Colloids and Surfaces B: Biointerfaces 208 (2021) 112030

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Colloids and Surfaces B: Biointerfaces

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

Silver nanoparticle-protein interactions and the role of lysozyme as an

antagonistic antibacterial agent
M. Beatriz Espeche Turbay a, b, *, Valentina Rey a, b, Rita D. Dorado a, Marcelo C. Sosa a,
Claudio D. Borsarelli a, b, *
a
Instituto de Bionanotecnología del NOA (INBIONATEC), CONICET, Universidad Nacional de Santiago del Estero (UNSE), RN9, km 1125, G4206XCP, Santiago del
Estero, Argentina
b
ICQ – Facultad de Agronomía y Agroindustrias, UNSE, Av. Belgrano (S) 1912, Santiago del Estero, Argentina

A R T I C L E I N F O A B S T R A C T

Keywords: The photoreductive synthesis and antibacterial activity of silver nanoparticles (AgNP) prepared in the presence
Silver nanoparticles of bovine serum albumin (BSA) and lysozyme (LZ) were evaluated. AgNP@BSA showed similar antibacterial
Antibacterial activity activity to those stabilized with citrate (AgNP@CIT) and to an AgNO3 solution, suggesting the releases of Ag+ as
Antagonistic effect
the mechanism of death. In contrast, AgNP@LZ solutions showed no activity, although LZ behaves as a
Protein corona
moderately antibacterial peptide. Furthermore, the addition of LZ to the AgNP@CIT or AgNP@BSA solutions
Lysozyme-silver interactions
induced their agglomeration and suppressed their original antibacterial efficacy. This antagonistic antibacterial
effect exerted by LZ on AgNPs is associated with electrostatic interactions exerted by LZ. Specific metal-LZ in­
teractions produce a harder protein corona on AgNP@LZ that retains Ag+, while LZ acts as a glue for AgNP@CIT
or AgNP@LZ due to its opposite electrical charge, besides strong binding to Ag+avoiding the bactericide effect.
Therefore, bactericidal effects of AgNP in biological media may be modulated by specific protein interactions.

1. Introduction
The irresponsible use of antibiotics has contributed to the prevalence
of super-resistant microorganisms capable of surviving classical drug
treatments, even those of the new generation, causing considerable
economic losses and deaths [1,2]. Hence, infectious diseases represent
one of the most dangerous health threats to overcome due to the
persistence of resistant pathogens [2].
Therefore, the search and development of new approaches to control
contaminations and infections with mechanisms of action that minimize
or avoid the development of antimicrobial resistance are crucial [3].
Regarding this, recent advances in nanotechnology point to new and
innovative alternative therapies that employ nanoparticles (NP) as
carriers for antibiotics or self-antimicrobial agents to reduce the toxic
effect of drugs, as well as to increase antimicrobial drug concentrations
at the target site [4,5]. Among the multiple nanotechnological appli­
cations of silver nanoparticles (AgNPs) [6], their use for the control and
treatment of bacterial infections, whether due to planktonic or biofilm
proliferation, represents an emerging field [7–10]. Silver-based
materials have been used for anti-infective treatments since ancient
times, basically by non-specific toxic effects exerted by the release of
silver ions, Ag+, and the overproduction of reactive species oxygen
(ROS), minimizing the possibility that pathogens can develop resistance
mechanisms [11].
However, in biological environments, the presence of other bio­
molecules can modulate the antibacterial efficacy of AgNPs [12,13]. For
example, proteins are the most abundant components in biological
fluids, so the understanding of protein-nanoparticle interactions is very
important, considering that the formation of a protein layer or corona
around the nanoparticle acts as an active biointerface between the
nanocores and cells [12–14]. The effect of the protein corona around
AgNPs using various proteins (e.g., ubiquitin, collagen, human or bovine
serum albumin, blood plasma, yeast extracts, and fetal calf) on the
cytotoxicity and antimicrobial effects of the bioconjugates have been
evaluated [12,15–18]. The data reported indicate that the properties
and functionalities of the protein corona depend on several factors, such
as the size and/or shape of the nanoparticles, the concentration of
proteins, the functionalization of the surface, etc., while some

* Corresponding authors at: Instituto de Bionanotecnología del NOA (INBIONATEC), CONICET, Universidad Nacional de Santiago del Estero (UNSE), RN9, km
1125, G4206XCP, Santiago del Estero, Argentina.
E-mail addresses: beaespeche@gmail.com (M.B. Espeche Turbay), cborsa@unse.edu.ar (C.D. Borsarelli).

https://doi.org/10.1016/j.colsurfb.2021.112030
Received 26 April 2021; Received in revised form 30 June 2021; Accepted 7 August 2021
Available online 10 August 2021
0927-7765/© 2021 Elsevier B.V. All rights reserved.
M.B. Espeche Turbay et al.
unexpected interactions of the protein corona with surrounding mole­
cules could modify the antimicrobial activity [12–14,19].
Regarding this, here we report the effect of globular proteins such as
bovine serum albumin (BSA) and lysozyme (LZ) on the formation and
antibacterial activity of AgNPs prepared by a photoreduction reaction
using Irgacure 2959 as a ketyl radical photoinitiator [20,21]. The for­
mation and specific protein interactions of the bionanocomposites
AgNP@BSA and AgNP@LZ, together with AgNP solutions stabilized
with citrate ions (AgNP@CIT), were monitored by various techniques
(UV–vis, fluorescence, FTIR, Raman, dynamic light scattering, zeta po­
tential, and transmission electron microscopy) [21], while the antibac­
terial activity of the different AgNPs was evaluated in Gram-positive and
Gram-negative bacteria using the zone of inhibition and minimum
inhibitory concentration methods [22].
The present results demonstrate that LZ, a recognized antibacterial
protein [23], induces the loss of the antibacterial efficacy of AgNPs, by
exerting intense specific interactions, either as a protein corona on the
silver nanocore of AgNP@LZ or by attaching to the coating layer of
AgNP@BSA or AgNP@CIT producing their agglomeration. This unan­
ticipated antagonistic antibacterial effect is associated with the strong
binding of the silver ions Ag+ to LZ, which dramatically reduces the
intrinsic toxic effect of AgNPs on the bacteria. To our knowledge, there
are still few studies on understanding the physicochemical aspects of
protein-metal/ion interfacial interactions that can modulate the bio­
logical activity of AgNPs [13,17]. In this sense, the present results may
help to design nanomaterials with greater antimicrobial efficacy in
biological media.
2. Experimental section
2.1. Materials and strains
Silver nitrate (AgNO3), sodium citrate dihydrate (C6H5Na3O7⋅2H2O),
sodium borohydride (NaBH4), 2-hydroxy-1-[4-(2-hydroxyethyl)
phenyl]-2-methyl-1-propanone (Irgacure®, I-2959), lysozyme (from
chicken egg white, approximately 58,100 units/mg, LZ) and bovine
serum albumin (≥98 %, BSA) of analytical grade were purchased from
Sigma-Aldrich Argentina and used without further treatment. The so­
lutions were prepared with ultra-pure water, provided from a MiliQ
system. Compressed ultrapure nitrogen (99.99 %) was purchased from
Indura Argentina. Staphylococcus (S.) aureus (ATCC 25923), Escherichia
(E.) coli O157H7, Serratia (S.) marcescens SmP9 and Klebsellia (Kb.)
pneunomiae CR1 strains were kindly supplied by CERELA-CONICET (S.
M. de Tucumán, Argentina). SmP9 and CR1 were isolated from clinical
samples and displayed multidrug-resistant (MDR) phenotypes [24].
Bacterial growth medium, Luria-Bertani (LB) medium, and antimicro­
bial sensitivity test were performed on Mueller-Hinton (MH) broth, and
agar was purchased from Laboratorios Britania, Argentina.
2.2. Synthesis of silver nanoparticles
Silver nanoparticles aqueous solutions stabilized with citrate
(AgNP@CIT), lysozyme (AgNP@LZ), or bovine serum albumin
(AgNP@BSA) were prepared by photoreduction of AgNO3 with I-2959
as previously reported [21], and is briefly described in section one of
Supplementary Material (SM1). AgNP solutions were stored at refrig­
erator temperature and their colloidal stability was confirmed by
monitoring the characteristic surface plasmon band (SPB) of AgNP be­
tween 300 and 700 nm by UV–vis spectroscopy as described below. In all
cases, almost no changes in absorbance of the SPB were observed after
one week of preparation of the solutions.
2.3. Spectroscopic measurements
UV–vis absorption spectra were registered using either an Ocean­
Optics USB2000 or Hewlett Packard 8453 spectrophotometers.
2
Colloids and Surfaces B: Biointerfaces 208 (2021) 112030
Fluorescence spectra were obtained with an Agilent Cary Eclipse
instrument.
Fourier-transform infrared (FTIR) spectra were acquired at room
temperature with a Jasco FTIR-4600 spectrometer equipped with the
ATR PRO ONE single reflection accessory under dry airflow to eliminate
water vapor distortions in the spectra. Four spectra for each sample were
averaged and processed with the Spectra Analysis™ software provided
by Jasco.
Raman spectra were recorded at room temperature with a Thermo
Scientific DXR Raman instrument equipped with a 532 nm CW laser of
10 mW as the excitation source. The samples were placed on gold-coated
sample slides and 100 acquisitions were accumulated with an exposure
time of 5 s for all samples to improve the signal-to-noise ratio.
2.4. AgNPs characterization
Hydrodynamic diameter (dh) and zeta-potential (ζ) of the different
AgNP solutions were determined with the Horiba SZ-100 particle size
analyzer using a 532 nm CW laser, focused at 173◦ relative to the
detection device. The data were analyzed with the Horiba NextGen
Proyect™ SZ-100 software.
Nanoparticle images were obtained with a Zeiss Libra 120 TEM at the
Integral Center for Electron Microscopy (CIME) of CONICET in
Tucumán, Argentina. A diluted aliquot of the sample solution was
evaporated on a carbon-coated copper mesh screen. Mean size with
standard deviation (SD) was calculated from at least four different re­
gions in randomized fields of view using the ImageJ™ free software.
2.5. Antibacterial activity
The viability tests of the strains mentioned above were performed
using the techniques recommended by the CLSI (https://clsi.org/s
tandards/). The agar diffusion method was used to detect the antimi­
crobial effect by placing a 50 μL aliquot of each sample in the well while
the minimum inhibitory concentration (MIC) was quantified using the
macrodilution technique [22].
2.6. Lysozyme (LZ) lytic activity assay
The lytic activity of LZ towards the cell walls of Micrococcus lyso­
deikticus (ATCC 4698) (Sigma-Aldrich) at different concentrations of
AgNO3 was monitored by the changes in the optical density (OD) of a
suspension of 0.15 mg.mL− 1 of the microorganism dispersed in ultra­
pure water.25
3. Results and discussion
3.1. Photochemical synthesis of AgNPs capped with BSA and LZ
Fig. 1 shows the UV–vis spectra of the surface plasmon band (SPB) of
AgNPs obtained after full reduction of Ag+ ions with ketyl radicals
generated by photo-fragmentation of the non-toxic benzoin I-2959 in the
presence of 1 mM citrate (AgNP@CIT), 10 μM LZ (AgNP@LZ), and 1 μM
BSA (AgNP@BSA), respectively. The concentration of each protein used
was minimal to obtain stable nano-colloidal solutions of AgNP@LZ [21]
or AgNP@BSA [26], respectively. In all cases, the position of the
maximum wavelength (λmax) of SPB was located at <410 nm, Table 1],
which suggests the formation of small spherical AgNPs with a core
diameter less than 10 nm [27]. Red-shift and broadening of the SPB of
AgNP@LZ and AgNP@BSA relative to that for AgNP@CIT are typical for
protein-stabilized AgNPs [15,16,28–33], due to dielectric changes pro­
duced by the formation of a protein corona that surrounds the metalcore
[12,14].
Table 1 also shows the hydrodynamic diameter (dh) and zeta po­
tential (ζ) values for the prepared AgNPs. For AgNP@CIT, dh = 3 ± 1 nm
was similar to dAgNP = 4 ± 1 nm as previously measured by TEM [21], as
M.B. Espeche Turbay et al.
Fig. 1. UV–vis absorption spectra of silver nanoparticle aqueous solutions
stabilized with 1 mM citrate (AgNP@CIT), 1 μM bovine serum albumin
(AgNP@BSA), and 10 μM lysozyme (AgNP@LZ) prepared by photochemical
reduction of 0.2 mM AgNO3 in the presence of 0.2 mM Igarcure 2959 (I-2959)
at room temperature. Inset: kinetic growth curves of the area under the curve
(AUC) for the formation of the surface plasmon band (SPB) of the AgNPs. Solid
black lines represent the non-linear fitting with the aggregation-growth or
KJMA model, Eq. (1).
can be expected for a nanoparticle capped with a small molecule. On the
other hand, for AgNP@LZ and AgNP@BSA, an average dh = 13.5 ± 4.0
nm was obtained, in agreement with the values previously reported for
these nanoparticles [21,31]. Then by comparison with the TEM di­
ameters [31,33], the thickness of the protein corona (rcorona) was esti­
mated ≈ 3.5 nm and 4.5 nm for AgNP@LZ and AgNP@BSA solutions,
respectively. These rcorona values can be expected for a monolayer of LZ
corresponding to an elongated ellipsoid of 3 × 3 × 4.5 nm [34], and for a
monolayer of BSA with an equilateral triangular prism shape of 8.4 × 8.4
× 8.4 × 3.2 nm [35], respectively. Therefore, the diameter of the
spherical metal core can be approximated to dAgNP = dh –2×rcorona, that
is, ≈5 nm and ≈6 nm for AgNP@BSA and AgNP@LZ, respectively. These
dAgNP values were used to calculate the AgNP concentration of each
solution as explained in the supplementary material (section SM2),
assuming the complete conversion of the precursors according to the
plateau observed in the growth kinetics of the photoreduction reaction
(inset of Fig. 1). The calculated concentrations are reported in Table 1
and were of the same order of magnitude as those calculated using the
Lambert-Beer law with a molar extinction coefficient of ≈2 × 108 M− 1
cm− 1 for AgNP@CIT with diameters <10 nm, as reported by Paramelle
et al. [27].
The ζ values of the photochemically synthesized AgNPs were similar
to those previously reported for each nanocolloid prepared by different
methods [31,32]. The large negative value ζ = − 45 mV for AgNP@CIT is
associated with the complete ionization of the citrate carboxylic groups
at neutral pH. The different ζ values observed for AgNP@BSA (− 19 mV)
and AgNP@LZ (+60 mV), confirm the formation of a protein corona
around the metal core that stabilizes the nanocolloid [12,14]. The sign
change of the zeta potential between AgNP@LZ and AgNP@BSA is
consistent with the net surface charges of each native protein at neutral
pH, due to isoelectric points of 11 and 4.8 for LZ and BSA, respectively
[36].
Table 1
Some properties of AgNP solutions obtained by UVA photolysis of aqueous solutions of 0.2 mM AgNO3 and 0.2 mM I-2959 in presence of citrate (CIT), lysozyme (LZ),
and bovine serum albumin (BSA) as capping agents. Check the text for the meaning of abbreviations.
Capping agent λmax (nm) Fwhm (nm) dh (nm) dAgNP (nm)
a
1 mM CIT 390 ± 1 63 ± 1 3±1 4±1
1 μM BSA 409 ± 1 76 ± 1 14 ± 4 5±2
10 μM LZ 409 ± 1 73 ± 1 13 ± 4 6±2
a
From TEM measurements [21].
3
Colloids and Surfaces B: Biointerfaces 208 (2021) 112030
The slower growth kinetics for the photoreduction reaction in the
presence of proteins than with citrate also provides evidence of the
formation of a protein corona around the AgNPs (inset of Fig. 1). The
sigmoidal kinetic curves can be interpreted according to the
Kolmogorov-Johnson-Mehl-Avrami (KJMA) or aggregate growth model,
Eq. (1), where kg is the first-order rate constant of particle growth and
nAv is the Avrami exponent [37].
[ ( )n ]
AUCt = AUC∞ 1 − exp − kg t Av (1)
In general, kg is strongly associated with the nanoparticle growth rate
rather than nucleation rate, while nAv is related to the nucleation
mechanism and shape of the growing nanoparticle [37]. The calculated
nAv values are shown in Table 1 and are in the typical range expected for
radical-mediated growth of spherical metal nanoparticles [21,37]. On
the other hand, the kg value for AgNP@CIT was almost two and four
times higher than for AgNP@BSA and AgNP@LZ, respectively, indi­
cating the role of the protein corona as a steric barrier for the growth
pathway. Interestingly, the kg value for AgNP@BSA was higher than for
AgNP@LZ, although BSA is almost five times as bulky as LZ. Previously,
we have shown that kg was independent of the initial concentration of LZ
used for the photochemical synthesis [21], and therefore, the larger kg
value in the formation of AgNP@BSA may suggest that BSA has a more
flexible structure than LZ, favoring its adsorption on the metal nanocore.
3.2. Lysozyme effect on the antibacterial activity of the AgNPs
Table 2 shows the diameters of the zone of inhibition (mm) observed
after overnight growth at 37 ◦ C of the bacterial strains containing 50 μL
aliquot of AgNP or control solutions, as shown in the inset of Fig. 2a.
Except for Kb. pneunomiae, which is a strain intrinsically resistant to the
toxic activity of Ag+ ions [38], both AgNP@CIT and AgNP@BSA solu­
tions showed efficient antibacterial activity against the rest of the
strains, and similar to that obtained with a 0.2 mM AgNO3 solution
taken as the positive control for the toxic effect of Ag+ ions [7,39].
Interestingly, AgNP@LZ solutions prepared with 1.4 μM or 10 μM LZ
were completely ineffective in inhibiting the growth of all bacteria, even
though a 10 μM LZ solution was able to partially prevent the growth of
S. aureus. On the other hand, aliquots of citrate, I-2959, or BSA solutions
added at the same concentrations used for the preparation of AgNP so­
lutions did not show antibacterial activity, Table 2.
Taking into account that at neutral pH both Gram-positive and Gram-
negative bacteria show negative ζ values [36,40], the toxic effect of
AgNP@CIT and AgNP@BSA, both with negative surface charge, may be
associated with the release of Ag+ ions from the silver nanocore, which
in aerobic conditions is favored by the dissolution of Ag2O formed on the
surface of the nanoparticle [7,41]. To confirm this mechanism of bac­
terial death, the inhibition zone produced by the different AgNPs on
S. aureus was analyzed as a function of the NaCl concentration, Fig. 2a.
The results indicated that at ~100 μM NaCl the antibacterial activity of
both AgNP@CIT and AgNP@BSA was lost, while for AgNP@LZ the in­
crease in ionic strength did not modify the initial absence of activity. It is
noteworthy that this threshold value for the complete loss of antibac­
terial activity was coincident with the minimal inhibitory concentration
(MIC = 100 μM) elicited by AgNO3 on both E. coli and S. aureus (Table S1
of SM).
It is well-known that chloride ions (Cl–) induce dissolution and
3
[AgNP] (nM) ζ (mV) kg/10− (s− 1) nAv
10.2 ± 1.5 –45 ± 5 11.0 ± 3.0 2.1 ± 0.2
5.2 ± 0.9 –19 ± 2 5.0 ± 0.1 1.1 ± 0.1
3.0 ± 0.5 +60 ± 1 2.9 ± 0.1 1.4 ± 0.1
M.B. Espeche Turbay et al. Colloids and Surfaces B: Biointerfaces 208 (2021) 112030

Table 2
Inhibition zone (mm)a for the inactivation of Gram-positive and Gram-negative bacteria produced by the addition of 50 μL of AgNP solutions stabilized with citrate
(CIT), lysozyme (LZ), or bovine serum albumin (BSA), and by different control solutions. [AgNPs] are those indicated in Table 1.
AgNP solution Control solution

Strains AgNP@CIT AgNP@BSA AgNP@LZb AgNP@CIT +10 AgNP@BSA +10 0.2 mM 0.2 mM I- 1 mM 10 МM 1 μM
МM LZ МM LZ AgNO3 2959 CIT LZ BSA

S. aureus 8.5 9.0 n.d. n.d. n.d. 9.0 n.d. n.d. 6.0 n.d.
E. coli 7.0 10.0 “ “ “ 7.0 “ “ n.d. “
S. marcescens 9.0 10.0 “ “ “ 8.0 “ “ “ “
K. pneumoniae n.d.c n.d. “ “ “ n.d. “ “ “ “
a
Relative standard deviation ±6 %.
b
Prepared either with 1.4 μM or 10 μM lysozyme solutions.
c
n.d. = not detected.

aggregation phenomena of AgNPs by stoichiometric reaction with the

released Ag+ to form insoluble AgCl(s) with a Ksp = 1.8 × 10− 11 [42].
This effect can be observed by the spectral changes of the SPB of the
AgNPs as a function of the NaCl concentration, as is shown for
AgNP@CIT in Fig. 2b. Each spectrum was recorded after 10 min of
addition of the NaCl aliquot, ensuring a constant change in the absor­
bance of the SPB. The strong decrease in SPB absorption together with
the increase in the spectral baseline due to the light scattering effect
confirms the dissolution of AgNP@CIT with the formation of insoluble
colloidal particles which is reflected in the color discoloration of the
solution (inset of Fig. 2b).
Comparison of the relative values of the area under the curve (AUC)
of the PBS of the AgNPs as a function of the NaCl concentrations in­
dicates that the saline effect was greater for AgNP@CIT, milder for
AgNP@BSA, while it was almost absent for AgNP@LZ, Fig. 2c. Consid­
ering the longer incubation time used for the antibacterial experiments
than for the spectroscopic ones, to confirm the differential release of
Ag+, the absorbance changes in the SPB of the aqueous solutions of the
different AgNPs were monitored during 44 h of incubation at 37 ◦ C,
observing redshifts of the SPB maximum of 22 nm, 5 nm and 0.8 nm for
AgNP@CIT, AgNP@BSA, and AgNP@LZ, respectively. These spectral
changes are produced by the adsorption of dissolved Ag+ on the surface
of AgNPs, which amount is proportional to the concentration of Ag+
released by the nanoparticle, and the concentration of adsorbed ions
[Ag+]ad can be estimated by Eq. (2) [43].
[( ) ]
2
λ
+
[Ag ]ad = [Ag]0 − 1 (2)
λ0

λ0 and λ are the absorption wavelength of the maximum SPB observed at

Fig. 2. a) Effect of NaCl concentration on the inhibition diameter for S. aureus
elicited by adding 50 μL of AgNP@CIT, AgNP@BSA, and AgNP@LZ solutions.
The arrow indicates the minimal inhibition concentration (MIC) of AgNO3 for
S. aureus. Inset: Agar well diffusion test for S. aureus in the absence and presence
of NaCl as a function of increasing amounts (μL) of AgNP@CIT. b) NaCl effect
on the surface plasmon band (SPB) of AgNP@CIT solution. Inset: color fading
produced in the AgNP@CIT solutions by NaCl. c) Variation of the relative area
under the curve (AUC) of the SPB of different AgNPs solutions with the con­
centration of NaCl.
the initial and final incubation times, respectively, while [Ag]0 repre­
sents the initial silver concentration in the colloidal solution, which is
assumed to be the same as that of the precursor (0.2 mM AgNO3). Thus,
[Ag+]ad was estimated as 23.2 μM for AgNP@CIT, 4.9 μM for
AgNP@BSA, and 0.8 μM for AgNP@LZ, confirming the trend in Ag+
release capacity as AgNP@CIT > AgNP@BSA>> AgNP@LZ.
These results confirm that the antibacterial mechanism of AgNP@­
CIT and AgNP@BSA is mainly due to the slow release of Ag+ in the
culture media, where both types of AgNP act as reservoirs of silver ions
[7], while the supply of Ag+ in AgNP@LZ is blocked by its protein
corona. A blocking effect for the release of Ag+ ions has been also re­
ported for metallic silver surfaces with adsorbed LZ at a concentration
greater than ≈70 μM, and was explained by the association of the silver
ions in the adsorbed protein layer together with electrostatic repulsion
effects on the transport of Ag+ through the positively charged pro­
tein/solution interface preventing their exit into the bulk solution [44].
Table 2 also indicates that the addition of 10 μM of LZ to the
AgNP@CIT or AgNP@BSA solutions resulted in the total loss of the
original antibacterial activity of both types of nanoparticles. In contrast,
the addition of 1 μM BSA to AgNP@CIT or AgNP@LZ solutions did not
modify the antimicrobial activity of each nanoparticle solution.

4
M.B. Espeche Turbay et al.
Furthermore, the loss of the antimicrobial effect induced by LZ to the
AgNP@CIT and AgNP@BSA solutions was parallel to the spectral
modifications of the SPB observed for each nanoparticle, as shown in
Fig. 3a for AgNP@CIT.
As the LZ concentration increased, a continuous broadening and
redshift occurred along with a reduction in the absorbance of the SPB.
These spectral changes are parallel to a strong decrease of the negative ξ
value of the colloidal solution (inset of Fig. 3a). In contrast, the addition
of BSA to the AgNP@CIT solution almost did not modify either the SPB
spectrum or ξ value (Fig. 3b).
Moreover, a different effect on the dh of AgNP@CIT was produced by
the addition of LZ or BSA (Fig. 3c). BSA only slightly increased the
particle dispersibility of the AgNP@CIT solution, probably due to the
presence of small aggregates of protein-AgNP@CIT and/or protein di­
mers, considering dh ≈ 7 nm for monomeric BSA [45].
Fig. 3. a,b) UV–vis spectral changes produced in 10.2 nM AgNP@CIT solution
upon addition of increasing amounts of native LZ or BSA, respectively. Insets:
Variation of the zeta potential (ξ) with the protein concentration. c) Hydro­
dynamic diameter (dh) of AgNP@CIT solutions in the absence and presence of 1
μM BSA or 10 μM LZ, respectively. Inset: TEM image of AgNP@CIT obtained
after the addition of 10 μM LZ.
5
Colloids and Surfaces B: Biointerfaces 208 (2021) 112030
Instead, the TEM image inserted in Fig. 3c confirms that the addition
of LZ to AgNP@CIT solution produces large aggregates of dh ≈1 μm, due
to the agglomeration of small individual AgNPs rather than Ostwald’s
nucleation-coalescence process to produce larger particles [37]. The
agglomeration effect of AgNP is driven by the strong reduction of ξ value
for negatively charged nanoparticles by positively charged LZ molecules
(ξ ≈+7 mV at neutral pH) [46], as shown in the inset of Fig. 3a for
AgNP@CIT. Therefore, LZ acts as an “electrostatic glue” of negatively
charged nanoparticles.
3.3. About the antagonistic antibacterial effect exerted by LZ
Taking into account the intrinsic antibacterial function of AgNPs [7]
and lysozyme [23], respectively, the present results suggest a surprising
antagonistic antibacterial effect exerted by LZ in solutions of negatively
charged AgNPs. An antagonistic effect occurs between two or more
molecules when the functionality of the mixture is weaker or absent than
that given by the sum of the individual contributions [47,48].
In the present case, the antagonistic antibacterial effect may be
associated with strong specific interactions of LZ with silver, which may
affect the release of the toxic species Ag+ into the medium. To explore
this possibility, Fig. 4 compares the normalized infrared absorption of
the Amide I band (1700− 1600 cm− 1) of each native protein with that for
AgNP solution stabilized with the same amount of protein. This band is
typically used to monitor changes in protein secondary structure, and for
typical globular proteins like LZ and BSA, the Amide I band shows a
narrow Gaussian shape with a maximum at 1658 cm-1 that is charac­
teristic of a predominant α-helix protein conformation [49,50]. In
contrast, for the AgNP@BSA and AgNP@LZ solutions, the Amide I band
of LZ was distorted showing an increase in absorbance around 1601
cm-1, 1625 cm-1. and in the region between 1675 and 1725 cm -1, as
shown in each differential spectrum of Fig. 4. These modifications are
evidence of a relative increment in the β-sheet content (1625 cm-1) and
of extended chains (1675− 1725 cm-1) as the result of a partial dena­
turation of the protein after the formation of the protein corona adsor­
bed on the silver nanocore [49–51].
Comparison of the Raman spectral changes of each native protein
and its AgNP@protein solution provides information on the strength of
the protein-metal core interaction, Fig. 5a and b. Native proteins showed
relatively low-intensity Raman spectra with a maximum at 1640 cm− 1
Fig. 4. a, b) Normalized ATR-FTIR spectra of the native protein (10 μM LZ and
20 μM BSA) and of the respective AgNP@Protein colloidal solution, together
with the calculated differential absorption spectra (ΔA = AAgNP – AProtein).
M.B. Espeche Turbay et al.
due to the mixing of the characteristic Amide I band (1660 cm− 1) and
the vibratory stretching of aromatic amino acids (Trp/Tyr) around 1615
cm− 1, together with a shoulder at 1400 cm− 1 associated with the sym­
metric stretching of carboxylic residues (COO–). Moreover, the weakest
band at 815 cm− 1 indicates the hydrogen bonding interactions of aro­
matic residues with water [52,53].
On the other hand, in both AgNP@protein solutions, the typical ef­
fect of surface-enhanced Raman scattering (SERS) was observed in the
spectrum of each protein induced by contact with the silver nanocore,
increasing the Raman intensity by several orders of magnitude [53].
Additionally, a new band at 235 cm− 1 due to the Ag-N(Protein) vibra­
tory mode was observed (insets of Fig. 5a and b), together with the
relative decrease of the band at 815 cm− 1 due to the breakdown of the
hydrogen-bonding network between aromatic residues and water mol­
ecules, suggesting that these residues interact with the metal surface
[53,54].
The SERS effect in the 1700− 1100 cm− 1 region was almost 30-times
more intense in AgNP@LZ than for AgNP@BSA solutions, confirming a
much stronger interaction of LZ than BSA with the silver nanocore and,
Fig. 5. a, b) Raman spectra of the native protein (10 μM LZ and 20 μM BSA)
and of the respective AgNP@Protein colloidal solution; and c) of 10 μM LZ in
the absence and presence of 200 μM AgNO3, respectively. Note the difference of
intensity scale due to the SERS effect. Insets: SERS band of the Ag–N(Protein)
vibrational mode around 235 cm− 1.
6
Colloids and Surfaces B: Biointerfaces 208 (2021) 112030
therefore, LZ can produce a harder protein corona on AgNPs than BSA.
Ultracentrifugation experiments for LZ and AgNP@LZ solutions using a
20 kDa cut-off filter combined with absorption and fluorescence detec­
tion confirmed that only the solution of native LZ passed completely
through the cut-off filter, but no free protein was collected for the
AgNP@LZ solution (Fig. S2 of SM). Taken together, these results
strongly suggest that the LZ molecules are tightly attached to the AgNPs.
The accepted model for the formation of protein corona on NPs in­
volves a dynamic process in which free protein molecules in solution are
constantly exchanged with those adsorbed on the surface of the NPs
[19]. Therefore, proteins with a high attraction for the nanoparticle
surface will form a hard corona with tightly bound proteins in the
innermost layer, and with a slow protein exchange lifetime of several
hours. On the other hand, proteins with lower affinity will form a softer
corona, with faster protein exchange rates occurring in a few seconds to
minutes [12,14,19,51].
Therefore, different protein corona dynamics can be expected for
AgNP@LZ and AgNP@BSA solutions. In the first case, the release of Ag+
from the inner core of AgNP is blocked by the strongly adsorbed LZ
corona, with very slow molecular exchange, preventing the toxic effect
during the incubation of the strains. In the case of AgNP@BSA, the
formation of a softer protein corona with faster molecular exchange rate
is anticipated, allowing the oxidation of silver on the AgNP surface and
subsequent release of Ag+ in the medium, which eventually induce
bacterial death.
The Raman spectrum of Fig. 5c indicates that also native LZ strongly
interacts in the presence of a molar excess of dissoved Ag+, since a
similar spectral fingerprint to that observed for AgNP@LZ was observed
[54], although the intensity enhancement is almost 10-times lower.
Nevertheless, the intense band at 240 cm− 1 attributed to the Ag+
interaction with the N-atom of the imidazole ring of residue His15 of LZ
was observed [55], with an association constant Ka = 1.9 μM− 1 deter­
mined by isothermal titration calorimetry [56]. Instead, the binding of
Ag+ to BSA is much weaker, since a Ka = 0.004 μM− 1 was determined by
BSA fluorescence quenching by AgNO3 measurements (Fig. S3 of SM).
As a consequence of the strong Ag+–LZ interaction, the lytic activity
of native LZ decreased with increasing AgNO3 concentration (Fig. S4 of
SM), in a similar way to that observed by Wu et al. [56]. This result
confirms that the binding of Ag+ ions modifies the conformational
structure at the specific site for LZ lytic activity, in particular, the Glu35
and Asp52 residues which are known to be directly responsible for
enzyme action cleavage of glycosidic bond in the alternating β-linked
(1-4) copolymer of the N-acetyl-D-muramic acid. [25,55].
4. Conclusions
In this work, we have demonstrated the suitability of the photore­
duction method of Ag+ ions using I-2959 as the photoiniator for pre­
paring thermodynamically stable AgNP solutions in the presence of
citrate or proteins (LZ and BSA) as stabilizers, as shown in Scheme 1.
The negatively charged AgNP@CIT and AgNP@BSA showed efficient
antibacterial activity, mainly due to their capability to act as an nano-
reservoir for the release of the toxic Ag+ ions. However, the prepared
AgNP@LZ was completely ineffective for bacterial inactivation, despite
the intrinsic antimicrobial function of LZ [23]. In addition, the presence
of native LZ in both AgNP@CIT and AgNP@BSA resulted in the com­
plete loss of the original antibacterial activity of these nanoparticles.
This antagonistic antibacterial effect exerted by positively charged
LZ is modulated by the strength of specific silver-lysozyme interactions,
resulting in the formation of a harder protein corona in AgNP@LZ, while
inducing electrostatic-mediated agglomeration of both AgNP@CIT and
AgNP@BSA, along with the strong association of free Ag+. These effects
decrease the viability of Ag+ ions to kill bacteria, Scheme 1.
These results were also in opposition to previous reports in which an
antibacterial effect of AgNP@LZ was observed [32,57]. However, in
these cases, the bionanocomposite was chemically synthesized under
M.B. Espeche Turbay et al.
strong protein denaturation conditions, such as using methanol as a
solvent [32], or highly alkaline media [57]. Therefore, it can be spec­
ulated that the synthesis conditions and the presence of other chemical
agents in the preparation of AgNPs can modify the strength of the
silver-protein interactions and, later, the antibacterial efficacy of the
biocomposite.
In conclusion, the present results point to the relevant role of the
specific interactions of AgNPs with proteins for the fate of their bio­
logical function, which may result in unexpected behaviors, such as the
antagonistic antibacterial effect that is reported here.
CRediT authorship contribution statement
M. Beatriz Espeche Turbay: Validation, Investigation, Writing -
Review & Editing. Valentina Rey: Validation, Investigation. Rita D.
Dorado: Validation, Investigation. Marcelo C. Sosa: Validation,
Investigation. Claudio D. Borsarelli: Conceptualization, Resources,
Writing - Review & Editing, Visualization, Supervision, Project admin­
istration, Funding acquisition.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
The authors thanks for the financial support to the following
Argentinean Institutions: Universidad Nacional de Santiago del Estero
(UNSE, grant #23A/254), Consejo Nacional de Investigaciones Científ­
icas y Tecnológicas (CONICET, grant #PUE-2018-035) and Fondo para
la Investigación Científica y Tecnológica (FONCyT grant #PICT-2019-
02052).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.colsurfb.2021.112030.
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7
Colloids and Surfaces B: Biointerfaces 208 (2021) 112030
Scheme 1. Pathways related to the antago­
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