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Cite This: ACS Appl. Mater. Interfaces 2018, 10, 36805−36813
Lipid-Polymer Bilaminar Oxygen Nanobubbles for Enhanced Photodynamic Therapy of Cancer Ruyuan Song,†,⊥ Dehong Hu,‡,⊥ Ho Yin Chung,§ Zonghai Sheng,*,‡ and Shuhuai Yao*,†,§
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†
Bioengineering Graduate Program, Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Hong Kong, China ‡ Paul C. Lauterbur Research Center for Biomedical Imaging, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China § Department of Mechanical and Aerospace Engineering, The Hong Kong University of Science and Technology, Hong Kong 999077, China S Supporting Information *
ABSTRACT: Hypoxia in solid tumors may be a hindrance to effective treatments of tumors in achieving their therapeutic potential, especially for photodynamic therapy (PDT) which requires oxygen as the supplement substrate. Oxygen delivery using perfluorocarbon emulsions or lipid oxygen microbubbles has been developed as the agents to supply endogenous oxygen to fuel singlet oxygen generation in PDT. However, such methods suffer from premature oxygen release and storage issues. To address these limitations, we designed lipid-polymer bilaminar oxygen nanobubbles with chlorin e6 (Ce6) conjugated to the polymer shell as a novel oxygen self-supplement agent for PDT. The resultant nanobubbles possessed excellent stability to reduce the risk of premature oxygen release and were stored as freeze-dried powders to avoid shelf storage issues. In vitro and in vivo experimental results demonstrated that the nanobubbles exhibited much higher cellular uptake rates and tumor targeting efficiency compared to free Ce6. Using the oxygen nanobubbles for PDT, a significant enhancement of therapeutic efficacy and survival rates was achieved on a C6 glioma-bearing mice model with no noticeable side effects, owing to the greatly enhanced singlet oxygen generation powered by oxygen encapsulated nanobubbles. KEYWORDS: oxygen nanobubbles, lipid-polymer bilaminar shell, chlorin e6, photodynamic therapy, hypoxic tumors
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INTRODUCTION
To address hypoxia-associated resistance to PDT, several strategies have been attempted to oxygenate the tumor microenvironment via hyperbaric oxygen therapy, hemoglobin, and perfluorocarbon emulsions, etc. For example, photosensitizers were coencapsulated with hemoglobin molecules, serving as oxygen carriers to fuel the ROS production of PDT for treatment of hypoxia tumors.13,14 Alternatively, by leveraging the excellent oxygen affinity of perfluorocarbons (40−50% v/v of oxygen solubility in perfluorocarbons),15 an oxygen self-supplement agent for PDT was constructed, which consisted of perfluorocarbons as the core and a lipid monolayer dispersed with a near-infrared (NIR) photosensitizer (e.g., IR780, indocyanine green) and saturated with oxygen before administration.16−18 However, the oxygensaturated PFC nanoemulsions would release oxygen upon injection into the bloodstream, which may lead to premature release before reaching the tumor targets. Therefore, hyperoxic breathing was adopted after administration of PFC nanoemulsions to enhance the oxygen delivery efficacy. 19
Photodynamic therapy (PDT) is a noninvasive and effective treatment that utilizes light-excited photosensitizers to convert dissolved oxygen into cytotoxic singlet oxygen that destroys nearby cells.1,2 Due to its minimal invasiveness and selectivity, PDT has become a promising therapeutic treatment for certain cancers, such as skin, neoplastic cancer, esophageal cancer, brain cancer, and nonsmall cell lung cancer.3−5 According to the mechanism of photosensitizer-mediated reactive oxygen species (ROS) generation, sufficient oxygen supply is critical to the efficacy of PDT. However, hypoxia is very common in solid tumors, such as glioma, pancreatic adenocarcinoma, prostate cancer, and cervix carcinoma (oxygen levels less than 1 mmHg).6−9 The hypoxia arises from abnormal vasculature perfusions, irregular cell proliferation, and dysfunctional lymphatic system in tumor tissues,10,11 which significantly jeopardizes the therapeutic outcome of PDT. Furthermore, the PDT worsens the hypoxia in the tumor microenvironment due to oxygen consumption and the vascular shut down effect.12 As a result, tumor metastasis, tumor residues, and even tumor recurrence may occur in all likelihood after the treatment of PDT. © 2018 American Chemical Society
Received: September 7, 2018 Accepted: October 9, 2018 Published: October 9, 2018 36805
DOI: 10.1021/acsami.8b15293 ACS Appl. Mater. Interfaces 2018, 10, 36805−36813
Research Article
ACS Applied Materials & Interfaces
(Ce6) was purchased from Frontier Technology (Logan, UT, USA). Dichloromethane (DCM), methanol, glycerol, diethyl ether (DEE), and hexane were obtained from Acros Organics (USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), antibiotics (penicillin/streptomycin), Pluronic F68, singlet oxygen sensor green (SOSG), and phosphate buffer saline (PBS) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Oxygen gases were purchased from Jietong Gas Technology (Guangzhou, China). All aqueous solutions were prepared in MilliQ deionized water (18 MΩ/cm, Millipore CO., USA). Synthesis of PFO-PCL-CE6. The PFO-PCL-Ce6 was synthesized by two steps. The fluorocarbon capped polymer PFO-PCL was synthesized via a ring-open polymerization of ε-CL using 1H−1H perfluoro-octan-1-ol (PFO) as the initiator and Sn(Oct)2 as the catalyst at first,37 and the PFO-PCL and Ce6 were coupled via the Steglich esterification method to obtain PFO-PCL-Ce6,38 as shown in Figure S1b. Briefly, ε-CL was dried over CaH2 to remove the trace of water molecules and recovered by distillation. A 200 mg portion of PFO and 5 μL of Sn(Oct)2 were added into a 25 mL Schlenk tube with a magnetic stirring bar. The tube was vacuumed and charged with nitrogen. Then, 5 mL of dried ε-CL and 5 mL of anhydrous toluene were injected into the tube via a syringe needle and mixed well via magnetic stirring. The solution was heated to 115 °C to activate the reaction. After 12 h, the solution became viscous and was allowed to cool to the room temperature. A 5 mL portion of DCM was added into the viscous solution, and the crude product was purified by precipitation in the cold methanol twice. The precipitates were dried under vacuum for 4 h to obtained the final product. Regarding the second step synthesis, 30 mg of Ce6, 30 mg of DCC, and 9 mg of DMAP were added into a 25 mL Schlenk tube. The tube was vacuumed and charged with nitrogen. A 6 mL portion of anhydrous DMSO was injected into the tube via a syringe needle to start the reaction, where the carboxyl groups of Ce6 were activated. After 2 h, a mixture of 400 mg of PFO-PCL dissolved in 4 mL anhydrous DMSO was added into the tube dropwise. The reaction continued for another 24 h. The solution was added into the dialysis tube (MWCO: ∼3000) and dialyzed against methanol/water solution (2/8, v/v) for 48 h. The dialyzed solution was lyophilized for 2 days to obtain green powders as the final product. The chemical structure was examined by the 1H NMR spectrum of the final product dissolved in CDCl3 (Figure S1b). Preparation of Lipid-Polymer Bilaminar Oxygen Nanobubbles. The lipid-polymer bilaminar oxygen nanobubbles were prepared using a combination of emulsion−solvent evaporation and internal phase separation.39−41 Briefly, 10 mg of DSPC, 5 mg of DPPG, and 5 mg of DSPE-PEG were dissolved in 5 mL of chloroform in a glass vial. A white thin film was obtained on the glass vial by removing the solvent using a nitrogen stream. A 5 mL portion of 10% (wt/v) glycerol solution was added into the glass vial to hydrate the lipid film followed by incubation at 65 °C for 30 min. The formed lipid solution was allowed to cool to the room temperature. An organic solution was prepared using 40 mg of PFO-PCL-Ce6 and 400 μL of decane dissolved in 1 mL of DCM. The organic solution was then added into 4 mL of the lipid solution, incubated in an ice−water bath, and sonicated for 1 min to obtain nanoemulsions in an ultrasonic cell disruptor (SFX150, Branson, USA) at an amplitude of 100% and 1 s/1 s on/off. A diluted lipid solution was dropwise added into the nanoemulsion solution with magnetic stirring (300 rpm). The nanoemulsion solution was stirred for 2 h to completely remove DCM and solidify PFO-PCL-Ce6 around the decane cores. The nanocapsule solution was purified by washing followed by a centrifuge process three times. Then nanocapsules were dispersed in 16 mL of a cryoprotectant solution, which was composed of 25 mM glycerol, 0.2% F-127, 3% (wt/v) mannitol, and 5% (wt/v) PEG. The resultant solution was evenly distributed into glass vials (2 mL per vial) and frozen in liquid nitrogen for 5 min. The frozen glass vials were subjected to lyophilization (FreeZone 4.5, Labconco, USA) for 72 h to remove water and sacrificial decane cores. The glass vials containing green lyophilized powders were sealed with a rubber septum, and the air in the headspace was replaced by oxygen using a
Furthermore, the administration of liquid PFCs causes microvascular vasoconstriction which may even worsen the hypoxia by shunting the blood away.20 And the shelf-storage of volatile PFC emulsions is another challenge for clinical transition of the PFC emulsions for therapy.21,22 Besides, lipid-coated oxygen microbubbles (LOMs) have emerged as a new potent agent to supply oxygen to the hypoxic microenvironment of solid tumors specifically with the aid of delicate control of ultrasound activation.23−27 LOMs with photosensitizer conjugated lipid shells have demonstrated a remarkable enhancement in therapeutic efficacy on hypoxic tumor models.27,28 Despite of the merits of excellent biocompatibility and effectiveness and large oxygen capacity (e.g., >70% v/v) of LOMs,29 several inherent limitations still hinder their way to clinical transitions. For instance, like other oxygen delivery systems, LOMs also encounter the premature oxygen release problem upon intravenous injection due to the high diffusivity and solubility of oxygen molecules. 30 Furthermore, LOMs suffer from similar storage issues as PFC-based oxygen delivery systems including dissolution, coalescence, and Ostwald ripening, during shelf storage which may result in severe product loss and size shift.31 Therefore, it is highly demanded to develop a PDT agent with a high oxygen carrying capacity, easy shelf storage, and low risk of premature release. In this study, we developed a method to produce lipidpolymer bilaminar oxygen nanobubbles with Ce6 conjugated to the polymer shell. Compared to phospholipids or surfactants used in LOMs or PFC-based oxygen delivery systems, polymers with higher mechanical strength provide a more robust barrier against gas dissolution which may avoid the premature oxygen release to the bloodstream.29,32−34 Besides, polymer shells could withstand higher Laplace pressure to make the bubbles in the nanoscale and maintain good stability for passive tumor-targeting by taking advantage of the enhanced permeability and retention effect.35 Polymer oxygen nanobubbles can be stored as freeze-dried powders and easily reconstituted prior to use so that storage issues could be circumvented. To make them biocompatible, PEG conjugated lipids were used as a surface coating for nanobubbles. To further render the constructed nanobubbles as a PDT agent, Ce6 was selected as a photosensitizer by virtue of its effectiveness and inherent merits for clinical use, such as activation by NIR wavelengths and relatively deep penetration.36 Therefore, our developed oxygen nanobubbles were synthesized using a mixture of Ce6-conjugated polymers and PEG lipids to provide a lipid-polymer bilaminar shell for the nanobubbles. Both in vitro and in vivo experiments were conducted to evaluate the tumor-targeting efficiency and PDT efficacy of the Ce6-conjugated nanobubbles.
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MATERIALS AND METHODS
Materials. Lipids of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (DPPG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). 1H−1H perfluoro-octan-1-ol (PFO), ε-caprolactone (ε-CL), tin(II) acetate (Sn(Oct)2), sucrose, mannitol, Poloxamer 188, Pluoronic F127, decane, polycaprolactone (MW: ∼10 000, PCL), polyethylene glycol (MW: ∼4000, PEG), 1,3-dicyclohexyl carbodiimide (DCC), 4dimethylaminopyridine (DMAP), anhydrous dimethyl sulfoxide (DMSO), anhydrous toluene, and chloroform-d (CDCl3) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chlorin e6 36806
DOI: 10.1021/acsami.8b15293 ACS Appl. Mater. Interfaces 2018, 10, 36805−36813
Research Article
ACS Applied Materials & Interfaces
Figure 1. Illustration of preparation of lipid-polymer bilaminar oxygen nanobubbles with Ce6 conjugated and their applications in vivo. The nanobubbles were prepared following the procedures of emulsification, internal phase separation, freeze-drying, and oxygen infusion. A DCM solution containing PFO-PCL-Ce6 (shell materials) and decane (nonsolvent) is emulsified in the DPPC/DSPE-PEG (surface coating materials) solution to obtain nanoemulsions. With the removal of the DCM, the internal phase separation occurs and core−shell nanocapsules are obtained. After the removal of the decane cores by freeze-drying and oxygen infusion, the nanobubbles yield. The nanobubbles are administrated intravenously and get retained in tumor tissues. Upon laser irradiation, the photosensitizer transfers energy to the oxygen encapsulated in nanobubbles, generating abundant singlet oxygen and leading to improved tumor inhibition. homemade gas-exchange system, which was controlled by a three-way valve that connected with the vacuum line, the sample bottle, and the oxygen gas bottle, respectively. The prepared oxygen nanobubbles (NBs-O2) were stored in dark at 4 °C overnight to achieve the gas equilibrium prior to use. The nitrogen nanobubbles (NBs-N2), in which the air in the glass vial was replaced with nitrogen, were made as a control. Characterization of Oxygen Nanobubbles. The size and size distribution of oxygen nanobubbles in the PBS solution were measured by Zeta Plus (Brookhaven Instruments Corp., USA) at a scattering angle of 90° in a cuvette (Eppendorf UVette) three times. The morphology and structure of oxygen nanobubbles were characterized by transmission electron microscopy (TEM) using a JEM-2100F instrument (JEOL, Tokyo, Japan). To prepare the samples on the copper grid for TEM imaging, a drop of nanobubble solution was deposited on the plasma-treated copper grid for 2 min, and afterward, the excess solution was removed using filter papers. The grid was air-dried overnight before the observation. Measurement of Oxygen Gas Release Kinetics. The oxygen release of NBs-O2 was evaluated in the hypoxic aqueous solution in comparison with LOMs. The oxygen partial pressure (pO2) of the hypoxic solution was adjusted to the hypoxic condition (pO2 = 4.0 mg/L) by the nitrogen purge.42 A 3 mL portion of oxygen nanobubbles and LOMs were injected into 40 mL of aqueous solution, incubated at 37 °C. The oxygen release kinetics was monitored for 10 min using an oximeter (JPB-607A, INESA Scientific Instrument, China), and the dissolved oxygen concentrations in each group were measured at specific time points. Measurement of Singlet Oxygen Generation in Vitro. The singlet oxygen generation of the nanobubbles was indicated by the fluorescent intensity of the oxidized SOSG. A 1 mL portion of NBsQ2, NBs-N2, and free Ce6 solutions at concentrations of Ce6 or equivalent Ce6 from 12.5 to 100 μg/mL were mixed with 10 μL of 50 μM SOSG. Then, the mixed solutions were added into quartz cuvettes and subjected to laser irradiation at 670 nm and 300 mW for 5 min. The fluorescence intensity of the oxidized SOSG was measured by fluorescence spectrometer (F900, Edinburgh Instruments Ltd.) at λex = 504 nm and λem = 525 nm. The singlet oxygen production was quantified by the increase of the SOSG fluorescence intensity compared to the background signal. Cell Culture. C6 glioma cells were cultured in DMEM, supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin under an atmosphere of 5% CO2 at 37 °C. The cells were passaged
using 0.25% (w/v) trypsin-EDTA at a split ratio of 1:5 about every 3 days when the culture reached ∼80% confluence. Cellular Uptake. C6 glioma cells were seeded in 10 mm culture dishes (1 × 104 cells per dish), covered with glass slips, and cultured overnight. After that, the cultured cells were treated with 0.5 mL of free Ce6 (∼50 μg/mL) and 0.5 mL of NBs-O2 (equivalent Ce6: ∼50 μg/mL) for 4 h, respectively. Then, the cells were washed twice with PBS and fixed with 1 mL of 4% paraformaldehyde for 10 min and subsequently incubated with DAPI (200 μL, 1 μg/mL) for another 10 min to stain the nuclei. After that, the cells were washed with PBS three times and then examined using a confocal microscope (Zeiss LSM 710, Germany). In Vitro PDT. C6 glioma cells (1 × 104) were seeded onto a 24well plate for 24 h. Then, the culture medium was replaced by a fresh medium containing PBS, NBs-N2 (equivalent Ce6: ∼50 μg/mL), NBs-O2 (equivalent Ce6: ∼50 μg/mL), respectively. After 4 h dark incubation, the cells were exposed to a 670 nm laser of 300 mW for 5 min. The cells were incubated in the dark for another 4 h, washed with PBS twice, and stained with the calcein-AM and propidium iodide (PI) solutions for visualization of the PDT efficiency. Cytotoxicity of Nanobubbles. The biocompatibility of nanobubbles was evaluated on the C6 glioma cells. A 100 μL portion of the C6 glioma cell suspensions (∼60 000 cells/mL) was added into each well of a 96-well plate and cultured overnight. Subsequently, the culture medium was replaced by a fresh medium containing NBs-O2 or free Ce6 of different concentrations (0.6−40 μg/mL). The cells were cultured in the dark for 24 h. After that, 100 μL of a fresh medium containing 10 μL Cell Counting Kit-8 (CCK-8, Dojindo, Japan) was added and the cells were incubated for 4 h. To assess the cell viability, the absorbance of each well in the 96-well plate was measured by a multimode microplate reader (Varioskan LUX, Thermo Fisher, USA) at 450 nm. Animal and Tumor Xenograft Model. Mice employed in this study were purchased from Guangdong Medical Experimental Animal Center and treated in accordance with protocols approved by the animal usage and care regulations of Shenzhen Institutes of Advanced Technology (SIAT). The mice were anesthetized using pentobarbital and then 100 μL of PBS containing C6 glioma cells (∼1 × 106) was subcutaneously injected in the bottom back of each mouse. The tumor sizes reached an average volume of ∼160 mm3 after approximately 2 weeks post injection. The tumor size was measured by a Vernier caliper and the tumor volume was calculated using the modified ellipsoid formula (i.e., 0.5(length × width2)). 36807
DOI: 10.1021/acsami.8b15293 ACS Appl. Mater. Interfaces 2018, 10, 36805−36813
Research Article
ACS Applied Materials & Interfaces
Figure 2. (a) Size distribution of precursor decane nanocapsules and resultant NBs-O2 with the removal of decane cores and oxygen infusion. (b) Transmission electron microscopy (TEM) images of the nanobubbles. (c) Average gray values of ultrasound images of deance nanocapsules (NCs, 2.5 mg/mL) and NBs-O2 at various concentrations (2.5, 5, and 10 mg/mL). (insets) Corresponding ultrasound images (scale bar: 5 mm). Ultrasound images were acquired at a frequency of 9 MHz and an MI of 0.2. (d) Cell viability of C6 glioma cells incubated with free Ce6 and nanobubbles at various concentrations for 24 h. In Vivo and in Vitro Fluorescence Imaging. To assess the distribution of free Ce6 and NBs-O2 in tissues and tumors, free Ce6 (i.v. dose: 0.5 mg/kg Ce6) in PBS (150 Mm, pH 7.4) and NBs-O2 solutions (i.v. dose: 0.5 mg/kg equivalent Ce6) were injected intravenously into the tumor-bearing mice via the tail vein, respectively. The fluorescent images of the nude mice at the specific time intervals (0.5, 3, 6, and 24 h) were captured by an IVIS fluorescence imaging system (PerkinElmer Health Sciences Inc.) using excitation/emission of 630/700 nm and an integration time of 1 ms. After 24-h post injection, the mice were euthanized to collect hearts, livers, spleens, lungs, kidneys, and tumors. The fluorescent images of the excised tissues were also acquired by the IVIS fluorescence imaging system at the same parameters. The mean fluorescence intensity of tumors and organs was quantified by ImageJ. In Vivo PDT. The PDT efficiency of NPs-O2 was evaluated on the C6 glioma tumor models. The C6 glioma tumor-bearing mice were randomly subdivided into six groups (six mice per group), including (1) PBS (150 μL) without laser irradiation; (2) PBS (150 μL) with laser irradiation; (3) NBs-N2 (i.v. dose: 2 mg/kg equivalent Ce6) without laser irradiation; (4) NBs-O2 (i.v. dose: 2 mg/kg equivalent Ce6) without laser irradiation; (5) NBs-N2 (i.v. dose: 2 mg/kg equivalent Ce6) with laser irradiation; and (6) NBs-O2 (i.v. dose: 2 mg/kg equivalent Ce6) with laser irradiation. For the laser irradiation group, a 670 nm laser (300 mW, 30 min) was exerted after 3 h of i.v. injection. The tumor size and the whole body weight of each mouse were measured every 3 days. After 4 h of the laser irradiation, one of the mice in each group were sacrificed to collect the tumor tissues for histology analysis. The excised tumors were wash with saline three times and fixed in the 10% neutral-buffered formalin. The formalinfixed tumors were stained with hematoxylin and eosin (H&E) in accordance with the standard protocol and examined by an optical microscope (Nikon Eclipse 90i microscope). The survival rate was calculated from the intervention day until the day when each mouse was sacrificed based on the Kaplan−Meier curves.43 To evaluate the safety of the NBs-O2, the mice from groups with laser irradiation were sacrificed at day 17. Tissues, including hearts, livers, spleens, lungs, and kidneys were collected and stained with H&E for the histology analysis.
Statistical Analysis. Statistical analysis of all data was performed using OriginPro 2016 and Microsoft Excel. The significance between groups was analyzed by a two-tailed Student’s t test. In all cases, the data were indicated with (*) for p < 0.05 and (**) for p < 0.001, respectively; and a p value (
