeks of the experiment, and the rate of wound healing was measured. Topical application of ZnO₂-NPs in the group of rabbits receiving ZnO₂-NPs enhanced the rate of wound healing significantly (0.3±0.1 cm), compared with the other groups. It was observed that the rabbits in this group had no ulcers compared with positive control and sulfadiazine-receiving. Both groups 2 and 4 showed large open burn wounds with no significant increase in wound diameter of 1.1±0.2 cm and 1.3±0.1 cm, respectively. In the healing process, the damaged, burned tissues were restored to their normal status through several clinical mechanisms, including the contraction of wound that helped shrinkage of the damaged wound area. In addition, the rate of healing depended on the ability of the damaged tissues to repair themselves. The commercial synthetic ointment (silver sulfadiazine) in the case of burn treatment proved to be an effective antibacterial and antifungal agent; however, its drawback is that it delays the healing process by inhibiting the proliferation of the fibroblast and keratinocytes; this also leads to impairment of the healing process.^81,82 According to the antibiogram patterns of the isolated polymicrobial strains of P. aeruginosa and A. niger from patients of the infected burn wounds, all the bacterial and fungal strains were MDR, and according to the results of ZnO₂-NPs activities, PA6 and AN4 strains were selected as a mixed inoculum for induction of infection during in vivo evaluation of ZnO₂-NPs as promising antimicrobial agents against MDR pathogens in infected burn wounds. According to our results, the silver sulfadiazine ointment-receiving group showed no improvement in the treatment of infected burn, and this may be due to no eradications of PA6 and AN4 from the burn wounds that were more serious than rabbits of group 2 (positive control), which were infected by PA6 and AN4 strains without burn treatment. On the other hand, ZnO₂-NPs-receiving group exhibited a significant reduction in the burn surface area as well as skin contraction, which was almost complete. Furthermore, the rabbits of group 1 (negative control, burnt and without induction or treatment) were better than those of positive control group.

Histopathological microscopic examinations of all rabbit groups are shown in Figure 7 and in Supplementary materials (Table S1). Histopathological examination of wound healing in the ZnO₂-NPs-receiving group compared with silver sulfadiazine-receiving group revealed that the surface ulceration significantly decreased in the rabbit group receiving ZnO₂-NPs, whereas the seventh day of treatment showed ulcer healing with creeping of epithelium tissue by new healthy tissue to cover the ulcerated area. In addition, after 21 days of treatment, the ZnO₂-NPs-receiving group showed complete healing with a complete surface re-epithelialization, and no evidence of surface ulceration was observed (Figure 7A and B). On the other hand, after 21 days of treatment of burned skin in the silver sulfadiazine-receiving group, microscopic examination revealed delayed re-epithelialization, surface ulceration, inflammatory infiltrate, and congested vessels (Figure 7C and D). According to these results, ZnO₂-NPs have been proven to have a great potential toward MDR microorganisms-infecting burns. Therefore, we suggest the use of ZnO₂-NPs as an alternative or additional method of treatment of burn wounds infected with polymicrobial pathogens, such as P. aeruginosa and A. niger. Indeed, further studies should be conducted to well establish the efficiency of ZnO₂-NPs in clinical practices.

Figure 7 Histopathological examination of burn wounds after 7 days (A, C) and 21 days (B, D) after treatment protocols with ZnO2-NPs (A, B) compared to silver sulfadiazine (C, D). Note: Magnification power (200×). Abbreviation: NPs, nanoparticles.

Conclusion

The synthesis of a nanotechnology-based antimicrobial system is an expanding research area, which can contribute to the development of novel technologies, especially against MDR pathogens. ZnO₂-NPs were synthesized via co-precipitation method by oxidation of precipitated ZnO particles in the presence of H₂O₂ as oxidizing agent, and glycerol as a surface modifier to prevent agglomeration of NPs. Also, XDR pattern of ZnO₂-NPs confirmed a pure single phase with cubic ZnO₂ crystal, which compared with JCPDS card No 13-0311. FT-IR spectrum does not show any of absorption bands of −COO or −CH₃ groups of zinc acetate, suggesting the purity of synthetic ZnO₂-NPs. The characterization techniques using TEM revealed synthesis of the pure phase of non-agglomerated ZnO₂-NPs having spherical shape with sizes in the range of 15–25 nm. In addition, ZnO₂-NPs have a transition temperature of 211°C. Antimicrobial activities of ZnO₂-NPs increased with the increase of concentrations. Moreover, our results reported the reduced virulence and pathogenicity of both MDR strains, PA6 and AN4, by showing a reduction in their elastase and keratinase activities. Furthermore, the anti-inflammatory activity of ZnO₂-NPs was comparable with standard aspirin. Our findings provide scientific evidence to support clinical uses of ZnO₂-NPs, which have a great promising potential for the development of an anti-inflammatory agent. Indeed, in vivo histopathological results exhibited that ZnO₂-NPs may be effective as promising antimicrobial agents against MDR P. aeruginosa and A. niger inhabiting burn wounds. More investigations are needed on human volunteers with skin burns to confirm the efficacy of ZnO₂-NPs as a novel antimicrobial drug in wound healing, especially after the success of ZnO₂-NPs in the treatment of burn wounds of the experimental animal models compared with silver sulfadiazine ointment.

Acknowledgments

The authors are thankful to Prof Safenaz El Shorbagy from Faculty of Medicine, Tanta University, for her kind assistance and monitoring the data of histopathological examination. Also, we thank Dr Mohamed El-Shetehy from Plant Pathology Department, College of Agriculture at University of Kentucky, USA for his effort in English revision of this manuscript.

Author contributions

Conceived and designed the experiments: SSA, RM, NAZ, and MFF. Performed the experiments: SSA, RM, and NAZ. Analyzed the data: SSA, RM, NAZ, MFF, and MB. Contributed reagents/materials/analysis tools: SSA, RM, NAZ, and MB. Contributed to the writing of the manuscript: SSA, RM, NAZ, MFF, and MB.

Disclosure

The authors report no conflicts of interest in this work.

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Supplementary materials

Figure S1 Microscopic (A) and macroscopic (B) examination of Aspergillus niger. Notes: For microscopic examination, magnification power is (40×) and scale bar is 2 mm.

Table S1 In vivo evaluation of inflammation, ulcer formation, and re-epithelialization in different rabbit groups Notes: −, absent; +, present. Abbreviation: NPs, nanoparticles.