Biological Performances of Plasmonic Biohybrids Based on Phyto-Silver/Silver Chloride Nanoparticles

Abstract

Silver/silver chloride nanoparticles (Ag/AgClNPs), with a mean size of 48.2 ± 9.5 nm and a zeta potential value of −31.1 ± 1.9 mV, obtained by the Green Chemistry approach from a mixture of nettle and grape extracts, were used as “building blocks” for the “green” development of plasmonic biohybrids containing biomimetic membranes and chitosan. The mechanism of biohybrid formation was elucidated by optical analyses (UV–vis absorption and emission fluorescence, FTIR, XRD, and SAXS) and microscopic techniques (AFM and SEM). The aforementioned novel materials showed a free radical scavenging capacity of 75% and excellent antimicrobial properties against Escherichia coli (IGZ = 45 mm) and Staphylococcus aureus (IGZ = 35 mm). The antiproliferative activity of biohybrids was highlighted by a therapeutic index value of 1.30 for HT-29 cancer cells and 1.77 for HepG2 cancer cells. At concentrations below 102.2 µM, these materials are not hemolytic, so they will not be harmful when found in the bloodstream. In conclusion, hybrid systems based on phyto-Ag/AgClNPs, artificial cell membranes, and chitosan can be considered potential adjuvants in liver and colorectal cancer treatment.

Full text

nanomaterials
Article
Biological Performances of Plasmonic Biohybrids Based on
Phyto-Silver/Silver Chloride Nanoparticles
Yulia Gorshkova 1,2 , Marcela-Elisabeta Barbinta-Patrascu 3,* , Gizo Bokuchava 1, Nicoleta Badea 4,
Camelia Ungureanu 4, Andrada Lazea-Stoyanova 5, Mina Răileanu 3,6, Mihaela Bacalum 6,
Vitaly Turchenko 1,7 , Alexander Zhigunov 8and Ewa Juszy ´nska-Gał ˛azka 9


Citation: Gorshkova, Y.;
Barbinta-Patrascu, M.-E.; Bokuchava,
G.; Badea, N.; Ungureanu, C.;
Lazea-Stoyanova, A.; R˘aileanu, M.;
Bacalum, M.; Turchenko, V.;
Zhigunov, A.; et al. Biological
Performances of Plasmonic
Biohybrids Based on
Phyto-Silver/Silver Chloride
Nanoparticles. Nanomaterials 2021,11,
1811. https://doi.org/10.3390/
nano11071811
Academic Editors: Henrich
Frielinghaus and Lyudmila
M. Bronstein
Received: 28 May 2021
Accepted: 8 July 2021
Published: 12 July 2021
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with regard to jurisdictional claims in
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iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Joint Institute for Nuclear Research, Frank Laboratory of Neutron Physics, Joliot-Curie, 6,
141980 Dubna, Russia
; Yulia.Gorshkova@jinr.ru (Y.G.); gizo.bokuchava@jinr.ru (G.B.); turchenko@jinr.ru (V.T.)
2Institute of Physics, Kazan Federal University, 16a Kremlyovskaya Street, 420008 Kazan, Russia
3Department of Electricity, Solid-State Physics and Biophysics, Faculty of Physics, University of Bucharest,
405 Atomistilor Street, P.O. Box MG-11, 077125 Magurele, Romania; raileanumina@gmail.com
4General Chemistry Department, Faculty of Applied Chemistry and Materials Science,
University “Politehnica” of Bucharest, 1-7, Polizu Street, 011061 Bucharest, Romania;
nicoleta.badea@gmail.com (N.B.); ungureanucamelia@gmail.com (C.U.)
5National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street,
077125 Magurele, Romania; andrada@infim.ro
6Department of Life and Environmental Physics, Institute for Physics and Nuclear Engineering,
Horia Hulubei National, Reactorului, 30, 077125 Magurele, Romania; bmihaela@nipne.ro
7Department of Crystal Growth Laboratory, South Ural State University, 76, Lenin Aven.,
454080 Chelyabinsk, Russia
8Institute of Macromolecular Chemistry AS CR, Heyrovskeho nam. 2, 162 06 Prague 6, Czech Republic;
zhigunov@imc.cas.cz
9Institute of Nuclear Physics, Polish Academy of Sciences, ul. Radzikowskiego 152, 31-342 Krakow, Poland;
ewa.juszynska-galazka@ifj.edu.pl
*Correspondence: marcela.barbinta@unibuc.ro
Abstract:
Silver/silver chloride nanoparticles (Ag/AgClNPs), with a mean size of 48.2
±
9.5 nm
and a zeta potential value of
−
31.1
±
1.9 mV, obtained by the Green Chemistry approach from a
mixture of nettle and grape extracts, were used as “building blocks” for the “green” development of
plasmonic biohybrids containing biomimetic membranes and chitosan. The mechanism of biohybrid
formation was elucidated by optical analyses (UV–vis absorption and emission fluorescence, FTIR,
XRD, and SAXS) and microscopic techniques (AFM and SEM). The aforementioned novel materials
showed a free radical scavenging capacity of 75% and excellent antimicrobial properties against
Escherichia coli (IGZ = 45 mm) and Staphylococcus aureus (IGZ = 35 mm). The antiproliferative activity
of biohybrids was highlighted by a therapeutic index value of 1.30 for HT-29 cancer cells and 1.77
for HepG2 cancer cells. At concentrations below 102.2
µ
M, these materials are not hemolytic, so
they will not be harmful when found in the bloodstream. In conclusion, hybrid systems based on
phyto-Ag/AgClNPs, artificial cell membranes, and chitosan can be considered potential adjuvants in
liver and colorectal cancer treatment.
Keywords:
silver/silver chloride nanoparticles; “green” synthesis; chlorophyll-a-labeled bio-inspired
membranes; biohybrids; antioxidant, antibacterial, and antiproliferative activities
1. Introduction
Nowadays, the use of natural resources and, in particular, plants has experienced a
great importance, thanks to the impressive number of bioactive ingredients with a strong
beneficial impact on human health. The intensive development of new hybrid lipid–
nanoparticle complexes, including inorganic nanoparticles (NPs), lipid assemblies, and
lipid–polymer complexes, is caused by numerous potential biomedical applications [
1
,
2
].
Soy lecithin liposomes represent a promising drug delivery system, especially in the
Nanomaterials 2021,11, 1811. https://doi.org/10.3390/nano11071811 https://www.mdpi.com/journal/nanomaterials

Nanomaterials 2021,11, 1811 2 of 25
fight against serious illnesses such as tuberculosis and malaria [
3
]. Modification of the
surface of liposomes changes their physicochemical and biophysical properties that are
able to overcome the limitations of conventional liposomes. An important requirement
for the development of modified liposomes for drug delivery is the stability of such
systems, excluding their premature fusion with each other, on the one hand, and their
activation upon reaching a target such as viruses or bacteria, on the other. Polymers play
an important role in stabilizing liposomes. An alternative method to develop hybrid
nanomaterials for biomedical and pharmaceutical applications is the modification of the
liposomes using the natural-derived polymer chitosan (CTS), which is recognized as a
universal biomaterial because of its nontoxicity, low allergenicity, biocompatibility, and
biodegradability [
4
]. Chitosan is a chitin-derived polymer with a linear and semi-crystalline
structure bearing three functional groups on its main backbone: one amino (NH
2
) group
and two hydroxyl (OH) groups [
5
]. This positively charged polysaccharide [
6
] was used to
develop chitosan–silver composites [
7
] for wound-dressing applications [
8
] due to its low
cytotoxicity and high antimicrobial properties. Additionally, some successful results with
chitosan-coated lecithin liposomes and phytosomes were reported by the research teams
of Filipovi´c-Grci´c [
9
] and Barbinta-Patrascu [
10
], respectively. An alternate strategy is the
stabilization of the liposomes via surface-bound inorganic metal nanoparticles, for example
with silver nanoparticles (AgNPs) [
11
]. AgNPs have the advantage of possessing a great
antibacterial effect [
12
]. Other authors reported the improved stability, compatibility, and
antibacterial properties of one-step synthesis of AgNPs-stabilized liposomes compared to
AgNPs alone [13].
In recent decades, “green” syntheses of AgNPs from plant extracts have also been
developed to reduce the toxicity of solvents and stabilized agents. Generally, the synthesis
of nanoparticles from plant extracts has the advantage of being a low-cost, environmen-
tally friendly, rapid, and facile method. Phytofabricated eco-friendly silver nanoparticles
showed higher bactericidal, antioxidant, and anti-inflammatory activities [
14
]. In addition,
several authors reported the fabrication of silver chloride nanoparticles (AgClNPs) or
hybrid Ag/AgClNPs from plant extracts [
15
–
21
]. Such Ag/AgCl plasmonic hybrids have
antibacterial properties with low cytotoxicity, which may be attributed to the solubility
equilibrium of Ag
+
controlled by AgCl that allows for a low Ag
+
level to be released into
the environment [
18
,
19
]. Silver/silver chloride nanoparticles were also synthesized by
Kota et al. [
21
] by using aqueous leaf extract of Rumex acetosa; these nanoparticles demon-
strated good antimicrobial and antioxidant properties, as well as cytotoxicity against the
tested human osteosarcoma cell lines.
The challenge of our study is to demonstrate that silver/silver chloride nanoparticles
fabricated according to a “green” protocol, as well as the biohybrids based on Ag/AgClNPs,
have a high potential for biomedicine. The aim of this study was to improve the antimicro-
bial, antioxidant, and anticancer properties of Ag/AgClNPs, in combination with artificial
cell membranes, and chitosan. In this work, an aqueous extract of a mixture of nettles
and grapes to create hybrid nanoparticles was used. Three types of silver-based biohy-
brids with and without chitosan were produced. These nettle and grape extracts contain
biocompounds with excellent biological value (such as antioxidant and antibacterial proper-
ties) [
22
]. Physicochemical properties of the obtained phyto-based materials were evaluated
through optical (UV–vis, fluorescence emission, and FTIR spectroscopy), structural (XRD
and SAXS), and microscopic (AFM and SEM) investigations. The physical stability of the
suspensions of the developed materials was estimated through zeta potential measure-
ments. Moreover, the antioxidant, antibacterial, and antiproliferative activities were tested
to assess the bioperformances of our biodeveloped materials.
2. Materials and Methods
2.1. Materials
Tris (hydroxymethylaminomethane base), HCl, H
2
O
2
, luminol (5-amino-2,3-dihydro-
phthalazine-1,4-dione), silver nitrate (AgNO
3
), KH
2
PO
4
, and Na
2
HPO
4
were supplied

Nanomaterials 2021,11, 1811 3 of 25
from Merck (Darmstadt, Germany). Standard hemoglobin, soybean lecithin, NaCl, Drabkin
reagent, ethidium homodimer-1 (EthD-1), and acridine orange (AO) were purchased from
Sigma-Aldrich (Darmstadt, Germany). The yeast extract was supplied from BioLife, and
the agar was obtained from Fluka (Switzerland). Chlorophyll awas extracted in our
laboratory from fresh spinach leaves according to the method of Strain and Svec [
23
]. The
nettle leaves and the grapes were acquired from a local market.
Bacteria culture test
. Antimicrobial activity of the samples was tested against pathogenic
Gram (-) bacteria Escherichia coli ATCC 8738 and Gram (+) bacteria Staphylococcus aureus ATTC
25923. These bacterial cultures were maintained at 4
◦
C. All chemicals used for antibacterial
investigations were purchased from VWR (Darmstadt, Germany).
Cell culture and reagents
. Three different cell lines were used for the
in vitro
studies.
Human fibroblast BJ cells (ATCC CRL-2522, Manassas, VA, USA) and human colorectal
adenocarcinoma HT-29 cells (ATCC, Manassas, VA, USA) were grown in Minimal Essential
Medium (MEM) supplemented with 2 mM L-Glutamine, 10% fetal calf serum (FCS),
100 units/mL of penicillin, and 100
µ
g/mL of streptomycin at 37
◦
C in a humidified
incubator under an atmosphere containing 5% CO
2
. Human hepatocarcinoma HepG2
cells (ATCC, Manassas, VA, USA) were grown in DMEM supplemented with similar
reagents. All cell cultivation media and reagents were purchased from Biochrom AG
(Berlin, Germany). Drabkin reagent and standard hemoglobin were purchased from Sigma-
Aldrich (Darmstadt, Germany).
2.2. Preparation of Nanosilver-Based Biohybrids
2.2.1. Phytogeneration of Silver/Silver Chloride Nanoparticles
Two types of aqueous extracts of nettle leaves and grapes were prepared as described
in [
22
]. A mixture of (0.24 mL of nettle extract + 0.06 mL of grape extract) was added to a
volume of 30 mL of 1 mM AgNO
3
aqueous solution, under continuous magnetic stirring,
and then placed at room temperature for one day. This suspension was diluted 1.62 times
with phosphate-buffered saline (PBS, KH
2
PO
4
/Na
2
HPO
4
/NaCl, pH 7.4) and furthermore
subjected to an ultrasound treatment in an ultrasonic bath (BRANSON 1210, Marshall
Scientific, Hampton, NH, USA) for 30 min. The resulting Ag/AgClNPs were diluted in the
biodispersant PBS to a final silver content of 0.61 mM.
2.2.2. Preparation of Artificial Cell Membranes
Artificial cell membranes labeled with Chlawere obtained by the hydration of soybean
lecithin thin as previously described [
24
]. The resulting liposome suspensions were divided
into two parts: without and with chitosan. An acidic solution (0.4% acetic acid v/vin
distilled water) of 1% (w/v) chitosan (CTS) was added to a liposomal suspension, to a
final CTS concentration of 0.01% (w/v); this mixture was then strongly stirred for 15 min
(200 rpm, VIBRAX stirrer, Milian, OH, USA). These two types of biological entities were
further diluted in PBS to a final lipid concentration of 0.34 mg/mL.
2.2.3. Bottom-up “Green” Design of Plasmonic Biohybrids
Three types of hybrid silver-based systems were prepared based on suspensions of
Ag/AgClNPs and liposomes, with and without chitosan solution, according to Table 1
(see Samples P4–P6) by using strong stirring (VIBRAX stirrer, Milian, OH USA 200 rpm)
for 15 min and ultrasound treatment in an ultrasonic bath (BRANSON 1210, Marshall
Scientific, Hampton, NH, USA) for 30 min.
All these biohybrids were diluted in PBS to a final lipid concentration of 0.34 mg/mL
and with a final silver content of 0.61 mM.
Figure 1shows a diagram representing the “green” development of biohybrids gener-
ated from the aqueous extract of a mixture of nettle and grape extracts.

Nanomaterials 2021,11, 1811 4 of 25
Table 1. Codes and descriptions of the samples.
Sample Code Description CLiposomes
(mg/mL)
CAg
(mM)
CCTS
(% w/v)
P1 Liposomes 0.34 - -
P2 Liposomes—CTS 0.34 0 0.01
P3 Ag/AgClNPs - 0.61 -
P4 Ag/AgClNPs—CTS (Biohybrid I) - 0.61 0.01
P5 Ag/AgClNPs—Liposomes (Biohybrid II) 0.34 0.61 -
P6 Ag/AgClNPs–Liposomes–CTS (Biohybrid III) 0.34 0.61 0.01
Nanomaterials 2021, 11, x FOR PEER REVIEW 4 of 25
Table 1. Codes and descriptions of the samples.
Sample Code Description C
Liposomes
(mg/mL)
C
Ag
(mM)
C
CTS
(% w/v)
P1 Liposomes 0.34 - -
P2 Liposomes—CTS 0.34 0 0.01
P3 Ag/AgClNPs - 0.61 -
P4 Ag/AgClNPs—CTS (Biohybrid I) - 0.61 0.01
P5 Ag/AgClNPs—Liposomes (Biohybrid II) 0.34 0.61 -
P6 Ag/AgClNPs–Liposomes–CTS (Biohybrid III) 0.34 0.61 0.01
Figure 1 shows a diagram representing the “green” development of biohybrids gen-
erated from the aqueous extract of a mixture of nettle and grape extracts.
Figure 1. Schematic representation of the “green” development of biohybrids generated from nettle and grape extracts.
2.3. Physicochemical and Biological Characterization of the Developed Bioentities
2.3.1. Spectral and Morphological Characterization
The absorption spectra of the samples were recorded (at the resolution of 1 nm) from
200 to 800 nm on a double-beam Jasco V-670 UV–vis–NIR spectrophotometer (Jasco, To-
kyo, Japan).
The fluorescence emission spectra of chlorophyll-a-based samples were collected us-
ing a LS55 Perkin Elmer fluorescence spectrometer (Waltham, MA, USA) in the wave-
length range of 600–800 nm by illuminating the samples with a 430 nm excitation light.
Fourier-transform infrared (FTIR) spectra of initial components AgNPs, liposomes,
and biohybrid complexes were recorded on an Excalibur FTS–3000 FTIR spectrometer
(Bio-Rad, Munich, Germany), in the wavenumber range of 4000–500 cm
−1
. Each spectrum
was averaged over 128 scans, and the spectral resolution was 0.4 cm
−1
at a constant tem-
perature of 40 ± 1 °C. The samples were thin films, placed between two ZnSe window
discs. During the recorded spectra, the spectrometer was purged with dry nitrogen. For
all obtained spectra, the baseline correction and normalization were applied.
For atomic force microscopy (AFM) experiments, the sample solutions (50 µL ali-
quots) were deposited onto freshly cleaved 15 mm × 15 mm mica squares, left to adsorb
for 3 min, and rinsed with Millipore water added dropwise to remove redundant sam-
ples. The surface was air dried (3 h) in a dust-free enclosure at room temperature, then
imaged using AFM. Atomic force microscopy NTEGRA PRIMA was provided by NT-
MDT Spectrum Instruments (Zelenograd, Russia). AFM images were recorded in tap-
ping mode with commercial NSG01 tips of a 10 nm curvature radius (NT-MDT Spec-
trum Instruments, Zelenograd, Russia) at room temperature. Images were taken contin-
uously at a scan rate of 0.3−0.5 Hz. Both the height image and the phase image were
Figure 1. Schematic representation of the “green” development of biohybrids generated from nettle and grape extracts.
2.3. Physicochemical and Biological Characterization of the Developed Bioentities
2.3.1. Spectral and Morphological Characterization
The absorption spectra of the samples were recorded (at the resolution of 1 nm)
from 200 to 800 nm on a double-beam Jasco V-670 UV–vis–NIR spectrophotometer (Jasco,
Tokyo, Japan).
The fluorescence emission spectra of chlorophyll-a-based samples were collected using
a LS55 Perkin Elmer fluorescence spectrometer (Waltham, MA, USA) in the wavelength
range of 600–800 nm by illuminating the samples with a 430 nm excitation light.
Fourier-transform infrared (FTIR) spectra of initial components AgNPs, liposomes,
and biohybrid complexes were recorded on an Excalibur FTS–3000 FTIR spectrometer (Bio-
Rad, Munich, Germany), in the wavenumber range of 4000–500 cm
−1
. Each spectrum was
averaged over 128 scans, and the spectral resolution was 0.4 cm
−1
at a constant temperature
of 40
±
1
◦
C. The samples were thin films, placed between two ZnSe window discs. During
the recorded spectra, the spectrometer was purged with dry nitrogen. For all obtained
spectra, the baseline correction and normalization were applied.
For atomic force microscopy (AFM) experiments, the sample solutions (50
µ
L aliquots)
were deposited onto freshly cleaved 15 mm
×
15 mm mica squares, left to adsorb for
3 min, and rinsed with Millipore water added dropwise to remove redundant samples. The
surface was air dried (3 h) in a dust-free enclosure at room temperature, then imaged using
AFM. Atomic force microscopy NTEGRA PRIMA was provided by NT-MDT Spectrum
Instruments (Zelenograd, Russia). AFM images were recorded in tapping mode with
commercial NSG01 tips of a 10 nm curvature radius (NT-MDT Spectrum Instruments,
Zelenograd, Russia) at room temperature. Images were taken continuously at a scan rate of
0.3
−
0.5 Hz. Both the height image and the phase image were recorded. The images were
flattened using the NT-MDT Spectrum Instruments Image Analysis P9 software.

Nanomaterials 2021,11, 1811 5 of 25
The surface morphology of the samples presented in this paper was analyzed us-
ing a scanning electron microscope (SEM), a FEI Inspect Model S50 apparatus (Hills-
boro, OR, USA). The apparatus is equipped with a secondary electrons (SE) detector. The
SEM images were obtained at a 10 mm working distance, a 10 kV acceleration voltage, and
for 50 up to 50,000
×
magnifications. Before SEM investigations, all the samples were coated
with a thin Au layer (~10 nm). The Au layer was obtained using a sputtering Cressington
108 auto sputter coater apparatus (Cressington Scientific Instruments UK, Watford, England
(UK)), equipped with a Cressington MTM-20 thickness controller. The ImageJ program
was used to estimate the size of the developed materials from the magnified SEM images.
The hydrodynamic diameters (Zav) of samples were measured by the dynamic light
scattering (DLS) technique (Zetasizer Nano ZS, Malvern Instruments Ltd., Worcester-
shire, U.K.), at a scattering angle of 90
◦
by the Stokes–Einstein equation. The mean values
were calculated from three individual experiments, so the average values (
±
standard
deviation, SD) were further reported. The polydispersity indexes (PdI—the indicator
of the width of the particle size distribution) were also determined from 3 individual
measurements using intensity distribution.
Zeta potential (
ξ
) measurements were carried out at 25
◦
C in triplicate, and the
mean values were reported on Malvern Zetasizer Nano ZS (Malvern Instruments Inc.,
Worcestershire, UK) by measuring, in an electric field, the electrophoretic mobility of the
prepared samples.
The crystallographic structure and phase composition of the sample series P3–P6 con-
taining Ag/AgClNPs were examined by X-ray diffraction (XRD) by using an EMPYREAN
diffractometer (PANalytical, Almelo, The Netherlands) with Cu-K
α
incident radiation.
Samples P3–P6 were extracted from the PBS solution by centrifuging (15,000
×
g, 30 min)
at 4
◦
C. The separated upper layer (supernatant) was removed. The liquid sediment was
placed on quartz glass (2.5 cm
×
2.5 cm) and was evaporated in a vacuum chamber for
12 h at room temperature. The XRD spectra were measured with an exposition time of 12 h
for each sample. The average sizes of nanoparticles for each phase were estimated using
the Scherrer equation:
D=
Kλ
βcos ϑ, (1)
where Dis the average crystallite size, Kis a dimensionless shape factor close to unity,
λ
is
the X-ray wavelength,
θ
is the Bragg angle, and
β
is the peak width at half the maximum
intensity (FWHM) after subtracting the instrumental line broadening.
SAXS experiments were performed using a pinhole camera (MolMet, Rigaku, Japan,
modified by SAXSLAB/Xenocs) attached to a microfocused X-ray beam generator (Rigaku
MicroMax 003) operating at 50 kV and 0.6 mA (30 W). The camera was equipped with
a vacuum version of the Pilatus 300 K detector. Calibration of primary beam position
and sample-to-detector distances was performed using a AgBehenate sample. For the
measurements, samples were sealed into borosilicate capillaries. Two experimental setups
were used to cover the qrange of 0.005–0.5 Å
−1
. The scattering vector, q, is defined as
q=(4
π
/
λ
)sin(
Θ
), where
λ
is the wavelength and 2
Θ
is the scattering angle. Homemade
software based on the PyFAI Python library [25] was used for data reduction.
2.3.2. In Vitro Antioxidant Activity
The chemiluminescence (CL) technique (Turner Design TD 20/20 USA Chemilu-
minometer) was used to evaluate the antioxidant properties of the samples by using a
free radical generator system based on 10
−5
M luminol and 10
−5
M H
2
O
2
in TRIS-HCl
buffer (pH 8.6). The in vitro antioxidant activity (AA%) of each sample was calculated, in
triplicate, as:
AA = [(I0−I)/I0]·100%, (2)
where I
0
and I are the maximum CL intensities at t = 5 s for the control (i.e., the reaction
mixture without the sample) and for each sample, respectively [26].

Nanomaterials 2021,11, 1811 6 of 25
2.3.3. Antibacterial Activity
Bacteria stock cultures (Staphylococcus aureus and Escherichia coli) were subcultured
onto Luria Bertani Agar acc. Miller (LBA) plates at 37
◦
C. To evaluate the antibacterial
properties of the tested samples, the antibacterial activity was determined by the agar well
diffusion method as previously described in [
27
]. Briefly, the LBA surface was inoculated
by spreading a volume of the bacteria inoculum. Using a sterile Durham tube 6 mm in
diameter, the wells were made and inoculated with 50
µ
L of each sample; then, the Luria
Bertani Agar plates were incubated at 37
◦
C for 24 h. The antimicrobial agent diffused
into the LBA and inhibited the growth of the test bacteria, and then the diameters of the
inhibition growth zones (IGZs) were measured.
2.3.4. Cell Viability
The biocompatibility of the biohybrids was evaluated using MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay as follows. Cells (BJ,
HT-29, and HepG2) were seeded in 96-well plates and cultured for 24 h in medium. The next
day, the medium was changed, and different concentrations of biohybrids were added for
24 h. Cells grown only in medium were used as negative control. Following incubation, the
medium was changed, and 1 mg/mL of MTT solution was added to each well and incubated
for an additional 4 h at 37
◦
C. Finally, the medium was collected, and DMSO was used to
dissolve the insoluble formazan product. The absorbance of the samples was recorded at
570 nm using a Mithras 940 plate reader (Berthold, Germany). The data were corrected for the
background, and the percentage of viable cells was obtained using the following equation:
% Viable cells = [(A570 of treated cells)/(A570 untreated cells)]·100%, (3)
The NP concentration that reduced the viability of the cells by half (IC
50
) was obtained
by fitting the data with a logistical sigmoidal equation using the software Origin 8.1
(Microcal Inc., Northampton, MA, USA.). The therapeutic index (TI) was calculated as
the ratio of the dose that produces toxicity to the dose needed to produce the desired
therapeutic response [28].
2.3.5. Evaluation of Cellular Morphology
Cells were grown on coverslips and treated for 24 h with two different concentrations
for the biohybrids. Afterwards, the cells were washed with PBS, fixed for 15 min with
3.7% formaldehyde dissolved in PBS, and washed again with PBS buffer. Sequentially,
cells were stained with 20
µ
g/mL AO solution for 15 min, then immediately washed with
PBS, followed imaging using an Andor DSD2 Confocal Unit (Andor, Belfast, Northern
Ireland), mounted on an Olympus BX-51 epifluorescence microscope (Olympus, Ham-
burg, Germany), equipped with a 40
×
objective and an appropriate filter cube (excitation
filter, 466/40 nm; dichroic mirror, 488 nm; and emission filter, 525/54 nm).
2.3.6. Hemocompatibility
The hemolytic activity of the new hybrids was determined using an adapted protocol
based on the ASTM F 756-00 standard previously described [
29
]. Briefly, fresh blood was
collected on heparin from healthy volunteers and diluted with PBS to a final hemoglobin
concentration of ~10 mg/mL. The blood was incubated with the highest concentration of
the samples for 4 h at 37
◦
C under constant shaking. Finally, the supernatant was collected
and mixed with an equal amount of Drabkin reagent (Sigma-Aldrich, Darmstadt, Germany).
After 15 min, the absorbance of the samples was read at 570 nm using a plate reader. As
negative and positive controls, the human red blood cells (hRBCs) in PBS and distilled
water, respectively, were used. The experimental values were corrected for background
dilution factors and used to calculate the percentage of hemolysis (i.e., hemolytic index),
according to the equation:
% Hemolysis = (AS/AT) 100%, (4)

Nanomaterials 2021,11, 1811 7 of 25
where A
S
is the corrected absorbance of the hemoglobin released in the supernatant
after treatment with nanoparticles, and A
T
is the corrected absorbance of the total re-
leased hemoglobin.
2.3.7. Statistical Analysis
Unless stated otherwise, all data were expressed as the mean value
±
standard
deviation of three individual experiments. Statistical significance was estimated using the
Student’s t-test (Microsoft Excel 2010) to determine the significant differences among the
experimental groups, and values of p< 0.05 were considered statistically significant.
3. Results and Discussion
3.1. Optical Characterization of the Developed Materials
The developed bionanosilver-based hybrid materials were optically characterized by
UV–vis absorption, fluorescence emission, and FTIR spectroscopy, in order to acquire deep
insights about the nanobiointeraction between components of biohybrids and also about
the biohybrids’ formation. Chlorophyll a(Chla) incorporated in artificial cell membranes
was used as a spectral biosensor to monitor the formation of plasmonic biohybrids.
In the absorption spectra of the samples containing artificial cell membranes, the
spectral signature of Chlaat ~669 nm was observed. Moreover, the SPR band at ~431 nm,
characteristic for Ag/AgClNP formation, was identified in the UV–vis absorption spectra
of the silver-based materials (see Figure S1 in the Supplementary Materials).
The formation of plasmonic biohybrids was also confirmed by fluorescence emission
spectra of Chla-containing samples (
λexcitation
= 430 nm), which underwent considerable
emission quenching (see Figure S2 in the Supplementary Materials) at the interaction
between the porphyrinic ring of Chlaand the surface of phyto-Ag/AgClNPs.
FTIR spectra allow for the identification of functional groups present in the compo-
nents of the systems under investigation. The observation of the vibration spectrum of the
biohybrid nanocomplexes allows evaluating the type of interaction that occurs between
the Ag/AgClNPs and lecithin liposomes in the presence of chitosan or without it, as the
vibrations of the atoms involved in this interaction can suffer changes in frequency and
intensity (see Figure S3). These FTIR results indicate the involvement of hydroxyl and
carbonyl groups (belonging to polyphenols and proteins, respectively) in the generation of
biohybrids P5 and P6.
More details regarding the optical characterization of the developed materials are
presented in the Supplementary Materials.
3.2. Estimation of Particle Size and Zeta Potential of the Obtained Biohybrids
The mean hydrodynamic diameters of the developed particles were estimated by DLS
measurements (see Figure S4 in the Supplementary Materials). As observed, the addition of
chitosan to Liposomes, Ag/AgClNPs, and Ag/AgClNPs–Liposomes resulted in a size in-
crease. This aspect will be further confirmed by SAXS experiments and microscopic studies.
The physical stability of the suspensions of the developed particles is related to the
particle electrical charge, which was quantified by zeta potential (
ξ
) measurements via the
electrophoretic mobility of the particles in an electric field [
30
,
31
]. Great
ξ
magnitude is
indicative of a stable system [
32
,
33
], being related to high electrical interparticle repulsion
due to the surface charge, thus indicating the high stability of the suspension. Figure 2
displays the zeta potential values of the bio-based materials developed in this study.

Nanomaterials 2021,11, 1811 8 of 25
Nanomaterials 2021, 11, x FOR PEER REVIEW 8 of 25
electrophoretic mobility of the particles in an electric field [30,31]. Great ξ magnitude is
indicative of a stable system [32,33], being related to high electrical interparticle repulsion
due to the surface charge, thus indicating the high stability of the suspension. Figure 2
displays the zeta potential values of the bio-based materials developed in this study.
Figure 2. Evaluation of the physical stability of the obtained bio-based materials.
Chlorophyll-labeled artificial cell membranes exhibited moderate stability (ξ
P1
=
−20.17 ± 0.49 mV), and after chitosan addition, the zeta potential value increased to −8.45
± 0.27 mV (for P2) due to the positive charge of amino functional groups in chitosan.
“Green” synthesized silver nanoparticles alone and embedded in biomimetic membranes
registered more accentuated electronegative values (ξ
P3
= −31.1 ± 1.9 mV and ξ
P5
= −32.57
± 1.5 mV, respectively), thus good stability. In the presence of chitosan, these values
shifted to positive values: ξ
P4
= +17.5 ± 1.07 mV and ξ
P6
= +18.1 ± 0.79 mV, respectively. The
surface positivity of “green” developed nanoparticles can be attributed to their stabiliza-
tion with the positively charged chitosan [34].
3.3. Structural Characterization of the Biohybrid Complexes
XRD and SAXS analyses were performed to investigate the structure of the obtained
materials.
3.3.1. X-ray Diffraction
Phase characterization and chemical composition of the liquid sediments, separated
from the PBS solution by centrifugation at 4 °C, followed by evaporation at room temper-
ature, were performed for all samples, which included hybrid Ag/AgCl nanoparticles
phytogenerated from nettles and grapes, i.e., for Samples P3–P6 (Figure 3). All XRD spec-
tra demonstrate the presence of two phases: Ag and AgCl, which have a face-centered
cubic (FCC) structure with a 𝐹𝑚3
𝑚 space group and lattice parameters a
Ag
= 0.40895 nm
and a
AgCl
= 0.55487 nm, correspondingly. Moreover, it resulted from the peak intensities
that the AgCl phase substantially dominated in all the studied systems. This phenome-
non can be explained by two reasons. First, we demonstrate that hybrid Ag/AgCl nano-
particles are the result of the synthesis from biocompounds of nettle and grape aqueous ex-
tracts (see Section 3.1 and Supplementary Materials), as were observed in the case of the
various plant extracts mediated [35,36]. Second, because of the effect of the PBS buffer,
when the anions of Cl
−
bond to Ag
+
, the AgCl phase becomes dominant in the composition
of Ag/AgClNPs.
Two phases of NaCl and sodium hydrogen phosphate hydrate (SHPH) are remaining
byproducts arising from the PBS buffer. The NaCl phase has a face-centered cubic (FCC)
structure with a 𝐹𝑚3
𝑚 space group and a lattice parameter a
NaCl
= 0.5640 nm. The SHPH
Sample
Figure 2. Evaluation of the physical stability of the obtained bio-based materials.
Chlorophyll-labeled artificial cell membranes exhibited moderate stability
(
ξP1
=
−
20.17
±
0.49 mV), and after chitosan addition, the zeta potential value increased
to
−
8.45
±
0.27 mV (for P2) due to the positive charge of amino functional groups in
chitosan. “Green” synthesized silver nanoparticles alone and embedded in biomimetic
membranes registered more accentuated electronegative values (
ξP3
=
−
31.1
±
1.9 mV
and
ξP5
=
−
32.57
±
1.5 mV, respectively), thus good stability. In the presence of chitosan,
these values shifted to positive values:
ξP4
= +17.5
±
1.07 mV and
ξP6
= +18.1
±
0.79 mV,
respectively. The surface positivity of “green” developed nanoparticles can be attributed to
their stabilization with the positively charged chitosan [34].
3.3. Structural Characterization of the Biohybrid Complexes
XRD and SAXS analyses were performed to investigate the structure of the ob-
tained materials.
3.3.1. X-ray Diffraction
Phase characterization and chemical composition of the liquid sediments, separated
from the PBS solution by centrifugation at 4
◦
C, followed by evaporation at room tem-
perature, were performed for all samples, which included hybrid Ag/AgCl nanoparticles
phytogenerated from nettles and grapes, i.e., for Samples P3–P6 (Figure 3). All XRD spectra
demonstrate the presence of two phases: Ag and AgCl, which have a face-centered cubic
(FCC) structure with a
Fm3m
space group and lattice parameters a
Ag
= 0.40895 nm and
a
AgCl
= 0.55487 nm, correspondingly. Moreover, it resulted from the peak intensities that
the AgCl phase substantially dominated in all the studied systems. This phenomenon can
be explained by two reasons. First, we demonstrate that hybrid Ag/AgCl nanoparticles
are the result of the synthesis from biocompounds of nettle and grape aqueous extracts
(see Section 3.1 and Supplementary Materials), as were observed in the case of the various
plant extracts mediated [
35
,
36
]. Second, because of the effect of the PBS buffer, when
the anions of Cl
−
bond to Ag
+
, the AgCl phase becomes dominant in the composition of
Ag/AgClNPs.
Two phases of NaCl and sodium hydrogen phosphate hydrate (SHPH) are remaining
byproducts arising from the PBS buffer. The NaCl phase has a face-centered cubic (FCC)
structure with a
Fm3m
space group and a lattice parameter a
NaCl
= 0.5640 nm. The SHPH
phase with chemical formula Na
2
HPO
3
(H
2
O)
5
has an orthorhombic structure (space group
Pmn2
1
) with lattice parameters a = 0.7170 nm, b = 0.6360 nm, and c = 0.9070 nm [
37
]. The
average crystallite sizes of the nanoparticles (D
XRD
) for each phase, estimated using the
Scherrer equation (Equation (1)), are collected in Table 2.

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phase with chemical formula Na2HPO3(H2O)5 has an orthorhombic structure (space group
Pmn21) with lattice parameters a = 0.7170 nm, b = 0.6360 nm, and c = 0.9070 nm [37]. The
average crystallite sizes of the nanoparticles (DXRD) for each phase, estimated using the
Scherrer equation (Equation (1)), are collected in Table 2.
Figure 3. XRD patterns for samples with hybrid Ag/AgCl nanoparticles phytogenerated from nettles
and grapes: P3—Ag/AgClNPs (light blue line), P4—Ag/AgClNPs–CTS (black line), P5—
Ag/AgClNPs–Liposomes (magenta line) and P6—Ag/AgClNPs–Liposomes–CTS (dark yellow line).
The identification of XRD peaks was done according to Inorganic Crystal Structure Database (ICSD)
files as follows: (1) SHPH—ICSD Entry: 16138; (2) NaCl—ICSD Entry: 28948; (3) AgCl—ICSD Entry:
64734; (4) Ag—ICSD Entry: 64994.
Table 2. Mean sizes of crystallites (DXRD, nm) estimated from diffraction line broadening.
Silver-Based Samples’ Codes Ag AgCl NaCl SHPH
P3—Ag/AgClNPs 16 62 459 149
P4—Ag/AgClNPs–CTS 17 25 448 –
P5—Ag/AgClNPs–Lip 23 47 351 –
P6—Ag/AgClNPs–Lip–CTS 14 33 – –
3.3.2. SAXS Results
Small-angle X-ray scattering was used to study the structural changes of the Ag/AgCl
nanoparticles in the biohybrid complexes P3–P6 in excess of PBS medium at the nanoscale
level.
The proposed model (Guinier–Porod) allows for the determination of the size and
dimensionality of the objects (see Supplementary Materials). Scattering curves for P3, P4,
and P5 are quite similar [38]. The SAXS experimental data for the initial components and
30 40 50 60 70 80
0
1
2
3
(031) SHPH
(022) SHPH
(210) SHPH
(111) Ag
(420) AgCl
(331) AgCl
(222) AgCl
(311) AgCl
(220) AgCl
(200) AgCl
(111) AgCl
(222) NaCl
(220) NaCl
(200) NaCl
(111) Ag
(400) NaCl
Phases
SHPH
NaCl
AgCl
Ag
I×10
4
, a.u.
2Θ, °
(400) AgCl
Figure 3.
XRD patterns for samples with hybrid Ag/AgCl nanoparticles phytogenerated from
nettles and grapes: P3—Ag/AgClNPs (light blue line), P4—Ag/AgClNPs–CTS (black line), P5—
Ag/AgClNPs–Liposomes (magenta line) and P6—Ag/AgClNPs–Liposomes–CTS (dark yellow line).
The identification of XRD peaks was done according to Inorganic Crystal Structure Database (ICSD)
files as follows: (1) SHPH—ICSD Entry: 16138; (2) NaCl—ICSD Entry: 28948; (3) AgCl—ICSD Entry:
64734; (4) Ag—ICSD Entry: 64994.
Table 2. Mean sizes of crystallites (DXRD, nm) estimated from diffraction line broadening.
Silver-Based Samples’ Codes Ag AgCl NaCl SHPH
P3—Ag/AgClNPs 16 62 459 149
P4—Ag/AgClNPs–CTS 17 25 448 –
P5—Ag/AgClNPs–Lip 23 47 351 –
P6—Ag/AgClNPs–Lip–CTS 14 33 – –
3.3.2. SAXS Results
Small-angle X-ray scattering was used to study the structural changes of the Ag/AgCl
nanoparticles in the biohybrid complexes P3–P6 in excess of PBS medium at the nanoscale level.
The proposed model (Guinier–Porod) allows for the determination of the size and
dimensionality of the objects (see Supplementary Materials). Scattering curves for P3, P4,
and P5 are quite similar [
38
]. The SAXS experimental data for the initial components and
their complexes are shown in Figure 4. The obtained parameters from the fitting procedure
on the experimental SAXS data are presented in Table 3.

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Nanomaterials 2021, 11, x FOR PEER REVIEW 10 of 25
their complexes are shown in Figure 4. The obtained parameters from the fitting proce-
dure on the experimental SAXS data are presented in Table 3.
Table 3. Structural parameters derived from the analysis of SAXS data and the calculated diameter (DSAXS = 2(5/3)1/2Rg) of
the Ag and Ag/AgCl nanoparticles for silver-based samples (P3–P6).
Silve
r
-Based Samples’
Codes Rg2 (nm) s2 m2 DSAXS2 (nm) Rg1 (nm) s1 m1 DSAXS1
(nm)
P3—Ag/AgClNPs 33.7 ± 1.8 0.06 ± 001 2.2 ± 0.003 86.9 ± 1.8 10.1 ± 2.8 0.27 ± 0.08 4.2 ± 0.05 26.1 ± 2.8
P4—Ag/AgClNPs–CTS 35.3 ± 0.2 0.1± 0.03 2.9 ± 0.001 91.1 ± 0.2 12.4 ± 0.3 0.08 ± 0.02 4.1 ± 0.04 32.0 ± 0.3
P5—Ag/AgClNPs–Lip 43.4 ± 0.3 0.01 ± 0.005 2.5 ± 0.01 112.0 ± 0.3 15.7 ± 0.01 0.01 ± 0.03 4.1 ± 0.02 40.5 ± 0.01
P6—Ag/AgClNPs–Lip–CTS 23.6 ± 1.2 0.002 ±
0.0002 2.2 ± 0.02 60.9 ± 1.2 15.3 ± 0.7 0.01 ± 0.05 4.3 ± 0.05 39.5 ± 0.7
Figure 4. SAXS curves for Samples P1–P6 in excess PBS medium: P1—Lip (purple), P2—Lip–CTS
(violet), P3—Ag/AgClNPs (blue), P4—Ag/AgClNPs–CTS (green), P5—Ag/AgClNPs–Lip (cyan)
and P6—Ag/AgClNPs–Lip–CTS (magenta). Symbols are experimental data, and lines are fits by the
generalized Guinier–Porod model (Equation (1) in the Supplementary Materials). For better visual-
ization, the curves are spaced with a coefficient of 10 relative to each other in the intensity scale from
the bottom to the top.
Best fits are presented in Figure 4 as lines. The presence of two structural levels is
clearly seen from the SAXS curves. The scattering objects were tentatively divided into
two parts: smaller nanoparticles with Rg1 ranging from 10.1 nm up to 15.7 nm and larger
nanoparticles with Rg2 ranging from 33.7 nm up to 43.4 nm. Based on the dimensionality
value(s), we can assume a spherical shape of NPs. Then, the average diameter can be cal-
culated as DSAXS = 2(5/3)1/2Rg. The obtained values are presented in Table 3.
0.1 1
R
g1
P3
P4
P5
P6
P2
Intensity, arb. units
Q, nm
-1
P1
R
g2
Figure 4.
SAXS curves for Samples P1–P6 in excess PBS medium: P1—Lip (purple), P2—Lip–CTS
(violet), P3—Ag/AgClNPs (blue), P4—Ag/AgClNPs–CTS (green), P5—Ag/AgClNPs–Lip (cyan)
and P6—Ag/AgClNPs–Lip–CTS (magenta). Symbols are experimental data, and lines are fits by
the generalized Guinier–Porod model (Equation (1) in the Supplementary Materials). For better
visualization, the curves are spaced with a coefficient of 10 relative to each other in the intensity scale
from the bottom to the top.
Table 3.
Structural parameters derived from the analysis of SAXS data and the calculated diameter (D
SAXS
= 2(5/3)
1/2
R
g
)
of the Ag and Ag/AgCl nanoparticles for silver-based samples (P3–P6).
Silver-Based
Samples’ Codes Rg2 (nm) s2m2DSAXS2 (nm) Rg1 (nm) s1m1DSAXS1 (nm)
P3—Ag/AgClNPs 33.7 ±1.8 0.06 ±001 2.2 ±0.003 86.9 ±1.8 10.1 ±2.8 0.27 ±0.08 4.2 ±0.05 26.1 ±2.8
P4—Ag/AgClNPs–
CTS 35.3 ±0.2 0.1±0.03 2.9 ±0.001 91.1 ±0.2 12.4 ±0.3 0.08 ±0.02 4.1 ±0.04 32.0 ±0.3
P5—Ag/AgClNPs–
Lip 43.4 ±0.3 0.01 ±0.005 2.5 ±0.01 112.0 ±0.3 15.7 ±0.01 0.01 ±0.03 4.1 ±0.02 40.5 ±0.01
P6—Ag/AgClNPs–
Lip–CTS 23.6 ±1.2 0.002 ±0.0002 2.2 ±0.02 60.9 ±1.2 15.3 ±0.7 0.01 ±0.05 4.3 ±0.05 39.5 ±0.7
Best fits are presented in Figure 4as lines. The presence of two structural levels is
clearly seen from the SAXS curves. The scattering objects were tentatively divided into
two parts: smaller nanoparticles with R
g1
ranging from 10.1 nm up to 15.7 nm and larger
nanoparticles with R
g2
ranging from 33.7 nm up to 43.4 nm. Based on the dimensionality
value(s), we can assume a spherical shape of NPs. Then, the average diameter can be
calculated as DSAXS = 2(5/3)1/2Rg. The obtained values are presented in Table 3.
SAXS and XRD results both confirm the presence of two types of nanoparticles in
the system: AgNPs and hybrid Ag/AgClNPs. The findings achieved through XRD and

Nanomaterials 2021,11, 1811 11 of 25
SAXS analyses support the results obtained by UV–vis absorption spectroscopy. Thus, the
mixture with CTS (Sample P4) increased to a larger diameter by 5 nm, while in the sample
with liposomes (Sample P5), we observed an increase by 25 nm. Interestingly, the scattering
curve for Sample P6 slightly differs from other complexes with Ag/AgClNPs. The size of
the small nanoparticles is identical to the values determined for the previously discussed
samples, while the larger nanoparticles are much smaller than observed for Samples P3-P5.
Note that the small NPs containing Ag crystallites have a diffusive interface [
39
], resulting
in higher anisotropy objects and thus higher values of the dimensionality parameter. For
larger Ag/AgClNPs, the power-law exponent implies that scattering occurs on the mass
fractals with a fractal dimension of ~3 [39–42].
3.4. Morphological Characterization of the Developed Bio-Based Materials
The surface characteristics of the biohybrids were studied using AFM and SEM methods.
3.4.1. AFM Analysis of the Developed Materials
AFM topology of the hybrid Ag/AgClNPs is shown in Figure 5(left). The NPs
have a spherical shape, which is in good agreement with the results obtained from the
approximation of the SAXS curves (see Figure 4). The average size of Ag/AgClNPs is
48.2 ±9.5
nm. Silver/silver chloride nanoparticles in the presence of chitosan are involved
in the polymer network, as is clearly seen in Figure 5(right).
Nanomaterials 2021, 11, x FOR PEER REVIEW 12 of 25
Figure 5. Topologies of Ag/AgClNPs (P3, left) and Ag/AgClNPs–CTS Biohybrid I (P4, right) with size distributions (in
the middle) for both systems, obtained using Image Analysis software (version 3.5) (https://nexus.ntmdt.ru/dl_3687; ac-
cessed on 04.03.2020). Bottom figures (from left to right): height analysis in the selected direction (red dashed line) and
magnified 3D visualization of the selected blue area for the P3 system, cropped 3D image of the selected green area, and
height analysis in the selected direction (red dashed line) for the P4 system.
(a) (b)
Figure 5.
Topologies of Ag/AgClNPs (P3,
left
) and Ag/AgClNPs–CTS Biohybrid I (P4,
right
) with size distributions (in
the
middle
) for both systems, obtained using Image Analysis software (version 3.5) (https://nexus.ntmdt.ru/dl_3687;
accessed on 4 March 2020).
Bottom
figures (from left to right): height analysis in the selected direction (red dashed line) and
magnified 3D visualization of the selected blue area for the P3 system, cropped 3D image of the selected green area, and
height analysis in the selected direction (red dashed line) for the P4 system.
Ag/AgClNPs coated with chitosan have an average size of 56.9
±
19.5 nm. The size
distribution includes aggregates ranging in size from 75 to 100 nm. Biohybrid Complex I
consists of NPs with a chitosan shell, indicated by the following items: (1) an increase in NP
size; (2) an increase in surface roughness (R
a_P3
= 0.5 nm, R
a_P4
= 0.7 nm, R
q_P3
= 0.57 nm,
and R
q_P4
= 0.9 nm); and (3) the appearance of a rough surface of Ag/AgClNPs in the

Nanomaterials 2021,11, 1811 12 of 25
presence of CTS compared to the smooth surface of Ag/AgClNPs without CTS, as can
be clearly seen from the height analysis and magnified 3D images for P3 and P4 systems
(Figure 5, bottom figures).
Low height is, according to AFM, a flattening effect that occurs during the adsorption
of the nanoparticles on mica and their drying. More details regarding the sonication time
effect on liposome formation are given in Figure S5 in the Supplementary Materials.
AFM images for biohybrid complexes II (P5) and III (P6) are demonstrated in Figure 6.
In both cases, the presence of Ag/AgClNPs on the surface of the liposomes was detected
(green crops from scan images). Obviously, the morphology of the liposomes is changed:
they have an ellipsoidal shape with an apparent width (W, in its short axis) of
∼
341 nm and
length (L, in its long axis) of
∼
516 nm for the P5 system, and W
≈
480 nm and L
≈
660 nm
for the P6 system, while most liposomes in the AFM images of the P1 and P2 systems appear
regularly spherical and not deformed (see Figure S5 in the Supplementary Materials).
Nanomaterials 2021, 11, x FOR PEER REVIEW 12 of 25
Figure 5. Topologies of Ag/AgClNPs (P3, left) and Ag/AgClNPs–CTS Biohybrid I (P4, right) with size distributions (in
the middle) for both systems, obtained using Image Analysis software (version 3.5) (https://nexus.ntmdt.ru/dl_3687; ac-
cessed on 04.03.2020). Bottom figures (from left to right): height analysis in the selected direction (red dashed line) and
magnified 3D visualization of the selected blue area for the P3 system, cropped 3D image of the selected green area, and
height analysis in the selected direction (red dashed line) for the P4 system.
(a) (b)
Figure 6.
(
a
) AFM image of Ag/AgClNPs–Liposomes (P5—Biohybrid II) with the size distribution of the “free” silver/silver
chloride nanoparticles. The cropped green fragment illustrates the association of Ag/AgClNPs with chlorophyll-a-labeled
soybean lecithin liposomes. (
b
) AFM image of Ag/AgClNPs–Liposomes–CTS (P6—Biohybrid III); magnification of the
areas with “free” Ag/AgClNPs and Ag/AgClNPs bonded to liposomes are presented in the cropped orange and green
fragments, respectively.
The sizes of hybrid Ag/AgClNPs associated with liposomes (highlighted by circles in
green crops) are 64–77 nm and 97–172 nm for biohybrids P5 and P6, respectively.
The average size of Biohybrid P5 obtained from the AFM image (Figure 6a) is
72.6
±
18.2 nm, and this value is in good agreement with 76.3 nm obtained from SAXS (see
Supplementary Materials).
A close examination of the area highlighted in orange in Figure 6b indicates the
presence of “free” silver/silver chloride nanoparticles with an almost spherical shape
and sizes between 42 and 78 nm, which is consistent with the values of 39.5 and 60.9 nm
obtained from the SAXS method. The central NP has a dimension of 112 nm. Such larger

Nanomaterials 2021,11, 1811 13 of 25
nanoparticle sizes, from which SAXS scattering was not detected due to method limitations,
are present over the entire studied area (Figure 6b, top image), and we assume they are
nanoparticle aggregates.
3.4.2. SEM Analysis of the Developed Bio-Based Materials
Figure 7presents the SEM images of Samples P1 up to P6 and a magnified area of
3
×
3
µ
m
2
. As one can observe, the samples are not identical in their morphologies. They
present various structures with different shapes and sizes. For instance, Sample P1 shows
individual spherical shape structures of lipid vesicles, whereas Sample P2 exhibits chain-
like structures due to the presence of chitosan, including spherical structures in the mesh of
the network. The silver nanoparticles without (Sample P3) and with (Sample P4) chitosan
are spread all over the silicon plate surface. However, Sample P5 shows micrometric
structures, and Sample P6 shows nano- and micrometric entities. This behavior can be
explained by the diversity in the synthesis procedure of the samples, as explained in
Table 1
.
As observed, the addition of chitosan resulted in increasing size of the samples, in good
agreement with UV–vis absorption spectra, DLS results, SAXS data, and AFM analysis.
Nanomaterials 2021, 11, x FOR PEER REVIEW 13 of 25
Figure 6. (a) AFM image of Ag/AgClNPs–Liposomes (P5—Biohybrid II) with the size distribution of the “free” silver/silver
chloride nanoparticles. The cropped green fragment illustrates the association of Ag/AgClNPs with chlorophyll-a-la-
beled soybean lecithin liposomes. (b) AFM image of Ag/AgClNPs–Liposomes–CTS (P6—Biohybrid III); magnification of
the areas with “free” Ag/AgClNPs and Ag/AgClNPs bonded to liposomes are presented in the cropped orange and green
fragments, respectively.
The average size of Biohybrid P5 obtained from the AFM image (Figure 6a) is 72.6 ±
18.2 nm, and this value is in good agreement with 76.3 nm obtained from SAXS (see Sup-
plementary Materials).
A close examination of the area highlighted in orange in Figure 6b indicates the
presence of “free” silver/silver chloride nanoparticles with an almost spherical shape and
sizes between 42 and 78 nm, which is consistent with the values of 39.5 and 60.9 nm ob-
tained from the SAXS method. The central NP has a dimension of 112 nm. Such larger
nanoparticle sizes, from which SAXS scattering was not detected due to method limita-
tions, are present over the entire studied area (Figure 6b, top image), and we assume
they are nanoparticle aggregates.
3.4.2. SEM Analysis of the Developed Bio-Based Materials
Figure 7 presents the SEM images of Samples P1 up to P6 and a magnified area of 3
× 3 µm2. As one can observe, the samples are not identical in their morphologies. They
present various structures with different shapes and sizes. For instance, Sample P1 shows
individual spherical shape structures of lipid vesicles, whereas Sample P2 exhibits chain-
like structures due to the presence of chitosan, including spherical structures in the mesh
of the network. The silver nanoparticles without (Sample P3) and with (Sample P4) chi-
tosan are spread all over the silicon plate surface. However, Sample P5 shows micrometric
structures, and Sample P6 shows nano- and micrometric entities. This behavior can be
explained by the diversity in the synthesis procedure of the samples, as explained in Table
1. As observed, the addition of chitosan resulted in increasing size of the samples, in good
agreement with UV–vis absorption spectra, DLS results, SAXS data, and AFM analysis.
Figure 7. SEM images of the developed materials. Magnified SEM images of 3 × 3 µm2 are also presented, in which various
particle sizes are measured.
Figure 7.
SEM images of the developed materials. Magnified SEM images of 3
×
3
µ
m
2
are also presented, in which various
particle sizes are measured.
3.5. Mechanism of Biohybrid Formation
Summarizing our optical, structural, and morphological investigations, as well as
the stability study of the biohybrid complexes and their components, a mechanism of
Ag/AgClNP fabrication and biohybrid formation, can be discussed.
Biohybrid Complex I (Ag/AgClNPs–CTS) with the proposed interaction model be-
tween the chitosan matrix and silver/silver chloride nanoparticles is shown schematically
in Figure 8. The silver ions’ interaction takes place through the amino and hydroxyl groups
of chitosan [
43
]. Moreover, it could be assumed that the formation of the chitosan-capped
Ag/AgClNPs was confirmed by zeta potential measurements and by SAXS, SEM, and
AFM studies.

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3.5. Mechanism of Biohybrid Formation
Summarizing our optical, structural, and morphological investigations, as well as the
stability study of the biohybrid complexes and their components, a mechanism of
Ag/AgClNP fabrication and biohybrid formation, can be discussed.
Biohybrid Complex I (Ag/AgClNPs–CTS) with the proposed interaction model be-
tween the chitosan matrix and silver/silver chloride nanoparticles is shown schematically
in Figure 8. The silver ions’ interaction takes place through the amino and hydroxyl
groups of chitosan [43]. Moreover, it could be assumed that the formation of the chitosan-
capped Ag/AgClNPs was confirmed by zeta potential measurements and by SAXS, SEM,
and AFM studies.
Figure 8. Mechanism of silver-based biohybrid formation.
Biohybrid II (Ag/AgClNPs–Lip) is a multilamellar vesicle (MLVs) with a surface
coated with nanoparticles (Figure 8B). This output was obtained from a mixture of soybean
lecithin liposomes and silver/silver chloride nanoparticles in PBS after sonication of the
Figure 8. Mechanism of silver-based biohybrid formation.
Biohybrid II (Ag/AgClNPs–Lip) is a multilamellar vesicle (MLVs) with a surface
coated with nanoparticles (Figure 8B). This output was obtained from a mixture of soybean
lecithin liposomes and silver/silver chloride nanoparticles in PBS after sonication of the
suspension for 5 min. Such a short time of treatment of the suspension with ultrasound
does not lead to the formation of unilamellar vesicles (ULVs) and excludes with a high
degree of probability the creation of other possible systems: (1) liposomes with bilayer-
embedded nanoparticles, (2) liposomes with core-encapsulated nanoparticles, and (3)

Nanomaterials 2021,11, 1811 15 of 25
lipid-bilayer-coated nanoparticles. The obtained complex is a very stable system, as
indicated by the zeta potential value of
−
33 mV. In addition, this value is very close to
−
31.7 mV for Ag/AgClNPs, and this result indicates the adhesion of nanoparticles on the
liposome surface. At the same time, the interaction of Ag/AgClNPs with liposomes causes
a change in liposome morphology. The ellipsoidal shape was observed by the AFM method,
while the pure soybean lecithin liposomes were almost spherical, as clearly seen on the
microscopic images (Figure S5a–c in the Supplementary Materials and Figure 6a). A similar
effect was detected for Biohybrid III (Ag/AgClNPs–Lip–CTS): liposomes surrounded by
chitosan have a spherical shape, and liposome–nanoparticles systems involved in the 3D
chitosan network have an ellipsoidal shape (Figure S5d,e in the Supplementary Materials
and Figure 6b). Even though this complex is less stable (
ξ
= +18.4 mV) than previously
described, it has high antioxidant activity and antibacterial effectiveness, as will be further
presented in Section 3.6.
3.6. Evaluation of Biological Activities of the Developed Bio-Based Materials
The
in vitro
bioactivities of materials phytogenerated from an aqueous extract of a
mixture of nettle and grapes were tested by assessing: the antioxidant activity, the antibac-
terial properties against S. aureus and E. coli, the cytotoxicity, the antiproliferative activity,
and the hemocompatibility. A therapeutic index was also calculated for each sample.
The biological performances of the developed materials were closely related to their
zeta potential values, size, morphology, and structure.
The samples showed good antioxidant activity, ranging from 62 to 75% (
in vitro
tested
through the chemiluminescence method; see Figure 9).
Nanomaterials 2021, 11, x FOR PEER REVIEW 15 of 25
suspension for 5 min. Such a short time of treatment of the suspension with ultrasound
does not lead to the formation of unilamellar vesicles (ULVs) and excludes with a high
degree of probability the creation of other possible systems: (1) liposomes with bilayer-
embedded nanoparticles, (2) liposomes with core-encapsulated nanoparticles, and (3) li-
pid-bilayer-coated nanoparticles. The obtained complex is a very stable system, as indi-
cated by the zeta potential value of −33 mV. In addition, this value is very close to −31.7
mV for Ag/AgClNPs, and this result indicates the adhesion of nanoparticles on the lipo-
some surface. At the same time, the interaction of Ag/AgClNPs with liposomes causes a
change in liposome morphology. The ellipsoidal shape was observed by the AFM method,
while the pure soybean lecithin liposomes were almost spherical, as clearly seen on the
microscopic images (Figure S5a–c in the Supplementary Materials and Figure 6a). A sim-
ilar effect was detected for Biohybrid III (Ag/AgClNPs–Lip–CTS): liposomes surrounded
by chitosan have a spherical shape, and liposome–nanoparticles systems involved in the
3D chitosan network have an ellipsoidal shape (Figure S5d,e in the Supplementary Mate-
rials and Figure 6b). Even though this complex is less stable (ξ = +18.4 mV) than previously
described, it has high antioxidant activity and antibacterial effectiveness, as will be further
presented in Section 3.6.
3.6. Evaluation of Biological Activities of the Developed Bio-Based Materials
The in vitro bioactivities of materials phytogenerated from an aqueous extract of a
mixture of nettle and grapes were tested by assessing: the antioxidant activity, the anti-
bacterial properties against S. aureus and E. coli, the cytotoxicity, the antiproliferative ac-
tivity, and the hemocompatibility. A therapeutic index was also calculated for each sam-
ple.
The biological performances of the developed materials were closely related to their
zeta potential values, size, morphology, and structure.
The samples showed good antioxidant activity, ranging from 62 to 75% (in vitro tested
through the chemiluminescence method; see Figure 9).
The good effectiveness of Ag/AgClNPs-based materials in the scavenging activity of
reactive oxygen species (ROS) is explained by: (i) their composition (especially the pres-
ence of “green” Ag/AgClNPs carrying antioxidant molecules such as polyphenols, chlo-
rophyll, etc., arising from vegetal extracts) and (ii) their nanosized dimensions (which of-
fer more reaction centers for ROS scavenging) [27].
Figure 9. The in vitro antioxidant activity of the developed plasmonic composites and their “build-
ing blocks”, evaluated by the chemiluminescence method.
Antibacterial effectiveness of the developed biohybrids was tested against Staphylo-
coccus aureus ATTC 2592 (as a representative Gram (+) bacteria) and Escherichia coli ATCC
8738 (as a representative Gram (-) bacteria) studied by the agar well diffusion method (see
Figure 10).
Figure 9.
The
in vitro
antioxidant activity of the developed plasmonic composites and their “building
blocks”, evaluated by the chemiluminescence method.
The good effectiveness of Ag/AgClNPs-based materials in the scavenging activity of
reactive oxygen species (ROS) is explained by: (i) their composition (especially the presence
of “green” Ag/AgClNPs carrying antioxidant molecules such as polyphenols, chlorophyll,
etc., arising from vegetal extracts) and (ii) their nanosized dimensions (which offer more
reaction centers for ROS scavenging) [27].
Antibacterial effectiveness of the developed biohybrids was tested against Staphylo-
coccus aureus ATTC 2592 (as a representative Gram (+) bacteria) and Escherichia coli ATCC
8738 (as a representative Gram (-) bacteria) studied by the agar well diffusion method
(see Figure 10).

Nanomaterials 2021,11, 1811 16 of 25
Nanomaterials 2021, 11, x FOR PEER REVIEW 16 of 25
The need for creating new antibacterial systems is an important research area in the
medical field. Staphylococcus aureus and Escherichia coli are the most important human
pathogens associated with nosocomial and community-acquired infections, causing seri-
ous health problems [44].
Our “green” obtained Ag/AgClNPs are more efficient against the two pathogens S.
aureus and E. coli (IGZ
S. aureus
= 20 mm; IGZ
E. coli
= 13 mm) as compared to the AgClNPs
prepared by Trinh et al. [45], who obtained IGZ values less than 11 mm for both strains.
Kashyap et al. [20] prepared Ag/AgCl particles showing a zone of inhibition of 7.5 mm
against E. Coli. Moreover, AgClNPs synthesized by Kota et al. [21] proved to be less potent
against S. aureus (IGZ
S. aureus
= 18.5 mm) as compared to our silver nanoparticles.
Figure 10. Antibacterial activity expressed as a diameter of the inhibition growth zone (IGZ) against
Staphylococcus aureus ATTC 2592 and Escherichia coli ATCC 8738.
The biohybrids containing “green” Ag/AgClNPs and Chla-Liposomes (Biohybrid II)
exhibited high antibacterial properties against Staphylococcus aureus ATTC 2592 (IGZ = 25
mm) and Escherichia coli ATCC 8738 (IGZ = 40 mm) (see Sample P5 in Figure 10), as com-
pared to Biohybrid I, which showed an IGZ value of 20 mm against E. coli and 22 mm
against S. aureus (see Sample P4 in Figure 10).
Co-loading of phyto-derived Ag/AgClNPs, biomimetic membranes, and chitosan
(Biohybrid III) resulted in impressive antibacterial effectiveness against S. aureus (IGZ =
30 mm) and E. coli (IGZ = 45 mm) (see Sample P6 in Figure 10), these remarkable properties
being due to their composition.
These developed materials showed different behavior against two bacterial strains
due to the differences in the bacterial cell wall structures. Gram (+) bacteria possess a thick
peptidoglycan layer and no outer lipid membrane, while Gram (-) bacteria have an outer
lipid membrane with pores and a unique periplasmic space with a thin peptidoglycan
layer [46].
Direct contact of our developed materials with the bacterial cells resulting in pertur-
bation/deterioration of cell walls and membranes leading to cell death is the most plausi-
ble antibacterial mechanism [47–49].
Figure 10.
Antibacterial activity expressed as a diameter of the inhibition growth zone (IGZ) against
Staphylococcus aureus ATTC 2592 and Escherichia coli ATCC 8738.
The need for creating new antibacterial systems is an important research area in the
medical field. Staphylococcus aureus and Escherichia coli are the most important human
pathogens associated with nosocomial and community-acquired infections, causing serious
health problems [44].
Our “green” obtained Ag/AgClNPs are more efficient against the two pathogens
S. aureus and E. coli (IGZ
S. aureus
= 20 mm; IGZ
E. coli
= 13 mm) as compared to the AgClNPs
prepared by Trinh et al. [
45
], who obtained IGZ values less than 11 mm for both strains.
Kashyap et al. [
20
] prepared Ag/AgCl particles showing a zone of inhibition of 7.5 mm
against E. Coli. Moreover, AgClNPs synthesized by Kota et al. [
21
] proved to be less potent
against S. aureus (IGZ S. aureus = 18.5 mm) as compared to our silver nanoparticles.
The biohybrids containing “green” Ag/AgClNPs and Chla-Liposomes (Biohybrid II)
exhibited high antibacterial properties against Staphylococcus aureus ATTC 2592
(IGZ = 25 mm)
and Escherichia coli ATCC 8738 (
IGZ = 40 mm
) (see Sample P5 in Figure 10), as compared to
Biohybrid I, which showed an IGZ value of 20 mm against E. coli and 22 mm against S. aureus
(see Sample P4 in Figure 10).
Co-loading of phyto-derived Ag/AgClNPs, biomimetic membranes, and chitosan (Bio-
hybrid III) resulted in impressive antibacterial effectiveness against S. aureus (
IGZ = 30 mm
)
and E. coli (IGZ = 45 mm) (see Sample P6 in Figure 10), these remarkable properties being
due to their composition.
These developed materials showed different behavior against two bacterial strains
due to the differences in the bacterial cell wall structures. Gram (+) bacteria possess a thick
peptidoglycan layer and no outer lipid membrane, while Gram (-) bacteria have an outer
lipid membrane with pores and a unique periplasmic space with a thin peptidoglycan
layer [46].

Nanomaterials 2021,11, 1811 17 of 25
Direct contact of our developed materials with the bacterial cells resulting in perturba-
tion/deterioration of cell walls and membranes leading to cell death is the most plausible
antibacterial mechanism [47–49].
Cell viability results are reported in Figure 11 for all three cell lines investigated,
highlighting the dependence of cell viability on the silver content and the type of cell line.
Low doses of AgNPs had less cytotoxicity on the growth of normal cell lines (BJ cells), but
enhanced cytotoxicity was observed with increasing doses. The results obtained are in
correlation with those reported in the literature [
50
,
51
]. It was found that at concentrations
less than 25.5
µ
M, the samples do not show a toxic effect, the cell viability being higher than
80% in the case of BJ cells. Chitosan addition to the AgNPs did not improve the efficiency
of the NPs but only increased the overall concentration where the effect was observed. A
better efficiency was observed for the samples prepared with the liposomes. Thus, the most
efficient proved to be the NPs prepared with liposomes, P5 and P6, especially Sample P6,
which, over the entire range, tested displayed less toxicity to normal cells and increased
toxicity to cancer cells.
Nanomaterials 2021, 11, x FOR PEER REVIEW 17 of 25
Cell viability results are reported in Figure 11 for all three cell lines investigated,
highlighting the dependence of cell viability on the silver content and the type of cell line.
Low doses of AgNPs had less cytotoxicity on the growth of normal cell lines (BJ cells), but
enhanced cytotoxicity was observed with increasing doses. The results obtained are in
correlation with those reported in the literature [50,51]. It was found that at concentrations
less than 25.5 µM, the samples do not show a toxic effect, the cell viability being higher
than 80% in the case of BJ cells. Chitosan addition to the AgNPs did not improve the effi-
ciency of the NPs but only increased the overall concentration where the effect was ob-
served. A better efficiency was observed for the samples prepared with the liposomes.
Thus, the most efficient proved to be the NPs prepared with liposomes, P5 and P6, espe-
cially Sample P6, which, over the entire range, tested displayed less toxicity to normal
cells and increased toxicity to cancer cells.
Figure 11. Cell viability of the silver-based materials (P3, P4, P5, and P6) against BJ, HT-29, and HepG2 cells after 24 h of
treatment.
The values of IC
50
and the therapeutic index (TI) are reported in Table 4. IC
50
values
were used further to calculate the TI, which is an indicator of the treatment efficiency
against cancer cells. A value higher than one of TI indicates that the anticancer activity is
higher compared with the cytotoxic activity of the samples investigated against the nor-
mal cells (BJ cells) [28].
Figure 11.
Cell viability of the silver-based materials (P3, P4, P5, and P6) against BJ, HT-29, and HepG2 cells after 24 h
of treatment.
The values of IC
50
and the therapeutic index (TI) are reported in Table 4. IC
50
values
were used further to calculate the TI, which is an indicator of the treatment efficiency
against cancer cells. A value higher than one of TI indicates that the anticancer activity is

Nanomaterials 2021,11, 1811 18 of 25
higher compared with the cytotoxic activity of the samples investigated against the normal
cells (BJ cells) [28].
Table 4. IC50 and therapeutic index (TI) of the silver-based materials developed in our study.
IC50 TI
BJ HT-29 HepG2 HT-29 HepG2
P3 36.31 43.37 28.03 0.84 1.30
P4 49.31 56.26 50.04 0.88 0.99
P5 34.64 30.89 33.55 1.12 1.03
P6 35.7 27.52 20.15 1.30 1.77
From Table 4, one can see that Samples P3, P5, and P6 are more efficient against HepG2
cells, while P5 and P6 against HT-29 cells. The results indicated that Samples P3 and P4,
which have in their composition only Ag and Ag and chitosan, respectively, are more toxic
for normal cells as compared to cancerous cells. However, after the addition of lipids to
the samples (P5 and P6), their efficacy increased, mainly by observing an increase in the
toxicity against cancer cells.
Further, we investigated the effect induced by the highest concentration of the samples
tested before against the red blood cells. At 102.2
µ
M, for each sample, the hemolysis
percentage of new silver-based hybrids (Figure 12) is far below 5% [
29
], meaning that these
samples are slightly hemolytic, but they will not be harmful when found in the bloodstream
at smaller concentrations.
Nanomaterials 2021, 11, x FOR PEER REVIEW 18 of 25
Table 4. IC
50
and therapeutic index (TI) of the silver-based materials developed in our study.
IC
50
TI
BJ HT-29 HepG2 HT-29 HepG2
P3 36.31 43.37 28.03 0.84 1.30
P4 49.31 56.26 50.04 0.88 0.99
P5 34.64 30.89 33.55 1.12 1.03
P6 35.7 27.52 20.15 1.30 1.77
From Table 4, one can see that Samples P3, P5, and P6 are more efficient against
HepG2 cells, while P5 and P6 against HT-29 cells. The results indicated that Samples P3
and P4, which have in their composition only Ag and Ag and chitosan, respectively, are
more toxic for normal cells as compared to cancerous cells. However, after the addition of
lipids to the samples (P5 and P6), their efficacy increased, mainly by observing an increase
in the toxicity against cancer cells.
Further, we investigated the effect induced by the highest concentration of the sam-
ples tested before against the red blood cells. At 102.2 µM, for each sample, the hemolysis
percentage of new silver-based hybrids (Figure 12) is far below 5% [29], meaning that
these samples are slightly hemolytic, but they will not be harmful when found in the
bloodstream at smaller concentrations.
Figure 12. Hemolysis of the silver-based samples (P3, P4, P5, and P6) against hRBCs after 4 h of
treatment at 102.2 µM.
Figure 13 Figure 14 Figure 15 present the morphological changes induced by the
treatment as compared to the control BJ cells. To determine how the presence of the NPs
affects the morphology of the cells, we chose two different concentrations: a concen-
tration not affecting the cells’ viability (6.4 µM) and a concentration above the IC
50
val-
ues (51.1 µM). Similar concentrations were used for all the samples and all the cells that
are further investigated.
Figure 12.
Hemolysis of the silver-based samples (P3, P4, P5, and P6) against hRBCs after 4 h of
treatment at 102.2 µM.
Figures 13–15 present the morphological changes induced by the treatment as com-
pared to the control BJ cells. To determine how the presence of the NPs affects the mor-
phology of the cells, we chose two different concentrations: a concentration not affecting
the cells’ viability (6.4
µ
M) and a concentration above the IC
50
values (51.1
µ
M). Similar
concentrations were used for all the samples and all the cells that are further investigated.

Nanomaterials 2021,11, 1811 19 of 25
Nanomaterials 2021, 11, x FOR PEER REVIEW 19 of 25
Figure 13. Morphology of control BJ cells (A) and cells after treatment with 6.4 µM and 51.1 µM of
P3 (B,C), P4 (D,E), P5 (F,G), and P6 (H,I). Images are obtained with a 40× objective and using the
epifluorescence mode.
Figure 13.
Morphology of control BJ cells (
A
) and cells after treatment with 6.4
µ
M and 51.1
µ
M of
P3 (
B
,
C
), P4 (
D
,
E
), P5 (
F
,
G
), and P6 (
H
,
I
). Images are obtained with a 40
×
objective and using the
epifluorescence mode.

Nanomaterials 2021,11, 1811 20 of 25
Nanomaterials 2021, 11, x FOR PEER REVIEW 20 of 25
Figure 14. Morphology of control HT-29 cells (A) and cells after treatment with 6.4 µM and 51.1 µM
of P3 (B,C), P4 (D,E), P5 (F,G), and P6 (H,I). Images are obtained with a 40× objective and in an
epifluorescence mode.
Figure 14.
Morphology of control HT-29 cells (
A
) and cells after treatment with 6.4
µ
M and 51.1
µ
M
of P3 (
B
,
C
), P4 (
D
,
E
), P5 (
F
,
G
), and P6 (
H
,
I
). Images are obtained with a 40
×
objective and in an
epifluorescence mode.

Nanomaterials 2021,11, 1811 21 of 25
Nanomaterials 2021, 11, x FOR PEER REVIEW 21 of 25
Figure 15. Morphology of control HepG2 cells (A) and cells after treatment with 6.4 µM and 51.1
µM of P3 (B,C), P4 (D,E), P5 (F,G), and P6 (H,I). Images are obtained with a 40× objective and in an
epifluorescence mode.
Figure 15.
Morphology of control HepG2 cells (
A
) and cells after treatment with 6.4
µ
M and 51.1
µ
M
of P3 (
B
,
C
), P4 (
D
,
E
), P5 (
F
,
G
), and P6 (
H
,
I
). Images are obtained with a 40
×
objective and in an
epifluorescence mode.

Nanomaterials 2021,11, 1811 22 of 25
The control BJ cells normally present an elongated shape and when treated with the
smallest concentration of the sample, it could be observed that the morphology of these
cells was not affected (Figure 13). However, at 51.1
µ
M silver content, the cells’ morphology
was drastically affected: the cells’ branches were reduced, as well as the cell body. The
results are in correlation with those reported for cell viability, where at 51.1
µ
M, the viability
decreased by around 30–40% for all the samples investigated.
Figure 14 presents the morphological changes of HT-29 cells induced by different
treatments. Control HT-29 cells grow in clusters as observed in Figure 14A. When treated
with 6.4
µ
M, we can observe that the morphology of the cells was nonsignificantly affected.
At 51.1
µ
M, the cells’ morphology is affected as well as the reduction of the number of cells.
In Figure 15 are presented the results for HepG2 cells. Control HepG2 cells grow in
clusters, as observed in Figure 15A. When treated with 6.4
µ
M, we can observe that the
morphology of the cells is not affected so much. However, at 51.1
µ
M, the number of cells
is reduced. For both HT-29 and HepG2 cells, the results reported are in good concordance
with the results reported for cell viability.
To summarize, the results obtained regarding the biological activity of the prepared
materials are encouraging, taking into account that good bioperformances were obtained
at a lower silver content compared to the data previously reported by our research
group [10,27].
4. Conclusions
Vegetal wastes of grapes and nettle leaves were used to generate biohybrid entities by
using the Green Chemistry principles.
The natural porphyrin chlorophyll a, inserted in biomimetic membranes, acted as a
spectral sensor to monitor the generation of the biohybrids. The biointeraction between
bionanometals and artificial cell membranes was detected by Chlaat the nano level, through
changes in its spectral fingerprints.
The optical, structural, and morphological investigations correlated well, and the
mechanism of Ag/AgClNP fabrication and biohybrid formation was discussed.
Microscopical investigations by AFM and SEM images gave information about the
(quasi)spherical morphology and nanoscaled size of the samples.
XRD and SAXS investigations revealed the structural changes of the Ag/AgCl nanopar-
ticles in the biohybrid complexes, highlighting the copresence of AgNPs and hybrid
Ag/AgClNPs in the obtained silver-based systems. Particular attention was paid to the
study of Biohybrid III (Ag/AgClNPs–Liposomes–CTS) because during the formation
of biohybrid complexes, namely liposomes NPs, there is a high probability that small
nanoparticles, such as AgNPs, may pass through the cell membranes, thereby affecting
their functionality. However, in our case, it can be assumed that silver chloride nanoparti-
cles (AgClNPs) or hybrid Ag/AgClNPs, being large in size, do not disturb the integrity of
the membrane and accumulate on their outer leaflet, shown by the mechanism of biohybrid
formation proposed by us.
Coincorporation of Chla-labeled mimetic biomembranes, Ag/AgClNPs and chitosan,
resulted in significant bioperformances. The obtained silver-based biohybrids showed good
antioxidant properties by scavenging 71% of reactive oxygen species and also presented
high antimicrobial activity against Escherichia coli (IGZ = 45 mm) and Staphylococcus aureus
(IGZ = 30 mm). The silver/biomimetic membrane biohybrids with and without chitosan
were hemocompatible and presented efficiency against HepG2 and HT-29 cancer cells. The
therapeutic index of Ag/AgClNPs–Liposomes–CTS samples supports the antitumor effect;
these biohybrids exhibited a TI value of 1.30 for HT-29 cancer cells and 1.77 for HepG2
cancer cells, highlighting their antitumor effect against two types of cell lines.
These results demonstrate that silver-containing hybrid entities could be used as
building blocks to design new materials with many bioapplications, especially in the
treatment of liver and colorectal cancer.

Nanomaterials 2021,11, 1811 23 of 25
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/
10.3390/nano11071811/s1 at optical characterization (discussions; Figure S1. UV–vis absorption
spectra of the developed materials; Figure S2. Fluorescence emission spectra of chlorophyll-labeled
materials (
λexcitation
= 430 nm); Figure S3. FTIR spectra; Particle size estimated by DLS measurements
(Figure S4. Mean particle size (Zav, nm) and polydispersity index (PdI) of the samples, estimated by
DLS measurements); SAXS comments—Guinier–Porod model; AFM discussions (Figures S5 and S6).
Author Contributions:
Conceptualization, M.-E.B.-P.; methodology, M.-E.B.-P., Y.G., N.B. and G.B.;
formal analysis, M.-E.B.-P., Y.G., N.B. and G.B.; investigation, M.-E.B.-P., Y.G., G.B., V.T., A.Z., E.J.-G.,
N.B., M.R., M.B., C.U. and A.L.-S.; data curation, M.-E.B.-P., A.Z. and E.J.-G.; writing—original
draft preparation, M.-E.B.-P., Y.G. and G.B.; writing—review and editing, M.-E.B.-P., Y.G., G.B., N.B.,
M.B., A.L.-S. and C.U.; visualization, M.-E.B.-P., Y.G., N.B., G.B. and A.L.-S.; supervision, M.-E.B.-P.;
funding acquisition, M.-E.B.-P., Y.G., N.B. and M.B. All authors have read and agreed to the published
version of the manuscript.
Funding:
M.-E.B.-P. and Y.G. acknowledge the funding through Projects JINR–RO 2021 No. 38/2021
and No. 68/2021 (IUCN Order No. 365/11.05.2021) and Grant JINR–RO 2021 No. 19/2021 (IUCN
Order No. 367/11.05.2021). A.L.-S. acknowledges the financial support by the Romanian Ministry
of Education and Research, under the Romanian National Nucleu Program LAPLAS VI, Contract
No. 16N/2019. M.B and M.R. acknowledge the financial support of the Nucleu Programme at
IFIN-HH, Contract No. PN 19 06 02 03/2019. SAXS measurements were performed in the framework
of collaboration of the Institute of Macromolecular Chemistry AS CR with the Frank Laboratory of
Neutron Physics of the Joint Institute of Nuclear Research (JINR Project No. 04-4-1121-2015/2020
“Investigation of Condensed Matter by Modern Neutron Scattering Methods”). The financial support
is gratefully acknowledged.
Data Availability Statement:
The data is included in the main text and/or the supplementary materials.
Conflicts of Interest: The authors declare no conflict of interest.
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