A cerium-based MOFzyme with multi-enzyme-like activity for the disruption and inhibition of fungal recolonization

Article abstract of DOI:10.1039/d0tb00894j

A cerium-based metal-organic framework (Ce-MOF, denoted as AU-1) was synthesized using a solvothermal method by employing 4,4′,4′′-nitrilotribenzoic acid (H3NTB) as the linker and cerium clusters as the metal center. The material was considered as a MOFzy

Full text of DOI:10.1039/d0tb00894j

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A cerium-based MOFzyme with multi-enzyme-like
activity for the disruption and inhibition of fungal
Cite this:DOI: 10.1039/d0tb00894j
recolonization†
b
Hani Nasser Abdelhamid, *a Ghada Abd-Elmonsef Mahmoud
and
Walid Sharmoukc
A cerium-based metal–organic framework (Ce-MOF, denoted as AU-1) was synthesized using a solvothermal
method by employing 4,40,400-nitrilotribenzoic acid (H3NTB) as the linker and cerium clusters as the metal
center. The material was considered as a MOFzyme based on the peroxidase-like activity of Ce-MOF
that could eliminate and kill fungi, such as Aspergillus flavus, Aspergillus niger, Aspergillus terreus,
Candida albicans, and Rhodotorula glutinis. Ce-MOF showed high antifungal activity against airborne
opportunistic human pathogens isolated from the outside of a hospital. The antifungal activity of CeMOF
was evaluated using the colony-forming units, dry mass method, soluble proteins, and microscopic
imaging. It exhibited an inhibition efficiency of 93.3–99.3% based on the colony-forming unit method.
The Ce-MOF caused extensive deformation of the conidiophores, vesicles, and phialides with growth
inhibition between 7.55–77.41% (based on the dry mass method). Ce-MOF showed different efficiencies
in inhibiting the different fungal species. The biological activity of Ce-MOF was due to its enzymatic
activity, such as those of antioxidants, catalase, superoxide dismutase, and peroxidase. Ce-MOF
exhibited excellent enzymatic activity towards the fungal cells. Our results may facilitate the design of a
MOFzyme system and pave the way for more profound applications of nanozymes.
Received 4th April 2020,
Accepted 16th June 2020
DOI: 10.1039/d0tb00894j
rsc.li/materials-b
activities that could combat biofilms.24 Cerium (Ce)-doped zeolitic
imidazolate framework-8 (ZIF-8) showed simultaneous antibacter-
Introduction
Metal–organic frameworks (MOFs)1,2 are fascinating porous materi- ial and anti-inflammatory capabilities.25 A mixed-valence Ce-MOF
als with unique properties compared with other families of porous (Ce-BPyDC) was applied for ascorbic acid (AA) detection using a
materials.3–12 They have several advanced applications,13–21 sensitive and selective colorimetric method.26
including MOF-based nanozymes (MOFzymes). MOFzymes are
Fungi are a heterogeneous group of microorganisms, which
promising for applications, such as sensing and cancer inhabit various environments, such as plants, debris, soils,
therapy,22 and biomimetic catalysts.3 MOFzymes demonstrate rivers, lakes, seas and even extreme environments.27 They
significant advantages, such as facile synthesis procedure, low release their spores into the air and thus can be called as
price, long-term storage stability, and high environmental airborne fungi.28 Inhalation of hyphal fragments, spores, and
stability.23 Several MOFs, including cerium-based MOFs, have by-products of fungi by human leads to serious respiratory
been reported with enzymatic properties. It has been reported problems, and may cause asthma.29 The airborne fungal spores
that ligand metal charge transfer (LMCT) is favorable only in pose a huge risk to human health, especially immunocompro-
Ce-based MOFs.4 A Ce-based MOF has been reported as an mised individuals and allergy-prone people,29 especially hospi-
enzyme with deoxyribonuclease (DNase)- and peroxidase-mimetic talised patients, who are very susceptible to the infections
caused by the spores.30 Aspergillus is a well-known opportunis-
tic human pathogen associated with aspergillosis, allergy,
asthma, rhinitis, and conjunctivitis.31 Aspergillus strains, such
as Aspergillus flavus, have been reported as causal agents of
aspergillosis, keratitis, otitis, invasive sinusitis, and other sys-
temic infections.32 Yeasts, such as Candida albicans, colonize
the skin and gastrointestinal tract of human as opportunistic
pathogens.33 Nanotechnology has been employed in several
advanced applications, including the development of antimicrobial
a Advanced Multifunctional Materials Laboratory, Department of Chemistry, Faculty
of Science, Assiut University, Assiut 71516, Egypt.
E-mail: hany.abdelhamid@aun.edu.eg
b Department of Botany & Microbiology, Faculty of Science, Assiut University,
Assiut 71516, Egypt
c National Research Centre, Department of Inorganic Chemistry, Tahrir St, Dokki,
12622 Giza, Egypt
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0tb00894j
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agents.34–41 Nanoparticles exhibit high potential in the treat- Then, the reaction mixture was stirred for 12 h at room tempera-
ment of gastrointestinal tract42 and as antimicrobial materials ture. The aqueous layer was extracted with CH2Cl2 (3 ꢀ 150 mL)
against resistant microbial infections.
and brine. The organic layers were dried over anhydrous Na2SO4.
Herein, a new cerium-based MOF, AU-1 (referring to Assiut The solution was concentrated under reduced pressure to give the
University-1), was synthesized using a solvothermal method. crude product, which was purified using flash column chromato-
The crystallinity and phase purity of the synthesized material graphy on silica gel with hexane/CH2Cl2 (1 : 10) as the eluent. The
was confirmed using X-ray diffraction (XRD). The chemical purified material was obtained a yellow solid with a yield of 92%
1
bonds and coordination within the frame were verified via Fourier (2.8 g). H NMR (400 MHz, CDCl3) chemical shift (d) 7.90 ppm
transform infrared spectroscopy (FT-IR) and X-ray photoelectron (d, 6H, J = 8 Hz), 7.14 ppm (d, 6H, J = 8 Hz), and 2.58 ppm (s, 9H)
spectroscopy (XPS). The morphology and particle size were esti- (Fig. S1, ESI†). 13C NMR (100 MHz, CDCl3) d: 196.5 ppm, 150.3 ppm,
mated using transmission electron microscopy (TEM), high- 132.8 ppm, 130.1 ppm, 123.9 ppm, and 26.4 ppm (Fig. S2, ESI†).
resolution TEM (HR-TEM), and scanning electron microscopy
4,40,400-nitrilotribenzoic acid (H3NTB). A solution of 4,40,400-
(SEM). According to the thermal analysis performed using ther- triacetyltriphenylamine (1.7 g, 4.7 mmol) was prepared in 1,4-
mogravimetric analysis (TGA) and differential thermal analysis dioxane (50 mL). The solution was cooled down on an ice bath.
(DTA), AU-1 exhibited high thermal stability. The Ce-MOF showed Bromine (2.4 mL, 47.2 mmol) was added dropwise to a solution
enzyme-mimicking activities and offered promising biological of NaOH (6.2 g, 156.0 mmol) in H2O (30 mL) cooled on an ice
activity against airborne opportunistic fungi. It caused deforma- bath with stirring for 20 min and then added dropwise to the
tion and oxidative stress in the investigated fungal cells.
above solution. The reaction mixture was stirred at room
temperature for 12 h at 60 1C. Then, the mixture was cooled
on an ice bath. A saturated solution of hydroxylamine hydro-
chloride was added. The solution was acidified with HCl. A
solid precipitate was filtered and dried under vacuum to afford
the pure product as a yellowish-white solid. Proton nuclear
magnetic resonance (1H NMR, 400 MHz, DMSO-d6) chemical
shifts (d) of 12.8 ppm (bs, 3H), 7.92 ppm (d, 6H, J = 8 Hz), and
7.16 ppm (d, 6H, J = 8 Hz, Fig. S3, ESI†). 13C NMR (100 MHz,
DMSO-d6) d: 167.2 ppm, 150.3 ppm, 131.6 ppm, 126.4 ppm,
124.2 ppm (Fig. S4, ESI†).
Materials and methods
Aluminum chloride anhydrous (AlCl3), triphenylamine, CH2Cl2,
Na2SO4, 1,4-dioxane, bromine, cerium nitrate, and sodium
hydroxide were purchased from Sigma-Aldrich (Germany). All
chemical reagents were used without purification.
Synthesis of organic linker
The synthesis of the organic linker is systematically presented
in Fig. 1a. The procedure involved two steps.
Synthesis of the cerium-MOF
Synthesis of 4,40,400-Triacetyltriphenylamine (1,10,100-[nitrilotri
(4,1-phenylene)]tri(ethan-1-one)). A solution of aluminum chlor- The cerium MOF, AU-1, was synthesized using a solvothermal
ide anhydrous (AlCl3, 3.6 g, 26.9 mmol) was prepared in dry method (Fig. 1b). Ce(NO3)3ꢁ6H2O (0.14 g) and 4,40,400-
CH2Cl2 (150 mL). The solution was cooled down to 273 K and nitrilotribenzoic acid (H3NTB, 0.12 g) were dissolved in a mixture
stirred for 20 min. A solution of triphenylamine (2.0 g (8.1 mmol) of DMF (10 mL), H2O (1 mL), and HCl (0.1 M, 2 mL) in a sealed
was dissolved in 30 mL of dry CH2Cl2) was added dropwise to the glass vial (Fig. 1b). The solution was sonicated for 20 min. Then, it
solution of AlCl3. The color of the solution turned deep blue. After was heated at 85 1C for 16 h. A yellow crystalline powder was
that, a solution of acetyl chloride (prepared via mixing 3.9 mL obtained using filtration. The product was characterized using
(56.2 mmol) in 30 mL of dry CH2Cl2) was added dropwise at 273 K. XRD before and after washing with DMF and ethanol (3 ꢀ 25 mL).
Fig. 1 Schematic representation of the synthesis of the (a) organic linker and (b) Ce-MOF AU-1.
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Characterization instruments
0.22 mm membrane filter was added as a bacteriostatic. AU-1 (0,
10, 20, 30 and 40 mg mLꢂ1) was tested in Czapek’s broth (50
mL). The inoculums (1%, v/v) were added and incubated at 28
ꢃ 1 1C in a rotary shaker (150 rpm) for 7 days. After fungal
growth, the Aspergillus biomass was filtered using Whatman
filter paper and washed twice with distilled water for drying at
70 1C in a hot air oven for dry mass estimation.44 For yeast, the
biomass was collected using centrifugation (4000 rpm) for 10 min.
The harvested biomass was then washed with distilled water
(2 ꢀ 50 mL) to remove residues. The experiments were repeated
at least three times to ensure repeatability and reproducibility.
X-ray diffraction (XRD) was recorded using Phillips 1700 X’Pert
(Cu Ka radiation). The structure solution and simulation of the
XRD pattern were carried out using EXPO2014 and the
diffraction-crystal module of the mercury (Hg) program version
3.1, which is available free of charge (http://www.ccdc.cam.ac.
uk). The X-ray photoelectron spectra (XPS) were recorded using
a Thermo Fisher (K-alpha) instrument with monochromated
micro-focused Al Ka radiation (1486.6 eV). The C1s peak at
284.4 eV was used as the reference. The transmission electron
microscopy (TEM) and high-resolution TEM (HR-TEM) images
were recorded using TEM-2100 (JEOL, Japan, operated at accel-
erating voltage 200 kV). Scanning electron microscopy (SEM)
was performed using JSM-6010LV (JEOL, Japan). The Ce-MOF
sample was coated with Pt-Pd using a JEC-3000FC auto fine
coater (JEOL, Japan). Fourier transform infrared spectroscopy
(FT-IR) was carried out using a Nicolet spectrophotometer (model
6700). Thermogravimetric analysis (TGA) and differential thermal
analysis (DTA) were performed using a TA 60 thermal analyzer
(Shimadzu, Japan) at a heating rate of 10 1C minꢂ1 and an airflow
rate of 30 mL minꢂ1. The proton (1H) and carbon (13C) analyses of
the organic linker were conducted on Bruker 400 MHz instru-
ments using the residual signals for CDCl3 at chemical shifts (d) of
7.26 ppm and 77.0 ppm, and for DMSO-d6 at d = 2.50 ppm and
39.4 ppm.
The effect of AU-1 on the morphology of pathogenic fungi
All the cultures at different concentrations were examined
using a microscope (Olympus CX41, Japan) for changes in cell
morphology. Typically, samples from the growing cultures were
trapped using a needle, stained with lactophenol cotton blue,
and directly subjected to the microscopic imaging. The effect of
the amount of Ce-MOF on the viability of the spores at the
highest Ce-MOF concentration (40 mg mLꢂ1) was recorded by
inoculating the spore suspension from the Czapek’s broth
culture in sterilized Czapek’s agar medium and determining
the viability and ability of these spores to grow again. Mean-
while, the total counts of the yeast cells and fungal spores were
determined via microbial counting on a hemocytometer.
Oxidative stress generated by AU-1 on the opportunistic fungi
Microorganisms and inoculum preparation
The culture flasks containing AU-1 and inoculums were pre-
pared in the broth medium, as described previously. Afterward,
the biomass was collected using filtration (for Aspergillus) or
centrifugation (for C. Albicans, and R. glutinis). The separated
biomass was washed with double distilled water (3 ꢀ 50 mL)
and disrupted mechanically via grinding in a cold mortar
containing liquid nitrogen to ensure complete cellular break-
age. The extracted cells were treated using potassium phos-
phate buffer supplemented with ethylenediaminetetraacetic
acid (EDTA) and polyvinyl pyrrolidone (PVP, pH = 7.8). The
microbial extracts were centrifuged at 4 1C (18000 rpm, 10 min).
The supernatants were collected for the following biological tests:
total antioxidant activity (TA), soluble proteins (SP), superoxide
dismutase (SOD), catalase (CAT), and peroxidase (POD).45
Aspergillus flavus (Link, Af-10), Aspergillus niger (van Tieghem,
An-14), Aspergillus terreus (Thom, At-40), Candida albicans
(Robin, Berkhout, Ca-6) and Rhodotorula glutinis (Fresenius,
Harrison, Ro-39) were isolated from the air spores present
outside of a public hospital in the Assiut Governorate using
Czapek’s dextrose agar medium supplemented with chloram-
phenicol (250 mg mLꢂ1) as the bacteriostatic. The climatic
conditions were 17.2 km hꢂ1 wind speed and 36% relative
humidity with the minimum and maximum temperatures of
22 1C and 26.7 1C, respectively. The plates containing the
medium were maintained open for 10 min outside the hospital
in different places, then transferred directly to the laboratory
and incubated at 28 ꢃ 1 1C in aerobic conditions for 7 days. The
developed isolates were identified and stored at 4 ꢃ 1 1C on
potato dextrose agar (PDA) slants.43 Before the experiment, the
microbial inoculums were prepared via scraping of 5 and 3 days
for Aspergillus sp. and C. Albicans or R. glutinis, respectively. The
cells were cultured on PDA and suspended in sterilized distilled
water fortified with 0.1% Triton X100 to at a concentration of
Analysis of enzymatic activity
The total antioxidants were estimated using the phosphomo-
lybdenum method at 695 nm. The results were calculated as
mg gꢂ1 soluble protein using a standard curve of ascorbic
acid.46 The soluble proteins were measured using the Folin reagent
method (colorimetric method using absorbance at 750 nm). The
amount of soluble protein (mg gꢂ1) was calculated using a
standard curve of bovine serum albumin. Superoxide dismutase
(EC: 1.15.1.1) was assayed using the epinephrine autoxidation
2 ꢀ 105 spore mLꢂ1
.
The effect of AU-1 on the growth of opportunistic pathogenic
fungi
The Czapek’s broth medium (g Lꢂ1) was prepared using glucose method at 480 nm for 1 min. Catalase (EC: 1.11.1.6) was evaluated
(30.0 g), KH2PO4 (1.0 g), yeast extract (5.0 g), MgSO4ꢁ7H2O (0.5 via the measurement of hydrogen peroxide consumption for 1 min
g), NaNO3 (3.0 g), KCl (0.5 g), FeSO4ꢁ7H2O (0.01 g) and distilled at 240 nm. Peroxidase (EC: 1.11.1.7) was determined using guaiacol
water (1 L) and autoclaved for 20 min at a pressure of 1.5 atm (1.5 mM) at 470 for 1 min. All colorimetric assays were performed
and a temperature of 121 1C. Chloramphenicol sterilized using a using a T60 UV spectrophotometer split beam.
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The effect of H2O2 on the UV-Vis absorption of Ce-MOF was O1s spectrum showed peaks at the binding energies of 531.1 eV
recorded using FLAME-S-UV-VIS (200-850 nm, Rochester, NY and 533.1 eV representing CQO and C–O, respectively (Fig. 3d).
14607 USA). Ce-MOF (8 mg) was dispersed in deionized water The XPS spectra of Ce3d indicated the presence of a mixed-
(8 mL) using ultrasonication. Hydrogen peroxide (0.05 mL) valence state (Ce3+/Ce4+) in the Ce-MOF, and the ratio of Ce4+/
was added. A UV-cuvette was filled with 4 mL of the solution Ce3+ was found to be 6% (Fig. 3e).48 The peaks at the binding
and the UV-Vis spectra of the solution were recorded over time energy values of 881.5 eV, 886.9 eV, 900.0 eV, and 912.3 eV were
(0–80 min).
associated with Ce4+, while the peaks at 885.0 eV and 903.7 eV
were associated with Ce3+.48 The XPS analysis confirmed the
successful formation of Ce-MOF (Fig. 3f).
The FT-IR spectra of the organic linker H3NTB and AU-1
confirmed the formation of coordination bonds within the frame-
work (Fig. S6, ESI†). The FT-IR spectrum of H3NTB showed a strong
vibrational peak at the wavenumber of 1693 cmꢂ1 referring to the
carbonyl group CQO (Fig. S7, ESI†). After coordination with Ce3+,
the organic linker exhibited strong absorption peaks at wavenum-
bers 1595 cmꢂ1 and 1410 cmꢂ1 corresponding to the asymmetric
and symmetric stretching vibrations of COOꢂ (Fig. S7, ESI†). The
results from the FT-IR analysis confirmed the complete activa-
tion of the compound (Fig. S7, ESI†). AU-1 had a hexagonal
nanorod morphology, as confirmed by the TEM image (Fig. S8,
ESI†). The material weakly diffracted in electron diffraction
(Fig. S9, ESI†). The morphology of the Ce-MOF was confirmed
using SEM images (Fig. S10, ESI†). The SEM images showed
rods with a hexagonal morphology at the end (Fig. S10, ESI†).
To investigate the thermal stability of AU-1, TGA was per-
formed in the range of 25–700 1C under an air atmosphere
(Fig. 2b). It could be concluded that the material (empirical
Results and discussion
Material characterization
The solvothermal reaction between cerium nitrate and the
tritopic carboxylate linker H3NTB (Fig. 1a) in DMF formed a
Ce-MOF, namely AU-1 (Fig. 1b). The XRD of the as-synthesized
Ce-MOF contained a strong diffraction Bragg’s peak at 2y
around 25.31, which was attributable to the linker H3NTB
molecules residing inside the pores (Fig. S5, ESI†). The XRD
pattern of the purified and activated frameworks showed no
peak for the occluded linker molecules (Fig. 2). The synthesized
crystals were small-sized, making it very difficult to directly
determine their crystal structure using single-crystal diffraction
measurements. Therefore, we relied on powder X-ray diffrac-
tion (PXRD) analysis to elucidate the Ce-MOF structure (Fig. 2).
Based on the data, the AU-1 structure was first indexed to the
trigonal R32 space group.47 AU-1 crystallized in the hexagonal space
group P63/mcm with the following cell parameters: lengths – a =
24.803 Å and c = 13.33 Å and angles – a = b = 901 and g = 1201.47
The elemental composition and chemical oxidation state
of the Ce-MOF was investigated using XPS spectra (Fig. 3).
The XPS survey spectrum showed peaks related to the elements
Ce, N, C, and O (Fig. 3a). The analysis of the C1s region showed
peaks at the binding energies of 284.2 eV, 285.7 eV, and
287.9 eV corresponding to C–C, C–O–C, and C–CQO, respec-
tively (Fig. 3b). The spectrum of N1s showed a peak at the
binding energy of 399.5 eV for C–N (Fig. 3c). The analysis of the
Fig. 2 (a) Simulated XRD and experimental XRD patterns of AU-1, and (b)
thermal analysis using TGA and DTA. Note that the low resolution of the
lab X-ray data precluded the use of Rietveld refinement to determine the
atomic coordinates further accurately.
Fig. 3 XPS analysis of the Ce-MOF: (a) element survey, (b) C1s, (c) N1s, (d)
O1s, (e) Ce3d5, and (f) the chemical structure of the Ce-MOF.
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formula, C21H12CeNO7, and molecular weight 530.44 g molꢂ1
)
change in dry mass was higher for A. flavus, A. niger, and A. terreus,
was stable up to 390 1C. The first weight loss (40–183 1C, 12 wt%) compared to C. Albicans and R. glutinis (Fig. 4a). The decrease in dry
occurred because of the removal of the guest molecules. In the mass was due to the antifungal activity of Ce-MOF, which inhibited
temperature range of 300–330 1C, the second weight loss took fungal growth and disrupted their components. AU-1 showed high
place due to the removal of the occluded DMF molecules. Above antifungal activity and inhibited the growth of the pathogenic fungi
the temperature of 370 1C, the material started to decompose due with inhibition efficiencies between 7.55–77.41% (Fig. 4a).
to the loss of the framework linker. The residual value 32.2%
The soluble proteins of the fungi after treatment with different
corresponded to CeO2 (calculated value = 32.5%). The TGA curve concentrations of AU-1 were estimated (Fig. 4b). The amount of
confirmed the chemical formula of AU-1 (C21H12CeNO7). The soluble proteins (mg gꢂ1 fresh weight) increased with an increase
differential thermal analysis (DTA) showed a major exothermic in the concentration of AU-1 (Fig. 4b). The maximum amount of
band at 380 1C corresponding to linker volatilization (Fig. 2b).
soluble proteins was observed at the highest concentration of AU-1,
indicating the disruption of fungal cells due to the oxidative stress
generated by the Ce-MOF (Fig. 4b). Thus, the amount of soluble
Biological activity of AU-1
Antifungal activity of AU-1: disruption and inhibiting reco- proteins is increased upon treatment with Ce-MOF (Fig. 4b).
lonization of fungi. The antifungal properties of the Ce-MOF
The effect of the concentration (0–40 mg mLꢂ1) of AU-1 on
were investigated against five fungal pathogens, namely Can- the morphology of pathogenic fungi was tested (Fig. 5). There
dida albicans, Aspergillus niger, Aspergillus flavus, Aspergillus was no dramatic change in the morphology of A. niger, C.
terreus, Candida albicans, and Rhodotorula glutinis. C. Albicans Albicans and R. glutinis at all concentrations. On the other
is a dimorphic fungus i.e. forms both yeast and filamentous cells. hand, changes in the morphology of A. flavus and A. terreus
A. niger is a filamentous Ascomycete fungus that can infect humans were observed at a high concentration of AU-1 (40 mg mLꢂ1
,
and plants and cause food contamination or fermentation. It can Fig. 5) in the conidiophores, vesicles, and phialides. These
be found in bakery products, cheeses, and preserved fruits. A. flavus morphological changes confirmed the disruption of cell wall
is a pathogenic fungus that can colonize cereal grains, legumes, and release of content due to the toxic effect of high concentra-
and tree nuts. A. terreus is a mold that can be found in soil. Both tions of the Ce-MOF. These changes also verified the alterations
A. flavus and A. terreus are saprotrophic fungi. R. glutinis colonizes in the physiology and metabolic activities of the cells. These
foods, animals, and environment-based materials. All these fungi fungal deformations were attributed to the toxic effects at high
are spread worldwide. Thus, they were used as models to investi- concentrations due to the accumulation of reactive oxygen
gate the antifungal activity of the Ce-MOF, AU-1.
species (ROS) in the cells, which may lead to cell death.
The colony-forming units (CFU) of the fungi were also used
The antifungal activity of AU-1 against A. niger, A. flavus,
A. terreus, C. Albicans and R. glutinis is presented in Fig. 4, 5 and to evaluate the cytotoxicity of the Ce-MOF (Fig. S11–S13, ESI†).
Fig. S11–S13 (ESI†). The bioactivity of AU-1 was evaluated based The spores generated after the treatment with Ce-MOF were
on dry mass (Fig. 4a), soluble proteins (SP, Fig. 4b), microscopic still viable and could grow again in the medium (Fig. S11, ESI†).
images (Fig. 5), and colony-forming units (CFU, Fig. S11–S13, However, the total counts of the spores and yeast cells decreased
ESI†). The activities were measured using different concentra- dramatically compared with those from the non-treated samples
tions of AU-1 (0–40 mg mLꢂ1).
(Fig. S13, ESI†). Data analysis for A. flavus, A. niger, A. terreus,
The dry mass of fungi upon incubation with AU-1 is reported C. Albicans, and R. glutinis showed the efficiencies of 93.3%,
(Fig. 4a). The dry mass decreased with an increase in the AU-1 96.3%, 99.3%, 93.3%, and 96%, respectively (Fig. S13, ESI†).
concentration (Fig. 4a). The dry mass of A. flavus (12.55 ꢃ 0.39 g Lꢂ1), The CFU method revealed that the Ce-MOF exhibited high anti-
A. niger (19.05 ꢃ 0.68 g Lꢂ1), A. terreus (15.45 ꢃ 0.17 g Lꢂ1), C. fungal activity (Fig. S13, ESI†), which varied between the different
Albicans (1.13 ꢃ 0.02 g Lꢂ1), and R. glutinis (1.06 ꢃ 0.03 g Lꢂ1
)
,
fungi due to their properties. However, the 6% difference in
efficiency among them was insignificant, indicating that the
decreased to 6.65 ꢃ 0.14 g Lꢂ1, 8.38 ꢃ 0.24 g Lꢂ1, 3.49 ꢃ 0.09 g Lꢂ1
0.97 ꢃ 0.05 g Lꢂ1, and 0.98 ꢃ 0.02 g Lꢂ1, respectively (Fig. 4a). The Ce-MOF is a broad-spectrum antifungal agent (Fig. S13, ESI†).
Fig. 4 (a) Dry mass and (b) soluble proteins of the fungi upon treatment with different concentrations of AU-1 (0–40 mg mLꢂ1).
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Fig. 5 Microscopic images of A. flavus in (a) the medium and (b–d) after incubation with AU-1 (10, 20, and 40 mg mLꢂ1, respectively); images of A. terreus
in (e) the medium and (f) after incubation with AU-1 (40 mg mLꢂ1). Co – conidiophore; Vi – vesicle; Ph – phialid; C – conidia.
Enzymatic activity of AU-1 against fungi: mechanism of
The total antioxidant values (mg gꢂ1 protein) reached the
antifungal activity. The antifungal activity of AU-1 could be maximum of 4.44 ꢃ 0.06 (30 mg mLꢂ1) for A. flavus, 4.55 ꢃ 0.09
due to the well-known enzymatic activity of cerium-based (40 mg mLꢂ1) for A. niger, 7.34 ꢃ 0.19 (40 mg mLꢂ1) for A. terreus,
nanomaterials. Thus, the enzymatic activity of AU-1 was 8.03 ꢃ 0.31 (30 mg mLꢂ1) for C. Albicans, 8.88 ꢃ 0.05 (40 mg mLꢂ1
)
recorded via measuring the total antioxidant (TA), catalase for R. glutinis, which were much lower compared with those of the
(CAT), superoxide dismutase (SOD), and peroxidase (POD) control samples: 2.08 ꢃ 0.16 mg gꢂ1, 2.53 ꢃ 0.22 mg gꢂ1, 2.63 ꢃ
activities (Fig. 6a–d).
0.11 mg gꢂ1, 6.64 ꢃ 0.35 mg gꢂ1, and 4.88 ꢃ 0.24 mg gꢂ1 protein,
Fig. 6 The multi-enzymatic activity of the Ce-MOF: (a) total antioxidant activity, (b) catalase enzyme, (c) superoxide dismutase enzyme, and
(d) peroxidase enzyme.
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reactions. The cerium center with two oxidation states (Ce4+
and Ce3+) was intrinsically formed during the synthesis of AU-1.
A mechanism explaining the efficiency of AU-1 is proposed in
Fig. 7 without involving the auto-regeneration of Ce3+ on AU-1.
To support the antioxidant activity, the UV-Vis absorption
Fig. 7 Superoxide dismutation in the AU-1 framework.
respectively (Fig. 6a). The maximum catalase enzyme (CAT) values spectra of Ce-MOF without and with a mild oxidant e.g. hydro-
were 0.46 ꢃ 0.002, 0.64 ꢃ 0.008, 0.82 ꢃ 0.009, 0.54 ꢃ 0.004, and gen peroxide (H2O2) were recorded (Fig. 8). The Ce-MOF
0.66 ꢃ 0.006 specific units for A. flavus, A. niger, A. terreus, solution displayed absorption at wavelengths 246 nm and
C. Albicans, and R. glutinis, respectively (Fig. 6b). The maximum 356 nm corresponding to p–p* and n–p*, respectively (Fig. 8a).53,54
superoxide dismutase enzyme (SOD) specific activity values were The absorption intensities of both peaks increased with time
0.88 ꢃ 0.03, 1.68 ꢃ 0.01, 1.23 ꢃ 0.21, 0.49 ꢃ 0.01, and 0.69 ꢃ 0.03 (Fig. 8b). The increment in the absorbance was due to the
for A. flavus, A. niger, A. terreus, C. Albicans, and R. glutinis, oxidation of the Ce nodes upon interaction with H2O2. The
respectively (Fig. 6c). The maximum peroxidase enzyme (POD) absorbance intensity reached a steady-state within a very short
activity of AU-1 values were 0.34 ꢃ 0.01, 0.51 ꢃ 0.01, 0.24 ꢃ 0.001, time (10 min). This observation indicated the fast response of
0.36 ꢃ 0.01, and 0.34 ꢃ 0.001 U gꢂ1 protein for A. flavus, A. niger, the Ce-MOF toward the oxidant, namely H2O2 (Fig. 8b).
A. terreus, C. Albicans, and R. glutinis, respectively (Fig. 6d). Thus,
MOFs are effective antifungal agents (Table 1). Copper-based
AU-1 generated oxidative stress in the fungal cells and further MOFs, such as Cu-BTC (BTC = 1,3,5-benzenetricarboxylate)55
caused generation of reactive oxygen species (ROS), which and HKUST-1,56 have been reported as antifungal agents
damaged the cell membrane.
against C. Albicans and Saccharomyces cerevisiae, respectively
There is no single mechanism that can support the redox (Table 1). Cu-BTC could inhibit the spores of C. Albicans,
regeneration mechanism of a Ce-MOF, such as AU-1.49 However, Fusarium oxysporum, and Aspergillus oryzae up to 100% and
X-ray photoelectron spectroscopy (XPS) revealed the presence of 30% (for F. oxysporum, A. oryzae), without significant activity
both Ce3+ and Ce4+ ions in the framework of AU-1.50 It has been against A. niger even after 7 days of treatment (Table 1).55 The
reported that cerium-based materials with a high ratio of Ce3+
/
antifungal activity of Co(II)-coordination polymers (Co-CPs)
Ce4+ exhibited better SOD activity.51,52 We also observed the containing glutarates and bipyridyl ligands was shown to
formation of H2O2, which is one of the products of SOD-catalyzed be the highest at an optimum concentration of 2.0 mg mLꢂ1
Fig. 8 (a) UV-Vis absorption spectra of Ce-MOF without and with H2O2 over time, and (b) the absorbance intensities at wavelengths 246 nm and
Table 1 A summary of MOF-based nanomaterials as antifungal agents
Optimal
conc.
Materials
Synthesis
Microorganism
Efficiency
Ref.
57
[Co2(Glu)2(m-bpa)2]ꢁ(H2O)4, Solvothermal synthesis, Candida albicans and Aspergillus niger
44–62% (C. albicans)
90–99.98% (A. niger)
2.0 mg
mLꢂ1
[Co4(Glu)4(m-bpp)2], and
[Co2(Glu)2(m-bpe)2]ꢁ(H2O)0.5
Cu-BTC
100 1C for 72 h
Solvothermal synthesis, C. Albicans, F. oxysporum, A. oryzae
110 1C for 24 h
100% (C. albicans)
30% (F. oxysporum and
A. oryzae)
500 ppm 55
V-ZIF
Stirred at ambient tem- C. albicans
perature for 30 min
Solvothermal synthesis, Aspergillus flavus, Aspergillus niger, Aspergil- 93.3–99.3%
100%
0.78 mg
mLꢂ1
40 mg
mLꢂ1
58
CeMOF, AU-1
This
study
85 1C for 16 h
lus terreus, Candida albicans, and Rhodotor-
ula glutinis
Notes: Glu, glutarate; bpy, 4,40-bipyridine; bpe, 1,2-bis(4-pyridyl)ethylene; bpymh, N,N0-bis((pyridine-4-ylmethylene)hydrazine).
356 nm.
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Journal of Materials Chemistry B
(Table 1).57 In this study, the reported inhibition rates were
44–62% and 90–99.98% for A. niger and C. Albicans (Table 1).57
The antifungal activity of Co-MOF was due to the generation of
reactive nitrogen oxide species (NO) and H2O2. The Ce-MOF
exhibited high activity at a comparatively low concentration of
4 X.-P. Wu, L. Gagliardi and D. G. Truhlar, J. Am. Chem. Soc.,
2018, 140, 7904–7912.
5 H. N. Abdelhamid, Mater. Today Chem., 2020, 15, 100222.
6 A. A. Kassem, H. N. Abdelhamid, D. M. Fouad and
S. A. Ibrahim, Int. J. Hydrogen Energy, 2019, 44, 31230–31238.
7 K.-J. Kim, H.-J. Kim, H.-G. Park, C.-H. Hwang, C. Sung,
K.-S. Jang, S.-H. Park, B.-G. Kim, Y.-K. Lee, Y.-H. Yang,
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X. Zou, Inorg. Chem., 2017, 56, 9139–9146.
9 H. N. Abdelhamid, Microchim. Acta, 2018, 185, 200.
10 H. E. Emam, H. N. Abdelhamid and R. M. Abdelhameed,
Dyes Pigm., 2018, 159, 491–498.
11 M. N. Goda, H. N. Abdelhamid and A. E.-A. A. Said, ACS
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40 mg mL .
ꢂ1 57 Voriconazole-incorporated zinc 2-methylimidazolates
frameworks (V-ZIF) exhibited high penetration of C. Albicans
biofilms with an antifungal efficiency of 100% (Table 1).58
MOF-based antifungal agents are promising and offer several
advantages, such as high efficiency, low agent loading, and the
possibility to be modified with other agents, such as nano-
particles (Table 1). AU-1 showed enzyme-like activities, including
peroxidase-like activity, and could kill fungi with 93.3–99.3%
efficiency (Table 1). Thus, it could prevent the recolonization of the
fungi. The cerium-based MOF exhibited excellent ROS-scavenging
activity and mimicked superoxide dismutase and catalase.59 Thus, it
can be used further for biomedical applications, such as bio-
catalysis, biosensing, and drug delivery.
12 A. A. Kassem, H. N. Abdelhamid, D. M. Fouad and S. A.
Ibrahim, Microporous Mesoporous Mater., 2020, 305, 110340.
13 H. N. Abdelhamid, A. M. El-Zohry, J. Cong, T. Thersleff,
M. Karlsson, L. Kloo and X. Zou, R. Soc. Open Sci., 2019,
6, 190723.
Conclusions
14 L. Valencia and H. N. Abdelhamid, Carbohydr. Polym., 2019,
213, 338–345.
The solvothermal synthesis of a Ce-MOF, AU-1, was achieved
using a tri-topic carboxylic linker and cerium as the metal center.
The material had a crystalline phase with good thermal stability
up to 380 1C. It contained Ce in mixed-valence states (Ce4+ and
Ce3+). The antifungal activity of AU-1 was evaluated using different
bioanalytical methods, including dry mass, soluble proteins,
microscopic images, and colony-forming units. The data revealed
high species-dependent antifungal activity of the Ce-MOF. The
antifungal activity of the Ce-MOF increased with an increase in
material concentration. AU-1 possessed good enzymatic activities,
such as superoxide dismutation, catalase, and peroxidase, and
may be useful as a new anticancer, antioxidant, and antimicrobial
platform for other biomedical applications. This work promotes
the application of MOFs in nanomedicine and biomaterials.
15 H. N. Abdelhamid, M. Wilk-Kozubek, A. M. El-Zohry,
´
´
A. Bermejo Gomez, A. Valiente, B. Martın-Matute, A.-V.
Mudring and X. Zou, Microporous Mesoporous Mater.,
2019, 279, 400–406.
16 A. F. Abdel-Magied, H. N. Abdelhamid, R. M. Ashour, X. Zou
and K. Forsberg, Microporous Mesoporous Mater., 2019, 278,
175–184.
17 S. Sultan, H. N. Abdelhamid, X. Zou and A. P. Mathew, Adv.
Funct. Mater., 2018, 1805372.
¨
18 H. N. Abdelhamid, M. Dowaidar and U. Langel, Microporous
Mesoporous Mater., 2020, 110200.
19 H. N. Abdelhamid, Macromol. Chem. Phys., 2020, 221, 2000031.
¨
¨
20 H. N. Abdelhamid, M. Dowaidar, M. Hallbrink and U. Langel,
Microporous Mesoporous Mater., 2020, 300, 110173.
21 H. N. Abdelhamid, Dalton Trans., 2020, 49, 4416–4424.
22 X. Zhang, G. Li, D. Wu, X. Li, N. Hu, J. Chen, G. Chen and
Y. Wu, Biosens. Bioelectron., 2019, 137, 178–198.
23 W. Song, B. Zhao, C. Wang, Y. Ozaki and X. Lu, J. Mater.
Chem. B, 2019, 7, 850–875.
Conflicts of interest
There are no conflicts to declare.
24 Z. Liu, F. Wang, J. Ren and X. Qu, Biomaterials, 2019, 208,
21–31.
25 X. Li, M. Qi, C. Li, B. Dong, J. Wang, M. D. Weir, S. Imazato,
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26 L. Luo, L. Huang, X. Liu, W. Zhang, X. Yao, L. Dou, X. Zhang,
Y. Nian, J. Sun and J. Wang, Inorg. Chem., 2019, 58, 11382–11388.
27 C. J. Alexopoulos, C. W. Mims and M. Blackwell, Introductory
Mycology, Wiley, New York, 4th edn, 1996, p. 868.
28 The Fifth Kingdom, http://www.mycolog.com/fifthtoc.html,
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Acknowledgements
The authors thank the Ministry of Higher Education and
Scientific Research (Egypt), Assiut University, and Institutional
Review Board (IRB) of the Faculty of Science at Assiut Univer-
sity, Egypt for support.
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