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COLSUB-6633; No. of Pages 7
Colloids and Surfaces B: Biointerfaces xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Green synthesis of bacterial gold nanoparticles conjugated to resveratrol as delivery vehicles C. Ganesh Kumar a,b,∗ , Y. Poornachandra a,b , Suman Kumar Mamidyala b a b
Academy of Scientific and Innovative Research, CSIR-Indian Institute of Chemical Technology, Uppal Road, Hyderabad 500007, India Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Uppal Road, Hyderabad 500007, India
a r t i c l e
i n f o
Article history: Received 9 May 2014 Received in revised form 12 September 2014 Accepted 15 September 2014 Available online xxx Keywords: Nanoparticles Resveratrol Nanocarrier Cancer therapy Green chemistry
a b s t r a c t Bio-directed synthesis of metal nanoparticles is gaining importance in view of their biocompatibility, low toxicity and eco-friendly characteristics. The present study describes the application of resveratrol conjugated gold nanoparticles as effective delivery vehicles. The green chemistry approach was used for the synthesis of gold nanoparticles by using the culture supernatant of Delftia sp. strain KCM-006. The synthesized gold nanoparticles were mono-dispersed, spherical in shape with an average size of 11.3 nm. They were found to be photoluminescent and crystalline in nature with a zeta potential of −25 mV, indicating their high stability. Resveratrol, an anticancer drug, was conjugated to these gold nanoparticles (RSVAuNP). The cell viability and immunocytochemistry analysis with human lung cancer cell line (A549) demonstrated that RSV-AuNPs were 65% more effective as drug when compared to resveratrol alone. In vitro observations on the drug release from these nanoparticles exhibited pH dependency; the release was significant (95%) under acidic conditions (pH 5.2) when compared to physiological conditions (pH 7.4). © 2014 Elsevier B.V. All rights reserved.
1. Introduction Nanomaterial preparations using different noble metals find use in a wide variety of biomedical applications as drug carriers for targeted delivery, for cancer treatment, molecular imaging, gene therapy, DNA analysis, magnetic resonance imaging, antibacterial agents and in other disciplines such as biosensors, catalysis and separation science due to their unique physical and chemical properties [1,2]. They can be conjugated to a wide variety of biomolecules and can be potential candidates for drug delivery systems in cancer therapy and related diseases [3,4]. Our laboratory has been exploring the possibility of ecofriendly green chemistry approach for the synthesis of nanoparticles using biomaterials of microbial origin which are biodegradable, non-toxic and costeffective [5]. Most of the cancers are controlled by chemotherapy; however, the multidrug resistance has become a limiting factor. One of the promising strategies to overcome this drug resistance is the
∗ Corresponding author at: Medicinal Chemistry and Pharmacology Division, CSIRIndian Institute of Chemical Technology, Uppal Road, Hyderabad 500007, India. Tel.: +91 40 27193105; fax: +91 40 27193189. E-mail addresses: [email protected], [email protected] (C. Ganesh Kumar).
targeted nanomaterial based drug delivery by conjugating drugs to nontoxic or neutral nanoparticles [6]. Gold nanoparticles are identified as promising candidates for drug delivery applications due to their unique dimensions, tunable surface functionalities, non-toxicity and controlled drug release [7]. They can potentially overcome physiological barriers, and transport the drug to specific cells or intracellular compartments by passive or ligand-mediated targeting approaches [8]. Resveratrol (3,5,4 -trihydroxystilbene) is a naturally occurring polyphenol in different medicinal plants and is rich in red wine produced from grapes. It protects plants from infectious pathogens, ultraviolet irradiation and various environmental stresses. Several biological studies revealed that resveratrol exhibits strong antioxidant activity, inhibits platelet aggregation and protects from cancer and cardiovascular diseases [9–11]. Preclinical and clinical studies have revealed that it exhibited no cytotoxic effects against normal cell lines and thus can be used as an ideal capping agent [12]. In the present study, the culture supernatant derived from Delftia sp. strain KCM-006 was used for the preparation of gold nanoparticles (AuNPs) without the addition of any reducing agent. The AuNPs exhibited excellent stability at room temperature. The anti-cancer agent, resveratrol, was conjugated to the AuNPs and the resulting delivery system was tested for their efficacy against the survival of A549 (lung carcinoma) cells. In vitro studies demonstrated that the release of resveratrol from the nanoparticles is pH-dependent.
http://dx.doi.org/10.1016/j.colsurfb.2014.09.032 0927-7765/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: C. Ganesh Kumar, et al., Green synthesis of bacterial gold nanoparticles conjugated to resveratrol as delivery vehicles, Colloids Surf. B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.09.032
G Model
ARTICLE IN PRESS
COLSUB-6633; No. of Pages 7
C. Ganesh Kumar et al. / Colloids and Surfaces B: Biointerfaces xxx (2014) xxx–xxx
2
2. Experimental 2.1. Materials All chemicals were of analytical grade and procured from Sigma–Aldrich, St. Louis, MO, unless otherwise stated. All culture media were purchased from HiMedia Laboratories Pvt. Ltd., Mumbai, India. 2.2. Medium and fermentation conditions Delftia sp. strain KCM-006, previously isolated in our laboratory from oil contaminated soil sample collected at a depth of 10 m from the Manuguru Coal Mines, Khammam, Andhra Pradesh, India, was cultured aerobically in modified glycerol peptone broth containing (g/L): glycerol, 30, peptone, 5 and meat extract, 5. Fermentation was carried out at 35 ◦ C in 1000 mL Erlenmeyer flasks containing 250 mL of modified glycerol peptone broth (adjusted to different pH values ranging from 6 to 9) with agitation at 150 rpm for 72 h in a New Brunswick Innova 43R shaker (Eppendorf North America, Hauppauge, NY, USA). The fermented medium was later subjected to centrifugation (Sorvall RC 5C Plus, Kendro Lab Products, Asheville, NC, USA) at 10,400 × g for 20 min to obtain a cell-free supernatant. 2.3. Preparation and characterization of gold nanoparticles The gold nanoparticles were synthesized using aqueous hydrogen tetrachloroaurate at different pH values. In the synthesis protocol, independent reactions were carried out using hydrogen tetrachloroaurate (10 mL of 10−2 M) separately added to 90 mL of cell-free supernatant cultured at different pH values, i.e. 6.0, 7.0, 8.0 and 9.0, and kept for continuous stirring on a magnetic stirrer at 200 rpm for 14 h at ambient temperature (28 ◦ C). The effect of temperature on nanoparticle synthesis was studied by performing the reactions at different temperatures, i.e. 35, 45, 55 and 100 ◦ C individually under earlier mentioned conditions using the cell-free supernatant (optimum pH 8.0). Sample aliquots (2 mL) were withdrawn at periodic intervals and monitored for the bioreduction of metal ions. The effect of pH and temperature on nanoparticle formation was examined by recording the UV–visible scan spectra of these sample aliquots from 300 to 800 nm on a UV–visible double beam spectrophotometer (Lambda 25, Perkin-Elmer, Shelton, CT, USA). Sample aliquots (2 mL) of the reaction mixture were withdrawn at periodic intervals and monitored for the kinetics of bioreduction of gold ions as a function of time by UV–visible spectroscopy. The synthesized nanoparticles were washed with distilled water, centrifuged at 8200 × g for 30 min, concentrated and later stored at 4 ◦ C for further use. Morphological analysis of gold nanoparticles and resveratrolconjugated gold nanoparticles was carried out using transmission electron microscope (TEM). Reaction mixtures were subjected to ultrasonication and a drop of each reaction mixture was dispersed on an amorphous carbon-coated copper grid and the samples were dried prior to the measurements using a 100 W infrared lamp (Philips, Holland). Micrographs were acquired on Technai-FE 12 (Philips, Holland) TEM instrument which was operated at an accelerating voltage of 120 kV. The histograms were constructed based on size distribution to determine the average size of nanoparticles. The crystalline structure of nanoparticles was determined based on X-ray diffraction (XRD) analysis. The XRD pattern of gold nanoparticles was recorded on Bruker AXS D-8 Advance powder X-ray diffractometer (Shimadzu, Japan) operated at a voltage of ˚ 40 kV and current of 30 mA using CuK␣ radiation ( = 1.5406 A). The data collection and its processing was performed by the Datascan software interfaced to the diffractometer and the set scan
parameters were scan rate 1.2◦ per minute and scan range, 2 = 0–80◦ . The dried powder of the synthesized nanoparticles was re-dispersed in deionized water by ultrasonication and was subjected to dynamic light scattering (DLS) measurements at 25 ◦ C to determine the hydrodynamic particle size and the nanoparticle charge quantified as zeta potential which was determined on a Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK) instrument equipped with a He–Ne laser operating at 632.8 nm and a scattering detector at 173◦ . DLS and nanoparticle charge measurements were determined using the same instrument at 25 ◦ C. Polydispersity index (PDI) was also quantified to determine the particle size distribution range. The concentration of gold ions in the aqueous gold nanoparticle solutions was determined using inductively coupled plasma optical emission spectrophotometer (ICP-OES, IRIS Intrepid II XDL, Thermo Jarrel Ash, USA). The concentration of gold ions in the nanoparticle sample solutions was calculated from the HAuCl4 standard graphs (1–50 ppm) plotted based on ICP-OES analysis. Gold nanoparticles synthesized via citrate reduction [13] were used as a positive reference control. The photoluminescence spectrum of gold nanoparticles dispersed in water and its control was recorded on Infinite M200 (Tecan Trading AG, Switzerland) spectrophotometer using a non-fluorescent 96 well microtitre plate at an excitation wavelength of 300 nm. The dried powder of gold nanoparticles was re-dispersed in water and further its stability was monitored by UV–visible spectroscopy at regular intervals over a period of 5 months. 2.4. In vitro stability studies of gold nanoparticles The stability of gold nanoparticles was studied in different liquid milieu including fetal bovine serum, phosphate buffer, Dulbecco phosphate buffered saline (DPBS), 5% NaCl and buffered saline adjusted to pH 6.0, 7.0 and 8.0. Each solution (200 L) was added to 800 L of gold nanoparticle solution and incubated at room temperature for 24 h to 3 weeks. The stability of nanoparticles was monitored by recording the absorbance at 520 nm. 2.5. Conjugation of resveratrol with gold nanoparticles The standard curve of resveratrol (RSV) was initially prepared using different concentrations of resveratrol ranging from 1 to 50 g mL−1 in 1% ethanol and the absorbance was measured at 304 nm. The standard curve was constructed based on concentration versus absorbance values. Resveratrol solution (10 g mL−1 ) was added drop by drop to the AuNP solution and kept for continuous stirring at 150 rpm for 2 h at room temperature to obtain the resveratrol-conjugated gold nanoparticles (RSV-AuNP). The prepared solution was centrifuged at 18,600 × g for 1 h and the pellets containing the RSV-AuNPs were dispersed in water and subjected to TEM and FT-IR analyses to confirm the conjugation. 2.6. Fourier-transform infrared spectroscopy The synthesized gold nanoparticles and resveratrol-conjugated gold nanoparticles were centrifuged at 12,000 × g for 10 min and the obtained pellet was washed thrice with deionized water to remove proteins and other components present in the solution and the nanoparticles were dried by lyophilization to obtain a powder. The FT-IR spectrum of the dried nanoparticle powder in the form of KBr pellets was recorded on Thermo-Nicolet Nexus 670 spectrometer at a resolution of 4 cm−1 in a wavenumber region of 400–4000 cm−1 .
Please cite this article in press as: C. Ganesh Kumar, et al., Green synthesis of bacterial gold nanoparticles conjugated to resveratrol as delivery vehicles, Colloids Surf. B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.09.032
G Model COLSUB-6633; No. of Pages 7
ARTICLE IN PRESS C. Ganesh Kumar et al. / Colloids and Surfaces B: Biointerfaces xxx (2014) xxx–xxx
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2.7. In vitro release of resveratrol The in vitro release of resveratrol from RSV-AuNPs was analyzed at two different pH conditions (pH 5.2 and pH 7.4) using the membrane dialysis method [14]. Briefly, the RSV-capped AuNPs were placed into the dialysis tube (MWCO of 10 kDa) in phosphate buffered saline (pH 5.2 and pH 7.4) at 37 ◦ C under perfect sink conditions and stirred at 100 rpm. Aliquots (1 mL) were withdrawn at periodic intervals of 30 min and monitored by absorbance measurements at 304 nm on a UV–visible double beam spectrophotometer for determining the quantity of resveratrol released.
2.8. Cell viability assay The cell viability of the AuNPs, resveratrol and RSV-AuNPs was assessed against A549 cell line derived from human lung carcinoma (ATCC no. CCL-185) and MRC-5 derived from normal human lung tissue (ATCC no. CCL-171) using MTT assay [15]. Briefly, the cells were grown in 96-well microtitre plates for 24 h. The cells were treated with test samples of different concentrations (1–10 M mL−1 ) and incubated for 48 h. Later, the cells were incubated with MTT (250 g mL−1 ) for 2 h. After incubation, the medium was replaced with 100 L of DMSO and the absorbance was measured at 570 nm using the TRIAD multimode reader (Dynex Technologies, Inc., Chantilly, VA). The IC50 values (50% inhibitory concentration) were calculated from the plotted absorbance data for the dose–response curves. Statistical analysis of the cell viability was performed using OriginPro 8 software. The data on IC50 values (in M) are expressed as means ± SD of three independent experiments. All experimental data were compared using Student’s t-test. In all comparisons, p
ARTICLE IN PRESS
COLSUB-6633; No. of Pages 7
Colloids and Surfaces B: Biointerfaces xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Green synthesis of bacterial gold nanoparticles conjugated to resveratrol as delivery vehicles C. Ganesh Kumar a,b,∗ , Y. Poornachandra a,b , Suman Kumar Mamidyala b a b
Academy of Scientific and Innovative Research, CSIR-Indian Institute of Chemical Technology, Uppal Road, Hyderabad 500007, India Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Uppal Road, Hyderabad 500007, India
a r t i c l e
i n f o
Article history: Received 9 May 2014 Received in revised form 12 September 2014 Accepted 15 September 2014 Available online xxx Keywords: Nanoparticles Resveratrol Nanocarrier Cancer therapy Green chemistry
a b s t r a c t Bio-directed synthesis of metal nanoparticles is gaining importance in view of their biocompatibility, low toxicity and eco-friendly characteristics. The present study describes the application of resveratrol conjugated gold nanoparticles as effective delivery vehicles. The green chemistry approach was used for the synthesis of gold nanoparticles by using the culture supernatant of Delftia sp. strain KCM-006. The synthesized gold nanoparticles were mono-dispersed, spherical in shape with an average size of 11.3 nm. They were found to be photoluminescent and crystalline in nature with a zeta potential of −25 mV, indicating their high stability. Resveratrol, an anticancer drug, was conjugated to these gold nanoparticles (RSVAuNP). The cell viability and immunocytochemistry analysis with human lung cancer cell line (A549) demonstrated that RSV-AuNPs were 65% more effective as drug when compared to resveratrol alone. In vitro observations on the drug release from these nanoparticles exhibited pH dependency; the release was significant (95%) under acidic conditions (pH 5.2) when compared to physiological conditions (pH 7.4). © 2014 Elsevier B.V. All rights reserved.
1. Introduction Nanomaterial preparations using different noble metals find use in a wide variety of biomedical applications as drug carriers for targeted delivery, for cancer treatment, molecular imaging, gene therapy, DNA analysis, magnetic resonance imaging, antibacterial agents and in other disciplines such as biosensors, catalysis and separation science due to their unique physical and chemical properties [1,2]. They can be conjugated to a wide variety of biomolecules and can be potential candidates for drug delivery systems in cancer therapy and related diseases [3,4]. Our laboratory has been exploring the possibility of ecofriendly green chemistry approach for the synthesis of nanoparticles using biomaterials of microbial origin which are biodegradable, non-toxic and costeffective [5]. Most of the cancers are controlled by chemotherapy; however, the multidrug resistance has become a limiting factor. One of the promising strategies to overcome this drug resistance is the
∗ Corresponding author at: Medicinal Chemistry and Pharmacology Division, CSIRIndian Institute of Chemical Technology, Uppal Road, Hyderabad 500007, India. Tel.: +91 40 27193105; fax: +91 40 27193189. E-mail addresses: [email protected], [email protected] (C. Ganesh Kumar).
targeted nanomaterial based drug delivery by conjugating drugs to nontoxic or neutral nanoparticles [6]. Gold nanoparticles are identified as promising candidates for drug delivery applications due to their unique dimensions, tunable surface functionalities, non-toxicity and controlled drug release [7]. They can potentially overcome physiological barriers, and transport the drug to specific cells or intracellular compartments by passive or ligand-mediated targeting approaches [8]. Resveratrol (3,5,4 -trihydroxystilbene) is a naturally occurring polyphenol in different medicinal plants and is rich in red wine produced from grapes. It protects plants from infectious pathogens, ultraviolet irradiation and various environmental stresses. Several biological studies revealed that resveratrol exhibits strong antioxidant activity, inhibits platelet aggregation and protects from cancer and cardiovascular diseases [9–11]. Preclinical and clinical studies have revealed that it exhibited no cytotoxic effects against normal cell lines and thus can be used as an ideal capping agent [12]. In the present study, the culture supernatant derived from Delftia sp. strain KCM-006 was used for the preparation of gold nanoparticles (AuNPs) without the addition of any reducing agent. The AuNPs exhibited excellent stability at room temperature. The anti-cancer agent, resveratrol, was conjugated to the AuNPs and the resulting delivery system was tested for their efficacy against the survival of A549 (lung carcinoma) cells. In vitro studies demonstrated that the release of resveratrol from the nanoparticles is pH-dependent.
http://dx.doi.org/10.1016/j.colsurfb.2014.09.032 0927-7765/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: C. Ganesh Kumar, et al., Green synthesis of bacterial gold nanoparticles conjugated to resveratrol as delivery vehicles, Colloids Surf. B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.09.032
G Model
ARTICLE IN PRESS
COLSUB-6633; No. of Pages 7
C. Ganesh Kumar et al. / Colloids and Surfaces B: Biointerfaces xxx (2014) xxx–xxx
2
2. Experimental 2.1. Materials All chemicals were of analytical grade and procured from Sigma–Aldrich, St. Louis, MO, unless otherwise stated. All culture media were purchased from HiMedia Laboratories Pvt. Ltd., Mumbai, India. 2.2. Medium and fermentation conditions Delftia sp. strain KCM-006, previously isolated in our laboratory from oil contaminated soil sample collected at a depth of 10 m from the Manuguru Coal Mines, Khammam, Andhra Pradesh, India, was cultured aerobically in modified glycerol peptone broth containing (g/L): glycerol, 30, peptone, 5 and meat extract, 5. Fermentation was carried out at 35 ◦ C in 1000 mL Erlenmeyer flasks containing 250 mL of modified glycerol peptone broth (adjusted to different pH values ranging from 6 to 9) with agitation at 150 rpm for 72 h in a New Brunswick Innova 43R shaker (Eppendorf North America, Hauppauge, NY, USA). The fermented medium was later subjected to centrifugation (Sorvall RC 5C Plus, Kendro Lab Products, Asheville, NC, USA) at 10,400 × g for 20 min to obtain a cell-free supernatant. 2.3. Preparation and characterization of gold nanoparticles The gold nanoparticles were synthesized using aqueous hydrogen tetrachloroaurate at different pH values. In the synthesis protocol, independent reactions were carried out using hydrogen tetrachloroaurate (10 mL of 10−2 M) separately added to 90 mL of cell-free supernatant cultured at different pH values, i.e. 6.0, 7.0, 8.0 and 9.0, and kept for continuous stirring on a magnetic stirrer at 200 rpm for 14 h at ambient temperature (28 ◦ C). The effect of temperature on nanoparticle synthesis was studied by performing the reactions at different temperatures, i.e. 35, 45, 55 and 100 ◦ C individually under earlier mentioned conditions using the cell-free supernatant (optimum pH 8.0). Sample aliquots (2 mL) were withdrawn at periodic intervals and monitored for the bioreduction of metal ions. The effect of pH and temperature on nanoparticle formation was examined by recording the UV–visible scan spectra of these sample aliquots from 300 to 800 nm on a UV–visible double beam spectrophotometer (Lambda 25, Perkin-Elmer, Shelton, CT, USA). Sample aliquots (2 mL) of the reaction mixture were withdrawn at periodic intervals and monitored for the kinetics of bioreduction of gold ions as a function of time by UV–visible spectroscopy. The synthesized nanoparticles were washed with distilled water, centrifuged at 8200 × g for 30 min, concentrated and later stored at 4 ◦ C for further use. Morphological analysis of gold nanoparticles and resveratrolconjugated gold nanoparticles was carried out using transmission electron microscope (TEM). Reaction mixtures were subjected to ultrasonication and a drop of each reaction mixture was dispersed on an amorphous carbon-coated copper grid and the samples were dried prior to the measurements using a 100 W infrared lamp (Philips, Holland). Micrographs were acquired on Technai-FE 12 (Philips, Holland) TEM instrument which was operated at an accelerating voltage of 120 kV. The histograms were constructed based on size distribution to determine the average size of nanoparticles. The crystalline structure of nanoparticles was determined based on X-ray diffraction (XRD) analysis. The XRD pattern of gold nanoparticles was recorded on Bruker AXS D-8 Advance powder X-ray diffractometer (Shimadzu, Japan) operated at a voltage of ˚ 40 kV and current of 30 mA using CuK␣ radiation ( = 1.5406 A). The data collection and its processing was performed by the Datascan software interfaced to the diffractometer and the set scan
parameters were scan rate 1.2◦ per minute and scan range, 2 = 0–80◦ . The dried powder of the synthesized nanoparticles was re-dispersed in deionized water by ultrasonication and was subjected to dynamic light scattering (DLS) measurements at 25 ◦ C to determine the hydrodynamic particle size and the nanoparticle charge quantified as zeta potential which was determined on a Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK) instrument equipped with a He–Ne laser operating at 632.8 nm and a scattering detector at 173◦ . DLS and nanoparticle charge measurements were determined using the same instrument at 25 ◦ C. Polydispersity index (PDI) was also quantified to determine the particle size distribution range. The concentration of gold ions in the aqueous gold nanoparticle solutions was determined using inductively coupled plasma optical emission spectrophotometer (ICP-OES, IRIS Intrepid II XDL, Thermo Jarrel Ash, USA). The concentration of gold ions in the nanoparticle sample solutions was calculated from the HAuCl4 standard graphs (1–50 ppm) plotted based on ICP-OES analysis. Gold nanoparticles synthesized via citrate reduction [13] were used as a positive reference control. The photoluminescence spectrum of gold nanoparticles dispersed in water and its control was recorded on Infinite M200 (Tecan Trading AG, Switzerland) spectrophotometer using a non-fluorescent 96 well microtitre plate at an excitation wavelength of 300 nm. The dried powder of gold nanoparticles was re-dispersed in water and further its stability was monitored by UV–visible spectroscopy at regular intervals over a period of 5 months. 2.4. In vitro stability studies of gold nanoparticles The stability of gold nanoparticles was studied in different liquid milieu including fetal bovine serum, phosphate buffer, Dulbecco phosphate buffered saline (DPBS), 5% NaCl and buffered saline adjusted to pH 6.0, 7.0 and 8.0. Each solution (200 L) was added to 800 L of gold nanoparticle solution and incubated at room temperature for 24 h to 3 weeks. The stability of nanoparticles was monitored by recording the absorbance at 520 nm. 2.5. Conjugation of resveratrol with gold nanoparticles The standard curve of resveratrol (RSV) was initially prepared using different concentrations of resveratrol ranging from 1 to 50 g mL−1 in 1% ethanol and the absorbance was measured at 304 nm. The standard curve was constructed based on concentration versus absorbance values. Resveratrol solution (10 g mL−1 ) was added drop by drop to the AuNP solution and kept for continuous stirring at 150 rpm for 2 h at room temperature to obtain the resveratrol-conjugated gold nanoparticles (RSV-AuNP). The prepared solution was centrifuged at 18,600 × g for 1 h and the pellets containing the RSV-AuNPs were dispersed in water and subjected to TEM and FT-IR analyses to confirm the conjugation. 2.6. Fourier-transform infrared spectroscopy The synthesized gold nanoparticles and resveratrol-conjugated gold nanoparticles were centrifuged at 12,000 × g for 10 min and the obtained pellet was washed thrice with deionized water to remove proteins and other components present in the solution and the nanoparticles were dried by lyophilization to obtain a powder. The FT-IR spectrum of the dried nanoparticle powder in the form of KBr pellets was recorded on Thermo-Nicolet Nexus 670 spectrometer at a resolution of 4 cm−1 in a wavenumber region of 400–4000 cm−1 .
Please cite this article in press as: C. Ganesh Kumar, et al., Green synthesis of bacterial gold nanoparticles conjugated to resveratrol as delivery vehicles, Colloids Surf. B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.09.032
G Model COLSUB-6633; No. of Pages 7
ARTICLE IN PRESS C. Ganesh Kumar et al. / Colloids and Surfaces B: Biointerfaces xxx (2014) xxx–xxx
3
2.7. In vitro release of resveratrol The in vitro release of resveratrol from RSV-AuNPs was analyzed at two different pH conditions (pH 5.2 and pH 7.4) using the membrane dialysis method [14]. Briefly, the RSV-capped AuNPs were placed into the dialysis tube (MWCO of 10 kDa) in phosphate buffered saline (pH 5.2 and pH 7.4) at 37 ◦ C under perfect sink conditions and stirred at 100 rpm. Aliquots (1 mL) were withdrawn at periodic intervals of 30 min and monitored by absorbance measurements at 304 nm on a UV–visible double beam spectrophotometer for determining the quantity of resveratrol released.
2.8. Cell viability assay The cell viability of the AuNPs, resveratrol and RSV-AuNPs was assessed against A549 cell line derived from human lung carcinoma (ATCC no. CCL-185) and MRC-5 derived from normal human lung tissue (ATCC no. CCL-171) using MTT assay [15]. Briefly, the cells were grown in 96-well microtitre plates for 24 h. The cells were treated with test samples of different concentrations (1–10 M mL−1 ) and incubated for 48 h. Later, the cells were incubated with MTT (250 g mL−1 ) for 2 h. After incubation, the medium was replaced with 100 L of DMSO and the absorbance was measured at 570 nm using the TRIAD multimode reader (Dynex Technologies, Inc., Chantilly, VA). The IC50 values (50% inhibitory concentration) were calculated from the plotted absorbance data for the dose–response curves. Statistical analysis of the cell viability was performed using OriginPro 8 software. The data on IC50 values (in M) are expressed as means ± SD of three independent experiments. All experimental data were compared using Student’s t-test. In all comparisons, p
