Green synthesis of silver nanoparticles from Delonix regia leaf extracts: In-vitro cytotoxicity and interaction studies with bovine serum albumin

Journal Pre-proof Green synthesis of silver nanoparticles from Delonix regia leaf extracts: In-vitro cytotoxicity and interaction studies with bovine serum albumin Md Abrar Siddiquee, Mehraj ud din Parray, Syed Hassan Mehdi, KhalidAhmed Alzahrani, Abdulmohsen Ali Alshehri, Maqsood Ahmad Malik, Rajan Patel PII:

S0254-0584(19)31305-7

DOI:

https://doi.org/10.1016/j.matchemphys.2019.122493

Reference:

MAC 122493

To appear in:

Materials Chemistry and Physics

Received Date: 24 August 2019 Revised Date:

24 November 2019

Accepted Date: 25 November 2019

Please cite this article as: M.A. Siddiquee, M.u.d. Parray, S.H. Mehdi, K. Alzahrani, A.A. Alshehri, M.A. Malik, R. Patel, Green synthesis of silver nanoparticles from Delonix regia leaf extracts: In-vitro cytotoxicity and interaction studies with bovine serum albumin, Materials Chemistry and Physics (2019), doi: https://doi.org/10.1016/j.matchemphys.2019.122493. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Graphical Abstract:

Green synthesis of silver nanoparticles from Delonix regia leaf extracts: In-vitro cytotoxicity and interaction studies with bovine serum albumin Md. Abrar Siddiqueea, Mehraj ud din Parraya, Syed Hassan Mehdib, Khalid Ahmed Alzahranic, Abdulmohsen Ali Alshehrid, Maqsood Ahmad Malikd and Rajan Patela*

a

Biophysical Chemistry Laboratory, Centre for Interdisciplinary Research in Basic

Sciences, Jamia Millia Islamia, New Delhi-110025, India b c

Department of Biosciences, Jamia Millia Islamia, New Delhi-110025, India

Department of Chemistry, King Abdulaziz University, Jeddah, 21589, Saudi Arabia

d

Surface Chemistry and Catalytic Studies Group, SCCS, Department of Chemistry,

King Abdulaziz University, Jeddah, 21589, Saudi Arabia

*Corresponding author. Tel.: +91 8860634100; fax: +91 11 26983409. Email address: [email protected], (Dr. R. Patel)

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Abstract Biogenic synthesis is an effective, nontoxic, clean, eco-friendly and cost-effective green chemistry approach to synthesize metal nanoparticles having significant properties and applications in various fields. Herein, we have approached an environment-friendly protocol for the biogenic synthesis of silver nanoparticles (AgNPs) using Delonix regia (D. regia) leaf aqueous extract as bio-reducing and capping agent. The initial reaction progress was observed by the colour change in the reaction mixture and the AgNPs formation was confirmed by the appearance of surface resonance plasmon (SRP) band at 455 nm by UV-visible spectroscopy. The morphology of the biosynthesized AgNPs was further confirmed by using different techniques such as transmission electron microscopy (TEM), selected area electron diffraction (SAED) and scanning electron microscopy (SEM). The crystalline nature of the biogenic AgNPs was analysed by X-ray diffraction (XRD) technique. We have further shown the in-vitro cytotoxicity of biogenic AgNPs against A459 and SiHa cell line. We have also investigated the esterase like activity of BSA with synthesized AgNPs. Fluorescence results suggested the contribution of static quenching in the complex formation between AgNPs and BSA. The AgNPs are seen to interact with BSA effectively resulting in structural changes of BSA confirmed by far UV-CD and FT-IR techniques.

Keywords: Silver nanoparticles; green synthesis; cytotoxicity; anticancer activity; esterase activity; bovine serum albumin.

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1. Introduction Nanotechnology is primarily related to the synthesis of nanoparticles in a variety of shapes, sizes and controlled dispersity for human benefits. It exhibits promising potential for remarkable impact on human society and different fields of science and technology[1-4]. Notably, silver nanoparticles (AgNPs) own narrow plasmon resonance, large surface to volume ratio, unique physicochemical, diverse application in, medical science, microelectronics and biological activities[5, 6]. Number of commercially and pharmacologically significant products have been explored in the production of nanoparticles specifically silver nanoparticles for various uses in, electronics, medicine, biology, environmental remediation, and material science[7-9]. Therefore,

AgNPs

gained

considerable

attention

among

different

metal

nanoparticles[10, 11]. Presently, various protocols are established for the nanomaterials synthesis such as chemical reduction, electrochemical method, photochemical approach and heat evaporation method [12]. Chemical synthesis routes involve the use of toxic materials and solvents which may lead capping of toxic chemicals on the surface of synthesized nanoparticles which are very harmful to the environment and mankind [13, 14]. Therefore, nanoparticles synthesis based on the principle of green chemistry emerged and consequently number of studies have appeared in the field of nanotechnology[15]. Green nanotechnology came into existence due to the combination of biology and nanotechnology which provided different approaches of biogenic synthesis of nanomaterials with enhanced applications in biomedical science and biotechnology. [16, 17]. Recently, the green methods for metal nanoparticles synthesis have attracted tremendous attention within the scientific community, owing to the mounting ecological contamination caused by the conventional methods [18, 19]. Therefore,

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research groups have moved towards alternative routes to produce clean, non-toxic, eco-friendly and biocompatible nanoparticles [20]. One of the major advantage using biological/green approach is that this method reduces the downstream processing of materials, which makes it more facile, economical and convenient to produce metal nanoparticles[21]. Green synthesis precursor includes plants, roots, bacteria and fungi which comprise of high concentrations of bio-organic molecules i.e. alkaloids, flavonoids, terpenoids, quinines, phenols, etc. which manage the reduction and stabilization of metal nanoparticles [22]. For example, Alfalfa Sprouts, Azadirachta (neem) leaf [23], Aloe vera plant leaves [24], broths of Geranium[23], Camellia sinensis [25], coffee tea, Morinda citrifolia extract[26] and Palm shell extract have been used to prepare metal nanoparticles. It is well documented that Delonix regia (D. regia) contains good biological and reducing properties due to the presence of abundant organic compounds in its extract[27, 28]. Nanoparticles interact with physiological surroundings once they are in contact with them. The first molecules, they often interact with, inside the body are proteins[29]. For the understanding of nanoparticle–protein interactions, AgNPs have used as a predictive tool as they show promising potential in biomedical applications. AgNPs have been used as probes in many bio-diagnostic systems in the targeted drug delivery of various carcinoma[30]. Although in biological medium, nanoparticles may interact with other biomolecules such as, nucleic acids, lipids etc. but their interaction with proteins is of prime importance as it forms the basis of nanoparticle-bioreactivity. The nanoparticlesprotein interaction leads to the formation of a structure called dynamic nanoparticles protein-corona. Furthermore, the secondary structure of protein may be distorted upon adsorption on the surface of the nanoparticle and several consequences can be speculated such as the overall bioreactivity of the nanoparticles may be affected [31].

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Thus, the binding interaction of biogenic AgNPs with bovine serum albumin (BSA) would be an excellent way to know the therapeutic action of the nanoparticles protein bioconjugate mechanism. It is a multifunctional and most abundant transport protein capable of binding, transporting and delivering a variety of exogenous and endogenous compounds, such as fatty acids, metal ions, nutrients, steroids, hormones and several classes of therapeutic drugs[32]. Cancer/tumor is the most common diseases which causes foremost mortality worldwide. From many decades treatment of this life-threatening disease has become challenging due to the occurrence of multiple drug resistance, damaging side effects and the lack

of pioneering approaches[33].

In this regard, nano-systems have

evolved as encouraging substitutes and therapeutic agents for the treatment of many diseases including cancer. In future, nanoparticles can also act as controlled delivery carriers with an ability to carry anticancer drugs to an encoded site and releasing them in a regular predetermined rate as they encounter no cellular barriers and interact strongly with the biomolecules [30, 34]. The research studies reveal that AgNPs have curative activities against several diseases. Recently, various research groups reported the biogenic synthesis of metal nanoparticles and evaluate their antimicrobial, antioxidant and anticancer activity [5, 35-37] . In this article, we have reported the synthesis of silver nanoparticles from D. regia leaf extract and their anticancer activity against cancerous cell lines (A549 and SiHa). In addition, the effect of these nanoparticles on the esterase like activity and structure of bovine serum albumin (BSA) were also evaluated. This study focuses on grasping a better understanding of the biophysical mechanism of interaction between the biosynthesized AgNPs and BSA using different types of spectroscopic techniques. To the best of our knowledge, this is the first ever report on the synthesis of silver nanoparticles using D. regia leaf

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extract. Hence, D. regia was chosen for the preparation and stabilization of silver nanoparticles. 2. Materials and Methods 2.1. Materials All chemicals and reagents including silver nitrate (AgNO3 ), bovine Serum Albumin (BSA), p- nitrophenyl acetate, mono and di sodium phosphate were purchased from Sigma-Aldrich USA. The stock solution of BSA was prepared in 10 mM phosphate buffer of pH 7.4. Cell lines: A549 and SiHa cells were procured from NCCS Pune. All other materials, reagents and solvents used were of analytical grade. Double distilled water was used in all the experiments. 2.2. Preparation of leaf extract D. regia leaves were collected from the university campus, Jamia Millia Islamia, New Delhi. Leaves were thoroughly washed with tap water and subsequently with Milli-Q water and were chopped into small pieces. To prepare the leaf extract, 5 g of chopped leaves were mixed with 200 mL of distilled water and boiled for 15 minutes at 60 °C The extract was then filtered before it was used for biosynthesis of silver nanoparticles. 2.3. Preparation of biogenic silver nanoparticles Biogenic silver nanoparticles were prepared according to standard protocol as mentioned elswhere, by mixing an appropriate amount of leaf extract with aqueous solution of 1 mM AgNO3 ( 20:80,V/V). The mixture was subjected to moderate stirring at room temperature (25 °C) for about 10 minutes to achieve the reduction of silver ions. After keeping the reaction mixture in a static condition for about 3 hours, the colour of the reaction mixture changes indicating the formation of AgNPs [38]. 2.4. Characteriztion of Biogenic AgNPs

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2.4.1. UV-visible spectral analysis The bio-reduction of silver ions to AgNPs was confirmed by measuring the UVvisible spectra of the reaction mixture at different time intervals by a double beam Specord-210 Analytik Jena spectrophotometer at a scanning range of 200-700 nm. The coloured reaction samples were diluted with deionized water and scan in UVvisible spectra were recorded by using with 1 cm Quartz cuvette at 25 ˚C. 2.4.2. X-ray diffraction studies(XRD) and high resolution transmission electron microscopy(HRTEM) X-ray diffraction (XRD) analysis has been utilized to confirm the crystallographic structure and crystalline phase of biosynthesized silver nanoparticles. The diffraction pattern for the biogenic AgNPs was recorded on X-ray diffractometer (Rigaku IV) at room temperature.The lyophilized powder samples of silver nanoparticles were used for XRD analysis with a scanning speed of 10° min-1 in the 2θ range between 20˚-80˚ angles with a tube current of 30 mA and a voltage of 40 kV. The average particle size of the biogenic AgNPs was determined by the Debye-Scherrer equation (1).


=



(1)



where, D denotes crystal size, k is the shape factor (0.94), the full width in radians at half maximum is denoted by β, λ represents the X-ray wavelength (λ = 1.5418 Å) and θ is the Bragg's angle. The morphological features including size, shape and other physical properties of biogenic AgNPs were analyzed by transmission electron microscopy (TEM) using an FEI Tecnai F20 instrument operating at an accelerating voltage of 120 kV. A drop of the purified AgNPs aqueous solution was poured on the carbon-coated copper grid and the grid was allowed to be air-dried at room temperature and clear TEM 7

microscopic images were collected in different ranges of magnification. Selected area electron diffraction (SAED) pattern was obtained to find out the crystalline nature of the biosynthesized AgNPs carried out by TEM. 2.4.3. Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis Scanning electron microscopy (SEM) analysis of biogenic AgNPs was performed by FESEM, ZEISS scanning electron microscope, model Sigma Version 5.05 to confirm the surface morphology. A thin layer of AgNPs sample was prepared on a carboncoated copper grid by placing a small sample quantity on the grid and the extra sample was removed by blotting paper. The grid was kept under mercury lamp for 5 minutes to achieve the complete dryness of the sample on the grid and the film was magnified at different resolution power for surface morphology observation of AgNPs.The elemental analysis was performed by Bruker/EDAX/Oxford detector by transmitting the AgNPs on the carbon-coated copper grid and the presence of AgNPs was confirmed by a strong silver element signals. 2.4.4. Fourier transform infrared (FTIR) spectroscopy The Fourier transform infrared spectroscopy (FTIR) spectra were collected by using Bruker TENSOR 37, equipped with nitrogen gas cylinder. A resolution detector of 1 cm-1 was employed to detect all the possible functional groups present in D. regia leaf extract derived nanoparticles. All spectral measurements were taken in the range of 400-4000 cm-1. Binding study of BSA (5µM) in the presence of AgNPs was also studied by using the infrared spectroscopy technique. Conformational changes in the secondary structure of BSA was studied with FT-IR spectra in the range of 1500 to 1700 cm-1 (amide region) in the presence of AgNPs. 2.4.5. Dynamic light scattering (DLS)

8

Dynamic light scattering (DLS) was carried out to determine the hydrodynamic radii and size distribution of the biosynthesized silver nanoparticles by using Laser Spectro scatter 201 (RiNA). All the experiments were recorded at 90º and incident wavelength was set at 689 nm. For each sample, five measurements were taken with an acquisition time of 20 seconds. 2.4.6. Stability study by UV-vis. and DLS In order to check the stability of nanoparticles we performed dynamic light scattering and UV-vis. absorption spectrosccopy along with the colour-change observation. The data is given in Table S1. 2.5. Evaluation of in-vitro cytotoxicity activity and Interaction of AgNPs with BSA 2.5.1. Evaluation of in-vitro cytotoxicity activity using green synthesised AgNPs. The cancerous cell lines (A549 and SiHa) were treated with different concentration of AgNPs to evaluate the cell viability by MTT (3-(4,5-dimethyl thiazole-2-yl)-2,5diphenyl tetrazolium bromide) assay. The cells were developed in minimal essential condition DMEM (Dulbecco Modified Eagles Medium) supplement containing 10% fetal bovines serum and 1% antibiotic solution of streptomycin and amphotericin B (Himedia) at 37 ºC in a humidified environment of 95% oxygen and 5% CO2. Briefly, the cell lines were seeded at a number of 2 × 104 per well in 96-well microplates (200 µl/well) in triplicate, allowed them to grow for 24 hrs at 37 ˚C. After 24 hrs of incubation, cells were exposed to different concentration of green synthesized AgNPs (2-64 µg/mL) for 48 hours. At the end of the treatment, the medium was removed, and the cells were incubated and 20 µl of MTT (5 mg/ml) in phosphate buffer saline (PBS-pH- 7.4) in a fresh medium in the dark for 4 hours at 37°C was added. After 4 hours, formazan crystals formed by mitochondrial reduction of MTT were dissolved in DMSO (150 µl/well), and the absorbance was measured at 570 nm

9

after a 10-min incubation on the Multiskan EX micro-plate reader (Thermo scientific, Germany). Percentage of cell viability was calculated as a fraction of control (without Malathion), and the cytotoxicity of AgNPs was expressed as IC50 according to standard protocol [39]. 2.5.2. Esterase like activity The effect of AgNPs on the esterase activity of BSA was assayed to find out the change in the activity of protein upon binding AgNPs with the substrate p-nitrophenyl acetate (PNPA), by following the releases p-nitrophenol on the addition of BSA. The action of BSA on PNPA was monitored by measuring the formation of p-nitrophenol at λabs= 405 nm on UV-visible spectrophotometer (Analytik Jena Specord-210 spectrophotometer) using a 1.0 cm cell. The reaction conditions were, 10.0 µM [BSA], whereas substrate [PNPA] varied from 50.0 to 400 µM, and different AgNPs concentration (47, 130 and 220 µM) in 10 mM phosphate buffer (pH =7.4) at temperature 37 ˚C. 2.5.3. Interaction of AgNPs with BSA. The interaction of AgNPs with BSA was studied by using different spectroscopic techniques viz; UV-visible, steady-state fluorescence and structural changes in BSA induce by AgNPs studied through FTIR and circular dichroism spectroscopy as prescribed in our previous publications [40-48]. 3. Results and discussion 3.1. Characteriztion of AgNPs 3.1.1. UV- visible analysis of Delonix regia plant mediated AgNPs synthesis UV-visible spectroscopy serves an excellent tool for the determination of reaction progress for green synthesized silver nanoparticles and the surface plasmon resonance (SPR).The UV-visible absorption spectra of colloidal silver nanoparticles showed an

10

intense peak at 455 nm which indicates the formation of silver nanoparticles. Fig.1. shows the UV-vis spectra of colloidal silver nanoparticles synthesized using D. regia leaf extract [16, 38]. As the metal solution of the silver nitrate was mixed with an aqueous solution of D. regia leaf extract, the reduction of the silver ions takes place resulting in the formation of AgNPs. During reduction, we analyzed the change in colour of mixture from colourless to brown within 3-hours[49]. The colour change in the aqueous solution is due to the excitation of surface plasmon resonance (SPR) band in the UV-vis region. After 3-hours further change in colour was not seen which indicates the complete formation of silver nanoparticles. Furthermore, the spectra were recorded after every 30 minutes interval and it was found that the absorption intensity increased regularly after every interval as shown in Fig.1. Similar results of AgNPs have already been reported in the literature [50]. The silver nanoparticles were formed when the aqueous solution of leaf extract snatch the silver ion (Ag+) from the aqueous AgNO3 surroundings and reduce the ions into their corresponding pure elemental state with the help of various organic and phenolic compounds present in the extract [36]. 3.1.2. X- ray diffraction analysis The XRD pattern of green synthesized nanoparticles are depicted in Fig. 2. The peaks were recorded at 2θ from 20˚ to 80˚ angle. The sharp peaks obtained at 26.87˚, 32.44˚, 46.13˚ 46.31˚ 64.31˚ and 77.55˚angles correspond to hkl planes - (111), (200), (400), (220), (311), (132) and (351), respectively. Aziz and Hamedi et al. [38, 51] reported the XRD patterns for the crystalline AgNPs, exhibited a sharp peak at 38.3°, 46.1°, 67.5°, and 76.5° which corresponds to (111), (200), (220) and (311) Miller indices, respectively. The production of highly pure AgNPs by the biogenic method was confirmed because there was no diffraction peak of any impurity or secondary

11

phase. All the XRD data were in agreement with a Joint Committee on Powder Diffraction Standard (JCPDS 870597) and validate the crystalline nature of synthesized silver nanoparticles. The average crystalline size of biosynthesized AgNPs was calculated by Debye-Scherrer equation (1) and the average size of biogenic AgNPs was found to be 43.5 nm, which makes these nanoparticles more active in different applications. 3.1.3. High resolution transmission electron microscopy (HRTEM), scanning electron microscope (SEM) and elerctron dispersive spectroscopy (EDS) analysis of AgNPs The morphology of biosynthesized AgNPs was investigated by HR-TEM as shown in Fig.3 A. The result indicates that the biogenic AgNPs exhibit non-uniform morphology. The aqueous leaf extract of D. regia used to synthesize AgNPs play a vital role in the capping/stabilization of the biogenic AgNPs and therefore can affect the morphology of the biogenic AgNPs. The selected area of electron diffraction pattern (SAED) reveals the presence of bright ring patterns, signifying the presence of various planes of AgNPs indicating the crystalline nature of the biogenic AgNPs (Fig.3 B). The patterns demonstrate fringes with bright circular rings related to the (111), (200) and (311) Bragg’s reflection planes of AgNPs which again indicates the crystallinity of nanoparticles [52]. The results acquired from XRD analysis are in good agreement with the selected area of the electron diffraction pattern, which suggests the crystalline nature of the biogenic AgNPs. The surface morphology of the biosynthesized AgNPs was carried out by scanning electron microscopy (SEM) as the technique is extensively used for the analysis of size, shape and dispersity of nanoparticles. The SEM image of biosynthesized AgNPs reveals that particles exhibit non-uniform morphology (Fig. 3 C). Energy dispersive Xray spectroscopy, EDX (Fig. 3 D ) is an excellent technique to carry out the chemical analysis and to detect the

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metal ions in the synthesized nanomaterials by using metallic signals. The strong elemental signal of Ag reveals the elemental composition of biosynthesized AgNPs and confirms the formation of AgNPs and their crystalline nature. The EDX spectra show two strong signals for silver at 0.2 and 3.0 keV. The appearance of aluminium peak at 1.5 keV due to the aluminum stub on to which our sample mounts for characterization. The presence of another peak in the spectra suggests that the AgNPs were capped by organic/biomolecule/constituents [38]. 3.1.4. AgNPs analysis by dynamic light scattering (DLS) Dynamic light scattering (DLS) is the most common analytical method used for nanoparticle size estimation. DLS analysis of green synthesized AgNPs are shown in Fig. S1 (A,B). DLS is utilized to determine the size distribution profile of nanomaterials present in the solution. It gives information about the hydrodynamic diameter of nanoparticles, the number-based size distribution and the polydispersity index (PDI). The hydrodynamic diameters of AgNPs were found to be 72.77 nm. The obtained value is well supported with the value already reported in the literature[53]. 3.1.5. Analysis of Fourier transform infrared (FT-IR) spectroscopy As the identification of possible organic and biomolecules responsible in the formation of nanoparticles through the reduction of silver ion, hence, to check the presence of such reducing and stabilizing agent FTIR measurements of D.regia extract was performed. Fig. 4 (A, B) shows the FTIR spectrum of D.regia leaf extract and silver nanoparticles, respectively. The spectra showed the emergence of the peak at 1680 cm-1 and 1532 cm-1 (Fig. 4A) corresponding to amide I and amide II band, respectively while an absorption band at 2961 cm-1 is due to C-H stretching vibration of hydrocarbon groups. A broadband near 3536 cm-1 is assigned to O-H stretching of

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phenolic group involving in hydrogen bonding. N-H primary amines show absorption at 3406 cm-1 [52]. The absorption band appears in the range of 1020 cm-1 and 1173 cm-1 corresponding to C-O vibrations of carboxylic acids, phenols, alcohols and maybe C-N vibrations present in amine. The band appearing at 1767 cm-1 corresponds to the presence of the internal carbonyl group. The band at 2024 cm-1 is due to the occurrence of C≡O stretching of terminal groups[38, 54]. The band at lower range of spectrum at 895 cm-1 is assigned to the appearance of C-H out of plane bending and amine, N-H wagging modes. The recorded FT-IR spectra of AgNPs (Fig. 4B) revealed the shifting or broadening of the fundamental band as compared to D.regia extract after bioreduction. The main absorption band appearing at 3728 cm-1, 2970 cm-1, 1673 cm-1

1523 cm-1, 1032 cm-1 in the FT-IR spectra of synthesized AgNPs

proves that the phenols, organic molecules and aliphatic amines are responsible in the formation and stabilizing the AgNPs. The presence of various water-soluble ingredients in the leaf extracts helps in the reduction of silver ion. 3.2. Biological activity 3.2.1. Cytotoxicity studies by MTT assay Cytotoxicity and cell viability are the characteristic parameters to find out the biocompatibility of silver nanoparticles. After successful completion of the characterisation of AgNPs, we have examined in-vitro study on A549 and SiHa cell line by MTT assay. AgNPs revealed a potent cytotoxic effect against A549 and SiHa cell lines. The results confirmed that the viability of the A549 and SiHa cell line was insulated by various concentrations of AgNPs. Fig. 5 (A, B) shows the cell viability after 48 hours of incubation in a medium containing 2µg/mL to 32 µg/mL of AgNPs of cell lines - A549 (5A) and SiHa (5B) . These figures clearly show the inhibitory effect and sensitivity of AgNPs on the A549 and SiHa cellular growth at different

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doses. Results obtained after the study were significant at p-value <0.05. The AgNPs exhibited an IC50 value after 48 h for A549 is 14.96 µg/mL and for SiHa 15.96 µg/mL, respectively. However, AgNPs exhibited better anti-proliferative activity in A549 cell line in comparison to SiHa cell line. Venugopal et al. have also reported the synthesis of AgNPs from Syzygium aromaticum and have performed their anticancer activity against MCF7 breast and A549 lung cell line [55]. They found the IC50 value for AgNPs against A549 was 50 µg/ml, which is quite higher than the IC50 value of AgNPs synthesised from D. regia, (as reported in this manuscript) by our group. Further, Mishra et al. found the IC50 value of neem mediated AgNPs against SiHa cell line was much lower (≤4.25 µg/ml ) than the IC50 values of AgNPs[56]. The cytotoxicity properties of AgNPs might be due to physiochemical interaction of AgNPs with functional groups present in BSA protein [39]. 3.2.2. Determination of esterase like activity In this experiment, the activity of BSA was checked in the absence and presence of different concentrations of AgNPs (47, 130 and 220 µM), employing various concentrations of substrate PNPA (Fig.6A). It is notable that AgNPs exhibit almost negligible absorbance at 405 nm. The effect of AgNPs on the esterase like activity is shown in comparison to the activity of BSA. One unit of activity is defined as the amount of BSA required to release 1µM of p-nitrophenol from PNPA per minute at 310 K [57]. The Michaelis-Menten equation (2) was used to calculate the kinetic parameter of BSA in presence and absence of AgNPs

v0 =

Vmax [ S ] K m + [S ]

(2)

15

where ν0, Vmax, Km, and [S] are the reaction’s initial velocity, maximum velocity, Michaelis-Menten constant, and molar concentration of substrate, respectively. Further, Kcat is calculated from equation (3)

Vmax = Kcat[E]

(3)

where [E] is the total enzyme concentration. The Michaelis-Menten plot and the Lineweaver-Burk plot of BSA in the absence and presence of AgNPs are shown in Fig 6 (A, B), respectively. BSA acts like an ‘esterase’ for PNPA and releases pnitrophenol from it as mentioned earlier. The different kinetic parameters are reported in table 1 and it can be seen that the values of Km and Vmax increase with increasing concentration of AgNPs which suggests the involvement of uncompetitive binding [58]. 3.3. Interaction and conformational changes in BSA in the presence of AgNPs 3.3.1. UV-visible absorption spectrophotometry The UV-visible absorption spectroscopy is the most reliable technique for the study of protein interaction with metal nanoparticles. To examine the impact of bioconjugation on the absorption spectra of BSA and AgNPs, the UV-visible absorption spectra of native BSA at various concentrations of AgNPs were taken and obtained absorption spectra are shown in Fig. (7A). The absorption spectra of the native form of BSA show an absorption band at 279 nm which arises due to the presence of aromatic amino acid residues (tryptophan and tyrosine) and disulfide bond. It is observed that the absorbance increases regularly on increasing the concentration of AgNPs. The results obtained from the absorption spectra revealed the complex formation between BSA and AgNPs[59]. The surface plasmon resonance (SPR) of AgNPs appears at λmax = 440 nm, a characteristics band of silver nanoparticles [6062]. Further, the absorption studies of AgNPs in the absence and presence of BSA 16

were also taken. Fig. (7B) shows the absorption spectra of AgNPs at various concentrations of BSA. AgNPs show the absorption maximum at 440 nm. With the increasing concentration of BSA, there is a gradual decrease in absorption intensity, indicating the complex formation between the two. BSA is negatively charged at pH 7.2 and it looks unusual that it will interact with negatively charged AgNPs [63, 64]. However, there are possibilities of hydrophobic interactions playing a crucial role in the bio-conjugation of AgNPs with BSA. The changes in SPR band of AgNPs in presence of BSA are attributed to the interaction of BSA with the surface of AgNPs [61]. Furthermore, bio-conjugation is also favoured by the presence of several functional groups such as disulfide bond(S-S), thiol group and cysteine residues which help in the formation of Ag-S linkage [64]. The results obtained from UVvisible spectral investigation are in good agreement with the reported literature about the conjugation of protein with nanoparticles [60]. 3.3.2. Fluorescence quenching mechanism Fluorescence emission spectroscopy technique is an extensive tool for binding studies of protein and nanoparticles. From these measurements, we can obtain extensive information about the polarity changes around the fluorophores. Tyrosine (Tyr), tryptophan (Trp) and phenylaniline (Phe) amino acid residues are the aromatic fluorophores that generate spectra among these residues, Trp residue is the most sensitive to the changes of the microenvironment, and it shows the strongest fluorescence intensity[65]. Fluorescence emission spectral analysis was performed to check the effect of AgNPs on the fluorescence emission intensity of BSA. The fluorescence quenching spectra of BSA in the presence of the varying concentration of AgNPs are shown in Fig. (8A). As shown in the figure, with a successive increase in the concentration of AgNPs, the fluorescence intensity of BSA gets quenched. The

17

progressive quenching in fluorescence intensity shows the formation of a complex between BSA and AgNPs. Quenching involves a wide range of processes including molecular interaction, molecular arrangements, complex formation, and energy transfer. Nanoparticles and protein interaction are associated with the change in the intrinsic emission intensity of bio-macromolecules, and AgNPs are known to display high quenching potential towards many chromophores[51, 66]. The excitation wavelength was set at 280 nm. The emission was taken from 290 to 450 nm. The maximum emission peak of BSA was obtained at 341 nm. A redshift of 9 nm in fluorescence emission of BSA was seen with an increasing concentration of AgNPs. The redshift showed that the increase in hydrophilicity and polarity around the fluorophore regions. The fluorescence quenching may be categorized into dynamics and static mechanisms; the former involves the collision between fluorophore and quencher in the excited state of fluorophore while the latter is an outcome of the ground state fluorophore-quencher complex formation[64]. The Stern-Volmer quenching constant (Ksv) and different parameters of the quenching mechanism were calculated by the following equation (4)[67, 68].  

= 1 +   = 1 +  ! 

(4)

where F0 and F are the fluorescence intensities of quencher in the absence and presence of quencher respectively, Ksv is the Stern-Volmer constant, [Q] is the concentration of quencher, kq is the quenching rate constant and τo is the average fluorescence lifetime of protein fluorophores in the absence of a quencher. Fig. (8B) represents the Stern-Volmer plot, between F0/F versus [Q]. The Ksv value was obtained from the slope of the Stern-Volmer plot and was found to be 14.54 × 103 L mol-1. The value of kq was found to be 14.548 ×1011 L mol-1s

-1

(table 2) which is

greater than 2.0 × 1010 L mol-1 s-1 suggesting that AgNPs quenching the fluorophore of 18

BSA through dynamic quenching mechanism. The results showed that AgNPs have a stronger ability to quench the fluorescence intensity of BSA. 3.3.3. Estimation of binding parameters and binding energy: Fluorescence quenching data was further exploited to calculate the various binding parameters such as binding constants (Kb) and n is the number of binding sites. Following equation (5) was used to evaluate the value of Kb and n for AgNPs and BSA interaction

log

F0 − F = logKb + n log[Q] F

(5)

where, F0 and F are fluorescence intensities of BSA in the absence and presence of NPs, respectively. The numerical values of Kb, and n are obtained from the plot of log (F0-F/F) versus log [Q] (Fig. S2) and the values are listed in table 2. The value of n for the BSA-NPs interaction is close to 1 at room temperature indicating the presence of a single binding site at BSA for NPs. The calculated value Kb is of the order of 104 justifying the existence of strong binding interaction of AgNPs with BSA. The value of ∆G was calculated by the following equation (6). "# = −%& '( )

(6)

The negative value of ∆G (table 2) indicates the spontaneity of the binding process. 3.3.4. Synchronous fluorescence spectroscopy A synchronous fluorescence measurement is an excellent technique to investigate the change in the microenvironment of Trp and Tyr residues in the vicinity of fluorophore molecules. It provides key information about the conformational changes in the microenvironment of protein when it interacts with nanoparticles. It gives the characteristics information of Trp and Tyr residues when ∆λ (λem- λex), is fixed at 60 nm and 15 nm respectively. The position-shift in λmax of Tyr and Trp facilities

19

information regarding the change in polarity around these amino acid residues [69]. The spectra are shown in Fig.S3 (A & B) for native BSA (5µM) and BSA with different concentrations of AgNPs at ∆λ = 60 nm and ∆λ=15 nm, for Trp and Tyr residues are shown. It is clear from the Fig.S3 (A & B) that the serial addition of AgNPs results in the decrease in the fluorescence intensity. The shift in the λem predicts the alteration in the polarity of the molecular environment surrounding Tyr/Trp residues[67, 70]. Also, it is clear from the synchronous spectra, the intensity variation at ∆λ = 60 nm is more than at ∆λ = 15 nm. This result further confirms that the effect of AgNPs is more pronounced for Trp residues than that of Tyr residues. There was a slight red shift at ∆λ = 60 of 4 nm in BSA-AgNPs and a blue shift at ∆λ = 15 of 3nm. These results showed that the microenvironment of both Tyr and Trp residues of BSA is perturbed in the presence of AgNPs. 3.3.5. Three-dimensional (3D) fluorescence measurements 3D fluorescence is the most reliable technique to consider the conformational changes of proteins. 3D fluorescence is known as excitation-emission matrix spectroscopy. It is a complete demonstration of fluorescence characteristics of the sample under simultaneous excitation and emission wavelength. To explore the effect of AgNPs on the conformational changes of BSA, 3D fluorescence was performed. Fig. 9 (A & B) shows the 3D fluorescence spectra of pure BSA and BSA in the presence of AgNPs and the peak positions are given in table 3. The spectra contain three emission peaks. One of the peaks corresponds to Raleigh scattering. Peak 2 gives the spectral characteristics of tryptophan and tyrosine residues, while peak 3 is the spectral outcome of polypeptide backbone of protein[40]. It can be seen from table 3 that there is a Stokes Shift of 6 nm in peak 2 whereas in peak 2 there is a Stokes Shift of 2 nm which confirmed that AgNPs induced the conformational changes in BSA structure.

20

The structural changes induce by AgNPs were further confirmed by FTIR and CD spectroscopic techniques. 3.3.6. FT-IR measurements for secondary structure analysis FTIR spectral analysis of protein is helpful to investigate the change in the secondary structure of the protein. It is based on spectral shift and variation in the amide band of protein[71]. The first amide band at 1700–1620 cm−1 is the characteristic band for the secondary structure of the protein which arises due to carbonyl (C=O) stretching vibration. Another band at 1680 cm-1 is specific for the anti-parallel β-sheet and the band at 1668 cm-1 is characteristic of β-turns. The band at1650 cm-1 is attributed to αhelix while the band at 1630 cm-1 is assigned to the random coil and a band at 1620 cm-1 is specific for β-sheet. A very weak band, amide II ranges from 1600 to 1500 cm1

. It contains the C-N stretching vibration and N-H vibration and characteristics of

deformation vibration of N-H bond[69, 72]. The synthesized nanoparticles were interacted with BSA and subjected to FTIR analysis to check the structural and conformational alteration (if any) in the protein (Fig S4). The above spectral region of FT-IR spectra for the amide I band of native BSA and BSA with AgNPs were deconvolution. The result is shown in Fig. 10 (A&B). Strong amide I region near 1647 cm−1 of free BSA solution (pH = 7.4 and phosphate buffer) was observed, which indicates that BSA is rich in α-helical conformation. On the addition of nanoparticles to BSA solution, a little alteration in both peak position and nature of the amide I band of free BSA was observed, confirming that the nanoparticles interact with BSA, and as such induces the change in the secondary structure of BSA. Table 4 summarises the total content of the α-helical, β-sheet, random coil, β-turn and βantiparallel, calculated for BSA and BSA with AgNPs. As can be seen in table 4 the content of β-antiparallel increases compared to free BSA which can be attributed to

21

the affinity of nanoparticles with BSA. In general, the reduction of α-helical content is accepted to be balanced by an increase in β-structures[73, 74]. 3.3.7. Far UV-CD measurements CD measurements provide very convenient information for assessing secondary structural change in proteins. The fluctuations in the CD signal serve as a probe to assess the secondary structural changes. BSA shows two negative bands, one at 208 nm which corresponds to π → π* transition and the second band at 222 nm which is attributed to n → π* transition in the ultraviolet region. The two negative bands at 208 nm and 222 nm in CD are the characteristic feature of alpha helical content [75]. The secondary structural changes were observed in BSA on the addition of AgNPs. The results are shown in Fig. 11. The ellipticity values are obtained from CD at 208 nm. CD signal was converted to concentration independent parameter, mean residual ellipticity (MRE) [76], utilising the following equation (7). * =

+ ,

(7)

-!./

where θλ represents the observed ellipticity in millidegrees at wavelength λ, M0 represents the mean residue weight of BSA, C stands for concentration of protein in mg ml-1 and l is the path-length of the cell in centimetres. The α-helical contents of pure BSA and BSA-AgNPs complex was estimated from MRE values at 208 nm by using equation (8) [76]. 0 − 12'34 =

5+6789 5:!!! ;;!!!5:!!!

× 100

(8)

where MRE208 is the observed ellipticity value at 208 nm. The α-helical content of pure BSA was found to be 60 percent. It decreases to 43.34 % on conjugation with AgNPs. The decrease in the α-helical content results in the perturbation of the secondary structure of the protein. It is noteworthy that the CD results corroborate with the results obtained from FTIR measurements. 22

3.3.8. Probing binding site using site markers BSA has two binding sites, site I and site II. The site I is in sub-domain IIA while site II is in sub-domain III A. It has been reported that site I and site II have the affinity for indomethacin (IND) and ibuprofen (IBU), respectively[77]. To determine the location of the binding site for synthesized AgNPs, the Ksv values of BSA-AgNPs system was determined in the presence and absence of IND and IBU (Fig.S5) using Stern Volmer equation (4) as shown in the table S2. The values of Ksv decrease for both IND and IBU suggesting the affinity of both site markers to either of the binding sites. However, the decrease in Ksv is more for IND than IBU (located in site II), which suggests that AgNPs have a greater affinity with site I of BSA[57]. 4. Conclusions The work shows a facile route for the biogenic fabrication of benign AgNPs using aqueous leaf extract of D. regia as an effective bio-reducing and capping agent. The biosynthesized AgNPs are characterized by different techniques such as UV-visible spectroscopy, TEM, SEM, XRD, EDX and are found crystalline in nature and have non-uniform spherical appearance. The MTT assay results showed that AgNPs exhibited better anti-proliferative activity in A549 cell line in comparison to SiHa cell line. It is noteworthy that biogenic AgNPs show anticancer activity against A459 and SiHa cell line. The results of esterase activity suggested uncompetitive binding between AgNPs and BSA. The CD results showed a decrease in the α-helical content results in the perturbation of the secondary structure of BSA. The binding was spontaneous as suggested by the negative sign of ∆G. The site marker experiments confirmed that AgNPs have a greater affinity with active the site I of BSA.

Notes The authors declare no competing financial interest. 23

Acknowledgements This project was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, under grant no. (DF-695-130-1441). The authors, therefore, acknowledge with thanks DSR for technical and financial support.

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Figure Captions: Fig.1. UV-Visible absorption spectra of formation of AgNPs at different time interval. 30

Fig.2. XRD patterns of biosynthesized AgNPs synthesized from Delonix regia leaf extract. Fig.3. (A) HRTEM micrographs (B) SAED diffraction pattern (C) SEM image and (D) Energy dispersive X-ray spectrum of

silver nanoparticles synthesized from

Delonix regia leaf extract. Fig.4. FT-IR spectra of leaf extract (A) and leaf extract derived AgNPs (B). Fig.5. Cytotoxicity effect of green synthesized AgNPs on A549 cell line (A) and SHIA cell line (B). Fig.6. (A) Michaelis-Menten plot of BSA-AgNPs with varying concentrations of substrate (p-NPA) and double reciprocal plot of 1/[υ] versus [1/S] in the absence and presence of AgNPs (B). Fig.7(A) UV-Visible absorption spectra of 5 µM BSA in the absence and presence of different concentrations of AgNPs (12.3, 24.4, 36.1, 47.6, 58.8 and 69.8 µM). (B)UVVisible absorption spectra of AgNPs (1mM) at different concentration of BSA (0.06, 0.12, 0.18, 0.23, 0.29 and 0.34 µM). Fig. 8 (A) Fluorescence quenching spectra of BSA in the absence and presence of green synthesized [AgNPs] (0.00 to 240 µM). (B) Stern-Volmer plot for BSA and green synthesized AgNPs. Fig.9. (A) 3D-Fluorescence spectra and the respective contours of BSA (5 µM) and (B) BSA in the presence of AgNPs (30µL). Fig.10. FT-IR spectra, curve fitted and deconvoluted spectra of BSA (5 µM) and BSA with AgNPs (1 mM) in the amide I band region at room temperature and pH 7.4. Fig.11. Far UV-CD spectra of BSA (5µM) in presence of AgNPs [47 µM and 220 µM].

Figures:

31

Fig.1. UV-Visible absorption spectra of formation of AgNPs at different time interval.

Fig.2. XRD patterns of biosynthesized AgNPs synthesized from Delonix regia leaf extract.

32

Fig.3. (A) HRTEM micrographs (B) SAED diffraction pattern (C) SEM image and (D) Energy dispersive X-ray spectrum of silver nanoparticles synthesized from Delonix regia leaf extract.

Fig.4. FT-IR spectra of leaf extract (A) and leaf extract derived AgNPs (B).

33

Fig.5. Cytotoxicity effect of green synthesized AgNPs on A549 cell line (A) and SHIA cell line (B).

Fig.6. (A) Michaelis-Menten plot of BSA-AgNPs with varying concentrations of substrate (p-NPA) and double reciprocal plot of 1/[υ] versus [1/S] in the absence and presence of AgNPs (B).

34

Fig.7(A) UV-Visible absorption spectra of 5 µM BSA in the absence and presence of different concentrations of AgNPs (12.3, 24.4, 36.1, 47.6, 58.8 and 69.8 µM). (B) UV-Visible absorption spectra of AgNPs (1mM) at different concentration of BSA (0.06, 0.12, 0.18, 0.23, 0.29 and 0.34 µM).

Fig. 8 (A) Fluorescence quenching spectra of BSA in the absence and presence of green synthesized [AgNPs] (0.00 to 240 µM). (B) Stern-Volmer plot for BSA and green synthesized AgNPs.

35

Fig.9. (A) 3D-Fluorescence spectra and the respective contours of BSA(5µM) and (B) BSA in the presence of AgNPs (30µL).

Fig.10. FT-IR spectra, curve fitted and deconvoluted spectra of BSA (5µM) and BSA with AgNPs (1mM) in the amide I band region at room temperature and pH 7.4.

36

Fig.11. Far UV-CD spectra of BSA (5µM) in presence of AgNPs [47 µM and 220 µM.

Table Captions: Table 1. Kinetic parameters for the hydrolysis of PNPA by BSA.

Table 2. The binding constants and thermodynamic parameters for AgNPs binding to BSA. Table 3. Three dimensional- fluorescence parameters of BSA and BSA in the presence of AgNPs. Table 4. The secondary structure values of BSA protein in the absence and presence of AgNPs obtained from deconvolution of FTIR data.

37

Tables: Table 1. Kinetic parameters for the hydrolysis of PNPA by BSA.

Sample

vmax (µMs-1)

Km (µM)

kcat (s-1)

kcat/ Km (µM-1 s-1)

BSA

9.84 × 10-3

89.40

9.84 × 10-4

1.10 ×10-5

BSA+SNPs (47 µM)

8.24 × 10-3

83.64

8.24 × 10-4

9.85 ×10-6

BSA+SNPs (130 µM)

7.42 × 10-3

77.06

7.42 × 10-4

9.62 ×10-6

BSA+SNPs (220 µM)

6.90 × 10-3

68.03

6.90 × 10-4

10.1 ×10-6

Table 2. The binding constants and thermodynamic parameters for AgNPs binding to BSA. Binding Parameter

Ksv (Lmol-1)

kq (L mol-1s-1)

Ka (L mol-1)

n

∆G (kJ/m)

BSA-AgNPs

14.54 ×103

14.54×1011

25.5×104

1.34

-30.84

Table 3. Three dimensional- fluorescence parameters of BSA and BSA in the presence of AgNPs. System

Peak 2 Peak Position

Peak 3

Relative intensity

Stokes Shift (nm)

225/343

809

118

BSA+AgNPs 225/349

610

124

(λex/λem) (nm) BSA

38

Peak position

Relative intensity

Stokes Shift (nm)

280/344

604

64

280/342

474

62

(λex/λem) (nm)

Table 4. The secondary structure values of BSA protein in the absence and presence of AgNPs obtained from deconvolution of FTIR data. Systems

α-helical (1650 cm-1) %

β-Sheet (1630 cm-1) %

Random Coil

β-turn

β-antiparallel

(1688 cm-1) %

(1620 cm-1) %

(1670 cm-1) %

BSA

66.14

5.44

6.89

13.18

8.33

BSA+ AgNPs

46.94

4.52

2.24

22.88

23.39

39

Highlights: 1. 2. 3. 4.

Silver nanoparticles (AgNPs) were synthesized using Delonix regia (D. regia) leaf extract. AgNPs show anticancer activity against A459 and SIHA cell line. AgNPs quenches the fluorophore of BSA through static quenching mechanism. These NPs induces the conformational changes in BSA.

Declaration of interest Statement The authors declare no competing financial interest.