Studies on Preparation of Photosensitizer Loaded Magnetic Silica Nanoparticles and Their Anti-Tumor Effects for Targeting Photodynamic Therapy
© to the authors 2009
Received: 13 November 2008
Accepted: 8 January 2009
Published: 31 January 2009
As a fast developing alternative of traditional therapeutics, photodynamic therapy (PDT) is an effective, noninvasive, nontoxic therapeutics for cancer, senile macular degeneration, and so on. But the efficacy of PDT was compromised by insufficient selectivity and low solubility. In this study, novel multifunctional silica-based magnetic nanoparticles (SMNPs) were strategically designed and prepared as targeting drug delivery system to achieve higher specificity and better solubility. 2,7,12,18-Tetramethyl-3,8-di-(1-propoxyethyl)-13,17-bis-(3-hydroxypropyl) porphyrin, shorted as PHPP, was used as photosensitizer, which was first synthesized by our lab with good PDT effects. Magnetite nanoparticles (Fe3O4) and PHPP were incorporated into silica nanoparticles by microemulsion and sol–gel methods. The prepared nanoparticles were characterized by transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy and fluorescence spectroscopy. The nanoparticles were approximately spherical with 20–30 nm diameter. Intense fluorescence of PHPP was monitored in the cytoplasm of SW480 cells. The nanoparticles possessed good biocompatibility and could generate singlet oxygen to cause remarkable photodynamic anti-tumor effects. These suggested that PHPP-SMNPs had great potential as effective drug delivery system in targeting photodynamic therapy, diagnostic magnetic resonance imaging and magnetic hyperthermia therapy.
Photodynamic therapy (PDT) is an effective, noninvasive and nontoxic therapeutics for cancer, senile macular degeneration, actinic keratosis, port-wine stains, rheumatoid arthritis, and so on [1, 2]. After bio-distribution, photosensitizer (PS) administered systemically or topically is activated by light of appropriate wavelength and dosage. The activated PS transfers its excited-state energy to nearby oxygen molecular to generate reactive oxygen species, such as singlet oxygen (1O2) or peroxides inducing oxidative damage to target tissue and blood vessels that feed them [1–4]. Due to minimal invasion and nontoxicity, PDT provides patients, weak or failed in traditional therapy, opportunities to be treated painlessly and repeatedly.
However, the PDT efficacy is compromised by insufficient selectivity and low solubility. Although several methods including drug delivery systems were investigated [3–9], developing a PS delivery system for higher selectivity and less dark toxicity is still a challenge [5, 6].
Magnetic drug delivery system is a promising drug delivery system, which can be steered to the target tissue simply by an external magnetic field [10, 11]. Silica nanoparticles, easily prepared with desired size, shape and porosity, are water-soluble, stable and biocompatible. More importantly, silica nanoparticles are permeable to small molecular such as singlet oxygen [4, 5], which is the key effector of PDT. Therefore, photosensitizer loaded silica nanoparticles are different from conventional delivery systems which need releasing of the loaded drug .
Previous investigations of fluorescent-magnetic nanoparticles mainly focused on the MRI imaging and fluorescence imaging for diagnosis; however, there are few studies on the multifunctional magnetic targeting drug delivery system for diagnosis and therapy [12, 13]. In the earliest study of magnetic targeting, a magnetic fluid was developed to which epirubicin was chemically bound to enable those agents to be directed within an organism by high-energy magnetic fields. In vitro and in vivo study of the epirubicin-magnetic fluid indicated biosafety and complete tumor response [10, 11], demonstrating the potential of magnetic targeting. Recently, the investigation of PS encapsulated magnetic silica nanoparticles (SMNPs) showed efficient cellular uptake  and obvious generation of singlet oxygen in vitro [15, 16], which indicated the potential of SMNPs as targeting drug delivery system.
Ferrous(II) sulfate heptahydrate (FeSO4 · 7H2O, 99%), ferric chloride hexahydrate (FeCl3 · 6H2O, 99%), anhydrous ethanol (99.7%), ammonium hydroxide (25.2–28.0%), 1-butanol (99.8%), dimethyl sulfoxide (DMSO, 99.8%), tetrahydrofuran (THF, 99.9%), hydrochloric acid (36%) and oleic acid (99%) were purchased from Sinopharm Co. (China). Surfactant aerosol OT (AOT, 98%), tetraethylorthosilicate (TEOS, 99.99%), (3-mercaptopropyl) trimethoxysilane (MPS, 95%),N,N-dimethyl-4-nitrosoaniline (RNO, 99%), imidazole (≥99%), trypsinase (0.25%), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Aldrich. The PS PHPP was synthesized by our lab with purity ≥98%. SW480 cell was available in the cell store of Chinese Academy of Science. Other materials for cell culture, unless mentioned otherwise, were purchased from GIBCO. All the above-mentioned chemicals were used without any further purification.
Preparation of Fe3O4Nanoparticles
In total, 2.51 g (9 mmol) FeCl3 · 6H2O and 1.25 g (4.5 mmol) FeSO4 · 7H2O were dissolved in 20 mL water. The solution was vigorously stirred, followed by adding 10 mL 1.5 mol L−1NH3 · H2O. The color of the solution was changed into black and the black solid produced was precipitated to the bottom. The Fe3O4nanoparticles were obtained after the precipitants were washed for five times with 20 mL distilled water and 20 mL ethanol alternatively to remove unreacted chemicals.
Preparation of Silica-Based Fe3O4Nanocarriers
In total, 1 g Fe3O4nanoparticles and 10 g oleic acid were mixed with 10 mL ethanol. The suspension was refluxed for 30 min. Fe3O4/OA nanoparticles were obtained after the excess oleic acid was scoured off with ethanol by the magnetic decantation.
Micelles were prepared by dissolving 0.90 g AOT and 1600 μL 1-butanol in 40 mL doubly distilled water by vigorous magnetic stirring. A solution of 60 μL PHPP (15 mmol L−1) in 1-butanol and 0.003 g Fe3O4/OA nanoparticles were added to above micellar system. After 30 min stirring, a new micellar system containing PHPP-Fe3O4/OA was formed. A total of 200 μL TEOS and 1.2 mL aqueous ammonia were added to the PHPP-Fe3O4/OA system prior to 1 h stirring. Then, 10 μL MPS was added, followed by continued 20 h stirring. The resultant was treated by magnetic separation and washed with ethanol until no PS could be detected in the supernatant by UV–Vis spectroscopy. All the above-mentioned experiments were conducted at room temperature. The products were dried at 60 °C for 3 h in vacuum oven.
The X-ray diffraction pattern of silica-based magnetic nanocarriers powders was obtained using D/max-2550PC (Geigerflex, Rigaku, Japan) with monochromated CuKα radiation operated at 40 kV and 100 mA. Transmission electron microscopy (TEM) was employed to determine the morphology and size of the aqueous dispersion of nanocarriers, using a HITACHIH-800 electron microscope, operating at an accelerating voltage of 200 kV. UV–Vis absorption spectra were recorded using a Jasco V-530 spectrophotometer, in a quartz cuvette with 1 cm path length. Fluorescence spectra were recorded on a HITACHIH FL-4500 spectrofluorimeter.
Encapsulation Efficiency Measurements
The UV–Vis measurements of the PHPP-SMNPs were carried out contrasted to other six groups: (a) PHPP; (b) Fe3O4 + PHPP; (c) PHPP + HCl; (d) Fe3O4 + HCl; (e) Fe3O4 + PHPP + HCl; (f) PHPP + |SMNPs + HCl. The amount of the mixed solvent was 0.1 mL the concentrated HCl and 2.9 mL ethanol. The absorbance at 409 nm was used to validate the PS presence and estimate the PS encapsulation efficiency. Each measurement was repeated three times.
The standard curve was established in the drug concentration range from 7.65 × 10−7 mol L−1to 1.02 × 10−5 mol L−1. Different concentrations of PHPP (7.65 × 10−7, 2.55 × 10−6, 5.10 × 10−6, 7.65 × 10−6, 1.02 × 10−5 mol L−1) were mixed with 0.1 mL concentrated HCl and 2.9 mL ethanol. The samples were measured at 409 nm wavelength. Each experiment was repeated three times.
Detection of Singlet Oxygen
The PHPP-SMNPs in phosphate buffer (pH = 7.4) were irradiated in the presence of imidazole (10 mmol L−1) and RNO (50 mmol L−1). The RNO bleaching by1O2was followed spectrophotometrically with observing the decrease in the 440 nm absorption peak of RNO as a function of irradiation time. The reaction mixture in a 1 cm spectrometric cuvette, placed at a distance of 12 cm, was continuously irradiated using 632.8 nm laser.
In Vitro Studies with Tumor Cells
Preparation of PHPP-SMNPs Solution
PHPP-SMNPs was diluted to 100 μmol L−1with 0.5% carboxymethylcellulose sodium. The solution was then diluted with RPMI-1640 medium (supplemented with 100 U mL−1penicillin, 10 U mL−1streptomycin and 10% calf serum) using a dilution factor of 5 to varied concentrations: 0, 0.03, 0.13, 0.64, 3.20, 16.00, and 80 μmol L−1.
SW480 carcinoma cells (3 × 103cells per well) were seeded in 96-well plates and incubated overnight at 37 °C in a humidified 5% CO2atmosphere. After being rinsed with PBS (pH 7.4), the cells were incubated with 100 μL varied concentration of PHPP-SMNPs prepared above for 24 h at 37 °C in the dark under the same conditions. Rinsed with PBS, the cells were incubated another 48 h. Cell viability was determined by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were rinsed with PBS and then incubated with culture medium containing 0.5 mg mL−1MTT reagent for 3 h. The medium was then removed and the formazan crystals formed were dissolved in 100 μL DMSO. The absorbance at 492 nm for each well was recorded by a microplate reader.
Photodynamic Activity Assay
Two plates were set up as dark control and experimental group for the MTT assay and these plates were seeded, exposed identically to the plates prepared for the biosafety assessment. The cells of experimental group were then rinsed again with PBS and immersed in 100 μL of fresh culture medium before being illuminated using a 488 nm argon-ion laser with energy density of 4.35 J/cm2from the underside of the culture plate. After 10 min illumination, cells were incubated 48 h in a 5% CO2, 95% air humidified incubator at 37 °C. Dark control group keep identical to experimental group except illumination. Photodynamic activity assay was also determined by MTT assay as mentioned above.
Cell viability was calculated using the following formula: averageA value of experimental group/averageA value of control group × 100%. Results were expressed as means ± SD. Comparisons between two groups were made by unpaired two-tailed Student’st-test using SPSS 15.0 software.P-value of less than 0.05 was taken to indicate statistical significance.
Results and Discussion
Preparation of Fe3O4Nanoparticles
Preparation of PHPP-SMNPs
Characterization of PHPP-SMNPs
Figure 4b illustrates the XRD pattern of PHPP-SMNPs. The (111) peak is derived from the amorphous mesoporous silica spheres and the characteristic (311), and (440) peaks are typical of a cubic structure. The result showed that the crystallinity has not changed after encapsulation.
Encapsulation Efficiency Measurements
The amount of drug entrapped within PHPP-SMNPs was determined by dissolving PHPP-SMNPs into hydrochloric acid to destroy the Fe3O4cores of PHPP-SMNPs for the release of PHPP. After ethanol addition, the absorbance of PHPP was detected and was performed by ultraviolet spectrophotometer at 409 nm.
Detection of Singlet Oxygen
The bleaching of RNO by 1O2 was followed spectrophotometrically with observing the decrease in the 440 nm absorption peak of RNO as a function of irradiation time.
In Vitro Studies with Tumor Cells: Cellular Uptake, Biosafety Assessment and Photodynamic Activity Assay
In Vitro Photodynamic Efficacy
Novel multifunctional silica-based magnetic nanoparticles containing photosensitizer PHPP were prepared. The PHPP-SMNPs were approximately spherical and 20–30 nm in diameter, achieving 20.8% encapsulation efficiency of PHPP. They showed no obvious toxicity without irradiation, but significant generation of singlet oxygen and remarkable photodynamic efficacy after irradiation. The PHPP-SMNPs were primarily distributed in the cytoplasm.
It can be concluded that the silica-based magnetic nanoparticles are of great value as effective drug delivery system in targeting photodynamic therapy. The potential of the magnetic core for magnetic resonance imaging and magnetic hyperthermia therapy could also be expected.
This work was supported by National Natural Science Foundation of China (grant nos. 30070862, 30271534), Shanghai Municipal Foundation (grant nos. 05ZR14002, 06PJ14001, 064319020).
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