Perylene Diimide-Grafted Polymeric Nanoparticles Chelated with

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Perylene Diimide-Grafted Polymeric Nanoparticles Chelated with Gd3+ for Photoacoustic/T1-Weighted Magnetic Resonance Imaging Guided 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 ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09633 • Publication Date (Web): 21 Aug 2017 Downloaded from http://pubs.acs.org on August 23, 2017

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ACS Applied Materials & Interfaces

Displays (KLOEID) & Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications; Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech) Fan, Quli; 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

ACS Paragon Plus Environment


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Perylene Diimide-Grafted Polymeric Nanoparticles Chelated with Gd3+ for Photoacoustic/T1-Weighted Magnetic Resonance Imaging Guided Photothermal Therapy

Xiaoming Hu,a Feng Lu,a Liang Chen,b Yufu Tang,a Wenbo Hu,a Xiaomei Lu,c Yu Ji,a Zhen Yang,a Wansu Zhang,a Chao Yin,a Wei Huang a,c and Quli Fan*a

a

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. E-mail: [email protected]; b

Department of Orthopedics, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071,

China; c

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.

KEYWORDS:

multifunctional

polymeric

nanoparticle,

photothermal

photoacoustic imaging, magnetic resonance imaging, gadolinium ion

1


therapy,

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ABSTRACT Developing versatile and easily prepared nanomaterials with both imaging and therapy properties have received significant attentions in cancer diagnostics and therapeutics. Here, we facilely fabricated

Gd3+-chelated

poly(isobutylene-alt-MAnh)

framework

pendent

with

perylene-3,4,9,10-tetracarboxylic diimide (PDI) derivatives and polyethylene 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 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 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 (EPR) effect. In addition, 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.

1. INTRODUCTION Photoacoustic imaging (PAI), a newly arising biomedical imaging technique which integrates optical absorption with ultrasonic detection, has great superiorities for the visualization of pathology and physiology with fine spatial resolution and deep tissue penetration.1-4 In 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, 2



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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 drawn more attention owing to their good biocompatibility and biodistribution. For instance, traditional NIR-absorptive dyes (for example, indocyanine green and methylene blue) have shown remarkable properties for 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 present 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. Despite of 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,

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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 NPs based on lanthanide ion Gd3+, served as T1 agents, have been the predominant in clinical MRI attributing to their relatively reliable physiological and anatomical information of disease tissue in vivo.25-27 3


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However, conventional clinical MRI contrast agents based on molecular gadolinium chelates, such as Magnevist, Dotarem, DOTA and DTPA, have certain limitations such as rapid elimination and low relaxivity coefficient especially at the higher magnetic fields.28 To possess preeminent magnetic relaxivity properties, immobilization of Gd-complexes onto macromolecules,27, 29-31 red blood cells,32 monoclonal antibody33 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 the 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 Gd3+-chelated polymeric NPs 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-MAnh) (PMA) framework can provide reactive sites for the easy modification of NIR absorptive materials (PDI) for PAI and PTT. As a typical organic semiconducting material, PDI derivatives have been successfully explored as efficient PAI agents in 4



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our previous work.13, 14 (ii) The abundant carboxyl groups of polymer framework are able to react with amino 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 nano-aggregates in aqueous solution. The formed molecular aggregation in the NPs is beneficial for strengthening PA signals and 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 approximate 60 nm exhibited good enhanced permeability and retention (EPR) effect. Furthermore, the passive targeting and precise synergistic diagnosis provided satisfactorily information to guide the photothermal ablation of tumors in living mice and prevent against the damage of the ambient normal tissues. All these make the versatile and easily prepared polymeric NPs an efficient theranostic nanomaterial for PAI/MRI dual-modal imaging guided PTT.

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Scheme 1. Schematic illustration of the preparation of PMA-PDI-PEG-Gd NPs and the process of dual-modal imaging guided photothermal therapy of Hela tumor in vivo by PMA-PDI-PEG-Gd NPs.

2. EXPERIMENTAL SECTION Chemicals and Instruments. All the reagents were bought from commercial suppliers and adopted directly. Poly(isobutylene-alt-MAnh) (Mw = 6 kDa) was purchased from Sigma-Aldrich and adopted without further purification. MALDI-TOF-MS was carried out on a Bruker Autoflex with matrix-assisted for mass spectra acquisition. NMR spectra were conducted on a 400 MHz spectrometer (Bruker Ultra Shield Plus,

13

C: 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 6



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microscopy (TEM) images at an accelerating 100 kV voltage. Dynamic light scattering (DLS) 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, U.S.). Cell apoptosis assay were conducted by Flow Sight 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 uesd for MRI. The photothermal experiments were performed by IRS E50 Pro Thermal Imaging Camera (IRS Systems Inc, Shanghai). Synthesis of PMA-PDI-PEG-Gd Nanoparticles. The synthesis of PMA-PDI-PEG was described in the Supporting Information. PMA-PDI-PEG-Gd NPs was prepared as previously described with modification.34 In brief, the 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) in water followed by a 2 h incubation at 38 oC. The obtained complexes were then refined by a PD-10 column to remove redundant gadolinium ion and other byproducts. Then, the centrifugal-filter (amicon device, MWCO = 30 kDa) was used for concentrating the final PMA-PDI-PEG-Gd NPs. The obtained NPs were reconstituted in PBS (phosphate-buffered saline) and filtrated through a 0.22-µm millipore filter for cell and animal experiments. Cell Viability Assay. The cytotoxicity and photothermal cytotoxicity of PMA-PDI-PEG-Gd NPs 7


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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 material.40, 41 Cell viability assay was performed by the decrease of the MTT reagent. 96-well plate was used to seed Hela cells at a density of 0.5×104 cells per well in DMEM medium containing 1% penicillin/streptomycin and 10% fetal bovine serum (FBS) at 37 oC 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 stochasticly 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 accessional 20 h was used for culturing these treated cells in completed medium. After that, each well was added 10 µL MTT (0.5 mg/mL) solution and then incubated for 4 h at 37 oC. Next, we removed the supernatant and added 200 µL dimethyl sulfoxide (DMSO) 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. Cell Apoptosis Assay. 6-well plate was used to culture Hela cells at a density of 5×104 cells per well as aforementioned method. PMA-PDI-PEG-Gd NPs (50 µg/mL) 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 Flow Sight Imaging Flow Cytometer for flow cytometer analysis. Subcutaneous Tumor Models. All nude mice born 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 8



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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 tumor. 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 NPs aqueous solutions (62.5, 12.5, 250, 500 and 1000 µg/mL) was acquired under the Nexus 128 PA tomography system (Endra). Then, to observe the maximum PA signal intensity peak at different excitation wavelength, the PA signal of PMA-PDI-PEG-Gd in aqueous solution at different excitation wavelength (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 (2mg/mL, 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 oC water. The obtained data was analyzed using the Vevo LAZR PAI System. And an identical region-of-interest (ROI) was analyzed for acquiring quantitative PA signal intensity. Furthermore, the graph of concentration-dependent PA signal intensity was acquired and PA spectrum was then obtained by plotting excitation wavelength vs PA signal intensity. The final imaging analysis and the quantified PA signals of tumor sites were performed using software OsiriX Lite. 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 9


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transferred into the 300 µL PCR tubes, anchored in the MR holder, and obtained the T1 and T2 MRI. Image analysis was carried out using Image J. Importantly, the relaxivity value of r1 or r2 was confirmed by fitting the Gd concentration vs 1/T1 or 1/T2 relaxation time and the gadolinium ion content of PMA-PDI-PEG-Gd NPs were measured by ICP-MS. As for in vivo MRI detection, the Hela tumor-bearing mice, injected with PMA-PDI-PEG-Gd NPs (2 mg/mL, 200 µL) into tail vein, were imaged at 7.0 T Micro-MRI using a mouse cradle and 35-mm birdcage coil and detected at different time. The image analysis was obtained through the similar method using Image J.. The parameters were TR/TE =1000 ms/10 ms, FOV = 35 mm × 35 mm, flip angle = 30°, matrix 256 × 256, slice thickness = 1 mm. In Vitro and In Vivo Photothermal Therapy. Firstly, heating curves of different concentrations of PMA-PDI-PEG-Gd NPs was obtained in vitro.42 In brief, a series of NPs concentrations ranging from 0 to 1 mg/mL in the 500 µL of PCR tubes were exposed to a 730 nm laser irradiation (laser power: 1.5 W cm−2) for 10 min. The variational temperature and photothermal imaging were measured by infrared thermal imager (FLIR E50, FLIR Systems OU, Estonia). Secondly, when the Hela tumor volume reached approximately 120 mm3, the photothermal therapy in living mice experiments were started to perform. PMA-PDI-PEG-Gd NPs (2 mg/mL, 200 µL) or saline (200 µL) were 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 region 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 vs irradiation time was considered as the heating curve. Finally, the treatment mice were dissected for major organs including tumor, kidney, 10



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liver, heart, lung, and spleen after 24 days and further evaluated the biocompatibility of PMA-PDI-PEG-Gd NPs by histological examination. 3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of PMA-PDI-PEG-Gd NPs The detailed synthetic process and characterization of PMA-PDI-PEG architecture was illustrated in Supporting Information (Scheme S1 and Figure S1-S9). In short, we first synthesized a modifiable PDI molecule by 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-MAnh) (Mw=6000) can react with the PDI molecule by 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 were 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 corresponding polymer and Figure S9 showed 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 NPs 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 a concentrated gadolinium ions solution to PMA-PDI-PEG aqueous solution.28, 34 The number of Gd3+ per NPs based on the mean size of 60 nm were about 1.92 × 105 through the theoretical calculation.43 Due to 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. Figure 1A, B and Figure S10 illustrated the change of size distribution before and after adding gadolinium ions. 11


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PMA-PDI-PEG NPs showed outstanding dispersity in water with 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 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 as-prepared PMA-PDI-PEG-Gd NPs possessed an average hydrodynamic diameter of 72.6 ± 2.4 nm (Figure S10B), and the TEM image indicated 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 cross-linking inside NPs through the strong chelating ability of 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 around 692 nm in aqueous solution (Figure 1C). And the extinction coefficient of the PMA-PDI-PEG-Gd NPs at 680 nm reached up to 2.84 × 108 M-1 cm-1 indicated the NPs were good NIR absorptive 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 revealed both NPs were almost nonfluorescent in water, which can be attributed to the photo-induced electron-transfer (PET) 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 Gd-chelating stability assay of PMA-PDI-PEG-Gd NPs during different incubation temperature. Only approximate 2% of Gd3+ were released from the NPs in PBS solution at room temperature even after 24 h incubation (Figure 1D), confirming the excellent stability of the chelating complex. In addition, the NPs were incubated in high temperature 12



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(50 and 60 oC) 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 one 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. What’s more, 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 commercial 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 absorption while the absorption of polymeric NPs almost remain 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.

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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). (D) Stability study of gadolinium ion-chelated PMA-PDI-PEG-Gd NPs during different incubation temperature (25, 50 and 60 oC) in PBS (pH = 7.4).

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) showed the PA spectral aspects of PMA-PDI-PEG-Gd NPs with the highest PA signal at 680 nm, which roughly accorded with the UV−visible absorption spectrum (Figure 1C). In addition, we acquired the PA images from aqueous solutions at different 14



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concentrations of PMA-PDI-PEG-Gd NPs under the 680 nm excitation (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 Figure 2E, the brighter MR images of the aqueous solution of PMA-PDI-PEG-Gd NPs were obtained with the increase of NPs concentrations. The linear correlation between Gd3+ concentration and MR signal intensity was calculated to be r1 = 13.95 mM-1s-1 (Figure 2D), demonstrating an excellent magnetic relaxivity property which was much stronger than the commercial MR agents such as Magnevist, Dotarem, Omniscan and ProHance (r1 = 2-5 mM-1s-1).28 The high magnetic relativity of PMA-PDI-PEG-Gd NPs was attributed to the strongly bonded Gd3+ ions to the abundant 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 vs. 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 the PAI of PMA-PDI-PEG-Gd NPs was more sensitive than the MRI.

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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 FEP tubing. (B) Linear relationship between concentration and PA signal of PMA-PDI-PEG-Gd NPs (R2 = 0.995). (C) PA 16



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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. (F) Temperature IR images of PMA-PDI-PEG-Gd NPs 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.

With strong NIR absorption, photothermal characteristic of the obtained polymeric NPs were firstly 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 to 2.5 W cm-2) to investigate the feasibility of photothermal agents for tumor ablation in vivo.46-49 Thus, in this paper we also used the continuous laser with power of 1.5 W cm-2 at 730 nm for PTT research and the following experiments 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 NPs 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, while water did not trigger the increase of temperature (Figure 2G). Interestingly, the NPs possessed the concentration-dependent thermal increase, and the induced hyperthermia is 17


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sufficient to kill tumor cells (above 42 oC).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 it was 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 the 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 number of early stage apoptosis and necrotic (Annexin V-FITC+/PI−) or later apoptosis (Annexin V-FITC+/PI+) Hela cells obviously increased after treated with the combining utilization of PMA-PDI-PEG-Gd NPs and laser irradiation. All these results indicated that 18



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PMA-PDI-PEG-Gd NPs can be developed as a safe and highly efficient photothermal agent for PTT in living mice.

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. (C) Cell apoptosis assay of Hela cell induced by PMA-PDI-PEG-Gd NPs mediated PTT.

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, obvious PA signal around the tumor sites was acquired after NP injection, while only weak signal of major blood vessels could be seen when pre-injection. In addition, after 3.5 h tail vein injection of 19


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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 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, causing 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 biodistribution assay. Images in Figure S18 revealed an absolute PA intensity of the organs, while the data in Figure 4C represented 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, 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 these proved the NPs were mainly cleared via hepatobiliary system. In order to test the MRI ability of PMA-PDI-PEG-Gd NPs in vivo, the mice with Hela tumor were administrated intravenously with 200 uL NPs (2 mg mL-1). Then, the tumor region of mice was imaged at different times (Pre-injection, 1 h, 3.5 h, 6 h, 12 h and 24 h) after injection. Figure 4D and 4E suggested the MR intensity and signal distributions as the time going 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 NPs injection and reached a maximum after 3.5 h post-injection. Afterwards, the 20



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quantitative MR signals in the tumor sites decreased gradually over 3.5 h post injection 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 high resolution pathogeny structure at unprecedented depth. Thus, PMA-PDI-PEG-Gd NPs holds great potential to provide satisfactorily accuracy and comprehensive information to guide the highly efficient photothermal ablation of tumors and prevent against the damage of the ambient normal tissues.

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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 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 22



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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 with at 0 h. (E) In vivo T1-weighted MRI of living mice at different time point after injection of PMA-PDI-PEG-Gd NPs. The yellow circles showed the position of the tumor.

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 mice were marked, weighed, and given three treatments: (1) saline (200 µL) + laser, (2) PMA-PDI-PEG-Gd NPs (2 mg/mL, 200 µL) and (3) PMA-PDI-PEG-Gd NPs (2 mg/mL, 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 post-injection 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 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 sites temperature rapidly reached to approximately 54 oC 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 PMA-PDI-PEG-Gd NPs injection merely appeared mildly increase to less than 40 oC within 10 min laser exposure (Figure 5A and 5B), further demonstrating that laser irradiation itself cannot cause damage to the tumors and is safe for 23


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animals.

Figure 5. In vivo photothermal therapy. (A) IR thermal images of mice tumor area 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 24



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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’ treatments.

To evaluate the PTT therapeutic effect, we monitored the tumor sizes of three treatment groups after PTT. Figure 5D indicated that the growth of tumor size after the treatment of PMA-PDI-PEG-Gd NPs + laser was significantly inhibited, while either NIR laser irradiation or PMA-PDI-PEG-Gd NPs alone did not inhibit tumor growth (Figure 5E). These results further confirmed 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’ treatment, mice were sacrificed with major organs including tumor, liver, heart, spleen, kidney and lung for 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 PMA-PDI-PEG-Gd NPs and laser showed dramatically necrosis and apoptosis of the tumor cells, indicating successful damage of the tumor cells. All in all, these results

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indicated the PMA-PDI-PEG-Gd NPs can be acted as an efficient theranostic platform for accurate PAI and MRI dual-modal imaging guided photothermal therapy.

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

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 high-performance MR contrast effect even at high magnetic field 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 insight on the location and size of the tumors, showing efficient passive tumor accumulation of the NPs through the EPR effect. Furthermore, in vivo photothermal cancer treatment was carried out taking advantages 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 photothermal therapy with little damage to the 26



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healthy tissue areas. Furthermore, we expect our straightforward synthetic strategy would stimulate further applications for bio-imaging and therapy.

ASSOCIATED CONTENT Supporting Information. The synthetic route to PMA-PDI-PEG, 1H-NMR spectra and

13

C-NMR

spectra of relevant compound. The size distribution of PMA-PDI-PEG and PMA-PDI-PEG-Gd NPs, the 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. The

UV–vis–NIR

absorption

and

fluorescence

spectra

of

PMA-PDI-PEG

NPs

and

PMA-PDI-PEG-Gd NPs in water. The detailed calculation method of the assembling number of PMA-PDI-PEG molecules per NPs and the number of Gd per NPs based on average size. The 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.

AUTHOR INFORMATION Corresponding Author *Email: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS 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. 27


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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, NY211003).

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