Four samples of modified titanium dioxide (TiO2), Fe/TiO2 (2 wt%), Fe/TiO2 (5 wt%), and 5-ALA/TiO2, were experimented in photodynamic therapy (PDT) on leukemia cells HL60, performing promising photocatalytic inactivation effect. Fe/TiO2 and 5-ALA/TiO2 were synthesized in methods of precipitation and ultrasonic methods, respectively. X-ray diffraction spectra and UV-Vis spectra were studied for the samples’ crystalline phase and redshift of absorption peak. Further, FTIR spectra and Raman spectra were obtained to examine the combination of 5-aminolevulinic (5-ALA) and TiO2 nanoparticles. The toxicity of these four kinds of nanoparticles was studied through darkroom experiments. And based on the concentration which caused the same toxic effect (90%) on HL60, PDT experiments of TiO2, Fe/TiO2 (2%), Fe/TiO2 (5%), and ALA/TiO2 were done, resulting in the fact that the photokilling efficiency was 69.7%, 71.6%, 72%, and 80.6%, respectively. Scanning electron microscope (SEM) images of the samples were also taken to study the morphology of HL60 cells before and after PDT, resulting in the fact the activation of the modified TiO2 from PDT was the main cause of cell apoptosis.
Titanium dioxide (TiO2) has been widely used in biomedical, industrial, environmental, and energy technology fields during the past two decades on account of its chemical and physical property [
In spite of its tremendous effect on killing cancer cells, the application of TiO2 in PDT was hindered by a few of its properties. TiO2, with the band gap of 3.23 eV for anatase, turned excited only when exposed to UV light; furthermore, the activated electron-hole pairs had a high speed rate of recombination, which would weaken the promising photocatalytic effect in PDT [
In this paper, four kinds of nanoparticles (including TiO2, Fe/TiO2 (2 wt%), Fe/TiO2 (5 wt%), and 5-ALA/TiO2) were applied in PDT experiments on leukemic HL60 cells. Each dose was used and tested separately for PDT and toxicity experiments, and the comparison of cell viability between each independent experiment has been specifically studied, and this possible use of the experimental nanoparticles will be discussed at the end of this paper.
HL60 cells were kindly provided by the Department of Medicine of Sun Yat-sen University. 5-aminolevulinic acid (ALA) was purchased from Shanghai Xianhui Pharmaceutical Co. Ltd. (CHN). TiO2 nanoparticles were purchased from Degussa (GER). RPMI medium 1640 was obtained from Gibco BRL (USA). Cell Counting Kit-8 (CCK-8) assays were purchased from Dojindo (Japan). Fe/TiO2 (2%) and Fe/TiO2 (5%) nanoparticles were synthesized in the method of precipitation by Department of Physics and Electronic Engineering of South China Normal University. 5-ALA/TiO2 nanoparticles were synthesized by author’s laboratory. All chemicals were of the highest purity commercially available. The tock solutions were well prepared in serum-free medium before use and materials stored in sealing space at a proper temperature.
The apparatus used included UV-2550 UV-visible spectrophotometer (Hitachi, Japan), HH.CP-TW80 CO2 Incubator, DG5031 ELISA Reader (Nanjing Huadong Electronics Group Medical Equipment Co., Ltd., China), BS124S Electronic Scales (Sartorius, GER), SK2510LHC Ultrasonic Cleaner (KUDOS, China), SW-CJ Standard Clean Bench (Suzhou Antai Airtech Co., Ltd., China), LPE-1A Laser Power Meter (Physcience Opto-Electronics, Beijing), Eppendof (Finland), lab-assembled PDT light reaction chamber, 96-well culture plates, cell counting boards, and so on.
Leukemia HL60 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS) and refreshed daily. They were stored in a humidified incubator with 5% CO2 at 37°C before use. In order to maximum the reliability of PDT effect, cells viability will be measured using a countess automated cell counter, verifying their sound concentration over 95%.
5-ALA/TiO2 nanoparticles were synthesized in a way of surface modification. 2.62 mg 5-ALA and 3.2 mg TiO2 nanoparticles were measured by an electronic scale and then dissolved in 20 mL deionized water contained with a beaker. Subsequently, the beaker was then sealed, vibrated using a magnetic stirrer, and placed in ultrasonic heater for an ultrasonic processing of 4 h. In this process, carboxyl groups on 5-ALA were believed to bond with the hydrogen bonds on TiO2 nanoparticles. In this way, the 5-ALA/TiO2 nanoparticles with a molar mass ratio (5-ALA : TiO2) of 2 : 1 were synthesized.
Ti(SO4)2, C18H29NaO3S (DBS), H2NCONH2, deionized water, CH3CH2OH, and concentrated H2SO4 were compounded together in doses of 6 g, 0.18 g, 32 g, 250 mL, 0.25 mL, and 0.25 mL, respectively, with a certain dose of Fe2(SO4)3; 0.1 g Fe2(SO4)3 was applied to obtain Fe/TiO2 (2%) and 0.5 g to Fe/TiO2 (5%). The beaker of the reactant was put in 80°C thermostatic bath, quickly stirred for 2 to 3 hours until the PH value of the reactant reached 2. Then another 24 hours were taken for its complete sedimentation. Subsequently,
Cell viability of the samples was evaluated by Cell Counting Kit-8 assays (CCK-8 assay) [
Samples of treated HL60 cells were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer solution (PBS) and freeze-dried in 6°C for 6 hours. Then they were washed three times by 0.1 M phosphate buffer solution and dehydrated by ethanol solution of 30%, 50%, 70%, 90%, and 100% successively. Isoamyl acetate was used to replace the ethanol solution in cell samples and subsequently removed by CO2 in critical point drying method. Finally, the samples were coated with platinum in automatic high vacuum coating system (QuorumQ150T ES) before observation in a ZEISS Ultra-55 scanning electron microscope.
Data are presented as means ± SD (standard deviation) from three independent groups. Statistical software SPSS11.5 is used in statistical analysis. Any data with value
X-Ray diffraction is a solid method in analyzing crystalline structural properties. Thus, XRD images were taken to investigate the crystalline phases of three samples, pure TiO2 used for the synthesis of 5-ALA/TiO2, Fe/TiO2 (2%), and Fe/TiO2 (5%); they were presented in Figures
X-ray diffraction of nanoparticles: (a) pure TiO2 nanoparticles used to synthesize 5-ALA/TiO2; (b) Fe/TiO2 (2%) nanoparticles; (c) Fe/TiO2 (5%) nanoparticles.
UV-Vis spectra of the nanoparticles were studied to investigate their photocatalytic performance. As is shown in Figure
UV-Vis spectra of TiO2, 5-ALA/TiO2, Fe/TiO2 (2%), and Fe/TiO2 (5%) nanoparticles.
As shown in Figures
FTIR spectra: (a) TiO2 nanoparticles; (b) 5-ALA/TiO2 nanoparticles.
Raman spectrum of 5-ALA/TiO2 was also taken to reaffirm the existence of carboxylic ester (–COOTi–). As shown in Figure
Raman spectrum of 5-ALA/TiO2 nanoparticles.
Low dark toxicity is known as one of the important features of photosensitive drugs. We ran a series of experiments to test the dark toxicity of our modified TiO2 nanoparticles on HL60 cells. Various concentrations (0
Effect of (a) TiO2 and Fe/TiO2 and (b) 5-ALA/TiO2 of different concentration on HL60 cell viability.
We learned that the viability of HL60 softly declines with the increase of drug concentration. That said, photosensitive drugs influenced HL60 cells viability without radiation, even though not much. In detail, TiO2 had relatively mild inhibitory effect on HL60 until the concentration went up to 500
HL60 cells in a concentration of 0.5 × 105/mL were inoculated into two 96-well plates marked as A and B. Plate A received light treatment (luminous power 5 mW/cm2, light dose 18 J/cm2, wavelength 403 ± 6 nm, period 1 hour) after 24-hour incubation in dark and then another 24-hour incubation in dark. Plate B, as control group, was incubated in dark for continuous 48 hours in the incubator. According to Figure
Effect of PDT of different nanoparticles on OD value of HL60 cells.
From Figure
The ultrastructural morphology of HL60 cell, the ultrastructural morphology of HL60 cell cultured with Fe/TiO2 and 5-ALA/TiO2, and the ultrastructural morphology of PDT-treated HL60 cell cultured with 5-ALA/TiO2 and 5-ALA/TiO2 were shown in Figures
Ultrastructural morphology of HL60 cells: (a) normal cultured cell; (b) cell cultured with Fe/TiO2 in darkroom; (c) cell cultured with 5-ALA/TiO2 in darkroom; (d) PDT-treated cell cultured with Fe/TiO2; (e) PDT-treated cell cultured with 5-ALA/TiO2.
In this paper, the modification of Fe and 5-ALA on TiO2 was explored in experiments. Fe/TiO2 (2%), Fe/TiO2 (5%) were synthesized by precipitation method and 5-ALA/TiO2 was synthesized by ultrasonic method. They were all used as experimental photosensitizer in photodynamic therapy. X-ray diffraction was used to verify their anatase phase; and the UV-Vis spectrum indicated that the modification of TiO2 leads to a redshift of absorption spectrum. Our experimental results showed that the modification of TiO2 promotes the absorption in visible light region of TiO2. TiO2 and modified TiO2 develop nearly no harm to HL60 cells when in darkroom with proper experimental doses but can still result in cell death in high concentration. Our experiment proved that modified TiO2 nanoparticles performed a high photocatalytic effect on HL60 cells than pure TiO2. And among all three kinds of them, 5-ALA/TiO2 had the greatest inhibitory effect on HL60 cells. Besides, Fe/TiO2 (5%) cause a higher inhibitory effect than Fe/TiO2 (2%), indicating that higher proportion of Fe+ in modified TiO2 performs better photocatalytic effect in light treatments. Three kinds of nanoparticles, including 5-ALA/TiO2 in 360
Scanning electron microscope was used to observe the ultrastructural morphology of HL60 cells before and after PDT and the images and PDT results together showed that the synthesized nanoparticles cause obvious damage on HL60 through PDT.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work has been financially supported by the National Natural Science Foundation of China (61072029) and Science and Technology Planning Project of Guangzhou City (2014J4100049).