Overcome the limitation of hypoxia against photodynamic therapy to treat cancer cells by using perfluorocarbon nanodroplet for photosensitizer delivery

Accepted Manuscript Overcome the limitation of hypoxia against photodynamic therapy to treat cancer cells by using perfluorocarbon nanodroplet for photosensitizer delivery Xiaolei Tang, Yuhao Cheng, Shiting Huang, Ahu Yuan, Yiqiao Hu, Jinhui Wu PII:

S0006-291X(17)30618-6

DOI:

10.1016/j.bbrc.2017.03.142

Reference:

YBBRC 37529

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 18 March 2017 Accepted Date: 26 March 2017

Please cite this article as: X. Tang, Y. Cheng, S. Huang, A. Yuan, Y. Hu, J. Wu, Overcome the limitation of hypoxia against photodynamic therapy to treat cancer cells by using perfluorocarbon nanodroplet for photosensitizer delivery, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/ j.bbrc.2017.03.142. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT 1

Overcome the Limitation of Hypoxia against Photodynamic Therapy to Treat

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Cancer Cells by Using Perfluorocarbon Nanodroplet for Photosensitizer Delivery

3 Xiaolei Tang a, ‡, Yuhao Cheng a, ‡, Shiting Huang a, Ahu Yuan a, Yiqiao Hu a,b,c,d,* and

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Jinhui Wu a,b,c,d,*

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a

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University, Nanjing 210093, China

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b

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State Key Laboratory of Pharmaceutical Biotechnology, Medical School, Nanjing

Jiangsu Key Laboratory for Nano Technology, Nanjing University, Nanjing 210093,

China

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Institute of Drug R&D, Medical School, Nanjing University, Nanjing 210093, China

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Jiangsu R&D Platform for Controlled & Targeted Drug Delivery, Nanjing

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University, Nanjing 210093, China

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Co-first authors: These authors contributed equally to this work.

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Co-corresponding authors.

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E-mail address: [email protected] (Y. Hu); [email protected] (J. Wu).

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‡

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ACCEPTED MANUSCRIPT Abstract:

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The low oxygen concentration limits the therapeutic efficacy of photodynamic

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therapy in treating cancer cells in hypoxia, since the cytotoxic 1O2 can’t be effectively

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generated in this condition. To overcome this, we load photosensitizer into

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perfluorocarbon nanodroplet, which has a high oxygen capacity to enrich O2 for

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photodynamic consumption. Under the well-controlled hypoxic condition, we test its

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efficacy both in vitro and in vivo. This method can be successfully used for destroying

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cancer cells in hypoxic condition.

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28 Keywords:

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Photodynamic Therapy, Perfluorocarbon, Drug Delivery System, Nanomedicine,

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Hypoxic Tumor

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ACCEPTED MANUSCRIPT 1. Introduction

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Photodynamic therapy (PDT) is a promising strategy which utilizes the cooperation of

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oxygen, photosensitizer and light to treat cancer. Under an appropriate laser

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irradiation, photosensitizers (PS) can be stimulated to the excited triplet state (3PS),

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which can subsequently transfer its energy to molecular oxygen (O2) and leads to the

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formation of singlet oxygen (1O2) for killing cancer cells [1].

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Although satisfied effect in treating superficial tumor has already been achieved, the

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photodynamic efficacy in hypoxic environment (commonly formed in solid tumor) is

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still impaired [2-4]. These hypoxia areas (usually pO2 < 1.3% [5]) are formed and

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aggravated due to the fast proliferation of tumor cells and the lack of tumor

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vasculatures [6]. The efficacy of 1O2 generation is fundamentally depended on the

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concentration of O2, thus low oxygen concentration would hampered the

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photodynamic treatment to cancer cells in hypoxia. Results suggested that full effect

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was reached at 5% O2; the half value of cell inactivation was found to be at 1% O2;

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nearly no cell kill was achieved under anoxic conditions [7].

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Ameliorating the hypoxia in tumor may contribute to the efficient 1O2 generation

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during irradiation. Previous trials such as hyperbaric oxygen inhaling [8-11], dividing

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irradiation into light-dark circles [12] or extending irradiation with a low fluence rate

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[13] were demonstrated. However, due to the intrinsic absence of tumor vasculature,

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the vascular shutdown effects by photodynamic treatment and the photodynamic

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oxygen consumption, these methods cannot effectively reverse the depletion of

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ACCEPTED MANUSCRIPT oxygen [14, 15]. Thus, it is essential to develop an effective approach to treat cancer

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cells in hypoxia.

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More recently, we reported a method called “oxygen self-enriching photodynamic

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therapy (Oxy-PDT)” to enhance the 1O2 generation of photosensitizer [16]. However,

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whether our technique can remove cancer cells in hypoxic tumor remains unclear.

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Herein we load photosensitizer into perfluorocarbon nanodroplet to be an effective

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photodynamic nanomedicine, which can get enhanced efficacy in hypoxia (Figure 1a).

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IR780, a hydrophobic near-infrared (NIR) photosensitizer was chosen in this

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demonstration (Figure 1b); Perfluorohexane (PFH), an FDA approved ultrasound

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contrast agent was chosen to be the perfluorocarbon (PFC) core. Due to the intrinsic

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high oxygen capacity of perfluorocarbon (Figure S1), we supposed that the oxygen

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concentration in PFC can maintain at a higher level than that in surrounding hypoxic

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tumor matrix. Thus during the laser irradiation, the 1O2 generation in PFC phase

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would be much more and faster. Subsequently, the generated 1O2 would be

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continually released into tumor matrix for tumor killing and the consumed O2 in PFH

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phase would be fast recovered, which is based on the Le Chatelier’s principle. By this

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way, although tumor was still in hypoxia, the photodynamic efficacy would be

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significantly increased by using PFH as a “sponge for oxygen” (Figure 1c).

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2. Materials and Methods

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2.1. Chemicals and Reagents 4 / 15

ACCEPTED MANUSCRIPT Lecithin and cholesterol were bought from Aladdin Industrial Corporation, and

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DSPE-PEG2000 was purchased from A.V.T. Pharm. Ltd. (Shanghai, China). IR780

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was obtained from Sigma-Aldrich Chemical Corporation. The 99% perfluorohexane

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was obtained from J&K Scientific Ltd. (Beijing, China). Singlet Oxygen Sensor

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Green was from Molecular Probes, Inc. Carboxy-H2DCFDA was supplied by

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Invitrogen (USA). Cell Counting Kit-8 (CCK-8) was bought from Dojindo

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Laboratories (Japan). HIF-1α monoclonal antibody was purchased from Abcam (UK).

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All the chemicals were used as supplied without further purification.

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2.2. Synthesis of LIP(IR780+PFH) and LIP(IR780)

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24.65 mg lecithin, 4.28 mg cholesterol, 3.79 mg DSPE-PEG2000 and specified IR780

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were dissolved in 5 ml dichloromethane. Dichloromethane was removed from 25 ml

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round flask by rotary evaporation to form lipid films. 1.4 ml pure water was added

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and the film was peeled off by using 10-min sonication. 0.6ml PFH were added

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gradually under high-speed dispersion (IKA, T25, German) at 24000 r/min in ice bath

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for 10 min to form 2 ml LIP(IR780+PFH) (30 v/v% PFH). LIP(IR780) can be formed

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by replacing PFH with pure water.

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2.3. Detection of Singlet Oxygen In Vitro

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0.1 ml samples and 0.02 ml of 50 µM SOSG were mixed in black 96-well plates

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(Costar). Then the plate was put into the 2.5 L GENbox Jar with GENbox Anaer

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(bioMérieux Inc., Shanghai, China) consuming oxygen, Z-1100 Oxygen Meter

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(MuCheng Corporation, Shanghai, China) indicating the concentration of oxygen.

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ACCEPTED MANUSCRIPT After irradiation (808-nm, 2 W/cm2), the oxidized SOSG was quantified by measuring

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the fluorescence intensity (excited at 504 nm and measured at 525 nm) using a

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multifunctional microplate reader (Safire, TECAN). All operations need to avoid light.

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The experiments for each group were run in triplicates. The CT26 cells were seeded

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with a density of 1× 104 per well in 96-well plates. After the cells were incubated for

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24 h, the medium were replaced with 100 µl fresh culture medium. Then 100 µl

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LIP(IR780) or LIP(IR780+PFH) were added into the well, respectively. The cells

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were further incubated for 30 min at 37 °C and 5% CO2. The final concentration of

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IR780 and PFH (v/v%) was 10 µg/ml and 7.5%. After washing once with PBS, the

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cells were incubated with 100 µl carboxy-H2DCFDA (25 µM). Then the plate was

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put into a chamber with 1% O2 for 30 min. Subsequently, the cells were irradiated

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with 808-nm laser (2 W/cm2) for 20 s each well. Finally, the cells were replaced with

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100 µl PBS. The fluorescence carboxy-H2DCFDA (Ex/Em = 495/529 nm) was

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immediately captured on a fluorescence microscope (Nikon ECLIPSE Ti).

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2.4. Cytotoxicity

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The CT26 cells were seeded into 96-well plate at a density of 8 × 103 cells per well.

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After incubation for 24 h, the cells were treated with LIP(IR780) or LIP(IR780+PFH)

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at different concentrations (finally concentrations of 20 µl samples mixed with 100 µl

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culture medium). Then the plate was put into GENbox Jar with GENbox Anaer

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consuming oxygen. When Oxygen Meter indicated 1% O2 in the Jar, the cells were

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irradiated with 808-nm laser (2 W/cm2) for 20 s per well. After co-incubation for 2 h,

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ACCEPTED MANUSCRIPT the drugs were removed and washed once with PBS. Finally, the mixed solution

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consisting of CCK-8 (10 µl) and fresh culture medium (100 µl) were added into each

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well, and incubated for an additional 2 h at 37°C and 5% CO2. The absorbance was

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measured at 450 nm using the microplate reader. The cells without any drugs or NIR

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irradiation were taken as the negative control. Furthermore, cells were stained with

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calcein-AM for visualization of live cells and with PI for visualization of dead/late

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apoptotic cells, according to the manufacturer’s suggested protocol (Invitrogen). The

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fluorescence calcein-AM (490/515nm) and PI (535/617nm) was immediately captured

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on a fluorescence microscope (Nikon ECLIPSE Ti).

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2.5 Animals and Tumor Model

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Age 4-6 week BALB/C male mice were purchased from Yangzhou University

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Medical Center (Yangzhou, China), and used in accordance with Institutional Animal

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Care and Use Committee (IACUC) of Nanjing University. Anesthesia was done by

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intraperitoneal injection of 1% pelltobarbitalum natricum (0.12 ml). Hairs on the

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flanks of the mice were removed before they received further treatments. Tumors

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were first developed in BALB/C mice by subcutaneously implanting 1 × 107 CT26

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cells suspended in 100 µl of serum-free RPMI 1640 in the lower flanks of the mice.

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When the tumor volume reached about 200 mm3, the tumor mass was removed and

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cut into small pieces about 2-6 mm3, subcutaneously implanted into other mice.

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2.6. HIF-1α Staining Assay

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ACCEPTED MANUSCRIPT The CT26 subcutaneous tumors were developed in 4-to 6-week-old BALB/C male

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mice. When the tumor volume reached 50mm3, the following study was performed.

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The groups were as follows: group 1: 21% O2; group 2: 7% O2. The group 2 mice

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were put into a chamber, adjusting oxygen and nitrogen flow rate to make the 7% O2

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in the chamber. Half an hour later, the mice were taken out and immediately killed.

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The tumors of two groups were removed and performed according to the HIF-1α

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immunocytochemistry instructions (Abcam).

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2.7. PDT on Tumor-bearing Mice by Intravenous Injection

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The CT26 subcutaneous tumors were first developed in the mice as described above.

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PDT treatment was then performed at 8 d after inoculation of the tumor pieces. The

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initial average tumor volume was 50 mm3. Testing groups were as follows: group 1:

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Saline; group 2: Saline+NIR; group 3: LIP(IR780); group 4: LIP(IR780)+NIR; group

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5:LIP(IR780+PFH); group 6: LIP(IR780+PFH)+NIR. 200 µl of LIP(IR780) (60

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µg/ml IR780), LIP(PFH+IR780) (60 µg/ml IR780, 10 v/v% PFH) or saline alone were

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intravenously injected. The mice in LIP(IR780+PFH) group were taken near-infrared

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images of IR780 accumulation in tumors at 0.5, 10, 24, 48 h and ultrasound images of

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tumor accumulation of PFH in tumor pre- and 24 h post-injection. 24 hours later, laser

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treatment was performed on groups 3 and 5 by irradiating the tumor region with an

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808-nm laser (2 W/cm2) for two consecutive exposures of 10 s each, 1-min time

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interval was added between the two irradiations for tumor cooling. Tumor size was

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measured every two days using a vernier caliper for 10 d after the first PDT treatment.

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ACCEPTED MANUSCRIPT Changes in the tumor volume as a function of time were determined for each mouse

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by normalizing the tumor volume at day X to their respective tumor volume at day 0

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after treatment. Mice were randomly selected in each group and tumor was

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photographed at day 0, 5 and 10. At 10 d after treatment the mice of each group were

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euthanized and the tumors were removed to photograph and weigh. Mice will be

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sacrificed if the tumor volume is larger than 2000 mm2.

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164 3. Results and Discussion

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The IR780 loaded perfluorohexane nanodroplet (LIP(PFH+IR780)) was prepared by

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emulsifying PFH with IR780 loaded lipids (including lecithin, cholesterol and

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DSPE-PEG2000). It maintained stable with an average diameter at about 250 nm

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(Figure S2) and a zeta potential at -39.6 mv. To demonstrate the photodynamic

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efficacy of LIP(IR780+PFH) in hypoxic condition, LIP(IR780) were also prepared for

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control and then samples were put in atmospheres with different hypoxic fractions. A

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fluorescent marker singlet oxygen sensor green (SOSG) were added to measure the

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1

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(808 nm) for 20 s, fluorescence of oxidized SOSG suggested that the 1O2 production

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is associated with the hypoxic fraction. More significant efficacy of LIP(IR780+PFH)

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than LIP(IR780) can be observed (p < 0.05). Besides, the 1O2 generation of

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LIP(IR780+PFH) in 1% O2 was even higher than LIP(IR780) in 21% O2, which

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demonstrated the superiority of using PFH nanodroplet as photodynamic medium. To

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O2 generation during laser irradiation (Figure 2a). After irradiated by the NIR laser

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ACCEPTED MANUSCRIPT determine whether this enhancement was caused by PFH, we diluted samples into

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different levels and tested their efficacy in hypoxia with 1% O2 (Figure 2b). We

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observed that PFH increased the photodynamic efficacy of LIP(IR780+PFH) in a dose

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dependent manner, which indicate that the PFH plays an major role in accelerating

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Subsequently, whether LIP(IR780+PFH) can generate more 1O2 to treat hypoxic

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cancer cells were tested in vitro. CT26 murine colon adenocarcinoma cells were

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incubated in hypoxic air (1% O2) before and during irradiation. The 1O2 generation

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was measured by carboxy-H2DCFDA (a ROS marker) staining (Figure 3a). Little

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fluorescence was observed in cells treated with LIP(IR780) and LIP(IR780+PFH)

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alone. After laser irradiation, signals in both LIP(IR780) and laser treated cells were

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still weak, but strong fluorescence of carboxy-H2DCFDA was observed in

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LIP(IR780+PFH) plus laser treated cells, which suggested a high level of 1O2

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production (p < 0.05) (Figure 3b).

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The superior cytotoxicity of LIP(IR780+PFH) in hypoxic condition (1% O2) was then

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tested. Firstly the PDT induced CT26 cellular death was examined by the dual

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fluorescence of calcein-AM / propidium iodide (PI), which was used to distinguish

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viable cells (green) from dead cells (red) (Figure 4a). Results suggested that most

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cells treated with LIP(IR780+PFH) or LIP(IR780) before irradiation were alive. After

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laser exposure, nearly no cells were killed by LIP(IR780)+NIR, however most cells

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were killed by LIP(IR780+PFH)+NIR. We further confirmed the cytotoxicity under

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O2 generation.

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ACCEPTED MANUSCRIPT 1% O2 by CCK-8 assays. No significant cytotoxicity was appeared by incubating with

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LIP(IR780) and LIP(IR780+PFH) alone, however after laser irradiation the

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LIP(IR780+PFH)+NIR shown significant therapeutic efficacy, while

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LIP(IR780)+NIR still shown no effects (P <0.05) (Figure 4b). These results

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demonstrated that hypoxia can highly hampered the therapeutic outcome of traditional

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photodynamic therapy, but didn’t limit the efficacy of photosensitizer loaded in

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perfluorocarbon droplet.

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To determine the effect of LIP(IR780+PFH) in treating hypoxic tumor, in vivo

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hypoxic CT26 subcutaneous tumor model was firstly established and confirmed. For

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ensuring tumors are hypoxic enough during photodynamic treatments, all mice were

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kept in an atmosphere with only 7% O2 for 30 min before irradiation. HIF-1α staining

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assay was carried out to evaluate the hypoxic level, revealed that the hypoxic

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breathing can successfully lower the intratumoral oxygen content (Figure 5a). Finally

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the therapeutic efficacy of LIP(IR780+PFH) and LIP(IR780) were tested in this

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hypoxic model by intravenous administration. When tumor grown up to about 50

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mm3, LIP(IR780) and LIP(IR780+PFH) (10 v/v% PFH, 60 µg/ml IR780) were

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injected into tail vein individually. After 24 h (day 1), the drugs has successfully

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accumulated in tumor due to the EPR (enhanced permeability and retention) effect of

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solid tumor (Figure S3). Then mice were put into the hypoxic box with an atmosphere

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containing only 7% oxygen for 30 min and immediately treated by laser. At day 4,

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drugs were dosed again and mice were breathed hypoxic air and irradiated again at

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ACCEPTED MANUSCRIPT day 5. The growths of tumors treated with LIP(IR780+PFH)+NIR were significantly

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inhibited (P < 0.05). However, nearly no efficacy can be achieved by

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LIP(IR780)+NIR in such an hypoxic condition (Figure 4b). This indicated that

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loading photosensitizer into perfluorocarbon nanodroplet can be successfully utilized

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to treating hypoxic tumor which resisted traditional PDT in vivo.

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Perfluorocarbons are believed to be bio-inert and safe, and have been clinically used

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for contrast agents and artificially blood. In this report, by loading photosensitizers

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into perfluorocarbon nanodroplet, we found the photodynamic efficacy of PS was

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significantly enhanced, even in extreme hypoxic condition (0.1 kpa O2). This

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phenomenon may reveal that the limitation of hypoxia may be overcome if admirable

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medium (such as PFC, rather than tumor matrix) can be utilized to support

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photodynamic 1O2 generation. In conclusion, we have successfully developed a safe,

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nano-scaled and perfluorocarbon based photosensitizer delivery system, we also

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tested its efficacy hypoxic condition, which can help photosensitizer achieve high

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photodynamic efficacy when treating hypoxic tumor.

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Acknowledgements

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This study was supported by National Natural Science Foundation No. 81202474,

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81273464 and 81473146.

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ACCEPTED MANUSCRIPT Figure 1. Photosensitizer loaded perfluorocarbon nanodroplet as an effective photodynamic nanomedicine. (a) Structure of the photosensitizer loaded nanodroplet. (b) Structure of the IR780 (the photosensitizer we utilized). (c) Schematic illustration

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of the accelerated 1O2 generation process.

Figure 2. Photodynamic behaviors of LIP(IR780+PFH) under hypoxic conditions. (a)

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Fluorescence of SOSG (a 1O2 probe, λEx = 504 nm, λEm = 525 nm). Samples were

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placed and irradiated (2 W per cm2 for 20 s) in different hypoxic condition (contain 0.1%, 1%, 7% and 21% O2). Data are means ± SD (n = 3), *p < 0.05. (b) Fluorescence of SOSG in diluted samples. Samples were kept in extreme hypoxic

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condition (1% O2) and been irradiated. Data are means ± SD (n = 3), *p < 0.05.

Figure 3. In vitro cellular ROS detection under 1% O2. (a) Intracellular ROS generation during photodynamic treatment was detected by using carboxy-H2DCFDA

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(Ex/Em = 495/529 nm). Photographs of bright filed indicated locations and quantities

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of the cells, scale bar 50 µm. (b) Average fluorescent intensity of SOSG per cell. Value of LIP(IR780+PFH) treated groups were set as 1. Data are means ± SD (n = 3), *p < 0.05.

Figure 4. In vitro cytotoxic assays under 1% O2. (a) Fluorescence images of calcein-AM / propidium iodide stained cells, scale bar 50 µm. (b) Cytotoxic study under 1% O2 measured by CCK-8 assay. Data are means ± SD (n = 6), *p < 0.05.

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Figure 5. In vivo photodynamic therapy in subcutaneous hypoxic tumor model by intravenous injection. (a) HIF-1α staining assay (yellow) to evaluate tumor hypoxic

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level, scale bar 100 µm. (b) Change of relative tumor volume (V/V0). Drugs were intravenously dosed at day 0 and day 4, 20 s laser irradiations were given at day 1 and

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day 5. Data are means ± SEM (n = 6), *p < 0.05.

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ACCEPTED MANUSCRIPT Development of a safe, nano-scaled and perfluorocarbon based photosensitizer delivery system. Demonstration of photosensitizer achieve significant photodynamic efficacy

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when treating hypoxic tumor. The problem of hypoxia can be overcome just by using a suitable drug delivery

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system.