Green Synthesis of Size-controlled Silver Nanoparticles and Their Anti- Cancer Potentiality

doi:10.21203/rs.3.rs-2694046/v1

Silver nanoparticles are now well recognized as one of the most prevalent kinds of materials that are put to use in a wide variety of biomedical applications, most notably as an anti-cancer agent. In the current investigation, silver nanoparticles (Ag NPs) were effectively produced by reducing silver + ions by employing the leaf extract of Artocarpus heterophyllus as a source of reducing and capping agents. By altering the quantity of the silver nitrate solution, we successfully produced three distinct kinds of Ag-nanoparticles that were mediated by Artocarpus heterophyllus leaf extract. The X-ray diffraction analysis first confirmed the formation of metallic silver, where peaks were found at fixed angles. The X-ray Diffraction (XRD) method was also used to validate the crystal geometry of the Ag-NPs, revealing that the Ag-NPs had a face-cantered cubic structure. The calculated average crystallite sizes of Sample-1 Ag-NPs, Sample-2 Ag-NPs, and Sample-3 Ag-NPs were found to be 20.34 nm, 16.99 nm, and 18.88 nm, respectively. Ag nanoparticles were also confirmed from EDX analysis, and firm Ag peaks, including several organic compound peaks. The nanoparticle’s range was between 120 nm and 220 nm, and the average particle size was near 170 nm, as found in the SEM image, and accumulation was observed in the SEM image. Using Fourier Transform Infrared (FT-IR) spectroscopy, our research group was able to determine the functional groups of organic compounds that might be responsible for reducing agents and the presence of capping agents on the surface of Ag NPs. The cell viability test was used to assess the cytotoxicity using the HeLa cell, a human carcinoma cell. The results revealed that the produced Ag NPs demonstrated toxicity against carcinoma cells.

Ag NPs

Artocarpus heterophyllus

cytotoxicity

HeLa cell

anti-cancer agent

Nanotechnology is a promising multidisciplinary branch of study that provides nanoscale materials with the potential for use in various scientific domains, including engineering, mechanical engineering, catalysis, photonics, atomic processing, and essential materials [1, 2]. The quantum size effect causes it to display new and enhanced features and a vast surface area to volume ratio [3]. This is the most significant property responsible for the widespread usage of nanomaterials [3].

A broad range of biological characteristics, such as antiviral, anticancer, antibacterial, and antifungal activities, are present in green produced AgNPs, making them both economical and environmentally benign [4]. Especially in the field of biology, nanoparticles play a significant role due to their attractive physicochemical properties [5]. If the barrier ability of the skin was compromised, then nanoparticles of silver (Ag) may potentially pass through it at a rate ranging from 0.2 to 2 percent [6]. Several investigations, both in vitro and in vivo, have shown that exposure to Ag NPs may cause toxicity in organisms [7, 8]. In addition, Ag NPs' cytotoxicity effects on A-549, NIH-3T3, PC12, and HepG2 cell lines have been published in the scientific literature [9]. Besides, each year, millions of people are diagnosed with cancer around the world [10]. The significant burden of most chemotherapeutic approaches to cancer treatment is that most of them are non-specific. Therapeutic, generally cytotoxic drugs are controlled intravenously, prompting total appropriation [11]. In this case, Ag-nanoparticles can play a significant role as anti-cancer agents by providing their cytotoxic effects [12]. In addition, the antibacterial activity in Ag NPs might facilitate their safe application in nutritional supplements, food packaging, cosmetics, and acne treatment products [13, 14]. Designing nanoparticles that contribute to the creation of effective nanomaterials is one of the most significant means of combating antibiotic resistance [14]. Because of their large surface area bound to cell surface, smaller Ag NPs have more antibacterial action [15]. As a result, developing a method for manufacturing silver nanoparticles (Ag NPs) that combines low toxicity with solid antibacterial activity is essential to ensure that these particles may be used without posing a risk to human health.

So many chemicals and physical processes have been established to synthesise Ag nanoparticles. Chemical reduction [13], electrochemical [16], and sonochemical [17] are some of such synthesis processes. All of these methods are popular for the synthesis of Ag nanoparticles. However, these technologies cannot circumvent the need to use hazardous compounds in the synthesis procedure, as that involves the generation of large amounts of dangerous by-products [18]. As a result of the widespread use of noble metal nanoparticles, such as silver and gold nanoparticles, in human-contact areas, it is of the utmost importance to develop what is known as "green chemistry," which entails the creation of nanoparticles in a way that is clean, non-toxic, and friendly to the environment [19].

As a result of slower kinetics, more significant excitation, control over crystal development and stability, and cost-effectiveness, biological synthesis techniques have paved the way for the "greener synthesis" of nanoparticles [20]. Compared to chemical and physical approaches, natural nanoparticle synthesis methods have several advantages, including being more affordable, environmentally benign, and suitable for large production [20, 21]. “Green synthesis” of Ag-NPs was primarily reported by Gardea-Torresdey et al. [22]. The thriving green synthesis might be a contender to replace the conventional chemical production of Ag- NPs. The biologically synthesized Ag-NPs are biocompatible and can be safely used for various therapeutic applications.

However, in this research, we used Artocarpus heterophyllus for synthesizing Ag-nanoparticles. Artocarpus heterophyllus is the national fruit of Bangladesh. It might contain organic compounds, namely vitamins A, C, and E, with high glucosinolate compounds [23]. Moreover, Artocarpus heterophyllus grows almost yearly in Bangladesh and the Indian subcontinent. Therefore, the leaf extract of Artocarpus heterophyllus might be a possible option for use as a source of reducing and capping agents in the environmentally friendly manufacture of silver nanoparticles (Ag NPs).

2.1: Chemicals and reagents

Analytical grade types of chemicals were utilized in this study without any additional processing. To create the necessary solutions for this task, deionized water, also known as DI water, which has a resistivity of 18 MΩ-cm, is employed as a solvent. The chemicals and reagents utilized in this study include AgNO₃ (Merck, Germany), Artocarpus heterophyllus (known as Jackfruit) leaf, Dulbacos enhanced eagle’s medium (DMEM) (Sigma, St. Louis, MO, USA), HeLa cell (American category culture collection, USA and Canada), etc.

2.2: Preparation of leaves extract

A few recent shed leaves of Artocarpus heterophyllus were gathered from the region immediately around the Dhaka University campus. After that, we removed the dust particles by washing them with tap water and rinsing them with deionized water. Furthermore, moisture was removed by drying them in an oven set at sixty degrees Celsius and crushed into a powder. The plant extract was made by combining 10 grams of plant extract with 200 ml of distilled water in a conical flask. Then the solution was mixed for one hour at a temperature of 80 degrees Celsius, then centrifuged at room temperature for thirty minutes at a speed of four thousand revolutions per minute. After separating the supernatant, the liquid was filtered using Whatman No. 1 filter paper. Finally, the solution was used to convert silver ions (Ag⁺) into nanoparticles of silver (Ag^o). The temperature of the leaf extract kept in the refrigerator was four degrees Celsius. At each stage of the experiment, sterile conditions were maintained to ensure that the findings were reliable and accurate to prevent the introduction of any contamination.

2.3: Silver nanoparticle (Ag NP) synthesis

An aqueous solution of 50 mM, 100mM, and 200mM silver nitrate was prepared in 200 mL conical flasks. For convenience, we marked the synthesized Ag-Nanoparticles using 50 mM silver nitrate solution as Sample-1, 100 mM solution as Sample-2, and 200 mM as Sample-3 silver nitrate solution. For a better reaction, we took the silver nitrate solution in a beaker, and 50ml leaf extract was added dropwise to reduce Ag⁺ ions for each type of silver nitrate solution. The mixture was stirred for 60 minutes with a magnetic stirrer at \({90}^{0}\) centigrade. Meanwhile, the gradual transformation of the mixture's colour from a pale yellowish-brown to a reddish-brown to a colloidal brown was seen and recorded at regular intervals. All the procedures were separately performed with each type of silver nitrate solution.

Complete reduction of silver ions (Ag⁺) to silver nanoparticles (Ag^o) was engrained by shifting colour from colourless to colloidal brown. After that, the colloidal solution was cooled to room temperature and preserved for 24 h to complete bio-reduction. The synthesized silver NPs were obtained by centrifugation at 4000 rpm for 20 min. After that, the particles were separated by centrifugation at a speed of 4000 revolutions per minute for 10 minutes. This procedure was repeated several times. In the following steps, separated silver NPs were dried and ground. That ground silver nanoparticles were then sealed and stockpiled correctly for future use. Figure 1 shows the whole process of silver nanoparticle synthesis.

3.1: X-ray diffraction (XRD) analysis.

Using the XRD method and the associated XRD patterns, the crystalline nature of the green-produced Ag-NPs was substantiated. Figure 2 showed the XRD patterns of Ag-NPs synthesized from the reduction of Ag⁺ using leaf extract of Artocarpus heterophyllus at three different concentrations of silver nitrate solution. The diffraction peaks attained around the 2θ values of 38.05°, 44.33°, 64.38°, and 77.33° were matched to the planes of (111), (200), (220), and (311), correspondingly demonstrating the face-centred cubic (fcc) silver in accordance to the reference standard JCPDS Card No. 87–0720 [24]. In some cases, the diffraction peaks were shifted slightly from the standard patterns, which may be due to the surface encapsulation of Ag-NPs [25]. The XRD patterns also showed that the relative intensity of the (111) diffraction peaks in Fig. 2 is higher than the other peaks. This result indicated that the synthesized Ag-NPs in our work were enriched in (111) facets. It is noteworthy that no unwanted peak was visualized in our synthesized Ag-NPs. This phenomenon indicates the formation of almost pure Ag-NPs. The structural parameters calculated from the XRD patterns for all three types of particles are summarized in Table 1. The crystallite size of Ag-NPs has been estimated using the Debye–Scherrer approximation: D=𝑘𝜆/𝛽𝐶𝑜𝑠𝜃 where “D is the average crystalline size, k is a geometric factor (0.9), λ is the wavelength of the X-ray radiation source, and β is the FWHM (full-width at half maximum) of the XRD peak at the angle of diffraction (θ)” [25]. The calculated average crystallite sizes of Sample-1 Ag-NPs, Sample-2 Ag-NPs, and Sample-3 Ag-NPs were found to be 20.34 nm, 16.99 nm, and 18.88 nm, respectively.

Table 1: The structural parameters of three different concentrated silver nitrate solutions-mediated synthesized Ag NPs were obtained from the XRD analysis.

Silver nanoparticles	Plane (h k l)	FWHM (deg.)	Crystallite Size, D (nm)	average Crystallite Size(nm)
Sample-1	111	0.4088	21.47	20.34
	200	0.4826	18.56
	220	0.4351	22.54
	311	0.5654	18.80
Sample-2	111	0.3882	22.61	16.99
	200	0.8501	10.54
	220	0.4993	19.64
	311	0.7005	15.17

Sample 3	111	0.3953	22.20	18.88
	200	0.7747	11.56
	220	0.4501	21.79
	311	0.5327	19.95

Table 2: Cytotoxicity of three different concentrated silver nitrate mediated synthesized Ag-NPs obtained from cell viability assay

Sample ID	Survival of HeLa Cells (%)	Remarks
Control	100%	Cytotoxicity was observed on the HeLa cell line
1	<5%
2	<5%
3	10%-20%

Table 3: FT-IR spectra peaks position of Artocarpus heterophyllus leaf aqueous extract mediated synthesized Ag-NPs of sample-1, sample-2, and sample-3 with corresponding functional group

Sample number	Peaks position Wave number (cm^-1)	Functional groups	References
Sample-1	1098 cm^-1, 1626 cm^-1, and 3433 cm^-1	stretching vibration bands of-C–O–C-, –C=O, and –OH respectively	[26, 27]
sample-2	1077 cm^-1, 1612 cm^-1, and 3440 cm^-1
sample-3	1084 cm^-1, 1625 cm^-1, and 3438 cm^-1
Sample-1	Weaker peaks appeared at 1366 cm^-1, 2386 cm^-1, and 2933 cm^-1	specifying the stretching vibration bands of -C–N of the amine, -C=C-, and-C–H, respectively	[28, 29]
sample-2,	Weaker peaks appeared at1358 cm^-1, 2367 cm^-1, and 2933 cm^-1
sample-3	Weaker peaks appeared at 1410 cm^-1, 2378 cm^-1, and 2927 cm^-1 for

3.2: EDX analysis

Strong indications of silver atoms were seen in the EDX profile. The existence of Ag NPs was shown by the prominent signal peak in the EDX spectrum at 3.25 KeV, which was characteristic of the absorption of silver Nano crystallites [24]. It is interesting to note that other components, including C, Cl, N, and O, were also found. The organic molecules from the extract that were absorbed on the surface of Ag NPs and played a critical role in Ag NPs reduction and stability were most likely connected with C and O [25].

The leaf extract solution also contained Cl [25–26]. Sample-1 and sample-3 had somewhat lower and greater concentrations, respectively. Only sample 2 was examined using EDS in this experiment. Since the three samples were all created using the same method, it seems sensible that they would have comparable components. By examining the sample-2 EDS result, it is simple to make predictions regarding sample-1 and sample-3.

3.3: SEM micrographs analysis:

Scanning electron microscopy (SEM) was used to observe the surface morphology of the artificially produced silver nanoparticles (sample-2), and the results were shown in Fig. 4. The scanning electron micrograph of silver nanoparticles (Ag NPs) showed unmistakably that the artificially produced Ag NPs were aggregated into dispersed clusters. The size distribution of the Ag NPs that were produced was roughly 120 nm to 220 nm, and the average particle size was in the vicinity of 170 nm. The formation of the big particles that were seen most likely included the aggregation of several smaller particles [26].

The massive particles may be eradicated using the heat treatment method. Other researchers also reported a similar phenomenon [26–27]. The fact that there was lower agglomeration may be because there are capping agents present on the surface of the Ag NPs; this was demonstrated both by the EDX and the FT-IR tests. Several characteristics working together typically determine the nanoparticle size range and size distribution. An essential factor to consider for size management is the silver nitrate solution (AgNO₃) concentration. On the other hand, the quantity of leaf extract solution used, and its concentration may both significantly impact the regulation of particle size. In this experiment, the scanning electron microscope (SEM) was only used to examine sample 2 since the concentration of samples 1 and 3 were, respectively, somewhat lower and higher. This lower and higher concentration might potentially increase or decrease the particle size. However, our primary objective was determining the cytotoxicity of concentration variation-mediated Ag NPs. The same method was used to generate all three samples, and the particle size distribution may be altered by adjusting the amount of AgNO₃ in the mixture. Finally, by analysing the results of the SEM experiment performed on sample 2, it is possible to make an accurate prediction about the nanoparticle size of samples 1 and 2.

3.4: Fourier transform infrared (FT-IR) spectroscopy

FT-IR spectra were recorded for three different samples of synthesized Ag-nanoparticles respectively, between wavenumbers of 400–4000 \({\text{c}\text{m}}^{-1}\), to recognize the conceivable biomolecules responsible for reducing Ag⁺ into Ag-NPs and their conjugation with the synthesized Ag-NPs that were shown in Fig. 5. In Table-3, Artocarpus heterophyllus leaf extract-mediated synthesized Ag-NPs, three strong peaks appeared for each sample which represent the stretching vibration bands of-C–O–C-, –C = O, and –OH, respectively [27, 28]. Moreover, Weaker peaks were also appeared which specified the stretching vibration bands of -C–N of the amine, -C = C-, and-C–H, respectively [29, 30].

These bands were similar to the bands of Artocarpus heterophyllus aqueous leaf extract [29]. The FT-IR spectra of the three different concentrated synthesized Ag-NPs indicate that similar functional groups are present in the leaf extract and all the synthesized Ag-NPs. However, the peaks were shifted slightly, and the peak intensities were found to be increased for Ag-NPs, suggesting the condensation of the biomolecules on the surface results in the encapsulation of Ag-NPs with bio-molecules.

3.5: Cytotoxicity of Ag-NPs against cancerous cell

The cell viability experiment assessed the cytotoxicity of the three different concentrated synthesized Ag-NPs. HeLa cell line, a human cervical carcinoma cell line, was taken as a model cell. The synthesized Ag-NPs were then exposed to the selected cell lines at a 100 mg/L concentration. The phase-contrast microscopic pictures of Ag-NPs persuaded cytomorphological variations, and growth inhibition of the cell lines with control and at three unlike concentrated synthesized Ag-NPs were shown in Fig. 6. From the investigation of the cytotoxicity assay, it was found that the synthesized Ag-NPs were toxic, which was evidenced by the change of cytomorphology, indicating the decrease in cell viability. The cytotoxicity of the sample-1 Ag-NPs. Sample-2 Ag-NPs and sample-3 Ag-NPs against carcinoma cells were slightly varied. In the present study, sample − 1, and sample-2 Ag-NPs were more toxic than sample-3 in table.2. Ag-NPs cytotoxicity has been widely studied. However, the specific processes by which Ag-NPs cause cell death are still largely unknown since cytotoxicity differs from cell to cell and NPs to NPs. Ag-NPs cytotoxicity has primarily been attributed to the disrupted antioxidant system and increased oxidative stress at the cellular level. Oxidative stress causes damage to the mitochondrial and plasma membranes, as well as to cellular proteins, lipids, and DNA, which ultimately results in cell death and electronic chain dysfunction. The cytotoxicity shown in the current investigation may result from cellular harm caused by oxidative stress [30]. Additionally, the interaction of the encapsulating chemicals with the carcinoma cell may cause sample 1 and sample 2s increased cytotoxicity. The nanoparticles' size may impact cell damage, and AgNO₃ concentration is strongly correlated with nanoparticle size. Maybe tiny particle size is ideal for lower AgNO₃ concentrations, and probably cancer cell destruction is better suited to small particle size.

In the current research, silver nanoparticles (Ag NPs) were effectively produced by the reduction of silver ions (Ag⁺) with the leaf extract of Artocarpus heterophyllus (Jackfruit), which served as the reducing agent. It is also used as both a capping and a stabilizing agent. Artocarpus heterophyllus leaf was chosen as it was available in the Indian Subcontinent and found around the year. Different biomolecules in the leaf extract, confirmed by the FT-IR analysis, acted as reducing and capping agents. The XRD analysis confirmed the crystallinity and formation of the biosynthesized Ag NPs and found them to be face-cantered cubic. Due to some constraints, the surface morphology, particle size, and size distribution could not be examined. From the cell viability measurements, it was found that Ag-NPs are toxic to HeLa cells. The synthesized Ag NPs were found to be effective as an anti-cancerous agent against human cancer cells. The toxicity increased with a particular concentration (50 mM and 100 mM) of Ag NPs. On the other hand, sample 3 has shown lower effectiveness than the other two samples. The Ag-NPs particle size-dependent cytotoxicity can impact future research, and the relationship between several parameters and cytotoxicity can be striking for future research. Cell-damaging mechanism study is quite important to know the actual role of Ag NPs in cytotoxicity, and it could be a unique research topic in the near future.

Acknowledgement

The authors would like to acknowledge Materials Science Division, Atomic Energy Centre Dhaka; Department of Physics, University of Dhaka; and Department of Glass and Ceramic Engineering, Bangladesh University of Engineering and Technology for their assistance for the characterization; School of Material Science, Japan Advanced Institute of Science and Technology (JAIST) and Kanazawa University for their cooperation.

Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sector.

Conflict of Interest: The authorsdeclare that they have no conflict of interest.

Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors

Authors contribution: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by, [Md. Faysal Kabir], [Md. Zakaria Rahman], [Jannatul Ferdousy], [ A.K.M. Atique Ullah] and [Md. Mizanur Rahman]. All the figures were drawn in origin by, [Md. Faysal Kabir], [Razzakul Islam] and [Jannatul Ferdousy]. The first draft of the manuscript was written by [Md. Faysal Kabir] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Availability of data and Materials: Not applicable

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No competing interests reported.

Green Synthesis of Size-controlled Silver Nanoparticles and Their Anti- Cancer Potentiality

Status:

Version 1

Abstract

Figures

1. Introduction

2. Methodology

3. Results And Discussion

Silver nanoparticles

Plane

(h k l)

FWHM

(deg.)

Crystallite Size, D

(nm)

average

Crystallite Size(nm)

Sample-1

111

0.4088

21.47

20.34

200

0.4826

18.56

220

0.4351

22.54

311

0.5654

18.80

Sample-2

111

0.3882

22.61

16.99

200

0.8501

10.54

220

0.4993

19.64

311

0.7005

15.17

Sample 3

111

0.3953

22.20

18.88

200

0.7747

11.56

220

0.4501

21.79

311

0.5327

19.95

4. Conclusions

Declarations

References

Additional Declarations

Status:

Version 1