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10.1186/s11671-024-04091-9
Research
Enhancing wound healing with zinc and silver nanocomposites synthesized with β-lactoglobulin: antimicrobial properties, collagen deposition, and systemic effects in a C57BL/6J mouse model
Rodzik Agnieszka 12
Pomastowski Paweł 1
Buszewska-Forajta Magdalena 34
Railean Viorica 15
Gołębiowski Adrian 12
Buszewski Bogusław 12
Niedojadło Katarzyna 6
Fijałkowski Paweł 2
Robotnik Kinga 1
Rafińska Katarzyna katraf@umk.pl

2
1 grid.5374.5 0000 0001 0943 6490 Centre for Modern Interdisciplinary Technologies, Nicolaus Copernicus University in Toruń, Wileńska 4, 87-100, Toruń, Poland
2 https://ror.org/0102mm775 grid.5374.5 0000 0001 0943 6490 Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University in Toruń, Gagarina 7, 87-100 Toruń, Poland
3 https://ror.org/0102mm775 grid.5374.5 0000 0001 0943 6490 Institute of Veterinary Medicine, Faculty of Biological and Veterinary Sciences, Nicolaus Copernicus University in Toruń, Lwowska 1, 87-100 Toruń, Poland
4 https://ror.org/05s4feg49 grid.412607.6 0000 0001 2149 6795 Department of Plant Physiology, Genetics, and Biotechnology, University of Warmia and Mazury in Olsztyn, 10-229 Olsztyn, Poland
5 https://ror.org/0102mm775 grid.5374.5 0000 0001 0943 6490 Department of Infectious, Invasive Diseases and Veterinary Administration, Institute of Veterinary Medicine, Nicolaus Copernicus University in Torun, Gagarina 7, 87-100 Toruń, Poland
6 https://ror.org/03sxjf271 grid.445394.b 0000 0004 0449 6410 Department of Cellular and Molecular Biology, Faculty of Biological and Veterinary Sciences, Nicolaus Copernicus University in Toruń, Lwowska 1, 87-100 Toruń, Poland
17 9 2024
17 9 2024
12 2024
19 1 15014 1 2024
22 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
This study explores the potential of zinc and silver nanocomposites, synthesized with β-lactoglobulin, a whey protein, in promoting wound healing, using the C57BL/6J mouse model. Our research is distinct in its dual focus: assessing the antimicrobial efficacy of these nanocomposites and their impact on wound healing processes. The antimicrobial properties were investigated through minimum inhibitory concentration (MIC) assessments and colony-forming unit (CFU) tests, providing insights into their effectiveness against wound-associated microorganisms. Notably, the formulation's effective antibacterial concentration did not exhibit toxicity to mouse fibroblasts. A key aspect of our methodology involved the use of a stereoscopic microscope for detailed monitoring of the wound closure process. Additionally, the distribution and potential systemic effects of the zinc and silver ions were analyzed using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). This analysis was crucial in evaluating metal ion absorption through the wound site and estimating any toxic effects on the body. Our findings are particularly significant in the field of regenerative medicine. Transmission electron microscopy (TEM) revealed that the tested nanocomposites notably enhanced collagen deposition, a vital component in the wound healing process. Furthermore, a reduction in glycogen levels in hepatocytes was observed following treatment with these metal-protein dressings. This novel finding warrants further investigation. Overall, our findings highlight the diverse roles of zinc and silver nanocomposites in wound healing. This study not only contributes to our understanding of metal-protein complexes in tissue regeneration but also opens new avenues for research into the delivery mechanisms of such treatments for hard-to-heal wounds.

Supplementary Information

The online version contains supplementary material available at 10.1186/s11671-024-04091-9.

Keywords

Wound healing
Zinc
Silver
Nanocomposites
Model C57BL/6J mouse
Collagen fibrils
Toxicity
National Science Centre2017/27/B/ST4/02628 2017/27/B/ST4/02628 2017/27/B/ST4/02628 2017/27/B/ST4/02628 2017/27/B/ST4/02628 2017/27/B/ST4/02628 2017/27/B/ST4/02628 2017/27/B/ST4/02628 Rodzik Agnieszka Pomastowski Paweł Buszewska-Forajta Magdalena Railean Viorica Gołębiowski Adrian Buszewski Bogusław Fijałkowski Paweł Robotnik Kinga issue-copyright-statement© Springer Science+Business Media, LLC, part of Springer Nature 2024
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pmcIntroduction

Regeneration of hard-to-heal wounds is a major therapeutic problem due to the complexity of the process and the lack of effective drugs. Open wounds expose tissues to external pathogens, increasing the risk of infection. The skin normally acts as a protective barrier against microbial invasion, so when it is compromised, infections can occur more easily and can impede the healing process, contributing to the chronicity of wounds. Chronic inflammation, caused by persistent infection, can further damage tissues and disrupt the normal healing stages (hemostasis, inflammation, proliferation, and remodeling). Some bacteria can form biofilms on the wound surface, which are protective layers that shield the bacteria from the immune system and antibiotics, making infections difficult to eradicate and a common cause of wound chronicity. Chronic wound infections can also lead to systemic effects like sepsis, where the infection spreads through the bloodstream, becoming life-threatening. Severe and persistent infections may lead to tissue necrosis, and in cases with compromised blood flow, such as in diabetics, amputation may be necessary to prevent the spread of infection and save the patient’s life. To prevent these severe outcomes, managing open wounds meticulously with appropriate antimicrobial therapies, regular debridement, and addressing underlying conditions that impair healing is crucial [1, 2] For this reason, scientific efforts are directed toward the search for new compounds with natural origins that could be used as drugs or evaluated for subsequent drug design [3]. For many years, the participation of many metal ions in wound healing has been confirmed, as they play crucial roles in various biological processes, including inflammation, cellular proliferation, and tissue remodeling. One of the key metal ions involved in wound healing is zinc (Zn2+) which is an essential trace element that plays a crucial role in immune function, protein synthesis (extracellular proteins, such as collagen and keratin), and cell division. It is involved in the synthesis and stabilization of the extracellular matrix, as well as in the regulation of enzymes, including metalloproteinases, which are important for wound healing [3]. Silver ions (Ag+) also have been known to play a role in wound healing, particularly due to their antimicrobial properties [1, 4]. Bacteria occurring in wounds can adversely affect the healing process, hence the increase in multi-resistant strains of bacteria is a cause for concern in the field of wound care. Silver is a useful tool in the treatment of infected wounds; however, the effectiveness of silver-based products depends on the form of silver used and the mechanism of delivery [5, 6]. It is important to note that the use of zinc or silver ions in wound healing should be carefully considered and monitored, as excessive exposure can be toxic. Therefore, the use of protein-ion complexes that will control the level of metal ions in the wound may be a good solution. Protein-ion complexes significantly impact wound healing, primarily through antimicrobial properties and promoting tissue repair, with metal ions like zinc and silver being particularly effective. While these ions disrupt microbial cell functions and enhance healing processes like collagen synthesis and cellular proliferation, other metals such as copper and iron may also be used, although their application is less common and requires careful consideration of their biological roles and potential toxicity.

In our previous studies we have developed the methods for synthesis metal ions—protein complexes which exhibit various biological activities. Buszewski et al. in their work indicated the inhibitory effect of Zn2+/ZnO-OVA hybrid complex based on egg white ovalbumin (OVA) against yeast strain Candida albicans ATCC10231 and bacteria Acetinobacter baumannii ATCC1605, Klebsiella pneumonia ATCC10031 [7]. The results obtained by Pomastowski et al. also demonstrate that the synthesized silver-lactoferrin hybrid complex can be used as an economical and environmentally friendly antimicrobial agent [8]. So far, we have focused on the immobilization of zinc (Zn2+) [9] and silver (Ag+) [10] ions on β-lactoglobulin (βLG), which is a small globular protein present in whey (a waste product of milk production). Zn2+ adsorption on βLG was aimed at obtaining metal complexes (ZnβLG). In turn, as a result of the Ag+ ion adsorption, reduction process was taking place and apart from the AgβLG metal complex, silver nanoparticles (AgNPs) were obtained, creating a hybrid system (AgβLG/AgNPsβLG). It turns out that depending on the structure of the protein itself as well as on the type of metal, its oxidation state, coordination numbers, and geometry, an infinite number of structures and conformations can be obtained, which have an impact on further properties of the particular complex/hybrid system [11].

The primary objective of this research is to elucidate the effects of our novel synthesized metal-protein systems: Zinc-β-lactoglobulin (ZnβLG) and Silver-β-lactoglobulin (AgβLG), on the wound healing process using a C57BL/6J mice model. These systems represent a unique approach in combining metal ions with β-lactoglobulin, a whey protein, to create nanocomposites with potential antimicrobial properties. This study is distinctive in its investigation of both ZnβLG and AgβLG as antimicrobial agents against microorganisms commonly found in bedsores. Our research methodically evaluates the antimicrobial efficacy of these nanocomposites through the determination of minimum inhibitory concentration (MIC) and colony-forming unit (CFU) tests. Furthermore, a significant aspect of our study involves measuring the accumulation of zinc and silver in the blood and liver of the treated mice. We extend our investigation to examine the impacts of these metal-protein complexes on wound healing and liver function at the ultrastructural level. This comprehensive approach allows us to understand not only the antimicrobial properties of these nanocomposites but also their overall effect on wound healing and systemic health. Through these multifaceted analyses, our study aims to contribute valuable insights into the application of metal-protein nanocomposites in medical therapeutics, specifically in the context of wound healing, and paves the way for further research in this innovative field.

Methods

Preparation and characteristics of ZnβLG and AgβLG nanocomposites

ZnβLG and AgβLG nanocomposites were synthesized by dispersing β-lactoglobulin (βLG) in a 0.09% sodium chloride solution at its isoelectric point (pI) of pH 4.6, following the batch method previously reported by Rodzik et al. [9, 10]. At this pH, βLG molecules possess no net charge and thus exhibit no electrophoretic mobility. In these experiments, zinc nitrate and silver nitrate solutions were prepared at concentrations of approximately 1 × 10−2mol/L (600 mg/L). The βLG solution, at a concentration of 5000 mg/L (∼3 × 10⁻4mol/L), was mixed with Zn2⁺ and Ag⁺ ions in a 1:1 volume ratio and incubated at room temperature for 24 h. The resulting ZnβLG and AgβLG nanocomposites were then centrifuged at 12,000 rpm for 10 min at room temperature, and the supernatant was removed. The precipitates were washed three times with double distilled water (ddH₂O) to purify the nanocomposites.

The concentrations of elemental silver and zinc in the nanocomposites were measured using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES, AVIO 220, PerkinElmer, Warsaw, Poland). For analysis, 1 mg of nanocomposites was dissolved in 1 mL of concentrated nitric acid (65% HNO₃) and then diluted with ddH₂O to create a 5% (v/v) HNO₃ solution.

The morphology and size of the ZnβLG and AgβLG nanocomposites were characterized using a Scanning Electron Microscope (SEM, LEO 1430 VP, Leo Electron Microscopy Ltd., Cambridge, United Kingdom) equipped with an Energy Dispersive X-ray (EDX) detector (XFlash 4010, Bruker AXS, Bremen, Germany) and transmission electron microscopy (TEM, model G2 F20X-Twin 200 kV, FEI) in combination with energy dispersive X-ray detector (EDX, Energy Dispersive X-ray, RTEM SN9577, 134 eV, Edax). The synthesis and characterization protocols for the ZnβLG and AgβLG nanocomposites used in this study were adapted from the methods detailed by Rodzik et al. [9, 10]

Antimicrobial potential of nanocomposites

Antimicrobial properties were studied by minimum inhibitory concentration (MIC) and by performing colony forming unit (CFU) assay. Zinc and silver complexes based on whey proteins: Zn–β-lactoglobulin (ZnβLG) and Ag–β-lactoglobulin (AgβLG) were tested against different microorganisms from the collection of the Centre for Modern Interdisciplinary Technologies (Nicolaus Copernicus University, Toruń: (I) Proteus mirabilis PX 220 86112 MLD, (II) Escherichia coli MB 11464 1 CHB, (III) Pseudomonas aeruginosa DSM 5007717 HAM, (IV) Staphylococcus aureus ATCC 33591 THL—isolated from the bedsores, (V) Candida albicans ATCC 10231 THL, (VI) Candida krusei CBS 2107 CBS—isolated from the diabetic foot, (VII) Lactobacillus paracasei DSM 2649 DSM, (VIII) Lactococcus lactis ATCC 10231—isolated from whey.

The study was performed in Miller Hilton (MH) broth medium according to Clinical and Laboratory Standards Institute (CLSI) procedures (with appropriate modifications) and by microdilution in 96-well plates using resazurin. In the first step, bacterial cells were inoculated in MH broth medium and incubated for 24 h at 37 °C. Zinc and silver complexes were also prepared in MH medium by serial dilution method in the concentration range of 6.25–200 μg/mL. Subsequently, the cultured bacterial strain (1 × 106 CFU/mL) and different concentrations of complexes were introduced into 96-well plates (Sigma Aldrich, Poznań, Poland) in a 1:1 ratio. After mixing, 12 μl of the redox indicator resazurin (Sigma-Aldrich, St. Louis, MO, USA) was added to each well. All samples were prepared in triplicate. The samples prepared in this manner were incubated for 24 h at 37 °C. The MIC value was visually determined by the color change of resazurin from blue to pink. The lowest concentration at which no color change was observed was considered as the MIC value.

The CFU assay procedure was similar to the MIC assay, except that after the incubation period, various dilutions of the bacterial suspension were prepared. Then, 100 μl of each sample was transferred to TSA tryptone-soy agar plates, incubated at 37 °C for 24 h, and the colonies were counted manually.

Cytotoxicity and wound healing scratch assay

The research was conducted on L929 cell line from the European Collection of Authenticated Cell Cultures (ECACC). These cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Once the cells reached 80% confluence, they were passaged using 0.25% trypsin/EDTA. For the experiments, cells were seeded in 96-well plates at a density of 2 × 10^5 cells/mL. After 24 h, the medium was replaced with fresh medium containing the tested silver and zinc complexes and incubated for an additional 24 h. Following this, 10% (v/v) of MTT solution (5 mg/mL in PBS) was added to each well and incubated for 4 h. The medium was then removed, and the formazan crystals were dissolved in DMSO for 10 min with mixing. Absorbance was measured using a microplate reader (Multiskan, ThermoFisher) at 570 nm with 650 nm as the background absorbance.

Moreover, wound healing assay was conducted to evaluate zinc and silver nanocomposites on wound closure efficiency. L929 cells were cultured in 24-well plates at a density of 1 × 10⁶ cells per well and incubated for 24 h. Linear scratches were then created in the confluent L929 monolayers using a sterile 100 µL pipette tip. The cells were immediately rinsed with PBS to remove cellular debris, and images of the scratches were captured (0 h). Following this, the cell cultures were exposed to AgβLG and ZnβLG corresponding to MIC concentration. Images of the scratches were taken again 24 and 48 h later.

Animal model

Ethics statement

All procedures complied with the regulations covering animal experimentation within EU (European Communities Council DIRECTIVE 2010/63/EU), reported in accordance with ARRIVE guidelines and were approved by the Local Animal Care and Use Committee in Bydgoszcz (BYD 21/2021, 26.04. 2021).

In the present study, C57BL/6J female mice aged 7–8 weeks were obtained from the Tri-City Academic Animal Laboratory and Service Center at the Medical University of Gdansk and divided into 8 animals in each group with an average weight of 21.53 ± 1.59 g. The size of the group was 8 individuals resulted from the value of the significance parameter determined by statistical tests and experience from previously conducted studies [12, 13]. The given estimate was based on the following aspects: (a) the experiments involved comparing the average measures from pairs of independent groups (t-test for independent groups); (b) a two-sided approach was desired; (c) a type I error of 5% was considered; (d) a power (type II error) of 80% was taken into account; (e) the variable, which according to our study has the most distinguishing aspect, concerns the measurement of wound size after several tested treatments; (f) the minimum expected difference between the sizes of wounds assessed for positive groups and corresponding control groups is 1 mm; (g) the expected maximum value of the standard deviation for this measure, within a group, is about 0.6 (for example, achieved by changing the size of the wound by ± 0.8 mm). Based on these assumptions, G*Power 3.1.9.7 [13] was used to calculate the statistical power analysis. Parameters (f) and (g) allowed for determining an effect size of about 1.67. The final result presents the sample size per group equal to 7. The charts mentioned are shown in Fig. 1 in Supplementary materials.Fig. 1 Schematic representation of the experimental design

Additionally, a 10% attrition rate was taken into account due to potential complications that could lead to the loss of animals. The experiments included making wounds, which in themselves cannot lead to the death of the animal. However, the application of the preparation can lead to the development of an infection, which can ultimately result in death. It is assumed that this is sufficient to cause a persistent infection, but not enough to quickly cause death from sepsis during the experiments. Therefore, the corrected sample size can be given by the formula: Corrected group size = [(actual sample size)/(1 − [% risk of death/100])]. As a result of such calculation, the number of 8 animals in the study group was obtained. It was used 6 groups, in which animals were treated with ZnβLG in an ointment base at a concentration corresponding to the MIC (ZnβLG(MIC)), AgβLG in an ointment base at a concentration corresponding to the MIC (AgβLG(MIC)), AgβLG in an ointment base at a concentration corresponding to the 110% MIC (AgβLG(110% MIC)). Additionally, control groups included no substance applied to the rodents (control), pure carrier applied to the mice (Ointment base) and commercially available wound-healing drug containing gentamicin applied (Gentamycin). Groups were observed for ten days. All the experimental procedures were approved by the Local Animal Care and Use Committee in Bydgoszcz (BYD 21/2021, 26.04. 2021).

Wound healing process: determination of acclimatization and body weight of mice, ANOVA analysis, wound formation, liver and blood organ collection

The procedure of the conducted experiment was carried out in six steps: (I) handling (adaptation for the laboratory condition in a period of 2 weeks), (II) wound formation, (III) application of an agent with potential therapeutic properties, (IV) observation of the wound healing process, (V) collection of biological material and euthanasia, (VI) analysis of zinc and silver distribution in tissues. The steps have been shown in Fig. 1.

First step of experiment covered the acclimatization of animals to the laboratory conditions. For this purpose, the animals were placed in cages according to the minimum living area for a single individual and kept under controlled environmental conditions with air temperature of 20–24 °C, humidity of 55 ± 10%, rate of air exchange in the room of 15–20 changes/1 h; an automated light cycle with a sequence of 12 h light/12 h darkness. Animals had provided access to food and water ad libitum. In the second step of the study, mice were given anesthesia with isoflurane (5%) by inhalation (flow rate of 3.5 l/min) (Combi-Vet Anesthesia Machine Rothacher-Medical GmbH, Bern, Switzerland). The state of anesthesia was maintained by continuous inhalation of isoflurane at a concentration of 3%. To create a wound, the skin on the back of the mouse was shaved and then disinfected with 70% ethanol. Round wounds (6 mm) were created by cutting the skin with surgical scissors. The thickness was 2 mm, which is in accordance with the thickness of the skin in the back part of rodents [14]. The wounds were made on the lateral sites of the rodent's shaved back, in the same location in all groups.

Each wound was measured and inspected at 10-day follow-up. In addition, photographic documentation was performed using a cold-light stereoscopic microscope (100 W-MST 132 Lab, Optika microscopes, Italy). During the third step, synthesized zinc and silver complexes were applied to the wounds once a day. The formulations were prepared according to the previous study by incorporating of tested substances to cocoa butter using gentle movements considering the minimum inhibitory concentration (MIC)—gentamicin 30 μg/mL (according to our previous studies), AgβLG 25 μg/mL and ZnβLG 200 μg/mL. For zinc complexes, both 100% MIC and 110% MIC were tested [15, 16].

After application, the wound was covered with a sterile plaster to reduce the risk of wound infection. In the fourth step, the effect of the applied complexes was monitored by measuring the weight and assessing wound closure using a stereoscopic microscope connected to a cold light source (100 W-MST 132 Lab, Optika microscopes Italy, Italy). Percentage of wound closuring was calculated in accordance to the equation: V [%] = (Vday,n/Vday,1)*100%. A free-to-access platform (https://goodcalculators.com/one-way-anova-calculator/%20accessed%2017.10.2022) was used to show changes. The fifth step involved the collection of biological material (blood, liver) for ICP-MS analysis. The collected liver samples were frozen and stored at −80 °C to deactivate the enzymatic reaction. Blood, in turn, was collected into a tube with the anticoagulant sodium citrate (3.2%). At the end of observation, the animals were euthanized by intraperitoneal injection of 18% sodium pentobarbital (200 mg/ml), at a dose of 200 mg/kg. Finally (step six), the amount of zinc and silver in the collected samples was determined.

Accumulation of zinc and silver in liver tissue and blood samples: inductively coupled plasma-mass spectrometry (ICP-MS)

In order to quantify the concentration of zinc and silver ions in liver and blood tissue samples, an ICP-MS technique (Shimadzu ICP-MS 2030, Shimadzu, Kyoto, Japan) with a collision cell with a helium flow rate of 6 mL/min was used. Both liver and blood were preheated for 4 h at 80 °C using a thermomixer (Thermomixer comfort, Eppendorf SE, Hamburg, Germany) in 150 µL HNO3 (1%) (Merck Suprapure, Merck, Darmstadt, Germany). Determinations were made for 66,67Zn and 45Sc, 107Ag and 103Rh, as an internal standard, using the external calibration method in three repetitions.

Microscopic analysis

The experimental material was fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate (pH 7.4) for 2 h, washed in buffer and then dehydrated in an ethanol series: 30, 50, 70, 90, 96 and 100% in each for 10 min. The step of post-fixation in OsO4 was omitted due to the possibility of measuring the silver or zinc content by EDS. The tissues were then embedded in LR White resin according to standard protocol and sectioned on an ultramicrotome (Leica Ultracut UC7). Finally, ultrathin sections were stained with 2.5% uranyl acetate and 0.4% lead citrate solutions, and examined on a JEOL 1010 transmission electron microscope at 80 kV.

Statistical analysis

Triplicate experiments were conducted to report data as mean ± SD. The results were further analyzed by Statistica DataMiner 7. One-way analysis of variance followed by Tukey post hoc test were applied to determine differences between the groups.

Data availability

The datasets used and analysed during the current study available from the corresponding author on reasonable request.

Results

Characteristics of ZnβLG and AgβLG nanocomposites

The synthesized zinc (ZnβLG) and silver (AgβLG) nanocomposites, derived from whey protein β-lactoglobulin (βLG), were prepared using batch methods. The AgβLG complex exhibited a silver content of 3.75 ± 0.12% (m/m). Remarkably, the AgβLG complex showed the formation of silver nanoparticles (AgNPs) due to the spontaneous reduction of silver ions, as illustrated in Fig. 2A. This reduction process significantly contributed to the formation of the AgβLG/AgNPsβLG hybrid system. The resulting spherical AgNPs, with an average size of 5 ± 1 nm, were uniformly dispersed within the protein matrix (Fig. 2A). Conversely, high-resolution scanning electron microscopy (HR-SEM) analysis of the ZnβLG complexes (Fig. 2B) revealed a compact and porous structure with a zinc content of 1.08 ± 0.11% (m/m). Unlike the AgβLG nanocomposites, the ZnβLG complexes did not contain metallic or metallic oxide nanoparticles. Previous spectroscopic and theoretical studies have played a crucial role in elucidating the interactions within these systems. In the AgβLG complexes, silver ions primarily interacted with the carboxylate groups of glutamic acid (Glu⁻) and aspartic acid (Asp⁻), as well as with lysine (Lys), tyrosine (Tyr), and methionine (Met). Quantum mechanical calculations based on density functional theory (DFT) simulations further clarified the reduction process of silver ions and the formation of AgNPs onto protein [8]. The binding of silver ions with Asp⁻ and Glu⁻, along with electrochemical processes, led to the immobilization and formation of the AgβLG/AgNPsβLG hybrid system [10]. In contrast, the ZnβLG nanocomposites exhibited a different interaction profile. Aspartic acid (Asp) and glutamic acid (Glu), along with the aromatic rings of tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe), played key roles in binding Zn2⁺ ions to the carboxyl groups, creating protein-metallocomplexes. These findings underscore the distinct mechanisms of metal ion binding and nanoparticle formation in the AgβLG and ZnβLG nanocomposites [9, 10].Fig. 2 High-resolution scanning electron microscopy (HR-SEM) analysis of Ag(βLG) (A) and B ZnβLG (B) nanocomposites

Antimicrobial potential of nanocomposites

For the study, four pathogens isolated from the bedsores, that are the cause of delayed healing and infections occurring in acute and chronic wounds were selected, such as Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis, and Escherichia coli. Successively, isolated from chronic wounds were fungi including Candida albicans and Candida krusei, which cause fungal infections in immunocompromised patients [13]. The antibacterial properties have also been tested on two probiotic lactic acid bacteria (LAB) Lactococcus paracasei, Lactococcus lactis, producing lactic acid, which is the end product of carbohydrate fermentation. They have health-promoting properties and also inhibit the growth of pathogenic microorganisms by showing strong antibacterial properties. For the ZnβLG complex, after 24 h incubation, for two fungal strains (C. krusei, C. albicans) and the probiotic strain L. paracasei, the MIC value was 25 μg/mL. Specific activity was noted for S. aureus whose MIC was 200 μg/mL isolated from the bedsores (Fig. 3). On the other hand, MIC of AgβLG for pathogens such as E. coli, P. mirabilis, L. lactis and S. aureus was 25 µg/mL. AgβLG complexes also presented greater inhibitory activity against L. paracasei, C. krusei, C. albicans and P. aeruginosa with MIC 12.5 µg/mL (Fig. 4).Fig. 3 Radar charts showing the full profile of the tested microorganisms treated with the synthesized zinc complexes –ZnβLG and silver complexes –AgβLG. MIC values are expressed in µg/mL

Fig. 4 Bacterial cell viability expressed as colony forming unit [%] of control

Furthermore, the experimental MIC values agreed with the CFU/mL values for each strain treated with the zinc and silver complexes (Fig. 4). In the case of ZnβLG complex, the inhibition did not even reach 50%. However, for AgβLG complexes, inhibition occurred in 99% against all microorganisms.

Cytotoxicity study and in vitro wound healing

The cytotoxicity of ZnβLG and AgβLG complexes was assessed using the MTT method on L929 mouse fibroblast cells (Fig. 5). The L929 cell line is commonly used to evaluate the biocompatibility of new materials, in accordance with ISO 10993-5 and ISO 10993-12 standards. Additionally, this cell line serves as an in vitro model for skin treatment formulations. The anti-proliferative effects were tested using colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). As shown in Fig. 5, the MTT assay results demonstrated that in L929 cells, AgβLG exhibited significant cytotoxicity at concentrations higher than 200 µg/mL. At concentrations up to this level, cell viability remained above 80% but drastically dropped to 40% at 400 µg/mL and about 7% at 800 µg/mL.Fig. 5 Viability of L929 cells treated with ZnβLG and AgβLG

ZnβLG complexes influenced the proliferation and viability of L929 cells in a completely different manner. At the lowest tested concentrations (6.25 and 12.5 µg/mL), ZnβLG did not significantly affect cell viability. However, in the range of 50–400 µg/mL, this complex promoted viability and proliferation by approximately 30%.

Using the scratch wound closure assay, it was found that supplementation of the culture medium with ZnβLG resulted in an increase in fibroblast migration after 24 h. However, complex AgβLG had no effect on the migration rate of fibroblasts; this process proceeded at a similar rate as for control cells (Fig. 6).Fig. 6 Migration of L929 fibroblasts treated with ZnβLG and AgβLG

Animal behavior and body weight of mice

During the observation period of the wound closure process, uncommon behavior of the mice was observed in the first four days. The animals showed lack of appetite, anemic behavior, and no desire to integrate. The mice's integration behavior, willingness to play, acclimatization and scratching at the bandage areas occurred in the second half of the experiment starting on the fifth day. Importantly, on all days of the experiment, the mice's behavior was correlated with their body weight characterized by weight loss up to the sixth day of the experiment (Fig. 7).Fig. 7 Body weights of animals represented in % depending on the day and the type of preparation used. Means with * are significantly different (p < 0.05) from first day control sample according to Tukey’s test

From the sixth day of observation, the animals' body weight began to increase, which was associated with gradual regeneration. The rodents reached their initial body weight on the eighth day of observation. In the case of the applied nanocomposites, the regeneration process occurred faster compared to the control. The change in weight and unnatural behavior of the mice in the present experiment may be the result of limited physical movement and discomfort after dressing application and itching of the wound site. The application of nanocomposites to wounds allowed observation of hair regrowth in contrast to controls. The nanocomposites showed a similar effect (hair regrowth) to gentamicin. This suggests that nanocomposites could be considered potential agents for treating hard-to-heal wounds.

Effect of nanocomposites on wound healing process, ANOVA analysis

On each day of observation, three independent measurements were taken of wound surface volume, which are presented as the mean ± SD. Figure 8 shows the process of wound closure. The ANOVA test showed no statistically significant differences in all studied groups (p = 0.591).Fig. 8 Wound closure in relation to the applied preparation (A), and wound volume as a percentage of closure area in reference to the wound size on the first day of the experiment (B)

The results indicate a gradual process of wound closure. An increased regeneration effect of about 30% occurred as early as the fourth day. For mice treated with zinc complexes of both ZnβLG (MIC) and ZnβLG (110% MIC), wound closure took place at about 50% on the sixth day already. For the same day, the AgβLG complex (MIC) had a slightly weaker effect, with a wound healing rate of about 40% and an effect worse than the control (45%). From day seven, for all complex’s, significantly accelerated wound closure was observed at about 73% for ZnβLG (MIC) and 77% for ZnβLG (110% MIC), so ZnβLG (110% MIC) showed a better effect. In contrast, wound closure was about 68% for AgβLG (MIC) complex. Similar observations were noted for gentamicin, which is an antibiotic often used to treat hard-to-heal wounds. The above values (day seven) indicate that the complexes performed much better in contrast to the control, for which the wound remained slightly open. Complete wound closure (approximately 100%) for the zinc and silver complexes occurred on the tenth day compared to the control, for which the wound remained open. The results indicate that a period of ten days is sufficient for wounds to heal.

Distribution of zinc and silver in tissues

The distribution of metals (Zn, Ag) in organs after treatment with ZnβLG (MIC), ZnβLG (110% MIC), and AgβLG (MIC) nanocomposites was assessed by ICP-MS (Fig. 9). The use of a zinc dressing ZnβLG (MIC) and ZnβLG (110% MIC) increased the level of this element in the blood compared to the level in control tissue. However, the level of zinc in the liver was comparable to the level in the liver of control mice. In presented study, the level of silver after treatment with AgβLG (MIC) in both blood and liver were at similar very low level.Fig. 9 Zinc and silver concentrations in liver and blood samples

Microscopic studies

The purpose of microscopic observations was to check the impact of the composites used on the structure of the scar and the potential accumulation and formation of agglomerates in the scar. Under histological evaluation, healed wounds from all tested groups were similar with almost normal hair follicles. Micrograph of a section of a 10-day wound show organization of collagen fibrils. In the control sections of the skin, small clusters of collagen fibers are visible. Cells that make up the hair follicle have the correct structure (Fig. 10A–D). In the skin in which healing was supported by AgβLG, the amount of collagen fibrils was higher. Similarly, their clusters were also larger and the arrangement more compact (Fig. 11A–D). A similar structure was visible in skin sections, the healing of which was supported by ZnβLG. In this group, fibroblasts with a large amount of rough endoplasmic reticulum were also visible, which may indicate still very active synthesis of extracellular matrix proteins for the progressing healing process (Fig. 11E–H). The presence of metal complexes was not detected in any of the analyzed sections.Fig. 10 Micrograph of a section of a 10-day control wound without treatment with ZnβLG and AgβLG. A—normal hair follicle; B—epithelial cells (E) with nuclei (Nu) are visible, along with collagen fibrils; C—fibroblast cell (F) with euchromatic nucleus (Nu), D—collagen fibrils marked with arrows

Fig. 11 Micrograph of a section of a 10-day wound treated with AgβLG (A–D) and ZnβLG (E–H). A, B—The cytoplasm of fibroblasts (F) is densely packed with cisternae of the endoplasmic reticulum (ER), which are often expanded and filled with a moderately electron-dense substance that has a fine filamentous structure. The cell nucleus (Nu) contains an abundance of euchromatin. In the surrounding extracellular matrix, there are collagen microfibrils, some aligned longitudinally and others oriented transversely. Area of intensive collagen fibrils synthesis is marked with a red border. Collagen fibrils are marked with arrows; C, D—clusters of collagen fibers arranged in different directions; E, F, G, H—Numerous fibroblasts (F) with endoplasmic reticulum (ER) cisternae, frequently enlarged. The cell's nucleus (Nu) is rich in euchromatin. In the adjacent extracellular matrix, bundles of collagen microfibrils are present, some arranged in a lengthwise direction and others crosswise. The region of active collagen fibril production is highlighted by a red boundary. Collagen fibrils are indicated with arrow markings

Sections of liver taken from control animals showed normal hepatocytes with smooth and rounded nucleus in which uniform euchromatin as well as nucleolus is present (Fig. 12A–D). Glycogen deposits are visible in the cytoplasm, near the smooth endoplasmic reticulum (SER). Mitochondria are also interspersed in this area (Fig. 12C–D).Fig. 12 A—Semithin section of liver cells from control mouse stained by methylene blue. The nuclei (Nu) of hepatocytes are notably spherical and often contain one or two nucleoli; B—Micrograph of a section of liver cell from control mouse showing spherical nucleus (Nu) with nucleolus (Nl). C, D—cytoplasm of control liver cells containing numerous mitochondria, endoplasmic reticulum (ER) and glycogen aggregates (arrows)

In liver section from mouse treated with ZnβLG and AgβLG, only minor changes were registered. Nuclei still had regular round shape and inside the main fraction of chromatin is euchromatin. Many mitochondria were located between endoplasmic reticulum membranes. The only significant difference was the lack of glycogen deposits (Fig. 13A–D, G–H).Fig. 13 Micrograph of a section of a liver in mice with 10-day wound treated with AgβLG and ZnβLG. A—Semithin section of liver cells from mouse treated with AgβLG stained by methylene blue. The nuclei of hepatocytes are spherical and contain one or two nucleoli. B—Micrograph of a section of liver cell from mice treated with AgβLG showing spherical nucleus (Nu) with two nucleoli (Nl); C, D—cytoplasm of liver cells from mouse treated with AgβLG that contain numerous mitochondria and endoplasmic reticulum (ER); E—Semithin section of liver cells from mice treated with ZnβLG stained by methylene blue. The nuclei of hepatocytes are spherical and contain several nucleoli; F—Micrograph of a section of liver cell from mice treated with ZnβLG showing spherical nucleus (Nu) with two nucleoli (Nl); G, H—cytoplasm of liver cells from mouse treated with ZnβLG wthat contain numerous mitochondria, endoplasmic reticulum (ER)

Discussion

Wound dressings have been conventionally used to protect the wound from external contamination, but they can also be functionalized with a variety of therapeutic complexes that are delivered to the wound site. Most traditional dressings (cotton bandages, cotton wool) have no active function in the healing process, but sophisticated dressings can be developed to participate in this process by incorporating active ingredients [16]. One key factor that promotes wound healing is preventing infections, which can hinder this process. Nonetheless, numerous studies suggest that bacteria are present in nearly all examined wounds and can influence all stages of wound healing, from inflammation to remodeling. Consequently, antibiotics such as gentamicin and clindamycin are commonly employed to reduce bacterial loads within wounds. Their extensive use presents several drawbacks, including mechanisms of action that are specific to certain bacterial groups and the rapid emergence of bacterial resistance. Nevertheless, the cytotoxicity of these antibiotics is less pronounced than that of other antiseptics. Thus, the initial phase of our research aimed to assess the antibacterial efficacy of silver and zinc complexes. It is challenging to compare the obtained results to published data. To the best of our knowledge, this research is pioneering and there is still a lack of wide knowledge regarding the biological effect of nanocomposites, composed of protein and metal. To date, two major preparations which are applied for the healing process are zinc oxide nanoparticles (ZnONPs) and silver nanoparticles (AgNPs) [17–20].

In our study, we demonstrated that AgβLG, in particular, exhibits an antibacterial effect against clinical bacterial strains isolated from bedsores, including S. aureus, P. aeruginosa, P. mirabilis, and E. coli. It also proved effective against fungi isolated from chronic wounds, C. albicans and C. krusei. Moreover, the concentrations at which AgβLG was effective against the tested bacterial strains are non-toxic for mouse fibroblasts and could be used for in vivo wound healing experiments. In this study, we tested a unique hybrid system where both silver ion immobilization and silver nanoparticle formation occur within the same molecule. The size of AgNPs in this system was only 5 nm, and their dispersion in the protein matrix was remarkably uniform. Similar, a study by Goswami and Vasilev demonstrated that a system based on ultrasmall nanoparticles (< 3 nm) evenly distributed in a biocompatible, thermosensitive hydrogel offers numerous advantages, including controlled silver release, long-term storage stability, and potent antibacterial activity [4]. They further showed that the AgNP hydrogel provides both antibacterial effects and wound healing properties. Using a murine model of wound infection, it was demonstrated that applying the AgNP hydrogel to the wound not only delivers superior bactericidal activity but also reduces inflammation, leading to accelerated wound closure and improved reepithelialization compared to the industry-standard silver sulfadiazine [21]. Similarly, our tested hybrid system with small nanoparticles and immobilized silver ions can act as a sustained-release reservoir, facilitating the continuous release of silver ions from the complex. This continuous release of silver ions from AgNPs is effective against a wide range of bacteria, including both gram-negative and gram-positive strains, while simultaneously ensuring low toxicity to wound tissue [1].

In contrast, the ZnβLG complex was less potent in reducing bacterial growth, showing no inhibitory effect on P. mirabilis and P. aeruginosa. Based on these findings, we conclude that AgβLG can potentially support healing primarily through its antibacterial activity, while the efficacy of ZnβLG is comparatively moderate.

In the study described, we observed differences in wound healing rates when using ZnβLG, AgβLG, and gentamicin. The wound closure progress for AgβLG was comparable to that of gentamicin. This similarity may arise from a shared mechanism of action, primarily through the inhibition of bacterial growth. Notably, accelerated wound healing was witnessed with ZnβLG, at both MIC and 110% MIC concentrations. These results imply that additional factors might influence the mechanism of action of ZnβLG. One of the can be fibroblast migration that is essential for wound healing as it aids in wound contraction, ECM deposition and tissue remodeling. In our study, we employed scratch assays to assess the impact of tested complexes on mouse fibroblast viability and migration. Results from this assay demonstrate a significant increase in fibroblast migration with ZnβLG and at the same time the lack of impact of AgβLG. The positive effect of zinc was also showed in human dermal fibroblasts. However its concentration play a main role, since higher amount of zinc due to its toxic properties, it may even limit the mobility of these cells [22]. Therefore, complexes ZnβLG can be a reservoir of zinc ions that are slowly released into the wound, where they support cells responsible for healing without causing cytotoxic effects. A factor to consider when developing new wound healing agents is their absorption through open wounds and potential systemic effects. It is a well-known phenomenon that absorption increases when the skin barrier is compromised. To ascertain the extent of metal-protein absorption during wound healing, we monitored the levels of metal ions in the blood and liver of treated mice.

Zinc is an essential trace element implicated in a myriad of physiological and biochemical functions and is naturally found in tissues. Utilizing the zinc dressing ZnβLG at a concentration equivalent to the MIC led to a slight increase in blood zinc levels compared to that seen after using ZnβLG at a 110% MIC concentration. This discrepancy might be attributed to an increased tendency for aggregation with growing nanoparticle concentration [23]. Aggregates typically have a smaller surface area in proportion to volume, hence they release fewer zinc ions. Conversely, metal complexes were not identified in the scars during micrograph analysis. This suggests that a specific quantity of metal ions is released during the healing process. However, the liver plays a crucial role in zinc (Zn) metabolism as it serves as a significant fast-exchangeable Zn reservoir and during overdose the level of zinc is growing in this organ. During an overdose, zinc levels in the liver can rise significantly, leading to potential toxicity and liver damage. However, in our study, we observed that zinc levels in the liver remained practically unchanged compared to the control group. This finding suggests that presented therapy does not cause an accumulation of zinc in the liver, indicating its safety and non-toxicity. By maintaining stable zinc levels, therapy avoids the adverse effects associated with zinc overdose, ensuring it is a safe option for further clinical applications [24].

The level of silver after treatment with AgβLG (MIC) in both blood and liver were at similar very low level. The concentrations of silver found in human tissues are extremely low [25]. In our earlier work, insignificant traces of silver were registered in both blood and liver samples, which were 9.3 µg/g and 0.022 µg/g, respectively [15]. In a study evaluating the viability of peripheral blood mononuclear cells (PBMCs) after treating a mouse model with an external preparation of LBPC-AgNCs, cell viability was found to be exceptionally high, exceeding 97%, even though minor amounts of silver were detected in the blood and liver. However, it is reported that silver can accumulate as nano-sized particles in various tissues. This accumulation can occur in organs such as the liver, spleen, and kidneys, potentially leading to long-term health effects. Additionally, silver released from implants and other medical devices tends to remain in the body for extended periods. The body primarily excretes silver through urine and feces, but the rate of excretion can be slow, leading to prolonged retention of silver particles. This extended presence raises concerns about potential chronic toxicity and the impact on organ function over time. Therefore, it is crucial to monitor and understand the long-term behavior of silver nanoparticles in the body to ensure the safety of silver-based medical therapies [26].

To gain deeper insights into the effects of ZnβLG and AgβLG on wound healing, we examined histological sections of the wounds. Our study revealed that AgβLG could positively influence the organization of collagen fibrils during the healing process. In skin treated with AgβLG, there was an increased amount of collagen fibrils. Furthermore, these fibril clusters were more substantial, and their alignment was denser compared to untreated skin. Currently, there is limited research on silver's impact on collagen fibril deposition. Future studies should consider factors that differentiate silver nanoparticles from nanocomplexes, such as their size, charge, coating, and stability. It's also crucial to correlate wound healing progress with the concentration of released silver ions and assess potential cytotoxicity. Prior research by Tian et al. has also shown that AgNPs play a role in regulating collagen deposition, leading to improved alignment during the wound healing process [27].

The findings presented in this study suggest that the presence of metal complexes can positively impact wound healing, not only by inhibiting bacterial infections but also by stimulating collagen synthesis. In a similar vein, research by Liu et al. demonstrated that chemically synthesized AgNPs can promote the differentiation of fibroblasts into myofibroblasts, which play a pivotal role in collagen fiber production [28]. Comprehensive studies by Kwan et al. further revealed that wounds treated with AgNPs exhibit a superior spatial distribution of collagen, which correlates with the mechanical properties of the healed skin [29].

In our study, effects similar to those observed for AgβLG were evident in wounds healed with ZnβLG. In these tissue sections, a high number of fibroblasts featuring an extensive membrane system was evident, suggesting intensive synthesis of extracellular matrix components. This represents one of the initial pieces of evidence highlighting the influence of Zn on collagen organization. Until now, only Khan et al. demonstrated, using Masson’s trichrome staining, that wounds treated with a PLGA/silk fibroin-based electrospun membrane loaded with zinc oxide nanoparticles exhibited a greater quantity of collagen with superior organization [30]. Zinc can play a multifaceted role in collagen fibril organization in wounds through its involvement in enzyme function, gene expression, direct interactions with collagen, and antioxidant properties. Zinc ions can directly interact with collagen molecules, stabilizing their triple-helical structure and promoting the formation of collagen fibrils. This interaction may contribute to the organization of collagen fibrils within the extracellular matrix. Adequate zinc levels are vital for maintaining the structural integrity and function of connective tissues in the body. However, more studies are needed to confirm the effect of zinc on fibroblast viability, their differentiation and organization of collagen fibrils.

The correct morphology of liver cells from mice treated with AgβLG and ZnβLG indicate that applied concentrations of nanocompomosites are not very toxic. However, the hepatocytes of these animals were characterized by the lack of glycogen deposits in the cytoplasm. Similar effect was observed in mice with elevated levels of heavy metals such as lead, cadmium or zinc [31]. It indicates that even low levels of silver ions or elevated level of zinc ions in the liver can lead to changes at the molecular level which have impact on different metabolic pathways for example through pathway involving Glycogen synthase kinase 3 (GSK-3).

Conclusion

Our studies highlight the therapeutic potential of AgβLG and ZnβLG complexes in wound healing. The wounds were reduced and healed in a relatively fast time for the tested mice at concentrations 25 µg/mL for AgβLG and 200 µg/mL for ZnβLG. The concentrations used in our study did not negatively affect the viability of L929 fibroblasts. Although AgβLG showed strong antibacterial properties, both complexes showed a positive effect on collagen synthesis and organization, which is crucial for wound healing. However, ZnβLG stimulates fibroblast proliferation and migration in a wound healing scratch assay. The study also indicates the absorption and systemic distribution of the released ions, highlighting the need for a comprehensive safety assessment. The diverse role of zinc in the organization of collagen and the functioning of fibroblasts is a new aspect that should be subjected to further research.

Supplementary Information

Supplementary Material 1.

Acknowledgements

This work was financially supported by the National Science Centre within the framework of Opus 14 Project No. 2017/27/B/ST4/02628 (2018-2021). K.R., P.P. and B.B. are members of Toruń Center of Excellence “Towards Personalized Medicine” operating under Excellence Initiative-Research University and V.R., M.B.F. are member of Emerging Fields “One Health—antimicrobial stewardship in human and veterinary medicine” operating under Excellence Initiative-Research University.

Author contributions

A.R.: Conceptualization, Methodology, Investigation—obtaining ZnβLG and AgβLG complexes, testing the antimicrobial properties of the complexes by MIC and CFU, carrying out studies of the wound healing process, Writing—Original Draft, Visualization, Data curation. P.P.: Conceptualization, Funding acquisition, Project administration, Writing—Review & Editing. M.B.F.: Investigation—carrying out studies of the wound healing process, Writing—Original Draft. V.R.: Methodology, Investigation—testing the antimicrobial properties of the complexes by MIC and CFU, Writing—Review & Editing, Visualization. A.G.: Investigation—quantify the concentration of zinc and silver ions in liver and blood tissue samples (ICP-MS) B.B.: Supervision, Resources. K.N.: Investigation—microscopic study. P.F.: Methodology, Investigation—cytotoxicity tests, Writing—Review & Editing. K.Ro: Methodology, Investigation—cytotoxicity tests, Writing—Review & Editing K.Ra.: Investigation—microscopic study, Writing—Original Draft (microscopic study), Writing—Review & Editing.

Data availability

The datasets generated and analyzed during the current study are available from the corresponding authors on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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