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A Donor−Acceptor Conjugated Polymer with Alternating Isoindigo Derivative and Bithiophene Units for Near-Infrared Modulated Cancer Thermo-Chemotherapy Dong-Dong Li,† Jun-Xia Wang,† Yan Ma,† Hai-Sheng Qian,† Dong Wang,‡ Li Wang,⊥ Guobing Zhang,*,‡ Longzhen Qiu,*,‡ Yu-Cai Wang,⊥ and Xian-Zhu Yang*,† †
School of Biological and Medical Engineering, Hefei University of Technology, Hefei, Anhui 230009, China Academy of Opto-Electronic Technology, Hefei University of Technology, Hefei, Anhui 230009, China ⊥ School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China ‡
S Supporting Information *
ABSTRACT: Conjugated polymers containing alternating donor/ acceptor units have strong and sharp absorbance peaks in near-infrared (NIR) region, which could be suitable for photothermal therapy. However, these polymers as photothermal transducers are rarely reported because of their water insolubility, which limits their applications for cancer therapy. Herein, we report the donor−acceptor conjugated polymer PBIBDF-BT with alternating isoindigo derivative (BIBDF) and bithiophene (BT) units as a novel photothermal transducer, which exhibited strong near-infrared (NIR) absorbance due to its low band gap (1.52 eV). To stabilize the conjugated polymer physiological environments, we utilized an amphiphilic copolymer, poly(ethylene glycol)-block-poly(hexyl ethylene phosphate) (mPEG-b-PHEP), to stabilize PBIBDF-BT-based nanoparticles (PBIBDF-BT@NPPPE) through a single emulsion method. The obtained nanoparticles PBIBDF-BT@NPPPE showed great stability in physiological environments and excellent photostability. Moreover, the PBIBDF-BT@NPPPE exhibited high photothermal conversion efficiency, reaching 46.7%, which is relatively high compared with those of commonly used materials for photothermal therapy. Accordingly, in vivo and in vitro experiments demonstrated that PBIBDF-BT@NPPPE exhibits efficient photothermal anticancer efficacy. More importantly, PBIBDF-BT@NPPPE could simultaneously encapsulate other types of therapeutic agents though hydrophobic interactions with the PHEP core and achieve NIR-triggered intracellular drug release and a synergistic combination therapy of thermo-chemotherapy for the treatment of cancer. KEYWORDS: conjugated polymer, donor−acceptor polymer, photothermal conversion, photothermal stability, synergistic combination therapy
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INTRODUCTION
have been developed more recently owing to their good biocompatibility and biodegradability.23,24 However, the serious photobleaching and low photothermal conversion efficiency hindered their anticancer efficacy. Therefore, development of efficient organic photothermal agents with high photothermal conversion efficiency and photostability is still highly desirable. Several groups have reported the use of conjugated polymers, including polyaniline,25,26 poly(3,4ethylenedioxythiophene):poly(4-styrenesulfonate),27,28 and polypyrrole (PPy),29−32 for photothermal cancer therapy. These polymers usually show a broad absorption band. Recently, MacNeill’s group demonstrated that the donor− acceptor (D−A) conjugated polymers, which have been widely used for field-effect transistors and organic photovoltaics, have sharper NIR absorbance peaks.33 They first reported the
Photothermal therapy (PTT), based on the principle of transducing light into local heat by a photothermal agent, has emerged as an efficient medical tool for treating various cancers.1−5 Compared with the conventional cancer therapies, PTT possesses a number of advantages such as high specificity, low toxicity to normal tissues, minimal invasiveness, and strong inhibition of tumor growth. Specifically, near-infrared (NIR) light-inducted PTT is a very attractive option due to the high transparency in vivo with NIR light in the range of 700−950 nm.6−9 Until now, various photothermal transducers with NIR absorbance have been explored for PTT applications. Among them, inorganic nanomaterials such as noble metal (e.g., Au, Ag, Pt) nanostructures,10−15 carbon nanomaterials,16−19 and copper sulfide nanoparticles20−22 were mostly used and exhibited great photothermal therapeutic efficacy in vitro and in vivo. However, these nonbiodegradable nanomaterials remain inside the body for very long periods of time, which could result in potential long-term toxicity. Organic NIR dyes © XXXX American Chemical Society
Received: May 8, 2016 Accepted: July 12, 2016
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DOI: 10.1021/acsami.6b05495 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
of the PBIBDF-BT at 811 nm against its concentration (in CHCl3). The obtained PBIBDF-BT@NPPPE nanoparticles was lyophilized and redissolved in CHCl3, and then the encapsulating efficacy was calculated according to this standard curve, reaching 98.3%. Infrared Thermal Imaging of PBIBDF-BT@NPPPE Nanoparticles. The PBIBDF-BT@NPPPE nanoparticles with different concentrations in Eppendorf tubes were irradiated by a 808 nm diode laser at a power density of 2.0 W/cm2 (New Industries Optoelectronics, Changchun, China) for 15 min. The infrared thermal images were acquired using an infrared camera (ICI7320, Infrared Camera Inc., Beaumont, TX, United States). Photostability of PBIBDF-BT@NPPPE Nanoparticles. The PBIBDF-BT@NPPPE nanoparticles (30.0 μg/mL) were exposed to an NIR laser (808 nm, 2.0 W/cm2, 15 min, laser on). Subsequently, the NIR laser was turned off for 15 min, and the solution was naturally cooled to room temperature (laser off). The laser on and laser off cycles were repeated four times, and the change in temperature was monitored as described above. Meanwhile, the absorbance spectrum of the irradiated samples was examined after the last NIR irradiation. In Vitro Photothermal Cytotoxicity of PBIBDF-BT@NPPPE Nanoparticles. Breast cancer MDA-MB-231 cells (American Type Culture Collection, Rockefeller, Maryland, United States) were seeded in a 96-well plate (1 × 104 cells per well) at 37 °C with 5% CO2 overnight. The medium was replaced by a fresh medium containing PBIBDF-BT@NPPPE at different concentrations of PBIBDF-BT. After incubation for 4 h, the cells were exposed to the NIR laser (808 nm, 1.0 W/cm2, 10 min) and further incubated for 24 h, and then the cell viability was analyzed by MTT assay. In addition, photothermal cytotoxicity of PBIBDF-BT@NPPPE nanoparticles at different power densities was also examined. The tumor cells were seeded in 96-well plates as described above. After incubation overnight, fresh medium containing PBIBDF-BT@NPPPE nanoparticles (30.0 μg/mL PBIBDF-BT) was added. After 4 h of incubation, the cells were exposed to different power density lasers for 10 min and further cultured for 24 h, and then the cell viability was analyzed. The tumor cells were seeded in 24-well plates at 5 × 104 cells per well and cultured for 24 h followed by treatment with PBIBDF-BT@ NPPPE at different concentrations of PBIBDF-BT as described above. After incubation for 4 h, the tumor cells were exposed to the NIR laser (808 nm, 1.0 W/cm2, 10 min) and subsequently rinsed by PBS twice and stained by a live/dead viability/cytotoxicity kit (Invitrogen, United States). Live and dead cells were then imaged with a fluorescence microscope (Nikon TE 2000-U, Japan). Additionally, the cells which were treated with PBIBDF-BT@NPPPE nanoparticles (30.0 μg/mL PBIBDF-BT) for 4 h and then exposed to the NIR laser at different power densities were also analyzed by the live/dead viability/ cytotoxicity kit. Animal and Tumor Model. Female BALB/c nude mice (20 ± 2 g, 6−8 weeks old) were purchased from Beijing HFK Bioscience Co. Ltd. The procedures were approved by the Hefei University of Technology Animal Care and Use Committee, and all animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. MDA-MB-231 cells (2 × 106 cells) were injected into the mammary fat pads of female Balb/c nude mice to establish a human breast cancer xenograft tumor model. The mice were used for in vivo experiments when the tumor volumes reached 60 mm3. In Vivo Photothermal Therapeutic Efficacy of PBIBDF-BT@ NPPPE. Mice bearing MDA-MB-231 tumors were intratumorally administrated with 40 μL of PBIBDF-BT@NPPPE nanoparticles at a PBIBDF-BT dose of 40 μg per mouse, and mice treated with the same volume of saline were used as the control. Mice with and without PBIBDF-BT@NPPPE nanoparticle injections were irradiated with the NIR laser (808 nm, 0.5 W/cm2, 10 min). The temperatures of the tumor sites were detected by an IR 7320 thermal camera. Additionally, the tumor volumes were measured by a caliper and calculated based on the following equation: tumor volume (mm3) = length × width2 × 0.5. After 2 weeks of treatment, the tumor tissues of killed mice were excised to measure their weight.
utilization of D−A conjugated polymer nanoparticles for PTT applications in vitro under NIR irradiation. However, their nanoparticles without stabilization could limit their application in vivo. To overcome this obstacle, Liu’s group achieved PEGylation of the D−A conjugated polymer PFTTQ-based nanoparticles using a PEGylated lipid of DSPE-PEG2000 and found that the obtained nanoparticles efficiently ablate tumors in vivo.34 These results demonstrated the great potential of D− A conjugated polymers for PTT cancer therapy, and the exploration of novel D−A conjugated polymer-based nanoparticles as organic photothermal agents is highly desired. Previously, we developed the D−A conjugated polymer PBIBDF-BT,35 which has a highly electron-deficient unit, bis(2oxoindolin-3-ylidene)-benzodifuran-dione (BIBDF), and a highly electron-rich unit, bithiophene (BT), in the backbone. The PBIBDF-BT polymer with a relatively narrow energy band gap, ensuring the strong and sharp absorbance peaks in the NIR region (maximal absorption peak at about 811 nm), could be suitable for photothermal therapy. However, PBIBDF-BT was insoluble in biological media. Herein, an amphiphilic diblock copolymer, poly(ethylene glycol)-block-poly(hexyl ethylene phosphate) (mPEG-b-PHEP), which has exhibited great anticancer efficacy as a drug delivery system, was used to stabilize the PBIBDF-BT-based nanoparticles through a single emulsion method. The obtained nanoparticles showed strong NIR absorbance at approximately 811 nm and high stability in physiological environments. In addition, their physiochemical characters, including size, photothermal conversion, and photostability, were systematically evaluated. Furthermore, the photothermal cytotoxicity and overall antitumor efficacy to MDA-MB-231 xenograft tumors were evaluated. Moreover, such nanoparticles could efficiently encapsulate other therapeutic molecules, such as chemotherapy drug doxorubicin (DOX), through their hydrophobic interaction with the polymeric PHEP core. The NIR-induced photothermal effect was capable of promoting the release of these chemotherapeutic agents, exhibiting NIR-triggered intracellular drug release and synergistic combination therapy of thermochemotherapy for cancer therapy.
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EXPERIMENTAL SECTION
Materials. Syntheses of conjugated polymer PBIBDF-BT and diblock copolymer mPEG-b-PHEP were reported previously.35,36 Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco BRL (Eggenstein, Germany). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, United States). Characterization. The size and size distribution of nanoparticles in aqueous solution were measured by dynamic light scattering (DLS) carried out with a NanoBrook-90 Plus instrument (Brookhaven Instrument Corporation, Holtsville, NY, United States). UV−vis−NIR spectra were detected using a UV-3802 (UNICO, Shanghai, China) spectrophotometer. The transmission electron microscope (TEM, JEOL-2010, Japan) measurements were carried out with an acceleration voltage of 200 kV. Preparation of PBIBDF-BT@NPPPE Nanoparticles. A CHCl3 solution (200 μL) containing mPEG-b-PHEP (10.0 mg), PBIBDF-BT (1.0 mg), and ultrapurified water (1.0 mL) were emulsified by sonication for 2 min (work 5 s and rest 2 s) at a 325 W output using a microtip probe sonicator (JY92-IIN, Scientz Biotechnology, Ningbo, China). The solution was further stirred under reduced pressure for 30 min to evaporate the organic solvent and then purified by being passed through a 0.45 μm filter (Millipore). No precipitation was found by this method. A standard curve was obtained by plotting the absorbance B
DOI: 10.1021/acsami.6b05495 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Scheme 1. Mechanism of PBIBDF-BT-Based Photothermal Therapy (A) and Schematic Illustration of the Preparation of PBIBDF-BT@NPPPE (B)a
a
The HOMO and LUMO are −5.55 and −4.03 eV, respectively. .
Figure 1. Particle size distribution (A) and absorption spectra (B) of PBIBDF-BT@NPPPE. Infrared thermal images (C) and temperature change curves (D) of PBIBDF-BT@NPPPE upon exposure to the NIR laser (808 nm, 2.0 W/cm2, 15 min). Preparation of DOX-Loaded Nanoparticles (PBIBDF-BT@ NPPPE/DOX). To prepare the DOX-loaded nanoparticles, PBIBDFBT (1.0 mg), DOX (1.0 mg), and mPEG-b-PHEP (10.0 mg) were mixed into 200 μL of a CHCl3/DMSO (3/1, v/v) component solvent and were then emulsified as described above. The obtained sample was further stirred under reduced pressure for 30 min to evaporate the CHCl3 and then transferred to a dialysis bag (cutoff molecular weight of 3500) and dialyzed against deionized water overnight. In addition, unloaded DOX was removed by a 0.45 μm filter (Millipore), and the DOX-loaded nanoparticle PBIBDF-BT@NPPPE/DOX was obtained. The DOX loading capacity was determined according to a previous method,36 reaching 2.1%. In Vitro Drug Release from PBIBDF-BT@NPPPE/DOX. One milliliter of the PBIBDF-BT@NPPPE/DOX sample containing about 100 μg of DOX was exposed to the NIR laser (808 nm, 1.0 W/cm2, 10 min). Then, the samples were transferred to the dialysis bag and submerged into 20 mL of PB buffer (pH 7.4). The PBIBDF-BT@ NPPPE/DOX sample without NIR irradiation was used as a control. At each predetermined point time, the external PB buffer was replaced
with an equal volume of fresh PB buffer and lyophilized, and the concentration of DOX was determined by HPLC analysis.36 MDA-MB-231 cells (5 × 104 cells per well) were seeded in 24-well plates for 24 h. Subsequently, the medium was removed, and fresh medium containing PBIBDF-BT@NPPPE/DOX (5.0 μg/mL of DOX) nanoparticles or free DOX (5.0 μg/mL) was added. After incubation for 4 h, the cells were washed and exposed to NIR irradiation (808 nm, 1.0 W/cm2) for 10 min. After further incubation for another 4 h, the tumor cells were trypsinized, collected, and suspended in PBS for fluorescence-activated cell sorting (FACS) analyses (Accuri C6 flow cytometer, BD Biosciences, United States). Additionally, for confocal laser scanning microscopy (CLSM) observation, the cells treated as described above were stained with DAPI for cell nuclei and then observed by CLSM (LSM 710, Carl Zeiss, Inc., Jena, Germany). The cells incubated with PBIBDF-BT@NPPPE/DOX but without NIR irradiation were used as control. Photothermally Enhanced Chemotherapy Using PBIBDFBT@NPPPE/DOX. The tumor cells (1 × 104 cells per well) were seeded in 24-well plates and cultured overnight followed by treatment with C
DOI: 10.1021/acsami.6b05495 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 2. (A) Temperature elevation of PBIBDF-BT@NPPPE nanoparticles over four laser on/off cycles of NIR irradiation. (B) The highest and lowest temperature of PBIBDF-BT@NPPPE recorded by IR thermal images during the four laser on/off cycles. (C) Absorption spectra of PBIBDFBT@NPPPE before and after four laser on/off cycles. (D) Temperature change curves of PBIBDF-BT@NPPPE upon exposure to an 808 nm laser after storage for 1, 3, and 7 days. free DOX, PBIBDF-BT@NPPPE, or PBIBDF-BT@NPPPE/DOX nanoparticles (30.0 μg/mL PBIBDF-BT). After incubation for 24 h, the cells were washed, exposed to NIR irradiation (808 nm, 1.0 W/cm2) for 10 min, and further cultured for 24 h. Then, the cell viability was analyzed by MTT assay. Statistical Analysis. The statistical significance of treatment outcomes was assessed using the Student’s t test: *p values
