Accepted Manuscript Green synthesized silver nanoparticles using Nelumbo Nucifera root extract for efficient protein binding, antioxidant and cytotoxicity activities T.V.M. Sreekanth, Sambandam Ravikumar, In-Yong Eom PII: DOI: Reference:
S1011-1344(14)00299-1 http://dx.doi.org/10.1016/j.jphotobiol.2014.10.002 JPB 9851
To appear in:
Journal of Photochemistry and Photobiology B: Biology
Received Date: Revised Date: Accepted Date:
12 July 2014 2 October 2014 6 October 2014
Please cite this article as: T.V.M. Sreekanth, S. Ravikumar, I-Y. Eom, Green synthesized silver nanoparticles using Nelumbo Nucifera root extract for efficient protein binding, antioxidant and cytotoxicity activities, Journal of Photochemistry and Photobiology B: Biology (2014), doi: http://dx.doi.org/10.1016/j.jphotobiol.2014.10.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
Green synthesized silver nanoparticles using Nelumbo Nucifera root extract for efficient protein binding, antioxidant and cytotoxicity activities. T.V.M. Sreekanth1, Sambandam Ravikumar2, In-Yong Eom1,3* 1
Department of life chemistry, Catholic University of Daegu, Gyeongsan-Si,
South Korea. 2
Department of Pharmaceutical Science and Technology, Catholic University of
Daegu,Gyeongsan-Si, South Korea. 3
Natural Science Research Institute, Catholic University of Daegu,Gyeongsan-Si, South
Korea. *
Corresponding author: In-Yong Eom, Tel: +82-53-850-3783; Fax: +82-53-850-3728 Email: [email protected]
Abstract Silver nanoparticles (AgNPs) with a mean particle size of ~16.7nm were synthesized using an eco-friendly reducing material, aqueous Nelumbo nucifera root extract. Rapid reduction resulted in the formation of polydispersed nanoparticles. The formation of AgNPs was characterized by surface plasmon resonance, which was determined by ultraviolet-visible (UV-Vis) spectroscopy (band at 412 nm), Fourier transform infrared spectroscopy, scanning electron microscopy- energy dispersive X-ray spectroscopy, transmission electron microscopy and X-ray diffraction. The interaction of the green synthesized AgNPs with bovine serum albumin (BSA) at various temperatures was investigated. Fluorescence quenching, synchronous and resonance light scattering spectroscopy along with UV-Vis absorption studies revealed the efficient binding between BSA and the AgNPs. In addition, the AgNPs exhibited moderate antioxidant and cytotoxicity activities against HeLa cell lines.
Keywords: Eco-friendly synthesis, AgNPs, Nelumbo nucifera, BSA, Antioxidant, Cytotoxicity.
1
1.
Introduction Over the past decade, there has been increasing interest in the interfacing of material
chemistry and biology with an emphasis on the non-covalent interactions of nanoparticles with proteins and DNA [1]. Studies of the interactions of nanoparticles (NPs) with proteins are facilitated by a large body of research on drug delivery [2, 3]. In addition, NPs can accumulate easily on a biomolecule surface and interact with proteins, which affects their conformation and subsequent functions. On the other hand, the introduction of NPs in the blood and their interactions with the proteins present in plasma is of key importance in their biomedical applications. The growing biosafety concerns of nanomaterials can be also be investigated by applying analogous techniques to probe the structural changes and aggregation of proteins during NPs interactions. Among the various types of metallic nanomaterials developed thus far, AgNPs have attracted substantial interest because of their wide range of applications in the chemical, physical and biological fields owing to their optical, chemical, photochemical, and electronic properties [4]. AgNPs can be synthesized by both chemical [5] and biological methods. Chemical methods generally involve toxic materials that are expensive and harmful to the environment [6]. The green synthesis of AgNPs is simple, inexpensive, less time consuming, and eco-friendly. Over the last few years, AgNPs were prepared using inexpensive biological resources, such as plants [7, 8], bacteria [9], fungi [10], yeast [11], and lotus seed extract [12], which reduce silver ions to AgNPs both extra- and intracellularly [13]. Several silver-based nanoparticles have been investigated in biological settings but the majority of those studies focused mainly on chemically-prepared metallic NPs rather than green synthesis, which focuses predominantly on biosafety. This paper reports the green synthesis of new AgNPs using N. nucifera root extracts for the reduction of Ag+ ions. N. nucifera is a large aquatic medicinal plant found in Asian countries, including Korea, Japan and China. N. nucifera root has many potential benefits including anti-diarrheal [14], psychopharmacological [15], diuretic [16], antipyretic [17], antifungal and anti-yeast activities [18], hypoglycemic activities [19], hypotension effects [20], and hypocholesterolemic effects [21]. Serum albumins are the most abundant proteins in plasma, and are used widely for the transportation of drugs and nutrition through the human body [22]. Therefore, it is of major importance to study the mechanism of the interaction between BSA and nanomaterials 2
to improve both the binding properties and conformational behavior of proteins. The photophysical properties of BSA are sensitive to conformational changes, and provide two tryptophan residues, Trp 212 and Trp 134, which possess intrinsic fluorescence. The present study focuses on understanding the biophysical mechanism of interaction between the greenly synthesized AgNPs and BSA using fluorescence spectroscopy. This study provides an important foundation in the design of new protein-NPs systems, which will motivate broader targets, including new therapeutics and biosensors. 2.
Results and discussion
2.1. Synthesis and characterization of AgNPs This study focused initially on the synthesis of AgNPs using an eco-friendly green synthesis method. The AgNPs were synthesized by reducing Ag+ ions using a N. nucifera root extract. A reaction of 1mM AgNO3 and N. nucifera root extract transformed a colorless or pale yellow solution to a strong wine red color due to the surface plasmon resonance of the AgNPs formed. This reaction was complete within 45 min at room temperature. The AgNPs solution was stable for 2 months at room temperature without any signs of precipitation. UV-Vis spectroscopy can be used to examine the size and shape of nanoparticles in aqueous suspensions [23]. In the UV-Vis spectra, the SPR bands of the AgNPs synthesized from N. nucifera root extract were centered between 400-500 nm, which confirmed the formation of AgNPs in solution. Fig. 1a shows no evidence of absorption in the range, 400500 nm, for the N. nucifera root extract only. An absorption peak at 414 nm was clearly observed when the aqueous plant extract was added to a 1mM AgNO3 solution (Fig. 1a). Fourier transform infrared (FTIR) spectroscopy was carried out to identify the possible biomolecules responsible for the reduction, capping and efficient stabilization of the AgNPs. Fig. 1b shows the FTIR spectra of the N. nucifera root extract and AgNPs. The N. nucifera root extract showed transmission peaks at 3309, 2926, 1637, 1411, 1148, and 1075 cm-1. The transmission peaks indicating the presence of AgNPs were located at 3337, 2937, 1620, 1339, 1148, and 1032 cm-1. These were peaks assigned to phenols and alcohols (Hydrogen- bonded O-H stretch); alkanes (C-H stretch); 1° amines (N-H bend); aromatics (C-H stretch (in-ring)); alcohols, carboxylic acids, esters, ethers (C-O stretch) and aliphatic amines (C-N stretch) respectively. Scanning electron microscopy (SEM) confirmed the AgNPs in the nano-size. The nanoparticles were predominantly spherical in shape with diameters below 100 nm, and a 3
mean size of ~16.7 nm (Fig. 2a). The EDX spectrum revealed a strong signal in the silver region and confirms the formation of AgNPs. Another strong signal for Si was observed, which was assigned to the Si wafer used as a substrate to prepare thin films. The remaining elemental signals were from the aqueous root extract of N. nucifera (Fig. 2b). The TEM images in Fig. 2c & 2d show more clearly that the AgNPs are commonly found in spherical, spherical triangle and uneven shapes. Fig. 3 shows X-ray diffraction (XRD) patterns of the AgNPs synthesized from the N. nucifera root extract. The Bragg reflections of 38.28, 44.38 and 64.54° 2θ were indexed to the (111), (200) and (220) planes, respectively. 2.2. BSA–AgNPs binding study UV–Vis spectroscopy was carried out on BSA in the absence and presence of AgNPs at different concentrations, as shown in Fig. 4. Upon the addition of AgNPs, the absorption band of BSA at 279 nm increased significantly with a blue shift of 2 nm (inset of Fig. 4). AgNPs do not display an absorption band at around 279 nm. Therefore, the increase in the intensity of the peak at 279 nm for BSA upon addition of AgNPs in the BSA was attributed to the formation of ground state complex formed by AgNPs–BSA interaction. In addition, the existence of an isosbestic point at approximately 290 nm in the UV spectrum (Fig. 4) indicates the conversion of free AgNPs to the BSA-bound AgNPs complex. Among several techniques to examine the interaction between NPs and BSA, the most convenient method was to study the fluorescence quenching of BSA. The main contribution to the fluorescence of BSA is by the tryptophan (Trp) moiety, which is quite sensitive to the local environment. Therefore, the fluorescence spectra (Fig. 5a) were recorded for BSA and mixed with different concentrations of AgNPs to examine the interactions between them. The figure clearly shows that BSA has a strong emission band at 343 nm when excited with a 279 nm wavelength. The intensity of this fluorescence band decreased gradually and the maximum of the peak was also shifted by 4 nm with increasing AgNPs concentration. This change in the fluorescence is characteristic of the BSA, which indicates the binding between AgNPs and BSA to form a certain complex. The RLS spectra were recorded, where the emission of BSA was measured at the same excited wavelength. This is a simple technique that allows the easy detection of aggregates in solution. Fig. 5b shows the RLS spectra of BSA, AgNPs and mixture of BSA and AgNPs with varying concentrations of AgNPs. BSA showed a very weak RLS signal over the entire wavelength range of 220-700 nm [23] and the AgNPs showed a peak at 4
445nm. In contrast, strong RLS signals was observed for the AgNPs-BSA mixtures with a maximum peak at approximately 450 nm. The generation of the RLS spectrum correlated with the formation of aggregates. The RLS intensity is affected primarily by the dimensions of the aggregate formed in solution. This suggests that the added BSA may interact with the AgNPs in solution to form a new BSA-AgNPs complex that would be expected to be an aggregate. Therefore, enhanced light scattering occurs under the given conditions [24]. Fluorescence quenching behaviors can be distinguished by examining fluorescence quenching at different temperatures. The fluorescence quenching data at different temperatures was analyzed using the well-known Stern–Volmer equation [25]. Fig. 6a presents the Stern–Volmer plot of F0 / F vs. [NPs]. The Stern–Volmer constant was obtained from the slope after linear regression of this plot (Table 1). The quenching constant increased with increasing temperature, indicating that fluorescence quenching occurs because of some specific binding between AgNPs and BSA by forming complexes, which were stabilized at higher temperatures. The value of Kq was calculated by considering the fluorescence life time of the biopolymer as 10−8 s (Table 1). The maximum scattering collision quenching constant of the various quenchers with the biopolymer was 2 × 1010 L mol−1 s−1. In the present case, the quenching constant, Kq, was in order of 1014 L mol−1s−1 and increased with increasing temperature (Table 1), confirming that the interaction between the BSA and AgNPs appears to be due to a mixed static and dynamic quenching process of the appropriate interaction and not to the collision effect. Fluorescence quenching of BSA also provides information on the binding constant (K) as well as the number of binding sites (n) between the quencher and BSA. By plotting log [(F0 − F)/ F)] vs. log[NPs] (Fig. 6b), the values of n and K were obtained from the slope of the line of best fit and Y axis intercept, respectively. Table 1 summarizes these values, as a function of temperature. The value of ‘n’ at the experimental temperature was equal to approximately one, indicating that there is a single binding site in BSA for AgNPs with respect to temperature and shows that the K values increase with increasing temperature, indicating the formation of a stable complex of BSA with AgNPs [26]. Normally, biological molecules bind with inorganic molecules by forming a hydrogen bond, or by van der Waals interactions, hydrophobic and hydrophilic interactions, electrostatic interaction, etc. An evaluation of the change in entropy (∆S°) and enthalpy (∆H°) of a binding reaction enables a determination of the type of interacting force. The binding 5
constant of BSA was determined at three different temperatures. A plot of ln(K) vs. 1/T was fitted linearly to obtain ∆S° and ∆H° from the slope and intercept, respectively. The ∆G° values were calculated from Eq. 4 and are listed in Table 1. The negative sign of the free energy (∆G°) indicates that the binding process occurs spontaneously. The positive values of ∆S° (57.35 J / mol. K) and the negative value of ∆H° (−12.26 KJ / mol) indicates the electrostatic interaction, which prefers the selective adsorption of NPs on the protein surface [27]. The conformational changes in BSA were examined by measuring the synchronous fluorescence intensity of the amino acid residues before and after adding the AgNPs. The environment of the fluorophore functional group was studied by measuring the possible shift in the maximum wavelength emission (λmax) in fluorescence. When ∆λ was kept at 15 nm or 60 nm, the synchronous fluorescence spectra provides characteristic information on the tyrosine or tryptophan residues, respectively [24]. Fig. 7a shows that the maximum emission wavelength at ∆λ =15 was red shifted by 2 nm, whereas Fig. 7b indicates that the maximum emission wavelength at ∆λ = 60 nm was blue shifted by 3 nm. This shows that the microenvironment close to both tyrosine and tryptophan residues of BSA was disturbed slightly and that the hydrophobicity of both residues was increased in the presence of AgNPs. Therefore, based on the results obtained from the binding study, it helped better understand the mechanism of the AgNPs interactions with BSA. On the other hand, BSA is the most abundant protein in plasma with exceptional properties to bind reversibly with NPs [28]. Therefore, the introduction of the green synthesized AgNPs in plasma would have great potential in the field of biomedical applications and also serves as a biosafety concern of nanoparticles. 2.3. Antioxidant and anticancer activity The radical–scavenging activity of the AgNPs was estimated by comparing the % inhibition of DPPH radicals (Fig. 8a). The AgNPs exhibited moderate antioxidant activity compared to butylated hydroxyanisole (BHA). The DPPH radical scavenging activity of AgNPs increased with increasing concentration (1 mM-45.59 %; 0.5 mM-41.63 %; 0.25 mM28.69 % and 0.125 mM- 25.67 %). The anticancer activity of the AgNPs was evaluated in-vitro against the HeLa cell line at different concentrations (1, 0.5, 0.25 and 0.125 mM). The AgNPs showed the maximum inhibition of 83.55 % at 1mM and the minimum inhibition of 56.5% at 0.125 mM (Fig. 8b), 6
the cytotoxicity of AgNPs showed a direct dose relationship. The cytotoxic effects of the AgNPs might be the result of an active physicochemical interaction of Ag atoms with the functional groups of the intracellular proteins, as well as with the nitrogen bases and phosphate groups in DNA [22]. 3.
Conclusion AgNPs (with ~ 16.7nm average size) were synthesized from an aqueous extract of N.
nucifera root quenched the fluorophore of BSA. The existence of single binding sites in BSA for AgNPs (n ~ 1) and the binding constants at different temperatures were identified by analyzing the fluorescence quenching data using the Stern-Volmer equation. In particular, the calculated thermodynamic parameters (∆G°, ∆H° and ∆S°) suggest that binding occurs spontaneously, involving electrostatic forces. The synchronous fluorescence spectra indicated that the microenvironment close to both the tyrosine and tryptophan residues of BSA was disturbed. The RLS spectra indicated the formation of aggregates of BSA-AgNPs. Finally, the green synthesis AgNPs exhibited moderate antioxidant and cytotoxicity activities against HeLa cell lines.
Supplementary data The details of the experiment, aqueous extract of N. nucifera root, synthesis, characterization and protein binding study of AgNPs can be found in the supplementary data. Notes The authors are declared no conflict of interest. Acknowledgments This work was supported by research grants from Catholic University of Daegu in 2014 (20141061). References [1]
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9
Figure 1. (a) UV-vis spectrum of AgNPs synthesized using 1 mM aqueous of AgNO3, solution with N. nucifera root extract after 45 min. (b) FTIR spectrum of N. nucifera root extract and AgNPs synthesized from N. nucifera root extract. 10
Figure 2. (a) SEM image of AgNPs synthesized from N. nucifera root extract. (b) EDX spectra of AgNPs synthesized from N. nucifera root extract. (c) & (d) TEM images of AgNPs synthesized from N. nucifera root extract.
11
Figure 3. XRD pattern of AgNPs synthesized from N. nucifera root extract.
Fig. 4. UV- visible absorption spectra of BSA and BSA in presence of 0.8 x 10-7, 1.6 x 10 -7, 2.4 x 10-7, 3.2 x 10-7 and 4.0 x 10 -7 molL-1 AgNPs. Inset shows the absorption intensity as a function of AgNPs concentration. 12
Figure 5. (a) Fluorescence spectra. (b) Resonance light scattering spectra of BSA in absence and in presence of AgNPs at different concentration.
Figure 6. (a) Stern Volmer plot for BSA and AgNPs. (b) Double-logarithm plot for the quenching of BSA by AgNPs at three different temperatures.
13
Figure 7. (a) Synchronous fluorescence spectra at ∆λ=15 (a) and ∆λ=60. (b) BSA in absence and in presence of 1.66 x 10-9, 3.33 x 10 -9, 5.0 x 10 -9, 6.66 x 10-9 and 8.33 x 10 -9 molL-1 AgNPs.
14
Figure 8. (a) DPPH assay of green synthesized AgNPs. (b) Cytotoxicity activity of green synthesized AgNPs.
15
Table 1. Temperature (K)
KSV (M-1)
Kq (M-1 S-1)
K (L mol-1)
n
∆G (KJ/mol)
281
7.18 x 10 6
7.18 x 10 14
1.23 x 10 7
0.90
-28.372
301
1.60 x 10
7
15
7
0.95
-29.519
310
3.04 x 10 7
8.04 x 10 8
1.07
-30.035
1.60 x 10
3.04 x 10 15
6.29 x 10
Binding parameters (quenching rate constant, binding constant and binding sites) and Gibbs free energy of AgNPs interacted with BSA.
16
Graphical abstract
17
Research Highlights AgNPs were developed using the root extract of Nelumbo nucifera. Green synthesis is a simple, rapid, clean, inexpensive and eco-friendly. The interaction of green synthesized AgNPs with Bovine Serum Albumin (BSA) Fluorescence quenching, synchronous and resonance light scattering spectroscopy inf luences the efficient binding between BSA and AgNPs. AgNPs showed moderate antioxidant and cytotoxicity activity against HeLa cell lines.
18
S1011-1344(14)00299-1 http://dx.doi.org/10.1016/j.jphotobiol.2014.10.002 JPB 9851
To appear in:
Journal of Photochemistry and Photobiology B: Biology
Received Date: Revised Date: Accepted Date:
12 July 2014 2 October 2014 6 October 2014
Please cite this article as: T.V.M. Sreekanth, S. Ravikumar, I-Y. Eom, Green synthesized silver nanoparticles using Nelumbo Nucifera root extract for efficient protein binding, antioxidant and cytotoxicity activities, Journal of Photochemistry and Photobiology B: Biology (2014), doi: http://dx.doi.org/10.1016/j.jphotobiol.2014.10.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
Green synthesized silver nanoparticles using Nelumbo Nucifera root extract for efficient protein binding, antioxidant and cytotoxicity activities. T.V.M. Sreekanth1, Sambandam Ravikumar2, In-Yong Eom1,3* 1
Department of life chemistry, Catholic University of Daegu, Gyeongsan-Si,
South Korea. 2
Department of Pharmaceutical Science and Technology, Catholic University of
Daegu,Gyeongsan-Si, South Korea. 3
Natural Science Research Institute, Catholic University of Daegu,Gyeongsan-Si, South
Korea. *
Corresponding author: In-Yong Eom, Tel: +82-53-850-3783; Fax: +82-53-850-3728 Email: [email protected]
Abstract Silver nanoparticles (AgNPs) with a mean particle size of ~16.7nm were synthesized using an eco-friendly reducing material, aqueous Nelumbo nucifera root extract. Rapid reduction resulted in the formation of polydispersed nanoparticles. The formation of AgNPs was characterized by surface plasmon resonance, which was determined by ultraviolet-visible (UV-Vis) spectroscopy (band at 412 nm), Fourier transform infrared spectroscopy, scanning electron microscopy- energy dispersive X-ray spectroscopy, transmission electron microscopy and X-ray diffraction. The interaction of the green synthesized AgNPs with bovine serum albumin (BSA) at various temperatures was investigated. Fluorescence quenching, synchronous and resonance light scattering spectroscopy along with UV-Vis absorption studies revealed the efficient binding between BSA and the AgNPs. In addition, the AgNPs exhibited moderate antioxidant and cytotoxicity activities against HeLa cell lines.
Keywords: Eco-friendly synthesis, AgNPs, Nelumbo nucifera, BSA, Antioxidant, Cytotoxicity.
1
1.
Introduction Over the past decade, there has been increasing interest in the interfacing of material
chemistry and biology with an emphasis on the non-covalent interactions of nanoparticles with proteins and DNA [1]. Studies of the interactions of nanoparticles (NPs) with proteins are facilitated by a large body of research on drug delivery [2, 3]. In addition, NPs can accumulate easily on a biomolecule surface and interact with proteins, which affects their conformation and subsequent functions. On the other hand, the introduction of NPs in the blood and their interactions with the proteins present in plasma is of key importance in their biomedical applications. The growing biosafety concerns of nanomaterials can be also be investigated by applying analogous techniques to probe the structural changes and aggregation of proteins during NPs interactions. Among the various types of metallic nanomaterials developed thus far, AgNPs have attracted substantial interest because of their wide range of applications in the chemical, physical and biological fields owing to their optical, chemical, photochemical, and electronic properties [4]. AgNPs can be synthesized by both chemical [5] and biological methods. Chemical methods generally involve toxic materials that are expensive and harmful to the environment [6]. The green synthesis of AgNPs is simple, inexpensive, less time consuming, and eco-friendly. Over the last few years, AgNPs were prepared using inexpensive biological resources, such as plants [7, 8], bacteria [9], fungi [10], yeast [11], and lotus seed extract [12], which reduce silver ions to AgNPs both extra- and intracellularly [13]. Several silver-based nanoparticles have been investigated in biological settings but the majority of those studies focused mainly on chemically-prepared metallic NPs rather than green synthesis, which focuses predominantly on biosafety. This paper reports the green synthesis of new AgNPs using N. nucifera root extracts for the reduction of Ag+ ions. N. nucifera is a large aquatic medicinal plant found in Asian countries, including Korea, Japan and China. N. nucifera root has many potential benefits including anti-diarrheal [14], psychopharmacological [15], diuretic [16], antipyretic [17], antifungal and anti-yeast activities [18], hypoglycemic activities [19], hypotension effects [20], and hypocholesterolemic effects [21]. Serum albumins are the most abundant proteins in plasma, and are used widely for the transportation of drugs and nutrition through the human body [22]. Therefore, it is of major importance to study the mechanism of the interaction between BSA and nanomaterials 2
to improve both the binding properties and conformational behavior of proteins. The photophysical properties of BSA are sensitive to conformational changes, and provide two tryptophan residues, Trp 212 and Trp 134, which possess intrinsic fluorescence. The present study focuses on understanding the biophysical mechanism of interaction between the greenly synthesized AgNPs and BSA using fluorescence spectroscopy. This study provides an important foundation in the design of new protein-NPs systems, which will motivate broader targets, including new therapeutics and biosensors. 2.
Results and discussion
2.1. Synthesis and characterization of AgNPs This study focused initially on the synthesis of AgNPs using an eco-friendly green synthesis method. The AgNPs were synthesized by reducing Ag+ ions using a N. nucifera root extract. A reaction of 1mM AgNO3 and N. nucifera root extract transformed a colorless or pale yellow solution to a strong wine red color due to the surface plasmon resonance of the AgNPs formed. This reaction was complete within 45 min at room temperature. The AgNPs solution was stable for 2 months at room temperature without any signs of precipitation. UV-Vis spectroscopy can be used to examine the size and shape of nanoparticles in aqueous suspensions [23]. In the UV-Vis spectra, the SPR bands of the AgNPs synthesized from N. nucifera root extract were centered between 400-500 nm, which confirmed the formation of AgNPs in solution. Fig. 1a shows no evidence of absorption in the range, 400500 nm, for the N. nucifera root extract only. An absorption peak at 414 nm was clearly observed when the aqueous plant extract was added to a 1mM AgNO3 solution (Fig. 1a). Fourier transform infrared (FTIR) spectroscopy was carried out to identify the possible biomolecules responsible for the reduction, capping and efficient stabilization of the AgNPs. Fig. 1b shows the FTIR spectra of the N. nucifera root extract and AgNPs. The N. nucifera root extract showed transmission peaks at 3309, 2926, 1637, 1411, 1148, and 1075 cm-1. The transmission peaks indicating the presence of AgNPs were located at 3337, 2937, 1620, 1339, 1148, and 1032 cm-1. These were peaks assigned to phenols and alcohols (Hydrogen- bonded O-H stretch); alkanes (C-H stretch); 1° amines (N-H bend); aromatics (C-H stretch (in-ring)); alcohols, carboxylic acids, esters, ethers (C-O stretch) and aliphatic amines (C-N stretch) respectively. Scanning electron microscopy (SEM) confirmed the AgNPs in the nano-size. The nanoparticles were predominantly spherical in shape with diameters below 100 nm, and a 3
mean size of ~16.7 nm (Fig. 2a). The EDX spectrum revealed a strong signal in the silver region and confirms the formation of AgNPs. Another strong signal for Si was observed, which was assigned to the Si wafer used as a substrate to prepare thin films. The remaining elemental signals were from the aqueous root extract of N. nucifera (Fig. 2b). The TEM images in Fig. 2c & 2d show more clearly that the AgNPs are commonly found in spherical, spherical triangle and uneven shapes. Fig. 3 shows X-ray diffraction (XRD) patterns of the AgNPs synthesized from the N. nucifera root extract. The Bragg reflections of 38.28, 44.38 and 64.54° 2θ were indexed to the (111), (200) and (220) planes, respectively. 2.2. BSA–AgNPs binding study UV–Vis spectroscopy was carried out on BSA in the absence and presence of AgNPs at different concentrations, as shown in Fig. 4. Upon the addition of AgNPs, the absorption band of BSA at 279 nm increased significantly with a blue shift of 2 nm (inset of Fig. 4). AgNPs do not display an absorption band at around 279 nm. Therefore, the increase in the intensity of the peak at 279 nm for BSA upon addition of AgNPs in the BSA was attributed to the formation of ground state complex formed by AgNPs–BSA interaction. In addition, the existence of an isosbestic point at approximately 290 nm in the UV spectrum (Fig. 4) indicates the conversion of free AgNPs to the BSA-bound AgNPs complex. Among several techniques to examine the interaction between NPs and BSA, the most convenient method was to study the fluorescence quenching of BSA. The main contribution to the fluorescence of BSA is by the tryptophan (Trp) moiety, which is quite sensitive to the local environment. Therefore, the fluorescence spectra (Fig. 5a) were recorded for BSA and mixed with different concentrations of AgNPs to examine the interactions between them. The figure clearly shows that BSA has a strong emission band at 343 nm when excited with a 279 nm wavelength. The intensity of this fluorescence band decreased gradually and the maximum of the peak was also shifted by 4 nm with increasing AgNPs concentration. This change in the fluorescence is characteristic of the BSA, which indicates the binding between AgNPs and BSA to form a certain complex. The RLS spectra were recorded, where the emission of BSA was measured at the same excited wavelength. This is a simple technique that allows the easy detection of aggregates in solution. Fig. 5b shows the RLS spectra of BSA, AgNPs and mixture of BSA and AgNPs with varying concentrations of AgNPs. BSA showed a very weak RLS signal over the entire wavelength range of 220-700 nm [23] and the AgNPs showed a peak at 4
445nm. In contrast, strong RLS signals was observed for the AgNPs-BSA mixtures with a maximum peak at approximately 450 nm. The generation of the RLS spectrum correlated with the formation of aggregates. The RLS intensity is affected primarily by the dimensions of the aggregate formed in solution. This suggests that the added BSA may interact with the AgNPs in solution to form a new BSA-AgNPs complex that would be expected to be an aggregate. Therefore, enhanced light scattering occurs under the given conditions [24]. Fluorescence quenching behaviors can be distinguished by examining fluorescence quenching at different temperatures. The fluorescence quenching data at different temperatures was analyzed using the well-known Stern–Volmer equation [25]. Fig. 6a presents the Stern–Volmer plot of F0 / F vs. [NPs]. The Stern–Volmer constant was obtained from the slope after linear regression of this plot (Table 1). The quenching constant increased with increasing temperature, indicating that fluorescence quenching occurs because of some specific binding between AgNPs and BSA by forming complexes, which were stabilized at higher temperatures. The value of Kq was calculated by considering the fluorescence life time of the biopolymer as 10−8 s (Table 1). The maximum scattering collision quenching constant of the various quenchers with the biopolymer was 2 × 1010 L mol−1 s−1. In the present case, the quenching constant, Kq, was in order of 1014 L mol−1s−1 and increased with increasing temperature (Table 1), confirming that the interaction between the BSA and AgNPs appears to be due to a mixed static and dynamic quenching process of the appropriate interaction and not to the collision effect. Fluorescence quenching of BSA also provides information on the binding constant (K) as well as the number of binding sites (n) between the quencher and BSA. By plotting log [(F0 − F)/ F)] vs. log[NPs] (Fig. 6b), the values of n and K were obtained from the slope of the line of best fit and Y axis intercept, respectively. Table 1 summarizes these values, as a function of temperature. The value of ‘n’ at the experimental temperature was equal to approximately one, indicating that there is a single binding site in BSA for AgNPs with respect to temperature and shows that the K values increase with increasing temperature, indicating the formation of a stable complex of BSA with AgNPs [26]. Normally, biological molecules bind with inorganic molecules by forming a hydrogen bond, or by van der Waals interactions, hydrophobic and hydrophilic interactions, electrostatic interaction, etc. An evaluation of the change in entropy (∆S°) and enthalpy (∆H°) of a binding reaction enables a determination of the type of interacting force. The binding 5
constant of BSA was determined at three different temperatures. A plot of ln(K) vs. 1/T was fitted linearly to obtain ∆S° and ∆H° from the slope and intercept, respectively. The ∆G° values were calculated from Eq. 4 and are listed in Table 1. The negative sign of the free energy (∆G°) indicates that the binding process occurs spontaneously. The positive values of ∆S° (57.35 J / mol. K) and the negative value of ∆H° (−12.26 KJ / mol) indicates the electrostatic interaction, which prefers the selective adsorption of NPs on the protein surface [27]. The conformational changes in BSA were examined by measuring the synchronous fluorescence intensity of the amino acid residues before and after adding the AgNPs. The environment of the fluorophore functional group was studied by measuring the possible shift in the maximum wavelength emission (λmax) in fluorescence. When ∆λ was kept at 15 nm or 60 nm, the synchronous fluorescence spectra provides characteristic information on the tyrosine or tryptophan residues, respectively [24]. Fig. 7a shows that the maximum emission wavelength at ∆λ =15 was red shifted by 2 nm, whereas Fig. 7b indicates that the maximum emission wavelength at ∆λ = 60 nm was blue shifted by 3 nm. This shows that the microenvironment close to both tyrosine and tryptophan residues of BSA was disturbed slightly and that the hydrophobicity of both residues was increased in the presence of AgNPs. Therefore, based on the results obtained from the binding study, it helped better understand the mechanism of the AgNPs interactions with BSA. On the other hand, BSA is the most abundant protein in plasma with exceptional properties to bind reversibly with NPs [28]. Therefore, the introduction of the green synthesized AgNPs in plasma would have great potential in the field of biomedical applications and also serves as a biosafety concern of nanoparticles. 2.3. Antioxidant and anticancer activity The radical–scavenging activity of the AgNPs was estimated by comparing the % inhibition of DPPH radicals (Fig. 8a). The AgNPs exhibited moderate antioxidant activity compared to butylated hydroxyanisole (BHA). The DPPH radical scavenging activity of AgNPs increased with increasing concentration (1 mM-45.59 %; 0.5 mM-41.63 %; 0.25 mM28.69 % and 0.125 mM- 25.67 %). The anticancer activity of the AgNPs was evaluated in-vitro against the HeLa cell line at different concentrations (1, 0.5, 0.25 and 0.125 mM). The AgNPs showed the maximum inhibition of 83.55 % at 1mM and the minimum inhibition of 56.5% at 0.125 mM (Fig. 8b), 6
the cytotoxicity of AgNPs showed a direct dose relationship. The cytotoxic effects of the AgNPs might be the result of an active physicochemical interaction of Ag atoms with the functional groups of the intracellular proteins, as well as with the nitrogen bases and phosphate groups in DNA [22]. 3.
Conclusion AgNPs (with ~ 16.7nm average size) were synthesized from an aqueous extract of N.
nucifera root quenched the fluorophore of BSA. The existence of single binding sites in BSA for AgNPs (n ~ 1) and the binding constants at different temperatures were identified by analyzing the fluorescence quenching data using the Stern-Volmer equation. In particular, the calculated thermodynamic parameters (∆G°, ∆H° and ∆S°) suggest that binding occurs spontaneously, involving electrostatic forces. The synchronous fluorescence spectra indicated that the microenvironment close to both the tyrosine and tryptophan residues of BSA was disturbed. The RLS spectra indicated the formation of aggregates of BSA-AgNPs. Finally, the green synthesis AgNPs exhibited moderate antioxidant and cytotoxicity activities against HeLa cell lines.
Supplementary data The details of the experiment, aqueous extract of N. nucifera root, synthesis, characterization and protein binding study of AgNPs can be found in the supplementary data. Notes The authors are declared no conflict of interest. Acknowledgments This work was supported by research grants from Catholic University of Daegu in 2014 (20141061). References [1]
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Figure 1. (a) UV-vis spectrum of AgNPs synthesized using 1 mM aqueous of AgNO3, solution with N. nucifera root extract after 45 min. (b) FTIR spectrum of N. nucifera root extract and AgNPs synthesized from N. nucifera root extract. 10
Figure 2. (a) SEM image of AgNPs synthesized from N. nucifera root extract. (b) EDX spectra of AgNPs synthesized from N. nucifera root extract. (c) & (d) TEM images of AgNPs synthesized from N. nucifera root extract.
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Figure 3. XRD pattern of AgNPs synthesized from N. nucifera root extract.
Fig. 4. UV- visible absorption spectra of BSA and BSA in presence of 0.8 x 10-7, 1.6 x 10 -7, 2.4 x 10-7, 3.2 x 10-7 and 4.0 x 10 -7 molL-1 AgNPs. Inset shows the absorption intensity as a function of AgNPs concentration. 12
Figure 5. (a) Fluorescence spectra. (b) Resonance light scattering spectra of BSA in absence and in presence of AgNPs at different concentration.
Figure 6. (a) Stern Volmer plot for BSA and AgNPs. (b) Double-logarithm plot for the quenching of BSA by AgNPs at three different temperatures.
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Figure 7. (a) Synchronous fluorescence spectra at ∆λ=15 (a) and ∆λ=60. (b) BSA in absence and in presence of 1.66 x 10-9, 3.33 x 10 -9, 5.0 x 10 -9, 6.66 x 10-9 and 8.33 x 10 -9 molL-1 AgNPs.
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Figure 8. (a) DPPH assay of green synthesized AgNPs. (b) Cytotoxicity activity of green synthesized AgNPs.
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Table 1. Temperature (K)
KSV (M-1)
Kq (M-1 S-1)
K (L mol-1)
n
∆G (KJ/mol)
281
7.18 x 10 6
7.18 x 10 14
1.23 x 10 7
0.90
-28.372
301
1.60 x 10
7
15
7
0.95
-29.519
310
3.04 x 10 7
8.04 x 10 8
1.07
-30.035
1.60 x 10
3.04 x 10 15
6.29 x 10
Binding parameters (quenching rate constant, binding constant and binding sites) and Gibbs free energy of AgNPs interacted with BSA.
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Graphical abstract
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Research Highlights AgNPs were developed using the root extract of Nelumbo nucifera. Green synthesis is a simple, rapid, clean, inexpensive and eco-friendly. The interaction of green synthesized AgNPs with Bovine Serum Albumin (BSA) Fluorescence quenching, synchronous and resonance light scattering spectroscopy inf luences the efficient binding between BSA and AgNPs. AgNPs showed moderate antioxidant and cytotoxicity activity against HeLa cell lines.
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