Applied Surface Science 463 (2019) 66–74

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Applied Surface Science

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

Full Length Article

Green tea extract mediated biogenic synthesis of silver nanoparticles: T

Characterization, cytotoxicity evaluation and antibacterial activity
Wallace R. Rolima,b, Milena T. Pelegrinoa,b, Bruna de Araújo Limac, Letícia S. Ferraza,b,
Fanny N. Costaa, Juliana S. Bernardesd, Tiago Rodiguesa,b, Marcelo Brocchic,
⁎
Amedea B. Seabraa,b,
a
Center for Natural and Human Sciences (CCNH), Federal University of ABC (UFABC), Santo André, SP, Brazil
b
Nanomedicine Research Unit (NANOMED), Federal University of ABC (UFABC), Santo André, SP, Brazil
c
Tropical Disease Laboratory, Department of Genetics, Evolution, Microbiology and Immunology, Institute of Biology, University of Campinas (UNICAMP), Campinas, SP,
Brazil
d
Brazilian Nanotechnology National Laboratory (LNNano), Brazilian Center for Research in Energy and Materials (CNPEM), Campinas, SP, Brazil

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

Keywords: Silver nanoparticles (AgNPs) are widely used in biomedical fields because of their potent antimicrobial activity.
Antibacterial activity Biogenic synthesis of nanoparticles has gained considerable attention due to its simplicity, low cost and absence
Biocompatibility of organic solvents. This work describes the one-pot one-minute biogenic synthesis of AgNPs with a commercial
Biogenic synthesis green tea extract (Camellia sinensis). The tea polyphenols acted as reducing and stabilizing agents for the na-
Green chemistry
noparticles. The surface of the biogenic AgNPs was further coated with polyethylene glycol (PEG) to enhance
Green tea
Silver nanoparticles
their dispersion and biocompatibility. The obtained nanoparticles were extensively characterized by ultraviolet-
visible spectroscopy (UV–vis), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), ther-
mogravimetric analysis (TGA), electronic and atomic force microscopy (AFM), X-ray photoelectron spectroscopy
(XPS), dynamic light scattering and inductively coupled plasma mass spectrometry (ICP-MS). The results de-
monstrated the formation of spherical nanoparticles of pure Ag° coated with tea polyphenols, at the nanoscale
and with moderate polydispersity. The nanoparticles did not exhibit significant toxicity to human keratinocyte
(HaCaT) cells. The antimicrobial efficacy of the biogenic nanoparticles was demonstrated against gram-positive
Staphylococcus aureus (ATCC 29213), gram-negative Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumo-
niae (ATCC 700603), Escherichia coli (ATCC 25922) and Salmonella enterica (ATCC 14028) bacterial strains.
Salmonella enterica was found to be the most sensitive strain to the nanoparticles, with a minimum inhibitory
concentration and minimum bactericidal concentration of 7 and 15 µg/mL, respectively. Interestingly, at the
concentration range in which the AgNPs showed an antibacterial effect, they were not toxic to HaCaT mam-
malian cells. Thus, green tea synthesized AgNPs could find important biomedical applications in the combat of
pathogenic bacteria with low cytotoxicity to normal cells.

1. Introduction due to their ability to disrupt the membrane of pathogenic organisms,

thus disturbing the microorganism’s enzymatic activities [5,6].
Silver nanoparticles (AgNPs) are one of the most engineered nano- Silver nanoparticles can be synthesized by different methods, such
particles used in several commercial areas, including medical devices, as thermal decomposition, chemical reduction of silver ions (Ag+),
healthcare products, cleaning agents, food storage, packing and textile electrochemical methods, pyrolysis, microwave irradiation and laser
coatings. They are also used for environmental applications because of ablation [7,8]. Among them, the chemical reduction is the most tradi-
their potent antimicrobial activity against bacteria, viruses and fungi tional route employed for the synthesis of AgNPs. The most employed
[1–4]. They have been extensively used in biomedical products, such as reducing agents are sodium borohydride and sodium citrate [7,9]. The
adding them to wound dressings, antiseptic sprays and topical creams traditional chemical synthesis protocol involves the presence of the

⁎
Corresponding author at: Center for Natural and Human Sciences (CCNH), Federal University of ABC (UFABC), Av. dos Estados 5001, CEP 09210-580 Santo
André, SP, Brazil.
E-mail address: [email protected] (A.B. Seabra).

https://doi.org/10.1016/j.apsusc.2018.08.203
Received 10 May 2018; Received in revised form 6 August 2018; Accepted 23 August 2018
Available online 24 August 2018
0169-4332/ © 2018 Elsevier B.V. All rights reserved.
W.R. Rolim et al. Applied Surface Science 463 (2019) 66–74

metal precursor (e.g. silver nitrate), the reducing agent (e.g. sodium characterized. The results demonstrated the successful formation of
borohydride) and the capping/stabilizer agent (usually a polymer, such plant-mediated AgNPs by a commercial green tea extract with a simple
as polyethylene glycol, polyvinyl alcohol, chitosan or alginate) to pre- green protocol. Interestingly, the antibacterial activity of the nano-
vent nanoparticle aggregation [4,10]. Although traditional routes to particles against different bacterial strains was demonstrated at con-
synthesize metallic nanoparticles are known to have superior control of centrations where the nanoparticles were not toxic to mammalian cells.
nanoparticle size distribution and reproducibility, these routes involve The work adds to the confirmation of previous reports on the bio-
high energy input, the presence of toxic chemicals, and in some cases, synthesis of nanometals that have potent antibacterial effects and good
the presence of controlled temperature and pressure, which might lead biocompatibility using plant leaf extracts.
to environmental contamination and high costs [11,12].
In contrast, biogenic (or biological) routes to synthesize metallic 2. Materials and methods
nanoparticles, including AgNPs, have been emerging as an economic-
ally feasible option in the “green chemistry” field [8,11–13]. Biogenic 2.1. Materials
synthesis of metallic nanoparticles is known to be simple, eco-friendly,
cost-effective and easily scaled up. It is also performed at room tem- Green tea powder (Camellia Sinensis) was obtained from Sumioka
perature and pressure, in an aqueous environment and in the absence of Shokuhin Kabushikikaisha, Hiraguti, Japan. Silver nitrate (AgNO3),
organic solvents [8,11–13]. Biogenic synthesis of metallic nanoparticles polyethylene glycol (PEG, MW 200), phosphate buffer saline (PBS), and
can be performed using biological systems, including plants, algae, nitric acid (HNO3) were obtained from Sigma Aldrich, St. Louis,
bacteria, yeast, diatoms and fungi [11,14,15]. Among them, plant- Missouri, USA. Sodium hydroxide (NaOH) was purchased from Synth,
mediated synthesis of metallic nanoparticles has gained attention due Diadema, SP, Brazil. All experiments were carried out using analytical
to its simplicity. Compared to other biogenic routes, the use of plant grade water from a Millipore Milli-Q Gradient filtration system
extracts eliminates the process of maintaining cell cultures and is a (Millipore, 18.2 MΏ, USA). For cytotoxicity experiments with HaCaT
lesser biohazard [16,17]. cells, Dulbecco's Modified Eagle's medium (DMEM), a high glucose
The use of a plant-extract as a reducing and capping agent for me- medium, and penicillin/streptomycin were acquired from Sigma-
tallic nanoparticles has drawn attention recently due to its advanta- Aldrich (USA). Fetal bovine serum was acquired from Gibco,
geous protocol because it is a fast, simple, eco-friendly and economic- Invitrogen, (USA). For antibacterial activity, the culture medium, sup-
ally feasible green technique [11,16,17]. Plant phytochemicals act as plements, antibiotics and bovine fetal serum were obtained from
strong reducing agents, leading to the formation of capped nano- Cultilab (Campinas, SP, Brazil). The Mueller-Hinton media (broth and
particles. Thus, a plant extract acts as a natural reducing and capping agar) were purchased from Difco (Franklin Lakes, USA) and the sodium
agent in a single-step and one-pot reaction, thus eliminating multiple chloride (NaCl, P.A. grade), used to obtain saline solution 0.85% w/v,
steps and reducing the costs and chemicals reagents [15–18]. In addi- was purchased from J.T. Baker (Ciudad del Mexico, Mexico).
tion, phytochemicals and aqueous environments replace chemical
compounds and organic solvents, respectively [18]. Nowadays, extracts 2.2. Synthesis of AgNPs with a green tea extract
of various parts of plants (such as seed powder, fruit, bran, peel, bark,
bran, flower and leaf) have been used for the synthesis of metallic na- The synthesis of AgNPs using green tea extract was adapted from a
noparticles [15–18]. Plant extracts have medical value owing to the previous publication [24]. First, the green tea extract was prepared by
presence of phenolic compounds (polyphenols, tannic acid, flavonoids, dissolving 2 g of green tea powder in 100 mL of deionized water, under
terpenoids), amino acids and vitamins [15–18]. stirring and heating at 60 °C. The extract suspension was filtered by
Green tea (Camellia Sinensis) is a rich source of polyphenolic com- Whatman No. 1 filter paper. To prepare AgNPs, 75 mL of AgNO3
pounds, and it has been exploited as a natural source in the synthesis of (0.1 mol/L) was added to 75 mL of green tea extract, and the pH of the
nanoparticles [19,20]. Green tea is mainly composed by epigalloca- final suspension was adjusted to 10.5 by dropping NaOH (1 mol/L)
techin, epigallocatechin-3-gallate (EGCG), epicatechin and epicatechin- [30]. The suspension was further stirred for 15 min and centrifuged at
3-gallate [20,21], which act as reducing and capping agents [22]. The 12000 rpm for 10 min. The supernatant was discarded and the pre-
medical benefits of green tea include anti-inflammatory and antioxidant cipitated AgNPs were washed twice and freeze dried using a lyophilizer.
effects, enhancement of immune function and intestinal protection
against food-borne pathogenic bacteria, among others [23]. Green tea 2.3. Preparation of PEG-AgNPs
phytochemicals have been used in cosmetic and therapeutic applica-
tions. The water-soluble green tea phytochemicals are mainly catechins AgNPs (250 mg) were dispersed in 10 mL deionized water while
and caffeine, which act as reducing agents of metal ions, leading to the 750 mg of PEG-200 was dissolved in 10 mL deionized water. The PEG
formation of capping metallic nanoparticles [24,25]. solution was added to the AgNPs suspension and vigorously stirred for
Polyethylene glycol (PEG) is a water-soluble, synthetic, biocompa- 24 h, leading to the formation of PEG-AgNPs with a ratio of 1:3 w/w
tible polymer used extensively for biomedical applications, mainly in AgNPs:PEG. The PEG-AgNPs were isolated by centrifugation, washed
the coating of nanoparticles to improve their dispersion in an aqueous with water several times and freeze dried.
medium, and also to enhance the nanoparticle penetration into the
mucus layer. In addition, PEG might protect the nanoparticles from 2.4. Characterization of AgNPs and PEG-AgNPs
immune system clearance by preventing the nanoparticle opsonization
in blood, reduce the nanoparticle toxicity and increase the lifetime of Both nanoparticles were characterized by different techniques, as
the nanomaterial in the circulation system [26–29]. described below.
The above-mentioned reasons led to the study of biogenic synthesis
of AgNPs with the one-pot green protocol using a commercial green tea 2.4.1. Ultraviolet–visible spectroscopy (UV–Vis) analysis
extract as a reducing and capping agent. The nanoparticles were further The UV–Vis spectral analysis of AgNPs and PEG-AgNPs was per-
coated with PEG in order to increase the potential applications for the formed by using UV–vis spectrophotometer Agilent 8454 (Palo Alto,
green tea synthesized AgNPs. Both AgNPs and PEG-AgNPs were ex- CA, USA), with a resolution of 1 nm from 200 to 800 nm.
tensively characterized by different materials analytical techniques.
Furthermore, the in vitro evaluation of the permeation of AgNPs, the 2.4.2. X-ray diffraction (XRD)
cytotoxicity and the antimicrobial activity of AgNPs against several The XRD analyses of AgNPs and PEG-AgNPs were performed in
gram-positive and gram-negative pathogenic bacterial strains were reflection geometry with a conventional Bruker D8 ADVANCE with

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DAVINCI design (CuKα radiation of 1.5418 Å and a graphite mono- 2.5. In vitro permeability study of AgNPs
chromator) coupled to a scintillation detector. The samples (approxi-
mately 200 mg powdered AgNPs or PEG-AgNPs deposited onto a glass The permeation assay for the AgNPs was performed using a Franz
substrate of 2 × 2 cm) were measured from 10° to 100° 2θ with a step vertical diffusion cell (Standard Model, 15 mm, 7 mL, Hanson Research
size of 0.02° and a counting time of 5 s per step. The grain size of the Corporation) using a hydrophobic membrane (Strat-M, Merck
AgNPs was calculated with the Debye–Scherrer equation [28]: Millipore) with a porosity ca. 30–50 nm (which mimics the human
D = (kλ)/(β cos θ ) skin). The diffusion cell had two compartments, namely one donor
(1)
chamber and one receptor chamber, separated by a membrane. The
where D is the diameter of crystallite size, λ is the wavelength for Cu donor compartment was filled with 2.5 mL of an aqueous suspension of
kα, β is the full width at half-maximum (FWHM), θ is the Bragg dif- AgNPs (1000 µg/mL). The receptor compartment was filled with
fraction angle and K is a constant (0.94 is used to correspond spherical 6.87 mL of PBS. The diffusion cell was kept at 32.5 °C (skin tempera-
crystallites with cubic symmetry). The XRD data were analyzed ac- ture) under constant magnetic stirring and protected from light. The
cording to the Rietveld Method [31,32]. The software used to perform amount of silver that permeated through the membrane from the donor
the Rietveld refinement was Topas-Academic v.5 [33]. to the receptor compartment was measured using inductively coupled
plasma-mass spectrometry (ICP-MS, Agilent 7900, Hachioji, Japan). An
2.4.3. Fourier-transform infrared spectroscopy (FT-IR) aliquot of 2 mL was withdrawn after 24 h. Subsequently, 40 µL of HNO3
The AgNPs, PEG-AgNPs and green tea extract powder were tritu- was added to the permeated suspension containing AgNPs, and the
rated with pure potassium bromide (KBr) powder. These mixtures were sample was analyzed on ICP-MS for silver detection and quantification.
ground into fine powders, pressed in a mechanical press to generate The experiment was performed in triplicate.
translucent pellets and analyzed in using a Bomem B-100 FT-IR spec-
trometer. A pure pellet of KBr was used for background. The FT-IR
2.6. Cell culture and cytotoxicity of AgNPs and PEG-AgNPs
spectra were recorded from 400 to 4000 cm−1 at a resolution of
4 cm−1.
Human keratinocytes (HaCaT) cells were grown in Dulbecco's
Modified Eagle's medium (DMEM), a high glucose medium (Sigma-
2.4.4. Thermogravimetric analysis (TGA)
Aldrich, USA), with a pH of 7.2. The medium was supplemented with
The TGA of the pure green tea extract, AgNPs and PEG-AgNPs were
10% fetal bovine serum (Gibco, Invitrogen, USA), 100 U/mL penicillin
carried out to determine the amount of plant residue and PEG on me-
and 100 μg/mL streptomycin in a 5% CO2 atmosphere at 37 °C
tallic surface of the AgNPs through thermal degradation. The analyses
(Panasonic MCO-19AIC, Japan). The cell line was tested to be myco-
were performed by using a TA Instruments Q500 TGA with approxi-
plasma-free by indirect staining with Hoechst 33258 (Thermo Fisher
mately 10 mg of the samples, under N2 atmosphere and with a heating
Scientific, USA) and was used within 3 months of thawing the frozen
rate of 5 °C min−1.
stock. The cytotoxicity of AgNPs and PEG-AgNPs was evaluated by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
2.4.5. Atomic force microscopy (AFM)
reduction assay at different nanoparticle concentrations (10–150 µg/
Topography and phase contrast images of AgNPs and PEG-AgNPs
mL). Cells were seeded for 24 h followed by the addition of nano-
were simultaneously obtained by using an Anasys nanoIR2s in tapping
particles in a supplemented DMEM medium and incubated for 24 h at
mode. A NanoWorld cantilever with K = 42 N m−1 and resonance fre-
37 °C in a 5% CO2 atmosphere. Next, a MTT solution (0.25 mg mL−1)
quency within 260–320 kHz were used. Before AFM imaging, 10 µL of
was added to each well and incubated for 4 h. Thereafter, 0.1 mL of
the of nanoparticle dispersions were deposited on a mica surface and
10% sodium dodecyl sulfate (SDS) (prepared in 10 mmol L−1 HCl) was
allowed to dry by slow evaporation at room temperature.
added and incubated overnight to dissolve the formazan crystals, and
the plates were read at 570 nm with 630 nm (BiochromAsys Expert Plus
2.4.6. Transmission electron microscopy (TEM)
Microplate Reader, Biochrom Ltd., UK). The control was performed in
Images of the green tea synthesized AgNPs were obtained with a
the absence of the nanoparticles and considered as 100%.
Carl Zeiss LIBRA® 120 TEM with an accelerating voltage of 120 kV
(Zeiss International, Oberkochen, Germany), operating at 80 kV using
the software iTEM. One drop of aqueous suspension of green tea syn- 2.7. Antibacterial activities of AgNPs and PEG-AgNPs
thesized AgNPs was deposited on 200 mesh holey carbon film sup-
ported on a copper grid and dried at room temperature. The minimal inhibitory concentration (MIC) and minimal bacter-
icidal concentration (MBC) of the AgNPs and PEG-AgNPs were carried
2.4.7. Dynamic light scattering (DLS) measurements out by microdilution assay as described by the Clinical and Laboratory
The average hydrodynamic diameter (assayed by % of number), Standards Institute (CLSI, 2017). The bacterial strains evaluated were
polydispersity index (PDI) and zeta potential of AgNPs and PEG-AgNPs the gram-positive strain Staphylococcus aureus ATCC 29213 (CLSI
were evaluated by DLS using the Zetasizer Nano ZS (Malvern standard) and the gram-negative bacterial strains (CLSI standard)
Instruments Co, UK). Measurements were performed at 25 °C using a Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and
fixed angle of 173° in disposable folded capillary zeta cells with a Salmonella enterica ATCC 14028. Initially, the bacteria were grown in a
10 mm path length in aqueous suspension. Mueller–Hinton solid medium at 37 °C to obtain isolated colonies.
Subsequently, the colonies were solubilized in a 0.85% (w/v) saline
2.4.8. X-ray photoelectron spectroscopy (XPS) solution and adjusted to the 0.5 index of the MacFarland scale
Chemical surface analyses of AgNPs and PEG-AgNPs were obtained (1.5 × 108 colony-forming units (cfu) mL−1). This solution was diluted
by XPS using a K-Alpha X-ray photoelectron spectrometer (Thermo in Mueller–Hinton broth and distributed in a 96-well plate at a density
Fisher Scientific, UK) equipped with hemispherical electron analyzer of 106 cfu/well. Each well was treated with different concentrations of
and monochromatic Al Kα (1486.6 eV) radiation. Full-range and high- AgNPs and PEG-AgNPs (2-fold dilution: 1000–0.1 µg/mL). The plates
resolution spectra for Ag were acquired using pass energy of 200 and were incubated for 18 h and the bacterial growth was measured after
50 eV, respectively. The data were analyzed using the Thermo Avantage this period. The MBC tests were performed after the MIC tests. After
Software (Version 5.921) and the XPS results presented in this work 24 h of incubation, drops of bacterial broth from the cavity with no
correspond to an average of three measurements performed on different visual growth were added to a Petri dish with the Mueller-Hinton solid
regions of the samples. medium, and bacterial growth was observed after 48 h.

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3. Results and discussion

3.1. Biogenic synthesis AgNPs and PEG-AgNPs

In this work, AgNPs were synthesized by a biogenic route. The use

of a plant extract to synthesize metallic nanoparticles, including AgNPs,
has several advantages including simplicity, low-cost and absence of
organic solvents and/or hazardous materials [1,11–25]. Among several
plants that can be used in the biogenic synthesis of AgNPs, green tea
(Camellia sinensis) was selected due to its rich content of polyphenolic
compounds (mainly catechin), which act as reducing and capping
agents [19,24,30]. The reduction of Ag+ to Ag0 occurred immediately
upon the contact of AgNO3 with the green tea extract and pH adjust-
ment. The formation of the AgNPs was indicated by the suspension
changing color from pale yellow to brown [24,30]. The possible me-
chanism of Ag+ reduction by the green tea is represented in Fig. SF1,
Supporting Information, in which tea polyphenols are ionized, trans-
ferring one electron to Ag+. As a result, Ag+ is reduced to Ag0 (AgNPs) Fig. 1. UV–Vis absorption spectra of AgNPs (black line) and PEG-AgNPs (red
while the tea polyphenols are oxidized to quinone [15,18]. In addition, line), showing the plasmonic band. (For interpretation of the references to color
polyphenols act as capping agent on the surface of biogenically syn- in this figure legend, the reader is referred to the web version of this article.)
thesized AgNPs. The presence of green tea polyphenols might act as an
antioxidant, preserving the nanoparticles [34]. The AgNPs were further results for PEG-AgNPs and AgNPs. The molar extinction coefficients of
capped with PEG to produce an additional protective shell. Several metallic nanoparticles might vary depending on nanoparticle size
synthetic and natural polymers have been used for capping AgNPs. [43,44]. Many factors as such particle size, shape of the nanoparticle,
Among them, PEG is one of the most employed polymers to coat na- chemical nature of the metallic material, wavelength of incident light,
noparticle surfaces due to its resistance to protein adsorption and bio- type of material, and the surrounding media might influence the ab-
compatibility [28,29]. The addition of PEG on nanoparticle surfaces sorption intensity and scattering processes of metallic nanoparticles
impairs the nanoparticle uptake by macrophages in in vivo applications [43]. The presence of PEG on the surface of AgNPs impacts the nano-
[35]. Therefore, the addition of PEG onto the nanoparticle surface particle size and the surrounding media leading to a difference on the
might increase the bioavailability of the nanoparticle, decrease the intensity of the (SPR) absorption band.
nanoparticle clearance by the reticuloendothelial system and improve
the pharmacokinetics of the nanomaterial [36]. Thus, in this work,
biogenically synthesized AgNPs were further coated with PEG to in- 3.2.2. XRD analysis
crease the nanoparticle biocompatibility. Fig. 2 shows the XRD patterns for the AgNPs and PEG-AgNPs. The
Supplementary data associated with this article can be found, in the XRD peak values at 2θ values of 38.16°, 44.44°, 64.55°, 77.41° and
online version, at https://doi.org/10.1016/j.apsusc.2018.08.203. 81.44° correspond to Ag0 (AgNPs) face centered cubic (FCC) planes
(1 1 1), (2 0 0), (2 2 0), (3 1 1) and (2 2 2), respectively [1,30,45]. These
3.2. Characterization of green tea synthesized AgNPs and PEG-AgNPs results were confirmed by using the Rietveld Method for crystal struc-
ture refinement [31,32]. Fig. SF2, Supporting Information, shows the
Biogenic synthesized AgNPs and PEG-AgNPs were characterized by results of the Rietveld refinements of the XRD data for the AgNPs and
different techniques, as described below. PEG-AgNPs. The results indicate the formation of single phase AgNPs
with a cubic structure, and the absence of silver oxide (Ag2O) and silver
3.2.1. UV–Vis spectroscopy analysis
The reduction of Ag+ to Ag0 by green tea can be observed by a color
change. The color of the AgNO3/green tea extract mixture changed
from light silver to brownish black due to the formation of AgNPs [37].
The formation of the AgNPs was confirmed by the detection of the
surface plasmon resonance (SPR) absorption band at ca. 410 nm in the
UV–vis region [30,38]. The formation of SPR was assigned to the re-
sonant oscillation of conducting electrons present on the surface of the
nanoparticle, caused by an interaction with electromagnetic waves
[30]. The yellowish-brown color formation was assigned to the ex-
citation of surface plasmon vibrations in the AgNPs [39]. Fig. 1 shows
the SPR absorption bands for the AgNPs and PEG-AgNPs. It can be
observed that both bands are similar, since no shift is observed in the
maximum absorption, suggesting that the particle size and shape did
not significantly change upon the addition of PEG onto the nanoparticle
surface [19]. In addition, the presence of a single SPR band in the
UV–vis spectrum as associated with the formation of uniformly sphe-
rical nanoparticles, since two or more absorption peaks are associated
with irregular shaped AgNPs [40]. As shown in Fig. 1, the maximum of
SPR band is located at 410 nm (relative low wavelength), suggesting the Fig. 2. X-ray powder diffraction patterns (XDR) of green tea synthesized AgNPs
formation of small nanoparticles [41]. In addition, Fig. 1 shows that the (black line) and PEG-AgNPs (red line) using a Cu source (l 1.5418 Å). Miller
intensity of the SPR absorption band is higher for AgNPs compared with indexes for all Bragg reflections are indicated in the figure. (For interpretation
PEG-AgNPs. The presence of PEG decreased the absorption intensity of of the references to color in this figure legend, the reader is referred to the web
the green tea synthesized AgNPs. Pinzaru et al. [42] reported similar version of this article.)

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W.R. Rolim et al. Applied Surface Science 463 (2019) 66–74

which gave stability to the nanoparticles.

3.2.4. TGA
There are very few reports of the TGA plot for the AgNPs prepared
using tea extracts. Thermal degradation of the pure green tea extract,
AgNPs and PEG-AgNPs heated from 25 °C to 900 °C under nitrogen at-
mosphere are shown in Fig. 4(a). It is possible to observe two major
weight loss ranges. The first one ranged from 3.4% to 4.9% and was
observed at ca. 130 °C due to the loss of adsorbed water [47,50]. The
second weight loss of 11.4–16.9% was found to be in the range of
130–150 °C and was assigned to the thermal degradation of phyto-
chemical compounds on the surface of the green tea synthesized AgNPs
and PEG-AgNPs [47,50]. The weight loss of ∼8.9–10% up to 900 °C
was probably due to thermal degradation of resistant aromatic struc-
tures of green tea [47]. Data from TGA revealed that the content of the
attached organic shell was found to be 23.7% for AgNPs and 30.4% for
PEG-AgNPs, suggesting the presence of 6.6% of PEG on PEG-AgNPs
surface, as represented in Fig. 4(b). Yallapa et al. [50] and Jia et al. [51]
Fig. 3. FT-IR spectra of pure green tea extract (red line), AgNPs (black line) and
reported a content of 30 – 35% of phytochemicals on the surface of
PEG-AgNPs (blue line). (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.) green tea synthesized AgNPs, whereas Sun et al. [47] reported 39% of
tea leaf extract on biogenically synthesized AgNPs. Therefore, the ob-
tained data agrees with the literature results. It should be noted that the
chloride (AgCl), indicating the formation of pure AgNPs with the green absence of weight gain in Fig. 4(a) suggests that the biogenically syn-
tea extract. The presence of PEG as a capping agent on the surface of the thesized AgNPs remained intact, with no oxidation under nitrogen at-
AgNPs did not lead to any AgNP phase change. mosphere [50].
In addition, the Debye-Scherrer equation was used to determine the
crystallite sizes for the AgNPs and PEG-AgNPs by taking into account 3.2.5. AFM analysis
the (1 1 1) Bragg reflection. Crystallite sizes of 2.17 nm and 2.18 nm for The AFM was used to verify the surface structure and the average
the AgNPs and PEG-AgNPs were obtained, respectively. size of green tea synthesized AgNPs and PEG-AgNPs. Topography and
phase contrast images from different areas were simultaneously ac-
3.2.3. FT-IR analysis quired, and some representative micrographs are depicted in Fig. 5. As
FT-IR measurements were carried out to confirm the presence of observed in Fig. 5(a) and (c), the nanoparticles were aggregated. In
polyphenols and caffeine derived from the green tea extract on the some regions of the phase contrast images, isolated particles were de-
surface of biogenically synthesized AgNPs and PEG-AgNPs. Fig. 3 shows tected and are indicated by blue arrows, as indicated in Fig. 5(b) and
the FT-IR spectra of pure green tea powder (red line), AgNPs (black (d). The average diameters obtained for AgNPs and PEG-AgNPs were
line) and PEG-AgNPs (blue line). Fig. 3 reveals similar FT-IR profile for found to be 3.9 ± 1.6 nm and 4.2 ± 1.3 nm, respectively. These re-
all samples. The bands at 3440 cm−1 and 2913 cm−1 are associated sults are in accordance with the crystallite sizes of the nanoparticles, as
with O-H stretching vibration assigned to eOH group of polyols such as described in Section 3.2.2. Another important feature revealed by AFM
catechins [46] and CeH and CH2 vibration of aliphatic hydrocarbons, is that a broken film was observed in the background for the nano-
respectively [1,24]. The band at 1630 cm−1 is associated to C]O vi- particle coated with PEG (Fig. 5(c) and (d)). This is a typical pattern
bration of bonded conjugated ketones, quinones, carboxylic acids and observed when thin films of macromolecules dwelled upon the sub-
esters [47,48]. The band at 1392 cm−1 is associated to CeN stretching strate during the drying process [52]. The obtained AFM results are in
vibration of aromatic amines, indicating the presence of water-soluble agreement with published reports [52,53].
caffeine, and the band at 1044 cm−1 is associated with CeOeC
stretching vibration [24,39]. These results are in accordance with 3.2.6. TEM analysis
previous reports [19,24,49] and demonstrated the presence of green tea The TEM was used to determine the size and morphology of the
polyphenols as capping agents on the surface of AgNPs and PEG-AgNPs, green tea synthesized AgNPs. A representative image of the AgNPs is

Fig. 4. TGA curves of AgNPs (black line), PEG-AgNPs (red line) and green tea powder (green line) (a); Schematic representation of the obtained AgNPs capped with
green tea extract compounds and PEG (b). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 5. Atomic Force Microscopy (AFM) images of topography (left) and phase-contrast images (right) of AgNPs (a, b) and PEG-AgNPs (c, d). The blue arrows indicate
the presence of isolated nanoparticles.

shown in Fig. SF3, Supporting Information. The abundance of spherical presented in the green tea extract (average hydrodynamic size of 44 nm,
shaped nanoparticles can be observed, with little agglomeration. These PDI value of 0.21 and zeta potential of – 35.9 mV for catechin-AgNPs).
results are in accordance with images of the nanoparticles obtained by In the case of PEG-AgNPs, the average hydrodynamic size dis-
field emission electron microscopy (data not shown). These results are tribution was 43.87 ± 3.08 nm, the average PDI was 0.25 ± 0.02 and
consistent with the earlier results of plant mediated synthesis of AgNPs, the average zeta potential was - 37.03 ± 1.49 mV. The hydrodynamic
including green tea [1,19,54]. size of the nanoparticles was found to be higher in comparison to the
average size observed at solid state. The presence of extra hydrate
3.2.7. DLS measurements layers, along with ions or molecules attached to the nanoparticle sur-
The DLS measurements revealed that the average hydrodynamic face in an aqueous environment, was responsible for the higher hy-
size of green tea synthesized AgNPs was 34.68 ± 4.95 nm, the average drodynamic sizes. An additional contributor, to a certain degree, was
PDI value was 0.28 ± 0.01 and the average zeta potential was - the aggregation, as previously reported [1,28,54]. The results indicated
35.5 ± 3.32 mV. Seabra et al. [22] reported similar DLS results for the formation of nanoparticles at the nanosize scale in an aqueous en-
biogenically synthesized AgNPs by catechin, the main polyphenol vironment, and PDI values that suggest moderate polydispersity. As

Fig. 6. X-ray photoelectron spectroscopy (XPS) results for AgNPs and PEG-AgNPs (a) surface composition; (b) high resolution Ag 3d spectra.

71
W.R. Rolim et al. Applied Surface Science 463 (2019) 66–74

expected, the hydrodynamic size of the nanoparticles increased by the

addition of PEG layer, as previous reported [28].
The negative values of zeta potential were assigned to the presence
of the negative charge of green tea polyphenols [22]. This result in-
dicates the presence of polyphenols, such as catechin, on the surface of
the nanoparticles. The addition of PEG coating on the surface of green
tea synthesized AgNPs maintained a negative zeta potential value.
Several reports demonstrated similar negative values of zeta potential
for metallic nanoparticles coated with different molecular weights of
PEG [28,55]. In addition, the magnitude of the obtained zeta potential
values observed in this work suggested the high stability of the obtained
colloidal nanoparticle suspension [47,56]. The obtained DLS results are
in accordance with similar reports based on biogenically synthesized
AgNPs [1,22,54] and PEG-coated metallic nanoparticles [28].
Fig. 7. HaCat cell viability after incubation with AgNPs and PEG-AgNPs at
3.2.8. XPS measurements different concentrations for 24 h. **, # Statistically different from the re-
The chemical surface composition of green tea synthesized AgNPs spective controls performed without NPs (p < 0.05).
and PEG-AgNPs acquired by XPS is shown in Fig. 6. Carbon, oxygen and
silver elements were all present on the surface of both nanoparticles, as
this was observed for PEG-AgNPs only at 150 µg/mL. Additionally, the
shown in Fig. 6(a). The atomic percentage of Ag atoms was similar for
comparison between the two nanoparticles revealed that PEG-AgNPs
the AgNPs with, and without, physio adsorbed PEG. On the other hand,
were found to be less toxic to the cells (Fig. 7).
the oxygen % reduced while the carbon content increased when PEG
Similarly, a previous study showed that AgNPs synthesized from
partially replaced the green tea extract capping agent. High-resolution
green tea extract at concentrations up to 100 µg/mL did not exhibit
spectra of the Ag for the two nanoparticles are shown in Fig. 6(b). The
cytotoxicity to HaCaT cells and did not induce changes in cellular
results indicate well separated spin-orbit components (Δ = 6.0 eV),
morphology [21]. Carrola et al. [59] evaluated the cytotoxicity of PEG-
asymmetric peaks and loss features (blue arrows), which are char-
coated AgNPs and citrate-coated AgNPs (at 10 and 40 µg/mL), and also
acteristic of Ag metal, suggesting that AgNPs were successfully syn-
proposed that PEG- and citrate-coated AgNPs did not present significant
thesized using the green tea extract [17].
impact on the cell viability. Other reports showed that PEG-coated
AgNPs were not cytotoxic at concentrations up to 100 µg/mL
3.3. In vitro permeability study of AgNPs
[27,59,60]. Furthermore, it was also reported that low AgNP con-
centrations (0.25–2.5 µg/mL) had beneficial effects on wound healing
The characterization of the permeation of the AgNPs was necessary
[61].
to evaluate the possible toxicity of them when in contact with human
Several parameters, such as nanoparticle size, shape, degree of ag-
skin, such as in topical applications, where the nanoparticles might
glomeration, chemical surface composition, charge, concentration and
have beneficial effects. For instance, the skin penetration of AgNPs is an
time of exposition might significantly influence the nanomaterial toxi-
important issue when designing suitable topical Ag-based nanomater-
city [5,12]. It is noteworthy that several reports described the cyto-
ials as antibacterial agents, with minimum systemic toxicity [57]. It has
toxicity of AgNPs, inducing protein alterations [5]. The use of tea
been demonstrated that AgNPs can penetrate through either damaged
polyphenols to synthesize AgNPs is responsible for the capping of the
or intact human skin [57]. In this study, the permeation of green tea
obtained nanomaterial with phytochemicals that act as potent anti-
synthesized AgNPs was evaluated by using a Franz diffusion cell, with a
oxidant and anti-inflammatory agents, reducing the potent toxicity of
hydrophobic membrane that mimics human skin. After 24 h of in-
the nanoparticles [62].
cubating the AgNPs (initial concentration of 1000 μg/mL) in the ver-
tical diffusion cell, a flux of 40.79 ± 10.03 μg/cm2 of silver was per-
meated, as detected by ICP-MS. This result indicates that AgNPs might 3.5. Antimicrobial activity
be able to permeate human skin; thus, their biocompatibility should be
further studied. Antimicrobial activity of the green tea synthesized AgNPs and PEG-
Huang et al. [58] showed that concentrations smaller than 40 µg/ AgNPs was evaluated against gram-positive Staphylococcus aureus
mL did not exhibit toxic effects to blood. Thus, the permeation flux (ATCC 29213) and gram-negative Pseudomonas aeruginosa (ATCC
studied is a safe concentration to blood. Tak et al. [57] characterized 27853), Klebsiella pneumoniae (ATCC 700603), Escherichia coli (ATCC
the in vitro and in vivo skin permeation for different shaped AgNPs 25922) and Salmonella enterica (ATCC 14028) bacterial strains at dif-
(spherical, rod and triangular nanoparticles). All shaped AgNPs were ferent concentrations. Table 1 shows the MIC and MBC values obtained
found to permeate the skin, and the triangle AgNPs displayed the after 24 h of bacterial incubation with the nanoparticles. As can be
highest penetration capability and accumulation on the dermal layer. observed, the antibacterial effect was dependent on the bacteria strain
As green tea synthesized AgNPs were found to permeate skin, the cy- and the chemical surface of the AgNPs. Salmonella enterica was found to
totoxicity of these nanoparticles was evaluated (next section). be the most sensitive strain to the nanoparticles, with MIC values of 7
and 60 µg/mL and MBC values of 15 and 60 µg/mL for AgNPs and PEG-
3.4. Cytotoxicity of AgNPs and PEG-AgNPs AgNPs, respectively. In contrast, Klebsiella pneumoniae was found to
have the highest MIC and MBC values (250–500 µg/mL) for both na-
The cytotoxicity of AgNPs and PEG-AgNPs was evaluated on human noparticles. Similar results have been reported for catechin-synthesized
epidermal keratinocytes cells (HaCaT) as a model to simulate the con- AgNPs [22], Swertia paniculata herbal extract synthesized AgNPs [1]
tact of nanoparticles with the skin. The HaCaT cells were incubated for and olive leaf extract synthesized AgNPs [63]. In all tested bacteria
24 h with green tea synthesized AgNPs and PEG-AgNPs, at concentra- strains, green tea synthesized AgNPs were found to have lower MIC and
tions ranging from 0 to 150 µg/mL. As observed in Fig. 7, both nano- MBC values compared with PEG-AgNPs (Table 1). This effect might be
particles did not promote a significant decrease in the HaCaT viability, explained by considering that the toxicity of AgNPs is dictated by
except for the higher concentrations. The cytotoxicity of AgNPs was several parameters such as nanoparticle size, the chemical nature of
statistically significant at concentrations higher than 50 µg/mL, while nanoparticle surface, surface charge, nanoparticle composition, and

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W.R. Rolim et al. Applied Surface Science 463 (2019) 66–74

Table 1 using the Franz diffusion cell with an artificial membrane that mimics
MIC and MBC values (µg/mL) for AgNPs and PEG-AgNPs tested using different human skin, the nanoparticles were not significantly toxic to the HaCaT
bacterial strains. cell line. Furthermore, AgNPs and PEG-AgNPs showed a potent anti-
Bacteria AgNPs PEG-AgNPs bacterial effect against several pathogenic gram-positive and gram-ne-
gative bacteria. Interestingly, the concentrations of AgNPs required to
MIC MBC MIC MBC (µ̫ g/ achieve an anti-bacterial effect towards Escherichia coli, Pseudomonas
(µ̫ g/mL) (µ̫ g/mL) (µ̫ g/mL) mL)
aeruginosa and Salmonella enterica were not cytotoxic to HaCaT cells.
Staphylococcus aureus 250 250 250 250 Therefore, plant mediated biogenic synthesis of AgNPs is a simple, ef-
ATCC 29213 ficient and cost-effective route to prepare AgNPs with considerable
Klebsiella pneumoniae ATCC 250 250 500 500 biocompatibility and potent antibacterial actions. These nanoparticles
700603
may find important biomedical applications.
Escherichia coli 15 15 60 60
ATCC 25922
Pseudomonas aeruginosa 30 30 125 125 Acknowledgments
ATCC 27853
Salmonella enterica 7 15 60 60 We would like to thank Proof Reading Service (Proof-Reding-
ATCC 14028
Service.com) for revising the manuscript. We have appreciated the
support from CNPq, FAPESP (2016/10347-6), INCT-INOMAT,
agglomeration state [5,11]. As the proposed mechanism of nanoparticle NanoBioss-SisNANO, MCTI) and the Brazilian Network on
toxicity towards bacterial involves the direct interaction of the nano- Nanotoxicology – Cigenanotox (CNPq/MCTI). We would like to thank
particle with the pathogen, the chemical surface of the nanomaterial is Prof. Bruno Lemos Batista for his assistance with ICP-MS measure-
important for the nanoparticle toxicity. The presence of PEG on the ments. We thank Douglas Soares da Silva (CNPq fellow Proc 371220/
surface of AgNPs increased the nanoparticle size (as demonstrated in 2017-3) for his assistance with the TEM measurements. The authors are
this work), which can affect the nanoparticle antimicrobial effect. In grateful to the Multiuser Central Facilities (UFABC) for the experi-
addition, the presence of PEG created an extra layer on the nanoparticle mental support.
surface, which interferes with the direct interactions between AgNP and
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