Polymerization-Enhanced Two-Photon Photosensitization for Precise

Feb 14, 2019 - Department of Chemical and Biomolecular Engineering, National University of Singapore , Singapore 117585 , Singapore. ‡ Singapore ...
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Polymerization-Enhanced Two-Photon Photosensitization for Precise Photodynamic Therapy Shaowei Wang, Wenbo Wu, Purnima Manghnani, Shidang Xu, Yuanbo Wang, Chi Ching Goh, Lai Guan Ng, and Bin Liu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b08398 • Publication Date (Web): 14 Feb 2019 Downloaded from http://pubs.acs.org on February 14, 2019

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Polymerization-Enhanced Two-Photon Photosensitization for Precise Photodynamic Therapy Shaowei Wang,1⊥ Wenbo Wu,1⊥ Purnima Manghnani,1 Shidang Xu,1 Yuanbo Wang,1 Chi Ching Goh,2 Lai Guan Ng,2 Bin Liu*,1 1Department

of Chemical and Biomolecular Engineering, National University of Singapore,

Singapore 117585, Singapore 2Singapore

Immunology Network (SIgN), Agency for Science Technology and Research

(A*STAR), Singapore 138648, Singapore Corresponding Author *Email: [email protected]

ABSTRACT Two-photon excited photodynamic therapy (2PE-PDT) has attracted great attention in recent years due to its great potential for deep tissue and high spatiotemporal precise cancer therapy. Photosensitizers (PSs) with high singlet oxygen (1O2) generation efficiency and large two-photon absorption (2PA) cross section are highly desirable, but the availability of such PSs is limited by challenges in molecular design. In this work, we report that polymerization of small molecule PSs with aggregation-induced emission (AIE) could yield conjugated polymer PSs with good brightness, high 1O2 generation efficiency and large 2PA cross section. Two conjugated polymer PSs were designed and synthesized and the corresponding AIE PS dots were prepared by nanoprecipitation, which exhibited outstanding 2PE-PDT performance in in vitro cancer cell ablation and in vivo zebrafish liver tumor treatment. Our work highlights a strategy to design highly

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efficient PSs for 2PE-PDT. KEYWORDS conjugated polymer, photosensitizer, two-photon, photodynamic therapy, aggregation-induced emission.

Photodynamic therapy (PDT) is a non-invasive and selective therapeutic method, which relies on the administration of photosensitizers (PSs) that are activated by specific light irradiation to generate cytotoxic reactive singlet oxygen (1O2), for the treatment of various cancerous tumors, skin diseases, and bacterial infections.1 Effective activation of PSs by light source that can penetrate deep in biological tissues plays an important role in PDT.2 However, most of the clinically approved PSs (e.g. porphyrin and its derivatives) have absorption in the visible region with limited light penetration.1,3,4 Developing PSs with long wavelength absorption in the phototherapeutic window (700-950 nm) is difficult because higher energy triplet state for PSs is required to ensure efficient energy transfer from PSs in triplet state to oxygen molecules.4,5 One strategy to shift the excitation wavelength of PSs to long wavelength region is via two-photon excitation, where the PSs are excited by simultaneous absorption of two photons of near-infrared (NIR) light.3,4,6,7 As compared to one-photon excitation, two-photon excited photodynamic therapy (2PE-PDT) can not only increase the light penetration and therapeutic treatment depth, but also provide better spatial control of PS activation in three dimensions during PDT treatment.6 This key feature of 2PE-PDT is attributed to the square dependence of two-photon fluorescence on the excitation power and only focused pulsed laser with high intensity can induce two-photon absorption (2PA) process at the focal point.8 These features endow 2PE-PDT with great potential

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for localized precision therapy, which is of significant importance for the treatment of diseases in important organs, such as brain tumor, capillary hemangioma, and retinopathy in eyes.3,4,7 The two main factors that determine the effectiveness of 2PE-PDT are the 2PA cross section (σ2) and singlet oxygen efficiency of PSs.3,4,7 Many one-photon PSs such as Photofrin®,9,10 5aminolevulinic acid (ALA),11,12 sulfonated aluminum phthalocyanine,13 and chlorophyll derivate PS,14 with good 1O2 production have been used in 2PE-PDT applications. Although they showed various 2PE-PDT effects, their σ2 values are relatively small (2-31 GM, 1 GM = 10−50 cm4 s photon−1),15 so that high light dose is needed for imaging and therapy. This could easily induce side effects on healthy tissue and reduce the signal-to-background ratio for in vivo imaging. To increase the σ2 values, a series of cyclic tetrapyrrolic PSs with extended π-conjugation was developed.15,16 Despite the fact that they possess high σ2 values, from several hundred to tens of thousands of GM based on molecules, the poor water solubility hampered their 2PE-PDT performance in biological applications. Their easy aggregation in aqueous media often leads to aggregation-caused quenching (ACQ) in fluorescence and reduction in 1O2 generation during practical applications.17-19 To address the intrinsic quenching problem faced by traditional PSs, we developed a series of PSs with both aggregation-induced emission (AIE) and high photosensitization efficiency in aggregate state.20-23 Due to their characteristic rotor structures, the non-radiative energy dissipation is largely prohibited in aggregate state, which allows us to fully utilize the excited state for fluorescence and 1O2 production. Therefore, when AIE PSs are fabricated into small-size nanoparticles (AIE PS dots), they offer targeted delivery with high brightness and strong photosensitization for image-guided surgery and therapy.24 Recently, by combining the advantages of two-photon excitation and AIE PSs, we reported an example of AIE PS dots for 2PE-PDT with

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moderate performance.25 As the photosensitization performance of most AIE PSs is high under one-photon excitation, the key is to enhance their capability in two-photon absorption without compromising photosensitization. Unfortunately, both rotor structures and the effective strategies to enhance the efficiency of intersystem crossing (ISC) (e.g. via HOMO and LUMO separation) for AIE PSs usually lead to limited conjugation within the molecule,21,22,26 not favorable for high 2PA cross section.27 In fact, simultaneous improvement in both 1O2 generation and 2PA cross section is very difficult to achieve, which is a challenge not limited to AIE PSs.28 Conjugated polymers are macromolecules with π-conjugated backbone structures. The special conjugated structures could endow them with good 2PA ability.7,29,30 Meanwhile, the energy levels in both singlet and triplet states of conjugated polymers are usually much denser than those of their small molecule analogues, which is beneficial to ISC process for 1O2 generation.31,32 As such, it is expected that conjugated polymer PSs may be able to show both high 1O2 generation efficiency and large 2PA cross section through precise molecular design. In this contribution, two conjugated polymer PSs of PTPEDC1 and PTPEDC2 were designed and synthesized based on a small molecule AIE PS of TPEDC. Subsequently, they were encapsulated by an amphiphilic polymer to yield nanoparticles for both in vitro cancer cell ablation and in vivo zebrafish liver tumor treatment via 2PE-PDT with high efficiency.

RESULTS AND DISCUSSION Molecular Design and Synthesis of AIE PS Dots The design of PTPEDC2 starts from TPEDC. TPEDC is an AIE PS with good 1O2 generation efficiency, high brightness and moderate 2PA cross section for 2PE-PDT.25 In the first step, a conjugated polymer of PTPEDC1 (Figure 1a) with the same donor and acceptor as TPEDC was

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designed to improve the 2PA cross section and 1O2 generation. In the second step, we further adjusted the linkage position between the donor and acceptor moieties to design PTPEDC2 to improve polymer conjugation, which is beneficial to enhance the 2PA cross section. PTPEDC1 and

PTPEDC2

were

prepared

through

Suzuki

coupling

polymerization

between

tetraphenylethene-based monomer M1 or M2 and dicyanovinyl-based monomer M3 (Scheme S1S2), in 57.1% and 85.5% yields, respectively. Both PTPEDC1 and PTPEDC2 show good solubility in common organic solvents, such as dichloromethane, chloroform, tetrahydrofuran, and toluene, etc. Their structures were further confirmed by NMR spectra (Figure S1). The average molecular weight of the two polymers was determined by gel permeation chromatography, with 10 800 for PTPEDC1 (Mw/Mn = 1.93) and 15 500 for PTPEDC2 (Mw/Mn = 1.77), respectively. TPEDC has an absorption peak at 310 nm with a shoulder at around 410 nm in tetrahydrofuran (THF)/water (1/9, v/v) mixture, whereas PTPEDC1 and PTPEDC2 show absorption peaks in the range of 300 to 350 nm (Figure S2). All three molecules exhibit AIE characteristics, which is attributed to the iconic AIEgen of tetraphenylethylene moiety (Figure S3). In the THF/water (1/9, v/v) mixture, all three have bright emissions in the far-red/NIR region (600 to 800 nm), which is desirable for in vivo biological imaging. To further endow PTPEDC1 and PTPEDC2 with good water dispersibility, 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[maleimide(poly(ethylene glycol))-2000] (DSPE-PEG-Mal) was utilized as an amphiphilic matrix to encapsulate both molecules via nanoprecipitation (Figure 1b). The yielded AIE PS dots exhibit spherical morphology and uniform hydrodynamic sizes of around 30 nm, which agree well with the TEM images (Figure S4). The UV-vis absorption and photoluminescence (PL) spectra of the three AIE PS dots in aqueous media are shown in Figure 1c. Their absorption and emission spectra are similar to those measured in THF/water (1/9, v/v).

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The slight blue shift of emission of PTPEDC1 dots in water is the result of molecular interactions between PTPEDC1 and the polymer matrix.

Figure 1. (a) Chemical structures of TPEDC, PTPEDC1 and PTPEDC2. (b) Schematic illustration for the synthesis of AIE PS dots and AIE-TAT PS dots using PTPEDC2 as an example. (c) Normalized absorption and photoluminescence (PL) spectra of AIE PS dots in aqueous media. (d) Normalized degradation percentages of ABDA in the presence of PS dots in aqueous media upon white light irradiation (400-700 nm, 50 mW cm-2). [PS dots] = 10 μM based on dye; [ABDA] = 50 μM. (e) Two-photon absorption cross section (2PACS) spectra of PS dots in aqueous solution. 1O 2

Generation Evaluation of AIE PS Dots

The 1O2 generation of these AIE PS dots in aqueous media was evaluated using 9,10anthracenediyl-bis-(methylene) dimalonic acid (ABDA) as an indicator. As shown in Figure 1d and Figure S5, with the increasing irradiation time of white light (400-700 nm, 50 mW cm-2), the

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absorbance of ABDA in the presence of AIE PS dots dramatically decreases, indicating that ABDA is decomposed by the generated 1O2. To compare the 1O2 generation capability of PS dots, their ABDA decomposition efficiencies were calculated (details see MATERIALS AND METHODS section). Upon light irradiation of 10 nmol AIE PS dots for 60 s, the amount of ABDA decomposed was 4 nmol for Ce6 dots, 4.65 nmol for TPEDC dots, 10.55 nmol for PTPEDC1 dots, and 25.5 nmol for PTPEDC2 dots, respectively. The 1O2 generation of PTPEDC2 dots was 548% and 637% more efficient than those of TPEDC dots and Ce6 dots, respectively. These results demonstrate that polymerization enhances the 1O2 generation efficiency of TPEDC. To further confirm the 1O2 generation of AIE PS dots, DCFDA, which can react with singlet oxygen, superoxide anion and hydroxyl radicals to generate DCF with green fluorescence, was also used as an indicator. As shown in Figure S6 and S7, when NaN3 (a well-know 1O2 scavenger) was added to the aqueous solutions of AIE PS dots, the absorbance of ABDA shows negligible changes (Figure S6) and the fluorescence intensity of DCF is immensely inhibited (Figure S7). These results confirm that singlet oxygen is the main component of reactive oxygen species (ROS) generated by these AIE PS dots upon light irradiation. Two-Photon Properties of AIE PS Dots Next, the two-photon absorption properties of the AIE PS dots were investigated. The logarithmic plots of the dependence of two-photon fluorescence (2PF) intensity on excitation power for all three AIE PS dots show good linear relationships and the slopes of the fitting curves are around 2, demonstrating nonlinear 2PA processes under femtosecond (fs) laser excitation (Figure S8). Next, the 2PA cross sections of the AIE PS dots were assessed by using 2PF imaging method in the wavelength range of 800 to 1040 nm at 20 nm intervals. The σ2 value of the AIE PS dots (based on dot molar concentration) was calculated by using Rhodamine 6G as the reference

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(details see the Experimental Section).33 The fluorescence quantum yields (η) of AIE PS dots in aqueous media were measured using 4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)4H-pyran (DCM) in methanol as reference (η = 43.5%) and the η values of TPEDC, PTPEDC1 and PTPEDC2 dots were calculated to be 11.2 ± 0.7%, 11.9 ± 0.9% and 3.1 ± 0.4%, respectively. The two-photon absorption spectra of the three AIE PS dots are plotted in Figure 1e. The maximal σ2 value of TPEDC dots was measured to be 1.13 × 105 GM with excitation wavelength at 840 nm. After polymerization, the peak σ2 value for PTPEDC1 dots increased to 3.56 × 105 GM upon 820 nm-fs laser excitation, which should be attributed to the improved conjugation length. After adjusting the linkage position, this value was further improved to 7.36 × 105 GM for PTPEDC2 dots. Mechanism of Polymerization-Enhanced Two-Photon Photosensitization To understand the differences among the three AIE PS dots in 1O2 generation and 2PA cross section, time-dependent density functional theory (TD-DFT) investigations were then performed using three model compounds 1-3, whose chemical structures, optimized 3D structures and HOMO-LUMO distributions are shown in Figure 2. For all the three model compounds, the HOMO is mainly distributed at the donor part, while the LUMO is mainly located at the acceptor part, indicating good HOMO-LUMO separation, which favors 1O2 generation (Figure 2c).26 In addition, the TD-DFT results clearly show that after polymerization, the differences between different energy levels become much smaller than before, indicating more ISC channels in PTPEDC1 and PTPEDC2, to facilitate 1O2 generation (Figure 2a). As compared to PTPEDC1, the energy levels of PTPEDC2 are denser, which agrees with the fact that the 1O2 generation efficiency of PTPEDC2 is higher than that of PTPEDC1. In addition, as shown in Figure 2b, the torsional angles between different units of PTPEDC2 are much smaller than those of TPEDC and

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PTPEDC1, indicating improved conjugation for PTPEDC2, which is favorable to yield a larger 2PA cross section.

Figure 2. (a) Chemical structures and singlet and triplet energy levels of three model compounds. (b) Optimized structures of the three model compounds. Green and red colors highlight the conjugated benzene rings within the same plane. (c) DFT-calculated HOMO and LUMO wave functions of the geometry optimized structures (Gaussian 09/B3LYP/6-31G(d)) of the three model compounds. Two-Photon Photosensitization Evaluation of AIE PS Dots in Aqueous Media

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From the above experiments, it is clear that higher 1O2 generation efficiency and larger 2PA cross section have been achieved for PTPEDC2 through polymerization of TPEDC. The combination of these two excellent properties would endow PTPEDC2 a promising candidate for 2PE-PDT. To directly evaluate the 2PE photosensitization of all the AIE PS dots in aqueous media, we specially designed a set-up suitable for multiphoton microscopy with the assistance of glass capillary to enable real-time monitoring of 2PE 1O2 generation. The schematic illustration of the method is shown in Figure 3a. The mixture solution of AIE PS dots and DCFDA (a two-photon dye serving as 1O2 generation indicator, which changes to DCF upon reaction with 1O2) was loaded in a glass capillary (0.5 mm diameter) using a micro-loader tip for 2PE imaging. As shown in Figures 3b-e and Figures S9-S10, the 2PE fluorescence of DCF gradually intensifies with the increased number of laser scans for each AIE PS dots suspension, indicating efficient 1O2 production upon 2PE. Among the evaluated AIE PS dots, PTPEDC2 dots show the brightest green emission of DCF (Figure S9d), which should be attributed to their large 2PA cross section and high 1O2 generation efficiency.

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Figure 3. In vitro 1O2 detection of AIE PS dots upon two-photon excitation. (a) Schematic illustration for the in vitro 1O2 detection of AIE PS dots in aqueous media under two-photon excitation. (b) 2PF image of PTPEDC2 dots in aqueous solution and loaded in the glass capillary. (c-e) 2PF images of DCF in the aqueous solution of PTPEDC2 dots after different fs laser scanning numbers. (f) Overlay of (b) and (e). (b-f) images share the same scale bar: 100 μm. Excitation: 820 nm. Emission: 635-675 nm (red, from AIE PS dots) and 510-535 nm (green, from DCF). Scanning laser: 820 nm, 6 mW, 5.33 s per scan. Intracellular Two-Photon Photosensitization Evaluation Next, the intracellular 1O2 generation of the three AIE PS dots was investigated. To enhance the cellular internalization of the AIE PS dots, a cell penetrating peptide (TAT-SH) was conjugated on their surface (Figure 1b). The obtained AIE-TAT PS dots (named as TPEDC-TAT, PTPEDC1TAT, and PTPEDC2-TAT dots) showed increases in Zeta potential after TAT-SH conjugation (Figure S11), indicating successful conjugation of the peptide. After incubation with HeLa cells for 4 h, bright 2PF was observed in all three AIE-TAT PS dots treated cells (Figure S12), while cells incubated with AIE PS dots showed negligible 2PF (Figure S13), indicating high effectiveness of TAT conjugation in improving cellular uptake efficacy. In addition, according to the 2PF images and confocal images of AIE-TAT PS dots treated HeLa cells, it is obvious that most of the dots are distributed in the cell cytoplasm (Figure S14). Once a selected area (364 × 364 μm2) was irradiated by 820 nm-fs laser, bright green 2PF (from DCF) was observed only in the irradiated area (Figure 4). With the increasing number of scans, the 2PF emission of DCF was intensified, which indicated sufficient intracellular 1O2 generation. As shown in Figure 4c, the irradiated area exhibited bright green fluorescence with only 10 scans for PTPEDC2-TAT dots treated cells, indicating that the PTPEDC2-TAT dots inside the cells produced sufficient 1O2 upon

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2PE. For TPEDC-TAT and PTPEDC1-TAT dots treated cells, the 1O2 generation is lower than that of PTPEDC2-TAT dots (Figure 4a and 4b), which agrees with the direct solution analysis results (Figure S9).

Figure 4. Intracellular 1O2 detection of (a) TPEDC-TAT dots, (b) PTPEDC1-TAT dots, and (c) PTPEDC2-TAT dots stained HeLa cells upon 2PE after different fs laser scanning numbers. The overlay image is the merge of images between the 1st and 4th columns. Excitation: 820 nm. Emission: 635-675 nm (red, from AIE PS dots) and 510-535 nm (green, from DCF). The white dashed line box in all the images indicates the laser exposure area (364 × 364 μm2). Scanning laser: 820 nm, 6 mW, 5.33 s per scan. All images share the same scale bar: 100 μm. Cancerous Cells Ablation by 2PE-PDT Subsequently, 2PE-PDT induced HeLa cell ablation was investigated (Figure 5a). The AIETAT PS dots showed negligible dark toxicity as the cell viability remained above 95% even at the

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dot concentration of 100 μg mL-1 (Figure S15). For in vitro cell 2PE-PDT, HeLa cells were incubated with AIE-TAT PS dots for 4 h and the selected area (400 × 400 μm2) was irradiated with 820 nm-fs laser for different scans. Afterwards, the cells were incubated for additional 5 h followed by fluorescein diacetate/propidium iodide (FDA/PI) staining for live/dead cell imaging. As shown in Figure 5d, for cells incubated with PTPEDC2-TAT dots and upon laser irradiation, some red dots (fluorescence from PI) were observed in the irradiated area as an indication of cell death. The number of red cells increases with scanning numbers, illustrating efficient 2PE-PDT effect. After 80 scans, nearly all the cells located in the irradiation area were killed, whereas cells located outside the irradiation area remained alive. Moreover, we tracked the morphology changes of HeLa cells incubated with PTPEDC2-TAT dots during two-photon laser irradiation. As shown in Figure S16, blebs were observed in cells, which became bigger with increased laser scanning numbers, as a result of the high 1O2 generation efficiency of PTPEPDC2-TAT dots upon two-photon excitation. The 2PE-PDT performance of PTPEDC2-TAT dots is substantially better than those of TPEDC-TAT dots and PTPEDC1-TAT dots (Figure 5b-5c), indicating high precision and good efficiency of PTPEDC2-TAT dots in 2PE-PDT cancer cell ablation. In addition, the live/dead cell image of HeLa cells without PS dots treatment reveals no obvious cell death after 80 scans by 820 nm-fs laser, which demonstrates negligible photothermal effects on cells upon two-photon excitation (Figure S17).

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Figure 5. (a) Schematic illustration of the AIE-TAT PS dots for in vitro 2PE cell PDT. (b-d) Confocal live/dead cell images of HeLa cells incubated with (b) TPEDC-TAT dots, (c) PTPEDC1TAT dots, and (d) PTPEDC2-TAT dots subjected to 2PE-PDT after different scanning numbers. Excitation: 488 nm (for fluorescein diacetate) and 559 nm (for propidium iodide). Emission: 500545 nm (green, live cells) and 575-620 nm (red, dead cells). The white dashed line box in all the

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images indicates the laser exposure area (400 × 400 μm2). Scanning laser: 820 nm, 6 mW, 5.33 s per scan. All the images share the same scale bar: 100 μm. 2PE-PDT for Zebrafish Liver Tumor Treatment As PTPEDC2 dots showed the best performance on 2PE-PDT in vitro among the three AIE PS dots, their in vivo 2PE-PDT effect was studied by using zebrafish liver tumor model (Figure 6a). The photostability of PTPEDC2 dots was demonstrated to be even better than commercial Qtracker 625 and Alexa Fluor 647, which are very stable fluorescent contrast agents with similar absorption and emission spectra as those of PTPEDC2 dots (Figures S18-S20). In addition, the stability of PTPEDC2 dots in aqueous media is well maintained after three-month storage, as illustrated by their steady UV-vis and PL spectra, as well as hydrodynamic size distributions (Figure S21). As the size of zebrafish liver tumor is relatively small, it is very suitable for in vivo 2PE-PDT using a multiphoton microscope.34-36 The detailed protocol for preparation of the liver tumor model with genetically engineered EGFP expression can be found in the experimental section. After intravenous microinjection of PTPEDC2 dots into zebrafish, 2PF imaging was conducted to evaluate distribution of the dots in liver tumors. 2PF images of liver tumor and PTPEDC2 dots at different time points reveal that accumulation of dots reaches their maximum at 24 h (Figure S22). As shown in Figure 6b, bright red dots are clearly observed under two-photon excitation, where the intense green emission indicates the liver tumor. The 3D reconstructed 2PF images showed good localization of PTPEDC2 dots within the green liver tissue, revealing good accumulation of dots in liver tumor, which should benefit 2PE-PDT. The in vivo 2PE-PDT was conducted by scanning the liver tumor within 300 × 300 × 100 (x × y × z) μm3 volume with 1 μm step along the z direction. The laser power at tumor surface was 6 mW, which was gradually increased to 12 mW at the tumor bottom. In total, 3 z-stacks were performed on each tumor. The measurement of the

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tumor size was conducted by confocal microscopy before and post 24 h of 2PE-PDT treatment. As shown in Figures 6c-e, the 3D reconstructed images revealed that the liver tumor size showed ~20% decrease after 2PE-PDT treatment, whereas the control group exhibited 45-55% increase in size. These results prove that PTPEDC2 dots have excellent performance in in vivo PDT upon twophoton excitation.

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Figure 6. 2PF imaging and 2PE-PDT of PTPEDC2 dots in zebrafish liver tumor model. (a) Schematic illustration of in vivo 2PE-PDT of PTPEDC2 dots in zebrafish liver tumor model. (b)

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2PF images of PTPEDC2 dots (PTPEDC2, red) distributed in zebrafish liver tumor (GFP, green). Excitation: 820 nm; Emission: 635-675 nm (red) and 510-535 nm (green). (c) Representative confocal 3D reconstructed images of zebrafish liver tumor (with PTPEDC2 dots injection and with laser irradiation) before (day 1) and after (day 2) 2PE-PDT treatment. (d) Representative confocal 3D reconstructed images of zebrafish liver tumor in control group (without dots injection and without laser irradiation). (e) The relative increase (%) in zebrafish tumor size after different treatments. N.S.: data are not significantly different; ** p < 0.01, n = 6. Laser: 820 nm fs laser. Scale bars in all the images are 50 μm.

CONCLUSION In summary, two conjugated polymer PSs with AIE characteristics were synthesized by polymerization of a small molecule PS to yield good brightness, high 1O2 generation efficiency and large 2PA cross sections for effective 2PE-PDT. Upon two-photon excitation, highly efficient 1O

2

generation was demonstrated both in aqueous solution and in cells for PTPEDC1 and

PTPEDC2 dots, as a result of enhanced ISC process and improved molecular conjugation. As compared to TPEDC dots, PTPEDC2 dots show 548% more efficient 1O2 generation efficiency and 651% larger 2PA cross section, which offered excellent 2PE-PDT performance in in vitro cancer cell ablation and in vivo zebrafish liver tumor treatment. As compared to the hybrid systems where a two-photon absorption dye serves as energy donor and a PS serves as the energy acceptor,37-39 our design is simpler and easier to synthesize. The demonstration of conjugated polymer PSs with enhanced two-photon photosensitization provides insight into developing highly efficient photosensitizers for two-photon excited photodynamic therapy.

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MATERIALS AND METHODS Materials and Instruments. The synthetic routes of monomers, M1,40 M2,20 and M3,31 are presented in Scheme S1, which have been reported in the literatures. Tetrahydrofuran (THF) was distilled under dry nitrogen prior to use from sodium benzophenone ketyl. All the other materials for organic synthesis were purchased from Sigma-Aldrich. Cyclic arginine-glycine-aspartic acid (TAT-SH, RKKRRQRRRC) was purchased from GL Biochem Ltd (Shanghai, China). 1,2Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene

glycol)-2000]

(DSPE-PEG-Mal) (MW = 2000) was supplied by Avanti Polar Lipid, Inc. (Alabama, USA). 9,10anthracenediyl-bis-(methylene) dimalonic acid (ABDA) and 2′,7′-dichlorofluorescin diacetate (DCFDA) were purchased from Sigma Aldrich. Qtracker 625 and Alexa Fluor 647 were purchased from Thermo Fisher Scientific. Milli-Q water was used in all the related experiments. All other chemicals were obtained from commercial sources and used as received without further purification. 1H

and

13C

NMR spectra were measured by a Bruker Avance 400 spectrometer using

tetramethylsilane (TMS; δ = 0 ppm) as internal standard. UV-vis absorption spectra were measured on a spectrometer (UV-1700, Shimadzu, Japan). Photoluminescence (PL) spectra were measured by a spectrofluorometer (Perkin-Elmer LS 55). Transmission electron microscope image of dots was captured by JEM-2010F (JEOL, Japan). The hydrodynamic size distributions and Zeta potentials of dots were recorded at room temperature by a Zetasizer Nano S (Malvern Instruments Ltd, UK). Preparation of AIE PS dots and AIE-TAT PS dots. The AIE PS dots were synthesized via nanoprecipitation. The polymer PTPEDC2 was used as an example. A mixture THF solution containing PTPEDC2 (1 mg) and DSPE-PEG-Mal (2 mg) was sonicated for 5 min in a bath

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sonicator and injected into 10 mL Milli-Q water. A 2-min sonication of the mixed water/THF solution was performed by a probe sonicator at 50% output power (VCX 130, 130 W, Sonics & Materials, Inc. USA). A clear mixture solution was obtained and stirred at 400 rpm min-1 for 12 h in fume hood to evaporate the THF. The obtained aqueous solution of PTPEDC2 dots was filtered by a PES filter (0.22 μm) to remove big aggregates and stored at 4 ℃ for further use. For the preparation of PTPEDC2-TAT dots, 50 μL of TAT-SH solution (0.5 mg, dissolved in DMSO) was added into the aqueous solution of PTPEDC2 dots (10 mL, 0.1 mg mL-1) and the solution was vortexed for 1 min and then stirred at 300 rpm min-1 at room temperature in the dark for 12 h. After dialysis (12000-14000 kDa) against Milli-Q water for 72 h to remove non-conjugated TAT-SH and DMSO, the final aqueous solution of PTPEDC2-TAT dots was filtered by a PES filter (0.22 μm) and stored at 4 ℃. The same protocols were used to prepare Ce6 dots, TPEDC dots, TPEDCTAT dots, PTPEDC1 dots, and PTPEDC1-TAT dots. In Vitro Evaluation of 1O2 Generation by ABDA. The 1O2 generation of PS dots (Ce6, TPEDC, PTPEDC1, and PTPEDC2 dots) was evaluated by using ABDA as an indicator. Briefly, 1 mL of the mixture aqueous solution of PS dots (10 μM) and ABDA (50 μM) was irradiated with a white light (400-700 nm, 50 mW cm-2). The absorption spectra of the mixture solution were measured before and after light irradiation for various durations. For the comparison of 1O2 generation of PS dots, the irradiation time was set to 60 s and the amount of PS dots and ABDA were fixed to be 10 nmol and 50 nmol in 1 mL aqueous solution. Upon light irradiation, the absorbance of ABDA at 378 nm was normalized to obtain the decomposition efficiency of ABDA, which multiplies by 50 nmol can result in the decomposed amount of ABDA. The comparison of 1O2 generation efficiency was calculated by taking the ratio of ABDA decomposition amounts of two PS dots.

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Measurement of Two-Photon Absorption Cross Section. The 2PA cross sections (σ2) of the AIE PS dots were assessed by using the 2PF imaging method. The σ2 value of the AIE PS dots (based on dot concentration) was calculated by using Rhodamine 6G in methanol as reference. Method for the calculation of the dot molar concentration can be found in the Supporting Information. The aqueous solution of AIE PS dots was loaded in a glass capillary (0.5 mm diameter) and fixed on a glass slide. The sample was put on the stage of a multiphoton microscope (Olympus, FVMPE-RS) and fs laser in the range of 800 to 1040 nm (Spectra-Physics, 80 MHz, 120 fs) was focused on the sample through a 25× objective (NA: 1.05). The two-photon fluorescence (2PF) signals were collected through a short pass filter (750 nm). The σ2 values of AIE PS dots were calculated by the following equation:41 𝜎21 𝜎20

=

𝐹1𝜂0c0𝑛0 𝐹0𝜂1c1𝑛1

where, σ2 stands for two-photon absorption cross section, F stands for two-photon fluorescence intensity, η stands for fluorescence quantum yield, c stands for molar concentration, n stands for the solvent refractive index, and the subscripts 1 and 0 denote the evaluated sample (AIE PS dots in aqueous solution) and the standard reference (Rhodamine 6G in methanol), respectively. 2PE 1O2 Generation Evaluation by DCFDA. To evaluate the 1O2 generation of the aqueous solution of the PS dots under two-photon excitation, DCFDA was used as an indicator. DCFDA is a cell permeant reagent and usually used as a 1O2 indicator in cell experiments. After incubation with viable cells, DCFDA would diffuse into cells and then deacetylated by nonspecific cellular esterases in the cytoplasm to a non-fluorescent compound. After reaction with the 1O2 generated by the photosensitizer, the non-fluorescent compound would be oxidized into 2′,7′dichlorofluorescin (DCF), which can emit bright green fluorescence upon laser excitation. To enable the use of the DCFDA in aqueous solution in the absence of cells, an alkaline hydrolysis

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method was adopted to deesterify DCFDA.42 One mL of 1 mM DCFDA solution (dissolved in methanol) was added into 4 mL of NaOH solution (10 mM) and left to react for 30 min in dark. Afterwards, the mixture solution was added into 20 mL PBS buffer (25 mM, pH 7.4). In a typical experiment for the evaluation of 1O2 generation, the PS dots (Ce6, TPEDC, PTPEDC1, and PTPEDC2 dots) was mixed with the DCFDA solution so that the final concentration of DCFDA was 20 μM and PS dots was 50 μg mL-1. The mixture solution was loaded into a glass capillary (0.5 mm diameter) by using a micro-loader tip (MicroloaderTM, Eppendorf). The capillary was fixed on a glass slide and put on the microscope stage. Two-photon excitation of the sample was performed by focusing the fs laser on the sample through a 25× objective and scanned for different scanning numbers (820 nm, 6 mW, 5.33 s per scan). The real-time two-photon fluorescence emitted from the capillary were captured through two filters: 635-675 nm (red, from PS dots) and 510-535 nm (green, from DCF). Intracellular 2PE 1O2 generation Detection. HeLa cells were seeded in a 35 mm cell well with a density of 2 × 105 cells per well and grew to a desired confluence. The cells were washed and fresh culture medium containing AIE-TAT PS dots (TPEDC-TAT, PTPEDC1-TAT, and PTPEDC2-TAT dots, 5 μg mL-1) was added in the well. After incubation for 4 h, the cells were washed and fresh culture medium containing DCFDA (20 μM) was added and incubated for 30 min. Afterwards, the culture medium was removed and cells were washed and fresh culture medium was added into the well. HeLa cells within an area of 364 × 364 μm2 were scanned by a two-photon fs laser (820 nm, 6 mW, 5.33 s per scan) for different scanning numbers. The laser dose per scan is around 24 J cm-2. The two-photon fluorescence emitted from the cells were captured through two filters: 635-675 nm (red, from AIE-TAT PS dots) and 510-535 nm (green, from DCF).

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Photostability of PTPEDC2 Dots under Two-Photon Excitation. To evaluate the photostability of PTPEDC2 dots under two-photon excitation, Qtracker 625 and Alexa Fluor 647 were used as references. A thin film was prepared by dropping a small drop of aqueous solution of PTPEDC2 dots or references (5 μg mL-1) on glass slide. The film was scanned by a two-photon fs laser with wavelength at 820 nm (4 μs per pixel, 5.33 s per scan) and the two-photon fluorescence was recorded through a filter (635-675 nm) and calculated for comparison. Live/Dead Cell Imaging of 2PE-PDT Treated HeLa Cells. HeLa cells with 85% confluence were incubated with AIE-TAT PS dots (TPEDC-TAT, PTPEDC1-TAT, and PTPEDC2-TAT dots, 5 μg mL-1) for 4 h and then the cells were washed and fresh culture medium was added. 2PE-PDT was performed on a multiphoton microscope (Olympus, FVMPE-RS) equipped with an InSight DS-OL pulsed IR laser system (Spectra-Physics, 80 MHz, 120 fs). The AIE-TAT PS dots treated HeLa cells were scanned within a 400 × 400 μm2 area by a fs laser at 820 nm through a 25× objective (XLPLN25XWMP2, NA: 1.05) for different scanning numbers (6 mW, 5.33 s per scan). The laser dose per scan is around 20 J cm-2. After the 2PE-PDT treatment, the HeLa cells were incubated for additional 5 h and then incubated with fluorescein diacetate (10 μg mL-1) and propidium iodide (10 μg mL-1) for 30 min. Afterwards, cells were washed and taken for confocal imaging on an inverted confocal microscope (Olympus, FV-1000) with excitation at 488 nm (for fluorescein diacetate) and 559 nm (for propidium iodide) and emissions within 500-545 nm and 575-620 nm ranges respectively. Zebrafish Preparation and Toxicity Test. Zebrafish embryos were screened and incubated in egg water (10% NaCl; 1.63% MgSO4·7H2O; 0.4% CaCl2; 0.3% KCl) containing 1-phenyl 2-thiourea (PTU) after 22 h post fertilization to prevent the generation of melanin to obtain optically transparent fish. To induce the cancerization of fish liver, EGFP:krasV12 transgenic zebrafish larvae were

incubated in egg water containing mifepristone (5 μM) and 1-phenyl 2-thiourea (PTU).35,43 For the

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in vivo toxicity assessment of the PTPEDC2 dots on zebrafish, zebrafish larvae at 5 days postfertilization were seeded in 96 well plate and soaked in 100 μL of dots (suspended in egg water) with

various concentrations for 24 and 48 h. The viability of the fish was recorded. In Vivo 2PE-PDT on Liver Tumor of Zebrafish. When the zebrafish liver tumor grew to a desired

size, the fish was immobilized in a mixture of agarose (1wt.%, low melting) and methyl cellulose (5wt.%) and intravenous microinjection of the PTPEDC2 dots was performed by using a nitrogen gas injector. The dots were loaded into the glass needle (20 μm) and the injection was conducted through the zebrafish retro-orbital in a continuous mode of the gas injector. The safe concentration of PTPEDC2 dots was evaluated to be 0.5 mg mL-1 (Figure S23) and the injection amount of the dots was estimated to be 5-7 nL. After 24 h injection, the zebrafish was mounted in a cell dish and imaged on an upright confocal microscope (Zeiss LSM 800) to record its liver tumor size before it was transferred to the multiphoton microscope stage for 2PE-PDT treatment. The microscope used for 2PE-PDT was an upright Olympus multiphoton microscope (FVMPE-RS) equipped with an InSight DS-OL pulsed IR laser system (Spectra-Physics, 80 MHz, 120 fs). The 820 nm-fs laser was focused on the fish liver tumor through a 25× objective (NA: 1.05). 3D Z-stack excitation was performed by scanning the liver tumor within 300 × 300 × 100 (x × y × z) μm3 volume with 1 μm step along the z direction. The time for one x-y frame is 5.33 s. The output laser power at the surface of the tumor was around 6 mW (35.5 J cm-2 per scan) and gradually increased to 12 mW (71 J cm-2 per scan) at the bottom of the tumor along with the increasing depth. Total 3 stacks were conducted on each tumor. After the 2PE-PDT treatment, the fish was released to egg water (containing mifepristone and 1-phenyl 2-thiourea) and incubated for additional 24 h followed by measurement of the tumor size by confocal microscopy. The tumor volume size was calculated in ImageJ software. Details for the measurement of the liver tumor size could be found in our previous report.43

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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. Synthetic routes to the conjugated polymers; AIE feature of the conjugated polymer photosensitizers; TEM images, size distributions and zeta potentials of AIE PS dots; Absorption spectra for 1O2 evaluation by ABDA; 2PA characterization of AIE PS dots; 2P photosensitization evaluation of AIE PS dots in aqueous solution; two-photon photostability evaluation data; toxicity evaluation data on cells and zebrafish.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (B. Liu) ORCID Shaowei Wang: 0000-0003-2773-4525 Wenbo Wu: 0000-0002-6794-217X Bin Liu: 0000-0002-0956-2777 Author Contributions ⊥S.

W. and W. W. contributed equally to this work.

ACKNOWLEDGMENTS The authors acknowledge the financial support from the Singapore NRF Competitive Research Program (R279-000-483-281), NRF Investigatorship (R279-000-444-281), and National University of Singapore (R279-000-482-133).

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Table of Contents A concept of polymerization-enhanced two-photon sensitization is reported, which yielded conjugated polymer photosensitizers with simultaneous enhancement in 1O2 generation efficiency (5.5-fold) and two-photon absorption cross section (6.5-fold) than those of the counterpart small molecule photosensitizers. This work provides insight into developing highly efficient photosensitizers for precise two-photon excited photodynamic therapy. Keywords: conjugated polymer, aggregation-induced emission.

photosensitizer,

two-photon,

photodynamic

therapy,

Polymerization-Enhanced Two-Photon Photosensitization for Precise Photodynamic Therapy Shaowei Wang, Wenbo Wu, Purnima Manghnani, Shidang Xu, Yuanbo Wang, Chi Ching Goh, Lai Guan Ng, Bin Liu*

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