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Antibacterial and cytotoxic effect of honey mediated copper nanoparticles synthesized using ultrasonic assistance
Materials Science & Engineering C 104 (2019) 109899
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Antibacterial and cytotoxic effect of honey mediated copper nanoparticles synthesized using ultrasonic assistance
T
Nur Afini Ismaila, Kamyar Shamelia, , Magdelyn Mei-Theng Wongb, Sin-Yeang Teowb, Jactty Chewc, Siti Nur Amalina Mohamad Sukria ⁎
a
Department of Environment and Green Technology, Malaysia-Japan International Institute of Technology, Universiti Teknologi Malaysia, Jalan Sultan Yahya Petra, 54100 Kuala Lumpur, Malaysia b Department of Medical Sciences, School of Healthcare and Medical Sciences, Sunway University, Jalan Bandar Sunway, 47500, Selangor Darul Ehsan, Malaysia c Department of Biological Sciences, School of Science and Technology, Sunway University, Jalan Bandar Sunway, 47500, Selangor Darul Ehsan, Malaysia
ARTICLE INFO
ABSTRACT
Keywords: Antibacterial Cytotoxicity assay Copper nanoparticle Honey mediated Sonochemical
In this study, a comparative study of effect using honey on copper nanoparticles (Cu-NPs) via simple, environmentally friendly process and inexpensive route was reported. Honey and ascorbic acid act as stabilizing and reducing agents with the assistance of sonochemical method. The products were characterized using UV–visible (UV–vis) spectroscopy, X-Ray Diffraction (XRD), High-Resolution Transmission Electron Microscopy (HRTEM), Field-Emission Scanning Electron Microscopy (FESEM) and Fourier Transform Infrared (FTIR) spectroscopy. The reddish brown colour demonstrated the formation of Cu-NPs and UV–visible proved the plasmon resonance of Cu-NPs. XRD also confirmed a highly pure Cu-NPs obtained with absence of copper oxide in which the structure is crystalline. The spherical size of the Cu-NPs was acquire in the presence of honey which is 3.68 ± 0.78 nm with narrow particle distribution. The antibacterial activity was seen against gram-positive and gram-negative bacteria which are Enterococcus faecalis (E. faecalis) and Escherichia coli (E. coli). At higher concentration of Cu-NPs, they were more effective in killing both bacteria. The Cu-NPs without and with honey exhibited toxicities toward normal and cancerous cells. However, Cu-NPs without honey showed more potent killing activity against normal and cancer cells.
1. Introduction
special characteristics such as low cost, less toxic, high surface area to volume ratio, heat transfer properties and high friction of atoms [11,12]. These characteristics are mainly resulted by their physical properties such as size, shape, composition, morphology and crystalline phase [13]. Cu-NPs also have abundant sources, easy preparation and flexibility in modifying into various shape of nano-sized dimension [14]. Cu-NPs can be implemented in several applications such as biomedical, optical, biosensor, catalytic and energy application [15–19]. In an important biomedical application which is drug delivery system and molecular doping, Cu-NPs play particularly important role as vehicles to deliver the compounds to the target sites [20]. Moreover, copper also is a multifunctional antibacterial agents [21]. Cu-NPs have been demonstrated to show potent inhibition toward various bacterial species [22–25]. Various studies showed that Cu-NPs could bind and conjugate with biomolecules like proteins, enzymes and DNA [20,26], indicating the potentials of Cu-NPs to be used as ‘nano-drugs’ [27]. There are various ways to synthesize Cu-NPs using physical and
Nanotechnology emerges as one of the alternative technologies in addressing problems that arise nowadays particularly pertaining to environmental issues [1]. It also has various medical and biomedical applications such as the production of nanoparticles and carbon nanotubes for bio-imaging, cancer therapy, drug delivery and antibacterial [2–4]. Nanoparticle production, particularly metallic and metal oxide nanoparticles, is attracting vast interest from scientists in recent years due to the unique characteristics such as having surface plasmon resonance (SPR) and optical properties [5]. They are also actively used for biosensor, electrical, catalytic and antimicrobial applications [6–9]. The size of nanomaterials typically ranges from 1 to 100 nm. Generally, nanoparticles could be classified as fine particles which has the size ranging from 100 to 2500 nm while ultrafine particles possess the size from 1 to 100 nm [10]. Gold-, platinum-, silver-, and copper-nanoparticles (Cu-NPs) are a few relatively well studied nanoparticles. Among them, Cu-NPs have
⁎
Corresponding author. E-mail address: [email protected] (K. Shameli).
https://doi.org/10.1016/j.msec.2019.109899 Received 24 January 2019; Received in revised form 11 June 2019; Accepted 15 June 2019 Available online 18 June 2019 0928-4931/ © 2019 Published by Elsevier B.V.
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chemical methods, such as laser ablation [28], gamma ray irradiation [29], chemical precipitation method [30], sol–gel methods [31], solidstate reaction [32] and sonochemical preparation [33,34]. However, the major problems of using these methods are producing toxic byproduct, costly and laborious process [34,35]. Green chemistry synthesis has drawn market attention nowadays as it is simple, inexpensive and environment friendly. Generally, there are three criteria to follow for green synthesis of nanoparticles, including the choice of solvent, the use of eco-friendly reducing agent and the use of nontoxic material as capping agent to stabilize the nanoparticles synthesis [36]. Honeymediated nanoparticle production, one of the examples of green synthesis is gaining attention in scientific community nowadays [37,38]. They are widely used in biological studies such as tissues engineering and cytotoxicity study [37,39]. Copper is highly unstable and it can easily oxidize at atmospheric condition which generates copper oxide (CuO) or copper (II) oxide (Cu2O). Hence, a surface-protecting stabilizing agent that can form complexes with the Cu-NPs is mandatory [40]. Honey is a natural food and a sweet viscous liquid consisting of carbohydrate, enzymes, vitamins, minerals and antioxidant [37,41]. The monosaccharides, proteins, amino acid and vitamin C in honey could help in reducing and stabilizing the nanoparticles [9], although little is known of the contributing factors for these capacities [38,42]. The use of honey as reducing and stabilizing agent is convenient as it does not require drying, extraction of plant materials and cell culture maintenance [41]. Ultrasonic irradiation-assisted heating is gaining attention as a common method for rapid synthesis of organic and inorganic compounds [43]. A few advantages using this method are, it could further reduce the size of particles and reduce the time of reaction [44,45]. The effects of ultrasonic irradiation on reaction are due to acoustic cavitation within collapsing bubbles. During bubble collapse, unique hot spots are produced at extreme conditions (high temperatures above 5000 K, pressures of about 1000 atm, and heating and cooling rates of approximately 1010 Ks−1) [43]. In this study, honey-mediated Cu-NPs were synthesized by green method assisted by ultrasonic irradiation. Honey was used as reducing and stabilizing agent along with the ascorbic acid (vitamin C) as supporting reducing agent. The size and morphology of Cu-NPs were characterized by UV–vis, XRD, HRTEM, FESEM and FTIR. The potential biomedical application of Cu-NPs was then evaluated by an antibacterial assay against two gram positive and gram negative bacteria as well as a cytotoxicity assay against two mammalian cell lines.
stirred until homogenized. After that, 0.6 M sodium hydroxide was added dropwise into the mixture solution while continuously stirring for 10 min until approximately pH 7.5. Next, 15 ml ascorbic acid (1 M) was added into the mixture solution and underwent ultrasound irradiation for 10 min with the amplitude of 80%, pulse on 1 s and pulse off 1 s. Lastly, the solution was washed with distilled water twice and dried in oven. These steps were repeated without honey to synthesize Cu-NPs without honey. 2.3. Characterization method and instrumentations The Cu-NPs were characterized using Ultraviolet-visible (UV–vis) spectroscopy (UV-2600, SHIMADZU). The clean quartz solution cells were used. The sample solution with desired concentration and a blank sample (water) were used for this experiment. The samples were homogenized in the quartz solution cells and placed into the UV–vis chamber. The UV–vis spectra were recorded over the range of 220 nm to 1000 nm. The structure of Cu-NPs were evaluated using X-ray diffraction (XRD, Philips, X'pert, Cu Ka) in the small-angle range of 20° to 80° (2θ). High Resolution Transmission Electron Microscopy (HRTEM) (model JEM-2100F) was used to examine the size of Cu-NPs. To analyse the morphology and elemental analysis of the Cu-NPs, JOEL-FESEM (model JSM 7600F FESEM) attached to Energy-Dispersive X-ray spectroscopy was used. Fourier Transform Infrared (FTIR) spectrum was utilized to examine the functional groups present in the synthesized compound. Finally, FTIR spectra were recorded over the range of 400–4000 cm−1 using Attenuated Total Reflectance (ATR), IRTracer100 spectrophotometer (Shimadzu, Malaysia). 2.4. Broth micro-dilution and MTT assay for antibacterial application A broth micro-dilution method and MTT assay were used to determine the minimum inhibitory concentration (MIC) values and minimum bactericidal concentration (MBC) of the Cu-NPs using the Clinical and Laboratory Standards Institute (CLSI) protocols. Grampositive (Enterococcus faecalis- ATCC 33186) and gram-negative (Escherichia coli- MTCC 710859) bacterial species were used in this study. Single colony of fresh bacterial culture (12–18 h) was isolated from Mueller Hinton agar (MHA) plates and inoculated into sterile Mueller Hinton broth (MHB). The culture was grown overnight (12–18 h) prior to the experiments. Next day, the bacterial concentration was standardized to an optical density (OD) of 600 nm (approximately 108 CFU/ml) with MHB. Two-fold serial dilutions of nanoparticles (Cu-NPs with and without honey) were prepared in 96 well plates to give final test concentrations of 0, 7.8, 15.6, 31.3, 62.5, 125, 250 and 500 μg/ml per well. 10 μl of bacterial suspension equivalent to 106 CFU/ml of exponentially growing bacterial cells were added to the wells. The plates were incubated at 35 ± 2 °C for 18 h. Following the overnight incubation, the plate was then read for the absorbance using microplate reader (GloMax Discover Instrument, Promega) to determine the MIC50 values. Positive control, imipenem antibiotic (1 μg/ ml), and negative controls (blank, without bacterial inoculum) were included in all experiments. Then, the wells were filled with 10 μl of MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] which were prepared in PBS (pH 7.2) and the wells were incubated for 30 min at 28 °C. Then the clear wells without colour changing with the MTT were plated on the agar to observe the MBC of the samples toward the bacteria strain.
2. Materials and methods 2.1. Materials Copper II nitrate (Cu(NO3)2.3H2O, ~99%), ascorbic acid (C6H8O6, ~99%), sodium hydroxide (NaOH) were purchased from R&M chemical, United Kingdom. Pure honey was purchased from Capilano Honey Limited, Australia where the source of flowers are from Australian eucalyptus and ground flora. Double distilled water was used as solvent throughout the experiment. All chemicals used were analytical grade without further purification. Imipenem was purchased from GoldBio (USA). Sterile Mueller-Hinton agar and broth (Becton Dickinson, USA) were used to culture and maintain the bacterial strains. 96-well plates for antibacterial application were purchased from Nest Biotech Co., Ltd., China. MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was purchased from Sigma Aldrich.
2.5. Cytotoxicity assay
2.2. Green synthesis of copper nanoparticles
To determine the cellular killing effect of nanoparticles, cell proliferation assay (Promega) was performed according to the manufacturer's instruction with slight modification. Briefly, 5,000 human colorectal cancer cell line (HCT116) (ATCC CCL-247) and human normal colon cell (CCD112) (ATCC CRL-1541) cells per well (100 μl/well) were
The Cu-NPs were prepared using chemical reduction assisted by ultrasonic irradiation. Briefly, copper nitrate trihydrate (0.025 mol) was dissolved in 100 ml of double distilled water. Then, 10% w/v concentration of honey was added into the copper nitrate solution and 2
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Fig. 1. Diagram showing Cu(NO3)2, [Cu(Honey)]2+ complex, honey‑copper hydroxide complex ([Cu(OH)2/Honey]), and Cu-NPs solutions (a–d). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Schematic illustration of Cu-NPs stabilized by fructose and glucose under ultrasonic irradiation.
Fig. 3. UV–vis spectroscopy of honey, and Cu-NPs without and with honey. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. X-ray diffraction patterns of Cu-NPs: (a) Cu-NPs without honey (b) CuNPs with honey.
and incubated for additional 3 h at 37 °C in the 5% CO2 incubator. Optical density (OD) was then measured at 490 nm using a multimode microplate reader (Tecan). The dose-response graph was plotted by calculating the percent cell viability using Eq. (1). The images of cells treated with the nanoparticles were captured using an inverted microscope attached to a camera system (IM3 Phase contrast, Optika, Italy).
seeded onto a 96-well plate and incubated at 37 °C overnight in a 5% CO2 humidified incubator. Next day, 2-fold serially diluted nanoparticles (500, 250, 125, 62.5, 31.3, 15.6, 7.8, 0 μg/ml) (100 μl/well) were added into the wells and the plate was incubated for 72 h at 37 °C in the 5% CO2 humidified incubator. Then 20 μl MTS (3-(4,5Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium) reagent (Promega) per well was added into the plate 3
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Fig. 5. HRTEM image for green synthesis of (a) Cu-NPs without honey and; (b) Cu-NPs with honey, the histograms on the right shows the distribution of particle size. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
shown in Eqs. (2)–(4).
%Viability = OD of sample well (mean)/OD of control well (mean) × 100 (1)
Cu2 + (aq) + Honey [Cu(Honey)]2 + (aq)
3. Results and discussion Comparison between Cu-NPs without and with honey was carried out by characterizing both samples using UV–vis, XRD, HRTEM, FESEM-EDX and FTIR to determine the effect of honey on Cu-NPs synthesis. The occurrence of reaction was first observed by the colour changes of the solution. During the synthesis, the colour of solution changed from light blue to light yellow, then to blue, and lastly turned to reddish brown (Fig. 1). This is consistent with the observation by Mardiansyah et al. for the biosynthesis of Cu-NPs [46]. The light blue colour (Fig. 1a) is the precursor solution which is copper II nitrate solution, while the light yellow (Fig. 1b) is [Cu(Honey)]2+ complex solution. On the other hand, the blueish solution (Fig. 1c) is the honey‑copper hydroxide complex ([Cu(OH)2/Honey]) and Fig. 1d shows the reddish brown Cu-NPs solution after 10 min of ultrasonic irradiation [47]. The proposed mechanism for the Cu-NPs productions is as follows: copper nitrate solution dissociated to form Cu2+, then the Cu2+ reacted with the negative charge of the hydroxyl group of fructose and glucose in honey and formed [Cu(Honey)]2+. With the addition of sodium hydroxide, the reaction occurred and formed ([Cu(OH)2/Honey]). After the addition of ascorbic acid, it yielded Cu-NPs following the ultrasonic irradiation process. This step is important to reduce the reaction time for Cu-NPs production. It is proposed that the positive charge on the surface of Cu-NPs formed electrostatic bond with the negative charge of hydroxyl group from the fructose and glucose of honey. The proposed mechanism is illustrated in Fig. 2. The chemical reaction equation are
Room temperature,Stir
[Cu(Honey)]2 + (aq)
NaOH (0.6 M) Room temperature,Stir
[Cu(OH)2/Honey](aq)
[Cu(OH)2/Honey](aq)
Ascorbic acid (1 M) Ultrasonic irradiation (10 min)
(2) (3)
[Cu/Honey](aq) (4)
Honey is a weak reducing agent [48]. It can reduce the Cu2+ to Cu+ but it will take a longer time to reduce the Cu2+ to Cu0. Thus, ascorbic acid was used enhance the reduction process of Cu2+ and shorten the reaction time. With ultrasonic assistance, the reaction time could be further reduced compared to the conventional method [34]. 3.1. UV–visible (UV–vis) absorption UV–vis absorption confirmed the successful synthesis of Cu-NPs without and with honey as peaks were observed at 613 and 583 nm respectively (Fig. 3). These peaks could be observed due to the surface plasmon resonance of Cu-NPs [44]. There is a blue shift from 613 nm (Fig. 3a) to 583 nm (Fig. 3b) for the Cu-NPs with honey which indicates the decreased size of nanoparticles [44]. This could be due to the presence of protein and monosaccharide in honey that act as stabilizing agent and prevent the Cu-NPs from the agglomeration as compared to the Cu-NPs without honey. According to the HPLC test done by Enrico et al. [49] for four different honey including pure honey and Australian honey, the percentage of glucose and fructose were 31% and 50% respectively. Thus, it is confirmed that the highest percentage composition in raw honey are glucose and fructose. Therefore, it should exhibit the same results in term of size as based on paper that producing Cu4
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Fig. 6. Lattice fringes d-spacing of Cu-NPs with honey and SAED image Cu-NPs with honey, respectively (a–b).
NPs using different concentration of glucose as capping agent, the particle size are decreasing as the amount of glucose increasing [50]. For honey solution alone (Fig. 3c), there is an intense peak around 300 nm which might be due to the geographic region and the aging honey itself [51].
and the distribution of particle size. Comparatively, there is a significant size difference between the two. The Cu-NPs without honey have the size of approximately 100 nm while the mean size of the CuNPs with honey is 3.68 ± 0.78 nm as shown in Fig. 5. The histogram in Fig. 5b shows an even distribution of the particle size. This might be due to the polyhydroxyl group of glucose and fructose in honey that acts as stabilizer to prevent the formation of large agglomerates from the nanoparticles. The sole effect of honey might have the similar effect produced by using glucose or fructose as it exhibit the same trend in term of size reduction in Cu-NPs production where the particle size decrease as concentration of glucose increase [50]. After comparing and analysing the histogram, it can be concluded that honey can act as a stabilizing agent as the distribution of the histogram is much more narrower compared to the histogram shown by Cu-NPs without honey [45]. The lattice d-spacing of Cu-NPs with honey was calculated to be 0.20 nm which is similar with the selected area diffraction (SAED) at 111 plane (Fig. 6).
3.2. X-ray diffraction (XRD) structure analysis Highly pure copper from honey was obtained as characterized by XRD depicted in Fig. 4. The XRD graphs showed only the copper without other compounds including copper oxide. Both Cu-NPs had a similar diffraction profile and XRD peaks at 2θ of 43.30°, 50.40° and 74.12° could be attributed to the crystal plane of 111, 200 and 220. All diffraction patterns are in agreement with the reference pattern of facecentred cubic (FCC) phase of copper (JCPDS 04-0836). Furthermore, the synthesized Cu-NPs were stored for 4 months and re-characterized using the same method. The XRD result was consistent without the peak of copper oxide which suggests the retained purity of copper.
3.4. Field-emission scanning electron microscopy and energy dispersive Xray spectroscopy (FESEM-EDX) analysis
3.3. High-resolution transmission electron microscopy (HRTEM) analysis Fig. 5 shows the HRTEM images of Cu-NPs without and with honey,
The morphology of Cu-NPs was characterized using FESEM-EDX. 5
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Fig. 7. FESEM micrographs and EDX spectra of Cu-NPs without honey (a, b) and Cu-NPs with honey (c, d) respectively.
Fig. 8. FTIR spectrum of honey, ascorbic acid, Cu-NPs without honey and CuNPs with honey after 10 min ultrasonication respectively (a-d). Table 1 Minimum inhibitory concentration inhibiting 50% bacterial growth (MIC50) of Cu-NPs without and with honey samples against E. coli and E. faecalis. Samples
MIC50 value of nanoparticles (μg/ml) Bacterial strains
Cu-NPs without honey Cu-NPs with honey
E. coli (gram negative)
E. faecalis (gram positive)
288 250
31.3 15.6
Fig. 9. Percentage viability of treated bacteria (a) E. coli and; (b) E. faecalis with Cu-NPs without and with honey at various concentrations.
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(a)
CCD112
Percentage viability (%)
100
Cu-NPs with honey Cu-NPs without honey
80 60 40 20 0 0
7.8
15.6
31.3
62.5
125
250
500
Concentration (µg/mL)
HCT116 Percentage viability (%)
(b)
100
Cu-NPs with honey Cu-NPs without honey
80 60 40 20 0 0
7.8
15.6
31.3
62.5
125
250
500
Concentration (µg/mL) Fig. 10. Cytotoxic effects of Cu-NPs without and with honey on (a) colon normal cells (CCD112) and (b) colorectal cancer cells (HCT116) after 72 h of treatment.
Fig. 7 shows the difference between the morphology and shape of the Cu-NPs synthesized without honey and with honey. The shape of CuNPs without honey was inconsistent compared to the one with honey. This may suggest that the agglomeration of Cu-NPs without honey might occur faster than the one using honey [34]. The size of the nanoparticles with honey was smaller than the one without honey. This finding is consistent with the HRTEM analysis (Fig. 5). Besides that, EDX analysis shows that there is small percentage of oxygen element in the Cu-NPs without honey, this might be due to the oxidation that occurred on the surface of the nanoparticles. However, this oxygen element is absent in the Cu-NPs with honey. This could support that honey can act as a capping agent to prevent oxidation. The carbon element might come from either ascorbic acid or compounds in honey as it was detected in both Cu-NPs as shown by EDX spectra (Fig. 7b and d).
honey, pure ascorbic acid, Cu-NPs without and with honey after 10 min of ultrasonic assistance. The peaks observed between 3100 and 3500 cm−1 might be due to the hydroxyl group (–OH) and primary amine –NH bonded group of carboxylic acid –COOH. However, the peak for the Cu-NPs without honey was less intense compared to CuNPs with honey, this might due to the absence of polyhydroxyl group from honey compared to the ascorbic acid alone. Next, the peak for the alkane CeH stretching observed around 2928 cm−1 for the Cu-NPs with honey but this was absence in Cu-NPs without honey. This is because of the sp3 hybridized CH3 for methyl which is not present in the ascorbic acid structure [52]. Besides, the peaks might shifting a little bit indicating reaction occur. According to Rajesh et al. [18], the differences in the peak locations indicate that the protein obligation for the synthesis of Cu-NPs is diverse. These protein molecules act as surface coating molecules that keep away the internal agglomeration of the particles. A peak was seen for Cu-NPs around 2887–2905 cm−1 which might due to the CueH bonding [28]. Meanwhile, the peaks were observed between 1780 cm−1 and 1607 cm−1 for the Cu-NPs with and without honey respectively which denote the C]C stretching aromatic rings, NeH vibration of amine, carbonyl group for amide and carboxylic groups. The peaks of amide I and amide II bands of protein
3.5. Fourier-transform infrared (FTIR) analysis The main constituents in honey are fructose, glucose, proteins, minerals and vitamins. The purpose of FTIR measurement is to identify the possible biomolecules that responsible for capping and stabilizing the Cu-NPs synthesized using honey. Fig. 8 shows the IR spectra of the 7
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Table 1 represents the minimum inhibitory concentrations that inhibit 50% bacterial growth (MIC50) measured in this study for both Cu-NPs against E. coli and E. faecalis. Fig. 9 shows the dose-response curves in which the percent viability of bacteria was calculated using Eq. (1). The data suggested that both Cu-NPs without and with honey exhibit antibacterial properties. As shown in Fig. 9a, the percent viability decreased following the increasing concentration. The MIC50 of both Cu-NPs without and with honey for E.coli are 288 and 250 μg/ml (Table 1). Ruparelia et al. [55] reported that the MIC of Cu-NPs for E.coli is around 300 μg/ml which is almost similar with the one that obtained in this study. The MIC might be dissimilar with some results that obtained at lower concentration in existed study where the difference are due to the size of nanoparticles and also the initial bacterial concentration. In this study, a high CFU/ml of bacteria were used. Besides, the MIC50 for Cu-NPs with honey against E. faecalis (15.6 μg/ml) shows > 2-fold lower than the one without honey (31.3 μg/ml). The value is quite acceptable as the previous study reported that the MIC for E. faecalis are between 31.3 and 150 μg/ml [56,57]. The difference in MIC value is possibly due to the surface of Cu-NPs with honey which have larger surface area to volume to react with the bacterial cell membrane as the size is smaller in this study compare to the existing study [56]. The MIC50 for Cu-NPs toward E. faecalis strain is much lower compared to E.coli and this is consistent with a study reported by Dinda et al. [58] in which gram positive bacteria were more susceptible to the Cu-NPs compared to the gramnegative bacteria. This could be due to the multiple membrane layers and complex structure of gram-negative bacteria. Meanwhile, the minimum bactericidal concentration (MBC) for E. faecalis strain for
Table 2 IC50 value of nanoparticles treated with normal and cancerous cell lines. Samples
IC50 value of nanoparticles (μg/ml) CCD112 (normal)
HCT116 (cancer)
8.23 44.07
16.32 46.11
Cu-NPs without honey Cu-NPs with honey
might overlap with the groups of 1780–1607 cm−1. This is due to the carboxyl stretching and NeH deformation vibration in the amide linkages of protein [53]. Rasouli et al. [53] have speculated that the free amine group or carboxylate ion of amino acid residue might be binding to the NPs. Besides that, the peaks of C]C appeared around 1607 cm−1 and 1611 cm−1 for Cu-NPs with and without honey respectively as the stretching of the C]C occur. Normally, if the conjugation occurs, the C]C stretching would shift to lower frequencies. The peaks around 1400–1300 cm−1 and 1000–1100 cm−1 are for the CeH deformations of –CH2 or –CH3 groups (lignin) in aliphatic and CeO of alcohol group or ester respectively [54]. These might be also due to the CeO stretching band of protein in honey arising from the C-O-C symmetric stretching and C-O-H bending vibration that are expected to occur around 1073 cm−1. 3.6. Antibacterial effects of Cu-NPs Minimum inhibitory concentration (MIC) is defined as the lowest antibiotic concentration that prevents visible growth of bacteria.
(a)
Control normal cell
(d)
Control cancer cell
(b)
Cu-NPs without honey
(e)
Cu-NPs without honey
(c)
Cu-NPs with honey
(f)
Cu-NPs with honey
Fig. 11. Microscopic images of Cu-NPs at concentration of 31 μg/ml on CCD112 [control, treated cell Cu-NPs without honey and Cu-NPs with honey (a–c)] and HCT116 [control, treated cell with Cu-NPs without honey and Cu-NPs with honey (d–f)] respectively. 8
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both Cu-NPs are observed at concentration of 125 μg/ml. However for E.coli, the MBC cannot be determined as at concentration of 500 μg/ml, the bacterial growth still can be seen on the agar plate. It is normal for the MBC to occur at high concentration of MIC where it can be concluded that the Cu-NPs were bacteriostatic at lower concentration but bactericidal at higher concentration [59]. At 1 μg/ml of imipenem which is the positive control, it killed > 70% bacteria. As described by Mahmoodi et al. [60], there are several ways for the Cu-NPs to kill bacteria which include the interaction of CuNPs with the bacterial cell membrane through electrostatic interaction, increasing the reactive oxygen species (ROS) in the cell, effect of NPs on the protein structure on the cell membrane, and the interaction of NPs with the phosphorus and sulphur-containing molecules such as DNA.
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Wang, X. Wang, L. Wang, X. Hou, W. Liu, C. Chen, Interaction of gold nanoparticles with proteins and cells, Sci. Technol. Adv. Mater. 16 (2015) 1–15, https:// doi.org/10.1088/1468-6996/16/3/034610. [21] G. Grass, C. Rensing, M. Solioz, Metallic copper as an antimicrobial surface, Appl. Environ. Microbiol. 77 (2011) 1541–1547, https://doi.org/10.1128/AEM. 02766-10. [22] A.K. Chatterjee, R. Chakraborty, T. Basu, Mechanism of antibacterial activity of copper nanoparticles, Nanotechnology 25 (2014) 1–12, https://doi.org/10.1088/ 0957-4484/25/13/135101. [23] I.M. Chung, A.A. Rahuman, S. Marimuthu, K. Vishnu, K. Anbarasan, P. Padmini, G. Rajakumar, Green synthesis of copper nanoparticles using Eclipta prostrata leaves extract and their antioxidant and cytotoxic activities, Exp. Ther. Med. 14 (2017) 18–24, https://doi.org/10.3892/etm.2017.4466.
3.7. Cytotoxic effects of Cu-NPs As shown in Fig. 10, the cytotoxicity of both Cu-NPs were performed on colon normal and cancerous cells (Fig. 10a and b). The cells were treated with Cu-NPs without and with honey at various concentrations (0–500 μg/ml) and incubated at 37 °C, 5% CO2 for 72 h. From Fig. 10a, 60% killing by Cu-NPs without honey was seen at 7.8 μg/ml, while the cells treated with Cu-NPs in the presence of honey retained their viability (> 90%). In both cells, Cu-NPs without honey killed most of them (approximately 90%) at high concentration (> 31 μg/ml). On the other hand, Cu-NPs with honey showed gradual killing toward both cells, about 90% killing activity was also seen at 250 μg/ml. The IC50 value for the Cu-NPs was determined and summarized in Table 2. Surprisingly, the larger Cu-NPs exhibited higher cytotoxicity than the smaller Cu-Nps where Cu-NPs without honey has higher killing activities than Cu-NPs with honey against both cells. This data is consistent with the microscopic examination as shown in Fig. 11. As reported by, Wongrakpanich et al. [61], it is possible that these two types of NPs have different modes of entry or rates of uptake because of their difference in size, which consequently may affect the levels of Cu2+ ions accumulating within the cells. Unfortunately, both NPs did not show their specificity toward the cancerous cells compared to the normal cells. This data suggests that the NPs are not selective enough to be used as anticancer compound. Thus, there is a possibility that further modifications of the NPs are required to improve the NPs' specificity. 4. Conclusions To conclude, highly pure Cu-NPs without and with honey have been successfully synthesized. Honey was proven effective in reducing and stabilizing the Cu-NPs as smaller size of Cu-NPs with honey, approximately 3.68 ± 0.78 nm was produced compared to the Cu-NPs without honey. The protein and carbohydrate of honey might contribute as the stabilizing and reducing agent for the Cu-NPs. The Cu-NPs are more effective toward gram positive (E. faecalis) compared to gram-negative bacteria (E. coli). Cu-NPs with honey exhibited higher inhibition against both bacteria compared to Cu-NPs without honey. As for the cytotoxicity, Cu-NPs possess the ability to kill the tested cell lines but Cu-NPs without honey has much more higher killing activity toward normal and cancer cells. Further modifications of the NPs are required to improve the selectivity of compound toward the cancer cells. Acknowledgements The authors wish to acknowledge funding by the Ministry of Higher Education, Malaysia under the Tier 1 grants (Grant no. #20H33 and #20H55) and express gratitude to the Research Management Centre (RMC) of UTM and Malaysia-Japan International Institute of Technology (MJIIT) for providing an excellent research environment and facilities. 9
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Antibacterial and cytotoxic effect of honey mediated copper nanoparticles synthesized using ultrasonic assistance
T
Nur Afini Ismaila, Kamyar Shamelia, , Magdelyn Mei-Theng Wongb, Sin-Yeang Teowb, Jactty Chewc, Siti Nur Amalina Mohamad Sukria ⁎
a
Department of Environment and Green Technology, Malaysia-Japan International Institute of Technology, Universiti Teknologi Malaysia, Jalan Sultan Yahya Petra, 54100 Kuala Lumpur, Malaysia b Department of Medical Sciences, School of Healthcare and Medical Sciences, Sunway University, Jalan Bandar Sunway, 47500, Selangor Darul Ehsan, Malaysia c Department of Biological Sciences, School of Science and Technology, Sunway University, Jalan Bandar Sunway, 47500, Selangor Darul Ehsan, Malaysia
ARTICLE INFO
ABSTRACT
Keywords: Antibacterial Cytotoxicity assay Copper nanoparticle Honey mediated Sonochemical
In this study, a comparative study of effect using honey on copper nanoparticles (Cu-NPs) via simple, environmentally friendly process and inexpensive route was reported. Honey and ascorbic acid act as stabilizing and reducing agents with the assistance of sonochemical method. The products were characterized using UV–visible (UV–vis) spectroscopy, X-Ray Diffraction (XRD), High-Resolution Transmission Electron Microscopy (HRTEM), Field-Emission Scanning Electron Microscopy (FESEM) and Fourier Transform Infrared (FTIR) spectroscopy. The reddish brown colour demonstrated the formation of Cu-NPs and UV–visible proved the plasmon resonance of Cu-NPs. XRD also confirmed a highly pure Cu-NPs obtained with absence of copper oxide in which the structure is crystalline. The spherical size of the Cu-NPs was acquire in the presence of honey which is 3.68 ± 0.78 nm with narrow particle distribution. The antibacterial activity was seen against gram-positive and gram-negative bacteria which are Enterococcus faecalis (E. faecalis) and Escherichia coli (E. coli). At higher concentration of Cu-NPs, they were more effective in killing both bacteria. The Cu-NPs without and with honey exhibited toxicities toward normal and cancerous cells. However, Cu-NPs without honey showed more potent killing activity against normal and cancer cells.
1. Introduction
special characteristics such as low cost, less toxic, high surface area to volume ratio, heat transfer properties and high friction of atoms [11,12]. These characteristics are mainly resulted by their physical properties such as size, shape, composition, morphology and crystalline phase [13]. Cu-NPs also have abundant sources, easy preparation and flexibility in modifying into various shape of nano-sized dimension [14]. Cu-NPs can be implemented in several applications such as biomedical, optical, biosensor, catalytic and energy application [15–19]. In an important biomedical application which is drug delivery system and molecular doping, Cu-NPs play particularly important role as vehicles to deliver the compounds to the target sites [20]. Moreover, copper also is a multifunctional antibacterial agents [21]. Cu-NPs have been demonstrated to show potent inhibition toward various bacterial species [22–25]. Various studies showed that Cu-NPs could bind and conjugate with biomolecules like proteins, enzymes and DNA [20,26], indicating the potentials of Cu-NPs to be used as ‘nano-drugs’ [27]. There are various ways to synthesize Cu-NPs using physical and
Nanotechnology emerges as one of the alternative technologies in addressing problems that arise nowadays particularly pertaining to environmental issues [1]. It also has various medical and biomedical applications such as the production of nanoparticles and carbon nanotubes for bio-imaging, cancer therapy, drug delivery and antibacterial [2–4]. Nanoparticle production, particularly metallic and metal oxide nanoparticles, is attracting vast interest from scientists in recent years due to the unique characteristics such as having surface plasmon resonance (SPR) and optical properties [5]. They are also actively used for biosensor, electrical, catalytic and antimicrobial applications [6–9]. The size of nanomaterials typically ranges from 1 to 100 nm. Generally, nanoparticles could be classified as fine particles which has the size ranging from 100 to 2500 nm while ultrafine particles possess the size from 1 to 100 nm [10]. Gold-, platinum-, silver-, and copper-nanoparticles (Cu-NPs) are a few relatively well studied nanoparticles. Among them, Cu-NPs have
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Corresponding author. E-mail address: [email protected] (K. Shameli).
https://doi.org/10.1016/j.msec.2019.109899 Received 24 January 2019; Received in revised form 11 June 2019; Accepted 15 June 2019 Available online 18 June 2019 0928-4931/ © 2019 Published by Elsevier B.V.
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chemical methods, such as laser ablation [28], gamma ray irradiation [29], chemical precipitation method [30], sol–gel methods [31], solidstate reaction [32] and sonochemical preparation [33,34]. However, the major problems of using these methods are producing toxic byproduct, costly and laborious process [34,35]. Green chemistry synthesis has drawn market attention nowadays as it is simple, inexpensive and environment friendly. Generally, there are three criteria to follow for green synthesis of nanoparticles, including the choice of solvent, the use of eco-friendly reducing agent and the use of nontoxic material as capping agent to stabilize the nanoparticles synthesis [36]. Honeymediated nanoparticle production, one of the examples of green synthesis is gaining attention in scientific community nowadays [37,38]. They are widely used in biological studies such as tissues engineering and cytotoxicity study [37,39]. Copper is highly unstable and it can easily oxidize at atmospheric condition which generates copper oxide (CuO) or copper (II) oxide (Cu2O). Hence, a surface-protecting stabilizing agent that can form complexes with the Cu-NPs is mandatory [40]. Honey is a natural food and a sweet viscous liquid consisting of carbohydrate, enzymes, vitamins, minerals and antioxidant [37,41]. The monosaccharides, proteins, amino acid and vitamin C in honey could help in reducing and stabilizing the nanoparticles [9], although little is known of the contributing factors for these capacities [38,42]. The use of honey as reducing and stabilizing agent is convenient as it does not require drying, extraction of plant materials and cell culture maintenance [41]. Ultrasonic irradiation-assisted heating is gaining attention as a common method for rapid synthesis of organic and inorganic compounds [43]. A few advantages using this method are, it could further reduce the size of particles and reduce the time of reaction [44,45]. The effects of ultrasonic irradiation on reaction are due to acoustic cavitation within collapsing bubbles. During bubble collapse, unique hot spots are produced at extreme conditions (high temperatures above 5000 K, pressures of about 1000 atm, and heating and cooling rates of approximately 1010 Ks−1) [43]. In this study, honey-mediated Cu-NPs were synthesized by green method assisted by ultrasonic irradiation. Honey was used as reducing and stabilizing agent along with the ascorbic acid (vitamin C) as supporting reducing agent. The size and morphology of Cu-NPs were characterized by UV–vis, XRD, HRTEM, FESEM and FTIR. The potential biomedical application of Cu-NPs was then evaluated by an antibacterial assay against two gram positive and gram negative bacteria as well as a cytotoxicity assay against two mammalian cell lines.
stirred until homogenized. After that, 0.6 M sodium hydroxide was added dropwise into the mixture solution while continuously stirring for 10 min until approximately pH 7.5. Next, 15 ml ascorbic acid (1 M) was added into the mixture solution and underwent ultrasound irradiation for 10 min with the amplitude of 80%, pulse on 1 s and pulse off 1 s. Lastly, the solution was washed with distilled water twice and dried in oven. These steps were repeated without honey to synthesize Cu-NPs without honey. 2.3. Characterization method and instrumentations The Cu-NPs were characterized using Ultraviolet-visible (UV–vis) spectroscopy (UV-2600, SHIMADZU). The clean quartz solution cells were used. The sample solution with desired concentration and a blank sample (water) were used for this experiment. The samples were homogenized in the quartz solution cells and placed into the UV–vis chamber. The UV–vis spectra were recorded over the range of 220 nm to 1000 nm. The structure of Cu-NPs were evaluated using X-ray diffraction (XRD, Philips, X'pert, Cu Ka) in the small-angle range of 20° to 80° (2θ). High Resolution Transmission Electron Microscopy (HRTEM) (model JEM-2100F) was used to examine the size of Cu-NPs. To analyse the morphology and elemental analysis of the Cu-NPs, JOEL-FESEM (model JSM 7600F FESEM) attached to Energy-Dispersive X-ray spectroscopy was used. Fourier Transform Infrared (FTIR) spectrum was utilized to examine the functional groups present in the synthesized compound. Finally, FTIR spectra were recorded over the range of 400–4000 cm−1 using Attenuated Total Reflectance (ATR), IRTracer100 spectrophotometer (Shimadzu, Malaysia). 2.4. Broth micro-dilution and MTT assay for antibacterial application A broth micro-dilution method and MTT assay were used to determine the minimum inhibitory concentration (MIC) values and minimum bactericidal concentration (MBC) of the Cu-NPs using the Clinical and Laboratory Standards Institute (CLSI) protocols. Grampositive (Enterococcus faecalis- ATCC 33186) and gram-negative (Escherichia coli- MTCC 710859) bacterial species were used in this study. Single colony of fresh bacterial culture (12–18 h) was isolated from Mueller Hinton agar (MHA) plates and inoculated into sterile Mueller Hinton broth (MHB). The culture was grown overnight (12–18 h) prior to the experiments. Next day, the bacterial concentration was standardized to an optical density (OD) of 600 nm (approximately 108 CFU/ml) with MHB. Two-fold serial dilutions of nanoparticles (Cu-NPs with and without honey) were prepared in 96 well plates to give final test concentrations of 0, 7.8, 15.6, 31.3, 62.5, 125, 250 and 500 μg/ml per well. 10 μl of bacterial suspension equivalent to 106 CFU/ml of exponentially growing bacterial cells were added to the wells. The plates were incubated at 35 ± 2 °C for 18 h. Following the overnight incubation, the plate was then read for the absorbance using microplate reader (GloMax Discover Instrument, Promega) to determine the MIC50 values. Positive control, imipenem antibiotic (1 μg/ ml), and negative controls (blank, without bacterial inoculum) were included in all experiments. Then, the wells were filled with 10 μl of MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] which were prepared in PBS (pH 7.2) and the wells were incubated for 30 min at 28 °C. Then the clear wells without colour changing with the MTT were plated on the agar to observe the MBC of the samples toward the bacteria strain.
2. Materials and methods 2.1. Materials Copper II nitrate (Cu(NO3)2.3H2O, ~99%), ascorbic acid (C6H8O6, ~99%), sodium hydroxide (NaOH) were purchased from R&M chemical, United Kingdom. Pure honey was purchased from Capilano Honey Limited, Australia where the source of flowers are from Australian eucalyptus and ground flora. Double distilled water was used as solvent throughout the experiment. All chemicals used were analytical grade without further purification. Imipenem was purchased from GoldBio (USA). Sterile Mueller-Hinton agar and broth (Becton Dickinson, USA) were used to culture and maintain the bacterial strains. 96-well plates for antibacterial application were purchased from Nest Biotech Co., Ltd., China. MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was purchased from Sigma Aldrich.
2.5. Cytotoxicity assay
2.2. Green synthesis of copper nanoparticles
To determine the cellular killing effect of nanoparticles, cell proliferation assay (Promega) was performed according to the manufacturer's instruction with slight modification. Briefly, 5,000 human colorectal cancer cell line (HCT116) (ATCC CCL-247) and human normal colon cell (CCD112) (ATCC CRL-1541) cells per well (100 μl/well) were
The Cu-NPs were prepared using chemical reduction assisted by ultrasonic irradiation. Briefly, copper nitrate trihydrate (0.025 mol) was dissolved in 100 ml of double distilled water. Then, 10% w/v concentration of honey was added into the copper nitrate solution and 2
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Fig. 1. Diagram showing Cu(NO3)2, [Cu(Honey)]2+ complex, honey‑copper hydroxide complex ([Cu(OH)2/Honey]), and Cu-NPs solutions (a–d). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Schematic illustration of Cu-NPs stabilized by fructose and glucose under ultrasonic irradiation.
Fig. 3. UV–vis spectroscopy of honey, and Cu-NPs without and with honey. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. X-ray diffraction patterns of Cu-NPs: (a) Cu-NPs without honey (b) CuNPs with honey.
and incubated for additional 3 h at 37 °C in the 5% CO2 incubator. Optical density (OD) was then measured at 490 nm using a multimode microplate reader (Tecan). The dose-response graph was plotted by calculating the percent cell viability using Eq. (1). The images of cells treated with the nanoparticles were captured using an inverted microscope attached to a camera system (IM3 Phase contrast, Optika, Italy).
seeded onto a 96-well plate and incubated at 37 °C overnight in a 5% CO2 humidified incubator. Next day, 2-fold serially diluted nanoparticles (500, 250, 125, 62.5, 31.3, 15.6, 7.8, 0 μg/ml) (100 μl/well) were added into the wells and the plate was incubated for 72 h at 37 °C in the 5% CO2 humidified incubator. Then 20 μl MTS (3-(4,5Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium) reagent (Promega) per well was added into the plate 3
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Fig. 5. HRTEM image for green synthesis of (a) Cu-NPs without honey and; (b) Cu-NPs with honey, the histograms on the right shows the distribution of particle size. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
shown in Eqs. (2)–(4).
%Viability = OD of sample well (mean)/OD of control well (mean) × 100 (1)
Cu2 + (aq) + Honey [Cu(Honey)]2 + (aq)
3. Results and discussion Comparison between Cu-NPs without and with honey was carried out by characterizing both samples using UV–vis, XRD, HRTEM, FESEM-EDX and FTIR to determine the effect of honey on Cu-NPs synthesis. The occurrence of reaction was first observed by the colour changes of the solution. During the synthesis, the colour of solution changed from light blue to light yellow, then to blue, and lastly turned to reddish brown (Fig. 1). This is consistent with the observation by Mardiansyah et al. for the biosynthesis of Cu-NPs [46]. The light blue colour (Fig. 1a) is the precursor solution which is copper II nitrate solution, while the light yellow (Fig. 1b) is [Cu(Honey)]2+ complex solution. On the other hand, the blueish solution (Fig. 1c) is the honey‑copper hydroxide complex ([Cu(OH)2/Honey]) and Fig. 1d shows the reddish brown Cu-NPs solution after 10 min of ultrasonic irradiation [47]. The proposed mechanism for the Cu-NPs productions is as follows: copper nitrate solution dissociated to form Cu2+, then the Cu2+ reacted with the negative charge of the hydroxyl group of fructose and glucose in honey and formed [Cu(Honey)]2+. With the addition of sodium hydroxide, the reaction occurred and formed ([Cu(OH)2/Honey]). After the addition of ascorbic acid, it yielded Cu-NPs following the ultrasonic irradiation process. This step is important to reduce the reaction time for Cu-NPs production. It is proposed that the positive charge on the surface of Cu-NPs formed electrostatic bond with the negative charge of hydroxyl group from the fructose and glucose of honey. The proposed mechanism is illustrated in Fig. 2. The chemical reaction equation are
Room temperature,Stir
[Cu(Honey)]2 + (aq)
NaOH (0.6 M) Room temperature,Stir
[Cu(OH)2/Honey](aq)
[Cu(OH)2/Honey](aq)
Ascorbic acid (1 M) Ultrasonic irradiation (10 min)
(2) (3)
[Cu/Honey](aq) (4)
Honey is a weak reducing agent [48]. It can reduce the Cu2+ to Cu+ but it will take a longer time to reduce the Cu2+ to Cu0. Thus, ascorbic acid was used enhance the reduction process of Cu2+ and shorten the reaction time. With ultrasonic assistance, the reaction time could be further reduced compared to the conventional method [34]. 3.1. UV–visible (UV–vis) absorption UV–vis absorption confirmed the successful synthesis of Cu-NPs without and with honey as peaks were observed at 613 and 583 nm respectively (Fig. 3). These peaks could be observed due to the surface plasmon resonance of Cu-NPs [44]. There is a blue shift from 613 nm (Fig. 3a) to 583 nm (Fig. 3b) for the Cu-NPs with honey which indicates the decreased size of nanoparticles [44]. This could be due to the presence of protein and monosaccharide in honey that act as stabilizing agent and prevent the Cu-NPs from the agglomeration as compared to the Cu-NPs without honey. According to the HPLC test done by Enrico et al. [49] for four different honey including pure honey and Australian honey, the percentage of glucose and fructose were 31% and 50% respectively. Thus, it is confirmed that the highest percentage composition in raw honey are glucose and fructose. Therefore, it should exhibit the same results in term of size as based on paper that producing Cu4
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Fig. 6. Lattice fringes d-spacing of Cu-NPs with honey and SAED image Cu-NPs with honey, respectively (a–b).
NPs using different concentration of glucose as capping agent, the particle size are decreasing as the amount of glucose increasing [50]. For honey solution alone (Fig. 3c), there is an intense peak around 300 nm which might be due to the geographic region and the aging honey itself [51].
and the distribution of particle size. Comparatively, there is a significant size difference between the two. The Cu-NPs without honey have the size of approximately 100 nm while the mean size of the CuNPs with honey is 3.68 ± 0.78 nm as shown in Fig. 5. The histogram in Fig. 5b shows an even distribution of the particle size. This might be due to the polyhydroxyl group of glucose and fructose in honey that acts as stabilizer to prevent the formation of large agglomerates from the nanoparticles. The sole effect of honey might have the similar effect produced by using glucose or fructose as it exhibit the same trend in term of size reduction in Cu-NPs production where the particle size decrease as concentration of glucose increase [50]. After comparing and analysing the histogram, it can be concluded that honey can act as a stabilizing agent as the distribution of the histogram is much more narrower compared to the histogram shown by Cu-NPs without honey [45]. The lattice d-spacing of Cu-NPs with honey was calculated to be 0.20 nm which is similar with the selected area diffraction (SAED) at 111 plane (Fig. 6).
3.2. X-ray diffraction (XRD) structure analysis Highly pure copper from honey was obtained as characterized by XRD depicted in Fig. 4. The XRD graphs showed only the copper without other compounds including copper oxide. Both Cu-NPs had a similar diffraction profile and XRD peaks at 2θ of 43.30°, 50.40° and 74.12° could be attributed to the crystal plane of 111, 200 and 220. All diffraction patterns are in agreement with the reference pattern of facecentred cubic (FCC) phase of copper (JCPDS 04-0836). Furthermore, the synthesized Cu-NPs were stored for 4 months and re-characterized using the same method. The XRD result was consistent without the peak of copper oxide which suggests the retained purity of copper.
3.4. Field-emission scanning electron microscopy and energy dispersive Xray spectroscopy (FESEM-EDX) analysis
3.3. High-resolution transmission electron microscopy (HRTEM) analysis Fig. 5 shows the HRTEM images of Cu-NPs without and with honey,
The morphology of Cu-NPs was characterized using FESEM-EDX. 5
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Fig. 7. FESEM micrographs and EDX spectra of Cu-NPs without honey (a, b) and Cu-NPs with honey (c, d) respectively.
Fig. 8. FTIR spectrum of honey, ascorbic acid, Cu-NPs without honey and CuNPs with honey after 10 min ultrasonication respectively (a-d). Table 1 Minimum inhibitory concentration inhibiting 50% bacterial growth (MIC50) of Cu-NPs without and with honey samples against E. coli and E. faecalis. Samples
MIC50 value of nanoparticles (μg/ml) Bacterial strains
Cu-NPs without honey Cu-NPs with honey
E. coli (gram negative)
E. faecalis (gram positive)
288 250
31.3 15.6
Fig. 9. Percentage viability of treated bacteria (a) E. coli and; (b) E. faecalis with Cu-NPs without and with honey at various concentrations.
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(a)
CCD112
Percentage viability (%)
100
Cu-NPs with honey Cu-NPs without honey
80 60 40 20 0 0
7.8
15.6
31.3
62.5
125
250
500
Concentration (µg/mL)
HCT116 Percentage viability (%)
(b)
100
Cu-NPs with honey Cu-NPs without honey
80 60 40 20 0 0
7.8
15.6
31.3
62.5
125
250
500
Concentration (µg/mL) Fig. 10. Cytotoxic effects of Cu-NPs without and with honey on (a) colon normal cells (CCD112) and (b) colorectal cancer cells (HCT116) after 72 h of treatment.
Fig. 7 shows the difference between the morphology and shape of the Cu-NPs synthesized without honey and with honey. The shape of CuNPs without honey was inconsistent compared to the one with honey. This may suggest that the agglomeration of Cu-NPs without honey might occur faster than the one using honey [34]. The size of the nanoparticles with honey was smaller than the one without honey. This finding is consistent with the HRTEM analysis (Fig. 5). Besides that, EDX analysis shows that there is small percentage of oxygen element in the Cu-NPs without honey, this might be due to the oxidation that occurred on the surface of the nanoparticles. However, this oxygen element is absent in the Cu-NPs with honey. This could support that honey can act as a capping agent to prevent oxidation. The carbon element might come from either ascorbic acid or compounds in honey as it was detected in both Cu-NPs as shown by EDX spectra (Fig. 7b and d).
honey, pure ascorbic acid, Cu-NPs without and with honey after 10 min of ultrasonic assistance. The peaks observed between 3100 and 3500 cm−1 might be due to the hydroxyl group (–OH) and primary amine –NH bonded group of carboxylic acid –COOH. However, the peak for the Cu-NPs without honey was less intense compared to CuNPs with honey, this might due to the absence of polyhydroxyl group from honey compared to the ascorbic acid alone. Next, the peak for the alkane CeH stretching observed around 2928 cm−1 for the Cu-NPs with honey but this was absence in Cu-NPs without honey. This is because of the sp3 hybridized CH3 for methyl which is not present in the ascorbic acid structure [52]. Besides, the peaks might shifting a little bit indicating reaction occur. According to Rajesh et al. [18], the differences in the peak locations indicate that the protein obligation for the synthesis of Cu-NPs is diverse. These protein molecules act as surface coating molecules that keep away the internal agglomeration of the particles. A peak was seen for Cu-NPs around 2887–2905 cm−1 which might due to the CueH bonding [28]. Meanwhile, the peaks were observed between 1780 cm−1 and 1607 cm−1 for the Cu-NPs with and without honey respectively which denote the C]C stretching aromatic rings, NeH vibration of amine, carbonyl group for amide and carboxylic groups. The peaks of amide I and amide II bands of protein
3.5. Fourier-transform infrared (FTIR) analysis The main constituents in honey are fructose, glucose, proteins, minerals and vitamins. The purpose of FTIR measurement is to identify the possible biomolecules that responsible for capping and stabilizing the Cu-NPs synthesized using honey. Fig. 8 shows the IR spectra of the 7
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Table 1 represents the minimum inhibitory concentrations that inhibit 50% bacterial growth (MIC50) measured in this study for both Cu-NPs against E. coli and E. faecalis. Fig. 9 shows the dose-response curves in which the percent viability of bacteria was calculated using Eq. (1). The data suggested that both Cu-NPs without and with honey exhibit antibacterial properties. As shown in Fig. 9a, the percent viability decreased following the increasing concentration. The MIC50 of both Cu-NPs without and with honey for E.coli are 288 and 250 μg/ml (Table 1). Ruparelia et al. [55] reported that the MIC of Cu-NPs for E.coli is around 300 μg/ml which is almost similar with the one that obtained in this study. The MIC might be dissimilar with some results that obtained at lower concentration in existed study where the difference are due to the size of nanoparticles and also the initial bacterial concentration. In this study, a high CFU/ml of bacteria were used. Besides, the MIC50 for Cu-NPs with honey against E. faecalis (15.6 μg/ml) shows > 2-fold lower than the one without honey (31.3 μg/ml). The value is quite acceptable as the previous study reported that the MIC for E. faecalis are between 31.3 and 150 μg/ml [56,57]. The difference in MIC value is possibly due to the surface of Cu-NPs with honey which have larger surface area to volume to react with the bacterial cell membrane as the size is smaller in this study compare to the existing study [56]. The MIC50 for Cu-NPs toward E. faecalis strain is much lower compared to E.coli and this is consistent with a study reported by Dinda et al. [58] in which gram positive bacteria were more susceptible to the Cu-NPs compared to the gramnegative bacteria. This could be due to the multiple membrane layers and complex structure of gram-negative bacteria. Meanwhile, the minimum bactericidal concentration (MBC) for E. faecalis strain for
Table 2 IC50 value of nanoparticles treated with normal and cancerous cell lines. Samples
IC50 value of nanoparticles (μg/ml) CCD112 (normal)
HCT116 (cancer)
8.23 44.07
16.32 46.11
Cu-NPs without honey Cu-NPs with honey
might overlap with the groups of 1780–1607 cm−1. This is due to the carboxyl stretching and NeH deformation vibration in the amide linkages of protein [53]. Rasouli et al. [53] have speculated that the free amine group or carboxylate ion of amino acid residue might be binding to the NPs. Besides that, the peaks of C]C appeared around 1607 cm−1 and 1611 cm−1 for Cu-NPs with and without honey respectively as the stretching of the C]C occur. Normally, if the conjugation occurs, the C]C stretching would shift to lower frequencies. The peaks around 1400–1300 cm−1 and 1000–1100 cm−1 are for the CeH deformations of –CH2 or –CH3 groups (lignin) in aliphatic and CeO of alcohol group or ester respectively [54]. These might be also due to the CeO stretching band of protein in honey arising from the C-O-C symmetric stretching and C-O-H bending vibration that are expected to occur around 1073 cm−1. 3.6. Antibacterial effects of Cu-NPs Minimum inhibitory concentration (MIC) is defined as the lowest antibiotic concentration that prevents visible growth of bacteria.
(a)
Control normal cell
(d)
Control cancer cell
(b)
Cu-NPs without honey
(e)
Cu-NPs without honey
(c)
Cu-NPs with honey
(f)
Cu-NPs with honey
Fig. 11. Microscopic images of Cu-NPs at concentration of 31 μg/ml on CCD112 [control, treated cell Cu-NPs without honey and Cu-NPs with honey (a–c)] and HCT116 [control, treated cell with Cu-NPs without honey and Cu-NPs with honey (d–f)] respectively. 8
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both Cu-NPs are observed at concentration of 125 μg/ml. However for E.coli, the MBC cannot be determined as at concentration of 500 μg/ml, the bacterial growth still can be seen on the agar plate. It is normal for the MBC to occur at high concentration of MIC where it can be concluded that the Cu-NPs were bacteriostatic at lower concentration but bactericidal at higher concentration [59]. At 1 μg/ml of imipenem which is the positive control, it killed > 70% bacteria. As described by Mahmoodi et al. [60], there are several ways for the Cu-NPs to kill bacteria which include the interaction of CuNPs with the bacterial cell membrane through electrostatic interaction, increasing the reactive oxygen species (ROS) in the cell, effect of NPs on the protein structure on the cell membrane, and the interaction of NPs with the phosphorus and sulphur-containing molecules such as DNA.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.msec.2019.109899. References [1] N. Wilson, Nanoparticles: environmental problems or problem solvers? BioScience 68 (2018) 241–246, https://doi.org/10.1093/biosci/biy015. [2] K.S. Mekheimer, W.M. Hasona, R.E. Abo-Elkhair, A.Z. Zaher, Peristaltic blood flow with gold nanoparticles as a third grade nanofluid in catheter: application of cancer therapy, Phys. Lett. A 382 (2018) 85–93, https://doi.org/10.1016/j.physleta.2017. 10.042. [3] W. Ahmed, A. Elhissi, V. Dhanak, K. Subramani, Carbon nanotubes: Applications in cancer therapy and drug delivery research, Micro and Nanotechnologies, Emerging Nanotechnologies in Dentistry, Second edition, Elsevier Inc., 2018, pp. 371–389, , https://doi.org/10.1016/B978-0-12-812291-4.00018-2. [4] L.L. Wang, C. Hu, L.Q. Shao, The antimicrobial activity of nanoparticles: present situation and prospects for the future, Int. J. Nanomedicine 12 (2017) 1227, https://doi.org/10.2147/IJN.S121956. [5] K. Harish Kumar, V. Nagasamy, B. Himangshu, K. Anuttam, Metallic nanoparticle: a review, Biomed. J. Sci. Tech. Res. 4 (2018) 1–11. https://doi.org/10.26717/BJSTR. 2018.04.001011. [6] F. Meng, H. Sun, Y. Huang, Y. Tang, Q. Chen, P. Miao, Peptide cleavage-based electrochemical biosensor coupling graphene oxide and silver nanoparticles, Anal. Chim. Acta 1047 (2019) 45–51, https://doi.org/10.1016/j.aca.2018.09.053. [7] M.N. Hossain, W. Jiali, A. Chen, Unique copper and reduced graphene oxide nanocomposite toward the efficient electrochemical reduction of carbon dioxide, Sci. Rep. 7 (2017) 3184, , https://doi.org/10.1038/s41598-017-03601-3. [8] M.T. Islam, H. Jing, T. Yang, E. Zubia, A.G. Goos, R.A. Bernal, C.E. Botez, M. Narayan, C.K. Chan, J.C. Noveron, Fullerene stabilized gold nanoparticles supported on titanium dioxide for enhanced photocatalytic degradation of methyl orange and catalytic reduction of 4-nitrophenol, J. Environ. Chem. Eng. 6 (2018) 3827–3836. [9] A.M. Hosny, M.T. Kashef, S.A. Rasmy, D.S. Aboul-Magd, Z.E. El-Bazza, Antimicrobial activity of silver nanoparticles synthesized using honey and gamma radiation against silver-resistant bacteria from wounds and burns, Adv. Nat. Sci. Nanosci. Nanotechnol. 8 (2017) 1–7, https://doi.org/10.1088/2043-6254/aa8b44. [10] S. Kamble, B. Utage, P. Mongle, S. Kamble, S. Hese, B. Dawane, R. Gacche, Evaluation of curcumin capped copper nanoparticles as possible inhibitors of human breast cancer cells and angiogenesis: a comparative study with native curcumin, AAPS PharmSciTech 17 (2016) 1030–1041. [11] H. Khalid, S. Shamaila, N. Zafar, S. Shahzadi, Synthesis of copper nanoparticles by chemical reduction method, Forensic Sci. Int. 27 (2015) 3085–3088 https://doi. org/10.13140/RG.2.1.1085.0643. [12] J.B. Fathima, A. Pugazhendhi, M. Oves, R. Venis, Synthesis of eco-friendly copper nanoparticles for augmentation of catalytic degradation of organic dyes, J. Mol. Liq. 260 (2018) 1–8, https://doi.org/10.1016/j.molliq.2018.03.033. [13] S.P. Prabhavathi, J. Punitha, P.S. Rajam, R. Ranjith, G. Suresh, N. Mala, D. Maruthamuthu, Simple methods of synthesis of copper oxide, zinc oxide, lead oxide and barium oxide nanoparticles, J. Chem. Pharm. Res. 6 (2014) 1472–1478. [14] M.B. Gawande, A. Goswami, F. Felpin, T. Asefa, X. Huang, R. Silva, X. Zou, R. Zboril, R.S. Varma, Cu and Cu-based nanoparticles: synthesis and applications in catalysis, Chem. Rev. 116 (2016) 3722–3811, https://doi.org/10.1021/acs. chemrev.5b00482. [15] N.V. Suramwar, S.R. Thakare, N.T. Khaty, One pot synthesis of copper nanoparticles at room temperature and its catalytic activity, Arab. J. Chem. 9 (2016) 1807–1812. [16] A.H. Keihan, H. Veisi, H. Veasi, Green synthesis and characterization of spherical copper nanoparticles as organometallic antibacterial agent, Appl. Organomet. Chem. 31 (2017) 1–7, https://doi.org/10.1002/aoc.3642. [17] D. Manoj, R. Saravanan, J. Santhanalakshmi, S. Agarwal, V.K. Gupta, R. Boukherroub, Towards green synthesis of monodisperse Cu nanoparticles: an efficient and high sensitive electrochemical nitrite sensor, Sensors Actuators B Chem. 266 (2018) 873–882, https://doi.org/10.1016/j.snb.2018.03.141. [18] K.M. Rajesh, B. Ajitha, A.K. Reddy, Y. Suneetha, P.S. Reddy, Assisted green synthesis of copper nanoparticles using syzygium aromaticum bud extract: physical, optical and antimicrobial properties, Optik 154 (2018) 593–600, https://doi.org/ 10.1016/j.ijleo.2017.10.074. [19] T. Hayat, M.I. Khan, S. Qayyum, A. Alsaedi, Entropy generation in flow with silver and copper nanoparticles, Colloids Surf. A Physicochem. Eng. Asp. 539 (2018) 335–346, https://doi.org/10.1016/j.colsurfa.2017.12.021. [20] P. Wang, X. Wang, L. Wang, X. Hou, W. Liu, C. Chen, Interaction of gold nanoparticles with proteins and cells, Sci. Technol. Adv. Mater. 16 (2015) 1–15, https:// doi.org/10.1088/1468-6996/16/3/034610. [21] G. Grass, C. Rensing, M. Solioz, Metallic copper as an antimicrobial surface, Appl. Environ. Microbiol. 77 (2011) 1541–1547, https://doi.org/10.1128/AEM. 02766-10. [22] A.K. Chatterjee, R. Chakraborty, T. Basu, Mechanism of antibacterial activity of copper nanoparticles, Nanotechnology 25 (2014) 1–12, https://doi.org/10.1088/ 0957-4484/25/13/135101. [23] I.M. Chung, A.A. Rahuman, S. Marimuthu, K. Vishnu, K. Anbarasan, P. Padmini, G. Rajakumar, Green synthesis of copper nanoparticles using Eclipta prostrata leaves extract and their antioxidant and cytotoxic activities, Exp. Ther. Med. 14 (2017) 18–24, https://doi.org/10.3892/etm.2017.4466.
3.7. Cytotoxic effects of Cu-NPs As shown in Fig. 10, the cytotoxicity of both Cu-NPs were performed on colon normal and cancerous cells (Fig. 10a and b). The cells were treated with Cu-NPs without and with honey at various concentrations (0–500 μg/ml) and incubated at 37 °C, 5% CO2 for 72 h. From Fig. 10a, 60% killing by Cu-NPs without honey was seen at 7.8 μg/ml, while the cells treated with Cu-NPs in the presence of honey retained their viability (> 90%). In both cells, Cu-NPs without honey killed most of them (approximately 90%) at high concentration (> 31 μg/ml). On the other hand, Cu-NPs with honey showed gradual killing toward both cells, about 90% killing activity was also seen at 250 μg/ml. The IC50 value for the Cu-NPs was determined and summarized in Table 2. Surprisingly, the larger Cu-NPs exhibited higher cytotoxicity than the smaller Cu-Nps where Cu-NPs without honey has higher killing activities than Cu-NPs with honey against both cells. This data is consistent with the microscopic examination as shown in Fig. 11. As reported by, Wongrakpanich et al. [61], it is possible that these two types of NPs have different modes of entry or rates of uptake because of their difference in size, which consequently may affect the levels of Cu2+ ions accumulating within the cells. Unfortunately, both NPs did not show their specificity toward the cancerous cells compared to the normal cells. This data suggests that the NPs are not selective enough to be used as anticancer compound. Thus, there is a possibility that further modifications of the NPs are required to improve the NPs' specificity. 4. Conclusions To conclude, highly pure Cu-NPs without and with honey have been successfully synthesized. Honey was proven effective in reducing and stabilizing the Cu-NPs as smaller size of Cu-NPs with honey, approximately 3.68 ± 0.78 nm was produced compared to the Cu-NPs without honey. The protein and carbohydrate of honey might contribute as the stabilizing and reducing agent for the Cu-NPs. The Cu-NPs are more effective toward gram positive (E. faecalis) compared to gram-negative bacteria (E. coli). Cu-NPs with honey exhibited higher inhibition against both bacteria compared to Cu-NPs without honey. As for the cytotoxicity, Cu-NPs possess the ability to kill the tested cell lines but Cu-NPs without honey has much more higher killing activity toward normal and cancer cells. Further modifications of the NPs are required to improve the selectivity of compound toward the cancer cells. Acknowledgements The authors wish to acknowledge funding by the Ministry of Higher Education, Malaysia under the Tier 1 grants (Grant no. #20H33 and #20H55) and express gratitude to the Research Management Centre (RMC) of UTM and Malaysia-Japan International Institute of Technology (MJIIT) for providing an excellent research environment and facilities. 9
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