High Density Glycopolymers Functionalized Perylene Diimide

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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 Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01029 • Publication Date (Web): 29 Aug 2017 Downloaded from http://pubs.acs.org on August 30, 2017

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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,‡ 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 & Telecommunications, 9 Wenyuan Road, Nanjing 210023, 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. KEYWORDS: Active-target, Glycopolymers, ASGP-R, PA imaging, Photothermal therapy

ABSTRACT: Near-infrared (NIR) absorbing nanoagents with functions of photoacoustic imaging (PAI) and photothermal therapy (PTT) have received great attention for cancer therapy.

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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 tumor-targeted 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 anti-tumor therapy.

INTRUDUCTION 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,


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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 with FI or PAI have been widely developed for imaging-guided therapy.13,14 Compared to PDT which is hindered by the rapid oxygen over-consumption of tumor cells and hypoxic features of the tumor microenvironment, PTT is more promising due to its oxygen-independant 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 NIR-absorbing 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 small molecule dyes mostly encounter the issues of debatable long-term toxicity and photo bleaching, 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


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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 post-conjugation 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 begining to be deployed as targeted drug delivery system and PDT nanoagents for in vitro anticancer cells 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


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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-3,4,9,10-tetracarboxylic diimide (PDI) derivative, as the NIR-absorbing material. In our previous works, PDI has been proved to be an efficient PAI contrast for lightening the brain tumor and early thrombus in living mice due to its strong light absorption in 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 obtainging 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 is good for increasing the NPs stability, photothermal conversion efficiency and PA signal strength.33 (3) Asialoglycoprotein receptors (ASGP-R) is overexpressed in HepG2、Caco-2、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 aboundant 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.


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Taken together, our results revealed that the nanomaterials surface-functionalized with glycopolymers could serve as robust nanoplatforms for targeted cancer theranostics.

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 cells 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


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Characterization NMR spectra were recorded on a Bruker Ultra Shield Plus 400 MHz spectrometer (1H, 400 MHz) using tetramethylsilane (TMS) as the internal standard. The UV-visible absorption spectra were recorded on a Shimadzu UV-3600 UV-vis-NIR 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 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 3 times, then


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heated to 35 °C under an argon atmosphere. After stirred for 5 minutes, PMDETA (0.082 g, 0.5 mmol) was added to start the reaction. 3 hours later, quenched the reaction with liquid nitrogen. 10 mL DCM was added to dilute the mixture. Removed copper salts 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 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 hours. After the reaction, cooled to the room temperature, filtered to remove the inorganic salts, concentrated and sedimentated in the water. Repeated this process for 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. 1H 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 for 3 times, heated to 45 °C under an argon atmosphere. After stirred for 5 minutes, added PMDETA (28 μL) quickly to start the reaction. 24 hours later, dialysis in the water (intercept molecular weight 3.5 Kda) to remove unreacted


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alkynyl sugar and copper salts. Obtained the final powder 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, 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 vs 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 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 the 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% feat 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


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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 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, USA) at an intensity of 2 × 104 cells mL-1, respectively. After 24 h incubation at 37 °C, then further inculcated 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 and 100 μL 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, USA) at an intensity of 2 × 104 cells mL-1, respectively. After 24 h incubation at 37 °C, then further inculcated in medium containing different doses of PLAC-PDI NPs for 4 h in the dark (Blocking group, HepG2 cells were


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(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 cells viability were calculated using MTT assay. Assessment of Photothermal Effect In Vitro by Confocal Imaging HepG2 Cells were seeded in CLSM culture dishes (Costar, IL, USA) 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 six 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 PBS (4 × 106) into the left armpit of each mice. The tumor volume was calculated as V=0.5 LW2, 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 pre-injection with lactose (100 μL, 2.00 mg mL-1) and then after 4 h


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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 high-performance 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 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, (e) mice pre-injection with lactose (400 μg) and then treated with PLAC-PDI NPs (0.50 mg mL-1, 100 μL) with laser irradiation. Injected after 8h, 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. 24 hours later, selected 3 mice from each group, 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. 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


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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 < 0.05 were considered significant).

RESULTS AND DISCUSSION Synthesis and Characterization of PLAC-PDI NPs

Scheme 1. Synthesis procedures for the glycopolymers PLAC-PDI. The synthetic route of the amphiphilic PLAC-PDI is illustrated in Scheme. 1 The PLAC-PDI was synthesized via ATRP and click reaction in sequence. First, poly(glycidyl methacrylate) modified PDI (PGMA-PDI) was obtained via controllable ATRP using Br-PDI as a


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macro-initiator (Figure S1).39 PGMA-PDI had 17 repeating units (glycidyl methacrylates) in every molecule calculated by 1H NMR (Figure S6) and narrow polydispersity of 1.2 determined by gel permeation chromatography (GPC, Figure S9). The molecular weight of PGMA-PDI was thus calculated to be 3.4  103 g mol-1. The subsequent ring opening of the pendent epoxide group in the PGMA segments with NaN3 provided the azide-containing polymer bearing one azide group on each repeating unit (PGMA- N3-PDI, 1H NMR in Figure S7). PGMA- N3-PDI was further clicked with the alkyne-modified sugar (1-(2’-propargyl)-D-lactose) to produce the final product PLAC-PDI (1H NMR in Figure S8). The total molecular weight of PLAC-PDI was 1.1  104 g mol-1.

Scheme 2. Schematic illustration of the preparation of PLAC-PDI NPs and its specfically PAI/PTT applications to HepG2 tumor.


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Figure 1. Characterization of PLAC-PDI NPs. a) The hydrodynamic radius (Rh) of PLAC-PDI NPs measured by dynamic light scattering (DLS). b) Transmission electron microscopy images of PLAC-PDI NPs, scale bars represent 200 nm. c) UV-vis-NIR spectra of PLAC-PDI NPs in water at different concentrations. d) The mole extinction coefficient of the PLAC-PDI NPs at 700 nm. PLAC-PDI NPs were then fabricated via self-assembly of PLAC-PDI molecules in aqueous solution (Scheme 2). Upon directly dispersed in water, PLAC-PDI quickly self-assembled into NPs through hydrophobic-hydrophobic interaction with hydrophobic PDI segments as the core


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and hydrophilic glycopolymer segments PLAC as the protective shell. The molecular weight (Mw) of PLAC-PDI NPs measured by static light scattering was 1.6  107 g mol-1 (Figure S10), indicating the assembling number of PLAC-PDI molecule in one PLAC-PDI NPs was about 1450.40 In consideration of 17 repeating lactoses in one PLAC-PDI molecule, there existed 24 650 lactoses with high density in one PLAC-PDI NPs. Such abundant lactoses were expected to provide efficient targeting ability to HepG2 cells through “cluster glycoside effect’’. PLAC-PDI NPs can be well-dispersed in water, PBS buffer, Dulbecco’s minimum essential medium (DMEM) cell culture medium and fetal bovine serum. The diameter and morphology of PLAC-PDI NPs in aqueous solution were determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The DLS results revealed that the hydrodynamic radius (Rh) of PLAC-PDI NPs was 35 nm (the average diameter was 70 nm, Figure 1a). TEM imaging showed that PLAC-PDI NPs possessed a well-dispersed spheres structure and the mean size is around 60 nm (Figure 1b). PLAC-PDI NPs exhibited excellent colloidal stability, which is evidenced by the unaltered size after being stored in dark for 30 days as well as after 24 h incubation in fetal bovine serum at 37 °C. Moreover, only slightly size change was found after 30 min laser irradiation at 730 nm and 500 mW cm-2 (Figure S11). Such a good colloidal stability of PLAC-PDI NPs can be ascribed to the strong π-π and hydrophobic interactions among planar PDI molecules in the NPs core and the high density PLAC in the NPs surface. The strong NIR-light absorption is essential for ideal PAI/PTT theranostic nanoagents. We thus studied the optical properties of PLAC-PDI NPs and its UV-vis-NIR spectrum was shown in Figure 1c. The NPs displayed two strong absorption peak in NIR region from 660 to 700 nm. The absorption of PLAC-PDI NPs was linearly strengthened with the increase of NPs concentrations. Considering the molecular weight of PLAC-PDI NPs is 1.6107 g mol-1 and its


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extinction coefficient was 0.97 L g-1 cm-1 at 700 nm (Figure 1d), PLAC-PDI NPs exhibited excellent NIR-absorption property with the extinction coefficient of 1.5107 M-1 cm-1 at 700 nm. PLAC-PDI NPs also showed good photostability since no obvious change of absorption intensity was observed after 30 min of continuous irradiation at 730 nm and 500 mW cm-2 (Figure S12). Thus, the good NIR-absorption property and phtostability of PLAC-PDI NP make it a promising candidate for PAI and PTT.

Figure 2. Photoacoustic characterization of PLAC-PDI NPs. a) PAI of PLAC-PDI NPs in an agar phantom at concentrations of 0.12, 0.20, 0.25, 0.40, and 0.60 mg mL-1. b) The PA signal of PLAC-PDI NPs at different wavelengths. c) Linear relationship between the PA signal intensity and PLAC-PDI NPs at different concentrations.


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The PA properties of PLAC-PDI NPs were evaluated in vitro (Figure 2a). Their PA signals in aqueous solution were monitored at various excitation wavelengths (Figure 2b). The obtained PA spectrum showed a maximum PA signal at 700 nm, which is consistent with its UV-vis-NIR absorption spectrum. The PA amplitudes of PLAC-PDI NPs at 700 nm were further determined at different concentrations in an agar phantom. As shown in Figure 2a and 2c, the brightness of PAI and the strength of PA signals of PLAC-PDI NPs were linearly enhanced with increasing concentration.

Figure 3. Photothermal-conversion efficiency of PLAC-PDI NPs. Temperature elevation of pure water and the aqueous solution of PLAC-PDI NPs at different concentrations under irradiation from a 730 nm laser with a power of a) 100 mW cm-2 and b) 500 mW cm-2 as a function of


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irradiation time (0-11 min). c) The photothermal response of the aqueous solution of PLAC-PDI NPs for 500 s with laser (730 nm, 500 mW cm-2) and then the laser was shut off. d) Linear time data versus -lnθ obtained from the cooling period of Figure 3c. The photothermal performances of PLAC-PDI NPs in aqueous solutions at different concentrations (0.30 and 0.60 mg mL-1) were studied under 730 nm laser irradiation with 100 and 500 mW cm-2 power intensity, respectively. As shown in Figure 3a and 3b, the temperatures of both PLAC-PDI NPs aqueous solutions were elevated with the increase of NPs concentrations and the irradiation power intensity, while laser irradiation did not trigger the obvious temperature increase for pure water. Typically, the temperature of NP aqueous solution (0.60 mg mL-1) swiftly increased to 70 °C only after laser irradiation for 10 min. By using a previously reported calculation method (Figure S13),41,42 the photothermal conversion efficiency of PLAC-PDI NPs was determined to be 42% (Figure 3c and 3d). This results is much higher than those widely used PTT agents such as gold nanomaterials (21%)43 and Bi2Se3 nanosheets (34%)44, suggesting that PLAC-PDI NPs have an excellent photothermal effect for thermal ablation in cancer treatment.


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In vitro Cancer Cells Targeting Ability and Photothermal Therapy

Figure 4. In vitro cell targeting ability of PLAC-PDI NPs and PTT to cancer cells. a) The PA intensity of pure HepG2 cells, pure Hela cells, HepG2 cells and Hela cells incubated with PLAC-PDI NPs, and HepG2 cells blocked with excess amount of lactose before incubation with PLAC-PDI NPs. b) Viabilities of HepG2 cells (with or without blocking) and Hela cells after PTT incubated with different concentrations of PLAC-PDI NPs. Error bars are based on the standard deviations of five parallel samples. The cytotoxicity of PLAC-PDI NPs to HepG2 cells and Hela cells was first evaluated using MTT assay (Figure S14). The cells retained 95% viability even at a maximum concentration of 0.5 mg mL-1. Such a low cytotoxicity of PLAC-PDI NPs was attributed to the biologically originated nature of the glycopolymers surface. Carbohydrate recognition receptors widely and selectively express on many kinds of cell surfaces, which can selectively interaction with carbohydrate.45,46 As a typical carbohydrate recognition receptor overexpress in HepG2 cells, ASGP-R can specifically bind with galactose and lactose.47,48 In this part, we investigate the in vitro targeting specificity of PLAC-PDI NPs to ASGP-R by PAI. HepG2 cells highly expressing


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ASGP-R were chosen as a target cells model and Hela cells lacking ASGP-R expression were selected as a negative control. After incubation with PLAC-PDI NPs, HepG2 and Hela cells were collected for PAI. As shown in Figure 4a, strong PA signals in the ASGP-R-positive HepG2 cells were observed and it was enhanced with the increase of the NPs concentration (Figure S15), while the PA signal in ASGP-R-negative Hela cells was negligible. Such a significant difference of PA intensity between the HepG2 and Hela cells (7:1) clearly demonstrated the high selectivity of PLAC-PDI NPs toward HepG2 cells. In addition, a competition assay was performed, HepG2 cells were first incubated with a certain amount of free lactose to block the ASGP-R and then incubated with PLAC-PDI NPs for PAI. A dramatically weakened PA signal was observed compared to HepG2 cells without ASGP-R blocking (1:3). These PAI results proved that the glycopolymers PLAC on the surface of PDI NPs has strong binding ability to ASGP-R in HepG2 cells. Inspired by the high photothermal conversion efficiency and strong targeting specificity of PLAC-PDI NPs, we investigated their PTT effect on tumor cells in vitro. The therapeutic effects of PLAC-PDI NPs toward HepG2 and Hela cells were examined by the MTT assay. As shown in Figure 4b, upon 500 mW cm-2 of laser irradiation at 730 nm for 10 min, the viabilities of HepG2 cells decreased with the increase of NPs concentration and 90% of the cells were dead at a NPs concentration of 0.10 mg mL-1. In contrast, without laser irradiation, PLAC-PDI NPs alone did not affect the survival of HepG2 cells. Moreover, the cells of the negative control groups, including ASGP-R-negative Hela cells and ASGP-R-positive HepG2 cells pre-blocked with lactose, showed high viability over 65% after laser treatment. Furthermore, the PTT efficacy of PLAC-PDI NPs in HepG2 cells and Hela cells was also confirmed by calcein-AM and propidium iodide (PI) co-staining. Live/dead cells were differentiated by calcein-AM (live cells, green


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fluorescence) and PI (dead cells, red fluorescence). As shown in Figure S16, all Hela cells with different treatments and those HepG2 cells treated with laser-irradiation only or PLAC-PDI NPs only displayed green fluorescence, showing no cells death. In contrast, all HepG2 cells were killed when incubated with PLAC-PDI NPs (0.10 mg mL-1) and exposed to the NIR laser at 500 mW cm-2 for 10 min. All the above results verified that the targeting ability of PLAC-PDI NPs to ASGP-R over-expressed HepG2 cells can efficiently enhance the PTT effect. In vivo Tumor PAI and Photothermal Therapy

Figure 5. In vivo PAI and distribution. a) PAI of blood vessels in HepG2 tumor bearing (or Hela tumor bearing) mice that received intravenous injection of PLAC-PDI NPs (or pre-injection with


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lactose) at different time points. b) PA signal intensity of tumor tissues at different time points. c) The PAI of main organs after 8 h injection of PLAC-PDI NPs (pre-injection without/with lactose) and saline. d) The PA signal intensity of main organs after 8 h injection of PLAC-PDI NPs (pre-injection without/with lactose) and saline. For in vivo PAI and PTT application, the long circulation time is one key factor. Thus, the blood circulation time of PLAC-PDI NPs was examined by real-time measuring the PA signal intensity of blood after intravenous injection of NPs. As shown in (Figure S18), blood PA signal intensity increased from 1020 ± 20 before NPs injection to the maximum (3165 ± 61) at 4 h post-injection. Then PA signal intensity declined with time and decreased to half at 12 h post-injection. The long circulating time of PLAC-PDI NPs can be attributed to the electrically neutral glycopolymers surface which significantly reduces nonspecific protein adsorption with NPs and then prevents its rapid clearance by mononuclear phagocyte system. Given its good PAI performances and specific binding ability to HepG2 cells, the tumor selectivity of PLAC-PDI NPs was next investigated by PAI in subcutaneous tumor models. After PLAC-PDI NPs were intravenously injected into ASGP-R-positive HepG2 and ASGP-R-negative Hela tumor-bearing mice, the PA signals in the tumors were monitored with a Nexus-128 PA imaging system at designated time intervals. The in vivo PAI of blood vessels in the HepG2 and Hela tumor tissues were displayed in Figure 5a. PLAC-PDI NPs exhibited considerably higher PA intensity at the tumor site and allowed the visualization of the microscopic tumor vasculature with high contrast and resolution after 4 to 8 h NPs injection. In both tumor models, the PA signals intensities were gradually increased over time and reached a maximum at 8 h post-injection. PA signal intensities of the tumor tissues compared to that before injection were calculated as 2.54, 3.46, 5.11, 3.38 and 1.33 for HepG2 tumor and 1.43, 1.84, 2.56, 1.48, and 1.13 for Hela tumor at 2, 4, 8, 12 and


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24 h post-injection, respectively (Figure 5b). The PA signal intensity of HepG2 tumors is obviously higher than that of Hela tumors. For example, the PA signal in HepG2 tumors is about twice the strength of that in Hela tumors at 8 h post-injection. This significant difference in PA signal between HepG2 tumors and Hela tumors exhibited the good specific targeting ability of PLAC-PDI NPs to HepG2 tumors, which can be contribute to its strong binding ability to ASGP-R over-expressed in HepG2 tumors. To further prove this, ASGP-R blocking test was performed in vivo. Lactose as the blocking agent was intravenously injected 30 min before NPs injection. A suppressed enhancement of the PA signal intensity (2 h, 2.14; 4 h, 2.96; 8 h, 3.71; 12 h, 2.53; 24 h, 1.21) in HepG2 tumor was observed at each time point (Figure 5b). All these data manifested that PLAC-PDI NPs can be significantly enriched in HepG2 tumor via specific lactose-ASGP-R interaction. To better monitor the accumulation of PLAC-PDI NPs in HepG2 tumor, HepG2 tumor-bearing mice were sacrificed at 1 h, 4 h, 8 h and 24 h post-injection. The major organs and the HepG2 tumor were harvested and subjected to ex vivo PAI (Figure 5c and Figure S19). Not surprisingly, HepG2 tumor showed intense PA signals when the mice were injected with PLAC-PDI NPs. Meanwhile, lower PA signal was observed in HepG2 tumor when ASGP-R activity was restrained by lactose. These ex vivo results are in good agreement with in vivo PAI, manifesting the superb HepG2 tumor targeting ability of PLAC-PDI NPs. Additionally, the results suggested that the 8 h time point is the most appropriate for PTT because PLAC-PDI NPs displayed the highest accumulation in tumor at this time point. In addition to strong accumulation of PLAC-PDI NPs in HepG2 tumor, strong PA signal was also found in liver because ASGP-R is also widely expressed in hepatocytes and NPs can be nonspecifically absorbed by reticuloendothelial system in liver (as shown in Figure 5d and Figure S19).49-51


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Figure 6. In vivo PTT. a) Thermal images of HepG2 tumor-bearing tumor tissue samples from mice intravenously injected with PLAC-PDI NPs, blocking (by pre-injection with lactose), saline with 730 nm laser irradiation examined at the indicated time point (8 h). The laser power density was 500 mW cm-2. b) Quantitative analysis of temperature changes in the tumor area at different time points. c) Tunnel and H&E stained tumor sections collected from different groups of mice at the 24 h time point after irradiation.


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Figure 7. In vivo PTT. a) HepG2 tumor growth rate in each group after the indicated treatments. Tumor volumes were normalized to their initial size. Error bars represent the standard error of the mean. b) Images of HepG2 tumors from mice sacrificed after 14 days of therapy. For the therapeutic groups, mice were intravenously injected with PLAC-PDI NPs and subjected to 730 nm laser irradiation (500 mW cm-2) for 10 min at the 8 h time point. Four groups of mice (n= 6/group) were used as controls: saline without laser irradiation; With laser irradiation; Blocking (by pre-injection with lactose) with laser irradiation; PLAC-PDI NPs without laser irradiation. c) Tumor weight, sacrificed mice from the above mentioned five groups. d) Body weight curves of HepG2 tumor-bearing mice for each group.


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Figure 8. H&E staining of major mouse organs. PTT efficacy of PLAC-PDI NPs to HepG2 tumor in vivo was next assessed. The HepG2 tumor-bearing mice were randomly divided into five groups (n= 6/group): (a) mice treated with saline without laser irradiation, (b) mice treated with saline with laser irradiation, (c) mice treated with PLAC-PDI NPs without laser irradiation, (d) mice treated with PLAC-PDI NPs with laser irradiation, and (e) mice pre-injected with lactose and then treated with PLAC-PDI NPs and laser irradiation (blocking). HepG2 tumor-bearing mice in b, d, and e groups were subjected to similar laser irradiation (730 nm, 500 mW cm-2) for 10 min at 8 h post-injection. First, we verified the in vivo photothermal effect of PLAC-PDI NPs with an infrared thermal imaging camera to record the infrared thermal images and temperatures (Figure 6a). In the PLAC-PDI NPs-treated group


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(d group), the tumor temperature was rapidly raised to 55 °C during the laser irradiation. On the contrary, no significant temperature change was observed in the saline-treated group (b group). A moderate increase of temperature (to 43 °C) in the tumor was observed for the blocking group (e group) (Figure 6b). This result revealed that PLAC-PDI NPs have efficient photothermal conversion in vivo. In addition, the in vivo photothermal effect is in accordance with the accumulation of PLAC-PDI NPs in HepG2 tumors. In d group, HepG2 tumor showed more accumulation of PLAC-PDI NPs due to the combination of their specific binding ability and passive EPR effect. Meanwhile, blocking group showed insufficient accumulation because PLAC-PDI NPs accumulation in this case only depended on EPR effect. Next, we investigated the effective ablation of PLAC-PDI NPs to HepG2 tumor. The tumor tissue was collected at 24 h after the various treatments (Figure 6c) for hematoxylin and eosin (H&E) staining and tunnel staining. No obvious tumor necrosis was found in a, b and c groups. In comparison, almost whole tumor cells were severely destroyed in d group whereas only partially damaged in e group after laser irradiation. These differences was also explained by the efficient targeting ability of PLAC-PDI NPs through the specific PLAC and ASGP-R interaction. Therefore, all these data manifested that PLAC-PDI NPs can act as a powerful PTT agent for in vivo tumor ablation. To further evaluate the enhanced PTT effect of PLAC-PDI NPs to HepG2 tumor, the tumor volume and mouse body weight were also monitored after the various treatments and recorded every other day. As shown in Figure 7, the tumors were nearly eliminated by PLAC-PDI NPs with laser irradiation treatment for 14 days (the V/V0 only increased to 1.5). In marked contrast, no antitumor effect (the V/V0 increased to 15-17) was detected in the control groups (a, b, and c groups). Noteworthy, the blocking group (e group) showed the inhibited growth rate of HepG2 tumor to some extent. The reason is that PLAC-PDI NPs can highly accumulate in HepG2


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tumors through both active and passive targeting effect while in blocking group the NPs only accumulate in HepG2 tumors by EPR effect. Therefore, PLAC-PDI NPs displayed the enhanced PTT efficacy to HepG2 tumor in vivo. At last, the low cytotoxicity of PLAC-PDI NPs was examined. Major normal organs, including heart, liver, spleen, lung, and kidney of mice from different groups were collected for H&E staining after 14 d treatment. As shown in Figure 8, no significant damage or inflammation was found in the H&E stained images. Combined with the slight weight increase in each group during the treatment (Figure 7d), all results revealed that PLAC-PDI NPs appeared slightly acute toxicity to normal tissues. Collectively, the PLAC-PDI NPs is an ideal HepG2 tumor-targeting nanoagent with low cytotoxicity for PA/PTT application.

CONCLUSIONS In summary, we have developed a high density glycopolymers-functionalized organic semiconducting NPs for PAI/PTT. The obtained NPs showed high photothermal conversion efficiency, low cytotoxicity, and long circulating time. In vitro and in vivo experiments showed that PLAC-PDI NPs exhibited specific targeting ability to HepG2 cell and therefore exerted enhanced PAI/PTT effect on HepG2 tumor. Considering carbohydrate recognition receptors widely exist in cancer cells, such as mannose receptor over-expressed on MCF-7 breast cancer cells, our designed nanoplatform can efficiently target different tumors by simply changing the glycopolymers on the NPs surface. This study provided new insights for designing novel glycopolymers-based therapeutic nanoagents for efficient tumor imaging and anti-tumor therapy.

ASSOCIATED CONTENT Supporting information


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1

H-NMR spectras of fabricating PLAC-PDI. GPC of PGMA-PDI and PGMA-N3-PDI. Details of

measuring molecular weight. Stability of PLAC-PDI NPs. Details of calculating the photothermal conversion efficiency. In vitro and in vivo specific-binding experiments and phototherapy.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] ORCID Quli Fan: 0000-0002-9387-0165 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by the National Basic Research Program of China (973 Program, No. 2015CB932200 and 2012CB723402), the National Natural Science Foundation of China (No. 21604042, 61378081, 21574064, and 61136003), Synergetic Innovation Center for Organic Electronics and Information Displays, Jiangsu National Synergetic Innovation Center for Advanced Materials, the Natural Science Foundation of Jiangsu Province of China (No. BK20150843), and NUPTSF (No. NY215017, NY211003, and NY215080).

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Table of Contents Graphic

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,‡ Quli Fan†, *


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Scheme 1. Synthesis procedures for the glycopolymers PLAC-PDI. 333x180mm (96 x 96 DPI)


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Scheme 2. Schematic illustration of the preparation of PLAC-PDI NPs and its specfically PAI/PTT applications to HepG2 tumor. 248x200mm (96 x 96 DPI)


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Figure 1. Characterization of PLAC-PDI NPs. a) The hydrodynamic radius (Rh) of PLAC-PDI NPs measured by dynamic light scattering (DLS). b) Transmission electron microscopy images of PLAC-PDI NPs, scale bars represent 200 nm. c) UV-vis-NIR spectra of PLAC-PDI NPs in water at different concentrations. d) The mole extinction coefficient of the PLAC-PDI NPs at 700 nm. 190x167mm (96 x 96 DPI)


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Figure 2. Photoacoustic characterization of PLAC-PDI NPs. a) PAI of PLAC-PDI NPs in an agar phantom at concentrations of 0.12, 0.20, 0.25, 0.40, and 0.60 mg mL-1. b) The PA signal of PLAC-PDI NPs at different wavelengths. c) Linear relationship between the PA signal intensity and PLAC-PDI NPs at different concentrations. 159x112mm (220 x 220 DPI)


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Figure 3. Photothermal-conversion efficiency of PLAC-PDI NPs. Temperature elevation of pure water and the aqueous solution of PLAC-PDI NPs at different concentrations under irradiation from a 730 nm laser with a power of a) 100 mW cm-2 and b) 500 mW cm-2 as a function of irradiation time (0-11 min). c) The photothermal response of the aqueous solution of PLAC-PDI NPs for 500 s with laser (730 nm, 500 mW cm2) and then the laser was shut off. d) Linear time data versus -lnθ obtained from the cooling period of Figure 3c. 222x173mm (300 x 300 DPI)


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Figure 4. In vitro cell targeting ability of PLAC-PDI NPs and PTT to cancer cells. a) The PA intensity of pure HepG2 cells, pure Hela cells, HepG2 cells and Hela cells incubated with PLAC-PDI NPs, and HepG2 cells blocked with excess amount of lactose before incubation with PLAC-PDI NPs. b) Viabilities of HepG2 cells (with or without blocking) and Hela cells after PTT incubated with different concentrations of PLAC-PDI NPs. Error bars are based on the standard deviations of five parallel samples. 225x88mm (300 x 300 DPI)


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Figure 5. In vivo PAI and distribution. a) PAI of blood vessels in HepG2 tumor bearing (or Hela tumor bearing) mice that received intravenous injection of PLAC-PDI NPs (or pre-injection with lactose) at different time points. b) PA signal intensity of tumor tissues at different time points. c) The PAI of main organs after 8 h injection of PLAC-PDI NPs (pre-injection without/with lactose) and saline. d) The PA signal intensity of main organs after 8 h injection of PLAC-PDI NPs (pre-injection without/with lactose) and saline. 225x180mm (300 x 300 DPI)


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Figure 6. In vivo PTT. a) Thermal images of HepG2 tumor-bearing tumor tissue samples from mice intravenously injected with PLAC-PDI NPs, blocking (by pre-injection with lactose), saline with 730 nm laser irradiation examined at the indicated time point (8 h). The laser power density was 500 mW cm-2. b) Quantitative analysis of temperature changes in the tumor area at different time points. c) Tunnel and H&E stained tumor sections collected from different groups of mice at the 24 h time point after irradiation. 237x185mm (300 x 300 DPI)


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Figure 7. In vivo PTT. a) HepG2 tumor growth rate in each group after the indicated treatments. Tumor volumes were normalized to their initial size. Error bars represent the standard error of the mean. b) Images of HepG2 tumors from mice sacrificed after 14 days of therapy. For the therapeutic groups, mice were intravenously injected with PLAC-PDI NPs and subjected to 730 nm laser irradiation (500 mW cm-2) for 10 min at the 8 h time point. Four groups of mice (n= 6/group) were used as controls: saline without laser irradiation; With laser irradiation; Blocking (by pre-injection with lactose) with laser irradiation; PLAC-PDI NPs without laser irradiation. c) Tumor weight, sacrificed mice from the above mentioned five groups. d) Body weight curves of HepG2 tumor-bearing mice for each group. 210x129mm (220 x 220 DPI)


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Figure 8. H&E staining of major mouse organs. 366x230mm (96 x 96 DPI)


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High Density Glycopolymers Functionalized Perylene Diimide

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