Subscriber access provided by University of Pennsylvania Libraries
Article
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 ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b03060 • Publication Date (Web): 19 Feb 2016 Downloaded from http://pubs.acs.org on February 20, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Nano is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 24
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
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), Engesserstr. 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-Str. 13-17, 68167 Mannheim (Germany)
ACS Paragon Plus Environment
1
ACS Nano
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 24
ABSTRACT: The nanoparticulate inorganic photosensitizer β-SnWO4 is suggested for photodynamic therapy (PDT) of near-surface tumors via reiterated 5-minutes 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 materials complexity. Anti-tumor and anti-metastatic 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 anti-metastatic effect being similar to chemotherapeutic doxorubicin but–according to blood counts–at significantly reduced side effects. Based on low materials 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, anti-metastatic. Table of Content Figure.
ACS Paragon Plus Environment
2
Page 3 of 24
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
Photodynamic 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 non-damaging to healthy tissue. Thus, it may help to avoid the disadvantage of conventional chemotherapeutic agents, such as serious adverse reactions or multi-drug resistances of certain malignant cells. 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/or located illumination guarantee a selective tackling of malignant tumors, while preserving non-illuminated surrounding tissue.1-4 In principle, two classes of photosensitizers are discussed for PDT: molecular photosensitizers (most often porphyrine-based),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 (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).1117,33,34
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 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 rareearth 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
ACS Paragon Plus Environment
3
ACS Nano
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 24
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 longterm 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 Based on 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 waterbased synthesis of high-quality nanoparticles (mean particle diameter: 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 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 (SI: Figure S1). In difference 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 as-prepared 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 as-prepared βSnWO4 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,S3). As a cationic biopolymer protamine adheres easily on the
ACS Paragon Plus Environment
4
Page 5 of 24
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
negatively charged β-SnWO4 nanoparticles and naturally reduces the sum zeta potential to about −20 mV in the physiologically relevant pH-range (Figure 1c). 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 anti-tumor agents.39 All in vivo experiments described hereafter were performed with protamine-functionalized β-SnWO4 nanoparticles throughout.
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 asprepared and protamine-functionalized β-SnWO4 nanoparticles. With a band gap of 2.7 eV, β-SnWO4 can be excited at wavelength < 550 nm, thus, blue light (Figure 2a). This is advantageous in comparison to alternative inorganic photocatalysts such as TiO2 or ZnO being limited to UV-excitation (< 380 nm).11-17,38 Since the penetration of blue light into tissue is limited, PDT based on β-SnWO4 nanoparticles needs to address near-surface tumors, including skin cancer, esophageal/gastric/colon tumors as well as certain types of breast cancer.1-4,39,43-45 As a preliminary test regarding cell uptake, systemic toxicity (in the dark) and phototoxicity (under blue-light illumination) towards tumor cells, human liver carcinoma cells (HepG2) were cultivated and incubated with protamine-functionalized β-SnWO4 nanoparticles (Figure 2b). According to confocal laser scanning microscopy (CLSM), the nanoparticles are readily taken up as demonstrated by intense green fluorescence within the cytosolic compartment (λexc = 458 nm, λem = 530 nm),41 but do not show any toxic effect or impact on cell viability. Moreover, normal proliferation rates and organelle integrity were proven in previous studies if
ACS Paragon Plus Environment
5
ACS Nano
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 6 of 24
the β-SnWO4-treated HepG2 cells are kept in the dark.41 In contrast, the HepG2 cells exhibit unusual spherical shapes and debris indicating encumbered growth and beginning apoptosis if illuminated continuously with a blue-light LED for about 4 hours (LED with λmax = 465 nm; emission: 450-490 nm; luminous flux: 12 lm; irradiance: 0.2 W/cm2) (Figure 2c). Notably, bluelight LED illumination did not affect the cells if they were not transfected with β-SnWO4 nanoparticles in advance. In previous studies (under artificial-daylight illumination),41 MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays also pointed to a low systemic cytotoxicity of β-SnWO4 after transfection in HepG2 cells as well as to the absence of negative long-term effects.
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 protamine-functionalized β-SnWO4 nanoparticles in the dark and after blue-light LED illumination for 4 hours (1×104 cells, 5 µM β-SnWO4). 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 non-fluorescent 2´,7´-dichlorodihydrofluorescein (H2DCF), which is subsequently oxidized into the highly fluorescent dichlorofluorescein (DCF) if ROS are present.46 HeLa cells were incubated with
ACS Paragon Plus Environment
6
Page 7 of 24
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
10 µM of β-SnWO4 for 24 hours, either in the dark or under blue-light LED illumination (Figure 3a,b). Negative controls were incubated under the same conditions, but in absence of βSnWO4 nanoparticles (Figure 3d,e). HeLa cells treated with H2O2 for 4 hours served as positive control (Figure 3c). 45 minutes 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).
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 βSnWO4 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).
Pharmacokinetic studies Based on 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
ACS Paragon Plus Environment
7
ACS Nano
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 8 of 24
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 minutes after application via cardiocentesis. Microwave-assisted digestion of 2-550 mg of blood and extraction of the analytes tin and tungsten was performed at 140 °C using HCl, HNO3 (3:1) and small amounts of HF. Blood of groups treated with physiological saline (DPBS) served as a control, but did not include detectable amounts of tin and tungsten. As presented in Figure 4, the concentration of the β-SnWO4 nanoparticles over time can be approximated by a bateman-function. 15 minutes after drug administration (tmax), the highest levels of tin and tungsten were detected. However, only portions of the applied drug enter the blood stream (cmax ~ 9 mg Sn/kg of blood) as often observed for intraperitonial application.47 It is to be noted that−unlike the suspension itself−the blood sample did not harbor tin and tungsten in a 1:1 ratio as expected for β-SnWO4, but comprised−especially in the long run−slightly lower amounts of tungsten. Assuming that β-SnWO4 slowly splits into the binary oxides SnO2 and WO3 under physiological conditions,41 this effect might by explained by precipitation of small amounts of WO3, which – once hydrolyzed to soluble [WO4]2– – can be excreted. The main part of tin and tungsten is cleared out of the blood stream within 8 hours, and falls below detection limit within 24 hours, thus indicating a comparably short half-life, which can in general be compensated by repeated application of β-SnWO4. Based on these results, we concluded for the following in vivo studies to apply and illuminate several times. Moreover, the interval of illumination should take place within 15 minutes after application in order to achieve maximal tumor doses (compare detailed discussion on biodistribution in Figure 10).
ACS Paragon Plus Environment
8
Page 9 of 24
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
Figure 4. Blood concentration of tin and tungsten after application of β-SnWO4 (23 mg/kg bodyweight). Blood samples were taken for t = 5, 15, 30, 60, 120, 240, 480, 1440 minutes after application via cardiocentesis.
In vivo breast-adenocarcinoma mouse model As a conceptual study, an orthotopic breast cancer BALB/c mouse model was used. To this concern, female BALB/c-mice in the age of 8–12 weeks were orthotopically implanted with a murine 4T1 breast-adenocarcinoma mouse cell line in the breast tissue between the forelegs (SI: in vivo studies). This cell line was used as a model system, because tumor growth and metastatic spread of 4T1 cells very closely mimic human breast cancer and can be directly transplanted into immune competent recipients. Based on 4T1 cells (1×106 cells per mL), moreover, a spontaneously metastasizing breast tumor model was applied that also allows studying metastasis in addition to the primary tumor (Figure 5).
ACS Paragon Plus Environment
9
ACS Nano
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 10 of 24
Figure 5. Scheme illustrating the orthotopic breast cancer BALB/c mouse model for in vivo studies. (a) Inoculation of 4T1 cells (1×106) into the mammary fat pad of BALB/c mice (group 1−4). (b) Intraperitoneal injection of protamine-functionalized β-SnWO4 (23 mg/kg of body weight, in PBS buffer) into the abdominal cavity (group 1). (c) Local blue-light LED illumination at the tumor site (λmax = 465 nm, duration: 5 minutes, group 1 with n = 6). Treatment of control mice with doxorubicin (2.5 mg/kg of body weight, group 2 with n = 6) and DPBS (group 3 with n = 6). 11 days after tumor-cell transplantation, mice were separated into three groups and intraperitoneally injected into the abdominal cavity with: Group 1−protamine-functionalized βSnWO4 (23 mg/kg of body weight, in PBS buffer) (Figure 5); Group 2−the chemotherapeutic agent doxorubicin as a positive control (2.5 mg/kg of body weight, in PBS buffer); Group 3−physiological saline (DPBS) only as a negative control. For each animal, injections of the relevant type were applied every other day for a total of seven doses. Group 1 was illuminated locally just for 5 minutes at the position of the breast tumor after each injection using a blue-light LED (emission: 450-490 nm; λmax: 465 nm; luminous flux: 12 lm; irradiance: 0.2 W/cm2) (Figure 5). Doxorubicin as one of the most widely used chemotherapeutic agent was applied as a reference for a chemotherapeutic anti-tumor effect.48-50 Body weight and tumor growth of all animals were documented during the complete survey (SI: Figure S4). During treatment, all animals did not show any signs of abnormality or mortality, but normal behavior. Mice did also not loose body weight (SI: Figure S4). The tumor size of mice treated with physiological saline (DPBS, group 3) increased from day 1 to day 21, indicating that DPBS did not have any effect on the tumor growth (Figure 6). In contrast, β-SnWO4-treated mice
ACS Paragon Plus Environment
10
Page 11 of 24
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
(group 1) showed a fundamentally reduced growth of the primary tumor (Figure 6). 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 control groups treated with DPBS (group 3), and even as compared to doxorubicin (group 2). In both cases some tumors grew to volumes up to 6 cm³ (Figure 6). Notably, normal statistical spreading of the tumor volume is observed for DPBS (group 3) as well as for doxorubicin (group 2), whereas the size difference of the βSnWO4-treated group 1 is much more narrow.
Figure 6. Effect of β-SnWO4 nanoparticles on the growth of the primary tumor. Group 1− −treated with β-SnWO4 nanoparticles (23 mg/kg of body weight), subsequently illuminated for 5 minutes with blue-light LED (λmax = 465 nm) at the site of the tumor; Group 2−treated with doxorubicin (2.5 mg/kg of body weight); Group 3− −treated with DPBS (each value represents the mean of n = 6 animals in the respective group; SI: Figure S5 for detailed data on individual animals). Significance between the respective groups was determined according to student’s t-test with p < 0.05 (*). It is to be noted that broken-up tumors in the DPBS and doxorubicin group continuously lose blood and appear smaller than non-broken-up tumors, leading to certain spreading of the measured tumor volume.
Only the control group and the doxorubicin-treated group showed cortically visible, brokenup tumors whereas this was not the case for the β-SnWO4-treated group. As these broken-up tumors continuously lose blood, they appear smaller than non-broken-up tumors, which also explains the higher size spreading for the DPBS and doxorubicin group (Figure 6, SI: Figure S5). Obviously, the nanoparticle accumulation in the tumor tissue is sufficient even without specific
ACS Paragon Plus Environment
11
ACS Nano
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 12 of 24
targeting (e.g. antibodies). Surprisingly, β-SnWO4-treated mice that were not subjected to bluelight LED illumination also showed significantly decreased tumor volumes (SI: Figures S6-S8). Since living mice cannot be kept in the dark, this finding−similar to previous in vitro studies41−points to the fact that β-SnWO4 is also activated by daylight. Continuous daylight illumination (several hours) after injection obviously results in a similar effect as compared to short-timed (5 minutes), but more powerful blue-light LED illumination. For small sized tumors (
Article
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 ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b03060 • Publication Date (Web): 19 Feb 2016 Downloaded from http://pubs.acs.org on February 20, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Nano is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 24
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
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), Engesserstr. 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-Str. 13-17, 68167 Mannheim (Germany)
ACS Paragon Plus Environment
1
ACS Nano
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 24
ABSTRACT: The nanoparticulate inorganic photosensitizer β-SnWO4 is suggested for photodynamic therapy (PDT) of near-surface tumors via reiterated 5-minutes 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 materials complexity. Anti-tumor and anti-metastatic 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 anti-metastatic effect being similar to chemotherapeutic doxorubicin but–according to blood counts–at significantly reduced side effects. Based on low materials 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, anti-metastatic. Table of Content Figure.
ACS Paragon Plus Environment
2
Page 3 of 24
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
Photodynamic 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 non-damaging to healthy tissue. Thus, it may help to avoid the disadvantage of conventional chemotherapeutic agents, such as serious adverse reactions or multi-drug resistances of certain malignant cells. 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/or located illumination guarantee a selective tackling of malignant tumors, while preserving non-illuminated surrounding tissue.1-4 In principle, two classes of photosensitizers are discussed for PDT: molecular photosensitizers (most often porphyrine-based),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 (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).1117,33,34
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 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 rareearth 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
ACS Paragon Plus Environment
3
ACS Nano
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 24
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 longterm 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 Based on 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 waterbased synthesis of high-quality nanoparticles (mean particle diameter: 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 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 (SI: Figure S1). In difference 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 as-prepared 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 as-prepared βSnWO4 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,S3). As a cationic biopolymer protamine adheres easily on the
ACS Paragon Plus Environment
4
Page 5 of 24
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
negatively charged β-SnWO4 nanoparticles and naturally reduces the sum zeta potential to about −20 mV in the physiologically relevant pH-range (Figure 1c). 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 anti-tumor agents.39 All in vivo experiments described hereafter were performed with protamine-functionalized β-SnWO4 nanoparticles throughout.
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 asprepared and protamine-functionalized β-SnWO4 nanoparticles. With a band gap of 2.7 eV, β-SnWO4 can be excited at wavelength < 550 nm, thus, blue light (Figure 2a). This is advantageous in comparison to alternative inorganic photocatalysts such as TiO2 or ZnO being limited to UV-excitation (< 380 nm).11-17,38 Since the penetration of blue light into tissue is limited, PDT based on β-SnWO4 nanoparticles needs to address near-surface tumors, including skin cancer, esophageal/gastric/colon tumors as well as certain types of breast cancer.1-4,39,43-45 As a preliminary test regarding cell uptake, systemic toxicity (in the dark) and phototoxicity (under blue-light illumination) towards tumor cells, human liver carcinoma cells (HepG2) were cultivated and incubated with protamine-functionalized β-SnWO4 nanoparticles (Figure 2b). According to confocal laser scanning microscopy (CLSM), the nanoparticles are readily taken up as demonstrated by intense green fluorescence within the cytosolic compartment (λexc = 458 nm, λem = 530 nm),41 but do not show any toxic effect or impact on cell viability. Moreover, normal proliferation rates and organelle integrity were proven in previous studies if
ACS Paragon Plus Environment
5
ACS Nano
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 6 of 24
the β-SnWO4-treated HepG2 cells are kept in the dark.41 In contrast, the HepG2 cells exhibit unusual spherical shapes and debris indicating encumbered growth and beginning apoptosis if illuminated continuously with a blue-light LED for about 4 hours (LED with λmax = 465 nm; emission: 450-490 nm; luminous flux: 12 lm; irradiance: 0.2 W/cm2) (Figure 2c). Notably, bluelight LED illumination did not affect the cells if they were not transfected with β-SnWO4 nanoparticles in advance. In previous studies (under artificial-daylight illumination),41 MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays also pointed to a low systemic cytotoxicity of β-SnWO4 after transfection in HepG2 cells as well as to the absence of negative long-term effects.
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 protamine-functionalized β-SnWO4 nanoparticles in the dark and after blue-light LED illumination for 4 hours (1×104 cells, 5 µM β-SnWO4). 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 non-fluorescent 2´,7´-dichlorodihydrofluorescein (H2DCF), which is subsequently oxidized into the highly fluorescent dichlorofluorescein (DCF) if ROS are present.46 HeLa cells were incubated with
ACS Paragon Plus Environment
6
Page 7 of 24
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
10 µM of β-SnWO4 for 24 hours, either in the dark or under blue-light LED illumination (Figure 3a,b). Negative controls were incubated under the same conditions, but in absence of βSnWO4 nanoparticles (Figure 3d,e). HeLa cells treated with H2O2 for 4 hours served as positive control (Figure 3c). 45 minutes 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).
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 βSnWO4 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).
Pharmacokinetic studies Based on 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
ACS Paragon Plus Environment
7
ACS Nano
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 8 of 24
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 minutes after application via cardiocentesis. Microwave-assisted digestion of 2-550 mg of blood and extraction of the analytes tin and tungsten was performed at 140 °C using HCl, HNO3 (3:1) and small amounts of HF. Blood of groups treated with physiological saline (DPBS) served as a control, but did not include detectable amounts of tin and tungsten. As presented in Figure 4, the concentration of the β-SnWO4 nanoparticles over time can be approximated by a bateman-function. 15 minutes after drug administration (tmax), the highest levels of tin and tungsten were detected. However, only portions of the applied drug enter the blood stream (cmax ~ 9 mg Sn/kg of blood) as often observed for intraperitonial application.47 It is to be noted that−unlike the suspension itself−the blood sample did not harbor tin and tungsten in a 1:1 ratio as expected for β-SnWO4, but comprised−especially in the long run−slightly lower amounts of tungsten. Assuming that β-SnWO4 slowly splits into the binary oxides SnO2 and WO3 under physiological conditions,41 this effect might by explained by precipitation of small amounts of WO3, which – once hydrolyzed to soluble [WO4]2– – can be excreted. The main part of tin and tungsten is cleared out of the blood stream within 8 hours, and falls below detection limit within 24 hours, thus indicating a comparably short half-life, which can in general be compensated by repeated application of β-SnWO4. Based on these results, we concluded for the following in vivo studies to apply and illuminate several times. Moreover, the interval of illumination should take place within 15 minutes after application in order to achieve maximal tumor doses (compare detailed discussion on biodistribution in Figure 10).
ACS Paragon Plus Environment
8
Page 9 of 24
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
Figure 4. Blood concentration of tin and tungsten after application of β-SnWO4 (23 mg/kg bodyweight). Blood samples were taken for t = 5, 15, 30, 60, 120, 240, 480, 1440 minutes after application via cardiocentesis.
In vivo breast-adenocarcinoma mouse model As a conceptual study, an orthotopic breast cancer BALB/c mouse model was used. To this concern, female BALB/c-mice in the age of 8–12 weeks were orthotopically implanted with a murine 4T1 breast-adenocarcinoma mouse cell line in the breast tissue between the forelegs (SI: in vivo studies). This cell line was used as a model system, because tumor growth and metastatic spread of 4T1 cells very closely mimic human breast cancer and can be directly transplanted into immune competent recipients. Based on 4T1 cells (1×106 cells per mL), moreover, a spontaneously metastasizing breast tumor model was applied that also allows studying metastasis in addition to the primary tumor (Figure 5).
ACS Paragon Plus Environment
9
ACS Nano
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 10 of 24
Figure 5. Scheme illustrating the orthotopic breast cancer BALB/c mouse model for in vivo studies. (a) Inoculation of 4T1 cells (1×106) into the mammary fat pad of BALB/c mice (group 1−4). (b) Intraperitoneal injection of protamine-functionalized β-SnWO4 (23 mg/kg of body weight, in PBS buffer) into the abdominal cavity (group 1). (c) Local blue-light LED illumination at the tumor site (λmax = 465 nm, duration: 5 minutes, group 1 with n = 6). Treatment of control mice with doxorubicin (2.5 mg/kg of body weight, group 2 with n = 6) and DPBS (group 3 with n = 6). 11 days after tumor-cell transplantation, mice were separated into three groups and intraperitoneally injected into the abdominal cavity with: Group 1−protamine-functionalized βSnWO4 (23 mg/kg of body weight, in PBS buffer) (Figure 5); Group 2−the chemotherapeutic agent doxorubicin as a positive control (2.5 mg/kg of body weight, in PBS buffer); Group 3−physiological saline (DPBS) only as a negative control. For each animal, injections of the relevant type were applied every other day for a total of seven doses. Group 1 was illuminated locally just for 5 minutes at the position of the breast tumor after each injection using a blue-light LED (emission: 450-490 nm; λmax: 465 nm; luminous flux: 12 lm; irradiance: 0.2 W/cm2) (Figure 5). Doxorubicin as one of the most widely used chemotherapeutic agent was applied as a reference for a chemotherapeutic anti-tumor effect.48-50 Body weight and tumor growth of all animals were documented during the complete survey (SI: Figure S4). During treatment, all animals did not show any signs of abnormality or mortality, but normal behavior. Mice did also not loose body weight (SI: Figure S4). The tumor size of mice treated with physiological saline (DPBS, group 3) increased from day 1 to day 21, indicating that DPBS did not have any effect on the tumor growth (Figure 6). In contrast, β-SnWO4-treated mice
ACS Paragon Plus Environment
10
Page 11 of 24
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
(group 1) showed a fundamentally reduced growth of the primary tumor (Figure 6). 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 control groups treated with DPBS (group 3), and even as compared to doxorubicin (group 2). In both cases some tumors grew to volumes up to 6 cm³ (Figure 6). Notably, normal statistical spreading of the tumor volume is observed for DPBS (group 3) as well as for doxorubicin (group 2), whereas the size difference of the βSnWO4-treated group 1 is much more narrow.
Figure 6. Effect of β-SnWO4 nanoparticles on the growth of the primary tumor. Group 1− −treated with β-SnWO4 nanoparticles (23 mg/kg of body weight), subsequently illuminated for 5 minutes with blue-light LED (λmax = 465 nm) at the site of the tumor; Group 2−treated with doxorubicin (2.5 mg/kg of body weight); Group 3− −treated with DPBS (each value represents the mean of n = 6 animals in the respective group; SI: Figure S5 for detailed data on individual animals). Significance between the respective groups was determined according to student’s t-test with p < 0.05 (*). It is to be noted that broken-up tumors in the DPBS and doxorubicin group continuously lose blood and appear smaller than non-broken-up tumors, leading to certain spreading of the measured tumor volume.
Only the control group and the doxorubicin-treated group showed cortically visible, brokenup tumors whereas this was not the case for the β-SnWO4-treated group. As these broken-up tumors continuously lose blood, they appear smaller than non-broken-up tumors, which also explains the higher size spreading for the DPBS and doxorubicin group (Figure 6, SI: Figure S5). Obviously, the nanoparticle accumulation in the tumor tissue is sufficient even without specific
ACS Paragon Plus Environment
11
ACS Nano
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 12 of 24
targeting (e.g. antibodies). Surprisingly, β-SnWO4-treated mice that were not subjected to bluelight LED illumination also showed significantly decreased tumor volumes (SI: Figures S6-S8). Since living mice cannot be kept in the dark, this finding−similar to previous in vitro studies41−points to the fact that β-SnWO4 is also activated by daylight. Continuous daylight illumination (several hours) after injection obviously results in a similar effect as compared to short-timed (5 minutes), but more powerful blue-light LED illumination. For small sized tumors (
