Biofunctional TiO₂ nanoparticle-mediated photokilling of cancer cells using UV irradiation

Abstract

Cancer cell-specific photokilling was successfully demonstrated using antibody-immobilized TiO₂ nanoparticles with only 1 J cm⁻² UV irradiation.

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Introduction

In the context of cancer therapy, nanoparticles (NPs) such as platinum,¹ gold,² quantum dots and liposomes have attracted great interest.³ Compared with organic nanoparticles such as liposomes or chitosan, metal nanoparticles have the advantage that due to their photodynamic characteristics, their therapeutic effect is autonomous and, thus, does not require encapsulation of therapeutic agents. Among various metal nanoparticles, titanium dioxide (TiO₂) has a great generation property of reactive oxygen species (ROS) under ultraviolet (UV) light excitation depending on the sizes and morphologies of the particles.⁴ In spite of its high potential for photodynamic cancer therapy (PDT), the use of these particles has been hampered because of the difficulty of size control, aggregation in aqueous solution and nanotoxicity itself^5,6, and so far only studies on their antibacterial effect have been successful.⁷ Few studies on cancer cell-specific photokilling by TiO₂ are reported;⁸ however, they only focus on the immobilization method of the biomolecule or mechanical delivery method. To control this “friends and foes” photocharacteristic of TiO₂ under UV irradiation, a fundamental study about the relationship between TiO₂ concentration and UV irradiation strength is necessary. In addition, cancer cell-specific molecules have to be immobilized to reduce the side effect on normal cells around the cancer.

In this study, to expand the versatility of TiO₂ NP-mediated cell-specific photokilling method, we evaluated the cytotoxicity of TiO₂ NPs according to UV irradiation strength, and measured photokilling of target cells via active cell targeting using antibody-immobilized TiO₂ NPs.

Experimental

Preparation of antibody-immobilized TiO₂ nanoparticles

Two kinds of TiO₂ NPs were used in this study, with sizes of about 30 nm and 10 nm, as measured by dynamic light scattering. For surface modification of TiO₂ NPs, polyacrylic acids (PAA) were used to avoid TiO₂ NP aggregation under physiological conditions. PAA modification of each of the TiO₂ NPs was carried out according to previous reports.⁹ UV-driven hydroxyl radical generation from various concentrations of NPs was measured using aminophenyl fluorescein (APF), which has a fluorescent response to various ROS. Chemical modifications of biofunctional molecules on the PAA-modified TiO₂ NPs were made with COMPOUND LINKS

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Download mol file of compoundN-hydroxysuccinimide and COMPOUND LINKS

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Download mol file of compound1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride chemistries.⁹ The variable domain of a camelid heavy chain-only antibody (la), which has high affinity to EGFR and is prepared easily using Escherichia coli as a host,¹¹ was immobilized as a cell-specific homing molecule on TiO₂ NPs. Detailed experimental procedures are shown in ESI.†

Preparation and evaluation of antibody-immobilized TiO₂ nanoparticles

HeLa cells were incubated with 0.03 wt% PAA-TiO₂/la for 10 min and then directly irradiated with UV (3 J cm⁻² or 1 J cm⁻²). Cell toxicity was evaluated immediately or 24 h later using calcein-AM (live cells) and ethidium homodimer-1 (EthD-1, dead cells) fluorescent dye. Detailed experimental procedure are shown in ESI.†

Results and discussion

Evaluation of antibody-immobilized TiO₂ nanoparticles

Our strategy is shown in Fig. 1. First, we investigated the radical generation ability of TiO₂ and PAA-modified TiO₂ NPs with UV irradiation using fluorescence derived from APF as an indicator. TiO₂ NPs (30 nm) generated the highest fluorescence level when 0.3 and 0.03 wt% NPs were irradiated with 1.5 J cm⁻² (Fig. 2A), suggesting the appropriate conditions to generate high amount of radicals. Interestingly, the fluorescence intensity decreased at higher levels of UV exposure. For NPs of lower concentration (0.003 wt%), more UV exposure (5 J cm⁻²) was required to generate nearly the same fluorescence levels. After PAA modification, the highest fluorescence intensity was detected with 3 J cm⁻² of exposure with 0.03 wt% NPs (Fig. 2C). We found that UV irradiation and pH did not affect APF fluorescence. PAA modification slightly decreased the amount of generated radicals, because produced radicals or UV radiation may be blocked by PAA. Similar results were obtained for TiO₂ NPs of 10 nm, although lower fluorescence (i.e. a smaller amount of radicals) was observed (Fig. 2B). However, after PAA modification, the amount of radicals decreased, and a maximum peak was not reached even after UV irradiation up to 10 J cm⁻² (Fig. 2D). From these results, there was an optimal size for NPs for ROS generation with UV irradiation. TiO₂ NPs of 30 nm possessed better ROS generation potential whether the NPs were modified or not, which corresponds to a previous report.¹² For target cell-specific photokilling, a large amount of radicals should be produced with a lower NP concentration and with lower UV exposure levels. We assumed that PAA-modified TiO₂ NPs of 30 nm (hereafter named PAA-TiO₂) were optimal for protein modification and cell photokilling in further experiments.

Fig. 1 Schematic illustration of target cell photokilling using antibody-modified TiO₂ nanoparticles with UV irradiation.

Fig. 2 The amount of radicals from TiO₂ exposed to UV. (A) TiO₂ (30 nm). (B) TiO₂ (10 nm). (C) PAA-TiO₂ (30 nm). (D) PAA-TiO₂ (10 nm).

We modified PAA-TiO₂ by anti-epidermal growth factor receptor (EGFR) antibody because EGFR is over-expressed on the surface of 20–30% of cancer cell lines.¹⁰ The anti-EGFR antibody modification of TiO₂ NPs, named as PAA-TiO₂/la, was analyzed by SDS-PAGE. The band corresponding to PAA-TiO₂/la was observed at a much higher molecular weight, suggesting successful modification (ESI, Fig. S1†). We also confirmed the antigen-binding ability of PAA-TiO₂/la by ELISA (ESI, Fig. S2†). Anti-EGFR antibody (la) and PAA-TiO₂/la efficiently bound the HeLa cells, although PAA-TiO₂ did not bind the cell.

Cell-specific photokilling using antibody-immobilized TiO₂ NPs with UV irradiation

Encouraged by these findings, we next carried out cell-specific photokilling using PAA-TiO₂/la. HeLa cells were employed as a model of EGFR-expressing cells. The addition of PAA-TiO₂ and/or anti-EGFR antibody without UV exposure did not affect cell viability (Fig. 3, left column), suggesting low toxicity of TiO₂ NPs itself on cultured cells at this concentration. HeLa cells treated with PAA-TiO₂/la showed extensive cell death (about 50% estimated by cell counting) after UV irradiation (Fig. 3D). In contrast, there was no cell death after treatment with only PAA-TiO₂ and anti-EGFR antibody mixtures (Fig. 3B, C), or BSA-modified TiO₂ NPs after UV exposure (Fig. 3E, F). When MCF-7, which is a low EGFR expression cell line, was treated with PAA-TiO₂/la, cell toxicity was not observed even under UV exposure (Fig. 3G, H). This clearly shows that PAA-TiO₂/la NPs allow EGFR expressing cell-specific TiO₂ NP accumulation and cell-specific killing by radicals generated with UV irradiation. However, after further cultivation for 24 h following UV irradiation (3 J cm⁻²), cells without TiO₂ NPs were detected to be dead in the end. It suggests apoptosis might be induced with large amount of UV irradiation. Therefore, to optimize the UV irradiation level, we applied UV irradiation at 1 J cm⁻² followed by a further 24 h incubation, and then cell viability was evaluated. HeLa cells treated with PAA-TiO₂/la under UV exposure (1 J cm⁻²) showed major cell death after 24 h incubation (Fig. 4D). In contrast, only a few dead cells under other conditions were observed (Fig. 4A–C). We also evaluated cell death depending on the amount of TiO₂ NPs with 1 J cm⁻² UV exposure. HeLa cells were efficiently damaged (about 80% estimated by cell counting) with 0.03 and 0.003 wt% PAA-TiO₂/la (Fig. 4B, C), suggesting that accumulation around the cell surface enhanced cell toxicity caused by radical generation with UV exposure, even at a low TiO₂ NP concentration. Effective photokilling with a low TiO₂ concentration demonstrates the advantage of active targeting, and is also a desirable property in therapy.

Fig. 3 HeLa cell viability after treatment with anti-EGFR antibody-immobilized TiO₂ NPs followed by UV irradiation (3 J cm⁻²). Cell viability was examined immediately after UV irradiation by calcein AM/EthD-1 fluorescence staining, and representative images from each sample are shown. Cells with green fluorescence are considered alive, whereas those with red are considered dead. (A) Untreated (B) PAA-TiO₂ (C) anti-EGFR antibody (la) and PAATiO₂ mixtures. (D) PAA-TiO₂/la. (E) la and BSA mixture; (F) PAA-TiO₂/BSA (G) Untreated MCF-7 cells (COMPOUND LINKS

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Download mol file of compoundH) MCF-7 cells treated with PAA-TiO₂/la.

Fig. 4 HeLa cell viability after treatment with PAA-TiO₂/la followed by UV irradiation (1 J cm⁻²) and further incubation for 24 h. (A) Untreated (B) PAA-TiO₂ (C) anti-EGFR antibody (la) and PAATiO₂ mixtures. (D) 0.003 wt% of PAA-TiO₂/la. (E) 0.03 wt% of PAA-TiO₂/la. (F) 0.3 wt% of PAA-TiO₂/la.

Conclusions

We established a cell-specific photokilling method using antibody immobilized TiO₂ NPs. It allows target cell-specific killing with low TiO₂ concentrations and low UV exposure levels. Our strategy is applicable for other targeting molecules such as various antibodies or bioactive ligands, as well as photoactivating nanoparticles. Although our system can be applicable for tumors near the skin surface, one possible disadvantage is that UV irradiation cannot be exposed to deeper tissues such as the liver and pancreas. Hence, novel nanoparticles which generate ROS with some radial ray, such as X-rays or gamma-rays, should be developed in the future.

Acknowledgements

This research was supported in part by Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe), MEXT, Japan.

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Footnote

† Electronic supplementary information (ESI) available: Detailed experimental procedure, and Figure S1 and S2. See DOI: 10.1039/c0md00027b

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