Materials Science & Engineering C 104 (2019) 109977

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Materials Science & Engineering C

journal homepage: www.elsevier.com/locate/msec

Nanostructured zinc oxide on silica surface: Preparation, physicochemical T

characterization and antimicrobial activity
⁎
Anna Donnadioa, , Gianluigi Cardinalib, Loredana Latterinic, Luca Roscinib, Valeria Ambrogia
a
Department of Pharmaceutical Sciences, University of Perugia, via del Liceo 1, 06123 Perugia, Italy
b
Department of Pharmaceutical Sciences, - Microbiology, University of Perugia, Borgo XX giugno, 06121 Perugia, Italy
c
Department of Chemistry, Biology and Biotechnologies, University of Perugia, via Elce di Sotto 8, 06123 Perugia, Italy

A R T I C LE I N FO A B S T R A C T

Keywords: Zinc oxide nanoparticles were synthesized using two silica supports largely used in pharmaceutical field as
Zinc oxide excipients, Cab-O-Sil-H5 and Syloid 244 FP characterized by high surface area and different porosity. In order to
Silica evaluate the effects of different silica on nanoparticle chemical physical properties, composites (ZnO-SiO2)
Composites containing different amounts of ZnO nanoparticles were obtained and characterized by X-ray Powder Diffraction
Antibacterial activity
(XRPD), Transmission Electron Microscopy (TEM), Attenuated Transmission Reflectance (ATR), UV–vis spec-
Antifungal activity
troscopy and finally Photoluminescence (PL). Composites showed the presence of quite uniformly distributed
zinc nanostructures on the silica surface with size in the range of 30–50 nm with an estimated specific surface
area ranged from ca. 20 to 70 m2/g. The formation of a Zn-O–Si interface in ZnO-SiO2 was observed as well.
Photoluminescence studies revealed that ZnO-SiO2 samples based on Cab-O-Sil present a higher contribution of
oxygen vacancies per unit volume. Finally, the resulting composites were tested for antibacterial and antifungal
activities. Whereas silica supports did not show any antibacterial and antifungal activities, most of the prepared
composites, both with Cab-O-Sil-5H and Syloid 244 FP supports, resulted active against both bacteria and fungi.
In particular the contingency analysis showed that the amount of zinc oxide in the composites was partly related
to MIC results in bacteria (p = 0.059), whereas it showed an interesting p = 0.022 in yeast in the case of low
amount of ZnO (10%). Thus, the described ZnO-SiO2 composites can be proposed for the preparation of both
pharmaceutical formulations and medical disposals with antibacterial and antifungal activities.

1. Introduction has found potential applications in agricultural field [7], and because of
its antimicrobial properties in biomedical [8] and food fields as well
Nanotechnology is one of the foremost areas in the field of materials [9]. It has been reported that
science, as nanoparticles, in comparison to bulk materials, exhibit new nanostructured ZnO has good antibacterial activity due to a large
and improved properties connected to their small size, distribution and concentration of Zn2+ that can produce detrimental effects on cell
morphology. These new properties open a large variety of applications growth and tissue formation and to the formation of reactive oxygen
in different fields. Inorganic nanoparticles (such as metallic silver, zinc species (ROS) that can induce cell apoptosis [10–16].
oxide, copper oxide and titania nanoparticles) have been extensively However the main problem in nanoparticle preparation is their
studied and represent a new and promising tool for fighting the emer- tendency to coalesce and agglomerate and thus, with the aim of de-
ging infectious diseases and the increase of drug resistance among creasing this limiting aspect, many different strategies for ZnO nano-
bacteria and fungi [1–4]. Among inorganic nanoparticles, zinc oxide particle synthesis and successive surface modification have been pro-
shows many advantages because of its stability within wide range of posed [17]. One appealing strategy is based on the preparation of ZnO
temperature and moisture and good shelf life. Besides ZnO is largely nanomaterials on inorganic supports. In particular, silica are very ver-
used in cosmetic creams and lotions because its capacity of blocking satile materials widely applied in many industrial fields and have been
ultraviolet radiations [5] and in pharmaceutical topical formulations proposed as a support for ZnO nanoparticles [18–22]. In some of these
for the treatment of eczema and wound [6]. Because of its anti- papers, antibacterial activity was evaluated but no reference to anti-
microbial, astringent, wound healing and angiogenic properties, ZnO fungal activity could be found. Cab-O-Sil H5 and Syloid 244 FP are

⁎
Corresponding author.
E-mail address: [email protected] (A. Donnadio).

https://doi.org/10.1016/j.msec.2019.109977
Received 4 February 2019; Received in revised form 9 July 2019; Accepted 14 July 2019
Available online 17 July 2019
0928-4931/ © 2019 Elsevier B.V. All rights reserved.
A. Donnadio, et al. Materials Science & Engineering C 104 (2019) 109977

silica widely employed as excipients with multiple functions largely Table 1

used in pharmaceutical and cosmetic industries but no used till now for Composites based on Syloid 244 FP.
the preparation of ZnO nanoparticles. Cab-O-Sil H5 is a synthetic Sample Synthetic Zinc Syloid 244 ZnO amount
amorphous, silicon dioxide with extremely small particles size and large procedure acetate FP (%)
surface area (300 m2/g) and interparticle porosity [23]. Syloid 244 FP (g) (g)
is a micronized (diameter 2.5–3.7 μm) synthetic amorphous silica with
ZnO/S-A-10 Aqueous solution 0.1 0.9 11.4
highly developed network of randomly oriented mesopores, that pro- ZnO/S-A-20 Aqueous solution 0.2 0.8 20.4
vide access to the large surface area (slightly higher than 300 m2/g) ZnO/S-A-30 Aqueous solution 0.3 0.7 29.9
[24]. It is characterized by a pore volume of ca. 1.42 cm3/g and an ZnO/S-S-10 Solid state 0.1 0.9 14.4
average pore diameter of 19.0 nm. These silicas are used as pharma- ZnO/S-S-20 Solid state 0.2 0.8 24.8
ZnO/S-S-30 Solid state 0.3 0.7 31.4
ceutical excipients as glidants, adsorbents, anti-tacking and desiccant
agents, carriers for active pharmaceutical ingredient and thickening
and gelling agents. Recently, non-ordered mesoporous silicas (Syloid) Table 2
have been proposed also as excipients for dermal delivery of poorly Composites based on Cab-O-Sil-H5.
soluble drugs [25,26].
Sample Synthetic zinc acetate Cab-O-Sil- ZnO amount
Finally, the presence of silica could offer a benefit for the nano-
procedure (g) H5 (%)
particles whose uncontrolled dispersion in the environment could be (g)
reduced by the presence of the support.
Antibacterial and antifungal effects of metallic nanoparticles is a ZnO/C-A-10 Aqueous solution 0.1 0.9 14.5
quite popular topic of microbial literature and there are several studies ZnO/C-A-20 Aqueous solution 0.2 0.8 18.5
ZnO/C-A-30 Aqueous solution 0.3 0.7 31.6
reporting the ability of metallic nanoparticles, such as silver and sele- ZnO/C-S-10 Solid state 0.1 0.9 13.7
nium composites, against fungi [27–30]. However, the number of stu- ZnO/C-S-20 Solid state 0.2 0.8 23.2
dies dealing with the effect of ZnO nanoparticles alone against yeast is ZnO/C-S-30 Solid state 0.3 0.7 27.3
not so large, so it represents a quite new field of application of this type
of composites.
In this paper a simple and fast strategy for the preparation of sup- the silicas at solid state. The mixture was heated in a muffle at 500 °C
ported ZnO nanoparticles was reported by using Cab-O-Sil-H5 and for 2 h and stored in a desiccator with P2O5. The composites based on
Syloid 244 FP silica as a support with the aim of favoring nanoparticle Cab-O-Sil-H5 will be named, hereafter, as ZnO/C, while those based on
nucleation and growth and preventing their agglomeration. Moreover Syloid 244 FP as ZnO/S.
the method can be proposed for the preparation of both pharmaceutical Both procedures were performed with different weight ratios be-
formulations and medical disposals with antibacterial and antifungal tween silica and zinc acetate as reported in Tables 1 and 2.
activities with reduced risk of nanoparticle dispersion in the host and Pure ZnO, as a reference compound, was prepared in a muffle by
the environment. heating the Zn(CH3COO)2 at 500 °C for 2 h [31,32].
The preparation and characterization of ZnO nanoparticles on Cab-
O-Sil-H5 and Syloid 244 FP silica are described to evaluate the effects of
different silica on nanoparticle chemical physical properties. Finally, 2.3. Characterization of ZnO-SiO2 composites
the resulting composites were tested for antibacterial and antifungal
activities against two yeast strains and two bacterial strains belonging X-ray powder diffraction (XRPD) patterns were collected with a
to Candida albicans and Staphylococcus aureus species respectively. Philips X'Pert PRO MPD diffractometer with a Cu Ka radiation source
These two species were chosen as biosensors for the study due to their (k = 0.15406 nm) operating at 40 kV and 40 mA at a scanning step of
presence and to the hazard they represent for several fields, from the 0.033 in the 2θ° range 5–70. Transmission electron microscopy (TEM)
medical to the environmental one. analysis was carried out by a Philips 208 transmission electron micro-
scope, operating at an accelerating voltage of 100 kV. Powders were
2. Materials and methods dispersed in ethanol and sonicated for a few minutes, then supported on
copper grids (200 mesh) precoated with Formvar carbon films, and
2.1. Materials quickly dried. The Zn content in the composites was determined by
using an ICP Varian Liberty inductively coupled plasma-optical emis-
Cab-O-Sil-H5 and Syloid 244 FP were kindly provided by Cabot sion spectrometer (ICP-OES) with axial injection. A weighed amount of
(Italy) and Grace Italy S.r.L. (Italy) respectively. Zinc acetate dihydrate the samples was dissolved in 3 M HF (≈2 mL), few drops of con-
was purchased from Carlo Erba (Italy). Deionized water was obtained centrated HCl and then diluted with water. Absorption spectra of the
by reverse osmosis process with a MilliQ system (Millipore, Rome, samples were recorded by a Varian (Cary 4000) spectrophotometer,
Italy). equipped with a 150 mm integration sphere, for which a barium sul-
phate tablet was used as a reference. The recorded spectra were ana-
2.2. Synthesis of ZnO-SiO2 composites lyzed with the Kubelka–Munk equation in order to make the compar-
ison possible. Photoluminescence (PL) spectra, corrected for the
For the preparation of the ZnO-SiO2 composites, Cab-O-Sil-H5 and instrumental response, of the powder samples were recorded by use of a
Syloid 244 FP silica matrices were used according to the following two Fluorolog-2 (Spex, F112AI) fluorimeter, using a co-linear detection
different procedures: by aqueous solution and solid state mixing. The geometry.
synthesis of the hybrid ZnO-SiO2 via aqueous solution was carried out Attenuated Transmission reflectance (ATR) spectra were collected
by dissolving a weighted amount of Zn(CH3COO)2·2H2O in 80 mL dis- with a Gateway ATR accessory from SPECAC, using a six reflection
tilled water, followed by the addition of 1 g of the silica (Cab-O-Sil-H5 horizontal ATR sampling system equipped with a ZnSe crystal. The
or Syloid 244 FP) to the solution. Then the reaction mixture was stirred sample was placed on top of the ZnSe crystal at room temperature and
for 2 h at room temperature. Subsequently, the solvent was withdrawn each ATR spectrum was the average of fifty scans, measured with a
by rotavapor and the resulting solid was heated at 500 °C for 2 h and resolution of 2 cm−1 in the 400–4000 cm−1 range.
stored in a desiccator with P2O5. The synthesis of the hybrid ZnO-SiO2
via solid state route was achieved by mixing Zn(CH3COO)2·2H2O with

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2.4. Release studies

Zinc release studies from all composites were performed incubating

10 mg of sample in 4.5 mL of deionized water at 37 °C under stirring
(150 rpm). After 24 h the samples were ultra-centrifuged (20,000 rpm
for 10 min) and the zinc content in the supernatant was determined by
ICP. The tests were conducted in triplicate and data are reported as an
average of three determinations.

2.5. Antimicrobial activity

Two yeast strains belonging to Candida albicans species (CMC 1778

and CMC 2020) and two bacterial strain belonging to Staphylococcus
aureus species (PI 5639 and PI 6870) were employed in this study. All
strains were isolated in a clinical (medical) environment from patients'
blood cultures (yeasts) and from diabetic foot (bacteria). Isolates are
kept frozen at −80 °C in 17% glycerol. Short term storage was carried
out at 4 °C on YEPDA (YEPD added with 1.7% agarose) for yeasts and Fig. 1. XRPD pattern of pure ZnO.
BHI (Brain Heart Infusion) agar for bacteria. Yeast strains were grown
in YEPD (Yeast Extract 1%, Peptone 1%, Dextrose 1% - all products 3. Results and discussion
from Biolife (Italy) and bacterial strains on BHI (Biolife, Italy) at 37 °C
with 150 rpm shaking. The ZnO based composites were prepared following the procedures
Antimicrobial activity was assessed using a broth microdilution described in the experimental section using two different silica matrices
method for the determination of the Minimum Inhibiting Concentration as supports. All the composite materials will be hereafter indicated as
(MIC) of each compound. The assay was carried out following the reported in Tables 1 and 2. In the same tables the ZnO amount as de-
guidelines established by EUCAST [33]. termined by ICP measurements, is also shown.
Briefly, each strain was grown over night in bottles containing YEPD
(for yeasts) and BHI (for bacteria) medium, at 37 °C in an orbital shaker
at 150–180 rpm and then harvested and centrifuged at 3000 ×g for 3.1. XRPD and TEM analysis
5 min at 4 °C. The supernatant was removed, and the pellet was washed
twice with sterile distilled water. Washed yeasts and bacterial cells Fig. 1 shows the XRPD patterns of ZnO. The peaks at 2θ° = 31.7,
were then resuspended in RPMI-1640 with 2% glucose (Sigma Aldrich) 34.4, 36.3, 47.6, 56.6, 62.9, 66.3, 67.9 and 69.1 can be indexed to
and Mueller Hinton II (Biolife Italia) media respectively, in order to (100), (002), (101), (102), (110), (103), (200), (112) and (201) crystal
obtain a final density of 1.0 × 106 cells/mL. 100 μL of this standardized planes of wurtzite-type hexagonal ZnO (JCPDS, No. 36-1451), implying
cell suspension were seeded in each selected well of 96-well microtiter the formation of crystalline ZnO with space group p63mc (186) and
plate in which the selected concentrations of ZnO-SiO2 nanoparticle with lattice constants a = 3.249 and c = 5.206 Å. Figs. 2 and 3 show
samples (1 and 2 mg/mL) were already aliquoted. A negative control the XRPD patterns of ZnO-SiO2 composites with different ZnO amounts
well, consisting of microbial cells without nanoparticle samples, was prepared by the two synthetic procedures.
also prepared. No characteristic peaks from the intermediate species such as Zn
The microtiter plate was closed, sealed and incubated for 24 h at (OH)2 can be detected. In general, the X-ray powder diffraction (XRPD)
37 °C. The plate was then recovered and using a multichannel pipette, profile of the composite shows the presence of weak diffraction peaks,
20 μL of fresh 0.01% w/v resazurin aqueous solution were added to whose positions fully match those of the ZnO particles. In the cases of
each well of the plate, included the negative control wells, to assess composites containing the lowest amount of ZnO, these peaks are rather
MIC. After 1 h incubation at 37 °C, the plate was visually inspected to weak or even not detectable. The presence of weak XRPD peaks in the
highlight the presence of pink color gradient, resulting from the re- ZnO-SiO2 samples can be probably attributed to the small amount of
duction of the blue dye resazurin to the pink dye resorufin by living ZnO, but the formation of amorphous nanomaterials cannot be ex-
cells. MIC value was assessed as the ZnO-SiO2 concentration present in cluded. The average grain size of the ZnO particles at the surface of SiO2
the last well before a visible color change was detectable. is of ca. 30 nm as estimated by the Debye-Scherrer's equation on the
basis of the XRPD peak (100) at ca. 2θ =32°. The TEM images show
particles having an irregular morphology, with sizes in the range of
2.6. Contingency analysis 30–50 nm, which are larger than those estimated for the ZnO crystalline
domain size by X-ray analysis; some of the particles are fused together
Contingency analysis was carried out as described by Legendre & over the SiO2 surface, thus forming larger micrometric aggregates
Legendreè [34]. Contingency tables were prepared by comparing the especially in the composites containing larger amount of ZnO.
occurrences of MIC values at the concentrations ranging from 1000 to Moreover, in ZnO/C materials, ZnO crystals started to form at lower
2000 mgL−1 with the following composite properties: the physical form percent of ZnO (ZnO/C-S-10) compared to the ZnO/S where the peaks
(crystalline or amorphous form), the photoluminescence intensity ratio are more evident only at higher ZnO percentage. A possible explanation
(ratio of intensities at 460 and 400 nm for the ZnO-SiO2 samples), the could be the different porosity of the Cab-O-Sil-H5 in contrast to the
UV–vis absorption intensity, the zinc amount in the composite and the Syloid 244 FP. Therefore, in the case of Cab-O-Sil-H5 the surface can be
zinc ions release. saturated at much lower loadings resulting in the formation of crys-
After calculating the expected values, a chi squared test was carried talline phases at lower amounts of ZnO.
out, obtaining the probability to reject the null hypothesis, i.e. that all From the TEM analysis it can be reasonably inferred that the ZnO
combinations were randomly distributed and due only to the effect of particles in the ZnO-SiO2 composites are, on the whole, smaller than
the case. those observed in the neat ZnO sample (obtained without the SiO2); this
observation confirms that silica matrix avoids the aggregation of ZnO

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A. Donnadio, et al. Materials Science & Engineering C 104 (2019) 109977

Fig. 2. XRPD patterns of samples prepared by solid state reaction. On the right the samples based on Cab-O-Sil-H5, a) ZnO/C-S-10, b) ZnO/C-S-20, c) ZnO/C-S-30,
and on the left the samples based on Syloid FP 244, d) ZnO/S-S-10, e) ZnO/S-S-20, f) ZnO/S-S-30.

clusters at least for lower amounts of ZnO formed (see Fig. SI1) sug- 3.3. UV–vis spectrophotometry
gesting the dispersion effect of the support to some extent. In all cases
the images clearly reveal that ZnO nanoparticles form and grow on the Fig. 6 shows the UV–vis absorption spectrum of crystalline bulk ZnO
surface of SiO2 also for high ZnO concentration (Fig. 4). At the condi- synthesized as a control without the presence of SiO2 nanoparticles; in
tion referred to above, the presence of free ZnO particles cannot be the Figs. 7 and 8 the absorption spectra of the ZnO–SiO2 composites are
observed. reported. All samples show an absorption band at ca. 400 nm which can
be ascribed to the characteristic absorption of the ZnO semiconductor
[37]. The position of the absorption band edge of ZnO, determined as
3.2. ATR analysis the cross point between the x-axis and the tangent to the linear section
of the absorption band edge, is a fundamental characteristic of ZnO
Fig. 5 shows ZnO ATR spectrum and the spectra of some re- nanomaterials, which depends on the average size of the nanoparticles,
presentative samples of ZnO-SiO2 composites. The spectrum of crys- but is affected also by the presence of structural/electronic defects.
talline bulk ZnO particles shows characteristic vibrational peaks of ZnO The UV absorption of all samples presents similar features.
at 410 and 580 cm−1. In the spectra of ZnO–SiO2 composites, the ZnO However, the absorption intensity of the composite samples decreased
related peaks are retained and several additional modes appear, which with the amount of ZnO and the absorption edge shifted slightly to a
can be assigned to the SiO2 support itself or its binding with the ZnO higher energy for the composites containing the lowest amount of ZnO,
surface. The broad band in the range of 1000–1200 cm−1 is typical for for which ZnO nanocrystals with smaller dimensions were determined
IR spectra of SiO2. The band at 790 cm−1 originates from a symmetric by XRPD data. This band gap expansion is likely caused by the reduc-
SieO stretching [35]. The shoulder at 950 cm−1 in the ATR spectrum of tion in size of ZnO nanoparticles with respect to the as-prepared pure
ZnO–SiO2 composites can be assigned to a Zn–O–Si vibration clearly ZnO.
indicating the formation of a ZnO–silica interface in ZnO-SiO2 nanos- It is worth to be noted that the intensity of UV bands, which in-
tructured materials already at low ZnO amounts and regardless of the creases with increasing ZnO loading, can be related to ZnO crystalline
procedure used (Fig. 5) [36]. phase. For composites with the lowest ZnO loading the absorption band
is not observed, indicating that no crystalline ZnO phase exists, as was
observed in the XRPD pattern. Therefore, with these low loadings, a

Fig. 3. XRPD patterns of samples prepared by aqueous solution. On the right the samples based on Cab-O-Sil-H5, a) ZnO/C-A-10, b) ZnO/C-A-20, c) ZnO/C-A-30, and
on the left the samples based on Syloid FP 244, d) ZnO/S-A-10, e) ZnO/S-A-20, f) ZnO/S-A-30.

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A. Donnadio, et al. Materials Science & Engineering C 104 (2019) 109977

Fig. 4. TEM images of samples a) ZnO/S-A-30, b) ZnO/S-S-30, c) ZnO/C-A-30, d) ZnO/C-S-30.

finely dispersed amorphous ZnO phase is obtained, which is particu- the emission centered at 460 nm has been attributed to oxygen va-
larly true for ZnO/S-A-10 and ZnO/S-S-10. cancies.
The photoluminescence (PL) spectra of ZnO crystals and of the ZnO/
SiO2 composites obtained upon excitation at 340 nm are plotted in
3.4. Photoluminescence analysis Figs. 9–11.
In general, the PL spectrum of the samples under investigation
It is well established that ZnO-materials upon photoexcitation can presents both the so-called UV- and VIS-bands, although the relative
relax through radiative processes; the profile and the features of the contribution at 400 and 460 nm is different for the samples prepared in
photoluminescence spectra are strongly correlated to structural and different conditions. Since the comparison of the absolute intensities is
electronic properties of the materials. In general, the ZnO exhibits a UV affected by the fraction of absorbed light, the spectral properties of the
luminescence band in the 340–440 range (2.80–3.60 eV) associated to samples are correlate through the ratio of intensities at 400 and
the near band-edge emission and a visible emission band ranging from 460 nm, which are collected in Table 3.
440 to 600 nm connected to structural defects [38–40]. In particular,

Fig. 5. ATR spectra of (a) pure Cab-O-Sil-H5, ZnO, ZnO/C-10-S and ZnO/C-30-A and (b) pure Syloid FP 244, ZnO, ZnO/S-10-S and ZnO/S-30-A.

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surface area in comparison to spheres (ZnO/S-S-30).

3.6. Antimicrobial activity

Antibacterial and antifungal activities were carried out through

broth microdilution assays in order to determine MIC values (the lowest
concentration of an antimicrobial agent that inhibits the growth of a
microorganism after overnight incubation) for each composite against
Staphylococcus aureus and Candida albicans. The two silica, used as a
support, were tested as well. Results are reported in Table 4. As the
tested samples are not constituted by pure ZnO nanoparticles (Tables 1
and 2), MIC values are expressed for each composite in two different
ways: MIC values referred to the composite (MIC) and MIC values re-
ferred to the effective ZnO amount (MIC ZnO). MIC ZnO was determined
by the following formula:
MICZnO = MIC × ZnO%/100

whereas silica supports did not show any inhibitory activity, most of the
Fig. 6. UV absorption spectrum of crystalline bulk ZnO. prepared composites resulted active with MICZnO between 0.187 and
0.628 mg/mL against both bacteria and fungi. Results against Candida
The ratio of intensities is on average higher for the ZnO-SiO2 sam- were particularly interesting as ZnO has been largely evaluated for its
ples based on Cab-O-Sil, indicating that in these samples a higher antibacterial activity, but only a few papers investigated antifungal
contribution of defect states is present. Thus ZnO-SiO2 samples based on activity against Candida species [42–45]. Composites ZnO/S-A-10 and
Cab-O-Sil present a higher contribution of oxygen vacancies per unit ZnO/S-S-10, for which the Zn2+ ion concentrations in the surnatant
volume. were the lowest, did not show growth inhibitory activity against fungi.
This is in agreement with the assumption that probably Zn2+ ion re-
3.5. Release of Zn2+ ions lease is one of the factors that affects antimicrobial activity [1]. Another
consideration which can be drawn is that composites based on Cab-O-
The zinc ions in vitro release from all samples was performed and Sil were more active than those based on Syloid 244 FP (Table 4)
results are reported in table SI2. For composites based on Cab-O-Sil-H5, probably because they showed higher contribution of oxygen vacancies
the Zn amount released after 24 h increased with the increasing of the per unit volume with consequent higher production of radical oxygen
zinc content in the sample. For composites based on Syloid 244 FP a species (ROS).
different behavior was observed. In fact, supernatants obtained by
samples with intermediate Zn content (ZnO/S-A-20 and ZnO/S-S-20) 3.7. Contingency analysis
showed the lowest zinc concentration. This behavior can be explained
because Syloid 244 FP is a mesoporous silica and ZnO nanoparticles can In order to establish the existence of a relationship between the
grow inside the mesopores and on the external surface. For composites examined properties of the composites and MIC values, a two-way
ZnO/S-A-10 and ZnO/S-S-10 with low Zn loading, the formation of contingency analysis was carried out, following the rationale described
amorphous nanoparticles on the external surface of the silica is ob- in Legendre & Legendre [34]. Confirming what previously described,
tained (see XRPD patterns). Increasing the ZnO loading, a partial pore the analysis of the p values obtained for the two silica supports showed
blockage can take place [41] due to partial crystallization of the na- that they were not related to the presence of positive MIC in yeast and
noparticles as shown by XRPD patterns of ZnO/S-A-20 and ZnO/S-S-20 in bacteria (p > 0.13). High photoluminescence and amorphous
composites. This fact could be responsible of the lower Zn2+ release. In structure were related with low MIC values in bacteria, although in
the case of ZnO/S-A-30, the zinc concentration in the acceptor medium general the comparison of all MICs with structure and photo-
increased notwithstanding the larger size of the nanoparticles. It could luminescence had low p support (p = 0.126 and 0.212, respectively).
be due to its different morphology, similar to rods, which have a higher The amount of zinc oxide in the composites was partly related to MIC

Fig. 7. UV absorption spectra of samples based on Syloid FP 244, a) ZnO/S-A-10, b) ZnO/S-A-20, c) ZnO/S-A-30, d) ZnO/S-S-10, e) ZnO/S-S-20 and f) ZnO/S-S-30.

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Fig. 8. UV absorption spectra of samples based on Cab-O-Sil-H5, a) ZnO/C-A-10, b) ZnO/C-A-20, c) ZnO/C-A-30, d) ZnO/C-S-10, e) ZnO/C-S-20 and f) ZnO/C-S-30.

photoluminescence and the amount of the released Zn2+ ions

(p = 0.052 and p = 0.120). Interestingly, UV absorption and photo-
luminescence displayed a similar relation with the displayed anti-
microbial activity: the composites were in fact more active against fungi
than bacteria (p = 0.073 (UV) and p = 0.052 (PL) vs p = 0.134 (UV)
and p = 0.212 (PL), for yeast and bacteria respectively). The obtained
results suggest that more than one property effects the antimicrobial
activity of ZnO nanoparticles, and this could be a confirm of the me-
chanism of action of ZnO nanoparticles which seems the result of a
complex of factors such as direct contact of ZnO-NPs with cell walls,
resulting in destructing bacterial cell integrity, liberation of Zn2+ ions
and ROS formation [1].

4. Conclusions

In the present work ZnO-SiO2 composites have been synthesized

without the use of surfactant or other organic solvent and using simple
Fig. 9. PL spectrum of bulk crystalline ZnO. methods which imply the use of two supports largely used in pharma-
ceutical and cosmetic field. The main objective for this study was to
results in bacteria (p = 0.059), whereas it showed an interesting explore the feasibility of using a wet or a dry system to grow ZnO na-
p = 0.022 in yeast in the case of low amount of ZnO (10%) and high noparticles on SiO2 matrix and to investigate the antibacterial and
MIC (> 2 mg/mL). Similarly, composites with low UV absorption was antifungal activity of supported ZnO nanoparticles.
partly related to high MIC in yeast (p = 0.073). Moreover, other re- The investigated methods allowed to obtain composites containing
levant properties related to antifungal activity were the presence of small ZnO nanoparticles characterized by a covalent interface Zn-O-Si
which showed positive results especially in terms of antifungal activity.

Fig. 10. PL spectra of samples based on Syloid 244 FP. On the left, the sample prepared by aqueous solution, a) ZnO/S-A-10, b) ZnO/S-A-20, c) ZnO/S-A-30. On the
right, the samples prepared by solid state reaction, d) ZnO/S-S-10, e) ZnO/S-S-20, f) ZnO/S-S-30.

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Fig. 11. PL spectra of samples based on Cab-O-Sil-H5. On the left, the sample prepared by aqueous solution, a) ZnO/C-A-10, b) ZnO/C-A-20, c) ZnO/C-A-30. On the
right, the samples prepared by solid state reaction, d) ZnO/C-S-10, e) ZnO/C-S-20, f) ZnO/C-S-30.

Table 3 Declaration of Competing Interest

Ratio of intensities at 460 and 400 nm for the
ZnO-SiO2 samples. There is no conflict of interest for this manuscript.
Sample I460/I400

ZnO 1.2 Acknowledgments

ZnO/S-A-10 1.1
ZnO/S-A-20 1.0
ZnO/S-A-30 1.6 The work was financially supported by the University of Perugia
ZnO/S-S-10 2.7 (Italy), Fondo Ricerca di Base 2015, titled “Nanoparticelle ibride per il
ZnO/S-S-20 1.1 controllo del biofilm delle specie opportunistiche del genere Candida”.
ZnO/S-S-30 2.3 The authors acknowledge gratefully Dr. Carlo Tascini of First
ZnO/C-A-10 2.5
ZnO/C-A-20 1.9
Division of Infectious Diseases, Cotugno Hospital, Naples, Italy and Dr.
ZnO/C-A-30 3.0 Matteo Bassetti of Infectious Diseases Clinic, Santa Maria Misericordia,
ZnO/C-S-10 1.3 University Hospital of Udine, Italy for providing the fungal and bac-
ZnO/C-S-20 2.7 terial isolates and Prof. Paola Sassi of Chemistry, Biology and
ZnO/C-S-30 3.0
Biotechnologies Department of Perugia University for ATR measure-
ments.
A.D. acknowledges the Università degli Studi di Perugia and the
The fact that ZnO nanoparticles are covalently bonded to the surface of
Ministero dell' Istruzione dell'Università e della Ricerca Scientifica
silica can decrease the risk of nanoparticle dispersion in the environ-
(MIUR - Rome) for the financial support through the program
ment and in the employer/host with decreasing pollution and animal/
“Dipartimenti di Eccellenza 2018-2022” (grant DELPHI).
human toxicity. Moreover, ZnO nanoparticles resulted active against
the tested microorganisms, in particular Candida species, under normal
lighting conditions.
Appendix A. Supplementary data
Therefore, in the future, the described ZnO-SiO2 composites could
be used for the preparation of both pharmaceutical formulations and
Supplementary data to this article can be found online at https://
medical disposals with antimicrobial activity.
doi.org/10.1016/j.msec.2019.109977.

Table 4
MIC and MICZnO values of the prepared composites.
Sample MIC MICZnO MIC MICZnO
(mg/mL) (mg/mL) (mg/mL) (mg/mL)
Candida albicans Candida albicans Staphylococcus aureus Staphylococcus aureus

ZnO/S-A-10 >2 – 2 0.228

ZnO/S-A-20 2 0.409 2 0.409
ZnO/S-A-30 1 0.299 2 0.597
ZnO/S-S-10 >2 – 2 0.288
ZnO/S-S-20 1 0.248 >2 –
ZnO/S-S-30 1 0.314 2 0.628
ZnO/C-A-10 2 0.289 >2 –
ZnO/C-A-20 1 0.187 >2 –
ZnO/C-A-30 2 0.632 2 0.632
ZnO/C-S-10 2 0.260 2 0.26
ZnO/C-S-20 1 0.232 2 0.463
ZnO/C-S-30 1 0.273 2 0.546

8
A. Donnadio, et al. Materials Science & Engineering C 104 (2019) 109977

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