Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 161–166
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
The interaction between ionic liquids modified magnetic nanoparticles and bovine serum albumin and the cytotoxicity to HepG-2 cells Juan-Juan Xue, Qiu-Yun Chen ⇑ School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The interaction of two magnetic
nanoparticles to BSA was studied. The affinity of the ionic modified
nanoparticles to BSA was evaluated. Protein attached with Fe2 has higher
thermal stability than free BSA. Fe2 exhibits low cytotoxicity to
HepG-2 cells.
a r t i c l e
i n f o
Article history: Received 2 July 2013 Received in revised form 29 September 2013 Accepted 2 October 2013 Available online 16 October 2013 Keywords: Ionic liquid Magnetic nanoparticle BSA Interaction
a b s t r a c t The interaction between ionic liquids modified magnetic Fe3O4 (Fe2) and bovine serum albumin (BSA) is reported and is compared with ANH2 functionalized magnetic nanoparticles Fe3O4 (Fe1) based on the UV–visible spectrum, steady-state fluorescence measurements, synchronous fluorescence and DSC methods. The results indicate a static quenching mechanism operating in both nanoparticles. The binding constant of the Fe2-BSA complex calculated from fluorescence data shows that BSA has a low binding affinity for Fe2 than Fe1. DSC data reveal that the thermal stability process of BSA in the Fe2-BSA complex is semi-reversible. This demonstrates that the ionic liquid modified magnetic nanoparticles (Fe2) enhance the thermostability of BSA in the range of 20–40 °C, and protein attached Fe2 has higher thermal stability than free BSA. Moreover, the in vitro assay results show that Fe2 shows low cytotoxicity to HepG-2 cells. Ó 2013 Elsevier B.V. All rights reserved.
Introduction Magnetic Fe3O4 particles are versatile for use in biomedical and biotechnological applications, e.g. as drug carriers, for MR imaging agents or protein purification, as adsorbents or biosensors [1,2]. Surface modified magnetic nanoparticles are promising alternatives for active drug targeting since they can be concentrated and held in position or controlled tumor-specific delivery of nanoparticles by an external field [3,4]. Ideally, nanoparticles as drug ⇑ Corresponding author. Tel.: +86 0511 8879800; fax: +86 0511 88791602. E-mail address: [email protected] (Q.-Y. Chen). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.10.005
delivery or diagnostic agents must bind specifically to their targets to distinguish normal cells from diseased cells and to avoid systemic toxicity. When magnetic Fe3O4 particles come in the organism, non-specific interactions between particles and biological macromolecules will take place at once. The bio-safety of magnetic Fe3O4 particles receives immediate attention. The absorption, distribution, metabolism, excretion properties, stability and toxicity of the nanoparticles can be significantly affected by binding with proteins. The interactions between drugs and serum albumins are important to the evaluation of nanoparticles’ toxicity [5,6]. Since cell membranes are weakly negatively charged, ionic liquids modified nanoparticles are expected to easily anchor to cell
162
J.-J. Xue, Q.-Y. Chen / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 161–166
membranes through electrostatic interactions and are internalized into cells through a charge-mediated endocytosis process. Ionic liquids are lipophilic cations which can pass through phospholipid bilayers and accumulate in negatively-charged compartments such as the mitochondrial matrix, driven by the membrane potential. The purpose of ionic liquids modified nanoparticles is to induce mitochondrial membrane potential permeability and improve the solubility of nanoparticles in water. Recently, we find that the ionic modified nanoparticles can target mitochondria [7,8]. Kamran et al. found that 1-hexyl-3-methylimidazolium bromide modified Fe3O4 nanoparticles could be used as nano-adsorbents for the adsorption of proteins from aqueous solutions [9]. However, the toxicity and the interactions of ionic modified magnetic Fe3O4 particles to BSA have not been reported. Previously, we report that 4-acetyl-N-propenylpyridinium chloride is low cytotoxicity to HepG-2 cells. Notably, the acetyl group of 4-acetyl-N-propenylpyridinium chloride can be linked with the amine functionalized Fe3O4 by condensation reaction under mild conditions. Here, (4-acetyl-N-propenylpyridinium chloride) modified magnetic Fe3O4 (Fe2) (Scheme S1) were prepared, the character of ionic liquid modified magnetic Fe3O4 (Fe2) was studied and was compared with mesoporous magnetic nanoparticles Fe3O4 (Fe1) by the UV–visible spectroscopy technique, fluorescence measurement and thermal analysis methods. Experimental Materials Commercially prepared BSA (purity >99.0%) was obtained from Sinopharm Chemical Reagent Co., Ltd. and stored at 4.0 °C in refrigerator. 4-Acetyl-N-propenylpyridinium chloride and amine functionalized magnetic nanoparticles (Fe1) were synthesized as reported [7,10]. The Tris, HCl and NaCl were all of analytical reagent grade, and obtained from Sinopharm Chemical Reagent Co., Ltd. and double distilled water was used for all solution preparation. Reagent preparation The 4-acetyl-N-propenylpyridinium chloride (IL) modified magnetic nanoparticle (Fe2) was prepared as followed: The amine functionalized magnetic nanoparticle (Fe1) (0.120 g) was dispersed in ethanol (10 mL) and mixed with 4-acetyl-N-propenylpyridinium chloride (IL) (0.045 g), and then the mixture was allowed to reflux for 4 h at 70 °C. The resulting precipitate (Fe2) was collected by centrifugation, washed with water to remove the unreacted IL, and dried under vacuum. The content of Fe in nanoparticles is 3.2 103 mol g1. BSA and relevant Fe3O4 nanoparticles stock solutions were prepared in Tris–HCl–NaCl buffer solution ([Tris–HCl] = 5 mM; [NaCl] = 50 mM, pH = 7.10). The concentration of the obtained BSA solution was 35 lM, and the content of iron in suspensions of Fe3O4 nanoparticles was 9.40 lM and 4.70 lM for Fe1 and Fe2, respectively. To a 10.00 mL volumetric flask, the 0.50 mL BSA stock solution and required volume of magnetic nanoparticles stock solution were added and diluted to the mark with Tris–HCl–NaCl buffer solution. The final concentration of BSA was 1.75 lM. Apparatus and instruments The fluorescence measurements were performed on a fluorophotometer (Cary 300, Varian Company, USA) and the UV–visible absorption spectra were recorded with an UV–visible spectrophotometer (Shimadzu Corporation). The pH value of solutions was measured with a pH meter (PHS-3C, Shanghai Lida Instrument
Ltd., China). The content of iron in nanoparticles (Fe1 and Fe2) was measured by atomic absorption spectrum (TAS-986, Beijing Tongyong Instrument Ltd., China). TEM was performed at room temperature on a JEOL JEM-200CX transmission electron microscope using an accelerating voltage of 200 kV. FT-IR characterizations were performed using a Nicolet Nexus 470 FT-IR spectrophotometer in the range of 4000–400 cm1. The absorption spectra were recorded on UV–visible spectrophotometer using 10 mm path length quartz cuvettes in the range of 190–350 nm, while fluorescence measurements were taken on a Carry eclipse spectrofluorometer using 10 mm path length quartz cuvettes with slit width of 5 nm. The excitation wavelength of BSA studied in this work was 280 nm. Synchronous fluorescence spectra were acquired by the same spectrofluorometer. The difference between excitation wavelength and emission wavelength was kept constant (Dk = kem kex) [11]. The protein samples were incubated at 288 K, 295 K, 303 K or 310 K for 2 h before measurements. The DSC measurement experiments were performed in a Germany Netzsch calorimeter, model DSC-204F1, using 160 ll medium pressure crucibles with 120 ll of sample in sample cell and the corresponding buffer in reference cell for each particular condition; the instrument was calibrated with indium. Scanning calorimetry measurements were performed with the Phoenix evaluation program, at a heating rate of 2.5 K min1 in the range of 20–40 °C. After the end of heating round, the protein sample was quickly cooled to 20 °C, and rescanned after 5 min stabilization at 20 °C. The sample was heated at a low heating rate (
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
The interaction between ionic liquids modified magnetic nanoparticles and bovine serum albumin and the cytotoxicity to HepG-2 cells Juan-Juan Xue, Qiu-Yun Chen ⇑ School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The interaction of two magnetic
nanoparticles to BSA was studied. The affinity of the ionic modified
nanoparticles to BSA was evaluated. Protein attached with Fe2 has higher
thermal stability than free BSA. Fe2 exhibits low cytotoxicity to
HepG-2 cells.
a r t i c l e
i n f o
Article history: Received 2 July 2013 Received in revised form 29 September 2013 Accepted 2 October 2013 Available online 16 October 2013 Keywords: Ionic liquid Magnetic nanoparticle BSA Interaction
a b s t r a c t The interaction between ionic liquids modified magnetic Fe3O4 (Fe2) and bovine serum albumin (BSA) is reported and is compared with ANH2 functionalized magnetic nanoparticles Fe3O4 (Fe1) based on the UV–visible spectrum, steady-state fluorescence measurements, synchronous fluorescence and DSC methods. The results indicate a static quenching mechanism operating in both nanoparticles. The binding constant of the Fe2-BSA complex calculated from fluorescence data shows that BSA has a low binding affinity for Fe2 than Fe1. DSC data reveal that the thermal stability process of BSA in the Fe2-BSA complex is semi-reversible. This demonstrates that the ionic liquid modified magnetic nanoparticles (Fe2) enhance the thermostability of BSA in the range of 20–40 °C, and protein attached Fe2 has higher thermal stability than free BSA. Moreover, the in vitro assay results show that Fe2 shows low cytotoxicity to HepG-2 cells. Ó 2013 Elsevier B.V. All rights reserved.
Introduction Magnetic Fe3O4 particles are versatile for use in biomedical and biotechnological applications, e.g. as drug carriers, for MR imaging agents or protein purification, as adsorbents or biosensors [1,2]. Surface modified magnetic nanoparticles are promising alternatives for active drug targeting since they can be concentrated and held in position or controlled tumor-specific delivery of nanoparticles by an external field [3,4]. Ideally, nanoparticles as drug ⇑ Corresponding author. Tel.: +86 0511 8879800; fax: +86 0511 88791602. E-mail address: [email protected] (Q.-Y. Chen). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.10.005
delivery or diagnostic agents must bind specifically to their targets to distinguish normal cells from diseased cells and to avoid systemic toxicity. When magnetic Fe3O4 particles come in the organism, non-specific interactions between particles and biological macromolecules will take place at once. The bio-safety of magnetic Fe3O4 particles receives immediate attention. The absorption, distribution, metabolism, excretion properties, stability and toxicity of the nanoparticles can be significantly affected by binding with proteins. The interactions between drugs and serum albumins are important to the evaluation of nanoparticles’ toxicity [5,6]. Since cell membranes are weakly negatively charged, ionic liquids modified nanoparticles are expected to easily anchor to cell
162
J.-J. Xue, Q.-Y. Chen / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 161–166
membranes through electrostatic interactions and are internalized into cells through a charge-mediated endocytosis process. Ionic liquids are lipophilic cations which can pass through phospholipid bilayers and accumulate in negatively-charged compartments such as the mitochondrial matrix, driven by the membrane potential. The purpose of ionic liquids modified nanoparticles is to induce mitochondrial membrane potential permeability and improve the solubility of nanoparticles in water. Recently, we find that the ionic modified nanoparticles can target mitochondria [7,8]. Kamran et al. found that 1-hexyl-3-methylimidazolium bromide modified Fe3O4 nanoparticles could be used as nano-adsorbents for the adsorption of proteins from aqueous solutions [9]. However, the toxicity and the interactions of ionic modified magnetic Fe3O4 particles to BSA have not been reported. Previously, we report that 4-acetyl-N-propenylpyridinium chloride is low cytotoxicity to HepG-2 cells. Notably, the acetyl group of 4-acetyl-N-propenylpyridinium chloride can be linked with the amine functionalized Fe3O4 by condensation reaction under mild conditions. Here, (4-acetyl-N-propenylpyridinium chloride) modified magnetic Fe3O4 (Fe2) (Scheme S1) were prepared, the character of ionic liquid modified magnetic Fe3O4 (Fe2) was studied and was compared with mesoporous magnetic nanoparticles Fe3O4 (Fe1) by the UV–visible spectroscopy technique, fluorescence measurement and thermal analysis methods. Experimental Materials Commercially prepared BSA (purity >99.0%) was obtained from Sinopharm Chemical Reagent Co., Ltd. and stored at 4.0 °C in refrigerator. 4-Acetyl-N-propenylpyridinium chloride and amine functionalized magnetic nanoparticles (Fe1) were synthesized as reported [7,10]. The Tris, HCl and NaCl were all of analytical reagent grade, and obtained from Sinopharm Chemical Reagent Co., Ltd. and double distilled water was used for all solution preparation. Reagent preparation The 4-acetyl-N-propenylpyridinium chloride (IL) modified magnetic nanoparticle (Fe2) was prepared as followed: The amine functionalized magnetic nanoparticle (Fe1) (0.120 g) was dispersed in ethanol (10 mL) and mixed with 4-acetyl-N-propenylpyridinium chloride (IL) (0.045 g), and then the mixture was allowed to reflux for 4 h at 70 °C. The resulting precipitate (Fe2) was collected by centrifugation, washed with water to remove the unreacted IL, and dried under vacuum. The content of Fe in nanoparticles is 3.2 103 mol g1. BSA and relevant Fe3O4 nanoparticles stock solutions were prepared in Tris–HCl–NaCl buffer solution ([Tris–HCl] = 5 mM; [NaCl] = 50 mM, pH = 7.10). The concentration of the obtained BSA solution was 35 lM, and the content of iron in suspensions of Fe3O4 nanoparticles was 9.40 lM and 4.70 lM for Fe1 and Fe2, respectively. To a 10.00 mL volumetric flask, the 0.50 mL BSA stock solution and required volume of magnetic nanoparticles stock solution were added and diluted to the mark with Tris–HCl–NaCl buffer solution. The final concentration of BSA was 1.75 lM. Apparatus and instruments The fluorescence measurements were performed on a fluorophotometer (Cary 300, Varian Company, USA) and the UV–visible absorption spectra were recorded with an UV–visible spectrophotometer (Shimadzu Corporation). The pH value of solutions was measured with a pH meter (PHS-3C, Shanghai Lida Instrument
Ltd., China). The content of iron in nanoparticles (Fe1 and Fe2) was measured by atomic absorption spectrum (TAS-986, Beijing Tongyong Instrument Ltd., China). TEM was performed at room temperature on a JEOL JEM-200CX transmission electron microscope using an accelerating voltage of 200 kV. FT-IR characterizations were performed using a Nicolet Nexus 470 FT-IR spectrophotometer in the range of 4000–400 cm1. The absorption spectra were recorded on UV–visible spectrophotometer using 10 mm path length quartz cuvettes in the range of 190–350 nm, while fluorescence measurements were taken on a Carry eclipse spectrofluorometer using 10 mm path length quartz cuvettes with slit width of 5 nm. The excitation wavelength of BSA studied in this work was 280 nm. Synchronous fluorescence spectra were acquired by the same spectrofluorometer. The difference between excitation wavelength and emission wavelength was kept constant (Dk = kem kex) [11]. The protein samples were incubated at 288 K, 295 K, 303 K or 310 K for 2 h before measurements. The DSC measurement experiments were performed in a Germany Netzsch calorimeter, model DSC-204F1, using 160 ll medium pressure crucibles with 120 ll of sample in sample cell and the corresponding buffer in reference cell for each particular condition; the instrument was calibrated with indium. Scanning calorimetry measurements were performed with the Phoenix evaluation program, at a heating rate of 2.5 K min1 in the range of 20–40 °C. After the end of heating round, the protein sample was quickly cooled to 20 °C, and rescanned after 5 min stabilization at 20 °C. The sample was heated at a low heating rate (
