Near-Infrared Conjugated Polymer

Jul 11, 2016 - Far-red (FR)/near-infrared (NIR) photosensitizer is highly desirable in image-guided photodynamic cancer therapy. Herein, a new conjuga...
5 downloads 18 Views 5MB Size
Download PDF
Article pubs.acs.org/Macromolecules

A Photostable Far-Red/Near-Infrared Conjugated Polymer Photosensitizer with Aggregation-Induced Emission for ImageGuided Cancer Cell Ablation Wenbo Wu,†,‡ Guangxue Feng,† Shidang Xu,† and Bin Liu*,†,§ †

Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585 Department of Materials Science and Engineering, National University of Singapore, 7 Engineering Drive 1, Singapore 117574 § Institute of Materials Research and Engineering (IMRE), 2 Fusionopolis Way, Innovis, Singapore 138634 ‡

S Supporting Information *

ABSTRACT: Far-red (FR)/near-infrared (NIR) photosensitizer is highly desirable in image-guided photodynamic cancer therapy. Herein, a new conjugated polymer of poly(1,2-bis(4-((6-bromohexyl)oxy)phenyl)-1,2diphenylethene-co-alt-9,10-anthraquinone) (PTPEAQ) consisting of tetraphenylethylene (TPE), an iconic aggregation-induced emission (AIE) active group as the electron donor, and anthraquinone (AQ) as the acceptor, is prepared for the first time through one-pot Suzuki polymerization. Encapsulation of PTPEAQ with a block copolymer followed by surface functionalization with anti-Her2 affibody yields PTPEAQ-NP-HER2. It shows bright AIE-active FR/NIR emission and efficient singlet oxygen generation under visible light irradiation, which has been successfully used for photodynamic cancer cell ablation using SKBR-3 cells, a type of breast cancer cell with HER2 overexpression on cell membrane, as an example.

■

INTRODUCTION In recent decades, conjugated polymers (CPs) have been widely used for biosensing, bioimaging, and photodynamic therapy (PDT).1−5 The development of CPs with the desirable functions for image-guided therapy represents a new research direction.6−10 The combination of light emission and photosensitization in a single polymer chain has attracted great research interest in biocidal studies and cancer cell ablation.11−16 An ideal photosensitizer should emit photostable FR/NIR (in the region of 650−900 nm) fluorescence and generate sufficient reactive oxygen species (ROS) to kill cancer cells under light illumination. So far, most of the pure CP photosensitizers are emissive in the blue or green region, and the short emission wavelength makes them less suitable for in vivo applications.6,8,10,17 One strategy to partially address the problem is to link FR/NIR photosensitizers (e.g., porphyrin) into CP side chains or backbones.17−19 However, when the concentration of FR/NIR photosensitizers is high or the polymer exists in aggregate state, the ROS generation ability is largely reduced.17 In addition, the photostability of the current CP photosensitizers remains a problem, as the CP chains could be decomposed by the generated singlet oxygen (1O2) to some extent.6 It is desirable if one could overcome the limitations by developing CP photosensitizers with strong light absorption in the visible region, effective ROS generation, and stable NIR emission for image-guided therapy or cancer cell ablation. To realize CPs with FR/NIR emission, one of the most effective strategies is to introduce strong donor and acceptor units into the polymers, so that one can take advantage of the © 2016 American Chemical Society

strong charge transfer characteristics of the polymer backbones.20−22 In general, the quantum yields (QYs) of these FR/ NIR CPs are much lower than the blue or green ones.22,23 The QYs could be further reduced when they are brought to water because the strong charge transfer could induce fluorescence quenching in polar media.24 One of the effective strategies to solve the problem is to form nanoparticles (NPs) which encapsulate the polymer photosensitizers and bring them into aqueous media.1,22,25,26 In these NPs, the hydrophobic end of the polymer matrix will wrap the CPs and provide a protection shell to more or less separate them from oxygen and water invasion, yielding improved QYs in aqueous media. However, when CPs are encapsulated into NPs, the well-known aggregation-caused emission quenching (ACQ) effect comes into play, which quenches the overall fluorescence.27,28 The aggregation can also decrease the ROS generation ability of the polymer NPs. On the other hand, aggregation-induced emission (AIE) represents an opposite phenomenon: a series of very weakly emissive or nonemissive molecules in dilute solutions are induced to emit efficiently by aggregate formation.29,30 Most of hydrophobic AIE dyes can emit strong fluorescence as aggregates in aqueous media, regardless of their emission colors.31−33 In our recent work, we reported that the ROS generation ability of AIE photosensitizer NPs was also very Received: May 7, 2016 Revised: June 18, 2016 Published: July 11, 2016 5017

DOI: 10.1021/acs.macromol.6b00958 Macromolecules 2016, 49, 5017−5025

Article

Macromolecules

Scheme 1. Synthetic Routes to M1 (A), PTPEAQ (B), PTPEAQ-PEG-NP (C), and PTPEAQ-NP and PTPE-NP-HER2 (D)

5018

DOI: 10.1021/acs.macromol.6b00958 Macromolecules 2016, 49, 5017−5025

Article

Macromolecules high.34 This motivates us to design and synthesize an AIEactive FR/NIR CP photosensitizer to test whether it can inherit the good performance from FR/NIR AIE photosensitizers, but with improved materials processability. In this contribution, we report the design and synthesis of a new FR/NIR CP with both AIE characteristics and good ROS generation ability. Tetraphenylethylene (TPE) is one of the iconic AIE active groups, and almost all the CPs containing TPE moieties reported so far are AIE active (Chart S1 shows some examples).35−38 On the other hand, anthraquinone (AQ) has been developed to construct a series of FR/NIR dyes with very small ΔEst values (the energy gap between the lowest singlet state (S1) and the lowest triplet state (T1)) (Chart S2).39 The small ΔEst values will favor intersystem crossing (ISC) process, which is likely to generate efficient 1O2 upon light irradiation.40,41 In this contribution, by using TPE as the electron donor and AQ as the electron acceptor, a new CP of PTPEAQ has been prepared (Scheme 1). As expected, the strong charge transfer from TPE to AQ makes the polymer FR/ NIR emissive, and the polymer also inherits the AIE characteristics from TPE. Furthermore, the polymer is photostable and could generate efficient 1O2 under white light irradiation. Subsequently, two types of NPs of PTPEAQPEG-NP and PTPEAQ-NP are prepared to bring PTPEAQ into aqueous media for image-guided cancer cell ablation. We show that the two NPs have different fluorescence intensity and ROS generation ability, and anti-HER2 affibody functionalized PTPEAQ-NPs could selectively target to SKBR-3 cancer cells for image-guided cancer cell ablation.

■

vigorous stirring for 40 h under a nitrogen atmosphere. After cooling to room temperature, the mixture was poured into methanol. The obtained solid was dissolved in THF, and the insoluble solid was filtered out. The filtrate was concentrated and precipitated into methanol, and the obtained solid was then washed with acetone to yield PTPEAQ as a red solid (142.2 mg, 79.3% yield). Mw = 21 000, Mw/Mn = 1.67 (SEC, polystyrene calibration). 1H NMR (600 MHz, CDCl3, 298 K) δ: (TMS, ppm): 1.4−1.5 (−CH2−), 1,7−1.8 (−CH2 −), 1.8−1.9 (−CH2−), 3.3−3.5 (−CH2Br−), 3.8−4.0 (−OCH2−), 6.6−6.7 (ArH), 6.9−7.0 (ArH), 7.1−7.2 (ArH), 7.4− 7.6 (ArH), 7.9−8.0 (ArH), 8.2−8.4 (ArH), 8.4−8.5 (ArH). 13C NMR (150 MHz, CDCl3, 298 K) δ (ppm): 24.4, 25.3, 28.0, 29.1, 32.7, 33.8, 59.3, 67.6, 76.8, 77.0, 77.2, 113.9, 125.2, 126.6, 127.2, 128.0, 131.9, 132.2, 132.6, 134.0, 135.7, 139.7, 145.0, 157.8, 157.9. Synthesis of NPs. To fabricate PTPEAQ-NP, the THF mixture containing 1 mg of PTPEAQ and 2 mg of DSPE-PEG2000-Mal was poured into water with 10-fold dilution. The THF/water mixture was then sonicated for 2 min using a microtip ultrasound sonicator at 12 W output (XL2000, Misonix Inc., Farmingdale, NY). After THF evaporation by stirring the obtained suspension in fume hood overnight, the PTPEAQ-NP (10 mL, 0.1 mg/mL based on PTPEAQ mass concentration) was obtained by filtration through a 0.2 μm syringe driven filter. To synthesize PTPEAQ-PEG-NPs, pure THF solution of PTPEAQ-PEG was used following the same procedure for PTPEAQ-NPs, without the addition of DSPE-PEG-Mal. Synthesis of PTPEAQ-NP-HER2. To synthesize PTPEAQ-NPHER2, the prepared PTPEAQ-NP suspension (6 mL) was mixed with cysteine modified anti-HER2 affibody (500 μg/mL, 100 μL) and was kept at room temperature for 12 h. The click chemistry reaction occurs between the PTPEAQ-NP surface maleimide groups and the only thiol group on the affibody. After centrifugation using a centrifugal filter with molecular cutoff of 100 000 kDa to remove excess affibody, PTPEAQ-NP-HER2 (6 mL, 0.1 mg/mL based on PTPEAQ mass concentration) was collected for further study. Cell Culture. SKBR-3 and NIH-3T3 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS and 1% PS at 37 °C in a humidified environment with 5% CO2. Before experiments, the cells were precultured until confluence was reached. Cellular Imaging. NIH-3T3 and SKBR-3 cells were seeded in 8well chamber (LAB-TEK, Chambered Coverglass System) at 37 °C. After 80% confluence, the cells were washed twice with 1× PBS buffer. PTPEAQ-NP-HER2 (10 μg/mL) suspended in DMEM was then added into these wells. The cells were incubated for 2 h, after which the cells were washed twice with 1× PBS buffer. The cells were subsequently incubated with Hoechst 33342 (5 μg/mL) for 20 min. After twice washing with 1× PBS buffer, the cells were imaged by confocal laser scanning microscopy (CLSM). Cytotoxicity Studies. The viabilities of SKBR-3 and NIH-3T3 cells under different treatment were evaluated using methylthiazolyldiphenyltetrazolium bromide (MTT) assays. Both cells were seeded in 96-well plates (Costar, Chicago, IL) at an intensity of 4 × 104 cells/ mL. After 24 h culture, the cells were incubated with PTPEAQ-NPHER2 DMEM suspension at various concentrations for 2 h. The PTPEAQ-NP-HER2 suspensions were replaced by fresh DMEM containing 10% FBS and 1% PS. The selected wells were exposed to white light irradiation (60 mW/cm2) for 5 min. All the cells were further cultured for 24 h and then washed with 1× PBS buffer. Freshly prepared MTT solution at concentration of 0.5 mg/mL was added to 96-well plate at the volume of 100 μL per well. After 3 h incubation, the MTT solution was replaced by filtered DMSO (100 μL per well). The plate was gently shaken for 10 min to dissolve all the crystals formed. The cell viability will be accessed by the means of MTT absorbance at 570 nm measured by the microplate reader (Genios Tecan). The controlled cells only treated with culture medium was arbitrarily determined to have 100% cell viability. Live Cell Staining. SKBR-3 cells were cultured in 8-well chamber. After 80% confluence, the cells were treated with PTPEAQ-NP-HER2 (20 μg/mL) suspended cell culture medium for 2 h. The cells were then washed twice with 1× PBS buffer, and the selected wells were then exposed to white light irradiation (400−1000 nm, 60 mW/cm2)

EXPERIMENTAL SECTION

Materials and Instrumentation. 1,2-Distearoyl-sn-glycero-3phosphoethanolamine-N-[maleimide(poly(ethylene glycol))-2000] (DSPE-PEG2000-Mal) was provided by Avanti Polar Lipid, Inc. (Alabaster, AL). Hoechst 33342 and fluorescein diacetate (FDA) were purchased from Invitrogen. Fetal bovine serum (FBS) was provided by Gibco (Lige Technologies, Switzerland). Anti-HER2 Affibody was purchased from affibody (Solna, Sweden). Tetrahydrofuran (THF) was distilled from sodium benzophenone ketyl under dry nitrogen immediately prior to use. Ultrapure grade 10× phosphate-buffered saline (PBS) buffer with pH = 7.4 was purchased from first BASE Singapore. Milli-Q water was supplied by Milli-Q Plus System (Millipore Corporation, Bedford, MA). SKBR-3 breast cancer cells and NIH-3T3 normal fibroblast cells were purchased from American Type Culture Collection. All other chemicals and reagents were purchased from Sigma-Aldrich and used as received. 1 H and 13C NMR spectra were measured on a Bruker Avance 600 spectrometer using tetramethylsilane (TMS; δ = 0 ppm) as internal standard. Size exclusion chromatography (SEC) analysis was conducted with a Waters 996 photodiode detector and Phenogel gel permeation chromatography columns, using polystyrenes as the standard and THF as the eluent at a flow rate of 1.0 mL/min. Attenuated total reflectance−Fourier transform infrared spectroscopy (ATR-FTIR) was record on a Nicolet iS 50 FT-IR spectrometer in the range of 4000−400 cm−1 with resolution of 4 cm−1 averaging from 32 scans. UV−vis and photoluminescence spectra were recorded using a Shimadzu UV-1700 and a PerkinElmer LS 55 spectrometer, respectively. Hydrodynamic diameter and size distribution were measured by laser light scattering (LLS) with a Zetasizer Nano S (Malvern Instruments Ltd., Worcestershire, UK) at room temperature. Synthesis of Polymer PTPEAQ. Tetraphenylethylene-based monomer M1 (188.4 mg, 0.20 mmol), anthraquinone-based monomer M2 (72.2 mg, 0.20 mmol), tris(dibenzylideneacetone)dipalladium(0) (3.0 mg), and tri(o-tolyl)phosphine (6.0 mg) were dissolved in a mixture of toluene (8 mL) and 20% aqueous tetraethylammonium hydroxide (2 mL) under nitrogen. The mixture was refluxed with 5019

DOI: 10.1021/acs.macromol.6b00958 Macromolecules 2016, 49, 5017−5025

Article

Macromolecules

Figure 1. (A) UV−vis spectra of PTPEAQ in THF (0.01 mg/mL). (B) Photoluminescence (PL) spectra of PTPEAQ in THF and THF−hexane mixtures (0.01 mg/mL, λex = 440 nm). (C) Fluorescent images of solutions or suspensions of PTPEAQ (0.01 mg/mL) in THF/hexane mixtures under UV lamp (λ = 365 nm, 3 W). (D) Fluorescent image of PTPEAQ in solid state under UV lamp (λ = 365 nm, 3 W). with different irradiation. After light treatment, the cells were further incubated with FDA (100 μg/mL) for 10 min to staining alive cells. After twice washing with buffer, the cells were imaged by CLSM.

PTPEAQ chains and the hydrophilic segments (PEG chains) extend into the aqueous phase. Figure S1 in the Supporting Information shows the 1H NMR spectra of PTPEAQ, M1, and M2. After polymerization, there is a clear signal broadening, and the chemical shifts of all the characteristic peaks are nearly the same as those for the corresponding monomers M1 or M2, confirming that PTPEAQ is constructed by TPE and AQ moieties. In addition, the signals assigned to the protons of the boronic ester in monomer M1 at δ = 1.32 ppm disappeared in the spectrum of PTPEAQ, confirming successful Suzuki polymerization. After reaction with sodium azide, the original peak at 3.4−3.5 ppm of −CH2Br in the 1H NMR spectrum of PTPEAQ disappears, while the signal at 3.2−3.3 ppm enhances in that of PTPEAQN3 (Figure S3), indicating the quantitative formation of the desired azido groups. In the 1H NMR spectrum of PTPEAQPEG (Figure S4), the typical peak of PEG at 3.5 ppm is the direct evidence to show the success of click reaction. A similar phenomenon is also observed in their FT-IR spectra. As shown in Figure S5, in comparison with the spectrum of PTPEAQ, a new peak at about 2095 cm−1 associated with azido groups appears in the IR spectrum of PTPEAQ-N3, indicating that the nucleophilic substitution reaction is successful. The fact that the same peak disappears in the spectrum of PTPEAQ-PEG further confirms the successful conjugation with PEG chains. The molecular weight of PTPEAQ, determined by SEC using THF as solvent and monodisperse polystyrene as calibration standard, is 2.10 × 104 g/mol (weight-average molecular weights) or 1.26 × 104 g/mol (number-average molecular weights), with a polydispersity index (PDI) of 1.67. The number-average molecular weight of PTPEAQ-N 3 was calculated to be 1.35 × 104 g/mol, while after the click reaction, it increases to 4.80 × 104 g/mol for PTPEAQ-PEG, indicating the successful PEG conjugation. To understand whether the molecular design has an obvious impact on the ΔEst value of the polymers, the time-dependent density functional theory (TD-DFT) method43−45 was used to calculate the ΔEst values for the model compounds of PTPEAQ (see Figure S6), which are 0.094, 0.103, and 0.157 eV respectively for the repeat unit of PTPEAQ (model 1), two

■

RESULTS AND DISCUSSION Synthesis and Theoretical Calculation of ΔEst. The synthetic route to PTPEAQ is shown in Scheme 1. In the first step, benzophenone derivative S3 was prepared in 65.6% from (4-methoxyphenyl)boronic acid (S1) and 4-bromobenzaldehyde (S2) following a reported method.42 The methyl group in compound S3 was removed in HBr solution to yield compound S4, which was further modified by 1,6-dibromohexane to yield compound S5. The bromide-functionalized TPE S6 was subsequently synthesized via McMurry olefination in 68.6% yield. Further reaction between compound S6 and 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) yielded TPE-based monomer M1 as a white solid in 46.4% yield. In the last step, the target polymer PTPEAQ was prepared in 79.3% yield through palladium-catalyzed Suzuki polymerization between monomer M1 and AQ-based monomer M2. After purification by precipitation, PTPEAQ is readily soluble in common organic solvents, such as toluene, THF, etc. We also prepared another polymer with a similar structure (Scheme S1 in Supporting Information), but only insoluble precipitates were obtained. All the reaction conditions used in the polymerization were exactly the same, except that different TPE-based monomers were used. The position of the alkyl group in TPE units is the only difference between the two monomers, which indicates the importance of the alkyl group in the side chain of the CPs for adjusting the solubility. To endow PTPEAQ with water dispersity for biological applications, different NPs were prepared to bring it into aqueous media (Schemes 1C and 1D). In the first strategy, PEG was directly linked to the side chain of PTPEAQ to yield PTPEAQ-PEG, which self-assembles in aqueous media to form PTPEAQ-PEG-NP (Scheme 1C). In the second strategy, an amphiphilic polymer DSPE-PEG2000-Mal (chemical structure shown in Scheme 1D) was used as the matrix to encapsulate the PTPEAQ polymer, which yielded PTPEAQ-NP with the hydrophobic segments (DSPE moieties) entangled with 5020

DOI: 10.1021/acs.macromol.6b00958 Macromolecules 2016, 49, 5017−5025

Article

Macromolecules

Figure 2. UV−vis spectra (A) and PL spectra (B) of PTPEAQ (black line), PTPEAQ-PEG-NP (red line), and PTPEAQ-NP (blue line) in water/ THF mixture (v:v = 100:1) or water. [PTPEAQ] = 0.01 mg/mL, λex = 440 nm.

Figure 3. UV−vis spectra of ABDA in the presence of PTPEAQ (A), PTPEAQ-PEG-NP (B), PTPEAQ-NP (C), or Rose Bengal (D) under light irradiation (60 mW/cm2, 400−1000 nm). (E) Decomposition rates of ABDA by PTPEAQ, PTPEAQ-PEG-NP, PTPEAQ-NP, and Rose Bengal under light irradiation, where A0 and A are the absorbance at 378 nm before and after irradiation, respectively. (F) Decomposition rates of PTPEAQ, PTPEAQ-PEG-NP, PTPEAQ-NP, and Rose Bengal under light irradiation, where A0 and A are the absorbance at 450 nm (PTPEAQ, PTPEAQPEG-NP, PTPEAQ-NP) or 550 nm (Rose Bengal) before and after irradiation, respectively.

TPE units linked to one AQ core (model 2), and two AQ units linked to a TPE core (model 3). Optical Properties of PTPEAQ, PTPEAQ-PEG-NP, and PTPEAQ-NP. In THF solution, PTPEAQ shows an absorption maximum at 340 nm with a shoulder at 440 nm (Figure 1A), which can be assigned to the absorption of the π−π* transition of the conjugated polymer backbone and the change transfer from the donor to the acceptor, respectively. As shown in Figure 1B, the polymer is almost nonemissive in THF. Addition of n-hexane, a poor solvent of PTPEAQ, to its THF solution leads to gradual increase in polymer fluorescence. When the volume fraction of n-hexane is higher than 90%, the solution becomes turbid, and obvious precipitation is observed, indicating the formation of polymer aggregates. The corresponding photos are shown in Figure 1C. The clear fluorescence change of PTPEAQ with the increasing of nhexane contents reveals that PTPEAQ is AIE active. Furthermore, when PTPEAQ powders are illuminated with a UV lamp (λ = 365 nm), bright red fluorescence is observed (Figure 1 D), which is clearly visible to naked eye, further confirming its AIE feature.

As shown in Figure 2A, the UV−vis spectrum of PTPEAQ in water/THF mixture (v:v = 99:1) is similar to that in THF solution, with a 10 nm red-shift due to the increased solvent polarity from THF to water. However, the emission of PTPEAQ in water/THF mixture (v:v = 100:1) is almost not detectable (Figure 2B). Although water could induce PTPEAQ to aggregate to enhance the fluorescence due to its AIE property, the very polar solvent medium could quench the fluorescence due to the strong charge transfer characteristics of the polymer. In this case, the quenching effect is dominant, leading to dark fluorescence of PTPEAQ in water. To enhance the PTPEAQ fluorescence in water, we fabricated polymer NPs with the hope to more or less shield PTPEAQ from the solvatochromic effect. As shown in Schemes 1C and 1D, both self-assembly and nanoprecipitation strategies have been used to prepare the relative NPs, namely PTPEAQPEG-NP and PTPEAQ-NP. The optical properties of both NPs are summarized in Figure 2. The peak shapes of UV−vis spectra of these two types of NPs are similar to PTPEAQ in aqueous solution. However, their PL behaviors are quite different. At the same concentration of PTPEAQ, both NPs are 5021

DOI: 10.1021/acs.macromol.6b00958 Macromolecules 2016, 49, 5017−5025

Article

Macromolecules

To test the targeting effect of PTPEAQ-NP-HER2, SKBR-3 cancer cells and NIH-3T3 normal cells were chosen as positive and negative control, respectively. We incubated PTPEAQ-NPHER2 and Hoechst with SKBR-3 and NIH-3T3 cells, and the fluorescence was monitored by laser confocal fluorescence microscope. The corresponding confocal images are shown in Figure 4, where red fluorescence of PTPEAQ-NP-HER2 was

emissive in aqueous media (Figure 2B), and PTPEAQ-NP gives much higher emission than PTPEAQ-PEG-NP. 1 O2 Generation Ability of PTPEAQ, PTPEAQ-PEG-NP, and PTPEAQ-NP. To evaluate the ROS generation ability of PTPEAQ, PTPEAQ-PEG-NP, and PTPEAQ-NP, 9,10anthracenediyl-bis(methylene)dimalonic acid (ABDA), a commercially available singlet oxygen (1O2) indicator, was used in the experiments. As shown in Figures 3A−C, under white light irradiation (400−1000 nm, 60 mW/cm2), the presence of PTPEAQ (Figure 3A) or PTPEAQ-PEG-NP (Figure 3B) or PTPEAQ-NP (Figure 3C) at a fixed PTPEAQ concentration of 0.01 mg/mL based on polymer chain can lead to gradually decreased absorbance of ABDA (100 μmol/L) in water, indicating that ABDA is degraded by the generated 1O2. In addition, under the same test conditions, PTPEAQ-PEG-NP and PTPEAQ-NP could decompose ABDA more quickly than PTPEAQ, indicating enhanced ROS generation ability for PTPEAQ-PEG-NP and PTPEAQ-NP. As PTPEAQ has strong electron donor−acceptor structures in the backbone, the fast process of charge transfer induced fluorescence quenching in aqueous media46 competes with ROS generation. For both PTPEAQ-PEG-NP and PTPEAQ-NP, the reduced environmental interaction on the polymer leads to enhanced ROS generation ability, which is consistent with the trend in fluorescence enhancement. When Rose Bengal (RB), a popular photosensitizer with a 1O2 quantum yield of 0.75 (Figure 3D), was used as a reference to quantify the ROS generation ability of these NPs, the 1O2 quantum yields of PTPEAQ-NP, PTPEAQ-PEG-NP, and PTPEAQ were calculated to be 0.82, 0.34, and 0.22, respectively. It is also important to note that the decomposition rate of ABDA by RB decreases with time, while that of the polymer remains nearly constant throughout the tested time. This is because of the difference in photostability between RB and the polymer NPs. As shown in Figures 3A− C,F, under light irradiation, PTPEAQ is very stable, while RB could be easily decomposed by the generated singlet oxygen. Therefore, PTPEAQ-NP is a better photosensitizer than RB due to its superior performance in photostability and ROS generation capability. Synthesis of PTPEAQ-NP-HER2 and Its Application in Image-Guided Cancer Cell Ablation. As discussed above, PTPEAQ-NP demonstrated better performance than PTPEAQ-PEG-NP in fluorescence output and ROS generation; therefore, PTPEAQ-NP was chosen for further applications. To realize image-guided and targeted cancer cell ablation, targeting molecules are required to bind to PTPEAQNP. In this regard, anti-HER2 affibody was used as the targeting molecule, which can specifically target SKBR-3 cancer cells with HER2 overexpressed on cell membrane.21 Click reaction between the maleimide groups on the surface of PTPEAQ-NP and cysteine modified Anti-HER2 affibody yielded PTPEAQ-NP-HER2 (Scheme 1D). After anti-HER2 affibody conjugation, the zeta potential of the NPs changed from −2.3 mV of PTPEAQ-NP to −5.7 mV of PTPEAQ-NPHER2, indicating the successful click reaction. The Bradford assay was further used to quantify the anti-HER2 conjugation percentage, which revealed that the weight percentage of antiHer2 on PTPEAQ-NP-HER2 was 11.6% with a conjugation yield of 39.6%. The obtained PTPEAQ-NP-HER2 has uniform sizes of around 50 nm (Figure S7), which showed similar optical properties as compared to those of PTPEAQ-NP (Figure S8).

Figure 4. Confocal images of SKBR-3 cancer cells (top) or NIH-3T3 cells (bottom) after incubation of the cells with PTPEAQ-NP-HER2 NPs (10 μg/mL) for 2 h. The red fluorescence of PTPEAQ-NP-HER2 was collected above 560 nm upon excitation at 488 nm, and the blue fluorescence of Hoechst was collected between 430 and 470 nm upon excitation at 405 nm. All the images share the same scale bar of 30 μm.

collected above 560 nm upon excitation at 488 nm and blue Hoechst was collected between 430 and 470 nm upon excitation at 405 nm. Intense red fluorescence is clearly observed in SKBR-3 cancer cells, while nearly no signal is found in NIH-3T3 normal cells, indicating good selectivity of PTPEAQ-NP-HER2. The quantitative evaluation of the toxicity and the photodynamic cancer cell ablation effect of the PTPEAQ-NP-HER2 at different concentrations was studied by standard methylthiazolyldiphenyltetrazolium bromide (MTT) assay. As shown in Figure 5A, upon incubation of the cells with PTPEAQ-NPHER2 (100 μg/mL based on PTPEAQ) for 2 h and further culturing for 24 h, both NIH-3T3 and SKBR-3 cells show high cell viabilities of over 80%, indicating low cytotoxicity of the PTPEAQ-NP-HER2 toward the cells. The slight toxicity of PTPEAQ-NP-HER2 toward SKBR-3 cells is due to the anticancer effect of anti-Her2 affibody (Figure S9). However, under light irradiation for only 5 min, the PTPEAQ-NP-HER2 shows obvious cytotoxicity to SKBR-3 cells, with a half-maximal inhibitory concentration (IC50) of 5 μg/mL, while NIH-3T3 cells still shows high cell viabilities (Figure 5B). In addition, upon increasing the concentrations of NPs, NIH-3T3 cells maintained high viabilities over 80% under light irradiation even at a very high PTPEAQ concentration of 100 or 200 μg/ mL, while nearly no SKBR-3 is alive under the same condition. In addition, no obvious toxicity is observed for cells upon exposure to light irradiation for various time (Figure S10). These results indicate that the toxicity shown in Figure 6 is originated from the 1O2 produced by PTPEAQ-NP-HER2 under light irradiation. These results also show good selectivity and effective ROS generation ability of PTPEAQ-NP-HER2. The live staining experiment was also used to confirm the photodynamic cancer cell ablation effect of the PTPEAQ-NPHER2. Fluorescein diacetate (FDA) is a dye with green emission, which only stains live cells, and therefore could be used to confirm the result of photodynamic cancer cell ablation. 5022

DOI: 10.1021/acs.macromol.6b00958 Macromolecules 2016, 49, 5017−5025

Article

Macromolecules

Figure 5. Cell viability of PTPEAQ-NP-HER2 treated SKBR-3 and NIH-3T3 cells under dark (A) or light irradiation (B) (60 mW/cm2, 5 min).

cancer therapy. To the best of our knowledge, this is the first report on FR/NIR CP photosensitizer with AIE characteristics.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.6b00958. Synthesis of the polymers in details, 1H and 13C NMR spectra of polymers, the computational results for the model compounds, and some characterization data for NPs (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (B.L.). Author Contributions

Figure 6. Live and dead SKBR-3 cells stained by FDA after incubation with the cells with PTPEAQ-NP-HER2 NPs (20 μg/mL), followed by light irradiation for different duration: (A) 0, (B) 2, and (C) 5 min. All the images share the same scale bar of 200 μm.

W.W. and G.F. contributed equally to this work. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank the Singapore NRF Competitive Research Program (R279-000-483-281), NRF Investigatorship (R279-000-444281), National University of Singapore (R279-000-482-133), and the Institute of Materials Research and Engineering of Singapore (IMRE/14-8P1110) for financial support.

As shown in Figure 6, nearly all the SKBR-3 cells are stained by FDA after incubation with 20 μg/mL of PTPEAQ-NP-HER2. However, under light irradiation for 2 min, only few cells are stained. After 5 min, there is almost no green fluorescence observed, which confirms good ablation effect of PTPEAQ-NPHER2.

■

■

CONCLUSION In summary, a new photostable FR/NIR conjugated polymer photosensitizer PTPEAQ with AIE characteristics has been prepared by using TPE as donor and AQ as acceptor. Based on this polymer, two types of NPs were prepared, and DSPEPEG2000-Mal encapsulated polymer nanoparticles (PTPEAQNP) demonstrated much better performance in both fluorescence and ROS generation. PTPEAQ-NP showed a FR/NIR emission centered at 680 nm, with higher 1O2 quantum yield and better photostability than Rose Bengal as a photosensitizer. After conjugation of anti-HER2 affibody to the surface of PTPEAQ-NP, the obtained PTPEAQ-NP-HER2 has been used for targeted and image-guided photodynamic cancer ablation. PTPEAQ-NP-HER2 showed selective internalization into SKBR-3 cancer cells over NIH-3T3 normal cells, which could be used to selectively kill SKBR-3 cancer cells upon white light illumination. These results showed that PTPEAQ-NP-HER2 is a good candidate for photodynamic

REFERENCES

(1) Li, K.; Liu, B. Polymer-encapsulated organic nanoparticles for fluorescence and photoacoustic imaging. Chem. Soc. Rev. 2014, 43, 6570−6597. (2) Feng, L.; Zhu, C.; Yuan, H.; Liu, L.; Lv, F.; Wang, S. Conjugated polymer nanoparticles: preparation, properties, functionalization and biological applications. Chem. Soc. Rev. 2013, 42, 6620−6633. (3) Xu, L. G.; Cheng, L.; Wang, C.; Peng, R.; Liu, Z. Conjugated polymers for photothermal therapy of cancer. Polym. Chem. 2014, 5, 1573−1580. (4) Peng, H. S.; Chiu, D. T. Soft fluorescent nanomaterials for biological and biomedical imaging. Chem. Soc. Rev. 2015, 44, 4699− 4722. (5) Reisch, A.; Klymchenko, A. S. Fluorescent polymer nanoparticles based on dyes: seeking brighter tools for bioimaging. Small 2016, 12, 1968−1992. (6) Yuan, Y.; Liu, J.; Liu, B. Conjugated-polyelectrolyte-based polyprodrug: targeted and image-guided photodynamic and chemotherapy with on-demand drug release upon irradiation with a single light source. Angew. Chem., Int. Ed. 2014, 53, 7163−7168.

5023

DOI: 10.1021/acs.macromol.6b00958 Macromolecules 2016, 49, 5017−5025

Article

Macromolecules (7) He, R.; Hu, M.; Xu, T.; Li, C.; Wu, C.; Guo, X.; Zhao, Y. Conjugated copolymer-photosensitizer molecular hybrids with broadband visible light absorption for efficient light-harvesting and enhanced singlet oxygen generation. J. Mater. Chem. C 2015, 3, 973−976. (8) Geng, J. L.; Sun, C. Y.; Liu, J.; Liao, L. D.; Yuan, Y. Y.; Thakor, N.; Wang, J.; Liu, B. Biocompatible conjugated polymer nanoparticles for efficient photothermal tumor therapy. Small 2015, 11, 1603−10. (9) Yuan, Y. Y.; Min, Y. Z.; Hu, Q. L.; Xing, B. G.; Liu, B. NIR photoregulated chemo-and photodynamic cancer therapy based on conjugated polyelectrolyte−drug conjugate encapsulated upconversion nanoparticles. Nanoscale 2014, 6, 11259−11272. (10) Feng, L.; Zhu, C.; Yuan, H.; Liu, L.; Lv, F.; Wang, S. Conjugated polymer nanoparticles: preparation, properties, functionalization and biological applications. Chem. Soc. Rev. 2013, 42, 6620−6633. (11) Hill, E. H.; Pappas, H. C.; Evans, D. G.; Whitten, D. G. Cationic oligo-p-phenylene ethynylenes form complexes with surfactants for long-term light-activated biocidal applications. Photochem. Photobiol. Sci. 2014, 13, 247−253. (12) Parthasarathy, A.; Pappas, H. C.; Hill, E. H.; Huang, Y.; Whitten, D. G.; Schanze, K. S. Conjugated polyelectrolytes with imidazolium solubilizing groups. Properties and application to photodynamic inactivation of bacteria. ACS Appl. Mater. Interfaces 2015, 7, 28027− 28034. (13) Wilde, K. N.; Whitten, D. G.; Canavan, H. E. In vitro cytotoxicity of antimicrobial conjugated electrolytes: interactions with mammalian cells. ACS Appl. Mater. Interfaces 2013, 5, 9305−9311. (14) Wang, Y.; Chi, E. Y.; Schanze, K. S.; Whitten, D. G. Membrane activity of antimicrobial phenylene ethynylene based polymers and oligomers. Soft Matter 2012, 8, 8547−8558. (15) Shi, H. F.; Ma, X.; Zhao, Q.; Liu, B.; Qu, Q. Y.; An, Z. F.; Zhao, Y. L.; Huang, W. Ultrasmall phosphorescent polymer dots for ratiometric oxygen sensing and photodynamic cancer therapy. Adv. Funct. Mater. 2014, 24, 4823−4830. (16) Yuan, Y. Y.; Liu, B. Self-assembled nanoparticles based on PEGylated conjugated polyelectrolyte and drug molecules for imageguided drug delivery and photodynamic therapy. ACS Appl. Mater. Interfaces 2014, 6, 14903−14910. (17) Zhu, C.; Liu, L.; Yang, Q.; Lv, F.; Wang, S. Water-soluble conjugated polymers for imaging, diagnosis, and therapy. Chem. Rev. 2012, 112, 4687−4735. (18) He, R.; Hu, M.; Xu, T.; Li, C.; Wu, C.; Guo, X.; Zhao, Y. Conjugated copolymer−photosensitizer molecular hybrids with broadband visible light absorption for efficient light-harvesting and enhanced singlet oxygen generation. J. Mater. Chem. C 2015, 3, 973−976. (19) Xing, C.; Xu, Q.; Tang, H.; Liu, L.; Wang, S. Conjugated polymer/porphyrin complexes for efficient energy transfer and improving light-activated antibacterial activity. J. Am. Chem. Soc. 2009, 131, 13117−13124. (20) Liu, J.; Geng, J.; Liu, B. A bright far-red and near-infrared fluorescent conjugated polyelectrolyte with quantum yield reaching 25%. Chem. Commun. 2013, 49, 1491−1493. (21) Liu, J.; Feng, G.; Ding, D.; Liu, B. Bright far-red/near-infrared fluorescent conjugated polymer nanoparticles for targeted imaging of HER2-positive cancer cells. Polym. Chem. 2013, 4, 4326−4334. (22) Liu, P.; Li, S.; Jin, Y.; Qian, L.; Gao, N.; Yao, S. Q.; Huang, F.; Xu, Q.-H.; Cao, Y. Red-emitting DPSB-based conjugated polymer nanoparticles with high two-photon brightness for cell membrane imaging. ACS Appl. Mater. Interfaces 2015, 7, 6754−6763. (23) Lee, S. H.; Kömürlü, S.; Zhao, X.; Jiang, H.; Moriena, G.; Kleiman, V.; Schanze, K. S. Water-soluble conjugated polyelectrolytes with branched polyionic side chains. Macromolecules 2011, 44, 4742− 4751. (24) Pu, K. Y.; Liu, B. Conjugated polyelectrolytes as light-up macromolecular probes for heparin sensing. Adv. Funct. Mater. 2009, 19, 277−284. (25) Feng, G.; Li, K.; Liu, J.; Ding, D.; Liu, B. Bright single-chain conjugated polymer dots embedded nanoparticles for long-term cell tracing and imaging. Small 2014, 10, 1212−1219.

(26) Wu, C.; Chiu, D. T. Bright single-chain conjugated polymer dots embedded nanoparticles for long-term cell tracing and imaging. Angew. Chem., Int. Ed. 2013, 52, 3086−3109. (27) Chiang, C.-L.; Wu, M.-F.; Dai, D.-C.; Wen, Y.-S.; Wang, J.-K.; Chen, C.-T. Red-emitting fluorenes as efficient emitting hosts for nondoped, organic red-light-emitting diodes. Adv. Funct. Mater. 2005, 15, 231−238. (28) Shimizu, M.; Hiyama, T. Organic fluorophores exhibiting highly efficient photoluminescence in the solid state. Chem. - Asian J. 2010, 5, 1516−1531. (29) Mei, J.; Hong, Y.; Lam, J. W. Y.; Qin, A.; Tang, Y.; Tang, B. Z. Aggregation-induced emission: the whole is more brilliant than the parts. Adv. Mater. 2014, 26, 5429−5479. (30) Mei, J.; Leung, N. L. C.; Kwok, R. T. K.; Lam, J. W. Y; Tang, B. Z. Aggregation-induced emission: together we shine, united we soar! Chem. Rev. 2015, 115, 11718−11940. (31) Gao, Y.; Feng, G.; Jiang, T.; Goh, C.; Ng, L.; Liu, B.; Li, B.; Yang, L.; Hua, J.; Tian, H. Biocompatible nanoparticles based on diketo-pyrrolo-pyrrole (DPP) with aggregation-induced red/NIR emission for in vivo two-photon fluorescence imaging. Adv. Funct. Mater. 2015, 25, 2857−2866. (32) Qin, W.; Li, K.; Feng, G.; Li, M.; Yang, Z.; Liu, B.; Tang, B. Z. Bright and photostable organic fluorescent dots with aggregationinduced emission characteristics for noninvasive long-term cell imaging. Adv. Funct. Mater. 2014, 24, 635−643. (33) Li, K.; Qin, W.; Ding, D.; Tomczak, N.; Geng, J.; Liu, R.; Liu, J.; Zhang, X.; Liu, H.; Liu, B.; Tang, B. Z. Photostable fluorescent organic dots with aggregation-induced emission (AIE dots) for noninvasive long-term cell tracing. Sci. Rep. 2013, 3, 1150. (34) Yuan, Y.; Feng, G.; Qin, W.; Tang, B. Z.; Liu, B. Targeted and image-guided photodynamic cancer therapy based on organic nanoparticles with aggregation-induced emission characteristics. Chem. Commun. 2014, 50, 8757−8760. (35) Wu, W.; Ye, S.; Huang, L.; Xiao, L.; Fu, Y.; Huang, Q.; Yu, G.; Liu, Y.; Qin, J.; Li, Q.; Li, Z. A conjugated hyperbranched polymer constructed by carbazole and tetraphenylethylene moieties: convenient synthesis through one-pot “A2+B4” suzuki polymerization, aggregationinduced enhanced emission, and application as explosive chemsensors and PLED. J. Mater. Chem. 2012, 22, 6374−6382. (36) Wu, W.; Ye, S.; Yu, G.; Liu, Y.; Qin, J.; Li, Z. Novel functional conjugative hyperbranched polymers with aggregation-induced emission: synthesis through one-pot “A2 + B4” polymerization and application as explosive chemsensors and PLEDs. Macromol. Rapid Commun. 2012, 33, 164−171. (37) Shi, J.; Wu, Y.; Tong, B.; Zhi, J.; Dong, Y. Tunable fluorescence upon aggregation: photophysical properties of cationic conjugated polyelectrolytes containing AIE and ACQ units and their use in the dual-channel quantification of heparin. Sens. Actuators, B 2014, 197, 334−341. (38) Wu, W.; Ye, S.; Tang, R.; Huang, L.; Li, Q.; Yu, G.; Liu, Y.; Qin, J.; Li, Z. New tetraphenylethylene-containing conjugated polymers: facile synthesis, aggregation-induced emission enhanced characteristics and application as explosive chemsensors and PLEDs. Polymer 2012, 53, 3163−3171. (39) Zhang, Q.; Kuwabara, H.; Potscavage, W. J., Jr.; Huang, S.; Hatae, Y.; Shibata, T.; Adachi, C. Anthraquinone-based intramolecular charge-transfer compounds: computational molecular design, thermally activated delayed fluorescence, and highly efficient eed electroluminescence. J. Am. Chem. Soc. 2014, 136, 18070−18081. (40) Xu, S.; Yuan, Y.; Cai, X.; Zhang, C.-J.; Hu, F.; Liang, J.; Zhang, G.; Zhang, D.; Liu, B. Tuning the singlet-triplet energy gap: a unique approach to efficient photosensitizers with aggregation-induced emission (AIE) characteristics. Chem. Sci. 2015, 6, 5824−5830. (41) Zhao, J.; Wu, W.; Sun, J.; Guo, S. Triplet photosensitizers: from molecular design to applications. Chem. Soc. Rev. 2013, 42, 5323− 5351. (42) Li, H.; Xu, Y.; Shi, E.; Wei, W.; Suo, X.; Wan, X. Synthesis of arylketones by ruthenium-catalyzed cross-coupling of aldehydes with arylboronic acids. Chem. Commun. 2011, 47, 7880−7882. 5024

DOI: 10.1021/acs.macromol.6b00958 Macromolecules 2016, 49, 5017−5025

Article

Macromolecules (43) Zhang, Q.; Li, B.; Huang, S.; Nomura, H.; Tanaka, H.; Adachi, C. Efficient blue organic light-emitting diodes employing thermally activated delayed fluorescence. Nat. Photonics 2014, 8, 326−332. (44) Li, J.; Nakagawa, T.; MacDonald, J.; Zhang, Q.; Nomura, H.; Miyazaki, H.; Adachi, C. Highly efficient organic light-emitting diode based on a hidden thermally activated delayed fluorescence channel in a heptazine derivative. Adv. Mater. 2013, 25, 3319−3323. (45) Uoyama, H.; Goushi, K.; Shizu, K.; Nomura, H.; Adachi, C. Highly efficient organic light-emitting diodes from delayed fluorescence. Nature 2012, 492, 234−238. (46) Burdzinski, G.; Kubicki, J.; Maciejewski, A.; Steer, R. P.; Velate, S.; Yeow, E. K. L. Photochemistry and Photophysics of Highly Excited Valence States of Polyatomic Molecules: Nonalternant Aromatics, Thioketones, and Metalloporphyrins. In Organic Photochemistry and Photophysics; Ramamurthy, V., Schanze, K. S., Eds.; Taylor & Francis Group, LLC: 2006; pp 1−36.

5025

DOI: 10.1021/acs.macromol.6b00958 Macromolecules 2016, 49, 5017−5025