Bio-inspired synthesis and cytotoxic evaluation of silver-gold bimetallic nanoparticles using Kei-Apple (Dovyalis caffra) fruits

Inorganic Chemistry Communications 109 (2019) 107569

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Bio-inspired synthesis and cytotoxic evaluation of silver-gold bimetallic nanoparticles using Kei-Apple (Dovyalis caffra) fruits

T

Jerry O. Adeyemia,b, Elias E. Elemikec, , Damian C. Onwudiwea,b, Moganavelli Singhd ⁎

a

Material Science Innovation and Modelling (MaSIM) Research Focus Area, Faculty of Natural and Agricultural Science, North-West University (Mafikeng Campus), Private Bag X2046, Mmabatho, South Africa b Department of Chemistry, Faculty of Natural and Agricultural, Science, North-West University (Mafikeng Campus), Private Bag X2046, Mmabatho 2735, South Africa c Department of Chemistry, College of Science, Federal University of Petroleum Resources Effurun, Nigeria d Nano-Gene and Drug Delivery Laboratory, Department of Biochemistry, University of KwaZulu-Natal, Private Bag X54001, Durban 4000, South Africa

GRAPHICAL ABSTRACT

ARTICLE INFO

ABSTRACT

Keywords: Kei apple Silver Gold Nanoparticles Bimetallic Cytotoxicity

This work presents the synthesis and cytotoxic properties of monometallic nanoparticles (NPs) of Ag, Au and their AueAg bimetallic nanoparticles (BNPs) using Kei apple fruit extract. The BNPs were prepared by varying the concentrations of the plant extract and the precursor salts (AgNO3 and HAuCl4). UV–visible spectroscopic methods showed surface plasmon resonance around 430 and 532 nm for the Ag and Au NPs respectively. The plasmon bands of the BNPs were recorded around 540 nm. Powder X-ray diffraction (pXRD) analysis and transmission electron microscopic (TEM) studies showed the crystalline nature and monodispersity associated with the monometallic nanoparticles. However, a clear evidence of polydispersity was apparent in the case of the bimetallic nanoparticles. The anticancer assay of the synthesized nanoparticles showed promising activities with the BNPs exhibiting higher potential with respect to the reaction conditions. The BNPs synthesized using higher volume of the fruit extract (AueAg BNPs3) gave the best anticancer properties against breast cancer MCF7 cell line whereas the AuNPs gave an IC50 of 105 μM as the least potent nanoparticles.

1. Introduction In the last three decades, there has been enormous growth in the ⁎

study and applications of nanotechnology due to the interestingly surface characteristics, size and properties of materials in the nanoforms [1,2]. The applications of the nanomaterials spans across several fields

Corresponding author. E-mail address: [email protected] (E.E. Elemike).

https://doi.org/10.1016/j.inoche.2019.107569 Received 22 June 2019; Received in revised form 13 August 2019; Accepted 4 September 2019 Available online 05 September 2019 1387-7003/ © 2019 Elsevier B.V. All rights reserved.

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Fig. 1. Scheme of green synthesis for Au, Ag and AuAg nanoparticles.

inflammatory, anti-angiogenesis, anti-platelet, antimicrobial, antidiabetic and anticancer agents [14–19]. The use of biological substrates in the preparation and development of bio-medically useful inorganic nanoparticles is due to their non-toxic and eco-friendly properties. In addition, the biological method of synthesis is very simple, straightforward and the plant phytonutrients contribute to the enhancement of the bioactivity of the synthesized nanoparticles [20]. The green method of synthesis has become more prominent lately as the dangers associated with the conventional route became rife and chemical routes are discouraged in the interest of environmental friendliness [20]. Although, the conventional chemical approaches have the ability to easily control the shape of the synthesized nanoparticles to a certain degree, the use of toxic chemicals and cost have been the major drawback in their application as biological agent [10]. A large number of reports exist on the synthesis of Ag, Au and their bimetallic nanoparticles using leaves, root, stem, bark and fruits extracts of different plant materials [9,21–23]. Bimetallic nanoparticles are expected to exhibit enhanced optical, electronic and chemical or biological properties due to the bifunctional or synergistic effects of both metals constituents [24]. Muntingia calabura flower extract [18], Antigonon leptopus [25] and Stigmaphyllon ovatum leaf extract [17] are among the few reported plant materials employed in the synthesis of bimetallic AgeAu nanoparticles. The potentials of Kei-apple (Dovyalis caffra) towards synthesis of nanoparticles are yet to be developed. This plant is a unique and interesting one, native to the Kei river area of Eastern Cape, South Africa [26]. The fruits are edible but are rarely consumed, hence end up as waste. This study, therefore, focuses on the use of Kei apple fruit in the synthesis of silver‑gold nanoparticles, the effects of variation in parameters towards the physical, chemical and the cytotoxicity properties of the nanoparticles.

including catalysis, agriculture, energy, electronics and biomedicine [3]. Their applications in nanomedicine are trending due to the search for biomedical tools and drugs with improved potency. Nanomaterials are becoming generally acceptable at a molecular level by aiding both treatments and the study of pathogenic diseases [4]. Nanotechnology has been applied extensively in tissue regeneration, biosensors, diagnostic, targeted drug therapy and as tools in other biological fields compared to the conventional technologies [5–7]. Conventional drugs and therapies are already marred by adverse side effects resulting from the non-specificity of the drugs and improper or ineffective dosage formulation, which is especially common with cancer chemotherapy [4]. Different nanotechnology based treatment methods have been employed in cancer therapy. These include polymeric nanoparticles, nanoemulsion and nano-functionalized metal complexes [8]. Fullerenes, nanotubes, quantum dots, nanopores, dendrimers, liposomes, magnetic nanoprobes and radio controlled nanoparticles are some of the nanomaterials which have been developed and used for therapeutic purposes [4]. Metal nanoparticles are one of the classes of nanomaterials that have been extensively studied in recent times due to their useful biological properties, benign nature and excellent characteristics [9]. Their applications can be greatly influenced by changes in their physical and chemical properties [10]. The localized surface plasmon resonance (LSPR) properties associated with the noble metal nanoparticles influences their optical properties [11]. The strong absorption band of the noble metals and their high luminescent tendencies are due to the coherent oscillation of conduction band electrons on the nanoparticles surface [12]. The oscillating electrons interact with electromagnetic radiation of appropriate wavelength to give rise to surface plasmon resonance [13]. Such properties do not only enable their application in optoelectronics and photonics but contribute immensely towards sensing, photothermal therapy, drugs and gene delivery, molecular labelling, bioimaging and as enhancer in composites formation for various applications [13]. Specifically, the ease of bio-conjugation and low cytotoxic activity of the noble metals nanoparticles have made them to be highly desirable in bio-nanotechnology [12,13]. They are, therefore, sought after in the field of biomedicine including antiviral, anti-

2. Materials and methods 2.1. Materials All the chemical reagents (AgNO3), chloroauric acid (HAuCl4) and NaOH were procured from Sigma Aldrich and used without further 2

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Fig. 2. UV–visible absorption spectra of (a) AuNP, (b) AgNP, (c) AuAgNP1, (d) AuAgNP2, (e) AuAgNP3 mediated by extract of Kei apple.

purification. Ripe Kei apple fruits were obtained from Kei River area, Eastern Cape, South Africa and properly identified. The fruits were washed, and the mesocarp was blended after the removal of the seeds. Afterwards, the juice obtained was then filtered and kept for the synthesis of nanoparticles.

2.2. Synthesis of Ag, Au and AgeAu bimetallic nanoparticles The method of synthesis followed an already reported procedure [17]. In the synthesis of the monometallic nanoparticles, 50 mL extract of the Kei apple fruit was added to 250 mL of 1 mM of the respective metal salts (AgNO3 and HAuCl4) and stirred for 1 h at 85 °C. The reactions were monitored periodically using UV–vis spectrophotometry 3

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formazan crystals were dissolved in 100 μL of DMSO followed by the absorbance readings taken at 570 nm using DMSO as a blank. The experiment was carried out in triplicates. 3. Results and discussion 3.1. Synthesis and UV–vis spectral analysis The reduction of the metal salts by the biocomponents of the juice extract showed a gradual change in color of the solution as shown in the scheme of formation in Fig. 1. Generally, nanoparticles prepared from plant extracts proceed in three phases: activation phase, growth phase and termination phase [28]. In the activation phase, the constituents of the plant extracts, with reduction capabilities in the form of phytochemicals (bearing –OH groups), reduce the metal ion from their salt precursors to a zero-valent state followed by the nucleation of the metal atom [29]. As this biological reduction process continues, the separated metal atoms begin to associate together. This growth phase thus results in enhanced thermodynamic permanence of the nanoparticles and the accumulation of the synthesized nanoparticles, which consequently alters their morphologies [28]. Eventually, these nanoparticles achieve their maximum possible activity and a consistent morphology obtained which is capped by the plant metabolites [28]. The varying composition of the active components of the plant extracts can however affect the morphology of the synthesized nanoparticles. Hence, different plants and their respective parts, such as leaves, fruits, roots and stems, can affect the shape of the nanoparticles, as well as their biological usefulness [28,30]. In the UV–visible study, the rate of formation of the nanoparticles was monitored using UV–vis spectroscopy. This was achieved by taking aliquot samples at different time intervals. The absorption spectra are shown in Fig. 2a–e. The surface plasmon bands occurred around 430 and 532 nm for Ag and Au monometallic nanoparticles respectively (Fig. 2a and b). There was a steady increase in the intensity of the absorption spectra which reflects the continuous formation of nanoparticles with time [31]. However, there was no obvious red or blue shift in the wavelength of absorption [32]. The broadening nature of the band in AuNPs could be attributed to the absorption of some bioorganic products from the juice extract of Kei apple. Fig. 2c–e present the absorption spectra of Au-AgBNPs1, AuAgBNP2, and Au-AgBNPs3. In the first 20 min of the reaction, the plasmon bands appeared in the AueAg BNP1 around 539 nm, while in the spectrum of AueAg BNP3, the absorption band appeared around 541 nm after a relatively longer time of 40 min. Since there were no two peaks recorded within the region of Ag and Au (as the case of core-shell structure), the observed peak in the region of Au could imply more reduction of Au salts than Ag salts, leading to the formation of a nanoalloy [33]. As the reaction progressed, a red shift occurred from 539 to 542 nm for Au-AgBNPs1 and 541 to 550 nm for Au-AgBNPs3. This observation indicated an increase in the sizes of the nanoparticles with increase in reaction time, which might result to variation in particle sizes or polydispersity. Hence, the observed band movements indicated a continuous growth into larger particle sizes [17]. Slight humps were observed after 60 min around 458 and 560 nm in the spectrum of AuAgBNPs2, as shown in Fig. 2d, and could be attributed to the formation of core-shell nanoparticles. It is, therefore, possible that the separate reduction of the salts with the juice extract before mixing could possibly trigger off the formation of core-shell nanoparticles.

Fig. 3. XRD patterns of all the synthesized nanoparticles.

which was accompanied by changes in color of the solution. In the synthesis of the bimetallic nanoparticles (BNPs), three different syntheses were carried out by changing the volume of the extract and precursors used. For the AueAg BNPs1, 250 mL of 1 mM of the respective metal salts (AgNO3 and HAuCl4) were both added in a flask, followed by the addition of 50 mL of the fruit extract. This mixture was stirred for 1 h and at 85 °C. Similar procedure was followed for AueAg BNPs2, but in this case 100 mL of the extract was added to the mixture and stirred for 1 h at 85 °C. For the synthesis of AueAg BNPs3, 250 mL of 1 mM of AgNO3 was mixed with 50 mL fruit extract and stirred for 30 min at 85 °C. This was followed by the addition of a mixture of 250 mL of 1 mM of HAuCl4 and 50 mL fruit extract. These reactions were monitored using UV–vis spectrophotometer. 2.3. Characterization of the nanoparticles The absorption spectra with respect to time were obtained using the Perkin Elmer Lambda 20 UV–vis spectrophotometer. The crystalline phases of the nanoparticles were identified by X-ray diffraction (XRD) technique, with a scanning rate of 0.0018o min−1, using a Rőntgen PW3040/60 X'Pert Pro XRD diffractometer equipped with nickel filtered Cu Kα radiation (k = 1.5418 Å) at room temperature. The sizes and morphology of the nanoparticles were characterized using a TECNAI G2 (ACI) transmission electron microscopy (TEM) with an accelerating voltage of 200 kV. 2.4. Cytotoxicity evaluation using MTT assay The in-vitro anticancer studies using mammary cell line MCF7 was done according to reported standard procedures [27]. The cell lines obtained from the ATCC, Manassas, USA, were cultured in 25 cm2 tissue culture flasks in EMEM already containing 10% fetal bovine serum 100 μg mL−1 penicillin, and 100 μg mL−1 streptomycin. The MTT assay in a 96-well plate containing 2.5 × 102 cells/well in 100 μL EMEM cell were used to investigate viability of the MCF7 cells. These cells were incubated overnight at 37 °C, and the medium was later replaced, followed by the addition of the samples at various concentrations (20, 40, 80, and 100 μg mL−1). The cells were then incubated for 48 h at 37 °C, followed by the MTT assay. Untreated cells were used as positive control 1 and the untreated cell with DMSO were used as positive control 2, while 5-Fluorouracil was used as standard. Fresh medium containing 10% MTT reagent was used to replace the medium in the assay followed by incubation at 37 °C for 4 h. This was removed, and the insoluble

3.2. X-ray diffraction (XRD) analysis The XRD patterns of the Au, Ag, and AueAg bimetallic nanoparticles are presented in Fig. 3. The diffraction patterns of the AuNPs showed distinct diffraction peaks at 38.12°, 44.32°, 64.54° and 77.54° corresponding to [1 1 1], [2 0 0], [2 2 0] and [3 1 1] planes of facecentered cubic structure of Au (JCPD file no 00-004-0784). The strong 4

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Fig. 4. TEM images and size distribution histogram (inset) of the synthesized nanoparticles (a) AgNPs, (b) AuNPs, (c) AueAg BNPs1, (d) AueAg BNPs2, (e) Au-Ag BNPs3. 5

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Fig. 4. (continued)

[111] peak at 38.12° is attributed to the main reflection from the crystal plane of AuNPs [34,35]. Similar characteristic peaks obtained at 2θ values 38.08°, 64.40° and 76.51° for AgNPs could be indexed to [1 1 1], [2 2 0] and [3 1 1] lattice planes respectively which also corresponded to the face-centered cubic structure of Ag with JCPDS file no 00-0040783. The two weak peaks at 32.02° and 46.05° indexed as [200] and [220] respectively, in the diffraction pattern of AgNPs were attributed to AgCl with JCPDS file no 00-031-1238. The existence of AgCl alongside the AgNPs is a common occurrence in plant extract mediated synthesis of Ag nanoparticles and might be due to chloride ion from the plant material [17]. The average particle sizes were estimated using Debye–Scherrer equation and were obtained as 8.60 and 7.21 nm for AgNPs and AuNPs respectively. The obtained XRD patterns for the bimetallic nanoparticles showed broader peaks for each of the method used compared to the peaks obtained for the individual metallic nanoparticles. The BNPs were poorly crystallized, thus resulted in a less ordered structure similar to some earlier reported BNPs [36]. The obtained diffraction patterns for the AueAg BNPs are very similar to those of the individual metals nanoparticles with additional peaks due to AgCl which normally coexist with Ag. The diffraction patterns of the AueAg BNPs1 indicated better

crystallinity compared to the monometallics as could be deduced from the intensity and strength of its peaks. The average particle sizes estimated for these bimetallic nanoparticles were 11.60, 12.89 and 13.76 nm for AueAg BNPs1, AueAg BNPs2 and AueAg BNPs3 respectively. 3.3. TEM analysis The morphologies of all the synthesized nanoparticles showed predominantly spherical shape nanoparticles. A good degree of monodispersity was observed for the monometallics, and some level of agglomeration in the case of the BNPs. The particle sizes of the AuNPs, AgNPs and AueAg BNPs were estimated from the TEM images and their corresponding particle size distribution histogram are presented in Fig. 4a–e. The volume of the fruit extract used showed great influence on the particle size and shape of the nanoparticles. Some polydispersity were observed for the AueAg BNPs1 (Fig. 4c). The micrograph of AueAg BNPs2, presented in Fig. 4d, showed that the higher concentration of the extract resulted into agglomeration, perhaps due to Oswald ripening. Fig. 4e presents the AueAg BNPs3 images, which showed darker particles in the center than the periphery. This 6

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Table 1 Viabilities (%) of the MCF7 cell lines at different concentration of the nanoparticles. Samples

Control 1

Control 2

AuNPs AgNPs AuAgNP1 AuAgNP2 AuAgNP3 5FU

100.00 100.00 100.00 100.00 100.00 100 ±

100.59 100.59 100.59 100.59 100.59 100.59

± 2.04 ± 2.04 ± 2.04 ± 2.04 ± 2.04 0.71

± ± ± ± ± ±

3.16 3.16 3.16 3.16 3.16 0.87

100 μg mL−1

80 μg mL−1

40 μg mL−1

20 μg mL−1

IC50 μM

50.25 ± 2.82 42.29 ± 3.11 45.94 ± 6.90 36.05 ± 12.48 100.59 ± 3.15 29.74 ± 5.89

52.52 42.43 52.18 37.19 40.93 42.98

70.91 56.92 55.22 32.15 49.32 58.36

73.72 41.32 59.64 57.32 51.95 82.39

105 57 77.6 22 39.8 56.2

± ± ± ± ± ±

6.47 6.51 6.09 13.15 7.72 1.28

± ± ± ± ± ±

10.79 3.71 10.38 8.82 7.72 2.79

± ± ± ± ± ±

11.34 8.20 4.79 1.98 11.65 0.86

120 100

(% viability)

80 60 40 20 0 Control 1

AuNPs

Control 2

AgNPs

100 µg mL-1

80 µg mL-1

concentra!on (µg mL-1) AuAgNP1 AuAgNP2

40 µg mL-1

AuAgNP3

20 µg mL-1

5FU

Fig. 5. Plot of percentage (%) cell viability against concentration for the nanoparticles.

more than 5-FU. The lower activity of Au-AgBNPs1 compared to the other synthesized BNPs and also to AgNPs may be due to the differences in the preparation procedures. The AgNPs also showed almost equal potency in its activity similar to the standard drug, but the AuNPs clearly showed weaker activity. Elemike et al. have reported similar study using Hela cell lines with good cytotoxic activities for these group of particles using Stigmaphyllon ovatum leaf extract [16,17]. Their reports gave a similar trend showing that the bimetallic AgeAu BNPs are more potent than the AgNPs and followed by AuNPs. The observed low activity of the particles in this study may be attributed to the selectivity towards the different cell line used. Generally, It has been suggested that the inhibition activity of the nanoparticles could be attributed to their ability to undergo a Fenton-type of reaction which produces reactive oxygen species that leads to DNA damage and eventual cell death [17]. However, the mechanism of the activity of metal nanoparticles still remains evasive and unclear.

observation was inconsistent with the other images of the BNPs, suggesting Aucore -Agshell nanoparticles. Furthermore, certain aggregation in the bimetallic nanoparticles showed, to a certain degree, the formation of hexagonal structure [23]. The individual monometallic nanoparticles were more uniform compared to the bimetallic nanoparticles which reveals homogeneous electron density within the volume of the particles [23]. The nature of the AueAg BNPs1 reveals formation of alloy with lower volume of extract and simultaneous reduction of different metal salts yielding lesser agglomerated particles than the others. Thus, the various volumes of extract and preparation method used influenced the sizes and morphology of each of the bimetallic nanoparticles synthesized. The particle size distribution plot presented as an inset in each of the TEM images showed that the pure metal (Au and Ag NPs) and the bimetallic (AueAg BNPs) nanoparticles have an average particle sizes in the range 3–5 nm and 9–14 nm respectively, which agree with the estimated size observed in the XRD study.

4. Conclusion

3.4. In-vitro cytotoxicity studies

The application of biological extracts in nanosynthesis became a drive towards the use of Kei apple fruit extract in the synthesis of silver, gold and their bimetallic nanoparticles. The volume of the fruit extract used was an important condition in this study, as it either led to agglomeration or polydispersity in the nanoparticles formed. The method of preparation of the nanoparticles also influenced the sizes and behavior of the nanoparticles. The monometallic nanoparticles were precipitated individually, while the bimetallic nanoparticles (AueAg BNPs) were formed by mixing both precursor salts alongside the fruit extract. In the UV–vis spectrum, two bands in the region of 458 and 560 nm appear slightly in the absorption spectra as humps for the bimetallic nanoparticles (AueAg BNPs2), which indicates a core shell behavior, supported by the TEM images. The anticancer behavior of the nanoparticles against MCF7 breast cancer cell lines showed promising activities in the following trend: Au-AgBNPs2 > Au-

The cytotoxicity studies of the as-synthesized nanoparticles were carried out against the MCF7 breast cancer cell lines and compared to standard anticancer drug, 5-Fluorouracil. The minimum inhibitory concentration (IC50 level) of the nanoparticles were obtained and recorded as shown in Table 1, while the representative plot for the percentage (%) cell viability to concentration is presented in Fig. 5. From the results, a concentration dependent profile with interesting activities was revealed which could be compared to the standard drug used. The results from the table showed that the AuNPs had the least anticancer potency while the Au-Ag BNPs2 exhibited the highest potency. It is evident that the synergistic effects of the individual monometallics were indications to the improved activities of the bimetallic nanoparticles especially for Au-Ag BNP2 and Au-Ag BNP3. Interestingly, the activity of Au-Ag BNPs2 was observed to be 2.6 times 7

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AgBNPs3 > AgNPs > Au-AgBNPs1 > AuNPs. It is therefore worthwhile to develop this line of research in order to find solution to some invading ailments especially tumor cells.

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