Tin Tungstate Nanoparticles: A Photosensitizer for Photodynamic Tumor Therapy Carmen Seidl,† Jan Ungelenk,‡ Eva Zittel,† Thomas Bergfeldt,§ Jonathan P. Sleeman,∥ Ute Schepers,*,† and Claus Feldmann*,‡ †
Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany ‡ Institute of Inorganic Chemistry, Karlsruhe Institute of Technology (KIT), Engesserstraße 15, 76131 Karlsruhe, Germany § Institute of Applied Materials Physics, Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany ∥ Medical Faculty Mannheim of the University of Heidelberg, Centre for Biomedicine and Medical Technology Mannheim (CBTM), Ludolf-Krehl-Straße 13-17, 68167 Mannheim, Germany S Supporting Information *
ABSTRACT: The nanoparticulate inorganic photosensitizer β-SnWO4 is suggested for photodynamic therapy (PDT) of near-surface tumors via reiterated 5 min blue-light LED illumination. β-SnWO4 nanoparticles are obtained via water-based synthesis and comprise excellent colloidal stability under physiological conditions and high biocompatibility at low material complexity. Antitumor and antimetastatic effects were investigated with a spontaneously metastasizing (4T1 cells) orthotopic breast cancer BALB/c mouse model. Besides protamine-functionalized β-SnWO4 (23 mg/kg of body weight, in PBS buffer), chemotherapeutic doxorubicin was used as positive control (2.5 mg/kg of body weight, in PBS buffer) and physiological saline (DPBS) as a negative control. After 21 days, treatment with β-SnWO4 resulted in a clearly inhibited growth of the primary tumor (all tumor volumes below 3 cm3) as compared to the doxorubicin and DPBS control groups (volumes up to 6 cm3). Histological evaluations of lymph nodes and lungs as well as the volume of ipsilateral lymph nodes show a remarkable antimetastatic effect being similar to chemotherapeutic doxorubicin butaccording to blood countsat significantly reduced side effects. On the basis of low material complexity, high cytotoxicity under blue-light LED illumination at low dark and long-term toxicity, β-SnWO4 can be an interesting addition to PDT and the treatment of near-surface tumors, including skin cancer, esophageal/gastric/colon tumors as well as certain types of breast cancer. KEYWORDS: tin tungstate, nanoparticle, photodynamic therapy, tumor, antimetastatic (e.g., SiO2, Fe2O3).25−31 These types of photosensitizers exhibit specific disadvantages, such as limited cell uptake and membrane permeability, high systemic toxicity, severe adverse effects, long-term photosensitivity, and heavy agglomeration under physiological conditions due to strong hydrophobic interaction (e.g., porphyrins)6−10,32 or low colloidal stability (e.g., nanoparticles).11−17,33,34 To evade these limitations, strategies such as encapsulation in vesicles or liposomes as well as functionalization with hydrophilic capping ligands are well established.1−4,32,35−37 However, such strategies, on the one hand, enhance the materials complexity. On the other hand, any form of encapsulation/capping blocks the active surface of the photosensitizer. The inorganic oxide nanoparticles TiO2 and ZnO, finally, suffer from the fact that both
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hotodynamic therapy (PDT) is known as a promising addition to the conventional armory against cancer.1−4 PDT, in general, is considered to be minimal invasive and nondamaging to healthy tissue. Thus, it may help to avoid the disadvantages of conventional chemotherapeutic agents, such as serious adverse reactions or multidrug resistances of certain malignant cells. The background of PDT is a photosensitizer of low systemic toxicity (in the dark) that becomes significantly cytotoxic only under illumination due to the photoassisted generation of reactive oxygen species (ROS).4,5 Targeted delivery and located illumination guarantee a selective tackling of malignant tumors while preserving nonilluminated surrounding tissue.1−4 In principle, two classes of photosensitizers are discussed for PDT: molecular photosensitizers (most often porphyrinebased)1−4,6−10 and inorganic nanoparticles, including binary oxides (e.g., TiO2, ZnO)1−4,11−17 and rare-earth metal based up-converters.1−4,18−24 Moreover, molecular photosensitizers (e.g., porphyrines) can be encapsulated in an inorganic matrix © 2016 American Chemical Society
Received: May 20, 2015 Accepted: February 19, 2016 Published: February 19, 2016 3149
DOI: 10.1021/acsnano.5b03060 ACS Nano 2016, 10, 3149−3157
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Figure 1. Colloidal properties of the β-SnWO4 nanoparticles.41 (a) TEM image. (b) Photo of suspension in PBS buffer (1 g/L β-SnWO4, 8 g/L NaCl, pH = 7.4). (c) Zeta-potential of as-prepared and protamine-functionalized β-SnWO4 nanoparticles.
Figure 2. Optical properties of the β-SnWO4 nanoparticles. (a) UV−vis spectrum showing the optical absorption of β-SnWO4 (orange) as well as the emission of the applied blue-light LED (blue). (b,c) CLSM fluorescence overlay images of HepG2 cells after incubation with protaminefunctionalized β-SnWO4 nanoparticles in the dark and after blue-light LED illumination for 4 h (1 × 104 cells, 5 μM β-SnWO4).
8 ± 2 nm, Figure 1a); (ii) excellent colloidal stability in physiological media (zeta-potential/η: −45 mV at pH 4−9, Figure 1c); (iii) direct band gap, daylight-active semiconductor (band gap/Eg: 2.7 eV, Figure 2a). First studies evaluating the photocatalytic activity of β-SnWO4 nanoparticles were conducted with the degradation of organic dyes (e.g., methylene blue, methyl orange) and show an encouraging performance.40 Moreover, in vitro studies on HepG2 and HeLa cells demonstrated high phototoxicity under artificial-daylight illumination in the absence of long-term effects.41 This motivated us to start in vivo studies and to verify the potential impact of β-SnWO4 nanoparticles on PDT. Aiming at physiological conditions and in vivo studies, we needed to modify the synthesis of β-SnWO4 nanoparticles (Supporting Information (SI): Figure S1). In contrast to previously reported water-based suspensions,40 the β-SnWO4 nanoparticles were here suspended in phosphate-buffered saline (PBS, pH = 7.4) exhibiting the same osmotic pressure as human cells and the same pH as human blood. Herein, the asprepared β-SnWO4 nanoparticles still turned out as colloidally highly stable, even at high concentrations up to 5 g/L, within a wide pH range from 4−9, and at high salt concentrations up to 8 g/L of NaCl (Figure 1b,c). As a second measure, the asprepared β-SnWO4 nanoparticles were functionalized with protamine (i.e., protamine sulfate from herring) that is well known to improve membrane penetration and cell uptake (SI: Figure S1).42 The presence of protamine on the β-SnWO4 was validated by infrared spectroscopy and thermogravimetry (SI: Figures S2 and S3). As a cationic biopolymer protamine adheres easily on the negatively charged β-SnWO4 nanoparticles and naturally reduces the sum zeta potential to about −20 mV in the physiologically relevant pH-range (Figure 1c).
can only be activated by UV-light, having a limited penetration depth and being harmful to cells and tissue.11−17,38 Upconverting nanoparticles, on the other hand, require high photon-density excitation (i.e., lasers) to establish a sufficient excitation intensity via the narrow, parity-forbidden f−f transitions on rare-earth metals (e.g., Yb3+).18−24 Many photosensitizers, moreover, are yet subjected to in vitro studies only. In view of the different nature of tumors and the pros and cons of existing photosensitizers, alternative photosensitizers for PDT are to be welcomed to diminish the current restrictions and to favor the use and efficacy of PDT.1−4,39 A general problem, however, is the increasing complexity and heterogeneity of nanostructured agents for PDT as pharmacokinetics, short- and long-term side-effects, clearance from the body, and thus, clinical approval become more and more advanced and less controllable.1,2 Here, we suggest β-SnWO4 nanoparticles as a new inorganic photosensitizer for PDT. In vivo studies addressing the systemic materials-related toxicity, the acute phototoxicity of β-SnWO4 under blue-light LED illumination, and the long-term toxicity are conducted for the first time and based on an orthotopic breast cancer BALB/c mouse model.
RESULTS AND DISCUSSION Synthesis and Functionalization of β-SnWO4 Nanoparticles. On the basis of our previous studies addressing fundamental synthesis, material characterization, and in vitro cell studies under artificial-daylight illumination,40,41 β-SnWO4 nanoparticles, in principle, comprise several features that are highly promising for PDT. This includes: (i) a water-based synthesis of high-quality nanoparticles (mean particle diameter: 3150
DOI: 10.1021/acsnano.5b03060 ACS Nano 2016, 10, 3149−3157
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ACS Nano
Figure 3. Detection of ROS upon treatment with fluorogenic H2DCFH-DA (25 μM) (in the presence of ROS, highly fluorescent DCF is generated). (a) Treatment with β-SnWO4 nanoparticles (10 μM) under illumination with blue-light LED. (b) Treatment with β-SnWO4 nanoparticles (10 μM) in the dark. (c) Treatment with H2O2 (500 μM). (d) No treatment and incubation in the dark. (e) No treatment and illumination with blue-light LED (scale bar: 50 μm).
of β-SnWO4 after transfection in HepG2 cells as well as to the absence of negative long-term effects. To proof that the phototoxicity of protamine-functionalized β-SnWO4 under blue-light LED illumination (λexc = 458 nm) involves the generation of ROS, the fluorogenic dye 2′-7′dichlorodihydrofluoresceindiacetate (H2DCFH-DA) was used to qualitatively measure the ROS levels within the cells. Upon cellular uptake, H2DCFH-DA is converted to the nonfluorescent 2′,7′-dichlorodihydrofluorescein (H2DCF), which is subsequently oxidized into the highly fluorescent dichlorofluorescein (DCF) if ROS are present.46 HeLa cells were incubated with 10 μM of β-SnWO4 for 24 h, either in the dark or under blue-light LED illumination (Figure 3a,b). Negative controls were incubated under the same conditions, but in the absence of β-SnWO4 nanoparticles (Figure 3d,e). HeLa cells treated with H2O2 for 4 h served as positive control (Figure 3c). Then, 45 min before the end of treatment, H2DCFH-DA (25 μM) was added to each well. At the end of the treatment, ROS levels were evaluated by means of confocal microscopy. The treatment with protamine-functionalized β-SnWO4 nanoparticles fundamentally increases the intracellular ROS levels upon excitation with a blue-light LED as indicated by the intense green fluorescence (Figure 3a), which even exceeds the fluorescence after H2O2 treatment (Figure 3c). In contrast, the β-SnWO4 nanoparticles do not have any strong impact on the intracellular ROS levels without illumination (Figure 3b). Moreover, illumination itself did not influence the intracellular ROS concentration (Figure 3e). Pharmacokinetic Studies. On the basis of the promising results of the above qualitative in vitro experiments, in vivo studies were performed to determine whether the β-SnWO4 nanoparticles could be used to induce the same phototoxic effect in near-surface solid tumors and whether they can hamper tumor progression. To determine the pharmacokinetics of β-SnWO4, 8−12 week old female BALB/c mice were treated intraperitoneally with β-SnWO4 for a single dose (23 mg/kg bodyweight, ∼ 460 μg β-SnWO4/mouse, i.e., ∼ 149 μg Sn/ mouse and 230 μg W/mouse). Blood samples were collected for t = 5, 15, 30, 60, 120, 240, 480, 1440 min after application via cardiocentesis. Microwave-assisted digestion of 2−550 mg
Electrostatic stabilization (due to negative surface charge) and steric stabilization (due to protamine) still guarantee for excellent colloidal stability of the nanoparticles. Due to the fact that almost all surfaces in the body are negatively charged, negative charging at low absolute value (i.e., 10−20 mV) has been claimed as most advantageous for nanoparticulate antitumor agents.39 All in vivo experiments described hereafter were performed with protamine-functionalized β-SnWO4 nanoparticles throughout. With a band gap of 2.7 eV, β-SnWO4 can be excited at wavelength
