The Comparative PDT Experiment of the Inactivation of HL60 on Modified TiO<sub><b>2</b></sub> Nanoparticles

Lu, Kaiqi; He, Qiyun; Chen, Li; Ai, Baoquan; Xiong, Jianwen

doi:https://doi.org/10.1155/2015/540247

Journal of Nanomaterials

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Abstract Introduction Materials and Methods Results and Discussion Conclusion Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2015 | Article ID 540247 | https://doi.org/10.1155/2015/540247

The Comparative PDT Experiment of the Inactivation of HL60 on Modified TiO₂ Nanoparticles

Kaiqi Lu,¹Qiyun He,¹Li Chen,²Baoquan Ai,¹and Jianwen Xiong¹

Academic Editor: Paulo Cesar Morais

Received24 Feb 2015

Revised24 May 2015

Accepted10 Jun 2015

Published23 Jun 2015

Abstract

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.

1. Introduction

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 [1]. For instance, iron and chromium doped TiO₂ nanotube had a promising effect on the degradation of environmental and industrial pollutants [2]. Ag/TiO₂ nanoparticles showed great photocatalytic effect on the degradation of water pollutions [3]. Wang and his team pointed out that TiO₂ based photocatalytic process for purification of polluted water had bridged fundamentals to applications [4]. It has been proven that pairs of photo-induced electrons and holes generated on the surface of TiO₂ under the irradiation of ultraviolet (UV) light [5, 6] performed strong reduction and oxidation power and could react with hydroxyl ions and water to form various reactive oxygen species (ROS) which played an important role in photodynamic therapy (PDT) [7–9].

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 [10, 11]. Seeing its potential practical use, a wealth of researches had been done on the method of modification to enhance the efficiency of TiO₂ treatments on cancer cells and received sound results. Pt-TiO₂ has been demonstrated effective in treatment of localized tumors [12]. ZnPc-TiO₂ has been proved an excellent candidate as sensitizer in PDT against cancer and infectious diseases [13]. Water-dispersed TiO₂-polyethylene glycol compound had high antitumor effect in PDT [14]. Other modified TiO₂ nanoparticles such as N-TiO₂, Er³⁺-Yb³⁺-Fe³⁺ tridoped TiO₂, and Au/TiO₂ had been proved to have higher photocatalytic effect than pure TiO₂ on tumor cell [15–18]. Our laboratory had made some progress on tests of in vitro PDT based on modified TiO₂ such as CdS doped TiO₂, Fe-N codoped TiO₂, and TiO₂-xNx [19, 20].

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.

2. Materials and Methods

2.1. Chemicals and Apparatus

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.

2.2. Cell Culture

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%.

2.3. Preparation of Modified TiO₂ Nanoparticles

2.3.1. Synthesis of 5-ALA/TiO₂

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.

2.3.2. Synthesis of Fe/TiO₂

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, and the activator on the surface of DBS were washed away using deionized water until there was no BaSO₄ produced when BaCl₂ solution was tentatively added. CH₃CH₂OH was added to dehydrate the sample. After 3-hour desiccation in drying oven, our sample was grinded for 15 minutes in an automatic desktop grinder and then sintered for 30 minutes in a high temperature electric furnace at 450°C. When the sample cooled down, it was grinded again for 15 minutes and collected for use. The involved chemical equations were listed below:

2.4. CCK-8 Assay

Cell viability of the samples was evaluated by Cell Counting Kit-8 assays (CCK-8 assay) [21]. Cell suspension was seeded into 96-well plate. Each well contained 200 μL HL60 cell suspension and 20 μL CCK-8 solution. The 96-well plates then were placed in an incubator (37°C 5% CO₂) for 4 hours before the samples’ OD value at 490 nm was determined by the Model 680 Microplate Reader. In order to guarantee the accuracy, each sample was seeded into 3 wells to obtain a mean value. The cell viability was calculated by comparison with the OD value of untreated cells.

2.5. Scanning Electron Microscope

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.

2.6. Statistical Analysis

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 smaller than 0.05 are considered statistically significant.

3. Results and Discussion

3.1. Characterization of 5-ALA/TiO₂, Fe/TiO₂ Nanoparticles

3.1.1. X-Ray Diffraction Spectra

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 1(a), 1(b), and 1(c), respectively. Characteristic peaks in accordance with crystalline phases of (101) were found in all of the samples. Also there was no characteristic peak in (110) , indicating that the samples are primarily in anatase phase. TiO₂ had three basic crystal phases, anatase, rutile, and brookite. It has been demonstrated that the anatase phase TiO₂ showed highest photocatalytic efficiency among the three [22, 23]. By comparison, characteristic peaks in Figure 1(b) were less crystallized; but the ones in Figures 1(a) and 1(c) were observed sharper, indicating that TiO₂ and Fe/TiO₂ (5%) were more crystallized than Fe/TiO₂ (2%).

(a)

(b)

(c)

3.1.2. UV-Vis Spectra

UV-Vis spectra of the nanoparticles were studied to investigate their photocatalytic performance. As is shown in Figure 2, the spectrum of TiO₂ has a very sharp edge from 350 to 400 nm, presenting that TiO₂ nanoparticles absorb light primarily from ultraviolet region. UV-Vis spectra of 5-ALA/TiO₂, Fe/TiO₂ (2%), and Fe/TiO₂ (5%) were also observed; by comparison, we observed varying degrees of redshift, indicating that the light absorption of TiO₂ has been enhanced and expanded to visible light region, meaning that damage on normal cells from UV light during clinical treatments could be reduced. TiO₂ with a higher concentration of Fe ions absorbed more visible light. We also observed that the redshift of 5-ALA/TiO₂ was more obvious than the former; the absorption of visible light was largely promoted. Our result demonstrated that the modification of TiO₂ nanoparticles, especially with 5-ALA, helps enhance the visible light absorption. This phenomenon of redshift allowed photocatalytic activity of TiO₂ to be further activated by visible light irradiation.

3.1.3. FTIR Spectra of TiO₂ and 5-ALA/TiO₂

As shown in Figures 3(a) and 3(b), the FTIR spectra of TiO₂ and 5-ALA/TiO₂ were studied to characterize the modification of 5-ALA/TiO₂ nanoparticles. Characteristic peak of hydroxyl on surface of TiO₂ and 5-ALA/TiO₂ nanoparticles emerged in 3350 cm⁻¹. Further, characteristic peaks of carboxylic ester (–COOTi–) emerged in 1430 cm⁻¹ and 1730 cm⁻¹ in Figure 3(b). This finding illustrated that possible reaction, similar to the esters produced by chemical reactions of carboxylic acids and alcohols, might happen between 5-ALA and TiO₂. The weakening of peak 3350 cm⁻¹ in Figure 3(b) demonstrated again that the potential reaction consumed part of the hydroxyl. The potential chemical equation was presented as follows:

(a)

(b)

3.1.4. Raman Spectrum of 5-ALA/TiO₂

Raman spectrum of 5-ALA/TiO₂ was also taken to reaffirm the existence of carboxylic ester (–COOTi–). As shown in Figure 4, characteristic peaks of carboxylic ester could be observed more clearly in 1423 cm⁻¹ and 1728 cm⁻¹, which matched the finding from the FTIR spectra in Section 3.1.3.

3.2. Cytotoxicity of Modified TiO₂ Nanoparticles on HL60

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 μg/mL, 50 μg/mL, 100 μg/mL, 150 μg/mL, 200 μg/mL, 250 μg/mL, 500 μg/mL, and 1000 μg/mL) of TiO₂, Fe/TiO₂ (2%), Fe/TiO₂ (5%), and that (72.75 μg/mL, 145.50 μg/mL, 218.25 μg/mL, 291.00 μg/mL, 363.75 μg/mL, 436.50 μg/mL, 582.00 μg/mL, and 1164.00 μg/mL) of 5-ALA/TiO₂ were adopted in the test; the OD value of HL60 was measured after culturing for 48 hours, and the relative cell viability of HL60 was presented in Figure 5.

(a)

(b)

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 μg/mL, where cell viability dropped to 90%; the inhibitory effect was more obvious in groups of Fe/TiO₂ and Fe/TiO₂ (5%) appeared to have higher toxicity than Fe/TiO₂ (2%). Their cell viability dropped to 90% when the concentration of Fe/TiO₂ reached approximately 200 μg/mL. And note that the curves turned sharper at the concentration range over 200 μg/mL. From Figure 5(b), we can observe that the curve of Fe/TiO₂ (5%) dropped slightly faster than the one of Fe/TiO₂ (2%). The 5-ALA/TiO₂ group revealed that 5-ALA/TiO₂ nanoparticles were less toxic to HL60 cells than Fe/TiO₂, but more than pure TiO₂. The corresponding cell viability dropped to 90% when the concentration of 5-ALA/TiO₂ reached approximately 360 μg/mL.

3.3. Photodynamic Therapy on HL60

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 5, the final concentration we adopted was 200 μg/mL for TiO₂, Fe/TiO₂ (2%), and Fe/TiO₂ (5%) and 360 μg/mL 5-ALA/TiO₂, for under these concentrations; the viability of HL60 cells was the same 90% in darkroom experiments. The photocatalytic effect (indicated by Pe) was expressed by cell viability, which was calculated as follows:where the and are the mean absorption values at 490 nm for the treated and untreated samples, respectively. Overall results were presented in Figure 6.

From Figure 6, we found that the cells viability of control group remained stable relatively. As to PDT group, cell viability dropped remarkably. To state the obvious, cell viability under light treatment of TiO₂ decreased greatly to 30.3%; furthermore, the ones of modified TiO₂ happened to reach a greater decrease. This evidence revealed that modification of Fe ion and 5-ALA enhances photocatalytic inactivation effect of TiO₂. Specifically, cell viability under 5-ALA/TiO₂-PDT dropped to 19.4% and Fe/TiO₂ (2%) and Fe/TiO₂ (5%) to 28.4% and 28.0%, respectively. That said, Fe/TiO₂ nanoparticles did not present obviously higher photocatalytic effect in comparison to pure TiO₂ nanoparticles. However, 5-ALA/TiO₂ present promising photocatalytic effect. For the viability of HL60 cells of each darkroom group stayed above 80%, and the one of each radiance group nearly stayed all below 30%; we found that the activation of the modified TiO₂ from PDT was the main cause of cell apoptosis.

3.4. Scanning Electron Microscope (SEM) Image of the Treated HL60 Cells

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 7(a), 7(b), 7(c), 7(d), and 7(e), respectively. In Figure 7(a), HL60 cell was observed to remain morphologically intact. In Figures 7(b) and 7(c), we observed plump HL60 cells with smooth surface, attached or covered by nanoparticles. Based on experience, cell viability was slightly influenced in this stage. In Figure 7(d), the HL60 cell surface became hollow and deform; breakage of cytomembrane was observed clearly. In Figure 7(e), the same condition emerged in HL60 cell. From it, not only the breakdown of cytomembrane but also the damage of the inner cytoplasm was obviously observed. Damaged cytoplasm was observed to have entirely extruded out from cytomembrane in certain PDT-treated cells.

(a)

(b)

(c)

(d)

(e)

4. Conclusion

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 μg/mL, Fe/TiO₂ (2%) in 200 μg/mL, and Fe/TiO₂ (5%) in 200 μg/mL, were used in photodynamic therapy on HL60 cells for 1-hour light treatment in a luminous power of 5 mW/cm². Cell viability of each independent group was 19.4%, 28.4%, and 28.0% respectively, indicating that 5-ALA/TiO₂ nanoparticles had a higher inhibitory effect in the doses which caused the same dark toxicity, yet Fe/TiO₂ nanoparticles did not present obviously higher photocatalytic effect in comparison to pure TiO₂ nanoparticles.

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.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

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).

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Copyright © 2015 Kaiqi Lu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Journal of Nanomaterials

The Comparative PDT Experiment of the Inactivation of HL60 on Modified TiO2 Nanoparticles

Abstract

1. Introduction

2. Materials and Methods

2.1. Chemicals and Apparatus

2.2. Cell Culture

2.3. Preparation of Modified TiO2 Nanoparticles

2.3.1. Synthesis of 5-ALA/TiO2

2.3.2. Synthesis of Fe/TiO2

2.4. CCK-8 Assay

2.5. Scanning Electron Microscope

2.6. Statistical Analysis

3. Results and Discussion

3.1. Characterization of 5-ALA/TiO2, Fe/TiO2 Nanoparticles

3.1.1. X-Ray Diffraction Spectra

3.1.2. UV-Vis Spectra

3.1.3. FTIR Spectra of TiO2 and 5-ALA/TiO2

3.1.4. Raman Spectrum of 5-ALA/TiO2

3.2. Cytotoxicity of Modified TiO2 Nanoparticles on HL60

3.3. Photodynamic Therapy on HL60

3.4. Scanning Electron Microscope (SEM) Image of the Treated HL60 Cells

4. Conclusion

Conflict of Interests

Acknowledgments

References

Copyright

The Comparative PDT Experiment of the Inactivation of HL60 on Modified TiO₂ Nanoparticles

2.3. Preparation of Modified TiO₂ Nanoparticles

2.3.1. Synthesis of 5-ALA/TiO₂

2.3.2. Synthesis of Fe/TiO₂

3.1. Characterization of 5-ALA/TiO₂, Fe/TiO₂ Nanoparticles

3.1.3. FTIR Spectra of TiO₂ and 5-ALA/TiO₂

3.1.4. Raman Spectrum of 5-ALA/TiO₂

3.2. Cytotoxicity of Modified TiO₂ Nanoparticles on HL60