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Perylene Diimide-Grafted Polymeric Nanoparticles Chelated with Gd3+ for Photoacoustic/T1‑Weighted Magnetic Resonance ImagingGuided Photothermal Therapy Xiaoming Hu,† Feng Lu,† Liang Chen,‡ Yufu Tang,† Wenbo Hu,† Xiaomei Lu,§ Yu Ji,† Zhen Yang,† Wansu Zhang,† Chao Yin,† Wei Huang,†,§ and Quli Fan*,† †

Key Laboratory for Organic Electronics and Information Displays (KLOEID) & Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, 9 Wenyuan Road, Nanjing 210023, China ‡ Department of Orthopedics, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, China § Key Laboratory of Flexible Electronics (KLOFE) & 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: Developing versatile and easily prepared nanomaterials with both imaging and therapeutic properties have received significant attention in cancer diagnostics and therapeutics. Here, we facilely fabricated Gd3+-chelated poly(isobutylene-alt-maleic anhydride) (PMA) framework pendent with perylene-3,4,9,10-tetracarboxylic diimide (PDI) derivatives and poly(ethylene glycol) (PEG) as an efficient theranostic platform for dual-modal photoacoustic imaging (PAI) and magnetic resonance imaging (MRI)-guided photothermal therapy. The obtained polymeric nanoparticles (NPs) chelated with Gd3+ (PMA−PDI−PEG−Gd NPs) exhibited a high T1 relaxivity coefficient (13.95 mM−1 s−1) even at the higher magnetic fields. After 3.5 h of tail vein injection of PMA−PDI−PEG−Gd NPs, the tumor areas showed conspicuous enhancement in both photoacoustic signal and T1-weighted MRI intensity, indicating the efficient accumulation of PMA−PDI−PEG−Gd NPs owing to the enhanced permeation and retention effect. In addition, the excellent tumor ablation therapeutic effect in vivo was demonstrated with living mice. Overall, our work illustrated a straightforward synthetic strategy for engineering multifunctional polymeric nanoparticles for dual-modal imaging to obtain more accurate information for efficient diagnosis and therapy. KEYWORDS: multifunctional polymeric nanoparticle, photothermal therapy, photoacoustic imaging, magnetic resonance imaging, gadolinium ion PAI.9,10 Nevertheless, their application generally suffers from their intrinsic optical instability. Recently, organic/polymeric semiconducting materials with excellent photostability and strong light absorption have presented superior PAI properties in vitro and in vivo.11−14 Interestingly, PAI contrast agents featured with high photothermal conversion efficiency can also function as photothermal agents for photothermal therapy (PTT).15,16 Therefore, by integrating diagnostic and therapeutic functions within a single platform, such PAI/PTT theranostic nanomaterials have shown supernormal properties for relatively precise cancer therapy.

1. INTRODUCTION Photoacoustic imaging (PAI), a newly arising biomedical imaging technique, which integrates optical absorption with ultrasonic detection, has great superiority for the visualization of pathology and physiology with fine spatial resolution and deep tissue penetration.1−4 As such, near-infrared (NIR, 650− 900 nm) light-absorptive materials serving as contrast agents for PAI are more attractive due to the deeper tissue penetration of NIR light compared to visible light-absorptive materials. Thus far, plenty of inorganic nanomaterials, like copper,5 silver,6 gold,7 and graphene,8 have been developed as PAI contrast agents. Compared with inorganic nanomaterials, organic materials have been drawing more attention owing to their good biocompatibility and biodistribution. For instance, traditional NIR-absorptive dyes (e.g., indocyanine green (ICG) and methylene blue) have shown remarkable properties for © 2017 American Chemical Society

Received: July 4, 2017 Accepted: August 21, 2017 Published: August 21, 2017 30458

DOI: 10.1021/acsami.7b09633 ACS Appl. Mater. Interfaces 2017, 9, 30458−30469

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ACS Applied Materials & Interfaces

Scheme 1. Schematic Illustration of the Preparation of PMA−PDI−PEG−Gd NPs and the Process of Dual-Modal ImagingGuided Photothermal Therapy of Hela Tumor in Vivo by PMA−PDI−PEG−Gd NPs

Despite the superiority of PAI/PTT combination, PAI for high anatomical spatial resolution is still an imperative problem.17 Hence, combining PAI with other imaging modalities to provide complementary information and synergistic advantages is of significance for more accurate theranostics.18,19 Magnetic resonance imaging (MRI) is a highly desired approach owing to its capability to offer high physiological and anatomical resolution.20−22 In this field, MRI contrast agents are important and can be divided into two parts, T1 agents (shorten longitudinal proton relaxation time) and T2 agents (shorten transversal proton relaxation time).23 Compared to T2 agents which induce negative contrast, T1 agents with positive contrast are more advantageous because they can provide more satisfactorily accurate information.24 Molecular composites and nanoparticles (NPs) based on lanthanide ion Gd3+, served as T1 agents, have been predominant in clinical MRI, attributing to their relatively reliable physiological and anatomical information of disease tissue in vivo.25−27 However, conventional clinical MRI contrast agents based on molecular gadolinium chelates, such as Magnevist, Dotarem, tetraazacyclododecane-1,4,7,10-tetraacetic acid, and diethylenetriaminepentaacetic acid, have certain limitations, such as rapid elimination and low relaxivity coefficient, especially at the higher magnetic fields.28 To possess pre-eminent magnetic relaxivity properties, immobilization of Gd-complexes onto macromolecules,27,29−31 red blood cells,32 monoclonal antibody,33 etc. are the major approaches to slow down rotational motion, thereby enhancing the MRI sensitivity of gadolinium. However, such functionalized complexes or NPs generally result in complicated fabrication processes. To address this issue, biopolymers (e.g., human serum albumin, melanin, and

bovine serum albumin) and colloidal ionic polymers with intrinsic metal-ion binding ability are directly used to chelate to Gd3+ and exhibit terrific magnetic relaxivity properties both in vitro and in vivo.28,34−37 Besides, to integrate MRI and PAI/ PTT within a single formulation, traditional theranostic nanomaterials are developed by encapsulating various components with individual properties together.38,39 This preparation process generally suffers from the intricate synthetic pathways and the unstable nanostructures in living environments. Therefore, developing versatile and easily prepared theranostic nanomaterials with high stability, superior PAI, and T1 magnetic relaxivity properties, and PTT functions is of extraordinary significance in cancer diagnostics and therapeutics. Inspired by the aforementioned pioneering works, we herein facilely fabricated a Gd3+-chelated polymeric NP pendent with semiconducting materials as an efficient theranostic platform for PAI/MRI dual-modal imaging-guided PTT (Scheme 1). The design strategy is mainly considered as below: (i) The poly(isobutylene-alt-maleic anhydride) (PMA) framework can provide reactive sites for the easy modification of NIRabsorptive materials (perylene-3,4,9,10-tetracarboxylic diimide (PDI)) for PAI and PTT. As a typical organic semiconducting material, PDI derivatives have been successfully explored as efficient PAI agents in our previous work.13,14 (ii) The abundant carboxyl groups of the polymer framework are able to react with amino poly(ethylene glycol) (PEG) to enhance its water solubility and prolong its circulation time. With the featured molecular structure constituted of hydrophobic PDI and hydrophilic PEG and carboxyl groups, the obtained polymer can spontaneously self-assemble into nanoaggregates 30459


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material.40,41 The cell viability assay was performed by the decrease of the MTT reagent. Ninety six-well plate was used to seed Hela cells at a density of 0.5 × 104 cells per well in the DMEM medium containing 1% penicillin/streptomycin and 10% fetal bovine serum at 37 °C in 5% CO2 humidified atmosphere for 24 h. The cells were then cultivated in the medium with our different concentrations’ material for 4 h and stochastically divided into following groups: (1) without laser, (2) laser exposure (730 nm, 1.5 W cm−2) for 5 min, and (3) laser exposure (730 nm, 1.5 W cm−2) for 10 min. Then, an additional 20 h was used for culturing these treated cells in completed medium. After that, each well was added to 10 μL of MTT (0.5 mg mL−1) solution and then incubated for 4 h at 37 °C. Next, we removed the supernatant and added 200 μL of dimethyl sulfoxide into each well. The optical absorbance was then determined at 490 nm on a microplate reader. The untreated cells’ absorbance was adopted as the contrast reference group and regarded as 100% cellular viability. 2.4. Cell Apoptosis Assay. A 6-well plate was used to culture Hela cells at a density of 5 × 104 cells per well, as the aforementioned method. PMA−PDI−PEG−Gd NPs (50 μg mL−1) were then carefully added into each well. After cultivation for 4 h, the wells were washed with PBS and irradiated by a 730 nm laser with the power density of 1.5 W cm−2 for 10 min. Then, the cells were cultured in completed medium for an additional 20 h. After that, the cells were tinted by a mixture of Annexin V-FITC/propidium iodide (PI) and used the FlowSight Imaging Flow Cytometer for flow cytometer analysis. 2.5. Subcutaneous Tumor Models. All nude mice borne Hela tumor were bought from Jiangsu KeyGEN BioTECH Corp., Ltd. and used under the guideline of the Laboratory Animal Center of Jiangsu KeyGEN BioTECH Corp., Ltd. The Hela tumor-bearing mice were acquired by subcutaneously injecting Hela cells (1 × 106) into the object region of the nude mice (4−6 weeks). V = 0.5 × A2 × B was the calculation equation of the tumor volume; in this equation, A represents the transverse diameter and B is the longitudinal diameter of the tumor. 2.6. In Vitro and in Vivo Photoacoustic Imaging. To probe the PAI properties of PMA−PDI−PEG−Gd NPs, the PA signal intensities of different concentrations of NP aqueous solutions (62.5, 12.5, 250, 500, and 1000 μg mL−1) were acquired under the Nexus-128 PA tomography system (Endra Inc.). Then, to observe the maximum PA signal intensity peak at different excitation wavelengths, the PA signal of PMA−PDI−PEG−Gd in aqueous solution at different excitation wavelengths (680, 685, 690, 695, 700, 710, 730, 750, 780, 800, 850, and 900 nm) were recorded, respectively. For in vivo PAI, the Hela tumor-bearing mice were injected with PMA−PDI−PEG−Gd NPs (2 mg mL−1, 200 μL) into tail vein. The excitation wavelength was selected at peak absorption of PMA−PDI−PEG−Gd NPs (680 nm), with the laser power 5.02 mJ, and the nude mice anesthetized with 2% isoflurane in oxygen were placed in a receptacle containing 38 °C water. The obtained data was analyzed using the Vevo LAZR PAI System, and an identical region-of-interest was analyzed for acquiring quantitative PA signal intensity. Furthermore, the graph of concentration-dependent PA signal intensity was acquired and the PA spectrum was then obtained by plotting excitation wavelength versus PA signal intensity. The final imaging analysis and the quantified PA signals of tumor sites were performed using software OsiriX Lite. 2.7. In Vitro and in Vivo MRI Measurement. In vitro and in vivo MRI experiments were conducted in Molecular Imaging Research Center of Central Southeast University (Nanjing, China) and recorded at 7.0 T Micro-MRI (Bruker Biospin and PharmaScans, Germany). To evaluate the T1 and T2 relaxivity, PMA−PDI−PEG−Gd NPs with different concentrations of aqueous solutions were transferred into the 300 μL polymerase chain reaction (PCR) tubes, anchored in the MR holder, and the T1 and T2 MRI were obtained. Image analysis was carried out using ImageJ. Importantly, the relaxivity value of r1 or r2 was confirmed by fitting the Gd concentration versus 1/T1 or 1/T2 relaxation time and the gadolinium-ion content of PMA−PDI−PEG− Gd NPs were measured by inductively coupled plasma mass spectrometry. As for in vivo MRI detection, the Hela tumor-bearing mice, injected with PMA−PDI−PEG−Gd NPs (2 mg mL−1, 200 μL)

in aqueous solution. The formed molecular aggregation in the NPs is beneficial for strengthening photoacoustic (PA) signals and the photothermal effect. (iii) The ionic polymeric NPs can further actively chelate to Gd3+ ions by carboxyl groups for MRI. Importantly, the magnetic relativities of the polymeric NPs can be greatly improved because when the Gd3+ ions were immobilized onto macromolecules, their rotational motion was slowed down, which provides more efficient relaxation. In addition, such a strong Gd3+ chelating ability of the NPs can facilitate the Gd3+ retention, which can reduce the Gd3+ leakage in the bloodstream and ensure MRI efficacy at the target location. The resulting polymeric NPs (PMA−PDI−PEG−Gd NPs) with an average size of approximately 60 nm exhibited a good enhanced permeability and retention (EPR) effect. Furthermore, the passive targeting and precise synergistic diagnosis provided satisfactory information to guide the photothermal ablation of tumors in living mice and prevent against the damage of the ambient normal tissues. All of these make the versatile and easily prepared polymeric NPs an efficient theranostic nanomaterial for PAI/MRI dual-modal imaging-guided PTT.

2. EXPERIMENTAL SECTION 2.1. Chemicals and Instruments. All of the reagents were bought from commercial suppliers and adopted directly. Poly(isobutylene-altmaleic anhydride) (Mw = 6 kDa) was purchased from Sigma-Aldrich and adopted without further purification. Matrix-assisted laser desorption ionization time-of-flight mass was carried out on a Bruker Autoflex spectrometer with matrix assistance for mass spectra acquisition. NMR spectra were conducted on a 400 MHz spectrometer (Bruker UltraShield Plus, 13C: 100 MHz, 1H: 400 MHz), and the following abbreviations represent the multiplicities: m, multiplet; q, quartet; t, triplet; d, doublet; and s, singlet. A HT7700 transmission electron microscope was used to record the transmission electron microscopy (TEM) images at an accelerating 100 kV voltage. Dynamic light scattering was conducted on a particle size analyzer (90 Plus; Brookhaven). The UV−vis−NIR absorption spectra were measured on the UV−vis−NIR spectrophotometer (UV-3600; Shimadzu). The methyl thiazolyl tetrazolium (MTT) experiments were studied by a microplate reader (PowerWave XS/XS2; BioTek). The cell apoptosis assay was conducted by the FlowSight Imaging Flow Cytometer (Merck Millipore; Germany). The 730 nm laser was bought from the Optoelectronics Technology Co., Ltd. (Changchun, China). All photoacoustic imaging results were obtained in the PAI tomography system (Nexus-128; Endra Inc., Ann Arbor, MI), accompanied with an adjustable nanosecond pulsed laser (20 Hz pulse repetition frequency, 5 ns pulses, 680−950 nm). The 7.0 T Micro-MRI (Bruker Biospin and PharmaScans, Germany) with a mouse cradle and 35 mm birdcage coil was used for MRI. The photothermal experiments were performed by IRS E50 Pro Thermal Imaging Camera (IRS Systems Inc., Shanghai). 2.2. Synthesis of PMA−PDI−PEG−Gd Nanoparticles. The synthesis of PMA−PDI−PEG was described in the Supporting Information. PMA−PDI−PEG−Gd NPs were prepared as previously described, with modification.34 In brief, PMA−PDI−PEG (10 mg in 10 mL H2O) was chelated with gadolinium ion by addition of 200 μL of fresh GdCl3 (10 mg mL−1) in water followed by a 2 h incubation at 38 °C. The obtained complexes were then refined by a PD-10 column to remove redundant gadolinium ions and other byproducts. Then, the centrifugal filter (amicon device, molecular weight cut-off = 30 kDa) was used for concentrating the final PMA−PDI−PEG−Gd NPs. The obtained NPs were reconstituted in phosphate-buffered saline (PBS) and filtrated through a 0.22 μm millipore filter for cell and animal experiments. 2.3. Cell Viability Assay. The cytotoxicity and photothermal cytotoxicity of PMA−PDI−PEG−Gd NPs was probed by studying the viability of Hela cells after incubation with Dulbecco’s modified Eagle’s medium (DMEM) containing a variety of concentrations of our 30460


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Figure 1. In vitro characterization of PMA−PDI−PEG−Gd NPs: (A) TEM image of PMA−PDI−PEG NPs, (B) TEM image of PMA−PDI−PEG− Gd NPs, (C) UV−vis−NIR absorption spectrum of PMA−PDI−PEG−Gd NPs and (inset) photo of PMA−PDI−PEG−Gd NPs in PBS (pH = 7.4), and (D) the stability study of gadolinium-ion-chelated PMA−PDI−PEG−Gd NPs during different incubation temperatures (25, 50, and 60 °C) in PBS (pH = 7.4). into tail veins, were imaged at 7.0 T Micro-MRI using a mouse cradle and 35 mm birdcage coil and detected at different times. The image analysis was obtained through a similar method using ImageJ. The parameters were TR/TE = 1000/10 ms; field of view = 35 mm × 35 mm; flip angle = 30°; matrix, 256 × 256; and slice thickness = 1 mm. 2.8. In Vitro and in Vivo Photothermal Therapy. First, heating curves of different concentrations of PMA−PDI−PEG−Gd NPs were obtained in vitro.42 In brief, a series of NP concentrations ranging from 0 to 1 mg mL−1 in the 500 μL PCR tubes were exposed to a 730 nm laser irradiation (laser power: 1.5 W cm−2) for 10 min. The variational temperatures and photothermal imaging were measured by an infrared thermal imager (FLIR E50; FLIR Systems OU, Estonia). Second, when the Hela tumor volume reached approximately 120 mm3, the photothermal therapy in living mice experiments were started to be performed. PMA−PDI−PEG−Gd NPs (2 mg mL−1, 200 μL) or saline (200 μL) was intravenously injected into the living mice, which were given the following treatments: (1) saline + laser, (2) PMA−PDI− PEG−Gd NPs, and (3) PMA−PDI−PEG−Gd NPs + laser. After 3.5 h, the tumor regions of (1) and (3) were exposed to the laser irradiation (730 nm, 1.5 W cm−2) for 10 min. Then, the photothermal imaging and temperature variation were monitored through the above similar method. Importantly, the measured variational temperature versus irradiation time was considered as the heating curve. Finally, the treatment mice were dissected for major organs, including tumor, kidney, liver, heart, lung, and spleen after 24 days, which were further evaluated for the biocompatibility of PMA−PDI−PEG−Gd NPs by histological examination.

introducing a hydroxy alkyl chain to one imide position of the PDI molecule and a long alkyl chain to another imide position. Then, poly(isobutylene-alt-maleic anhydride) (Mw = 6000) can react with the PDI molecule by the esterification reaction and the obtained abundant carboxyl groups of the polymer framework can provide reactive sites for the modification of PEG (Mw = 2000). Furthermore, the successful conjugation of PDI and PEG was determined by 1H NMR spectra and UV− vis−NIR absorption spectra of PMA−PDI−PEG. The conjugation ratio of PMA−PDI−PEG was characterized by comparing the integration value of PDI and PEG with that of the corresponding polymer, and Figure S9 shows that approximately 10 PDI molecules and 13 PEG chains are successfully conjugated to the polymer framework. The assembling number of PMA−PDI−PEG molecules in one NP was calculated to be approximately 1.05 × 104.13,14 To obtain the magnetic relaxivity properties, the Gd-chelated PMA−PDI−PEG NPs were prepared via simply adding concentrated gadolinium-ion solution to PMA−PDI−PEG aqueous solution.28,34 The number of Gd3+ per NP based on the mean size of 60 nm were about 1.92 × 105 through theoretical calculation.43 Because of the hydrophobic−hydrophilic groups of PMA− PDI−PEG, it was spontaneously self-assembled to form NPs in aqueous solution, which displayed a dark green color. Figures 1A,B and S10 illustrate the change of size distribution before and after adding gadolinium ions. PMA−PDI−PEG NPs showed outstanding dispersity in water, with an average hydrodynamic diameter of 101.9 ± 2.8 nm (Figure S10A), and the transmission electron microscopy (TEM) image showed an average particle size of about 75 ± 4.6 nm. The

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of PMA−PDI− PEG−Gd NPs. The detailed synthetic process and characterization of the PMA−PDI−PEG architecture was illustrated in the Supporting Information (Scheme S1 and Figures S1−S9). In short, we first synthesized a modifiable PDI molecule by 30461


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Figure 2. In vitro imaging and photothermal capacity of PMA−PDI−PEG−Gd NPs: (A) photoacoustic images of PMA−PDI−PEG−Gd NPs with different concentrations in fluorinated ethylene propylene tubing; (B) linear relationship between concentration and PA signal of PMA−PDI−PEG− Gd NPs (R2 = 0.995); (C) PA spectra of PMA−PDI−PEG−Gd NPs in water and in living mice; (D) relaxation rates 1/T1 (s−1) and 1/T2 (s−1) along with the change of Gd concentration of PMA−PDI−PEG−Gd NPs in PBS; (E) T1-weighted MR images of PMA−PDI−PEG−Gd NPs at different concentrations: 250.00, 125.00, 62.50, 31.25, 15.63, 7.81, and 0 μg mL−1; (F) temperature IR images of PMA−PDI−PEG−Gd NP aqueous solution under the 730 nm laser irradiation (1.5 W cm−2) imaged with an IR camera at a series of concentrations; (G) heating curves of a series of concentrations of PMA−PDI−PEG−Gd NPs exposed to a 730 nm laser irradiation for 10 min.

smaller particle size of the PMA−PDI−PEG NPs imaged under TEM should be due to the introduction of PEG and their shrinking in the dry state. After adding Gd3+ ions, the asprepared PMA−PDI−PEG−Gd NPs possessed an average hydrodynamic diameter of 72.6 ± 2.4 nm (Figure S10B) and the TEM image indicates that the NPs have a relatively constant diameter of approximately 60 ± 5.4 nm (Figure 1B).

The decreased NP size can be attributed to the formed crosslinking inside NPs through the strong chelating ability of the COOH group in PMA−PDI−PEG NPs to Gd3+ ions.44 In addition, the NPs showed NIR absorption, with a maximum absorption peak at 645 nm and a shoulder at around 692 nm in aqueous solution (Figure 1C), and the extinction coefficient of the PMA−PDI−PEG−Gd NPs at 680 nm reaching up to 2.84 30462


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Figure 3. In vitro cell viability experiments and photothermal therapy (730 nm laser exposure with power density of 1.5 W cm−2): (A) Relative viabilities of Hela cells for different treatment groups after being incubated with PMA−PDI−PEG−Gd NPs with different concentrations; (B) the cell viability of Hela cells cultured with PMA−PDI−PEG−Gd NPs, as a function of laser irradiation time; and (C) the cell apoptosis assay of Hela cell induced by PMA−PDI−PEG−Gd NPs-mediated PTT.

× 108 M−1 cm−1 indicated that the NPs were good NIRabsorptive materials for PAI and PTT. By acquiring the UV− vis−NIR absorption spectra of PMA−PDI−PEG NPs and PMA−PDI−PEG−Gd NPs (Figure S12), we can conclude that Gd3+ loading has no obvious effect on the optical properties of the polymeric NPs. Besides, Figure S13 reveals that both NPs were almost nonfluorescent in water, which can be attributed to the photoinduced electron-transfer from the electron-rich pyrrolidine to the electron-poor PDI. The PMA−PDI−PEG−Gd NPs were highly stable and can be stored in PBS for at least 12 months without any precipitation. We further conducted the Gd-chelating stability assay of PMA−PDI−PEG−Gd NPs during different incubation temperatures. Approximately only 2% of Gd3+ was released from the NPs in PBS solution at room temperature even after 24 h of incubation (Figure 1D), confirming the excellent stability of the chelating complex. In addition, the NPs were incubated in high temperature (50 and 60 °C) PBS solutions (imitating the physiological environment in vivo when it served as photothermal agents for PTT), less than 6% of Gd3+ were released from the NPs even after 1 day incubation (Figure 1D), indicating good thermostability of Gd-chelated PMA−PDI− PEG NPs, which can provide a great advantage for MRI-guided PTT. The excellent thermostability of PMA−PDI−PEG−Gd NPs can be ascribed to the strong chelating ability of abundant carboxyl groups in PMA−PDI−PEG NPs to Gd3+ ions. Moreover, we measured the hydrodynamic diameter of PMA−PDI−PEG−Gd NPs in serum during a certain period of time, and the limited change of the NPs’ size in serum indicated their good physiological stability (Figure S14). Compared with the size of PMA−PDI−PEG NPs in serum,

which was around 101.9 nm, the retained size of PMA−PDI− PEG−Gd NPs also indicated that little Gd3+ was released from serum. To further demonstrate the photostability of PMA− PDI−PEG−Gd NPs, the PMA−PDI−PEG−Gd NPs and commercially used ICG were exposed with a continuous laser irradiation at 730 nm (laser power: 1.5 W cm−2) for 60 min (Figure S15). The commercial materials (ICG) possessed obvious reduced absorptions, whereas the absorption of polymeric NPs almost remains the same. The high photostability of PMA−PDI−PEG−Gd NPs was derived from the PDI groups, which have been proven to be more stable than the traditional organic dyes.13 All in all, the excellent photostability, chemical stability, and physiological stability made it promising for further bioapplications in vivo. To explore the capacity of PMA−PDI−PEG−Gd NPs as an efficient PAI contrast agent, the PA spectrum of PMA−PDI− PEG−Gd NPs in aqueous solution was acquired at different excitation wavelengths. Figure 2C (red line) shows the PA spectral aspects of PMA−PDI−PEG−Gd NPs, with the highest PA signal at 680 nm, which roughly accorded with the UV−vis absorption spectrum (Figure 1C). In addition, we acquired the PA images from aqueous solutions at different concentrations of PMA−PDI−PEG−Gd NPs under the 680 nm excitation laser (Figure 2A). As we can see in Figure 2B, PMA−PDI− PEG−Gd NPs showed a linear relationship between PA signals and sample concentrations. Therefore, the PMA−PDI−PEG− Gd NPs as highly efficient photoacoustic agents have great promise for living imaging. Next, the MRI property of PMA−PDI−PEG−Gd NPs was studied by recording T1-weighted MR intensity in aqueous solution with different concentrations (Figure 2E). As shown in 30463


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Figure 4. In vivo dual-modal imaging of Hela tumor mice with PAI and MRI respectively: (A) (left) Photograph of Hela tumor-bearing mice. (Right) In vivo PAI of tumor mice recorded at changing time points after tail vein injection with PMA−PDI−PEG−Gd NPs. (B) Quantitative PA intensity in the tumor area at changing time after tail intravenous injection of PMA−PDI−PEG−Gd NPs. (C) Biodistribution of PMA−PDI−PEG− Gd NPs in mice at 24 h after injection. The relative PA intensities from the organs were determined by comparing PMA−PDI−PEG−Gd NPs with PBS. (D) Quantitative MR signal intensity in tumor sites at changing time after injection of PMA−PDI−PEG−Gd NPs compared to that at 0 h. (E) In vivo T1-weighted MRI of living mice at different time points after injection of PMA−PDI−PEG−Gd NPs. The yellow circles showed the position of the tumor.

magnetic relaxivity property, which was much stronger than that of the commercial MR agents, such as Magnevist, Dotarem, Omniscan, and ProHance (r1 = 2−5 mM−1 s−1).28 The high magnetic relativity of PMA−PDI−PEG−Gd NPs was attributed to the strongly bonded Gd3+ ions to the abundant

Figure 2E, the brighter MR images of the aqueous solution of PMA−PDI−PEG−Gd NPs were obtained with the increase of NP concentrations. The linear correlation between Gd3+ concentration and MR signal intensity was calculated to be r1 = 13.95 mM−1 s−1 (Figure 2D), demonstrating an excellent 30464


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number of early stage apoptosis, and necrotic (Annexin VFITC+/PI−) or later apoptosis (Annexin V-FITC+/PI+) Hela cells obviously increased after being treated with the combining utilization of PMA−PDI−PEG−Gd NPs and laser irradiation. All of these results indicated that PMA−PDI−PEG−Gd NPs can be developed as a safe and highly efficient photothermal agent for PTT in living mice. 3.3. In Vivo Dual-Modal Imaging. The in vivo properties of PMA−PDI−PEG−Gd NPs as highly efficient PA agents were evaluated on a tumor-bearing mouse via tail vein injection (100 μL, 2 mg mL−1). As shown in Figure 4A, an obvious PA signal around the tumor sites was acquired after NP injection, whereas only a weak signal of major blood vessels could be seen at preinjection. In addition, after 3.5 h of the tail vein injection of PMA−PDI−PEG−Gd NPs, the tumor regions reached a maximum PA signal enhancement, indicating the efficient accumulation of PMA−PDI−PEG−Gd NPs on account of the enhanced permeation and retention (EPR) effect in the tumor region. After that, the PA signal intensity decreased gradually over time, and nearly disappeared after 24 h (Figure 4B). Particularly, the relatively higher PA signal in red arrow was attributed to the minimal damage of tumor, which resulting from the injection of Hela cancer, caused much more accumulation of PMA−PDI−PEG−Gd NPs. After 24 h intravenous injection with PMA−PDI−PEG−Gd NPs, the major organs of the dissected mice were collected (Figure S18) for the biodistribution assay. Images in Figure S18 reveal the absolute PA intensity of the organs, whereas the data in Figure 4C represents the relative PA intensity of the organs after the injection of NPs compared with the injection of PBS. The results revealed that PMA−PDI−PEG−Gd NPs were less distributed in kidney, lung, heart, bone, and skin. By comparison, the obvious enhancement of PA intensity in tumor, liver, and spleen indicated a higher distribution of PMA−PDI−PEG−Gd NPs. In addition, given the strong background signals from liver and spleen, the enhancement of their absolute PA signals in tumor were much stronger than that of their relative PA signals. Therefore, all of these proved the NPs were mainly cleared via the hepatobiliary system. To test the MRI ability of PMA−PDI−PEG−Gd NPs in vivo, the mice with Hela tumor were administrated intravenously with 200 μL of NPs (2 mg mL−1). Then, the tumor regions of mice were imaged at different times (preinjection, 1, 3.5, 6, 12, and 24 h) after injection. Figure 4D,E suggests the MR intensity and signal distributions as the time goes on and little intrinsic contrast between the ambient tissue and tumors could be seen in tumor mice before the injection of NPs. As we can see in Figure 4E, MR signal intensity in the tumor areas increased at 1 h after PMA−PDI−PEG−Gd NP injection and reached a maximum after 3.5 h postinjection. Afterward, the quantitative MR signals in the tumor sites decreased gradually over 3.5 h postinjection of PMA−PDI−PEG−Gd NPs, which was consistent with the PA imaging result. Overall, with the aid of PMA−PDI−PEG−Gd NPs, MRI can detect more accurate anatomical date and real-time feedback information of tumor tissue in vivo. Meanwhile, the NPs serving as a PAI contrast agent can also be used to visualize tissue structures and acquire a high-resolution pathogenic structure at an unprecedented depth. Thus, PMA−PDI−PEG−Gd NPs holds great potential to provide satisfactory accuracy and comprehensive information to guide the highly efficient photothermal ablation of tumors and prevent the damage of the ambient normal tissues.

carboxylate groups, which can provide a strong hydrogen-bond interaction to water molecules.45 Hence, the easily prepared, greatly stable, and efficient PMA−PDI−PEG−Gd NPs exhibited significant advantages for in vivo MRI. Furthermore, we studied the relationship of MR versus PA signal intensity and they exhibited good linear relationship (Figure S16). Besides, the slope of increased MR signal intensity with different concentrations was lower than that of PA signal intensity, showing that the PAI of PMA−PDI−PEG−Gd NPs was more sensitive than the MRI. With strong NIR absorption, the photothermal characteristics of the obtained polymeric NPs were first explored in vitro. In this system, the continuous laser with power of 1.5 W cm−2 at 730 nm was adopted for PTT investigation. Although the maximum permissible exposure (MPE) for the continuous laser at 730 nm is 0.23 W cm−2, according to the guideline of the American National Standard for Safe Use of Lasers, for preclinical research, scientists generally use laser power at 730 nm, higher than the MPE (in the range of 0.75−2.5 W cm−2), to investigate the feasibility of photothermal agents for tumor ablation in vivo.46−49 Thus, in this article we also used the continuous laser with power of 1.5 W cm−2 at 730 nm for PTT research and in the following experiments there appeared little toxicity in vitro and in vivo after irradiation by the laser fluence. As shown in Figure 2F, temperature IR images of PMA−PDI− PEG−Gd NP aqueous solution under the 730 nm (1.5 W cm−2) laser irradiation were conducted with an IR camera at a series of concentrations. The PMA−PDI−PEG−Gd NPs exhibited an obvious increase of temperature after laser irradiation even at a low concentration, whereas water did not trigger the increase of temperature (Figure 2G). Interestingly, the NPs possessed the concentration-dependent thermal increase, and the induced hyperthermia is sufficient to kill tumor cells (above 42 °C).50 To further explore the photothermal conversion efficiency of the PMA−PDI−PEG− Gd NPs, the NPs were allowed to cool naturally, as depicted in Figure S17, when it was at a steady-state temperature. On the basis of the quantification method,15,51 the photothermal conversion efficiency value was calculated to be 40%, indicating the good PTT efficacy of PDI, which has been proved in our preceding study.52 3.2. In Vitro Cell Experiments. To apply the PMA−PDI− PEG−Gd NPs as photothermal therapy agents in living mice, it was necessary to explore their cytotoxicity and photothermal effect in vitro. The methyl thiazolyl tetrazolium (MTT) test was adopted to study the cytotoxicity of PMA−PDI−PEG−Gd NPs in Hela cells.40 The viable Hela cells cultured with PMA− PDI−PEG−Gd NPs were only slightly decreased even at high concentrations, demonstrating their excellent biocompatibility. Importantly, when they were exposed to 730 nm continuous laser radiation, the Hela cell viabilities decreased dramatically with the increasing concentrations of PMA−PDI−PEG−Gd NPs (Figure 3A) and laser irradiation time (Figure 3B). In addition, the laser exposure of Hela cells without PMA−PDI− PEG−Gd NPs was carried out and showed relatively no cytotoxicity (Figure 3B), suggesting the Hela cells were sufferable to the laser exposure. Furthermore, to further study the cell apoptosis during PTT, Annexin V-FITC/PI apoptosis cell detection kit was used to discriminate dead cells from viable cells of disparate stages.41 As shown in Figure 3C, plenty of Hela cells were viable (Annexin V-FITC−/PI−) under the condition of either laser irradiation or the PMA−PDI−PEG− Gd NPs alone. By contrast, the cell mortality rate, the total 30465


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Figure 5. In vivo photothermal therapy: (A) IR thermal images of mice tumor areas at 0, 0.5, 1, 2, 4, and 8 min under exposure to 730 nm laser at 1.5 W cm−2 after intravenous injection of PMA−PDI−PEG−Gd NPs; (B) temperature changes at the mice tumor area treated with PMA−PDI−PEG− Gd NPs and exposed to 730 nm laser at 1.5 W cm−2; (C) body weight changes of mice groups after different treatments; (D) tumor growth curves; (E) representative images of the Hela tumors of different groups after 24 days of treatments.

3.4. In Vivo Photothermal Therapy. Motivated by the highly efficient passive tumor targeting of PMA−PDI−PEG−

Gd NPs and the strong NIR absorption ability, a living mice study of photothermal therapy was performed. The Hela tumor 30466


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Figure 6. Images of H&E-stained slices from heart, liver, spleen, lung, kidney, and tumor in mice after PTT. Scale bar: 100 μm.

PMA−PDI−PEG−Gd NPs and laser showed dramatic necrosis and apoptosis of the tumor cells, indicating successful damage of the tumor cells. All in all, these results indicated that the PMA−PDI−PEG−Gd NPs can act as an efficient theranostic platform for accurate PAI and MRI dual-modal imaging-guided photothermal therapy.

mice were marked, weighed, and given three treatments: (1) saline (200 μL) + laser, (2) PMA−PDI−PEG−Gd NPs (2 mg mL−1, 200 μL), and (3) PMA−PDI−PEG−Gd NPs (2 mg mL−1, 200 μL) + laser. (Saline and agents were both injected into the Hela tumor mice by tail vein.) On account of above in vivo PAI and MRI results that PMA−PDI−PEG−Gd NPs can reach its maximum accumulation in tumor sites at 3.5 h postinjection of the NPs (Figure 4), the tumor area of the mice was irradiated under the 730 nm laser (1.5 W cm−2) for 10 min after 3.5 h of injection of PMA−PDI−PEG−Gd NPs (Figure 5A). As shown in Figure 5B, in the PMA−PDI−PEG−Gd NPs + laser group, the tumor site’s temperature rapidly reached to approximately 54 °C within 60 s, which was adequate to kill the tumor cells. The rapidly increasing temperature in the objective areas within a remarkably short period of time suggested that a large number of PMA−PDI−PEG−Gd NPs were accumulated at the objective tumor site, further confirming the excellent passive targeting ability of the NPs. In comparison, the control treatment group without the PMA−PDI−PEG−Gd NP injection merely appeared mildly increased to less than 40 °C within 10 min of laser exposure (Figure 5A,B), further demonstrating that laser irradiation itself cannot cause damage to the tumors and is safe for animals. To evaluate the PTT therapeutic effect, we monitored the tumor sizes of three treatment groups after PTT. Figure 5D indicates that the growth of the tumor size after the treatment of PMA−PDI−PEG−Gd NPs + laser was significantly inhibited, whereas either NIR laser irradiation or PMA− PDI−PEG−Gd NPs alone did not inhibit tumor growth (Figure 5E). These results further confirmed that the PMA− PDI−PEG−Gd NPs along with NIR laser irradiation possessed excellent therapeutic effectiveness. Besides, the body weight of the mice injected with PMA−PDI−PEG−Gd NPs or saline showed no apparent difference within 24 days of monitoring (Figure 5C), which indicated no obvious in vivo toxicity of all treatment groups and revealed a negligible side effect of PMA− PDI−PEG−Gd NPs. After 24 days of treatment, mice were sacrificed with major organs including tumor, liver, heart, spleen, kidney, and lung for the hematoxylin and eosin (H&E) staining assay to further evaluate the PTT efficacy. As seen in Figure 6, in the control treatment groups (PMA−PDI−PEG− Gd NPs or laser exposure alone), no obvious cell necrosis, apoptosis, or inflammation were observed in these organs, indicating no evident side effects of the NPs in living mice and further demonstrated the safety after irradiation by 730 nm laser at 1.5 W cm−2. In contrast, the tumor tissues treated with

4. CONCLUSIONS In summary, we successfully developed an easily prepared multifunctional polymer nanostructure (PMA−PDI−PEG− Gd), which possessed excellent biocompatibility and photostability, good water-solubility and low toxicity, strong PA signal intensity and a high-performance MR contrast effect even at high magnetic fields of modern MRI instruments. Then, Hela tumor-bearing mice via tail vein injection with PMA−PDI− PEG−Gd NPs are imaged by PAI and MRI, which greatly provided insights on the location and size of the tumors, showing efficient passive tumor accumulation of the NPs through the EPR effect. Furthermore, the in vivo photothermal cancer treatment was carried out, taking advantage of their strong NIR optical absorbance and perfect tumor ablation property, and then, histological examination suggested no obvious toxic side effects of PMA−PDI−PEG−Gd NPs to normal organs within the treatment days. Overall, our work provides a practical paradigm with accurate cancer diagnosing to preferably guide the photothermal therapy with little damage to the healthy tissue areas. Furthermore, we expect our straightforward synthetic strategy would stimulate further applications for bioimaging and therapy.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b09633. Synthetic route to PMA−PDI−PEG; 1H NMR spectra and 13C NMR spectra of the relevant compound; size distribution of PMA−PDI−PEG and PMA−PDI−PEG− Gd NPs; stability of PMA−PDI−PEG−Gd NPs during different incubation pH in PBS, photostability, physiological stability, and MR−PA signal intensities correlation of PMA−PDI−PEG−Gd NPs in vitro; UV−vis−NIR absorption and fluorescence spectra of PMA−PDI−PEG NPs and PMA−PDI−PEG−Gd NPs in water; detailed calculation method of the assembling number of PMA− PDI−PEG molecules per NPs and the number of Gd per 30467


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NPs based on average size; photothermal effect of PMA−PDI−PEG−Gd NPs aqueous solution and the biodistribution of PMA−PDI−PEG−Gd NPs in vivo after 24 h tail vein injection (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Xiaoming Hu: 0000-0003-0324-7255 Wenbo Hu: 0000-0001-5233-8183 Zhen Yang: 0000-0003-4056-0347 Wei Huang: 0000-0001-7004-6408 Quli Fan: 0000-0002-9387-0165 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We greatly appreciate the financial support from the National Basic Research Program of China (Grant No. 2015CB932200), the National Natural Science Foundation of China (Grant Nos. 21574064, 21674048, 21605088, and 61378081), the Natural Science Foundation of the Jiangsu Higher Education Institutions (Grant No. 16KJB150031), Synergetic Innovation Center for Organic Electronics and Information Displays, and the Natural Science Foundation of Jiangsu Province of China (Grant Nos. BK20160884, BM2012010, and NY211003).

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