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Antibiofilm and anticancer potential of β-glucan-binding protein-encrusted zinc oxide nanoparticles
Journal Pre-proof Antibiofilm and anticancer potential of β-glucan-binding protein-encrusted zinc oxide nanoparticles Mani Divya, Marimuthu Govindarajan, Sivashanmugam Karthikeyan, Elumalai Preetham, Naiyf S. Alharbi, Shine Kadaikunnan, Jamal M. Khaled, Taghreed N. Almanaa, Baskaralingam Vaseeharan PII:
S0882-4010(19)32227-2
DOI:
https://doi.org/10.1016/j.micpath.2020.103992
Reference:
YMPAT 103992
To appear in:
Microbial Pathogenesis
Received Date: 23 December 2019 Revised Date:
20 January 2020
Accepted Date: 21 January 2020
Please cite this article as: Divya M, Govindarajan M, Karthikeyan S, Preetham E, Alharbi NS, Kadaikunnan S, Khaled JM, Almanaa TN, Vaseeharan B, Antibiofilm and anticancer potential of β-glucan-binding protein-encrusted zinc oxide nanoparticles, Microbial Pathogenesis (2020), doi: https:// doi.org/10.1016/j.micpath.2020.103992. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Author statement
Mani Divya: Conceptualization, Methodology, Software; Marimuthu Govindarajan: Writing - Review & Editing; Sivashanmugam Karthikeyan: Methodology, Software; Elumalai Preetham: Methodology, Software; Naiyf S. Alharbi: Resources, Validation; Shine Kadaikunnan: Formal analysis; Jamal M. Khaled: Visualization, Validation; Taghreed N. Almanaa: Formal analysis; Baskaralingam Vaseeharan: Conceptualization, supervision, Writing- review and editing
Graphical abstract
1
Antibiofilm and anticancer potential of β-glucan-binding protein-encrusted zinc
2
oxide nanoparticles
3 4
Mani Divyaa, Marimuthu Govindarajanb,c, Sivashanmugam Karthikeyand, Elumalai
5
Preethame, Naiyf S. Alharbif, Shine Kadaikunnanf, Jamal M. Khaledf, Taghreed N. Almanaaf,
6
Baskaralingam Vaseeharana*
7 8
a
9
Nanopharmacology Division, Department of Animal Health and Management, Alagappa
Biomaterials
and
Biotechnology
in
Animal
Health
Lab,
10
University, Karaikudi-630004, Tamil Nadu, India
11
b
12
Annamalai University, Annamalainagar 608 002, Tamil Nadu, India
13
c
14
for Women (Autonomous), Kumbakonam 612 001, Tamil Nadu, India
15
d
16
e
17
Panangad, Kerala, India
18
f
19
Saudi Arabia
Nanobiosciences
and
Unit of Vector Control, Phytochemistry and Nanotechnology, Department of Zoology,
Unit of Natural Products and Nanotechnology, Department of Zoology, Government College
School of Bioscience & Technology, VIT University, Vellore, 632014, Tamil Nadu, India
School of Ocean Science and Technology, Kerala University of Fisheries and Ocean Studies,
Department of Botany and Microbiology, College of Science, King Saud University, Riyadh,
20 21
* Corresponding Author. Baskaralingam Vaseeharan. Tel: +91 4565 225682. Fax: +91 4565
22
225202. E-mail: [email protected]
23
24 25
Abstract β-Glucan-binding protein (βGBP) is important for the rational expansion of molecular
26
biology. Here, zinc oxide nanoparticle (ZnONP) was synthesized using βGBP from the crab
27
Scylla serrata (Ss-βGBP-ZnONP). Ss-βGBP-ZnONP was observed as a 100 kDa band on
28
sodium dodecyl sulfate polyacrylamide gel and characterized with UV-vis spectroscopy at
29
350 nm. X-ray diffraction analysis displayed values consistent with those for zincite. Fourier
30
transform infrared spectroscopy revealed the presence of functional groups, including amide,
31
alcohol, alkane, alkyl halide, and alkene groups. The zeta potential (−5.36 mV) of these
32
particles indicated their stability, and transmission electron microscopy revealed the presence
33
of 50 nm nanocones. Ss-βGBP-ZnONPs were tested at 100 µg/mL against the gram-positive
34
Enterococcus faecalis and gram-negative Pseudomanas aeruginosa using confocal laser
35
scanning microscopy and the bacterial viability assay was also performed. The growth of
36
MCF7 breast cancer cells was inhibited following treatment with 75 µg/mL Ss-βGBP-
37
ZnONPs. Thus, Ss-βGBP-ZnONPs have the ability to control the growth of pathogenic
38
bacteria and inhibit the viability of MCF7 breast cancer cell lines.
39 40
Keywords
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β-Glucan-binding protein; biofilm; confocal laser scanning microscopy; Pseudomanas
42
aeruginosa; cell line
43 44
45
1. Introduction
46
Nanotechnology has widespread applications in the field of science and medicine [1-6].
47
Nanoparticles (NPs) are used as imaging and therapeutic agents and their surfaces are often
48
coupled with biological molecules to reduce surface energy [7]. Metallic and metal oxide
49
NPs, including gold, silver, copper, titanium dioxide (TiO2), and zinc oxide (ZnO) particles,
50
play vital roles [8-11]. The ZnONPs have been used for various applications, including cell
51
imaging, drug delivery, and nanomedicine [12]. The ZnONPs are also used in goods such as
52
personal care products and are known to exhibit excellent antibacterial and UV blocking
53
properties. In vitro analyses have revealed the biocompatibility of ZnONPs [13]. The ZnONPs
54
serve as non-toxic, biocompatible, and safe drug carriers. Zinc is an important trace element
55
present in all body tissues, including the brain, muscle, bone, and skin [14]. Zinc is also
56
important for eukaryotes, as it is known to modulate several physiological functions [15]. In
57
biological systems, zinc has important roles in metabolic pathways such as the carbohydrate,
58
lipid, nucleic acid, and protein syntheses [16]. The NPs may interact with biomolecules such
59
as nucleic acids, proteins, and lipids, owing to their nano size [17]. Biological synthesis of
60
NPs is more labor- and time-intensive and expensive than physical and chemical methods
61
[18].
62
Cancer is an emerging disorder that is severely affecting human health. It is the second
63
leading cause of mortality worldwide. The prevalence of breast cancer is higher among
64
women [19] and corresponds to one-fifth of all cancer cases worldwide. Studies have
65
confirmed the cytotoxicity of ZnONPs on cancer cells, including HepG-2, MCF-7, Hela, U87,
66
and S91 cells [20]. To improve their targeting ability, these NPs are often coupled with
67
antibodies or peptides that bind to the receptors over expressed on cancer cells. Today,
68
different types of nanostructures, particularly ZnONPs, have gained significant attention
69
owing to their wider implications in cancer therapy [21]. The current anticancer
70
chemotherapies have restricted potential due to systemic toxicity, non-selective distribution,
71
and decreased bioavailability, which severely damage healthy tissues [22]. Some bioactive
72
compounds are used as chemopreventive agents against cancer [23]. Cancer therapies are
73
limited to radiation, surgery, and chemotherapy, all of which exert harmful effects on normal
74
cells. Nanotechnology has been employed for the targeted treatment of cancers. The
75
mechanism underlying ZnONP-induced toxicity involves oxidative stress and apoptosis in
76
selected cancer cells. The combination of macromolecules and NPs exerts strong effects on
77
biological systems, and ZnONPs have a potent activity against competent cancer cells [24].
78
The NPs have been applied in the field of cancer therapy to target useful anticancer drugs
79
in patients suffering from cancer [20]. β-1,3-Glucan-binding protein (β-GBP) is an abundant
80
protein in the hemolymph of invertebrates. It is involved in the response of invertebrates to
81
pathogenic microorganisms. β-GBP exerts phenoloxidase and peptidase activities [25].
82
However, the antimicrobial activity of β-GBP is yet undetermined. β-GBP has been purified
83
from a mixture of crustaceans, including Scylla serrata [26] and the mangrove crab
84
Episesarma tetragonum [27].
85
Here, β-GBP from S. serrata was used to coat ZnONP. The developed Ss-βGBP-ZnONPs
86
were purified, characterized, and functionally examined for their antibacterial activity against
87
gram-positive Enterococcus faecalis and gram-negative Pseudomanas aeruginosa. In
88
addition, cell viability of the selected bacterial strains was tested after treatment with 50 and
89
100 µg/mL Ss-βGBP-ZnONPs. We also determined the cytotoxicity of Ss-βGBP-ZnONPs to
90
MCF7 cancer cells. We show that Ss-βGBP-ZnONPs inhibited the growth of bacteria and
91
suppressed the proliferation of MCF7 breast cancer cells. Hence, Ss-βGBP-ZnONPs may
92
serve as effective agents for the treatment of pathogenic bacteria and cancer cells.
93 94
2. Materials and methods
95 96
2.1. Purification and synthesis of Ss-βGBP-ZnONPs βGBP from S. serrata was purified as previously reported [26]. Sodium dodecyl sulfate
97
polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the size of Ss-βGBP-ZnONPs to
98
be 100 kDa. For Ss-βGBP-ZnONP synthesis, 2.1 g of 0.02 M zinc acetate was dissolved in
99
500 mL distilled water and stirred for 20 min with a stirrer. The purified form of Ss-βGBP (5
100
mL) was added to the zinc acetate solution and the mixture was stirred for 12 h. The final
101
sample was calcined for 30 min at 100 °C to obtain Ss-βGBP-ZnONPs. The dried powder was
102
stored in a sealed container for further analysis.
103 104
2.2. Characterization of Ss-βGBP-ZnONPs
105
The synthesized Ss-βGBP-ZnONPs (1 mL) were subjected to UV-Vis spectrum analysis
106
as a function of time on a spectrophotometer at 200-800 nm wavelength and 1 nm resolution.
107
The crystalline nature of Ss-βGBP-ZnONPs was determined by X ray diffraction (XRD)
108
analysis (Powder X-ray diffractometer 1 X’ pert pro-P analytic) with λ value of 1.5406 Å.
109
Scanning was carried out at a 2θ range from 30 to 90. Fourier transform infrared (FTIR)
110
spectrophotometry (Thermo scientific Nicolet 380) was used for the analysis of functional
111
sets. Finely powdered (150 mg) Ss-βGBP-ZnONPs were treated with potassium bromide and
112
subjected to vacuum treatment using a pelletizer. The infrared spectrum was recorded
113
between 4000 and 500 cm−1. Stabilized Ss-βGBP-ZnONPs were analyzed with a 90 plus
114
particle size analyzer (Brookhaven instruments corporation, Holtsville, NY, USA) under an
115
applied electric field, and zeta potential was recorded. Ss-βGBP-ZnONPs were placed on
116
copper stubs and their shape and size were observed by transmission electron microscopy
117
(TEM) (HITACHI; Model: S-3400N) under bright field. The Ss-βGBP-ZnONPs shape, size
118
and selected area electron diffraction (SAED) were confirmed using carbon coated grids. A
119
working voltage of 20 kV with secondary electron detectors at the elemental action was
120
indomitable with energy dispersive X-ray spectroscopy (EDX, Joel JSM-6510) analysis.
121 122
2.3. Potency of Ss-βGBP-ZnONP against bacterial biofilms
123
The effects of Ss-βGBP-ZnONPs on bacterial biofilms were evaluated using gram-
124
positive E. faecalis and gram-negative P. aeruginosa with an enzyme-linked immunosorbent
125
assay (ELISA) plate reader. Confocal laser scanning microscopy (CLSM) was used for the
126
analysis of biofilm assay. Selected bacteria were cultured on glass pieces (1 × 1 cm) in
127
nutrient broth supplemented with Ss-βGBP-ZnONPs (50 and 100 µg/mL). As a control, Ss-
128
βGBP-ZnONPs were replaced with an equal concentration of bovine serum albumin (BSA).
129
The cells were incubated for 2 days at 36 °C and then stained with acridine orange (0.4%).
130
The cells were rinsed with phosphate-buffered saline (PBS) and analyzed using CLSM with
131
488-nm argon laser and Zen software (Carl Zeiss, Germany).
132 133 134
2.4. Bacterial viability assay The bacterial viability assay was performed as reported by Velusamy et al. [28] using the
135
live/dead BacLight bacterial viability kit (Invitrogen, India). Live and dead bacterial cells
136
were labelled with SYTO®9 (green) fluorescence and propidium iodide (PI; red) fluorescence
137
staining agents, respectively. Overnight cultured bacterial cells were treated with Ss-βGBP-
138
ZnONPs (50 and 100 µg/mL) at 37 °C at 60 min. After treatment, the cells were centrifuged
139
for 10 min at 6000 ×g and the pellet was rinsed twice with PBS, followed by incubation with
140
5 µL of SYTO®9, PI, and dimethyl sulfoxide (DMSO) in 990 µL of PBS. The cells were
141
stained with 10 µL SYTO® 9 and 20 µL of PI (1:2 ratio) at 27 °C for 15 min in the dark, and
142
subjected to CLSM.
143 144
2.5. Anticancer activity
145
The inhibitory effect of Ss-βGBP-ZnONPs on MCF7 cancer cell growth was assessed
146
with the 3-4, 5-dimethyl thiazol-2yl-2,5-diphenyl tetrazolium bromide (MTT) assay [29]. The
147
cells were cultured in ELISA plates (5000 cells in each well) for 1 day in the presence of 5,
148
25, 50, and 75 µg/mL of test agent. The absorbance spectra were measured at a wavelength of
149
595 nm using a microplate reader. Cell viability (% of control) was calculated as (ODtest −
150
ODblank)/(ODcontrol − ODblank), where ODtest is the optical density of the cells exposed to the
151
ZnO sample, ODcontrol is the optical density of the control sample, and ODblank is the optical
152
density of the wells without MCF7 cells.
153 154
3. Results
155
3.1. Synthesis and characterization of Ss-βGBP-ZnONPs
156
ZnONPs were synthesized using the purified form of β-GBP from S. serrata following 30
157
min incubation. The treatment with zinc acetate resulted in a pale white precipitate, indicative
158
of the formation of Ss-βGBP-ZnONPs. The size of the synthesized Ss-βGBP-ZnONPs was
159
confirmed to be 100 kDa by SDS-PAGE analysis (Fig.1). UV-vis spectrophotometry analysis
160
was performed at a wavelength of 350 nm (Fig.2). In brief, 0.1 mL of sample was prepared in
161
deionized H2O in a cuvette, and the change from bluish to pale white precipitate was
162
indicative of the synthesis of ZnONPs.
163
The diffraction peaks showed various Bragg’s expression at 2.8 (100), 2.5 (002), 2.4
164
(101), 1.9 (102), 1.6 (110), 1.4 (103), 1.3 (112), and 1.2 (202). The above mentioned values
165
were consistent with the values for zincite of ZnO phase (Joint committee on Powder
166
Diffraction Standards, JCPDS 75-0576; Fig.3). The ZnONPs present in Ss-βGBP were
167
determined with FTIR spectroscopy. The FTIR spectrum of Ss-βGBP-ZnONPs confirmed
168
strong bands at 1644, 1022, and 892 cm−1 belonging to C = O, C-F, and =C-H, respectively.
169
Weak bands were observed at 1556 and 1407 cm−1 that corresponded to amide and alkene
170
groups, respectively. However, the broad spectrum of strong bands of alcohol at 3418 cm−1
171
was associated with O-H stretching.
172
Overall, the analysis demonstrated the presence of amide, alcohol, alkane, alkyl halide,
173
and alkene groups (Fig.4). Zeta potential value revealed the surface charge and stability of Ss-
174
βGBP-ZnONPs; its conductivity was 0.233 mS/cm and zeta potential, −5.36 mV (Fig.5).
175
These values demonstrate the complete stabilization of NPs as well as their efficiency to serve
176
as capping materials. TEM of Ss-βGBP-ZnONPs revealed the presence of nano cones of 50
177
nm diameter (Fig.6a&b). Nature of Ss-βGBP-ZnONPs and lattice spacing were observed with
178
selected area electron diffraction (Fig.6c). Zinc elemental composition of Ss-βGBP-ZnONPs
179
was 40.78% and the weight % of oxygen was 6.54% (Fig.6d).
180 181 182
3.2. Effects of Ss-βGBP-ZnONPs on bacterial biofilms CLSM analysis confirmed the inhibitory effects of Ss-βGBP-ZnONPs on the biofilm
183
formation ability of both gram-positive E. faecalis and gram-negative P. aeruginosa. Both
184
bacteria produced well-ordered biofilms in the control group. Ss-βGBP-ZnONP-treated
185
groups showed the incompetent structure with separate bacterial cells. The biofilm formation
186
ability was considerably lower in the treatment groups than in the control group. The effect
187
was stronger with 100 µg/mL than that with 50 µg/mL Ss-βGBP-ZnONPs (Fig.7).
188 189 190
3.3. Bacterial viability assay The effects of Ss-βGBP-ZnONPs on bacterial cells were assessed with CLSM using the
191
live/dead BacLight bacterial viability kit. The green and red fluorescence corresponded to
192
live and dead bacterial cells, respectively. The bacterial cells treated with 50 and 100 µg/mL
193
Ss-βGBP-ZnONPs for 60 min were found to be dead (Fig.8). The red fluorescence signal was
194
higher after treatment with 100 µg/mL Ss-βGBP-ZnONPs than that observed following
195
treatment with 50 µg/mL Ss-βGBP-ZnONPs.
196 197 198
3.4. Anticancer activity The cytotoxicity study revealed no morphological alterations in MCF7 cells from the
199
control group. However, the growth of Ss-βGBP-ZnONP-treated cells was affected at a
200
concentration of 25 µg/mL. MCF7 cells treated with 50 µg/mL Ss-βGBP-ZnONP were
201
ruptured (Fig. 9). The cytotoxicity of Ss-βGBP-ZnONPs against MCF7 breast cancer cells
202
was higher at 75 µg/mL concentration than at 25 and 50 µg/mL (Fig. 10).
203 204
4. Discussion
205
Zinc plays an important role in metabolism and protein synthesis. Biological synthesis of
206
NPs in combination with immune molecules may reduce toxicity. In the present study,
207
ZnONPs were synthesized with the purified form of β-GBP from S. serrata (Ss-βGBP-
208
ZnONPs). The synthesized Ss-βGBP-ZnONPs were confirmed to have a molecular mass of
209
100 kDa. The purified form of βGBP from S. serrata has been previously described [26]. Ss-
210
βGBP-ZnONPs were characterized with UV-vis spectroscopy; the spectrum exhibited
211
absorption peaks at 350 nm, consistent with the results of Anjugam et al. [32] for ZnONPs
212
coated with β-GBP (Portunus pelagicus). Chaudhuri et al. [33] showed that ZnONPs obtained
213
from the leaf extract of Calotropis gigantean exhibited an absorption peak near 350 nm.
214
These results confirm the synthesis of ZnNPs. The present findings demonstrate the
215
crystalline nature of Ss-βGBP-ZnONPs; FTIR spectrum reveals the strong and weak bands
216
associated with the presence of amide, alcohol, alkane, alkyl halide, and alkene groups. The
217
stretching vibration reveals the presence of functional groups in ZnONPs. As reported by
218
Anjugam et al. [32], the FTIR spectrum of Ppβ-GBP-ZnONPs showed a peak at 3221 cm−1
219
corresponding to the N-H stretching vibrations of amide groups of the protein. The band at
220
1062 cm−1 was related to the presence of amine groups.
221
Ss-βGBP-ZnONPs had a zeta potential value of −5.36 mV. This elevated negative value
222
confirms the stability of these NPs. Moreover, previous reports on the biosynthesis of
223
ZnONPs from the leaf extract of Calotropis gigantean revealed a zeta potential value of −20.7
224
mV [34]. Gelatin-coated ZnONP also showed a zeta value of −6 mV, indicative of the surface
225
charge of ZnONPs [35]. TEM images showed the nanocone structure of Ss-βGBP-ZnONPs
226
and a standard particle size of 50 nm. This observation is in line with that reported by Kavitha
227
et al. [30], wherein a ZnO nanopowder was found to exhibit a cone shape and an uneven
228
surface. The calcification at 550 °C allows customization of the morphology to nanocones.
229
TEM images of ZnO nanocrystal illustrated the individual cone-shaped structure with an
230
average size of 70 nm [31]. Thus, the synthesized ZnO had a cone structure owing to thermal
231
oxidation.
232
Ss-βGBP-ZnONPs at 100 µg/mL concentration suppressed the bacterial biofilm formation
233
ability. Similar results were reported with Ppβ-GBP-ZnONPs at a concentration of 50 µg/mL
234
[32]. Ishwarya et al. [34] found that the plasma of fish fed with Phβ-GBP-SeNWs could
235
prevent the growth of bacterial colonies and suppress biofilm formation. Anjugam et al. [32]
236
demonstrated the effective anticancer activity of Ppβ-GBP-AgNPs against HeLa cells (50
237
µg/mL). Wahab et al. [21] used a very low concentration of NPs against MCF-7 cancer cells.
238
In the present study, Ss-βGBP-ZnONPs suppressed the viability of MCF7 cells at 75 µg/mL.
239 240 241
5. Conclusion Ss-βGBP-ZnONPs were synthesized and characterized with UV, XRD, FTIR, zeta
242
potential, and TEM. Ss-βGBP-ZnONPs effectively inhibited the biofilm formation ability of
243
gram-positive E. faecalis and gram-negative P. aeruginosa at 100 µg/mL concentration. The
244
bacterial viability of the same tested bacteria was suppressed at 100 µg/mL concentration. The
245
dropping ability of the bacterial biofilm could uplift all fields of research as in valuable. The
246
proliferation of MCF7 breast cancer cells was reduced after treatment with Ss-βGBP-ZnONPs
247
at 75 µg/mL. The particular property of this Ss- βGBP-ZnONPs without difficulty can be
248
elucidate into an effectual therapeutic for cancer therapy without causing dangerous effect to
249
ordinary tissues. Thus, Ss- βGBP-ZnONPs have the ability to inhibit the pathogenic bacteria
250
as well as to control the proliferation of MCF7 breast cancer cells. It provely Ss- βGBP-
251
ZnONPs used as a nanotheraputic agent in feature.
252 253
Conflict of interest
254
The authors declare no conflict of interest.
255 256
Acknowledgements
257
The authors extend their appreciation to the Researchers Supporting Project number
258
(RSP-2019/70), King Saud University, Riyadh, Saudi Arabia. This work was funded by
259
RUSA phase 2.0 grant (ref-24 – 51/2014 – U, Policy) TN. Multi-Gen, Depart of Edn, Govt.
260
of
261
Technology, NGO Colony road, Nagarcoil, Tamil Nadu, India.
India.
The authors also thank to Fundation of Innovative research in Science and
262 263
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Fig 1. SDS-PAGE analysis of purified Ss-βGBP and the synthesized zinc oxide nanoparticle (Ss-βGBP-ZnONPs). Lane-I, molecular marker; Lane-II, purified Ss-βGBP; Lane- III, SsβGBP-ZnONPs (100 kDa).
Fig 2. UV-Visible spectra of Ss-βGBP-ZnONPs
Counts DSS
400
200
0 20
30
40
50
60
70
80
Position [°2Theta] (Copper (Cu))
Fig 3. X-ray diffraction outline shows the crystalline nature of Ss-βGBP-ZnONPs
Fig 4. Fourier transform infrared spectra showing the functional group of Ss-βGBP-ZnONPs
Fig 5. Zeta potential distribution for size and surface charge of Ss-βGBP-ZnONPs
Fig 6. Transmission electron microscopy (TEM) of Ss-βGBP-ZnONPs (a), single element of Ss-βGBP-ZnONPs (b), selected area electron diffraction (SAED) pattern (c), energy dispersive X-ray spectroscopy (EDX) elemental work of Ss-βGBP-ZnONPs (d).
Fig 7. Confocal laser scanning microscopy showing the inhibitory effect of Ss-βGBPZnONPs on E. faecalis and P. aeruginosa biofilms at two concentrations.
Fig 8. Live and dead cell assay with CLSM using pathogenic bacteria E. faecalis and P. aeruginosa after treatment with Ss-βGBP-ZnONPs at 50 and 100 µg/mL.
Fig 9. Morphology of MCF7 breast cancer cells after treatment with different concentrations of Ss-βGBP-ZnONPs under a phase-contrast microscope. Arrows point to the morphological changes in cells.
Fig 10. Effect of zinc, Ss-βGBP, and Ss-βGBP-ZnONPs on the viability of MCF7 cells as compared with the control group; T-bars designated typical errors; different letters on the top of bars indicate important distinctions with treatments (p < 0.05).
Highlights •
β-Glucan-binding protein-coated ZnONPs was synthesized from Scylla serrata
•
Ss-βGBP-ZnONPs was characterized with UV, XRD, FTIR, zeta potential, and TEM
•
Inhibition of biofilms by Ss-βGBP-ZnONPs was confirmed at 100 µg/mL
•
Ss-βGBP-ZnONPs at 75 µg/mL effectively inhibited MCF7 breast cancer cells
S0882-4010(19)32227-2
DOI:
https://doi.org/10.1016/j.micpath.2020.103992
Reference:
YMPAT 103992
To appear in:
Microbial Pathogenesis
Received Date: 23 December 2019 Revised Date:
20 January 2020
Accepted Date: 21 January 2020
Please cite this article as: Divya M, Govindarajan M, Karthikeyan S, Preetham E, Alharbi NS, Kadaikunnan S, Khaled JM, Almanaa TN, Vaseeharan B, Antibiofilm and anticancer potential of β-glucan-binding protein-encrusted zinc oxide nanoparticles, Microbial Pathogenesis (2020), doi: https:// doi.org/10.1016/j.micpath.2020.103992. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Author statement
Mani Divya: Conceptualization, Methodology, Software; Marimuthu Govindarajan: Writing - Review & Editing; Sivashanmugam Karthikeyan: Methodology, Software; Elumalai Preetham: Methodology, Software; Naiyf S. Alharbi: Resources, Validation; Shine Kadaikunnan: Formal analysis; Jamal M. Khaled: Visualization, Validation; Taghreed N. Almanaa: Formal analysis; Baskaralingam Vaseeharan: Conceptualization, supervision, Writing- review and editing
Graphical abstract
1
Antibiofilm and anticancer potential of β-glucan-binding protein-encrusted zinc
2
oxide nanoparticles
3 4
Mani Divyaa, Marimuthu Govindarajanb,c, Sivashanmugam Karthikeyand, Elumalai
5
Preethame, Naiyf S. Alharbif, Shine Kadaikunnanf, Jamal M. Khaledf, Taghreed N. Almanaaf,
6
Baskaralingam Vaseeharana*
7 8
a
9
Nanopharmacology Division, Department of Animal Health and Management, Alagappa
Biomaterials
and
Biotechnology
in
Animal
Health
Lab,
10
University, Karaikudi-630004, Tamil Nadu, India
11
b
12
Annamalai University, Annamalainagar 608 002, Tamil Nadu, India
13
c
14
for Women (Autonomous), Kumbakonam 612 001, Tamil Nadu, India
15
d
16
e
17
Panangad, Kerala, India
18
f
19
Saudi Arabia
Nanobiosciences
and
Unit of Vector Control, Phytochemistry and Nanotechnology, Department of Zoology,
Unit of Natural Products and Nanotechnology, Department of Zoology, Government College
School of Bioscience & Technology, VIT University, Vellore, 632014, Tamil Nadu, India
School of Ocean Science and Technology, Kerala University of Fisheries and Ocean Studies,
Department of Botany and Microbiology, College of Science, King Saud University, Riyadh,
20 21
* Corresponding Author. Baskaralingam Vaseeharan. Tel: +91 4565 225682. Fax: +91 4565
22
225202. E-mail: [email protected]
23
24 25
Abstract β-Glucan-binding protein (βGBP) is important for the rational expansion of molecular
26
biology. Here, zinc oxide nanoparticle (ZnONP) was synthesized using βGBP from the crab
27
Scylla serrata (Ss-βGBP-ZnONP). Ss-βGBP-ZnONP was observed as a 100 kDa band on
28
sodium dodecyl sulfate polyacrylamide gel and characterized with UV-vis spectroscopy at
29
350 nm. X-ray diffraction analysis displayed values consistent with those for zincite. Fourier
30
transform infrared spectroscopy revealed the presence of functional groups, including amide,
31
alcohol, alkane, alkyl halide, and alkene groups. The zeta potential (−5.36 mV) of these
32
particles indicated their stability, and transmission electron microscopy revealed the presence
33
of 50 nm nanocones. Ss-βGBP-ZnONPs were tested at 100 µg/mL against the gram-positive
34
Enterococcus faecalis and gram-negative Pseudomanas aeruginosa using confocal laser
35
scanning microscopy and the bacterial viability assay was also performed. The growth of
36
MCF7 breast cancer cells was inhibited following treatment with 75 µg/mL Ss-βGBP-
37
ZnONPs. Thus, Ss-βGBP-ZnONPs have the ability to control the growth of pathogenic
38
bacteria and inhibit the viability of MCF7 breast cancer cell lines.
39 40
Keywords
41
β-Glucan-binding protein; biofilm; confocal laser scanning microscopy; Pseudomanas
42
aeruginosa; cell line
43 44
45
1. Introduction
46
Nanotechnology has widespread applications in the field of science and medicine [1-6].
47
Nanoparticles (NPs) are used as imaging and therapeutic agents and their surfaces are often
48
coupled with biological molecules to reduce surface energy [7]. Metallic and metal oxide
49
NPs, including gold, silver, copper, titanium dioxide (TiO2), and zinc oxide (ZnO) particles,
50
play vital roles [8-11]. The ZnONPs have been used for various applications, including cell
51
imaging, drug delivery, and nanomedicine [12]. The ZnONPs are also used in goods such as
52
personal care products and are known to exhibit excellent antibacterial and UV blocking
53
properties. In vitro analyses have revealed the biocompatibility of ZnONPs [13]. The ZnONPs
54
serve as non-toxic, biocompatible, and safe drug carriers. Zinc is an important trace element
55
present in all body tissues, including the brain, muscle, bone, and skin [14]. Zinc is also
56
important for eukaryotes, as it is known to modulate several physiological functions [15]. In
57
biological systems, zinc has important roles in metabolic pathways such as the carbohydrate,
58
lipid, nucleic acid, and protein syntheses [16]. The NPs may interact with biomolecules such
59
as nucleic acids, proteins, and lipids, owing to their nano size [17]. Biological synthesis of
60
NPs is more labor- and time-intensive and expensive than physical and chemical methods
61
[18].
62
Cancer is an emerging disorder that is severely affecting human health. It is the second
63
leading cause of mortality worldwide. The prevalence of breast cancer is higher among
64
women [19] and corresponds to one-fifth of all cancer cases worldwide. Studies have
65
confirmed the cytotoxicity of ZnONPs on cancer cells, including HepG-2, MCF-7, Hela, U87,
66
and S91 cells [20]. To improve their targeting ability, these NPs are often coupled with
67
antibodies or peptides that bind to the receptors over expressed on cancer cells. Today,
68
different types of nanostructures, particularly ZnONPs, have gained significant attention
69
owing to their wider implications in cancer therapy [21]. The current anticancer
70
chemotherapies have restricted potential due to systemic toxicity, non-selective distribution,
71
and decreased bioavailability, which severely damage healthy tissues [22]. Some bioactive
72
compounds are used as chemopreventive agents against cancer [23]. Cancer therapies are
73
limited to radiation, surgery, and chemotherapy, all of which exert harmful effects on normal
74
cells. Nanotechnology has been employed for the targeted treatment of cancers. The
75
mechanism underlying ZnONP-induced toxicity involves oxidative stress and apoptosis in
76
selected cancer cells. The combination of macromolecules and NPs exerts strong effects on
77
biological systems, and ZnONPs have a potent activity against competent cancer cells [24].
78
The NPs have been applied in the field of cancer therapy to target useful anticancer drugs
79
in patients suffering from cancer [20]. β-1,3-Glucan-binding protein (β-GBP) is an abundant
80
protein in the hemolymph of invertebrates. It is involved in the response of invertebrates to
81
pathogenic microorganisms. β-GBP exerts phenoloxidase and peptidase activities [25].
82
However, the antimicrobial activity of β-GBP is yet undetermined. β-GBP has been purified
83
from a mixture of crustaceans, including Scylla serrata [26] and the mangrove crab
84
Episesarma tetragonum [27].
85
Here, β-GBP from S. serrata was used to coat ZnONP. The developed Ss-βGBP-ZnONPs
86
were purified, characterized, and functionally examined for their antibacterial activity against
87
gram-positive Enterococcus faecalis and gram-negative Pseudomanas aeruginosa. In
88
addition, cell viability of the selected bacterial strains was tested after treatment with 50 and
89
100 µg/mL Ss-βGBP-ZnONPs. We also determined the cytotoxicity of Ss-βGBP-ZnONPs to
90
MCF7 cancer cells. We show that Ss-βGBP-ZnONPs inhibited the growth of bacteria and
91
suppressed the proliferation of MCF7 breast cancer cells. Hence, Ss-βGBP-ZnONPs may
92
serve as effective agents for the treatment of pathogenic bacteria and cancer cells.
93 94
2. Materials and methods
95 96
2.1. Purification and synthesis of Ss-βGBP-ZnONPs βGBP from S. serrata was purified as previously reported [26]. Sodium dodecyl sulfate
97
polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the size of Ss-βGBP-ZnONPs to
98
be 100 kDa. For Ss-βGBP-ZnONP synthesis, 2.1 g of 0.02 M zinc acetate was dissolved in
99
500 mL distilled water and stirred for 20 min with a stirrer. The purified form of Ss-βGBP (5
100
mL) was added to the zinc acetate solution and the mixture was stirred for 12 h. The final
101
sample was calcined for 30 min at 100 °C to obtain Ss-βGBP-ZnONPs. The dried powder was
102
stored in a sealed container for further analysis.
103 104
2.2. Characterization of Ss-βGBP-ZnONPs
105
The synthesized Ss-βGBP-ZnONPs (1 mL) were subjected to UV-Vis spectrum analysis
106
as a function of time on a spectrophotometer at 200-800 nm wavelength and 1 nm resolution.
107
The crystalline nature of Ss-βGBP-ZnONPs was determined by X ray diffraction (XRD)
108
analysis (Powder X-ray diffractometer 1 X’ pert pro-P analytic) with λ value of 1.5406 Å.
109
Scanning was carried out at a 2θ range from 30 to 90. Fourier transform infrared (FTIR)
110
spectrophotometry (Thermo scientific Nicolet 380) was used for the analysis of functional
111
sets. Finely powdered (150 mg) Ss-βGBP-ZnONPs were treated with potassium bromide and
112
subjected to vacuum treatment using a pelletizer. The infrared spectrum was recorded
113
between 4000 and 500 cm−1. Stabilized Ss-βGBP-ZnONPs were analyzed with a 90 plus
114
particle size analyzer (Brookhaven instruments corporation, Holtsville, NY, USA) under an
115
applied electric field, and zeta potential was recorded. Ss-βGBP-ZnONPs were placed on
116
copper stubs and their shape and size were observed by transmission electron microscopy
117
(TEM) (HITACHI; Model: S-3400N) under bright field. The Ss-βGBP-ZnONPs shape, size
118
and selected area electron diffraction (SAED) were confirmed using carbon coated grids. A
119
working voltage of 20 kV with secondary electron detectors at the elemental action was
120
indomitable with energy dispersive X-ray spectroscopy (EDX, Joel JSM-6510) analysis.
121 122
2.3. Potency of Ss-βGBP-ZnONP against bacterial biofilms
123
The effects of Ss-βGBP-ZnONPs on bacterial biofilms were evaluated using gram-
124
positive E. faecalis and gram-negative P. aeruginosa with an enzyme-linked immunosorbent
125
assay (ELISA) plate reader. Confocal laser scanning microscopy (CLSM) was used for the
126
analysis of biofilm assay. Selected bacteria were cultured on glass pieces (1 × 1 cm) in
127
nutrient broth supplemented with Ss-βGBP-ZnONPs (50 and 100 µg/mL). As a control, Ss-
128
βGBP-ZnONPs were replaced with an equal concentration of bovine serum albumin (BSA).
129
The cells were incubated for 2 days at 36 °C and then stained with acridine orange (0.4%).
130
The cells were rinsed with phosphate-buffered saline (PBS) and analyzed using CLSM with
131
488-nm argon laser and Zen software (Carl Zeiss, Germany).
132 133 134
2.4. Bacterial viability assay The bacterial viability assay was performed as reported by Velusamy et al. [28] using the
135
live/dead BacLight bacterial viability kit (Invitrogen, India). Live and dead bacterial cells
136
were labelled with SYTO®9 (green) fluorescence and propidium iodide (PI; red) fluorescence
137
staining agents, respectively. Overnight cultured bacterial cells were treated with Ss-βGBP-
138
ZnONPs (50 and 100 µg/mL) at 37 °C at 60 min. After treatment, the cells were centrifuged
139
for 10 min at 6000 ×g and the pellet was rinsed twice with PBS, followed by incubation with
140
5 µL of SYTO®9, PI, and dimethyl sulfoxide (DMSO) in 990 µL of PBS. The cells were
141
stained with 10 µL SYTO® 9 and 20 µL of PI (1:2 ratio) at 27 °C for 15 min in the dark, and
142
subjected to CLSM.
143 144
2.5. Anticancer activity
145
The inhibitory effect of Ss-βGBP-ZnONPs on MCF7 cancer cell growth was assessed
146
with the 3-4, 5-dimethyl thiazol-2yl-2,5-diphenyl tetrazolium bromide (MTT) assay [29]. The
147
cells were cultured in ELISA plates (5000 cells in each well) for 1 day in the presence of 5,
148
25, 50, and 75 µg/mL of test agent. The absorbance spectra were measured at a wavelength of
149
595 nm using a microplate reader. Cell viability (% of control) was calculated as (ODtest −
150
ODblank)/(ODcontrol − ODblank), where ODtest is the optical density of the cells exposed to the
151
ZnO sample, ODcontrol is the optical density of the control sample, and ODblank is the optical
152
density of the wells without MCF7 cells.
153 154
3. Results
155
3.1. Synthesis and characterization of Ss-βGBP-ZnONPs
156
ZnONPs were synthesized using the purified form of β-GBP from S. serrata following 30
157
min incubation. The treatment with zinc acetate resulted in a pale white precipitate, indicative
158
of the formation of Ss-βGBP-ZnONPs. The size of the synthesized Ss-βGBP-ZnONPs was
159
confirmed to be 100 kDa by SDS-PAGE analysis (Fig.1). UV-vis spectrophotometry analysis
160
was performed at a wavelength of 350 nm (Fig.2). In brief, 0.1 mL of sample was prepared in
161
deionized H2O in a cuvette, and the change from bluish to pale white precipitate was
162
indicative of the synthesis of ZnONPs.
163
The diffraction peaks showed various Bragg’s expression at 2.8 (100), 2.5 (002), 2.4
164
(101), 1.9 (102), 1.6 (110), 1.4 (103), 1.3 (112), and 1.2 (202). The above mentioned values
165
were consistent with the values for zincite of ZnO phase (Joint committee on Powder
166
Diffraction Standards, JCPDS 75-0576; Fig.3). The ZnONPs present in Ss-βGBP were
167
determined with FTIR spectroscopy. The FTIR spectrum of Ss-βGBP-ZnONPs confirmed
168
strong bands at 1644, 1022, and 892 cm−1 belonging to C = O, C-F, and =C-H, respectively.
169
Weak bands were observed at 1556 and 1407 cm−1 that corresponded to amide and alkene
170
groups, respectively. However, the broad spectrum of strong bands of alcohol at 3418 cm−1
171
was associated with O-H stretching.
172
Overall, the analysis demonstrated the presence of amide, alcohol, alkane, alkyl halide,
173
and alkene groups (Fig.4). Zeta potential value revealed the surface charge and stability of Ss-
174
βGBP-ZnONPs; its conductivity was 0.233 mS/cm and zeta potential, −5.36 mV (Fig.5).
175
These values demonstrate the complete stabilization of NPs as well as their efficiency to serve
176
as capping materials. TEM of Ss-βGBP-ZnONPs revealed the presence of nano cones of 50
177
nm diameter (Fig.6a&b). Nature of Ss-βGBP-ZnONPs and lattice spacing were observed with
178
selected area electron diffraction (Fig.6c). Zinc elemental composition of Ss-βGBP-ZnONPs
179
was 40.78% and the weight % of oxygen was 6.54% (Fig.6d).
180 181 182
3.2. Effects of Ss-βGBP-ZnONPs on bacterial biofilms CLSM analysis confirmed the inhibitory effects of Ss-βGBP-ZnONPs on the biofilm
183
formation ability of both gram-positive E. faecalis and gram-negative P. aeruginosa. Both
184
bacteria produced well-ordered biofilms in the control group. Ss-βGBP-ZnONP-treated
185
groups showed the incompetent structure with separate bacterial cells. The biofilm formation
186
ability was considerably lower in the treatment groups than in the control group. The effect
187
was stronger with 100 µg/mL than that with 50 µg/mL Ss-βGBP-ZnONPs (Fig.7).
188 189 190
3.3. Bacterial viability assay The effects of Ss-βGBP-ZnONPs on bacterial cells were assessed with CLSM using the
191
live/dead BacLight bacterial viability kit. The green and red fluorescence corresponded to
192
live and dead bacterial cells, respectively. The bacterial cells treated with 50 and 100 µg/mL
193
Ss-βGBP-ZnONPs for 60 min were found to be dead (Fig.8). The red fluorescence signal was
194
higher after treatment with 100 µg/mL Ss-βGBP-ZnONPs than that observed following
195
treatment with 50 µg/mL Ss-βGBP-ZnONPs.
196 197 198
3.4. Anticancer activity The cytotoxicity study revealed no morphological alterations in MCF7 cells from the
199
control group. However, the growth of Ss-βGBP-ZnONP-treated cells was affected at a
200
concentration of 25 µg/mL. MCF7 cells treated with 50 µg/mL Ss-βGBP-ZnONP were
201
ruptured (Fig. 9). The cytotoxicity of Ss-βGBP-ZnONPs against MCF7 breast cancer cells
202
was higher at 75 µg/mL concentration than at 25 and 50 µg/mL (Fig. 10).
203 204
4. Discussion
205
Zinc plays an important role in metabolism and protein synthesis. Biological synthesis of
206
NPs in combination with immune molecules may reduce toxicity. In the present study,
207
ZnONPs were synthesized with the purified form of β-GBP from S. serrata (Ss-βGBP-
208
ZnONPs). The synthesized Ss-βGBP-ZnONPs were confirmed to have a molecular mass of
209
100 kDa. The purified form of βGBP from S. serrata has been previously described [26]. Ss-
210
βGBP-ZnONPs were characterized with UV-vis spectroscopy; the spectrum exhibited
211
absorption peaks at 350 nm, consistent with the results of Anjugam et al. [32] for ZnONPs
212
coated with β-GBP (Portunus pelagicus). Chaudhuri et al. [33] showed that ZnONPs obtained
213
from the leaf extract of Calotropis gigantean exhibited an absorption peak near 350 nm.
214
These results confirm the synthesis of ZnNPs. The present findings demonstrate the
215
crystalline nature of Ss-βGBP-ZnONPs; FTIR spectrum reveals the strong and weak bands
216
associated with the presence of amide, alcohol, alkane, alkyl halide, and alkene groups. The
217
stretching vibration reveals the presence of functional groups in ZnONPs. As reported by
218
Anjugam et al. [32], the FTIR spectrum of Ppβ-GBP-ZnONPs showed a peak at 3221 cm−1
219
corresponding to the N-H stretching vibrations of amide groups of the protein. The band at
220
1062 cm−1 was related to the presence of amine groups.
221
Ss-βGBP-ZnONPs had a zeta potential value of −5.36 mV. This elevated negative value
222
confirms the stability of these NPs. Moreover, previous reports on the biosynthesis of
223
ZnONPs from the leaf extract of Calotropis gigantean revealed a zeta potential value of −20.7
224
mV [34]. Gelatin-coated ZnONP also showed a zeta value of −6 mV, indicative of the surface
225
charge of ZnONPs [35]. TEM images showed the nanocone structure of Ss-βGBP-ZnONPs
226
and a standard particle size of 50 nm. This observation is in line with that reported by Kavitha
227
et al. [30], wherein a ZnO nanopowder was found to exhibit a cone shape and an uneven
228
surface. The calcification at 550 °C allows customization of the morphology to nanocones.
229
TEM images of ZnO nanocrystal illustrated the individual cone-shaped structure with an
230
average size of 70 nm [31]. Thus, the synthesized ZnO had a cone structure owing to thermal
231
oxidation.
232
Ss-βGBP-ZnONPs at 100 µg/mL concentration suppressed the bacterial biofilm formation
233
ability. Similar results were reported with Ppβ-GBP-ZnONPs at a concentration of 50 µg/mL
234
[32]. Ishwarya et al. [34] found that the plasma of fish fed with Phβ-GBP-SeNWs could
235
prevent the growth of bacterial colonies and suppress biofilm formation. Anjugam et al. [32]
236
demonstrated the effective anticancer activity of Ppβ-GBP-AgNPs against HeLa cells (50
237
µg/mL). Wahab et al. [21] used a very low concentration of NPs against MCF-7 cancer cells.
238
In the present study, Ss-βGBP-ZnONPs suppressed the viability of MCF7 cells at 75 µg/mL.
239 240 241
5. Conclusion Ss-βGBP-ZnONPs were synthesized and characterized with UV, XRD, FTIR, zeta
242
potential, and TEM. Ss-βGBP-ZnONPs effectively inhibited the biofilm formation ability of
243
gram-positive E. faecalis and gram-negative P. aeruginosa at 100 µg/mL concentration. The
244
bacterial viability of the same tested bacteria was suppressed at 100 µg/mL concentration. The
245
dropping ability of the bacterial biofilm could uplift all fields of research as in valuable. The
246
proliferation of MCF7 breast cancer cells was reduced after treatment with Ss-βGBP-ZnONPs
247
at 75 µg/mL. The particular property of this Ss- βGBP-ZnONPs without difficulty can be
248
elucidate into an effectual therapeutic for cancer therapy without causing dangerous effect to
249
ordinary tissues. Thus, Ss- βGBP-ZnONPs have the ability to inhibit the pathogenic bacteria
250
as well as to control the proliferation of MCF7 breast cancer cells. It provely Ss- βGBP-
251
ZnONPs used as a nanotheraputic agent in feature.
252 253
Conflict of interest
254
The authors declare no conflict of interest.
255 256
Acknowledgements
257
The authors extend their appreciation to the Researchers Supporting Project number
258
(RSP-2019/70), King Saud University, Riyadh, Saudi Arabia. This work was funded by
259
RUSA phase 2.0 grant (ref-24 – 51/2014 – U, Policy) TN. Multi-Gen, Depart of Edn, Govt.
260
of
261
Technology, NGO Colony road, Nagarcoil, Tamil Nadu, India.
India.
The authors also thank to Fundation of Innovative research in Science and
262 263
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Fig 1. SDS-PAGE analysis of purified Ss-βGBP and the synthesized zinc oxide nanoparticle (Ss-βGBP-ZnONPs). Lane-I, molecular marker; Lane-II, purified Ss-βGBP; Lane- III, SsβGBP-ZnONPs (100 kDa).
Fig 2. UV-Visible spectra of Ss-βGBP-ZnONPs
Counts DSS
400
200
0 20
30
40
50
60
70
80
Position [°2Theta] (Copper (Cu))
Fig 3. X-ray diffraction outline shows the crystalline nature of Ss-βGBP-ZnONPs
Fig 4. Fourier transform infrared spectra showing the functional group of Ss-βGBP-ZnONPs
Fig 5. Zeta potential distribution for size and surface charge of Ss-βGBP-ZnONPs
Fig 6. Transmission electron microscopy (TEM) of Ss-βGBP-ZnONPs (a), single element of Ss-βGBP-ZnONPs (b), selected area electron diffraction (SAED) pattern (c), energy dispersive X-ray spectroscopy (EDX) elemental work of Ss-βGBP-ZnONPs (d).
Fig 7. Confocal laser scanning microscopy showing the inhibitory effect of Ss-βGBPZnONPs on E. faecalis and P. aeruginosa biofilms at two concentrations.
Fig 8. Live and dead cell assay with CLSM using pathogenic bacteria E. faecalis and P. aeruginosa after treatment with Ss-βGBP-ZnONPs at 50 and 100 µg/mL.
Fig 9. Morphology of MCF7 breast cancer cells after treatment with different concentrations of Ss-βGBP-ZnONPs under a phase-contrast microscope. Arrows point to the morphological changes in cells.
Fig 10. Effect of zinc, Ss-βGBP, and Ss-βGBP-ZnONPs on the viability of MCF7 cells as compared with the control group; T-bars designated typical errors; different letters on the top of bars indicate important distinctions with treatments (p < 0.05).
Highlights •
β-Glucan-binding protein-coated ZnONPs was synthesized from Scylla serrata
•
Ss-βGBP-ZnONPs was characterized with UV, XRD, FTIR, zeta potential, and TEM
•
Inhibition of biofilms by Ss-βGBP-ZnONPs was confirmed at 100 µg/mL
•
Ss-βGBP-ZnONPs at 75 µg/mL effectively inhibited MCF7 breast cancer cells