Marzena
Fandzloch
*a,
Adam W.
Augustyniak
b,
Joanna
Trzcińska-Wencel
c,
Patrycja
Golińska
c and
Katarzyna
Roszek
c
aInstitute of Low Temperature and Structure Research, Polish Academy of Sciences, Okólna 2, 50-422, Wrocław, Poland. E-mail: m.fandzloch@intibs.pl
bFaculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, 50-383, Wrocław, Poland
cFaculty of Biological and Veterinary Sciences, Nicolaus Copernicus University in Toruń, Lwowska 1, 87-100, Toruń, Poland
First published on 18th June 2024
Multifunctional materials that combine antimicrobial properties with the ability to stimulate bone formation are needed to overcome the problem of infected bone defects. As a novel approach, a new composite based on bioactive glass nanoparticles in a simple system of SiO2–CaO (BG) coated with NH4[Cu3(μ3-OH)(μ3-4-carboxypyrazolato)3] (Cu-MOF) with additionally anchored silver nanoparticles (AgNPs) was proposed. Ag@Cu-MOF@BG obtained by the spin coating approach in the form of a disc was characterized using PXRD, ATR-FTIR, XPS, ICP-OES, and TEM. Importantly, the material retained its bioactivity, although ion exchange in the bioactive glass administered as a disc is limited. Hydroxyapatite (HA) formation was identified in TEM images after 7 days of immersion of the composite in a physiological-like buffer (pH 7.4, 37 °C). The Cu and Ag contents of Ag@Cu-MOF@BG were as low as 0.013 and 0.018 wt% respectively, but the slow release of the AgNPs ensured its antibacterial nature. Ag@Cu-MOF@BG exhibited antibacterial activity against all tested bacteria (E. coli, S. aureus, P. aeruginosa, and K. pneumoniae) with the diameter of the inhibition zones of their growth between 8 and 10 mm and the reduction index determined to be ≥3. Moreover, the biocompatibility of the new composite has been demonstrated, as shown by cell culture assays with human dermal fibroblasts (HDFs). The results from the migration test also proved that the HDF cell's phenotypic properties were not changed, and the cell adhesion and migration ability were the same as in control indirect assays.
Infectious diseases can spread and develop through open fractured sites or blood transmission from adjacent sites or implants. Excessive inflammatory responses prevent bone tissue regeneration and significantly interfere with the bone healing process.3 In fact, bacteria can damage the extracellular matrix, inhibit osteogenic differentiation and function, stimulate the production of osteoclasts, cause excessive bone resorption and impaired osteogenic function, and even cause delayed healing and fracture failure.4
Traditional treatment strategies for infected bone defects include thoroughly clearing the wound, implanting antibiotic-impregnated cement spacers, and administering antibiotics.5 However, the efficacy of systemic antibiotic administration is significantly undermined by the infection-induced disruption of the local blood supply and the resulting onset of osteonecrosis. This severely limits the effective concentration of antibiotics at the site of infection. In addition, the prolonged and widespread use of antibiotics has led to the gradual evolution of bacterial resistance and the emergence of highly resistant strains of bacteria.5 Therefore, alternative materials with antibacterial properties are being investigated. Among them, metal–organic frameworks (MOFs) are becoming increasingly prominent in this aspect.6 These highly designable structure and functional materials, due to their high porosity (e.g. MIL-53(Fe), MOF-5, or ZIF-8) have been proposed as antibacterial drug delivery systems.7 On the other hand, MOFs can lead to the slow release of metal ions, giving rise to antibacterial behavior (e.g. Ti-MIL-125-NH2,8 Ag-MOFs,7 and Zn-containing MOFs7).
There is also an increasing number of Cu-MOFs being tested and used for their antimicrobial activity.9–11 A well-known representative is [Cu3(BTC)2]n (H3BTC = 1,3,5-benzenetricarboxylic acid), commonly known as Cu-HKUST-1 or MOF-199, which has remarkable antibacterial activity against S. aureus and E. coli.11–15 The progressive degradation of Cu-MOFs and the leaching of Cu2+ ions as well as the ligand, e.g.L-glutamic acid from [Cu(glum)]n,16 are likely to disrupt the bacterial cell membrane. In addition, highly synergistic antibacterial activity due to ROS generation and delayed Cu2+ release was reported for [Cu2(cit)(H2O)2]n nano-MOF (cit = citrate), which was effective against E. coli and B. subtilis.17 Interestingly, the contact of bacteria with the MOF surface without metal ion release is another possible mechanism for the antibacterial activity of Cu-MOFs, such as for [Cu2(Glu)2(μ-L)]·x(H2O) containing glutarates (Glu) and bipyridyl ligands (L).18 These MOFs exhibit strong antibacterial activities against both Gram-positive bacteria (S. aureus and MRSA) and Gram-negative bacteria (E. coli, K. pneumonia, and P. aeruginosa) at very low bactericidal concentrations. Meanwhile, Sheta et al. presented nanoparticles of the MOF [{Cu(SB)(H2O)2}(H2O)4]n, where H2SB = Schiff bases, with a size of 7–19 nm suitable to penetrate the bacterial cell through nanometric pores in the membrane by a passive diffusion mechanism.7,19
One of the strategies to further enhance the antimicrobial efficacy of Cu-MOFs could be the loading with AgNPs. To date, Cu-BTC20 or polyCu-MOF (a Cu-based polymer-metal–organic framework)21 have been efficiently used as carriers for silver nanoparticles. The controlled release of Ag+ and Cu2+ metal ions significantly affected their bactericidal performance against S. aureus and E. coli.
The above data indicate that Cu-MOFs, and in particular Ag@Cu-MOFs, can be effective antibacterial platforms and further extend the great potential of MOFs in the application of bone infections. The treatment of bone infections, however, is very demanding and requires multifunctional materials that combine therapy and regeneration.22 These properties, known as theragenerative or therepair, can be achieved by MOFs when combined with biomaterials.11,23,24 Among them, bioactive glasses seem to be very promising for orthopedic applications. Due to the formation of a bone-like hydroxyapatite layer on the surface, this type of material can form strong bonds with bone or soft tissue.25–28 In addition, specific concentrations of soluble Si and Ca ions released from bioactive glasses can stimulate the expression of several genes involved in osteogenesis and angiogenesis, resulting in rapid bone regeneration.29,30
To the best of our knowledge, two examples of composites based on bioactive glasses and MOFs have been developed to date to reduce bone infection.23,24 Both are based on zeolitic imidazolate framework-8 (ZIF-8) and ternary bioactive glass (SiO2–CaO–P2O5). One of these materials was obtained by deposition of vancomycin-loaded ZIF-8 (ZIF-8@VAN) on 3D-printed bioactive glass scaffolds. In addition to promoting the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells, this material has shown antibacterial activity against S. aureus.23 Meanwhile, Ahmed et al. proposed the use of a biocompatible composite based on a system of 60SiO2–10P2O5–(30−x)CaO–(x)ZIF-8 nanoparticles (x = 0 and 0.8 mol%) to solve the problem of infection related to orthopedic implant surgery.24
Recently, we demonstrated that the sol–gel-derived bioactive glasses in a simple binary system, depending on the size or amount of CaO, greatly enhanced the rate of hydroxyapatite formation.31 Based on bio- and hemocompatibility results, one of the nanoparticles of bioactive glasses (B_78S, herein referred to as BG) was found to be the most outstanding representative from a bioapplication viewpoint.
Taking into account this background and considering our previous results herein we present the use of bioactive glass nanoparticles (BG) as a component of a new MOF-based antibacterial composite. For the first time, we have used this bioactive glass in the structured form (discs), coated with a thin layer of a selected Cu-based MOF, namely NH4[Cu3(μ3-OH)(μ3-4-carboxypyrazolato)3] (denoted as Cu-MOF), with anchored silver nanoparticles to enhance the antibacterial effect. The physicochemical characterization of the new Ag@Cu-MOF@BG composite was extended to include bioactivity, in vitro biocompatibility, and antibacterial studies.
Cu-MOF, Ag@Cu-MOF and BG were analyzed by powder X-ray diffraction (PXRD) using an X'Pert PRO diffractometer (PANalytical) with Cu Kα radiation (λ = 1.5418 Å). The mid-infrared spectra in the region of 4000–530 cm−1 were acquired in attenuated total reflectance (ATR) mode using a Thermo Scientific Nicolet Summit X FTIR spectrometer. In each case, the discs were ground in a mortar before the measurement. The specific surface area of Ag@Cu-MOF was determined based on N2 sorption measurements. The N2 adsorption–desorption isotherms were obtained at 77 K using a Micromeritics 3Flex surface characterization analyzer. Prior to isotherm acquisition, the materials were activated and outgassed (150 °C, 1.3 kPa) for 12 h. MicroActive software was used to determine specific surface area according to the Brunauer–Emmett–Teller (BET) method and total pore volume at a relative pressure (p/p0) = 0.9. Transmission electron microscopy (TEM) images were acquired using a FEI Tecnai G2 20 X-TWIN microscope. Silver particle size was calculated from the size of 100 particles determined using ES Vision software from TEM images. Scanning electron microscopy (SEM) images were recorded using a Hitachi S-3400N microscope. The composition of powders and filtrates was characterized using an iCAP™ 7400 ICP-OES analyzer. To quantify the content of elements (Ca, Si, P, Cu, Ag) by ICP-OES, 1–3 mg of powders were degraded in the mixture of HF/HCl (1:1, 6 mL) (for BG and Ag@Cu-MOF@BG) or 2 mL of HCl (for Cu-MOF and Ag@Cu-MOF). Afterward, the samples were diluted with distilled water. XPS data were collected using a Kratos X-ray photoelectron spectrometer. The binding energy was calibrated according to the C 1s peak of 285 eV. The Ag 3d spectral regions were peak-fitted through CasaXPS processing software. BG and Ag@Cu-MOF@BG discs (Fig. S1†) of 8 mg powder with a diameter of 5 mm were prepared using an evacuable Pellet Die (Specac) and hydraulic press (Sirio) with a pressure of 1 ton. For thin layer deposition, KLM Spin-Coater SCI-40 (Schaefer Tec) was used.
To assess the minimal biocidal concentration (MBC) of Ag@Cu-MOF, 100 μL of the tested suspensions from wells with a concentration of ≥MIC were spread onto sterile tryptic soy agar (TSA, Becton Dickinson) or Sabouraud dextrose agar (SDA, Becton Dickinson) in Petri plates and incubated at 37 °C for 24 h. The lowest concentration of compounds that inhibited microbial growth ≥99.9% was determined as MBC values. All tests were performed in triplicate; the positive (inoculated medium) and negative (non-inoculated medium) controls were provided.34
The biocidal activity of Ag@Cu-MOF@BG discs was determined by placing them in 1 mL of the microbial inoculum (approx. 1.5 × 106 CFU per mL) in 24-well plates for 24 h at 37 °C under shaking conditions (100 rpm). After incubation, the suspensions were collected from each well into sterile 2 mL Eppendorf tubes and serially 10-fold diluted. The aliquot (100 μL) of each sample dilution was spread aseptically on the surface of TSA or SDA medium in Petri plates and incubated for 24 h at 37 °C. The colonies were counted and the reduction factor (R) was calculated according to the formula:
R = Ut − At |
The biocidal activity was determined if R ≥ 2 (99% reduction of microbial growth).
For the direct cytotoxicity test, 10 μL of suspension containing approximately 5 × 104 cells in culture media were seeded on each of the tested materials, i.e., different specimens in separate wells of a 12-well plate. The cells were left for 3 h for adhesion and then the culture medium was added and incubated for 24, 48, and 72 h. In an indirect test, the examined specimens were immersed in culture media for 72 h and then the suspension, as prepared, was added to a HDF cell culture seeded 24 hours prior to evaluation in a 12-well plate. The cell viability in both tests was assessed based on MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, Germany) assay, which shows the ability of the cells to reduce MTT. The absorbance of reduced formazan was measured at 570 nm using a Synergy HT Multi-detection reader (BioTek Instruments, Winooski, VT, USA).
In order to determine the rate of migration, HDF cells were seeded into 24-well plates and incubated for 24 h, then a scratch wound was created using a sterile pipette tip. Cells were washed once with the growth medium, and then the growth medium was added to the control sample, while the test samples contained medium obtained after 72 h-incubation with specimens (as in the indirect viability test). At time 0, after 6, 24, and 48 h, pictures were taken to measure the width of the wound using Motic Images Software. On the basis of the obtained results, the values were calculated for individual samples as (%) of the wound width relative to the control sample.
The Cu-based MOF chosen as a component of the new composite is an anionic 3D porous framework composed of trinuclear Cu3(μ3-OH) clusters linked to six others by μ3-4-carboxypyrazolato bridges.32 This forms tetrahedral cages containing two extra-framework NH4+ cations and crystallization water molecules. Interestingly, the ion-exchange processes of the extra-framework cations can modulate the textural properties and thus influence the adsorption selectivity of various separation processes of gases and vapors or open up new application possibilities.32,38 The synthesized NH4[Cu3(μ3-OH)(μ3-4-carboxypyrazolato)3] MOF was systematically characterized by PXRD and ATR-FTIR, confirming the purity of the isolated material (Fig. S2†). The treatment of this Cu-MOF with silver nitrate in a 3-fold molar excess in a mixture of methanol and water resulted in the formation of silver nanoparticles (AgNPs) anchored to the Cu-MOF. They were identified by the inter-planar spacing d = 0.232 nm corresponding to the (111) plane of the fcc silver crystals (Fig. S3†). Notably, AgNPs (10 ± 3 nm in size) were fairly dispersed on the MOF surface. In addition, its homogeneous distribution was confirmed by EDS mapping (Fig. S4†). The Ag content determined on the basis of ICP-OES was almost 13 wt%. The IR spectrum registered for Ag@Cu-MOF indicated that the in situ formation of AgNPs did not affect the Cu-MOF structure (Fig. S5a†). Further characterization of Ag@Cu-MOF by PXRD did not reveal any characteristic diffraction peaks of AgNPs, probably due to poor crystallinity or small particle size (Fig. S5b†).
On the other hand, the presence of AgNPs had a significant effect on changes in the textural properties of Ag@Cu-MOF compared to pristine Cu-MOF. The N2 adsorption–desorption isotherm recorded for Ag@Cu-MOF (Fig. S6†) was a combination of IUPAC's type I and IV isotherms. The presence of micro- and mesopores was also typical of pristine Cu-MOF.38 However, it can be noted that the anchored AgNPs markedly modulated the porosity of the Cu-MOF. The BET surface area and pore volume decreased twice from 476 m2 g−1 and 0.22 cm3 g−1 for Cu-MOF38 to 230 m2 g−1 and 0.10 cm3 g−1 for Ag@Cu-MOF, respectively.
The resulting Ag@Cu-MOF was deposited onto a disc of BG using a spin coater (Scheme 1 and Fig. S1†). TEM imaging and FFT analysis clearly confirmed the presence of a Cu-MOF layer with embedded Ag nanoparticles on spherical BG nanoparticles (Fig. 2a–c). AgNPs were identified again by the inter-planar spacing d = 0.234 nm. Furthermore, the EDS mapping showed a uniform distribution of Ag and Cu along with Ca and Si throughout the measured area (Fig. S7†). The surface state of Ag@Cu-MOF@BG was also investigated using XPS analysis. The survey spectrum (Fig. S8a†) indicated the presence of Ca, Si, C, O, Cu, and Ag in the composite. Two peaks at 368.7 eV (Ag 3d5/2) and 374.6 eV (Ag 3d3/2) were observed in the Ag 3d spectrum (Fig. 2d), corresponding to metallic Ag0.39 Subsequently, the Cu 2p spectrum showed a peak at 933.7 eV corresponding to Cu2+ sites of the MOF (Fig. 2e). Besides, peaks of Ca and Si due to BG with binding energies of 351.1 eV (Ca 2p1/2), 347.6 eV (Ca 2p3/2), and 103.3 eV (Si 2p3/2) were observed (Fig. S8b and c†). In contrast, the IR spectrum of Ag@Cu-MOF@BG only confirmed the characteristic bands for bioactive glass, which are related to stretching vibrations of Si–O–Si40 (at 1028 cm−1 and 793 cm−1) (Fig. S9†). The absence of bands from the MOF is due to the thin Ag@Cu-MOF layer deposited, as the Cu and Ag contents are limited to 0.015–0.018 wt%.
The bioactive glass used for the synthesis of Ag@Cu-MOF@BG, previously reported as B_78S, is well known for its excellent bioactivity.31 For this nanometric glass with a simple binary system of 78SiO2–22CaO (wt%), a CaP-rich layer was observed already after 2 h of immersion under physiological conditions. The change in the composition of B_78S was rapid, and after 4 hours of incubation the P content was equal to 18 at%, while the Si content decreased from 74 to 56 at%. However, to date, the bioactivity of this BG in a structured form (disc) has not been determined. A comparison of the bioactivity results of this bioactive glass tested in powder and disc form shows that their conversion rates to HA are significantly different. The more difficult exchange of ions in a compressed form is responsible for the slow changes in the composition of the BG disc. Nevertheless, as shown by TEM images, HA formation in the form of needle-like nanocrystallites was observed after 7 d of incubation for both BG and Ag@Cu-MOF@BG (Fig. 3c and Fig. S13†). Importantly, the presence of a thin Cu-MOF layer with anchored AgNPs did not hinder the formation of HA on the surface of the biomaterial.
For the composite, an additional analysis of the Ag content after immersion in DPBS was carried out, which showed the highest leaching of Ag during the first 4 h of incubation (Fig. S14†). At this time, the Ag content was reduced from 0.018 to 0.015 wt%, resulting in Ag leaching efficiency of approximately 15% given the initial content. The release of AgNPs in the initial stage will be important for the antibacterial effect of this material. After 24 h, the Ag content had increased to the initial level, suggesting that the released particles could be adsorbed back onto the BG surface.
Microorganisms | Cu-MOFa | Ag@Cu-MOF | ||
---|---|---|---|---|
MIC | MBC | MIC | MBC | |
a Published as Cu-capz;11 ND not determined. | ||||
Escherichia coli ATCC 25922 | 2 | 5 | 62.5* | 62.5* |
Klebsiella pneumoniae ATCC 700603 | ND | ND | 62.5* | 62.5* |
Pseudomonas aeruginosa ATCC 10145 | 5 | 6 | 31.125* | 125* |
Staphylococcus aureus ATCC 25923 | 2 | 3 | 125* | 125* |
Candida albicans ATCC 10231 | ND | ND | 125* | 250* |
Finally, the effect of a thin layer of Ag@Cu-MOF on the antimicrobial properties of the coated bioactive glass was tested. The results of the evaluation of antimicrobial activity based on the growth inhibition zone from agar diffusion assays and the determination of the reduction index are shown in Table 2 and Fig. 4. The antimicrobial activity of Ag@Cu-MOF@BG was favorable against all bacteria tested. The diameter of the inhibition zones of growth of test microorganisms was found to be between 8 and 10 mm, while the reduction index was determined to be ≥3. The lower sensitivity to Ag@Cu-MOF@BG discs was recorded for C. albicans, where the zone of inhibition of growth was determined with a diameter of 7 mm, and the reduction index was <1, showing a lack of biocidal activity against yeasts.
Microorganisms | Inhibition zone [mm] | Reduction index |
---|---|---|
Escherichia coli ATCC 25922 | 9 | ≥3 |
Klebsiella pneumoniae ATCC 700603 | 9 | ≥3 |
Pseudomonas aeruginosa ATCC 10145 | 10 | ≥3 |
Staphylococcus aureus ATCC 25923 | 8 | ≥3 |
Candida albicans ATCC 10231 | 7 | <1 |
MOFs can act as antimicrobial carriers by several routes. This can be mediated by their components or by agents loaded into the MOF and gradually released by diffusion or degradation of the MOF structure.44 Our results show that the antimicrobial activity of Cu-based MOFs is enhanced by the presence of small and uniformly distributed AgNPs synthesized in situ in the matrix. This is demonstrated by the significantly lower MIC and MBC values of Ag@Cu-MOF compared to Cu-MOF against all microorganisms tested. To date, several reports have revealed pleiotropic antimicrobial mechanisms of AgNPs related to the disruption of the ionic balance, interference with the cell membrane, impairment and damage to proteins or DNA, resulting in disruption of cell function and metabolism, leakage of cytoplasm, and cell death.34,45–47
The increase in the antimicrobial efficacy of Cu-based MOFs and AgNPs as a result of their incorporation has already been reported by other authors. For example, the synergistic effect of Cu-BTC and AgNPs against B. subtilis has been reported.48 Similarly, results presented by Soomro and coworkers revealed that copper(II) and guanosine 5′-monophosphate-based MOF (Cu/GMP) showed higher antimicrobial activity after impregnation with AgNPs (AgNPs@Cu/GMP).49 The inhibition zones for Cu/GMP and AgNPs@Cu/GMP were determined at 12 and 17 mm against S. aureus and 9 and 10 mm against E. coli, respectively. The MIC values for AgNPs@Cu/GMP were 18.75 μg mL−1 against S. aureus and 25 μg mL−1 against E. coli. In addition, ICP results suggested that antimicrobial activity of AgNPs@Cu/GMP was related to the pH-dependent release of metal ions from the composite. In another study, Guo et al. showed the biocidal activity of polyCu-MOF loaded with AgNPs (polyCu-MOF@AgNPs) against S. aureus and E. coli.21 This was due to the induction of damage to cell integrity through the generation of reactive oxygen species (ROS) and the disruption of bacterial metabolism.
To the best of our knowledge, the Ag@Cu-MOF system has not been combined with biomaterials to date. The results presented show that the activity of the new composite was slightly lower in the eukaryotic C. albicans cells than in the prokaryotic bacterial cells when Ag@Cu-MOF was combined with bioactive glass nanoparticles.
However, the demonstration of antibacterial properties of Ag@Cu-MOF@BG indicates the potential use of such composites for pathogen control as well as various implementations in the pharmaceutical or biomedical sectors. This is also supported by previous results for the Zn-MOF@bioactive glass in a ternary system (SiO2–CaO–P2O5), where the MOF is ZIF-8. This type of composite exhibited the presence of an inhibition zone against E. coli, S. aureus, and C. albicans equal to 17.66, 13.33, and 16.33 mm, respectively.24 After loading with gentamicin, the diameter of the inhibition zone increased significantly (up to 37.33 mm) against all of the microorganisms tested. On the other hand, ZIF-8@VAN@BG scaffolds showed significant antibacterial activity against S. aureus due to the inhibitory effect of the released vancomycin. Furthermore, with increasing ZIF-8@VAN deposition on the scaffolds, the antibacterial activity of ZIF-8@VAN@BG increased from 62.5% to 93.5%.23
Footnote |
† Electronic supplementary information (ESI) available: PXRD, ATR-FTIR, TEM, N2 adsorption–desorption isotherm, SEM-EDS, XPS, ICP-OES, and changes in the pH value. See DOI: https://doi.org/10.1039/d4dt01190b |
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