Green synthesis and characterization of silver and iron nanoparticles using Nerium oleander extracts and their antibacterial and anticancer activities

Medicinal plants can be used as reducing agents in the preparation of metal nanoparticles by green synthesis because of the chemotherapeutic and anti-infectious properties of natural compounds. Therefore, this paper reports the green synthesis of silver and iron nanoparticles from leaf and flower extracts of Nerium oleander and their capacity as anticancer and antimicrobial agents. Nanoparticle manufacturing and structural characterization of silver and iron nanoparticles are reported. The formation of nanoparticles is characterized by scanning electron microscopy with energy dispersive X-ray spectroscopy, UV-Vis and Fourier transform infrared (FTIR) spectroscopy. Nanoparticles formation was also investigated the surface charge, particle size, and distribution using zeta sizer analysis by DLS. Green synthesis of silver and iron nanoparticles using N. oleander showed different levels of selective cytotoxicity against K562 (human chronic myeloid leukemia cells) in low concentrations and were not cytotoxic to the HUVEC (human umbilical vein endothelial cells) in the same concentrations. Silver nanoparticles showed antibacterial activity against multidrug pathogens, while iron nanoparticles failed to show such activity. Results of the present research demonstrate the potential use of green synthesized nanoparticles in various biomedicine and pharmaceuticals fields in the future.


Introduction
Nanoparticles (NPs) are synthesized generally by expensive chemical synthesis methods that require the use of toxic chemicals. Thus, using biomolecules (i.e., bacteria, fungi, or plants) for NPs synthesis became a common method in past years that is safe, low-cost, and ecofriendly. Different plant extracts can be act as safe natural capping, reducing, and stabilizing agents without being a source of thermal or chemical hazards (Fedlheim & Foss, 2001;Arya, 2010). Nanoparticles formation can be a glimmer of hope for the production of drugs that can be used against infectious diseases and cancer.
Nerium oleander L. (Apocynaceae), grown in wetlands of the Mediterranean region, can be seen wild as well cultivated as an ornamental plant in parks and gardens. It is an evergreen shrub with pink and white flowers (Baytop, 1999). The plant grows up to 2-6 m tall. The leaves are in pairs or whorls of three, thick and leathery, dark-green, narrowly lanceolate, 5-21 cm long, and with an entire margin. Flowers develop in clusters at the end of branches, the diameter of each flower range about 2.5-5 cm with a deeply 5-lobed fringed corolla around the central corolla tube. The fruit is a long narrow capsule 5-23 cm long, which splits open at maturity to release numerous downy seeds (Baytop, 1999;Kiran & Prasad, 2014).
The leaves and flowers of N. oleander used in folkloric medicine among people in Turkey for rheumatism and urticaria (Bulut & Tuzlaci, 2013;Sağıroğlu et al., 2013). The latex of plant used for eczema (Gürdal & Kültür, 2013). Over the world, different parts of this plant are used traditionally for the treatment of various human ailments, including dermatitis, eczema, herpes, skin cancer, asthma, epilepsy, malaria, and tumors (Santhi, 2011). Nerium oleander is considered one of the most poisonous plants in the world which leads annually to the death of many people and animals (Rubini et al., 2019). This toxicity is due to toxins like oleandrin, oleandrigenin, and nerine, which belong to cardiac glycosides (Al-Badrani et al., 2008;Zibbu & Batra, 2010). In addition, the plant contains terpenoids and steroids (Santhi, 2011).
Despite the toxicity of plant, different scientific studies conducted on various parts of N. oleander showed its antibacterial (Chauhan et al., 2017), hepatoprotective and antioxidant (Singhal & Gupta, 2012), antiproliferative (Wong et al., 2011), antidiabetic (Sikarwar et al., 2009), anti-inflammatory (Erdemoglu et al., 2003, and anticancer (Pathak et al., 2000;Turan et al., 2006) activities. This study was aimed to synthesize Ag and Fe nanoparticles using leaves and flowers of N. oleander and then evaluate its antibacterial and anticancer activity against human chronic myeloid leukemia cell line.

Plant material
Nerium oleander leaves and flowers were collected from Servetiye Village, Sakarya Province, Turkey in June 2020. Plant was identified at the Herbarium of Faculty of Pharmacy, Istanbul University (voucher number -ISTE-117270).

Preparation of extracts
Collected dried leaves and flowers of N. oleander were washed thoroughly (three times) in distilled water and homogenized using a mortar and pestle. The shade dried leaves and flowers of N. oleander were powdered and then 10 g of both leaves and flowers were suspended in 100 ml of distilled water. Mixtures stirred for 20 min at 60 °C, then allowed to cool at room temperature, and then filtered using a Whatman no. 42 filter paper and centrifuged at × 2000 rpm for 20 min (Byrne et al., 2016). The extracts prepared were then transferred to a sterile container. The extracts were stored at 4 °C and freshly used.
Preparation of Nerium oleander silver nanoparticles (NO-AgNPs) 2.5 mL from the leaf (L) and flower (F) extract solutions were taken and mixed with 1 mM AgNO 3 in 47.5 mL deionized water and a solution of 50 mL in amount was obtained. The pH values of crude leaf and flower extracts, pH values of samples just after mixing with AgNO 3 metal source and after 24 h were measured ( Table 1). The pH of the prepared AgNO 3 solution was 5.28.

Preparation of Nerium oleander iron nanoparticles (NO-FeNPs)
Same sample preparation steps were followed for iron nanoparticles. Briefly, 5 mL from the leaf and flower extract solutions was taken and mixed with 0.2 M FeCl 3 in 45 mL deionized water and a solution of 50 mL in amount was obtained. The pH values of crude leaf and flower extracts, pH values of samples just after mixing with FeCl 3 and after 24 h were measured (Table 1). The pH of the prepared FeCl 3 solution was 2.25.

Characterization of Nerium oleander AgNPs and FeNPs
The synthesized nanoparticles were characterized through a UV-Vis spectrophotometer Shimadzu 2600. The reduction of nanoparticles was monitored by UV-spectrophotometer range of absorbance from 250-480 nm. The crystalline structures of the green-synthesized N. oleander (AgNP) and (FeNP) were examined by XRD Rigaku Flex 600 (600 models, with λ = 1.5406 and with a step size of 0.02 Å) at speed of 3 ° min -1 . Particle size and zeta potential were measured by Malvern Nano ZS. Morphology and elemental metal mapping were recorded using a high-resolution scanning electron microscope (SEM, Carl Zeiss Ultra Plus Gemini Fesem) were used to investigate 2D surface morphologies. The composition analyses of the samples were performed by EDX (EDX spectrometer attached to SEM). Fourier transformed infrared (FTIR) analyses were carried out on a liquid sample with Bruker Alpha FTIR spectrometer in the range from 4000-500 cm -1 . The device had a DTGS detector and ten scans were conducted for each spectrum with resolution four.
Cytotoxic assay K562 (human chronic myeloid leukemia cells) and HUVEC (human umbilical vein endothelial cells) cell lines were obtained from American Type Culture Collection (ATCC). Cells were cultured in DMEM with 10 % FBS and 1 % penicillin/streptomycin in a 5 % CO 2 humidified incubator, maintained at 37 °C. First, N. oleander nanoparticles were sterilized and diluted with DMEM to prepare four different dilutions which are 1, 1/2, 1/5, and 1/10. MTT assays were performed in 96-well plates. The plant extract and metal concentrations found in these nanoparticle dilutions are also shown in Table 2. K562 cells (about 105 cells per well) were seeded and incubated for 72 h. Then, supernatants were removed, and 10 µL (MTT -5 mg/mL) solution was added to each well. Following incubation at 37 °C for 3.5 h and kept dark in a humidified atmosphere at 5 % CO 2 in the air. Subsequently, the supernatant was discarded, and the precipitated formazan was dissolved in dimethyl sulfoxide (100 µL per well). The optical density of the solution was evaluated using a microplate spectrophotometer at a wavelength of 570 nm (Mosmann, 1983 Green synthesis and characterization of silver and iron nanoparticles using Nerium oleander  (VSEf)). The species were identified by using the Vitek 2 system (bioMerieux Vitek Inc.).
Antibacterial activity was detected by minimum inhibition concentrations (MICs), which were determined by serial microdilution method (MIC ranges -2.5-0.0012 mg/L for NOL-AgNPs and NOF-AgNPs, and 5.0-0.0024 mM for AgNO 3 ) following CLSI (2018). Briefly, 100 μL of each concentration were added to a well (96-well microplate) containing 100 μL of Mueller Hinton Broth (MHB) and 10 μL of inoculum (0.5 McFarland; 1.5 × 108 colony forming units/mL). Plates were then incubated at 37 °C for 24 h. Bacterial growth was determined by absorbance at 600 nm.   The existence of these constituents can be the main reason behind the biological activity.

Synthesis of Fe/Ag NPs by visual inspection
After 24 h of reaction, the reaction solution color changed from light to dark color, which can be seen in Fig. 1. The reduction of Fe3 + ions exhibits a dark color due to the excitation of surface plasmon vibration in a metal nanoparticle. Similarly, in the reduction of Ag + ions, the solution color change from light pink to light yellow. Visual photo images of the NOF-NPs are not reported due to similarity in colors with NOL-NPs.

SEM and DLS measurement
The scanning electron microscopy (SEM) technique was used to evaluate the morphology and size of the green synthesized NOL-AgNPs. Fig. 2 represents the surface images and DLS size distribution of green synthesized nanoparticles (NO-AgNPs). Specifically, the nanoparticles appear aggregated and spread uniform shapes. Iron nanoparticles seem also spherical with 70 nm average diameters.
In another study, silver nanoparticles from N. oleander flowers were synthesized (Bharathi & Shanthi, 2017). Silver particles of about 10 μm by SEM analysis are very small compared to ours, but we do not know their effectiveness as authors did not repot their bioactivity.
Besides, such small particles are not suitable for clinical use as they will be much easier to eliminate by the immune system (Bharathi & Shanthi, 2017).
The zeta potential is an indicator of surface charge potential, which is an important parameter for understanding the stability of nanoparticles in aqueous suspensions. Table 4 summarizes DLS size distribution measurements carried out on green synthesized NPs. For the NOF-FeNPs, the average particle size was 1872 nm with a polydispersity index of 0.69 (zeta potential -+5.3 mV). On the other hand, particle sizes of NOF-AgNPs were found 76 nm size with relatively homogenous distribution (polydispersity index -0.266, zeta potential -+8.1 mV). Secondly, For the NOL-FeNPs, the average particle size was 609 nm with a polydispersity index of 0.54 (zeta potential -+7.4 mV). On the other hand, particle sizes of NOL-AgNPs were found 93 nm size with relatively homogenous distribution (polydispersity index -0.364, zeta potential -+8.8 mV). No aggregation of the colloidal was observed for several months. Therefore, it may suggest that all synthesized NOF-NPs and NOL-NPs were highly stable when stored at the required temperature. It was also observed that produced NPs had positively charged on their surface with zeta potential values above 5 mV.

UV spectroscopy
The characterization of silver and iron nanoparticles by UV-spectrophotometer from the range of 350-900 nm was performed to monitor the reduction of metal ions and their   Green synthesis and characterization of silver and iron nanoparticles using Nerium oleander stability. The broad absorption peaks in the range from 326 to 432 nm were represented in Fig. 3. UV-Vis spectra were performed for NO-AgNPs and NO-FeNPs fabricated with leaf and flower extracts. To observe any shift, both crude NOL and NOF were also investigated. The absorption peaks of a plant extract with an organic mixture were not able to record since they are belonging to C-C and C-H electronic transitions (below 250 nm wavelength). The absorption peaks for NOF at 326 and 384 nm wavelengths belong to the n-π* and π-π* transitions (Wang et al., 2014). It is because this plant extract contains carbon-carbon double bonds, nitrogen-oxygen bonds, or cyclic aromatic structures. This absorption peak was only seen at 366 nm for NOL. While Ag nanoparticle synthesized with NOF gives an absorption peak at 432 nm as expected.
The synthesized Fe NP continued to interact with functional groups in the plant extract, and while a specific 435 nm absorption peak was observed in the region belonging to a typical metal nanoparticle, an absorption peak appeared at 377 nm due to its interaction with functional groups. Another possible explanation for this phenomenon is that the Fe nanoparticle can be in Fe 2 O 3 or Fe 3 O 4 structure types (Wang et al., 2014). UV-Vis spectra with the NOL nanoparticles showed much clearer absorption peaks of metal nanoparticles compared to a case in NOF. The Ag NPs absorption peak was observed at 437 nm whereas the Fe NPs was detected at 408 nm.

XRD analysis
The crystalline structures of the greensynthesized NOL-FeNPs, NOF-FeNPs NOF-AgNPs, and NOF-AgNPs were furtherly examined by XRD analysis. The obtained patterns were demonstrated in Fig. 4    Green synthesis and characterization of silver and iron nanoparticles using Nerium oleander

FTIR analysis
FTIR spectra of NOF and NOF-NPs are presented in Fig. 5. The absorption band at 3300 cm -1 was mainly attributed to OH vibration. The absorption peaks were assigned to the stretching vibration of C=C (1645 cm -1 ). Compared to NOF extract's FTIR, the disappearance of the most functional group is due to the successful reduction of metal ions. Three main bands were demonstrated in the FTIR spectrum of both NOF-FeNPs and NOF-AgNPs The presence of OH bonds and C=O functional groups on the NOF-AgNPs and NOF-AgNPs were presented at 3244 cm -1 and 1633 cm -1 , respectively. It was reported in the literature that FeNPs exhibit a characteristic stretching Fe-O vibration peak at 576 cm -1 (Wang et al., 2014). For NOF-AgNPs, stretching vibrations at 631 cm -1 can also be attributed to the reduction of Ag + to Ag. In similar green synthesis studies also reported observation of reduction of Ag + to Ag peak at around 538 cm -1 (Erdogan et al., 2019). The FTIR spectrums of NOL-Ag and NOL-Fe are similar to the FTIR spectrums of NOF-Ag and NOF-Fe. Therefore, there was no need to reinterpret the NOL-Ag and NOL-Fe spectrums. FeNPs (IC 50 -7 uM) are more variable than the NOL-FeNPs (IC 50 -48 uM). Our results suggested that NOL-Ag, NOF-Ag, NOF-Fe, and NOL-Fe are effective on the K562 cell line in low concentrations. Furthermore, we may conclude that NOF-Ag and NOF-Fe NPs have a cytotoxic effect on the K562 cell line in similar concentrations. However, NOL-Fe was cytotoxic at concentrations approximately 20 times higher than NOL-Ag (Fig. 6). In other studies, N. oleander conjugated gold nanoparticles were synthesized to investigate in vitro anticancer activity on MCF-7 cell lines. IC 50 values of these nanoparticles were found between 74.04 and 130.87 μg/mL. These values are much higher than ours (Barai et al., 2018).

Cytotoxicity assay
HUVECs were used in this study as a control. Ag and Fe NPs were not effective on HUVEC cells at the same concentrations. NOL-Ag (IC 50 -100 uM), NOF-Ag (IC 50 -100 uM), NOF-Fe (IC 50 -390 uM), and NOL-Fe (IC 50 -430 uM). The concentrations of Ag and Fe NPs, which are cytotoxic on HUVEC cells, are more than ten-fold higher compared to K562 cells. These results show that nanoparticles are harmless to normal cells when used at low doses, which are cytotoxic to leukemia cells (Fig. 6).

Antibacterial activity
Based on the results in Table 4, the tested bacteria were able to be killed at a low concentration of AgNO 3 (< 0.00976 mM). The green-synthesized NOL-Ag and NOF-Ag were able to inhibit bacteria including multidrug pathogens. As showed in Table 5, the MIC (mg/mL | mg/mM) values of NOL-Ag and NOF-Ag against Gram-negative bacteria were ranged from 0.019 | 0.039 to 0.3125 | 0.625 and 0.078 | 0.156, respectively. While the MIC (mg/mL | mg/mM) values of NOL-Ag and NOF-Ag against Gram-positive bacteria ranged from 0.078 | 0.156 to 0.3125 | 0.625 and 0.078 | 0.156 to 0.625 | 1.25, respectively. There is no significant difference observed between Gram-negative and Gram-positive bacteria including multi-resistant bacteria. The result of Fe-NPs was not given because the results were not effective.
Plant-derived essential oils and extracts have an antimicrobial effect with low toxicity and can be recommended as potential natural preservatives. According to Ríos & Recio (2005), extracts can be classified as significant (MIC < 100 mg/L), moderate (100 < MIC ≤ 512 mg/L), or weak (MIC > 512 mg/L) depending on their respective activities against the corresponding pathogens. An important advantage for the used metallic ions is that silver ions have relatively low toxicity to human cells while adversely affecting bacteria and fungi by different mechanisms, including binding to the thiol groups of protein and denaturing them, programmed cell death (apoptosis), and causing the DNA to be in the condensed form (Lansdown, 2006;Mohamed et al., 2020).