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Preparation of Tetraamino-phthalocyanine-Zinc-Loaded Silica Nanoparticles and Study of Their Cytotoxicity
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 39, Issue 10, October 2011 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2011, 39(10), 1567–1571.
RESEARCH PAPER
Preparation of Tetraamino-phthalocyanine-Zinc-Loaded Silica Nanoparticles and Study of Their Cytotoxicity ZHUANG Qian-Fen1,2, WANG Jin-E1, ZHU Zhi-Jun1, LI Feng2,*, WANG Zhen-Xin1 1
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China 2 College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, China
Abstract: Positively charged silica nanoparticles, entrapping the hydrophobic photosensitizer 2,9,16,23-tetraamino-phthalocyanine zinc (ZnPc(NH2)4), have been synthesized in the nonpolar core of micelles by hydrolysis of triethoxyvinylsilane and 3-aminopropyltriethoxysilane. The surface morphologies, charge state, solublility in water and stability have been characterized using transmission electron microscopy, dynamic light scattering technique, and ultraviolet-visible absorption spectra. The as-prepared nanoparticles are highly monodispersed spheres with uniform diameter (about 20 nm), which are stable in an aqueous system. Their average ȗ-potential value is (28.8 ± 2.79) mV, and they show strong absorption peaks at 714 nm. In addition, encapsulation of ZnPc(NH2)4 in silica nanoparticles prevents leakage of ZnPc(NH2)4 and enhances the resistance to photobleaching. The ZnPc(NH2)4-entrapped-silica nanoparticles (SiO2@ZnPc(NH2)4) can efficiently generate singlet oxygen, as measured using the chemical probe 1,3-diphenylisobenzofuran. The cytotoxicity of silica nanoparticles has been investigated by incubation of both the ZnPc(NH2)4-entrapped-silica nanoparticles and the nanoparticles without ZnPc(NH2)4 (SiO2-NH2) but containing living cancer cells (HeLa, U251 and PC-12). The experimental result shows that neither SiO2@ZnPc(NH2)4 nor SiO2-NH2 particles have significant cytotoxicity when the concentration of the particles is lower than 300 mg L–1. Key Words: Silica nanoparticles; Tetraamino-phthalocyanine-zinc; Microemulsion; Cytotoxicity
1
Introduction
In recent years, photodynamic therapy (PDT) has received increased attention as a novel treatment for cancer and degenerative macular diseases[1,2]. PDT is a light-activated treatment applied for cancer and other diseases, operated by using light-sensitive species or photosensitizers that can be preferentially localized in tumor tissues. On irradiation with visible light or near-infrared (NIR) light, these photosensitizers in the cells become excited; the excited photosensitizers transfer their excess energy to the surrounding molecular oxygen species to generate cytotoxic reactive oxygen species (ROSs), such as singlet oxygen (1O2). Subsequently, the ROSs can oxidize various cellular
macromolecules, such as lipids, nucleic acids, amino acids, and so on, resulting in the irreversible damage of the tumor cells[3]. Because photosensitizers are nontoxic without exposure to light, adjacent healthy tissues cannot be damaged unless they are exposed to light, and only the pathogenic tissues in the irradiated areas will be affected. The therapy exploits this principle of PDT to destroy pathogenic tissues in a selective and effective fashion, without damaging adjacent healthy tissues. Therefore, the overall efficacy of PDT depends greatly on the properties of the photosensitizers. Due to their strong absorption in the visible and NIR regions, low toxicity, and high efficiency of generating singlet oxygen, phthalocyanines have been considered to be a promising class of compounds for the development of second-generation
Received 2 March 2011; accepted 18 May 2011 * Corresponding author. Email: [email protected] This work was supported by the National Special Project of China (No. 21075178). Copyright © 2011, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(10)60476-8
ZHUANG Qin-Fen et al. / Chinese Journal of Analytical Chemistry, 2011, 39(10): 1567–1571
photosensitizers for PDT[4,5]. However, most of the photosensitizers, such as 2,9,16,23-tetraamino-phthalocyanine zinc (ZnPc(NH2)4) and silicon phthalocyanine-4 are hydrophobic, and almost insoluble, in addition to easily aggregating in an aqueous system under physiological conditions. These properties of phthalocyanine dramatically reduce its photodynamic activity and limit its potential clinical applications[6]. Recently, silica nanoparticles have been widely used as a carrier vehicle for delivery of drugs and genes due to their unique advantages, such as small yet uniform pore size, large specific surface area and pore volume, and good water solubility and biocompatibility[7]. However, the toxicity of silica nanoparticles to living cells or organisms has not yet been clearly understood[8]. The cytotoxicity of silica nanoparticles is related to the concentration, surface ligands, and the size of nanoparticles[9]. Compared with large-sized silica nanoparticles, small-sized nanoparticles easily circulate in vivo and infiltrate tumor and other tissues, thus being more suitable for in vivo applications[10]. In this work, positively charged 20-nm diameter silica nanoparticles, entrapping the hydrophobic photosensitizer ZnPc(NH2)4, have been synthesized using a water-in-oil microemulsion method by the hydrolysis of triethoxyvinylsilane (TEVS) and 3-aminopropyltriethoxysilane (APTES). In addition, we also synthesized 20-nm silica nanoparticles without ZnPc(NH2)4 (SiO2-NH2). The silica nanoparticles were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) technique, and ultraviolet-visible (UV-vis) absorption spectra. The stability of the ZnPc(NH2)4-entrappedsilica nanoparticles (SiO2@ZnPc(NH2)4) and their ability to generate singlet oxygen were also evaluated. The cytotoxicity of SiO2@ZnPc(NH2)4 and SiO2-NH2 nanoparticles was investigated by incubation of the nanoparticles with three types of living cancer cells (HeLa, U251 and C-12). The study aimed to obtain more information on the cytotoxicity of silica nanoparticles, which will provide a versatile platform for the future application of silica nanoparticles in the field of biochemistry.
2
TEVS (97%) and 1,3-diphenylisobenzofuran (DPBF) were obtained from Sigma-Aldrich Co. (USA). APTES (purity t 98%) was purchased from Fluka Co. (USA). 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich Co. (USA). 2,9,16,23tetraamino-phthalocyanine zinc (ZnPc(NH2)4) was obtained from Heilongjiang University, China. Heavy water (D2O) was purchased by Cambridge Isotope Lab. (UK). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco Co. (USA). Tween-80, n-butanol ( 99%), ammonia solution (25%), N,N-dimethylformamide (DMF), and other reagents were of analytical grade. Ultrapure water (18.2 Mȍ cm) from a Milli-Q system (Millipore Co., USA) was used for all experiments. 2.2 2.2.1
Preparation of SiO2@ZnPc(NH2)4 nanoparticles
The ZnPc(NH2)4-loaded nanoparticles (SiO2@ZnPc(NH2)4) were synthesized according to the procedures described in previous reports[6,10] with some modifications. Briefly, 600 ȝL of the cosurfactant n-butanol was added to 20 mL of 2% aqueous solution of Tween-80. Then, 8 ȝL of a solution of ZnPc(NH2)4 in DMF (10 mM) was added with vigorous magnetic stirring. After 20 min, 200 ȝL of TEVS were added to the system, and the resulting mixture was stirred for approximately 1 h. Then, 20 ȝL of ammonia solution were added to this mixture. After stirring for 30 min, APTES (30 ȝL) was added; the system was stirred for another 24 h in the dark. After the formation of the nanoparticles, the mixture was filtered through a 0.45-ȝm-cutoff membrane filter to remove the bigger aggregates. Next, the resulting nanoparticles were centrifuged at 13000 revolutions per minute for 30 min and then washed three times with ultrapure water to remove Tween-80, n-butanol, and unreacted molecules. The samples were dispersed in ultrapure water and stored at 4 °C for further use. The silica nanoparticles without ZnPc(NH2)4 (SiO2-NH2) were also prepared as a control.
Experimental 2.2.2
2.1
Experimental methods
Apparatus and reagents
A Hitachi H-600 transmission electron microscope (Hitachi, Japan) was used for morphology characterization. DLS measurements of the particle size and the ȗ potential were carried out using a light-scattering Zetasizer Nano-ZS lightscattering instrument (Malvern Instruments, Enigma Business Park, UK). Absorption spectra were measured with the UVmini-1240 (Shimadzu, Japan) and the Power WaveTM XS2 microplate spectrophotometers (BioTek Instruments, Inc., USA).
Photostability measurement of SiO2@ZnPc(NH2)4 nanoparticles
According to literature[6] the concentration of ZnPc(NH2)4 in SiO2@ZnPc(NH2)4 was estimated by the Lambert-Beer law. Thus, 5 ȝM solutions of SiO2@ZnPc(NH2)4 and ZnPc(NH2)4 were prepared in DMF. A 20-W iodine-tungsten lamp was used as the light source. On irradiation of the two solutions, the absorbance at 714 nm was measured at 2-min intervals between 0 and 36 min after irradiation. 2.2.3
Detection of singlet oxygen by a chemical method
ZHUANG Qin-Fen et al. / Chinese Journal of Analytical Chemistry, 2011, 39(10): 1567–1571
We used DPBF as a probe to evaluate the ability of SiO2@ZnPc(NH2)4 to release singlet oxygen[7,11]. DPBF is bleached by singlet oxygen to produce its corresponding endoperoxide. The reaction can be monitored by recording the decrease in absorption at 400 nm by UV-vis absorption spectrophotometry. Generally, 16.7 ȝL of a stock solution of DPBF (8 mM) was added to 1 mL of a 5-ȝM solution of SiO2@ZnPc(NH2)4 in D2O. Then, the solutions were irradiated with a 633.5-nm light, and the adsorptions at 400 nm were collected every minute, using a solution of nanoparticles in heavy water as a blank. In addition, SiO2-NH2 was used instead of SiO2@ZnPc(NH2)4 in a control experiment.
150 ȝL of dimethyl sulfoxide were added to solubilize the crystals. The absorbance at 555 nm in each well was collected on a Power WaveTM XS2 microplate spectrophotometer at room temperature. The results were plotted as 100% cell survival and compared with the corresponding results of the control experiments (cells not incubated with nanoparticles). Each experiment was repeated three times to ensure data reproducibility.
3
Results and discussion
3.1 3.1.1
2.2.4
Cell culture and tests for cytotoxicity and cell viability
HeLa (human cervical cancer cells), U-251 (human astrocytoma cells), and highly differentiated PC-12 (rat pheochromocytoma cells) cells were used in these studies. Cells were cultured in DMEM medium supplemented with 10% FBS and incubated at 37 °C in humidified air with 5% CO2. Before treatment with nanoparticles, the cells were seeded in 96-well plates and incubated overnight. The density of cells in the medium was 12000 cells per well. In the cytotoxicity test, different concentrations of nanoparticles (20, 50, 100 and 300 mg L–1) were suspended in DMEM and were ultrasonicated for 30 s to prevent agglomeration. After overnight incubation, the medium with 10% FBS was removed and replaced by 100 ȝL of the nanoparticle suspension at different concentrations. Control experiments were conducted by treating an equivalent volume of DMEM without any nanoparticles. After 4 h, 10 ȝL of serum were added into each well. Cells were incubated for 48 h in the dark at 37 °C with the test nanoparticles and washed three times with DMEM before testing for cell viability. Cell cytotoxicity was assessed using the colorimetric MTT assay[12]. Generally, the original medium containing 10% FBS present in the microplate well was removed; then, 10 ȝL of a 5.0-g L–1 solution of MTT in phosphate-buffered saline and 90 ȝL of fresh medium containing 10% FBS were added to each well. After incubation for 4 h, the medium was removed, and
Characterization of nanoparticles Size and shape of nanoparticles
We synthesized ZnPc(NH2)4-loaded silica nanoparticles (SiO2@ZnPc(NH2)4) by hydrolysis of TEVS and APTES in a micelle system using Tween-80 as the surfactant [6,10,13]. Silica nanoparticles without ZnPc(NH2)4 (SiO2-NH2) were also prepared. The morphology and size of the as-prepared silica nanoparticles were characterized using TEM. The TEM images (Fig.1) clearly showed that they appeared to be uniform and monodispersed spheres. Based on the measurement of 200 individual particles, the obtained average diameters of SiO2-NH2 and SiO2@ZnPc(NH2)4 nanoparticles were (18.51 ± 3.31) nm and (17.05 ± 2.50) nm, respectively. 3.1.2
Measurement of surface charge on nanoparticles
The zeta-potential values of the nanoparticles were characterized by DLS. The average zeta-potential values of SiO2-NH2 and SiO2@ZnPc(NH2)4 were (26.5 ± 1.90) mV and (28.8 ± 2.79) mV, respectively. These potential values were different from the values reported previously (5.35 mV for 20-nm to 25-nm SiO2-NH2 nanoparticles)[10]. This may be because the addition of excess APTES led to the presence of more amine functional groups on the nanoparticle surfaces. The high-density amine groups resulted in more number of positive charges on the surface under the present experimental conditions. In this work, the excess positive charges on the nanoparticle surfaces facilitated the adsorption of silica nanoparticles onto the negatively charged cell membranes.
Fig.1 TEM images of (a) SiO2-NH2 and (b) SiO2@ZnPc(NH2)4
ZHUANG Qin-Fen et al. / Chinese Journal of Analytical Chemistry, 2011, 39(10): 1567–1571
3.1.3
Spectroscopic characterization of nanoparticles
Figure 2 shows the UV-vis absorption spectra of SiO2@ZnPc(NH2)4, SiO2-NH2, and ZnPc(NH2)4 in 0.025% DMF aqueous solution. There were large differences in the UV-vis absorption spectra of SiO2@ZnPc(NH2)4 and ZnPc(NH2)4 at the same concentration. The spectrum of ZnPc(NH2)4 had weak absorption in the wavelength range studied, with two peaks at 350 and 714 nm, whereas that of SiO2@ZnPc(NH2)4 had strong absorption bands peaking at 350 and 714 nm. All these observations suggested that after ZnPc(NH2)4 was loaded in the silica nanoparticles, the solubility of ZnPc(NH2)4 in water was greatly increased. 3.2 3.2.1
Stability of nanoparticles Leakage test of nanoparticles
ZnPc(NH2)4 is hydrophobic and has low solubility in water, but when it is loaded onto silica nanoparticles, the SiO2@ZnPc(NH2)4 particles can form a stable aqueous dispersion and enter the cell easily in a site-specific manner. However, dyes have often been reported to leak from silica nanoparticles entrapping dyes[14]. Thus, we also examined the leakage of ZnPc(NH2)4 from SiO2@ZnPc(NH2)4. We recorded the UV-vis absorption spectra of the as-prepared SiO2@ZnPc(NH2)4 after storage for one month. The spectra were recorded before and after washing the nanoparticles thrice. After washing, the absorption intensity of SiO2@ZnPc(NH2)4 decreased by only less than 10%, suggesting that almost no leakage occurred in the SiO2@ZnPc(NH2)4. Thus, the as-prepared SiO2@ZnPc(NH2)4 had excellent stability. 3.2.2
Fig.2 Ultraviolet-visible absorption spectra of (a) SiO2@ZnPc(NH2)4, (b) ZnPc(NH2)4, and (c) SiO2-NH2
Photostability measurements of nanoparticles
Photosensitizers used in PDT have been proposed to undergo photobleaching, resulting in decrease of UV-vis absorbance and fluorescence[15]. Therefore, we examined the photobleaching of ZnPc(NH2)4 entrapped in silica nanoparticles. Figure 3 showed that after irradiation for 36 min with an iodine-tungsten lamp, the UV-vis absorbance of SiO2@ZnPc(NH2)4 was reduced by less than 9.5%, whereas the absorbance of ZnPc(NH2)4 decreased by more than 68%. This indicated that the silica nanoparticles enhanced the photostability of ZnPc(NH2)4, and shielded ZnPc(NH2)4 against the photobleaching process. 3.3
probe. Figure 4 shows that after irradiation of SiO2@ZnPc(NH2)4with a 633.5-nm light, a sharp decrease in the absorbance at 400 nm was observed, suggesting the generation of 1O2 with a high efficiency and the release of 1O2 from the nanoparticles. In comparison, there was no obvious decrease in the absorbance at 400 nm in the case of the bare silica nanoparticles. All these results confirmed that the bleaching of DPBF in the presence of ZnPc(NH2)4 was mainly caused by the singlet oxygen generated.
Fig.3 Photostability curves of SiO2@ZnPc(NH2)4 and ZnPc(NH2)4
Detection of singlet oxygen
Singlet oxygen (1O2) is a highly reactive species of oxygen and plays a key role in the process of PDT. To verify the generation of singlet oxygen, we used DPBF as the chemical
Fig.4 Photobleaching curves of DPBF
ZHUANG Qin-Fen et al. / Chinese Journal of Analytical Chemistry, 2011, 39(10): 1567–1571
3.4
Cell culture and phototoxicity study
Ideal photosensitizers not only have a high efficiency of generation of 1O2 after light exposure, but also show no or low toxicity in the absence of light irradiation. Therefore, we examined the toxicity of silica nanoparticles toward cells by incubation of the nanoparticles with three types of living cancer cells (HeLa, U251 and PC-12) without light irradiation. Cells were incubated for 48 h in the dark, and the phototoxicity was studied by the MTT assay. The experimental results in Fig.5 showed that bare SiO2-NH2 nanoparticles at a concentration of 300 mg L–1 had no significant toxicity toward these cells. Furthermore, SiO2@ZnPc(NH2)4 nanoparticles had low toxicity to HeLa and U251 cells with cell viability less than 90% and had almost no toxicity for PC-12. Moreover, experimental results also showed that both SiO2@ZnPc(NH2)4 and SiO2-NH2 particles at concentrations lower than 300 mg L–1 had almost no cytotoxicity, indicating that SiO2@ZnPc(NH2)4 had a great potential for applications in PDT of cancer.
irradiation has been investigated by the incubation of SiO2-NH2 and SiO2@ZnPc(NH2)4 nanoparticles with three types of living cancer cells (HeLa, U251 and PC-12). The experimental results showed that neither SiO2@ZnPc(NH2)4 nor SiO2-NH2 nanoparticles at concentrations lower than 300 mg L–1 had significant cytotoxicity. All these results provide some basis for the future application of silica nanoparticles in drug-and-gene delivery, in addition to paving a way for the future potential applications of these nanoparticles in PDT.
References [1]
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4
Conclusions
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SiO 2 @ZnPc(NH 2 ) 4 and SiO 2 -NH 2 nanoparticles were prepared by the reversible microemulsion method. The as-prepared nanoparticles were uniform and monodispersed spheres, with excellent solubility in water, biocompatibility, and stability. On irradiation with light, SiO2@ZnPc(NH2)4 nanoparticles can efficiently generate singlet oxygen. The toxicity of silica nanoparticles to cells in the absence of light
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Kumar R, Roy I, Ohulchanskyy T Y, Goswami L N, Bonoiu A C, Bergey E J, Tramposch K M, Maitra A, Prasad P N. ACS Nano., 2008, 2(3): 449–456
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[14]
Duan J H, Wang K M, He X X, Tan W H. J. Hunan Univ. (Nature Sciences), 2003, 30(2): 1–5
[15] Fig.5 Cell-viability assay with different silica nanoparticles
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Cite this article as: Chin J Anal Chem, 2011, 39(10), 1567–1571.
RESEARCH PAPER
Preparation of Tetraamino-phthalocyanine-Zinc-Loaded Silica Nanoparticles and Study of Their Cytotoxicity ZHUANG Qian-Fen1,2, WANG Jin-E1, ZHU Zhi-Jun1, LI Feng2,*, WANG Zhen-Xin1 1
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China 2 College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, China
Abstract: Positively charged silica nanoparticles, entrapping the hydrophobic photosensitizer 2,9,16,23-tetraamino-phthalocyanine zinc (ZnPc(NH2)4), have been synthesized in the nonpolar core of micelles by hydrolysis of triethoxyvinylsilane and 3-aminopropyltriethoxysilane. The surface morphologies, charge state, solublility in water and stability have been characterized using transmission electron microscopy, dynamic light scattering technique, and ultraviolet-visible absorption spectra. The as-prepared nanoparticles are highly monodispersed spheres with uniform diameter (about 20 nm), which are stable in an aqueous system. Their average ȗ-potential value is (28.8 ± 2.79) mV, and they show strong absorption peaks at 714 nm. In addition, encapsulation of ZnPc(NH2)4 in silica nanoparticles prevents leakage of ZnPc(NH2)4 and enhances the resistance to photobleaching. The ZnPc(NH2)4-entrapped-silica nanoparticles (SiO2@ZnPc(NH2)4) can efficiently generate singlet oxygen, as measured using the chemical probe 1,3-diphenylisobenzofuran. The cytotoxicity of silica nanoparticles has been investigated by incubation of both the ZnPc(NH2)4-entrapped-silica nanoparticles and the nanoparticles without ZnPc(NH2)4 (SiO2-NH2) but containing living cancer cells (HeLa, U251 and PC-12). The experimental result shows that neither SiO2@ZnPc(NH2)4 nor SiO2-NH2 particles have significant cytotoxicity when the concentration of the particles is lower than 300 mg L–1. Key Words: Silica nanoparticles; Tetraamino-phthalocyanine-zinc; Microemulsion; Cytotoxicity
1
Introduction
In recent years, photodynamic therapy (PDT) has received increased attention as a novel treatment for cancer and degenerative macular diseases[1,2]. PDT is a light-activated treatment applied for cancer and other diseases, operated by using light-sensitive species or photosensitizers that can be preferentially localized in tumor tissues. On irradiation with visible light or near-infrared (NIR) light, these photosensitizers in the cells become excited; the excited photosensitizers transfer their excess energy to the surrounding molecular oxygen species to generate cytotoxic reactive oxygen species (ROSs), such as singlet oxygen (1O2). Subsequently, the ROSs can oxidize various cellular
macromolecules, such as lipids, nucleic acids, amino acids, and so on, resulting in the irreversible damage of the tumor cells[3]. Because photosensitizers are nontoxic without exposure to light, adjacent healthy tissues cannot be damaged unless they are exposed to light, and only the pathogenic tissues in the irradiated areas will be affected. The therapy exploits this principle of PDT to destroy pathogenic tissues in a selective and effective fashion, without damaging adjacent healthy tissues. Therefore, the overall efficacy of PDT depends greatly on the properties of the photosensitizers. Due to their strong absorption in the visible and NIR regions, low toxicity, and high efficiency of generating singlet oxygen, phthalocyanines have been considered to be a promising class of compounds for the development of second-generation
Received 2 March 2011; accepted 18 May 2011 * Corresponding author. Email: [email protected] This work was supported by the National Special Project of China (No. 21075178). Copyright © 2011, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(10)60476-8
ZHUANG Qin-Fen et al. / Chinese Journal of Analytical Chemistry, 2011, 39(10): 1567–1571
photosensitizers for PDT[4,5]. However, most of the photosensitizers, such as 2,9,16,23-tetraamino-phthalocyanine zinc (ZnPc(NH2)4) and silicon phthalocyanine-4 are hydrophobic, and almost insoluble, in addition to easily aggregating in an aqueous system under physiological conditions. These properties of phthalocyanine dramatically reduce its photodynamic activity and limit its potential clinical applications[6]. Recently, silica nanoparticles have been widely used as a carrier vehicle for delivery of drugs and genes due to their unique advantages, such as small yet uniform pore size, large specific surface area and pore volume, and good water solubility and biocompatibility[7]. However, the toxicity of silica nanoparticles to living cells or organisms has not yet been clearly understood[8]. The cytotoxicity of silica nanoparticles is related to the concentration, surface ligands, and the size of nanoparticles[9]. Compared with large-sized silica nanoparticles, small-sized nanoparticles easily circulate in vivo and infiltrate tumor and other tissues, thus being more suitable for in vivo applications[10]. In this work, positively charged 20-nm diameter silica nanoparticles, entrapping the hydrophobic photosensitizer ZnPc(NH2)4, have been synthesized using a water-in-oil microemulsion method by the hydrolysis of triethoxyvinylsilane (TEVS) and 3-aminopropyltriethoxysilane (APTES). In addition, we also synthesized 20-nm silica nanoparticles without ZnPc(NH2)4 (SiO2-NH2). The silica nanoparticles were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) technique, and ultraviolet-visible (UV-vis) absorption spectra. The stability of the ZnPc(NH2)4-entrappedsilica nanoparticles (SiO2@ZnPc(NH2)4) and their ability to generate singlet oxygen were also evaluated. The cytotoxicity of SiO2@ZnPc(NH2)4 and SiO2-NH2 nanoparticles was investigated by incubation of the nanoparticles with three types of living cancer cells (HeLa, U251 and C-12). The study aimed to obtain more information on the cytotoxicity of silica nanoparticles, which will provide a versatile platform for the future application of silica nanoparticles in the field of biochemistry.
2
TEVS (97%) and 1,3-diphenylisobenzofuran (DPBF) were obtained from Sigma-Aldrich Co. (USA). APTES (purity t 98%) was purchased from Fluka Co. (USA). 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich Co. (USA). 2,9,16,23tetraamino-phthalocyanine zinc (ZnPc(NH2)4) was obtained from Heilongjiang University, China. Heavy water (D2O) was purchased by Cambridge Isotope Lab. (UK). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco Co. (USA). Tween-80, n-butanol ( 99%), ammonia solution (25%), N,N-dimethylformamide (DMF), and other reagents were of analytical grade. Ultrapure water (18.2 Mȍ cm) from a Milli-Q system (Millipore Co., USA) was used for all experiments. 2.2 2.2.1
Preparation of SiO2@ZnPc(NH2)4 nanoparticles
The ZnPc(NH2)4-loaded nanoparticles (SiO2@ZnPc(NH2)4) were synthesized according to the procedures described in previous reports[6,10] with some modifications. Briefly, 600 ȝL of the cosurfactant n-butanol was added to 20 mL of 2% aqueous solution of Tween-80. Then, 8 ȝL of a solution of ZnPc(NH2)4 in DMF (10 mM) was added with vigorous magnetic stirring. After 20 min, 200 ȝL of TEVS were added to the system, and the resulting mixture was stirred for approximately 1 h. Then, 20 ȝL of ammonia solution were added to this mixture. After stirring for 30 min, APTES (30 ȝL) was added; the system was stirred for another 24 h in the dark. After the formation of the nanoparticles, the mixture was filtered through a 0.45-ȝm-cutoff membrane filter to remove the bigger aggregates. Next, the resulting nanoparticles were centrifuged at 13000 revolutions per minute for 30 min and then washed three times with ultrapure water to remove Tween-80, n-butanol, and unreacted molecules. The samples were dispersed in ultrapure water and stored at 4 °C for further use. The silica nanoparticles without ZnPc(NH2)4 (SiO2-NH2) were also prepared as a control.
Experimental 2.2.2
2.1
Experimental methods
Apparatus and reagents
A Hitachi H-600 transmission electron microscope (Hitachi, Japan) was used for morphology characterization. DLS measurements of the particle size and the ȗ potential were carried out using a light-scattering Zetasizer Nano-ZS lightscattering instrument (Malvern Instruments, Enigma Business Park, UK). Absorption spectra were measured with the UVmini-1240 (Shimadzu, Japan) and the Power WaveTM XS2 microplate spectrophotometers (BioTek Instruments, Inc., USA).
Photostability measurement of SiO2@ZnPc(NH2)4 nanoparticles
According to literature[6] the concentration of ZnPc(NH2)4 in SiO2@ZnPc(NH2)4 was estimated by the Lambert-Beer law. Thus, 5 ȝM solutions of SiO2@ZnPc(NH2)4 and ZnPc(NH2)4 were prepared in DMF. A 20-W iodine-tungsten lamp was used as the light source. On irradiation of the two solutions, the absorbance at 714 nm was measured at 2-min intervals between 0 and 36 min after irradiation. 2.2.3
Detection of singlet oxygen by a chemical method
ZHUANG Qin-Fen et al. / Chinese Journal of Analytical Chemistry, 2011, 39(10): 1567–1571
We used DPBF as a probe to evaluate the ability of SiO2@ZnPc(NH2)4 to release singlet oxygen[7,11]. DPBF is bleached by singlet oxygen to produce its corresponding endoperoxide. The reaction can be monitored by recording the decrease in absorption at 400 nm by UV-vis absorption spectrophotometry. Generally, 16.7 ȝL of a stock solution of DPBF (8 mM) was added to 1 mL of a 5-ȝM solution of SiO2@ZnPc(NH2)4 in D2O. Then, the solutions were irradiated with a 633.5-nm light, and the adsorptions at 400 nm were collected every minute, using a solution of nanoparticles in heavy water as a blank. In addition, SiO2-NH2 was used instead of SiO2@ZnPc(NH2)4 in a control experiment.
150 ȝL of dimethyl sulfoxide were added to solubilize the crystals. The absorbance at 555 nm in each well was collected on a Power WaveTM XS2 microplate spectrophotometer at room temperature. The results were plotted as 100% cell survival and compared with the corresponding results of the control experiments (cells not incubated with nanoparticles). Each experiment was repeated three times to ensure data reproducibility.
3
Results and discussion
3.1 3.1.1
2.2.4
Cell culture and tests for cytotoxicity and cell viability
HeLa (human cervical cancer cells), U-251 (human astrocytoma cells), and highly differentiated PC-12 (rat pheochromocytoma cells) cells were used in these studies. Cells were cultured in DMEM medium supplemented with 10% FBS and incubated at 37 °C in humidified air with 5% CO2. Before treatment with nanoparticles, the cells were seeded in 96-well plates and incubated overnight. The density of cells in the medium was 12000 cells per well. In the cytotoxicity test, different concentrations of nanoparticles (20, 50, 100 and 300 mg L–1) were suspended in DMEM and were ultrasonicated for 30 s to prevent agglomeration. After overnight incubation, the medium with 10% FBS was removed and replaced by 100 ȝL of the nanoparticle suspension at different concentrations. Control experiments were conducted by treating an equivalent volume of DMEM without any nanoparticles. After 4 h, 10 ȝL of serum were added into each well. Cells were incubated for 48 h in the dark at 37 °C with the test nanoparticles and washed three times with DMEM before testing for cell viability. Cell cytotoxicity was assessed using the colorimetric MTT assay[12]. Generally, the original medium containing 10% FBS present in the microplate well was removed; then, 10 ȝL of a 5.0-g L–1 solution of MTT in phosphate-buffered saline and 90 ȝL of fresh medium containing 10% FBS were added to each well. After incubation for 4 h, the medium was removed, and
Characterization of nanoparticles Size and shape of nanoparticles
We synthesized ZnPc(NH2)4-loaded silica nanoparticles (SiO2@ZnPc(NH2)4) by hydrolysis of TEVS and APTES in a micelle system using Tween-80 as the surfactant [6,10,13]. Silica nanoparticles without ZnPc(NH2)4 (SiO2-NH2) were also prepared. The morphology and size of the as-prepared silica nanoparticles were characterized using TEM. The TEM images (Fig.1) clearly showed that they appeared to be uniform and monodispersed spheres. Based on the measurement of 200 individual particles, the obtained average diameters of SiO2-NH2 and SiO2@ZnPc(NH2)4 nanoparticles were (18.51 ± 3.31) nm and (17.05 ± 2.50) nm, respectively. 3.1.2
Measurement of surface charge on nanoparticles
The zeta-potential values of the nanoparticles were characterized by DLS. The average zeta-potential values of SiO2-NH2 and SiO2@ZnPc(NH2)4 were (26.5 ± 1.90) mV and (28.8 ± 2.79) mV, respectively. These potential values were different from the values reported previously (5.35 mV for 20-nm to 25-nm SiO2-NH2 nanoparticles)[10]. This may be because the addition of excess APTES led to the presence of more amine functional groups on the nanoparticle surfaces. The high-density amine groups resulted in more number of positive charges on the surface under the present experimental conditions. In this work, the excess positive charges on the nanoparticle surfaces facilitated the adsorption of silica nanoparticles onto the negatively charged cell membranes.
Fig.1 TEM images of (a) SiO2-NH2 and (b) SiO2@ZnPc(NH2)4
ZHUANG Qin-Fen et al. / Chinese Journal of Analytical Chemistry, 2011, 39(10): 1567–1571
3.1.3
Spectroscopic characterization of nanoparticles
Figure 2 shows the UV-vis absorption spectra of SiO2@ZnPc(NH2)4, SiO2-NH2, and ZnPc(NH2)4 in 0.025% DMF aqueous solution. There were large differences in the UV-vis absorption spectra of SiO2@ZnPc(NH2)4 and ZnPc(NH2)4 at the same concentration. The spectrum of ZnPc(NH2)4 had weak absorption in the wavelength range studied, with two peaks at 350 and 714 nm, whereas that of SiO2@ZnPc(NH2)4 had strong absorption bands peaking at 350 and 714 nm. All these observations suggested that after ZnPc(NH2)4 was loaded in the silica nanoparticles, the solubility of ZnPc(NH2)4 in water was greatly increased. 3.2 3.2.1
Stability of nanoparticles Leakage test of nanoparticles
ZnPc(NH2)4 is hydrophobic and has low solubility in water, but when it is loaded onto silica nanoparticles, the SiO2@ZnPc(NH2)4 particles can form a stable aqueous dispersion and enter the cell easily in a site-specific manner. However, dyes have often been reported to leak from silica nanoparticles entrapping dyes[14]. Thus, we also examined the leakage of ZnPc(NH2)4 from SiO2@ZnPc(NH2)4. We recorded the UV-vis absorption spectra of the as-prepared SiO2@ZnPc(NH2)4 after storage for one month. The spectra were recorded before and after washing the nanoparticles thrice. After washing, the absorption intensity of SiO2@ZnPc(NH2)4 decreased by only less than 10%, suggesting that almost no leakage occurred in the SiO2@ZnPc(NH2)4. Thus, the as-prepared SiO2@ZnPc(NH2)4 had excellent stability. 3.2.2
Fig.2 Ultraviolet-visible absorption spectra of (a) SiO2@ZnPc(NH2)4, (b) ZnPc(NH2)4, and (c) SiO2-NH2
Photostability measurements of nanoparticles
Photosensitizers used in PDT have been proposed to undergo photobleaching, resulting in decrease of UV-vis absorbance and fluorescence[15]. Therefore, we examined the photobleaching of ZnPc(NH2)4 entrapped in silica nanoparticles. Figure 3 showed that after irradiation for 36 min with an iodine-tungsten lamp, the UV-vis absorbance of SiO2@ZnPc(NH2)4 was reduced by less than 9.5%, whereas the absorbance of ZnPc(NH2)4 decreased by more than 68%. This indicated that the silica nanoparticles enhanced the photostability of ZnPc(NH2)4, and shielded ZnPc(NH2)4 against the photobleaching process. 3.3
probe. Figure 4 shows that after irradiation of SiO2@ZnPc(NH2)4with a 633.5-nm light, a sharp decrease in the absorbance at 400 nm was observed, suggesting the generation of 1O2 with a high efficiency and the release of 1O2 from the nanoparticles. In comparison, there was no obvious decrease in the absorbance at 400 nm in the case of the bare silica nanoparticles. All these results confirmed that the bleaching of DPBF in the presence of ZnPc(NH2)4 was mainly caused by the singlet oxygen generated.
Fig.3 Photostability curves of SiO2@ZnPc(NH2)4 and ZnPc(NH2)4
Detection of singlet oxygen
Singlet oxygen (1O2) is a highly reactive species of oxygen and plays a key role in the process of PDT. To verify the generation of singlet oxygen, we used DPBF as the chemical
Fig.4 Photobleaching curves of DPBF
ZHUANG Qin-Fen et al. / Chinese Journal of Analytical Chemistry, 2011, 39(10): 1567–1571
3.4
Cell culture and phototoxicity study
Ideal photosensitizers not only have a high efficiency of generation of 1O2 after light exposure, but also show no or low toxicity in the absence of light irradiation. Therefore, we examined the toxicity of silica nanoparticles toward cells by incubation of the nanoparticles with three types of living cancer cells (HeLa, U251 and PC-12) without light irradiation. Cells were incubated for 48 h in the dark, and the phototoxicity was studied by the MTT assay. The experimental results in Fig.5 showed that bare SiO2-NH2 nanoparticles at a concentration of 300 mg L–1 had no significant toxicity toward these cells. Furthermore, SiO2@ZnPc(NH2)4 nanoparticles had low toxicity to HeLa and U251 cells with cell viability less than 90% and had almost no toxicity for PC-12. Moreover, experimental results also showed that both SiO2@ZnPc(NH2)4 and SiO2-NH2 particles at concentrations lower than 300 mg L–1 had almost no cytotoxicity, indicating that SiO2@ZnPc(NH2)4 had a great potential for applications in PDT of cancer.
irradiation has been investigated by the incubation of SiO2-NH2 and SiO2@ZnPc(NH2)4 nanoparticles with three types of living cancer cells (HeLa, U251 and PC-12). The experimental results showed that neither SiO2@ZnPc(NH2)4 nor SiO2-NH2 nanoparticles at concentrations lower than 300 mg L–1 had significant cytotoxicity. All these results provide some basis for the future application of silica nanoparticles in drug-and-gene delivery, in addition to paving a way for the future potential applications of these nanoparticles in PDT.
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