Article pubs.acs.org/Biomac
High Density Glycopolymers Functionalized Perylene Diimide Nanoparticles for Tumor-Targeted Photoacoustic Imaging and Enhanced Photothermal Therapy Pengfei Sun,† Pengcheng Yuan,† Gaina Wang,† Weixing Deng,† Sichao Tian,† Chao Wang,† Xiaomei Lu,‡ Wei Huang,‡ and Quli Fan*,† †
Key Laboratory for Organic Electronics and Information Displays & Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts and Telecommunications, 9 Wenyuan Road, Nanjing 210023, People’s Republic of China ‡ Key Laboratory of Flexible Electronics (KLOFE) and Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing 211816, China S Supporting Information *
ABSTRACT: Near-infrared (NIR) absorbing nanoagents with functions of photoacoustic imaging (PAI) and photothermal therapy (PTT) have received great attention for cancer therapy. However, endowing them with multifunctions, especially targeting ability, for enhancing in vivo PAI/PTT generally suffers from the problems of synthetic complexity and low surface density of function groups. We herein report high density glycopolymers coated perylenediimide nanoparticles (PLAC−PDI NPs), self-assembled by poly(lactose)-modified perylenediimide (PLAC−PDI), as tumortargeted PAI/PTT nanoagents. Atom transfer radical polymerization and click reaction were used in sequence to prepare PLAC−PDI, which can accurately control the content of poly(lactose) (PLAC) in PLAC−PDI and endow PLAC−PDI NPs with high density PLAC surface. The high density PLAC surface provided NPs with long-time colloidal stability, outstanding stability in serum and light, and specific targeting ability to cancer cells and tumors. Meanwhile, PLAC−PDI NPs also presented high photothermal conversion efficiency of 42% by virtue of strong π−π interactions among perylenediimide molecules. In living mice, PAI experiments revealed that PLAC− PDI NPs exhibited effective targeting ability and enhanced PTT efficacy to HepG2 tumor compared with control groups, lactose blocking, and ASGP-R negative tumor groups. Overall, our work provids new insights for designing glycopolymers-based therapeutic nanoagents for efficient tumor imaging and antitumor therapy.
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process.15 Further considering that PAI can provide higher resolution and deeper tissue penetration in biological tissues than FI and PAI/PTT are both determined by photothermal conversion efficiency, PAI/PTT combination in a single NIRabsorbing material is an ideal pair for tumor imaging and therapy. Recently, people have been dedicated to development of PAI/PTT nanoagents to meet the increasing demands of cancer therapy applications. An ideal PAI/PTT nanoagents for cancer therapy applications should have the following properties: strong absorbance in the NIR wavelength region, high photothermal conversion efficiency, excellent photostability and biocompatibility, and high accumulation in tumor tissue. Thus far, various nanomaterials including graphene, metallic nanoparticles (NPs), and small molecule dyes, are widely investigated as PAI/PTT nanoagents.16,17 However, inorganic materials and
INTRODUCTION Recently, phototherapy including photothermal therapy (PTT) and photodynamic therapy (PDT) based on near-infrared (NIR) absorbing materials has received great attention.1−7 Since light is the only source needed, phototherapy has advantages superior to conventional chemotherapy and radiotherapy, such as controllability, high sensitivity, and low systemic toxicity.8−10 In addition, NIR light (650−950 nm) can efficiently penetrate through tissues with concurrent high depth of several centimeters and less tissues absorption. Meanwhile, NIR-absorbing materials can not only transform laser energy into heat for PTT or tranfer laser enerngy into molecular oxygen to generate reactive oxygen species for PDT, but also produce imaging signals, such as fluorescence imaging (FI) and photoacoustic imaging (PAI).11,12 Accordingly, PDT or PTT nanoagents accompanied by FI or PAI have been widely developed for imaging-guided therapy.13,14 Compared to PDT, which is hindered by the rapid oxygen overconsumption of tumor cells and hypoxic features of the tumor microenvironment, PTT is more promising due to its oxygen-independent © 2017 American Chemical Society
Received: July 20, 2017 Revised: August 24, 2017 Published: August 29, 2017 3375
DOI: 10.1021/acs.biomac.7b01029 Biomacromolecules 2017, 18, 3375−3386
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Biomacromolecules
strength.33 (3) Asialoglycoprotein receptors (ASGP-R) is overexpressed in HepG2, Caco-2, and HT-29 cells as an important element of some tumors and can be applied for tumor active-target therapy.34 Lactose has specific binding ability to asialoglycoprotein receptors (ASGP-R) on the surface of HepG2 cells.35−37 Thus, the formed PDI NPs coated with PLAC possessing abundant lactoses will give PLAC−PDI NPs high targeting specifity and accordingly enhanced PTT efficacy to HepG2 tumor. In this work, the as-prepared PLAC−PDI NPs presented a long-time colloidal stability and outstanding stability in serum and light. Also, they exhibited excellent photothermal conversion efficiency as high as 42%. Furthermore, in vitro and in vivo experiments proved that PLAC−PDI NPs presented specific targeting ability and enhanced PTT efficacy to HepG2 tumor. Taken together, our results revealed that the nanomaterials surface-functionalized with glycopolymers could serve as robust nanoplatforms for targeted cancer theranostics.
small molecule dyes mostly encounter the issues of debatable long-term toxicity and photobleaching, respectively. Recently, organic semiconducting nanoparticles (OSNPs) have received increasing attention due to their improved photostability, good biocompatibility, and higher absorption property.18−23 Take their hydrophobicity into account, various electrically neutral or anion hydrophilic polymers, such as polyethylene glycol (PEG)24−26 and polyacrylic-acid (PAA),27 are compulsorily explored for surface modification to enhance their biocompability and water-solubility. Unfortunately, these artificial hydrophilic polymers showed poor targeting ability to cells. Although further conjugating theranostic nanomaterials with targeting biomolecules, including folic acid, antibodies, and peptide, can enhance their cellular uptake ability, the postconjugation method has drawbacks such as synthetic complexity, lack of universality, low functionalization efficiency, and low surface ligand density. Therefore, it is highly desirable to develop a sort of hydrophilic polymer with easy chemical modification, excellent cancer-targeting capacity, and long-term stability to achieve the requirements of PAI/PTT applications. Glycopolymer is an electrically neutral water-soluble polymer with monosaccharides or disaccharides in its repeating units.28 Compared with other synthetic hydrophilic polymer, glycopolymer has better biocompatibility because carbohydrates are biomolecules widely found in natural biological systems. Specifically, glycopolymer displays high selectivity for carbohydrate-receptor interaction and enhanced targeting efficiency compared to monovalent carbohydrate−receptor interaction through “cluster glycoside effect’’. Hence, glycopolymer as an ideal water-soluble material can be applied for specific-target drug delivery, imaging, and cancer therapy.29 However, its application is still far behind traditional artificial hydrophilic polymers due to their synthetic difficulty. Most recently, with the development of polymerization techniques, some glycopolymers can be controllably synthesized and subsequently are beginning to be deployed as targeted drug delivery system and PDT nanoagents for in vitro anticancer cell therapy.30 However, despite their great potential for tumor imaging and therapy, glycopolymers have rarely been investigated for in vivo imaging-guided cancer therapy. Herein, we for the first time reported the successful development of glycopolymers-coated OSNPs with tumor targeting ability for efficient PAI/PTT. These NPs were constructed by self-assembly of amphiphilic poly(lactose)modified perylenediimide (PLAC−PDI), which was precisely synthesized via atom transfer radical polymerization (ATRP) and click reaction in sequence. Our synthetic PLAC−PDI NPs are in accordance to the following considerations: (1) We chose an organic semiconducting molecule, perylene-3,4,9,10tetracarboxylic diimide (PDI) derivative, as the NIR-absorbing material. In our previous works, PDI has been proven to be an efficient PAI contrast for lightening the brain tumor and early thrombus in living mice due to its strong light absorption in the NIR region and excellent photostability.31,32 (2) ATRP and click reaction were used to covalently bond hydrophilic glycopolymers poly(lactose) (PLAC) with accurate polymer degree and high proportion to hydrophobic PDI molecule. The glycoplolymer functionalization promotes the formation of PDI NPs in aqueous solution for obtaining good water-solubility, high density of grafted lactose, and low cytotoxicity. In addition, the strong π−π and hydrophobic interactions among planar PDI molecules in the NPs core are good for increasing the NPs stability, photothermal conversion efficiency, and PA signal
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EXPERIMENTAL SECTION
Materials and Instruments. Materials: 5,12-dibromoanthra [2,1,9-def:6,5,10-d′e′f′] diisochromene-1,3,8,10-tetraone (PDI, 70%) and 2-n-octyl-1-dodecylamine (98%) were purchased from Beijing HWRK Chemical Co., Ltd. Lactose was purchased from Shanghai Bangcheng chemical Co., Ltd. DMF (N,N-dimethylformamide) and DCM (dichloromethane) were dried and distilled under a nitrogen atmosphere before use. 2-Bromoisobutyryl bromide (BiBB, 98%), 1,1,4,7,7-pentamethyldiethylene triamine (PMDETA, 97%), and anisole were also purchased from J&K Scientific Ltd. CuBr (99.99%) and glycidyl methacrylate (GMA, 98%) were purchased from Sigma-Aldrich. Anisole was distilled from calcium hydride (CaH2) and stored under argon. GMA was passed through a column of alumina to remove inhibitor and dried over CaH2. It was then distilled under reduced pressure and stored under argon. Unless otherwise noted, all starting materials and organic solvents were obtained from commercial suppliers and used without further purification. HepG2 and HeLa cells were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Science (SLACCAS). Calcein-AM/propidium iodide (PI) cell apoptosis kit were purchased from Life Technologies Corporation. Dulbecco’s modified Eagle’s medium (DMEM, Gibco, America) was purchased from Gene Tech Co. (Shanghai, China). 1-(2′-Propargyl) D-lactose were synthesized according to the previous literatures.38 Characterization. NMR spectra were recorded on a Bruker Ultra Shield Plus 400 MHz spectrometer ( 1 H, 400 MHz) using tetramethylsilane (TMS) as the internal standard. The UV−visible absorption spectra were recorded on a Shimadzu UV-3600 UV−visNIR spectrophotometer. All the UV experiments were carried out at room temperature. Gel permeation chromatography (GPC) analysis of the neutral polymers was conducted on Shim-pack GPC-80 X columns with THF as the eluent and polystyrenes as the standard at a flow rate of 1.0 mL min−1. Transmission electron microscopy (TEM) images were performed on a HT7700 transmission electron microscope operating at an acceleration voltage of 100 kV. Micelle solutions were dropped onto Formvar-graphite-coated copper grids (300 mesh, Electron Microscopy Science) and air-dried for TEM imaging. Dynamic light scattering (DLS) studies were conducted using ALV/ CSG-3 laser light scattering spectrometers at scattering angle of 90°, CONTIN analysis was used for the extraction of Rh data. Static light scattering (SLS) studies were conducted using ALV/CSG-3 laser light scattering spectrometers. A 730 nm semiconductor laser was purchased from Changchun New Industries Optoelectronics Technology Co., Ltd. Cell imaging was conducted by confocal laser scanning microscope (CLSM) on an Olympus Fluoview FV1000 laser scanning confocal. The methyl thiazolyl tetrazolium (MTT) assay was measured using a PowerWave XS/XS2 microplate spectrophotometer (BioTek, Winooski, VT). PA data were collected by commercial 3376
DOI: 10.1021/acs.biomac.7b01029 Biomacromolecules 2017, 18, 3375−3386
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Biomacromolecules
mg mL−1) for 2 h, followed by incubated with fresh medium containing 0.10 mg mL−1 PLAC−PDI NPs. After 4 h incubation, the medium were washed twice with PBS and the cells were collected by trypsin treatment. The PAI were recorded using a PAI tomography system (Endra Inc., Ann Arbor, MI). Cytotoxicity Studies for PLAC−PDI NPs Alone. The MTT assay was used to determine the in vitro cytotoxicity of PLAC−PDI NPs in HepG2 cells and Hela cells. Cells were seeded in 96-well plates (Costar, IL, U.S.A.) at an intensity of 2 × 104 cells mL−1, respectively. After 24 h incubation at 37 °C, then further incubated in medium containing different doses of PLAC−PDI NPs for 4 h in the dark. After that, PLAC−PDI NPs suspensions were replaced by fresh DMEM and then washed with PBS buffer; 100 μL of freshly prepared MTT solution was added into each well. After 3 h incubation at 37 °C, the supernatant was removed and 200 μL of dimethyl sulfoxide (DMSO) was added, and the plate was gently shaken for 10 min at room temperature to dissolve all the precipitates formed. A PowerWave XS/XS2 microplate spectrophotometer was used to record the absorbance intensity at 490 nm. The cellular viability was expressed by the ratio of the absorbance of the cells incubated with PLAC−PDI NPs to that of the cells incubated with culture medium only. In Vitro Photothermal Ablation of Cells. Cells were seeded in 96-well plates (Costar, IL, U.S.A.) at an intensity of 2 × 104 cells mL−1, respectively. After 24 h incubation at 37 °C, then further incubated in medium containing different doses of PLAC−PDI NPs for 4 h in the dark (blocking group, HepG2 cells were preincubated with lactose (100 μL, 2.00 mg mL−1) for 2 h). After that, PLAC−PDI NPs suspensions were replaced by fresh DMEM, the selected wells were exposed to 730 nm laser light (500 mW cm−2, 10 min). The cells were further cultured for 24 h, and then cell viability was calculated using MTT assay. Assessment of Photothermal Effect In Vitro by Confocal Imaging. HepG2 cells were seeded in CLSM culture dishes (Costar, IL, U.S.A.) at an intensity of 1 × 105 cells mL−1, respectively. After 24 h incubation at 37 °C, then further inculcated in medium containing of PLAC−PDI NPs (0.50 mg mL−1) for 4 h in the dark. After that, PLAC−PDI NPs suspensions were replaced by fresh DMEM, the selected wells were exposed to 730 nm laser light (500 mW cm−2, 10 min). The cells were further cultured for 24 h, and then cells were incubated with calcein-AM/PI solution for 10 min. Then the cells were imaged by confocal laser scanning microscope (CLSM, Olympus Fluoview FV1000). Animals and Tumor Model. All animal experiments were in according with institutional animal use and care guidelines approved by by Jiangsu KeyGEN Bio TECH Corp., Ltd. Female BALB/c nude mice (aged five to 6 weeks) were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Science (SLACCAS). HepG2 tumor or Hela tumor model was established by subcutaneous injection of HepG2 cells or Hela cells suspended in 50 μL of PBS (4 × 106) into the left armpit of each mice. The tumor volume was calculated as V = 0.5LW2, in which L and W, respectively, represent the longitudinal and transverse diameter of tumor. When the tumor size reached volumes of approximately 150−200 mm3, HepG2 tumor bearing (or Hela tumor bearing) mice were intratumorally injected with PLAC−PDI NPs (100 μL, 0.50 mg mL−1). Meanwhile, HepG2 tumor bearing mice preinjection with lactose (100 μL, 2.00 mg mL−1) and then after 4 h intravenous injected with PLAC−PDI NPs (100 μL, 0.50 mg mL−1). The real-time in vivo PAI was performed using Endra Nexus 128 PA tomography system (Endra, Inc., Ann Arbor, MI). The excitation wavelength was fixed at the maximum absorption of PLAC−PDI NPs (700 nm) with laser energy ∼6.9 mJ cm−2 on the tumor surface, and 128 ultrasonic transducers with 5.8 MHz center frequency. A highperformance graphics unit (GPU) was used for volume reconstruction. The reconstructed raw data was analyzed using software OsiriX Lite. The quantitative PA signal intensity of tumor region was measured by using OsiriX Lite. In Vivo Photothermal Therapy. When the tumor volume reached 70−90 mm3, the HepG2 tumor-bearing mice were weighed, randomly divided into 5 groups (n = 9 per group), and given the
Nexus-128 PAI tomography system (Endra Inc., Ann Arbor, MI) equipped with a tunable nanosecond pulsed laser (680−950 nm, 5 ns pulses, 20 Hz pulse repetition frequency). Synthesis of PGMA−PDI. Br-PDI (0.5 g, 0.48 mmol), CuBr (cuprous bromide, 7.2 mg, 0.5 mmol), GMA (glycidyl methacrylate, 1.38 g, 9.7 mmol), and anisole (8 mL) was first freeze−thaw cycled for three times, then heated to 35 °C under an argon atmosphere. After stirring for 5 min, PMDETA (0.082 g, 0.5 mmol) was added to start the reaction. Three hours later, the reaction was quenched with liquid nitrogen. A total of 10 mL of DCM was added to dilute the mixture. Copper salts were removed by neutral alumina column chromatography, then sedimentated in the ether, concentrated, and filtered to get reseda solid. Repeated this process for three times to obtain the purified polymers (PGMA−PDI, yield: 0.43 g, 86%). 1H NMR (400 MHz, CDCl3, δ): 8.45−8.32 (m, 4H), 7.61 (d, 2H), 4.31, 3.81 (m, 68H), 3.23 (m, 34H), 2.83, 2.63 (m, 69H), 1.91−2.14 (d, 68H), 1.48 (m, 34 H), 0.80 (t, 6 H) ppm. 1H NMR spectrum of PGMA−PDI is presented in (Figure S6). Preparation of PGMA-N3-PDI. PGMA−PDI (0.2 g) was dissolved in 40 mL of DMF, then heated to 50 °C. After dissolved completely, added NaN3 (sodium azide, 0.25 g) and NH4Cl (ammonium chloride, 0.2 g), then stirred for 20 h. After the reaction, the mixture was cooled to the room temperature, filtered to remove the inorganic salts, concentrated, and sedimentated in the water. This process was repeated three times to obtain the purified polymers (PGMA-N3-PDI, yield: 0.16 g). 1H NMR (400 MHz, DMSO-d6, δ): 8.25−8.32 (m, 4H), 7.51 (d, 2H), 5.51−5.7 (m, 34H), 3.23 (m, 34H), 3.73−4.33 (m, 100H), 3.31−3.51 (d, 74H), 1.48−2.01 (d, 75 H) ppm. 1 H NMR spectrum of PGMA-N3-PDI is presented in (Figure S7). Preparation of PLAC−PDI. PGMA-N3-PDI (100 mg), CuBr (20 mg), and 1-(2′-propargyl) D-lactose (200 mg) were dissolved in DMF (20 mL) then freeze−thaw cycled 3× and heated to 45 °C under an argon atmosphere. After being stirred for 5 min, PMDETA (28 μL) was added quickly to start the reaction. A total of 24 h later, dialysis in the water (intercept molecular weight 3.5 Kda) to remove unreacted alkynyl sugar and copper salts. The final powder was obtained by the freeze-drying method (PLAC−PDI, yield: 84 mg). 1H NMR spectrum of PLAC−PDI is presented in (Figure S8). PA Spectrum and Comparison of PA Intensity. The PA spectrum of PLAC−PDI NPs was acquired via a point-to-point method. Briefly, the PA signal of PLAC−PDI NPs in aqueous solution at different excitation wavelength (680, 685, 690, 695, 700, 705, 710, 715, 720, 725,730, 735, 740, and 745 nm) were recorded, respectively. Then, PA signal intensities were measured by region of interest (ROI) analysis using the OsiriX. Finally, the diagram of PA signal intensity versus excitation wavelength was considered as PA spectrum. The diagram of concentration-dependent PA signal was obtained through the similar method. In Vitro Photothermal Conversion Efficiency. For measuring the photothermal conversion efficiency of NP-PDI-PLAC, 1.5 mL of aqueous dispersion of PLAC−PDI NPs with the concentration 0.05 mmol mL−1 was introduced in a quartz cuvette and irradiated with a 730 nm NIR laser at a power density of 500 mW cm−2 for 660 s. A thermocouple probe with an accuracy of 0.1 °C was inserted into the PLAC−PDI NPs aqueous solution perpendicular to the path of the laser. The temperature was recorded every 60 s by a digital thermometer with a thermocouple probe until it reached room temperature. The photothermal conversion efficiency of PLAC−PDI NPs was determined according to literature that has been reported. Detailed calculation was given in Supporting Information. Cell Culture and PAI of Cells. HepG2 and Hela cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS; Gibco), 100 U mL−1 penicillin (Gibco), 100 μg mL−1 streptomycin (Gibco), and 0.25 μg mL−1 Fungizone (BioSOURCE) under 37 °C in a humidified atmosphere of 5% CO2. HepG2 cells (1 × 105) were seeded in 6-well plates overnight, then cells were incubated in fresh medium with various concentrations of PLAC−PDI NPs at 37 °C. For the competition assay, the Hela cells were incubated in fresh medium containing 0.10 mg mL−1 PLAC−PDI NPs, the HepG2 cells were preincubated with lactose (100 μL, 2.00 3377
DOI: 10.1021/acs.biomac.7b01029 Biomacromolecules 2017, 18, 3375−3386
Article
Biomacromolecules Scheme 1. Synthesis Procedures for the Glycopolymers PLAC−PDI
Scheme 2. Schematic Illustration of the Preparation of PLAC−PDI NPs and Specifically PAI/PTT Applications to HepG2 Tumor
preinjected with lactose (400 μg) and then treated with PLAC−PDI NPs (0.50 mg mL−1, 100 μL) with laser irradiation. Injected after 8 h, the tumor region of the above-mentioned b, d, and e groups were exposed to 730 nm continuous laser for 10 min at a power density of 500 mW cm−2. A total of 24 h later, three mice were selected from
following treatments: (a) mice treated with saline (100 μL) without laser irradiation, (b) mice treated with saline (100 μL) with laser irradiation, (c) mice treated with PLAC−PDI NPs (0.50 mg mL−1, 100 μL) without laser irradiation, (d) mice treated with PLAC−PDI NPs (0.50 mg mL−1, 100 μL) with laser irradiation, and (e) mice 3378
DOI: 10.1021/acs.biomac.7b01029 Biomacromolecules 2017, 18, 3375−3386
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Biomacromolecules each group and sacrificed for their tumors, and the tumors were harvested for hematoxylin-eosin (H&E) and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining assay. The tumor volume was determined every other day for 14 days. A total of 14 days later, these mice were sacrificed for H&E staining of major organs. No noticeable abnormality was found in the heart, liver, spleen, lung, or kidney. Data Analysis. PA signal intensities were measured by region of interest (ROI) analysis using OsiriX. Results were expressed as the mean ± SD deviation unless otherwise stated. All statistical data were obtained using a two-tailed student’s t test and homogeneity of variance tests (p values
