PL-W18O49-TPZ Nanoparticles for Simultaneous Hypoxia-Activated

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PL-W O -TPZ Nanoparticles for Simultaneous HypoxiaActivated Chemotherapy and Photothermal Therapy Peichen Zhao, Shuangshuang Ren, Yumei Liu, Wei Huang, Chao Zhang, and Jian He ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17323 • Publication Date (Web): 09 Jan 2018 Downloaded from http://pubs.acs.org on January 9, 2018

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ACS Applied Materials & Interfaces

PL-W18O49-TPZ Nanoparticles for Simultaneous Hypoxia-Activated Chemotherapy and Photothermal Therapy

Peichen Zhao,† Shuangshuang Ren,⊥ Yumei Liu,⊥ Wei Huang,*,† Chao Zhang,*,§ and Jian He*,‡ †

State Key Laboratory of Coordination Chemistry, Nanjing National Laboratory of

Microstructures, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu, 210093, P. R. China. ‡

Department of Radiology, Nanjing Drum Tower Hospital, The Affiliated Hospital of

Nanjing University Medical School, Nanjing University, Jiangsu, 210008, P. R. China. §

Collaborative Innovation Center of Chemistry for Life Sciences, College of

Engineering and Applied Sciences, Nanjing University, Jiangsu, 210093, P. R. China. ⊥

Department of Periodontology, Nanjing Stomatological Hospital, Medical School of

Nanjing University, Nanjing, 210093, Jiangsu, P. R. China.

1

ACS Paragon Plus Environment

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ABSTRACT: The combination of W18O49 and Tirapazamine (TPZ) core has been first introduced into the preparation of PEG-PCL (PL) surrounded nanoparticles (NPs). The aim of using W18O49 is employing its capability of reacting with the absorbed O2 to generate reactive oxygen species (ROS) when exposed to a long wavelength laser at 808 nm to increase skin penetration and body tolerance. In this work, we have demonstrated that W18O49 unit gives rise to more hypoxic tumor microenvironment and activate the prodrug TPZ to achieve hypoxia-activated chemotherapy, which could be monitored by the intracellular ROS/hypoxia detection and in vivo PET imaging. In addition, the successful introduction of W18O49 into PL-W18O49-TPZ NPs could render the photothermal therapy under the irradiation of an 808 nm laser. As a result, in vivo antitumor results have clearly shown that PL-W18O49-TPZ NPs could efficiently erase the solid tumor tissues by means of simultaneous hypoxia-activated chemotherapy and photothermal therapy. In comparison with costly small-molecule photosensitizer Ce6 used in hypoxia-activated chemotherapy, W18O49 NPs have two advantages of large-scale preparation and additional photothermal therapy effect which could provide new insight into future clinical applications. Keywords:

nanoparticles,

W18O49,

TPZ,

hypoxia,

chemotherapy

2


photothermal

therapy,

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1. INTRODUCTION Hypoxia is generally defined as an insufficient oxygen supply and found to exist in most solid tumors because of the rapid proliferation of the tumor cells and the incomplete development of vasculature.1,2 Hypoxia not only induces the development and metastasis of the tumors3-7 but also severely reduces the sensitivity of the tumor tissues to conventional chemotherapy, radiotherapy (RT) and photodynamic therapy (PDT)8-17 leading to a major cause of cancer treatment failure. In PDT, ROS can be generated from a PDT agent illuminated by light to kill the cancer cells. However, most conventional PDT agents, such as chlorine e6 (Ce6) and indocyanine green (ICG), can only generate singlet oxygen (1O2) by reacting with the local O2. This process exacerbates the lack of oxygen and serious hypoxic degree inside a tumor, which makes the tumor cells less sensitive to the PDT treatment thereby enhancing their survival against PDT.18-24 Besides, traditional RT with low dosage can exacerbate the hypoxic condition resulting in the increase of the radiation inert to tumor cells and the decrease of the RT effect.25 Moreover, the tumor cells that survive the PDT or RT treatment are more resistant and malignant, and they proliferate throughout the entire solid tumors and contribute to a rapid local recurrence.26 In order to overcome the above-mentioned problems, oxygen loaded carriers are developed to supply gaseous oxygen to a solid tumor, enrich the intratumoral oxygen level and potentiate the PDT and RT effect.27-31 Different from the growing interest in reversing or alleviating the hypoxic microenvironment in solid tumors, Qian et al. reported anaerobe-inspired anticancer 3



nanovesicles as an alternative solution,32 which intensifies the hypoxic degree for tumor microenvironment triggering a prodrug TPZ to release the toxic oxidizing radicals for enhanced treatment efficacy. In addition, Feng et al. proposed a similar strategy by using AQ4N-hCe6-liposome for hypoxia-activated chemotherapy. These two papers are believed to provide a new strategy and open a new avenue for tumor treatment. In their system, Ce6 plays an important role in strengthening the hypoxic environment when exposed to the irradiation of a 664 nm laser. However, Ce6 is expensive and rapidly metabolic as a small molecule photosensitizer. On the other hand, the 664 nm laser has poor skin penetration and low body tolerance. Both disadvantages of Ce6 limit its future clinical applications. Recently, W18O49 NPs integrating X-ray computed tomography (CT) imaging and photothermal therapy (PTT) are extensively studied.33-40 It has been clear to us that W18O49 NPs could react with the absorbed O2 to generate 1O2 and ⋅O2−33,34,41 when exposed to an 808 nm laser. In addition, W18O49 NPs as an inorganic nanomaterial has a slow metabolic rate compared with Ce6, which makes it a more suitable candidate for better experimental operation, comparison and evaluation. It should be pointed out that an 808 nm laser irradiation is more conducive to deeper skin penetration and higher body tolerance. Based upon the afore-mentioned considerations, we have designed a kind of multi-functional PL-W18O49-TPZ NPs. Intracellular ROS/hypoxia detection and in vivo PET imaging have proved that PL-W18O49-TPZ NPs could result in more hypoxic tumor microenvironment. Further in vivo antitumor results have clearly 4


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demonstrated that these PL-W18O49-TPZ NPs could efficiently erase the solid tumor tissue with the help of TPZ. It is the first attempt making use of the core W18O49 structure in creating more hypoxic tumor microenvironment and making TPZ take effect under the irradiation of a longer wavelength laser at 808 nm, where hypoxia-activated chemotherapy and photothermal therapy have been achieved at the same time for PL-W18O49-TPZ NPs.

2. EXPERIMENTAL SECTION. 2.1 Materials. TPZ, tungsten hexachloride (WCl6), N,N’-dimethylformamide (DMF), ε-caprolactone (ε-CL) and diethylene glycol (DEG) were all obtained from Sigma-Aldrich (St. Louis, MO). All the chemicals were of analytical grade and used as received without further purification. The human cervical carcinoma cell line (HeLa) was obtained from the Nanjing Institute of Cell Biology (Nanjing, China).

2.2 Synthesis of PL-W18O49-TPZ NPs. W18O49 NPs and PEG-PCL (PL) NPs were first synthesized according to the previous report.35,42 After that, PL-W18O49-TPZ NPs were synthesized via an emulsion-solvent technique. Briefly, a mixture of deionized water (50 mL) containing PEG-PCL (50 mg) and a dichloromethane suspension (5 mL) containing W18O49 (5 mg) and TPZ (2 mg) was ultrasonicated for 0.5 h, and PL-W18O49-TPZ NPs were obtained after the centrifugation and washing with deionized water.

5



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2.3 Characterization of PL-W18O49-TPZ NPs. The morphology of PEG-PCL and PL-TPZ-W18O49 NPs were observed by transmission electron microscopy (JEOL, TEM-2100, TEM-100). A dynamic light scattering (DLS) technology (BI-9000AT, Brookhaven, USA) was used to monitor the size distribution. The results were carried out in triplicate. The UV-vis absorption spectra of TPZ, PEG-PCL

NPs,

W18O49

and

PL-W18O49-TPZ

were

recorded

on

a

spectrophotometer (UV-3100, Shimadzu, Japan). The tungsten concentrations were measured with an inductively coupled plasma-mass spectrometer (ICP-MS, NexION 300 D, Perkin-Elmer Corporation, USA). The data were presented as an average of three measurements. An 808 nm diode laser (LEO photonics Co. Ltd) was used to study the photothermal effect. The change of temperature of NPs exposed to an 808 nm laser was investigated in a tin foil-capped quartz cell to prevent the evaporation of water. The photothermal conversion efficiency (η) was calculated as following equations43 (c: specific heat capacity of PL-W18O49-TPZ NP solution, 4.2×103 J/(kg·K); m: mass of PL-W18O49-TPZ NP solution, 2.5×10-3 kg; ∆t: temperature increase of PL-W18O49-TPZ NP solution; w: the laser power, 1W; t: the laser irradiation time, 600s). η (%)=

2.4

In

௖௠∆௧ ௪௧

× 100% =

Vitro

ସ.ଶ×ଵ଴య ×ଶ.ହ×ଵ଴షయ ∆௧ ଵ×଺଴଴

Cytotoxicity

of

× 100%

PL-W18O49-TPZ

NPs.

A

MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was carried out 6


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to analyze the cytotoxicity of PL-TPZ-W18O49 NPs. Firstly, HeLa cells were seeded in 96-well plates (104 cells/well) and incubated for 12 h. The medium of each well was replaced with 100 mL fresh medium containing various concentrations of PL-TPZ NPs, PL-W18O49 NPs, or PL-W18O49-TPZ NPs after 24 h incubation with or without an 808 nm laser irradiation. Five replicates were performed on testing all concentrations. After that, the medium was replaced by a 25 mL of MTT solution (2 mg/mL in PBS) and further incubated for 4 h. After that, the medium was aspirated and washed with PBS. An iMark Enzyme mark instrument (BIO-RAD Inc., USA) was employed to measure the absorbance of each well at 490 nm after the collected cells were resuspended in 200 mL DMSO.

2.5 ROS/Hypoxia Detection in HeLa Cells. ROS/hypoxia level generated in HeLa cells was monitored by employing oxidative stress/hypoxia detection kit. Briefly, HeLa Cells were seeded in the confocal dish and incubated with different groups (saline group, PL-W18O49 without laser irradiation, PL-W18O49-TPZ without laser irradiation, PL-W18O49 assisted with laser irradiation, and PL-W18O49-TPZ assisted with laser irradiation) in the dark. Hypoxia/oxidative stress detection mixture was then added into the cells after 4 h and the cells were washed with PBS after 30 minutes’ culture and exposed to the irradiation of an 808 nm laser (1 W/cm2) for 5 min. After the irradiation, the HeLa cells were washed thrice with PBS and the confocal fluorescence imaging was carried out using a Confocal Laser Scanning Microscope (CLSM, LSM 710, Zeiss). The fluorescence signal of hypoxia was 7



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performed with the excitation wavelength of 561 nm and the emission wavelength of 600 nm. The ROS was detected with the excitation wavelength of 488 nm and the emission wavelength of 520 nm.

2.6 In Vivo Micro-PET Imaging. To assess the hypoxic condition in HeLa-tumor-bearing nude mice post different treatments, a saline suspension (100 µL) containing

18

F-MISO (75 µCi/mouse) was intravenously injected into the mice.

Micro-PET scans were carried out on Inveon PET/CT system (Siemens, Malvern, PA) 1 h post injection. All the PET scanners were cross-calibrated periodically.

2.7 Simultaneous Treatment Induced Tumor Blood Vasculatures Change. To explore

the

status

hypoxia-activated

of

the

tumor

chemotherapy

blood

and

vasculatures

photothermal

post

therapy

simultaneous rendered

by

PL-W18O49-TPZ, the HeLa tumor-bearing mice were intravenously injected with of PL-W18O49-TPZ at a dose of 50 mg/kg and exposed the irradiation of an 808 nm laser (1 W/cm2) for 8 min. The mice were then sacrificed after 4 h and the tumors were collected. The tumors slices were co-stained with rat-anti-mouse CD31 antibody and Alexa Fluo 488 conjugated donkey-anti-rat antibody and then imaged by employing the CLSM.

2.8 Bio-distribution of ICG and C3 in tumor-bearing mice. PL-W18O49-TPZ suspensions (50 mg/kg) were injected into tumor-bearing mice intravenously, and the 8


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mice were sacrificed (for each time point n = 5) 1, 2, 4, 8, 12 and 24 h after injection. The tumor, heart, liver, spleen, lung and kidney were excised and weighed. An inductively-coupled plasma mass spectrometer (ICP-MS, NexION 300 D, Perkin-Elmer Corporation, USA) was used to quantify the tungsten content after the collected organs were completely lysed in aqua regia. Dimethyl sulfoxide (DMSO) was used to extract TPZ

from the organs and TPZ content was calculated based on TPZ calibration curve obtained using a spectrophotometer (UV3100, Shimadzu, Japan), respectively.

2.9 In Vivo Antitumor Effect. HeLa-tumor-bearing mice were used to investigate the in vivo antitumor effect. When the tumor volume reached ~500 mm3, HeLa-tumor-bearing mice were randomly divided into four different groups with eight mice in each group (saline group, PL-W18O49 plus laser irradiation group, PL-TPZ plus laser irradiation group, and PL-W18O49-TPZ plus laser irradiation group). All groups were injected intravenously at a dose of 50 mg/kg and then irradiated with an 808 nm laser (1 W/cm2) for 8 min at 8 h after the injection. The tumor volume and body weight were monitored and recorded. 12 days post the treatments, the mice were sacrificed, and the tumors were collected, weighed, washed thrice with PBS and fixed in a 10 % neutral buffered formalin solution. Hematoxylin and eosin (H&E) staining and TUNEL apoptosis staining tests were carried out by Nanjing Google biotechnology Company and observed via fluorescence microscopy (IX71, Olympus).

2.10 Pathology Analysis. We carried out a time course of histological changes in the 9



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mice organs (heart, liver, spleen, lung and kidney) 48 h after the simultaneous hypoxia-activated

chemotherapy

and

photothermal

therapy

rendered

by

PL-W18O49-TPZ NPs. The organs collected from the mice were embedded in paraffin after immobilization in 4 % paraformaldehyde at 4 °C for 4 h, and then the sections were treated with hematoxylin and eosin (H&E) staining and observed by employing a light microscope. Finally, representative images were acquired.

2.11 Hematology and Biochemical Assay. Mouse blood was collected on day 1, day 7 and day 21 after the simultaneous hypoxia-activated chemotherapy and photothermal therapy rendered by PL-W18O49-TPZ NPs to conduct hematology and biochemical assays. To evaluate the biocompatibility and in vivo toxicity of PL-W18O49-TPZ NPs assisted with the simultaneous hypoxia-activated chemotherapy and photothermal therapy, serological liver function was analyzed by alanine aminotransferase (ALT), total bilirubin level (TBIL) and total protein (TP). Kidney function was detected by blood urea creatinine (CRE) and nitrogen (BUN), and spleen function was correlated with the platelet level. White blood cells (WBC), Red blood cells (RBC), Lymphocytes, Monocytes (MON) and Neutrophils (NEU) were counted by flow cytometry assay, and levels of hemoglobin (HB) were used to check the immune response and potential cytotoxic effect.

2.12 Statistics. Triplicate data were analyzed with Student’s t test by employing GraphPad Prism software (version 7.0). The significance level was p < 0.05 and the 10


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significant differences were expressed by asterisks in corresponding figures.

3. RESULTS AND DISCUSSION. 3.1 Characterization. The morphology of PEG-PCL and PL-W18O49-TPZ NPs was displayed in Figures S1 and 1A. PEG-PCL NPs and PL-W18O49-TPZ NPs both showed a homogeneously dispersed spherical morphology. A 40 nm mean diameter prior to the equipment of W18O49 and TPZ was observed in PEG-PCL and a 50 nm mean diameter after the load of W18O49 and TPZ was exhibited in PL-W18O49-TPZ NPs. A hydrodynamic diameter of approximately 80 nm mean diameter prior to the equipment of W18O49 and TPZ in PEG-PCL and 105 nm mean diameter after the load of W18O49 and TPZ in PL-W18O49-TPZ NPs as analyzed by dynamic light scattering were displayed in Figures S2-S3. As shown in Table S1, drug loading content and encapsulation efficiency of W18O49 were 2.99 % and 71.72 % while those of TPZ were 1.29 % and 77.41 %. UV-Vis spectra of PEG-PCL showed no apparent absorption in the NIR region (Figure 1B), while a typical absorption peak at 460 nm and a continuous high absorption intensity as a function of wavelength up to the NIR region were observed in TPZ and W18O49 respectively. After assembly and separation via three rounds of centrifugation, new peaks corresponding to TPZ and W18O49 appeared, indicating that TPZ and W18O49 were successfully loaded. Due to the strong NIR absorbance of the PL-W18O49-TPZ NPs, we next investigated the potency of these

nanoparticles

for

translating

the

absorbed

energy

into

heat.

The

temperature-time curves of saline and PL-W18O49-TPZ NPs are shown in Figure 1C. 11



When exposed to an 808 nm laser irradiation for 10 min, the temperatures of PL-W18O49-TPZ NPs solutions increased from 25.4 °C to 63.6 °C, whereas the control solution only showed a slight temperature increase. These data confirmed that PL-W18O49-TPZ NPs exhibited promising photothermal properties enabling for cancer treatment. As indicated in Figure S4, the conversion efficiency was calculated to be near 67 %, and this value did not experience remarkable reduction over repeated laser irradiation, confirming the stability of PL-W18O49-TPZ as photothermal agents. The high temperature would cause the system to be unstable and destroy the sphere spherical. From Figure 1D, we could find that little TPZ was released within 24 h without the irradiation of an 808 nm laser. However, near 80 % TPZ was released within 24 h post the 10 min 808 nm laser irradiation.

3.2 Proof of the Induction of Hypoxic Environment. Oxidative stress/hypoxia detection kit was used to investigate the generation of ROS/hypoxia in HeLa cells after different treatments (PL-W18O49 without laser irradiation, PL-W18O49-TPZ without laser irradiation, PL-W18O49 plus laser irradiation, PL-W18O49-TPZ plus laser irradiation). By the way, untreated cells were used as the negative control. As shown in Figure 2, the generation of ROS and the induction of hypoxia in PL-W18O49 or PL-W18O49-TPZ NPs co-cultured HeLa cells could be observed after the light-trigger. However, neither green nor red fluorescence was observed without the help of laser irradiation in PL-W18O49 or PL-W18O49-TPZ NPs incubated HeLa cells. All these results proved that W18O49 core could generate ROS and induce hypoxia at the same 12


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time under laser irradiation. Furthermore, in vivo PET imaging was determined to analyze the hypoxia condition after the treatment of PL-W18O49-TPZ assisted with irradiation of an 808 nm laser (1 W/cm2) or just the intravenous injection with saline as the control group. As indicated in Figure 3, I, II, III and IV in left panels showed 4 different layers from a mouse after the treatment of PL-W18O49-TPZ assisted with irradiation of an 808 nm laser, while I, II, III and IV in right panels illustrated 4 different layers from another mouse post the intravenous injection with saline. The hypoxia condition in the left and right panels depicted totally different degrees. Namely, high hypoxia signal could be found in every layer of tumor after the treatment, whereas comparatively small range of hypoxic zone was observed inside the tumor post the intravenous injection with saline (Figure 3, white circle). As mentioned above, the incomplete development of vasculature was the major cause of hypoxia.1,2 Thus, we have carefully examined the status of the tumor blood vasculatures after the treatment of PL-W18O49-TPZ assisted with the irradiation of an 808 nm laser (1 W/cm2) or just the intravenous injection with saline. After being treated, the blood vessels were significantly damaged compared with the control group (Figure S4), again demonstrating that PL-W18O49-TPZ assisted with irradiation of an 808 nm laser could induce hypoxia and thus open a new avenue for hypoxia-activated chemotherapy.

3.3 In Vitro Anti-Tumor Efficacy. From the above-mentioned ROS/hypoxia detection, in vivo Micro-PET imaging and in vitro infrared photothermal conversion

13



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study, we can conclude that PL-W18O49-TPZ NPs could achieve hypoxia-activated chemotherapy and photothermal therapy simultaneously. Next, we have explored the efficacy of this synchronous hypoxia-activated chemotherapy and photothermal therapy in the treatment of HeLa cells by the co-culture of PL-W18O49-TPZ NPs assisted with the irradiation of an 808 nm laser (8 min, 1 W/cm2) or without laser irradiation. PL-TPZ NPs and PL-W18O49 NPs with or without laser irradiation were used as controls to investigate the single therapy. The cell viability of HeLa cells was almost unaffected as a function of TPZ and W18O49 concentrations without the laser irradiation, and still around 85 % cell viability could be found when the concentration of TPZ and W18O49 reached 10 µg/mL and 23.2 µg/mL in all samples incubated HeLa cells (Figure 4A). However, the addition of the laser irradiation would contribute to a concentration-dependent cell apoptosis in PL-W18O49 NPs (photothermal therapy) and PL-W18O49-TPZ

NPs

(simultaneous

hypoxia-activated

chemotherapy

and

photothermal therapy) incubated HeLa cells (Figure 4B). Besides, we could also find that the chemotherapy- associated cytotoxicity activated by tumor-intrinsic hypoxia without the encapsulation of W18O49 in PL-TPZ was negligible (Figure 4B). However, with the encapsulation of W18O49 in PL-TPZ to form PL-W18O49-TPZ NPs, the photothermal therapy and the effective hypoxia-activated chemotherapy were introduced at the same time, causing approximately 87 % of cell death when the concentrations of TPZ and W18O49 reached 10 µg/mL and 23.2 µg/mL. The cell death was larger than the sum of the chemotherapy activated by tumor-intrinsic hypoxia in PL-TPZ and photothermal therapy in PL-W18O49, providing superior synergistic 14


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anti-tumor effect.

3.4 In Vivo Anti-Tumor Efficacy. After the in vitro anti-tumor assays, we next investigate the efficacy of this synchronous hypoxia-activated chemotherapy and photothermal therapy in the treatment of HeLa-tumor-bearing mice by the intravenous injection of PL-W18O49-TPZ NPs assisted with the irradiation of an 808 nm laser (8 min, 1 W/cm2). Before the in vivo anti-tumor assays were conducted, the in vivo distribution

profiles

of

PL-W18O49-TPZ

NPs

were

analyzed

in

HeLa-xenograft-bearing mice at 1, 2, 4, 8, 12 and 24 h post an intravenous injection of PL-W18O49-TPZ NPs at a dose of 50 mg/kg. As shown in Figure S6, a significant accumulation of tungsten and TPZ in tumors was observed. At 8 h post-injection, the tungsten and TPZ content in tumors reached a maximum proportion of the i.v. administrated-PL-W18O49-TPZ NPs. So we choose 8 h after intravenous injection to treat the cancer. The intravenous injection of saline into the HeLa-tumor-bearing mice was used as a negative control group. The intravenous injections of PL-W18O49 NPs and PL-TPZ NPs into the HeLa-tumor-bearing mice respectively along with the irradiation of an 808 nm laser were used as the positive control groups. All groups were randomly divided with eight mice per group and the dose in all groups was 50 mg/kg. The irradiation time and the laser power density in all groups were 8 min and 1 W/cm2. As depicted in Figure 5A, a rapid tumor growth could be noticed in the negative control group (saline group), and the tumor volume increased over 7-fold on day 12 compared with day 0. From Figure 5A, we could also find that 15



PL-W18O49-TPZ NPs plus laser irradiation, PL-W18O49 plus laser irradiation, PL-TPZ NPs plus laser irradiation all take effect in controlling the increase of the tumor volume. However, the treatment in the two positive control groups was not thorough because of the incomplete hypoxia tumor environment in PL-TPZ NPs plus laser irradiation group and the lack of pro-drug TPZ. On the contrary, PL-W18O49-TPZ NPs plus laser irradiation group dramatically decreases the tumor volume, indicating synergistic therapeutic effect on the ablation of solid tumors at the same time. Figure 5B shows the body weight of the mice, and no detective changes in the body weight of the mice were observed during the course of the study for all groups, suggesting that this synchronous hypoxia-activated chemotherapy and photothermal therapy in the treatment of HeLa-tumor-bearing mice would not lead to obvious acute toxicity. H&E and TUNEL staining was used to further elevate the therapeutic efficacy after the treatments in all groups. Tumor tissues in the PL-W18O49-TPZ NPs plus laser irradiation, PL-W18O49 plus laser irradiation, PL-TPZ NPs plus laser irradiation groups all presented remarkable necrosis and apoptosis. However, the degree of necrosis and apoptosis was the largest in the PL-W18O49-TPZ NPs plus laser irradiation group (Figures 5C and 5D). Therefore, we concluded that the combined hypoxia-activated chemotherapy and photothermal therapy at the same time could provide the best therapeutic effect on tumor treatment.

3.5 Safety Evaluation. Heart, liver, spleen, lung and kidney were harvested and weighed to evaluate the in vivo safety. As showed in Figure 6A, the mice treated with 16


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PL-W18O49-TPZ did not show any noticeable variation in the mass ratio of the organ to the body. Figure 6B displayed H&E staining images of major organs (heart, liver, spleen, lung and kidney) after the mice were treated with simultaneous hypoxia-activated chemotherapy and photothermal therapy. Compared to the images from saline group, no apparent necrosis, pulmonary fibrosis, histological abnormalities and inflammation lesions were seen in these organs, demonstrating good in vivo biocompatibility of PL-W18O49-TPZ NPs. Serum bio-chemical and hematology studies were performed to estimate the potential cytotoxicity of PL-W18O49-TPZ NPs on the body after the simultaneous hypoxia-activated chemotherapy and photothermal therapy. Serum levels of TP, ALT and TBIL were used to evaluate the liver function (Figure 7A), whereas serum levels of PLT, BUN and CRE were used to monitor the kidney and spleen function (Figure 7B). Negligible changes in statistical significance were found after the treatment compared with untreated mice (control group), supplying a solid evidence that the PL-W18O49-TPZ NPs based coinstantaneous hypoxia-activated chemotherapy and photothermal therapy did not breakdown metabolism. Hematotoxicity is relevant to the clinical application of nanoparticles. Thus, the counts of RBC, WBC and HB (Figure 7C) were recorded to analyze the hematopoietic and aerobic capacity and the data throughout the course of the treatment were similar compared with untreated mice (control group). Peripheral blood LYM, MON and NEU counts were recorded to investigate potential immune responses (Figure 7D). Negligible changes in statistical significance were noticed throughout the process of treatment when compared with 17



the control group, ensuring PL-W18O49-TPZ NPs a promising candidate in clinical applications.

4. CONCLUSION Inspired by the work of Qian et al. and Feng et al. on hypoxia-activated chemotherapy, we have designed and prepared multi-functional PL-W18O49-TPZ NPs in which the W18O49 core with photothermal therapy activity has been first introduced into PL-W18O49-TPZ NPs. The replacement of costly small-molecule photosensitizer Ce6 with W18O49 NPs capable of scaled-up production accomplishes the dual function in tumor treatment, namely simultaneous hypoxia-activated chemotherapy and photothermal therapy rendered by PL-W18O49-TPZ NPs under the irradiation of a longer wavelength laser at 808 nm. Further intracellular ROS/hypoxia detection and in vivo PET imaging indicate that the introduced W18O49 unit leads to severe tumor hypoxia, which is favorable for the activation of TPZ to fulfill the hypoxia-activated chemotherapy. Our final results on in vivo antitumor experiments manifest the effect of simultaneous hypoxia-activated chemotherapy and photothermal therapy, where the solid tumor tissues have been remarkably eliminated by PL-W18O49-TPZ NPs. To the best of our knowledge, the current work is the first report on introducing the W18O49 core in PL-W18O49-TPZ NPs in order to produce more hypoxic tumor microenvironment under the irradiation of a longer wavelength laser. The achievement of simultaneous hypoxia-activated chemotherapy and photothermal therapy is thought to give some promising examples and inspirations on tumor 18


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treatment.

ASSOCIATED CONTENT Electronic Supplementary Information (ESI) available: TEM image of PEG-PCL NPs. Hydrodynamic size distributions of PEG-PCL and PL-W18O49-TPZ. Drug loading content and encapsulation efficiency of W18O49 and TPZ in PL-W18O49-TPZ NPs. Temperature of PL-W18O49-TPZ NP solution after five cycles of NIR laser irradiation (1 W/cm2, 808 nm laser, 10 min)/cooling down (1 h) process. Ex vivo immunofluorescence staining images of HeLa tumor sections obtained from mice with i.v. injection of PL-W18O49-TPZ and then received an 808 nm (1 W/cm2) laser irradiation or not. Bio-distribution analysis of PL-W18O49-TPZ NPs. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; *E-mail: [email protected]; *E-mail: [email protected]. ORCID Wei Huang: 0000-0002-1071-1055 Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS

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This work was financially supported by the Natural Science Foundation of Jiangsu Province (No. BK20171334), Medical Science and Technology Development Foundation Nanjing Department of Health (ID: YKK15067), Jiangsu province key medical young talents, "13th Five-Year" health promotion project of Jiangsu province (JH.2016-2020).

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Figure 1. (A) TEM images of PL-W18O49-TPZ NPs. (B) UV-vis spectra of PEG-PCL dissolved in deionized water, TPZ dissolved in dichloromethane, W18O49 dissolved in dichloromethane, and PL-W18O49-TPZ NPs dissolved in deionized water. The concentrations of the samples were all 1 mg/mL. (C) Temperature-time curve of saline and PL-W18O49-TPZ NPs dissolved in deionized water (1mg/mL). (D) The release of TPZ from PL-W18O49-TPZ NPs was measured assisted with or without the 808 nm laser irradiation. The concentration of the PL-W18O49-TPZ NPs was 1 mg/mL. Error bars represent standard deviation of three independent experiments.

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Figure 2. Confocal fluorescence images of HeLa cells received different treatments with ROS/hypoxia detection probes as an indicator. Scale bar: 50 µm. Cells were exposed to the irradiation of an 808 nm laser at 1 W/cm2 for 5 min.

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Figure 3. PET images of mice after the treatment of PL-W18O49-TPZ assisted with irradiation of an 808 nm laser (1 W/cm2) or just the intravenous injection with saline as the control group. I, II, III and IV in left panels showed 4 different layers from a mouse after the treatment of PL-W18O49-TPZ assisted with irradiation of an 808 nm laser, while I, II, III and IV in right panels showed 4 different layers from another mouse post the intravenous injection with saline. Tumor lesions are highlighted with white circle. 12 h post the intravenous injection of PL-W18O49-TPZ or saline, 18

F-MISO was intravenously injected into the mice and representative PET images

were obtained at 1 h after the injection of 18F-MISO.

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Figure 4. (A) In vitro viability of HeLa cells co-cultured with different concentrations of PL-TPZ NPs, PL-W18O49 NPs, or PL-W18O49-TPZ NPs after 24 h incubation without the irradiation. (B) In vitro viability of HeLa cells co-cultured with different concentrations of PL-TPZ NPs, PL-W18O49 NPs, or PL-W18O49-TPZ NPs at different concentrations after 24 h incubation with an 808 nm laser irradiation. The results are represented as the mean ± SD. Error bars represent standard deviations of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.

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Figure 5. (A) Tumor growth curves of HeLa-tumor-bearing mice subjected to different treatments as displayed. The data are expressed as the mean ± SD (n = 8). * p < 0.05, ** p < 0.01. (B) Body weight of HeLa-tumor-bearing that mice subjected to different treatments. (C) Immunohistochemical staining images of HeLa tumor sections obtained from mice subjected to different treatments with H&E staining. (D) Immunohistochemical staining images of HeLa tumor sections obtained from mice subjected to different treatments with TUNEL staining.

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Figure 6. (A) Weight ratio of the main normal organs to the body of the mice after the treatment of PL-W18O49-TPZ assisted with irradiation of an 808 nm laser (1 W/cm2) or just the intravenous injection with saline as the control group. (B) Histological images of the HE-stained heart, liver, spleen, lung and kidney harvested from the mice after the treatment of PL-W18O49-TPZ assisted with irradiation of an 808 nm laser (1 W/cm2) or just the intravenous injection with saline as the control group.

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Figure 7. Serum biochemical study and hematology assay 1, 7 and 21 days post the treatment of PL-W18O49-TPZ assisted with the irradiation of an 808 nm laser (1 W/cm2) or just the intravenous injection with saline as the control group. (A) Serum bio-chemical test of alanine aminotransferase (ALT), total bilirubin (TBIL) and total protein (TP). (B) Creatinine (CRE), blood urea nitrogen (BUN) and hematology assay of platelets (PLT). (C) Red blood cells (RBC), hemoglobin (HB) and white blood cells (WBC). (D) Monocyte (MON), lymphocyte (LYM) and neutrophils (NEU). Level 1, 7, and 21 days post-treatment of PEG-PCL-C3-ICG rendered PTT/PDT.

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PL-W18O49-TPZ Nanoparticles for Simultaneous Hypoxia-Activated

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