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Near-Infrared Triggered Upconversion Polymeric Nanoparticles Based on Aggregation-Induced Emission and Mitochondria Targeting for Photodynamic Cancer Therapy Yue Guan, Hongguang Lu, Wei Li, Yadan Zheng, Zhu Jiang, Jialing Zou, and Hui Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b07768 • Publication Date (Web): 26 Jul 2017 Downloaded from http://pubs.acs.org on July 27, 2017

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ACS Applied Materials & Interfaces

Near-Infrared Triggered Upconversion Polymeric Nanoparticles Based on Aggregation-Induced Emission and Mitochondria Targeting for Photodynamic Cancer Therapy

Yue Guan, Hongguang Lu*, Wei Li, Yadan Zheng, Zhu Jiang, Jialing Zou, Hui Gao* Tianjin Key Laboratory of Organic Solar Cells and Photochemical Conversion, School of Chemistry and Chemical Engineering, Tianjin University of Technology, Tianjin 300384, (P. R. China)

*Corresponding author email: [email protected] or [email protected]

Keywords：aggregation-induced emission, upconversion nanoparticles, photodynamic therapy, cancer cells, mitochondria.

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ABSTRACT Photodynamic therapy (PDT) is an auspicious strategy for cancer therapy by yielding reactive oxygen species (ROS) under light irradiation. Here, we have developed near-infrared (NIR) triggered polymer encapsulated upconversion nanoparticles (UCNPs) based on aggregation-induced emission (AIE) characteristics and mitochondria target ability for PDT. The coated AIE polymer as a photosensitizer can be photoactivated by the up-converted energy of UCNPs upon 980 nm laser irradiation, which could generate ROS efficiently in mitochondria and induce cell apoptosis. Moreover, a “sheddable” poly(ethylene glycol) (PEG) layer was easily conjugated at the surface of NPs. The pH-responsive PEG layer shields the surface positive charges and shows stronger protein-resistance ability. In the acidic tumor environment, PEGylated NPs lose the PEG layer and show the mitochondria-targeting ability by responding to tumor acidity. A cytotoxicity study indicated that these NPs have good biocompatibility in the dark but exert severe cytotoxicity to cancer cells, with only 10% cell viability, upon being irradiated with an NIR laser. The AIE nanoparticles are a good candidate for effective mitochondria targeting photosensitizer for PDT.

INTRODUCTION Photodynamic therapy (PDT) has aroused tremendous interest because of its high therapeutic efficiency,1-3 as the excited photosensitizer, under light irradiation, can efficiently generate reactive oxygen species (ROS), which can be exploited to kill cells for cancer therapy. Compared to conventional radiotherapy and chemotherapy, PDT shows distinct advantages such as: a noninvasive nature, low systemic toxicity, precise controllability, negligible drug resistance, high spatiotemporal accuracy and confined side effects as its lesion is light controllable.4-6 However, PDT suffers from the extremely finite diffusion distance (< 0.02 µm) and short lifetime (< 0.04 µs) of ROS generated by the 2 ACS Paragon Plus Environment

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photosensitizer, which greatly hinder their biomedical applications.7,8 Furthermore, another defect of a conventional photosensitizer is the relative low penetration depth of the light source. Additionally, the widely used photosensitizers in PDT, such as porphyrin9 and phenylthiazinium derivatives,10 which have rigid planar and intrinsic hydrophobic structures, lead to aggregation-caused quenching (ACQ) with weaker fluorescence and reduced ROS generation in aggregates.11-13 Aggregation-induced emission (AIE), an outstanding characteristic that is opposite to ACQ, has attracted increasingly attention for biological applications.14-21 AIE luminogens (AIEgens) produce almost no emission in solution, but the fluorescence intensity could be improved greatly in the aggregate states due to the restriction of intramolecular rotations and prohibition of energy dissipation via nonradiative channels.22,23 AIE photosensitizers are expected to be image-guided PDT agents with a high signal-to-noise ratio for fluorescence imaging and enhanced ROS generation in aggregates.24-27 These unique properties for the accumulation or encapsulation of AIEgens have made them valuable in the development of novel biomedical materials. More importantly, the majority of AIE photosensitizers are excited by ultraviolet or visible light.2426

The light source shows limited penetration depth or causes photodamage to biological samples, as

well as rapid attenuation in tissue owing to severe absorption and scattering of photons.28,29 The upconversion nanoparticles (UCNPs) can transform near-infrared (NIR) excitation into ultraviolet or visible light. Since the NIR light can penetrate deep into tissue, the UCNPs have the potential for use as a noninvasive agent for PDT.30,31 The up-converted energy is used to activate the AIEgens for PDT in this manuscript. The fabrication of AIE polymer encapsulated UCNPs is required to effectively reduce the distance between AIEgens and UCNPs, which can maximize fluorescence resonance energy transfer (FRET) efficiency. Recently, poly(ethylene glycol) (PEG) has offered a significant way to improve the delivery of nanoparticles (NPs) to tumor tissues, increase the therapeutic efficiency and reduce the toxic side effects. PEGylated NPs can prolong the systemic circulation and tumor accumulation through the enhanced 3 ACS Paragon Plus Environment


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permeability and retention (EPR) property.32 Meanwhile, the PEG layer will inhibit cellular uptake and subsequent endosomal escape.33 Consequently, “sheddable” PEG-shelled NPs using the conjugation of AIE polymer and PEG with a pH sensitive benzoic imine bond were prepared. The PEG layer shields the surface positive charges and prolongs circulation time, which is benefit to improve stability and decrease cytotoxicity of NPs.34 Once the NPs arrive at tumor tissues through the EPR effect, the PEG is removed due to the acidic pH. In view of this, we have developed an NIR triggered mitochondria targeting photosensitizer for PDT by taking advantage of AIE photosensitizer, triphenylphosphine (TPP) and UCNPs. The Tm3+ doped UCNPs can emit upconversion luminescence (UCL) upon single NIR laser irradiation and activate AIE polymer to yield high ROS levels. The TPP, as a mitochondria-targeted group, can guide the NPs to specifically target mitochondria and then generate ROS in the specific locations, leading to mitochondrial collapse and cell apoptosis signaling (Scheme 1). This triple combination of ROS generation of an AIE polymer, subcellular targeted delivery and NIR triggered PDT is beneficial to optimize the therapeutic efficacy. Moreover, the pH-responsive PEG layer creates more potential for the NPs to demonstrate photodynamic cancer therapy. To the best of our knowledge, this is the first time that an NIR triggered photodynamic cancer therapy could be achieved by using an AIE photosensitizer to induce mitochondrial ROS collapse and cell apoptosis. RESULTS AND DISCUSSION Synthesis and characterization The monomeric AIEgen (AIEM) was synthesized as depicted in Scheme S1. Through a Knoevenagel reaction of 1 and 2, compound 3 was obtained. Compound 3 was deprotected to yield compound 4. Compound 4 reacted with iodoethane to yield compound 5. Then, compound 5 reacted with methacryloyl chloride to yield AIEM. AIEM is soluble in organic solvents such as chloroform, tetrahydrofuran, acetone and dimethyl sulphoxide (DMSO) but insoluble in water. The AIEgen4 ACS Paragon Plus Environment

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containing copolymers (PAIE), possessing the segments of AIEM, 2-aminoethyl methacrylate (AEMA) and N-(2-hydroxypropyl)methacrylamide (HPMA), were synthesized following the synthetic route (Scheme 2). The feed molar ratio of HPMA, AEMA and AIEM was 100: 10: 6. The use of HPMA is supposed to reduce the cytotoxicity of the polymer due to the good biocompatibility of poly[N-(2hydroxypropyl)methacrylamide]. The function of AEMA is to improve cellular uptake of NPs taking advantage of the cationic amine groups. The polymer PAIE was decorated with TPP as the mitochondria-target group to yield polymer PAIE-TPP. Then, water-dispersible UCNP@PAIE-TPP NPs were obtained by self-assembly. The surface of UCNP@PAIE-TPP NPs was easily functionalized through the conjugation of mPEG-CHO and AIE polymer with a benzoic imine bond. AIEM has a higher absorption peak at approximately 316 nm, which is attributed to a π-π* transition, and a weaker absorption appearing at 513 nm, which represents an intramolecular charge transfer transition (Figure 1A).17 Figure 1B shows the photoluminescence (PL) spectra of AIEM in DMSO/water mixtures. AIEM is non-emissive in a DMSO solution. The PL intensity is gradually enhanced with the increase of water fractions (fw). The emission is turned on when fw reaches to 80%, as a result of the heavy aggregation of the AIEM molecules in the DMSO/water mixtures. As fw is 95%, the PL intensity of AIEM is 870-fold higher than that in DMSO. The aggregations of AIEM in DMSO/water mixtures were confirmed by dynamic light scattering. Thus, AIEM exhibits remarkable AIE. The PL spectrum of PAIE NPs ranged from 550 nm to 750 nm with an emission maximum at 630 nm. The PL and absorption spectra of UCNP@PAIE NPs are similar to that of PAIE, indicating that the introduction of UCNPs did not affect the optical properties of PAIE NPs. Additionally, the UCL of UCNPs (Figure 1C) under excitation at 980 nm present peaks at 345 nm (1I6-3F4), 358 nm (1D2-3H6), 447 nm (1D2-3F4) and 475 nm (1G4-3H6). The UCL spectrum of UCNPs overlaps well with absorption spectrum of PAIE, indicating that these species are an acceptable FRET donor-acceptor pair. The UCL spectra in NIR range were collected on an Edinburgh FLS920 spectrometer with 980 nm laser as the



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excitation source. The UCNPs NPs and UCNP@PAIE-TPP NPs have strong UCL signals at 802 nm (Figure 2A). Significantly, the 802 nm emission can be used for UCL NIR bioimaging. UCNPs (NaYF4, 30 mol% Yb, 0.5 mol% Tm) was synthesized using oleic acid as the stabilizing agent according to the literature methods.35 The original UCNPs are highly dispersible in organic nonpolar solvents, such as toluene and hexane. UCNPs possess uniform spherical morphology with a diameter of nearly 8-12 nm (Figure 3A). The PAIE-TPP encapsulated UCNPs were prepared through self-assembly. A DMSO solution containing PAIE-TPP and UCNPs was injected slowly into water. Water dispersible UCNP@PAIE-TPP NPs were obtained due to the hydrophobic interaction between UCNPs and PAIE-TPP. The UCNP@PAIE-TPP NPs were easily modified with mPEG-CHO to yield the PEGylated NPs (UCNP@PAIE-TPP-PEG). The transmission electron microscopy (TEM) image of UCNP@PAIE-TPP-PEG NPs shows that UCNPs is well dispersed in the polymer matrix (Figure 3B). This nano-structure is beneficial to efficient energy transfer from the UCNPs donor to the AIEgen acceptor within the Förster distance (< 10 nm). ROS generation from upconversion polymeric NPs Under 980 nm excitation, along with the remarkably diminished emissions of UCNPs at 345 nm, 358 nm, 447 nm and 475 nm, there is no obvious emission from UCNPs@PAIE-TPP NPs in the 550-750 nm region (Figure 1C). However, the ROS generation from these NPs was obviously detected under 980 nm excitation (Figure 1D). The efficient and continuous ROS generation is very important for PDT. The ROS generation ability of UCNPs@PAIE-TPP NPs was evaluated by using a ROS-sensitive probe 2′,7′dichlorofluorescin diacetate (DCF-DA). The hydrolysate of DCF-DA is non-fluorescent but can be rapidly oxidized to dichlorofluorescein (DCF) by ROS, which emits green PL. As shown in Figure 1D, the PL spectra of the ROS-sensitive probe in the presence of the UCNP@PAIE-TPP NPs (100 µg mL-1) was activated by ROS under 980 nm laser irradiation (3 W cm-2). The emission of DCF raises with the increasing NIR illumination time, within 540 s. The PL intensity is 8 times higher than that with no irradiation, suggesting that the UCNPs@PAIE-TPP NPs generate ROS efficiently through FRET. 6 ACS Paragon Plus Environment

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The FRET efficiency from UCNPs to PAIE-TPP was calculated from the quenching of value of donor emission intensity as E = (I0 - I1)/I0, where I0 and I1 were the emission intensities at 475 nm (Figure 1C) of UCNPs and UCNP@PAIE-TPP NPs, respectively.36 Higher than 97% of FRET efficiency from UCNPs to PAIE-TPP is observed, indicating a very complete energy transfer. Such high FRET efficiency is desirable to improve ROS generation. To further discuss the FRET process of UCNP@PAIE-TPP upon 980 nm laser irradiation, the UCL lifetimes of the bare UCNPs and the UCNP@PAIE-TPP were measured. It was found that the lifetime of UCNPs monitored at 475 nm decreases from 0.16 to 0.12 ms (Figure S1). In UCNP@PAIE-TPP, the excited photons on the 1G4 state can be absorbed by PAIE-TPP resulting in the decreased lifetime, which indicates a FRET process between UCNPs and PAIE-TPP.37 Under the same laser irradiation, the individual UCNPs and PAIE-TPP NPs cannot generate ROS. Thus, the energy of UCNPs is capable of activating photosensitizer to produce cytotoxic ROS for PDT. The energy of PL emission and ROS generation from PAIE-TPP are both derived from the FRET donor UCNPs. In the UCNPs@PAIE-TPP NPs, no obvious enhancement of PL intensity from PAIE-TPP was detected, which is probably because the majority of energy transfering from UCNPs to PAIE was employed to generate ROS. Additionally, the ROS generation abilities of AIEM under white light irradiation and 980 nm laser irradiation were measured. As shown in Figure S2, the PL intensity of DCF in the presence of AIEM exhibits a quick time-dependent enhancement. The PL intensity was 9 times under white light irradiation within 450 s, indicating that the AIEM generates ROS very efficiently. However, the fluorescence signal of DCF has no obvious enhancement under 980 nm laser irradiation (3 W cm-2), suggesting that the AIEM cannot generate ROS under this laser irradiation. Similar results were obtained with the PAIE-TPP NPs, which can generate ROS under white light irradiation (Figure S3).



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The photostability of PAIE-TPP after irradiation for different times was measured. As shown in Figure 2B, a negligibly drop in absorbance is observed after continuous laser irradiation (3 W cm-2) for 30 min, suggesting the PAIE-TPP NPs has excellent photostability for PDT. Sensitivity of UCNP@PAIE-TPP-PEG NPs under acidic pH The UCNP@PAIE-TPP NPs was modified expediently with mPEG-CHO, which reacted with the amino group of NPs surface to produce a benzoic imine bond. At pH 7.4, the pH sensitive linker is stable, but it hydrolyzed under a mild acid condition to expose positively charged TPP and amino groups. Acid-responsive cleavage of the UCNPs@PAIE-TPP-PEG NPs was verified by 1H NMR spectra. It measured under three kinds of acidic conditions: pH 7.4, 6.8 and 5.4. The aldehyde proton peak (at 10.13 ppm) and benzene ring proton peaks (at 8.04-8.16 ppm) from mPEG-CHO was not detected in pH 7.4, indicating that all of the aldehyde groups reacted with UCNPs@PAIE-TPP NPs to form a benzoic imine bond (Figure 4). However, at pH 6.8 or 5.4, the aldehyde and benzene ring presented proton peaks presented, suggesting the hydrolysis of UCNPs@PAIE-TPP-PEG NPs. BSA adsorption At pH 7.4 (the physiological pH), the zeta potential of UCNPs@PAIE-TPP NPs was 14.0 ± 1.17 mV. In contrast, the positive charge of UCNP@PAIE-TPP-PEG NPs can be effectively shielded by the PEG layer with a zeta potential close to zero (2.96 ± 0.15 mV) at pH 7.4. The PEG layer shielding of positively charged NPs could reduce the nonspecific interactions of the NPs with proteins, thus potentially extending the blood circulation of NPs and improving accumulation in tumors. To verify this supposition, the interaction of UCNP@PAIE-TPP NPs or UCNP@PAIE-TPP-PEG NPs with proteins was determined using bovine serum albumin (BSA) as a model protein. The BSA adsorption percentage of the UCNP@PAIE-TPP NPs and UCNP@PAIE-TPP-PEG NPs are 23.3% and 8.4% at pH 7.4, respectively. Compared with UCNP@PAIE-TPP-PEG NPs, the UCNP@PAIE-TPP NPs interacted more strongly with the BSA. Therefore, the PEG layer shows a stronger protein-resistant characteristic. In addition, as shown in Figure S4, the UCNP@PAIE-TPP NPs and UCNP@PAIE-TPP-PEG NPs are 8 ACS Paragon Plus Environment

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well-dispersed in DMEM cell medium and PBS, and the size remained constant for 7 days at 37 oC, suggesting good physical stability of the NPs at the concentration used for PDT. Cancer cell imaging To achieve the specificity to mitochondria, mitochondria targeting group TPP was introduced. The ability of targeting mitochondria was evaluated using a confocal laser microscope system (CLMS) in A549 cell. Mito-Tracker Green (MTG), a commercial mitochondrial dye, was employed to label mitochondria before the imaging experiment. The red fluorescence signal (collected at a range of 662737 nm) of UCNPs@PAIE-TPP NPs is well-overlapped with the green fluorescence signal (collected at a range of 500-530 nm) from the MTG (Figure 5). The degree of overlapping is quantitatively evaluated using Pearson’s correlation coefficients. A high Pearson’s correlation coefficient of 0.91 was calculated, demonstrating the tremendous mitochondrial specificity of UCNPs@PAIE-TPP NPs. In a parallel experiment, UCNPs@PAIE NPs without TPP unit fails to show intense yellow images with MTG (Pearson’s correlation coefficient is 0.63), confirming that the mitochondria-targeting UCNPs@PAIETPP NPs could effectively accumulate at mitochondrial sites. Then, the mitochondria-targeted imaging of UCNP@PAIE-TPP-PEG NPs at pH 6.8 was evaluated. Similar to UCNP@PAIE-TPP, Figure 5 shows that UCNP@PAIE-TPP-PEG NPs (at pH 6.8) could also efficiently target mitochondria (Pearson’s correlation coefficient is 0.94) after the PEG shell was removed, suggesting the potential application of UCNP@PAIE-TPP-PEG NPs for mitochondrial targeting. In addition, UCL imaging of UCNP@PAIE-TPP-PEG NPs in A549 cells was performed (Figure S5). The red fluorescence signal (collected through a 750 nm lp filter) of UCNPs@PAIE-TPP NPs is acquired under 980 nm laser irradiation, indicating the potential application of UCNP@PAIE-TPP-PEG NPs for UCL NIR bioimaging. ROS generation in cell Cytotoxic ROS can cause damage to mitochondria in cells, resulting in cell death.38 The cellular ROS generation upon 980 nm laser irradiation in A549 cells was evaluated by using a DCF-DA probe. The 9 ACS Paragon Plus Environment


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A549 cells with DCF-DA and UCNPs@PAIE-TPP NPs without laser irradiation show negligible fluorescence. But, after the laser irradiation, there was a significant green fluorescence present inside the cells (Figure S6). When vitamin C (1 mg mL-1) is added as a scavenger of ROS, the fluorescence signal of DCF weakens significantly. The results suggest that the UCNPs@PAIE-TPP NPs can generate ROS in the complexity of biological systems upon 980 nm laser irradiation. Photodynamic cancer therapy The PDT effects of these AIE NPs were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. In the absence of light, the UCNPs@PAIE-TPP NPs and UCNPs@PAIE-TPP-PEG NPs are low cytotoxic even at a concentration of up to 200 µg mL-1, suggesting the good biocompatibility. However, a significant cytotoxicity was observed with A549 cells at different NPs concentrations after 980 nm laser irradiation. The ROS cytotoxicity of these NPs exhibit a concentration-dependent enhancement (Figure 6B). The cell viability of UCNPs@PAIE-TPP NPs (200 µg mL-1) and UCNPs@PAIE-TPP-PEG NPs (200 µg mL-1) decreased to approximately 16% and 10% after 10 min laser irradiation (3 W cm-2), respectively. This demonstrates that these NPs can exert severe toxicity to A549 cells upon 980 nm laser irradiation. The 50% inhibition concentration (IC50) of UCNP@PAIE-TPP-PEG NPs in vitro, which can reveal the PDT effect, was measured. The IC50 of 19.2 µg mL-1 is a relative low value, suggesting an efficient PDT effect.39 Additionally, the cytotoxicity of UCNPs@PAIE-TPP NPs is much higher than that of UCNPs@PAIE NPs without a TPP unit under the same laser irradiation, suggesting that the AIE photosensitizer producing ROS in mitochondria could improve PDT effect signally. It is worth noting that the pH responsive PEGylated NPs are capable of decreasing protein absorption at physiological pH and demonstrates the mitochondria-target group TPP with enhanced PDT effect under slightly acidic conditions. Moreover, the live/dead staining of UCNPs@PAIE-TPP-PEG NPs for killing A549 cell with fluorescein diacetate (FDA, green for live cells) and propidium iodide (PI, red for dead cells) was performed at pH 6.8. As shown in Figure 7, bright green fluorescence with negligible red emission is 10 ACS Paragon Plus Environment

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detected from A549 cells without NIR light irradiation, presenting good biocompatibility and low toxicity of the UCNPs@PAIE-TPP-PEG NPs in the dark. Upon 980 nm laser irradiation for 5 min or 10 min (3 W cm-2), the number of dead cells increased significantly. Red fluorescence was detected in A549 cells, suggesting that the cells can be effectively killed by UCNPs@PAIE-TPP-PEG NPs. Therefore, UCNPs@PAIE-TPP-PEG NPs exhibits outstanding PDT activity to trigger the malignant cell death under NIR laser irradiation.

EXPERIMENTAL SECTION Synthesis of polymer PAIE: The feed molar ratio of HPMA, AEMA and AIEM was 100: 10: 6. First, 87 mg of HPMA, 10 mg AEMA, 34 mg AIEM and 4 mg AIBN were dissolved in 0.5 mL DMF. Then, the mixture was stirred at 65 °C for 24 h under nitrogen. Polymer was precipitated into 15 mL of ether from the mixture solution. After filtration, the polymer was dissolved again in 1 mL of DMF and reprecipitated into 15 mL of ether to yield polymer PAIE (89 mg, yield: 68%). Mn = 14700, Mw = 26200, Mw/Mn = 1.78. 1H NMR (400 MHz, DMSO-d6, TMS, ppm) δ 7.22–8.13 (broad, aromatic protons), 4.74 (broad, -OH), 3.68 (broad, -CHOH-), 2.90 (broad, -CH(CH3)NH-), 1.23 (broad, -CH2-), 0.82-1.00 (broad, -CH3). Synthesis of polymer PAIE-TPP: First, the mixture of 1-bromo-2-(2-bromoethoxy)ethane (232.0 mg, 1 mmol) and tripheylphosphine (262.0 mg, 1 mmol) in acetonitrile (3 mL) was stirred under reflux for 12 h. Then, the mixture was cooled to room temperature. The solvent was then removed by evaporation under pressure, and the residue was recrystallized from a mixture of hexane/ethyl acetate to yield the product (2-(2-bromoethoxy)ethyl)triphenylphosphonium bromide. Yield: 440 mg (89%). 1H NMR (400 MHz, CDCl3, TMS, ppm): δ 7.66–7.90 (m, 15H), 4.27–4.32 (m, 2H), 4.06–4.09 (t, J = 5.6 Hz, 1H), 4.00–4.03 (t, J = 5.6 Hz, 1H), 3.58–3.61 (t, J = 5.6 Hz, 2H), 3.18–3.21 (t, J = 5.6 Hz, 2H). Polymer PAIE (20.0 mg), potassium carbonate (6.0 mg, 0.04 mmol) and the above product (20.0 mg, 0.04 mmol) were dissolved in DMF (1 mL), and the mixture was stirred at 60 °C for 12 h. Then, the mixture was cooled to room temperature. The solvent was further purified by dialysis (MWCO 3500 Da) against 11 ACS Paragon Plus Environment


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Milli-Q water for 2 days and then the solution was lyophilized to yield polymer PAIE-TPP (30 mg, yield: 73%). 1H NMR (400 MHz, DMSO-d6, TMS, ppm) δ 7.24–8.11 (broad, aromatic protons, 7.77– 7.89 from the TPP unit), 4.71 (broad, -OH), 3.68 (broad, -CHOH-), 2.90 (broad, -CH(CH3)NH-), 1.22 (broad, -CH2-), 0.82–1.00 (broad, -CH3). Mn = 20700, Mw = 38200, Mw/Mn = 1.85. Preparation of UCNP@PAIE-TPP-PEG: 5 mg of mPEG-CHO was added into the aqueous solution (5 mL) of the above-prepared NPs (5 mg) at pH 7.4 and then stirred for 2 h, yield the PEGylated NPs. The solution was centrifuged at 12000 rpm for 5 min. The precipitate was washed twice with deionized water. Cytotoxicity study: To determine the cytotoxicity of UCNP@PAIE NPs, UCNP@PAIE-TPP NPs and UCNP@PAIE-TPP-PEG NPs, an MTT test was performed. A549 cells were seeded in 96-well plates (5 × 103 cells per well) and the cells were cultured for 24 h. Then, the medium was replaced with NPs solution (0, 10, 25, 50, 100, 200 µg mL-1) and incubated at 37 °C. After incubation for 24 h, the cells in the culture medium of 100 µL were disposed in MTT solution at an MTT concentration of 0.5 mg mL-1. After 2 h of incubation, 100 µL of DMSO was added to dissolve all the precipitates formed. The absorbance rate was measured at 570 nm using a microplate reader (Epoch, BioTek, Genecompany Limited). The results were presented as a mean and standard deviation obtained from eight samples. To determine these NPs cytotoxicity upon laser irradiation, an MTT test was performed. Following incubation with NPs solution (0, 25, 50, 100, 200 µg mL-1) for 5 h, cells were washed twice with PBS and then upon 980 nm laser irradiation (3 W cm-2) for 10 min. After incubation for 24 h, the cells were disposed in MTT solution at MTT concentration of 0.5 mg mL-1 to measure cell survival. For UCNP@PAIE-TPP-PEG NPs, the pH of culture medium was 6.8 for incubation. Cell apoptosis: The MTT results were further confirmed by FDA and PI. Here, A549 cells were incubated with DMEM medium (pH 6.8) containing UCNPs@PAIE-TPP-PEG NPs (100 µg mL-1) at 37 °C in 5% CO2 for 5 h. After that, the cells were washed twice with PBS and then upon 980 nm laser irradiation (3 W cm-2) for 5 or 10 min. At the same time, the control group was put in dark without laser 12 ACS Paragon Plus Environment

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irradiation. Afterward, the cells were incubated with FDA for 10 min (10 µg mL-1). The cells were incubated with PI in the dark (20 µg mL-1) for another 10 min. Finally, cells were examined with CLMS. The green fluorescence from FDA was collected from 505 to 525 nm upon excitation at 488 nm. The red fluorescence from PI was collected from 570 to 610 nm upon excitation at 543 nm. CONCLUSION In summary, we have successfully constructed AIE polymer-encapsulated UCNPs with mitochondriatargeting ability and a pH-responsive PEG layer to achieve efficient ROS generation for PDT by using the up-converted energy from UCNPs upon 980 nm laser irradiation. A distinct nano-structure was observed by TEM and UCNPs is well dispersed in AIE polymeric NPs, which favours the efficient energy transfer from UCNPs donor to AIE polymer acceptor. The PDT characteristics of AIE polymer encapsulated upconversion NPs include the following: (1) high FRET efficiency, (2) UCL imaging ability and great PDT effect under the same NIR light irradiation, (3) mitochondria-targeting ability, which is beneficial to target mitochondria for subcellular bioimaging and beneficial to guide the NPs to specifically target mitochondria and then generate ROS in the specific location, improving PDT effect. Finally, the pH responsive PEG layer shields the surface positive charges and shows stronger proteinresistance ability, which is beneficial to improve the NPs stability in the bloodstream. Therefore, this NIR triggered UCNPs@PAIE-TPP-PEG NPs with good biocompatibility and mitochondria targeting can perform bioimaging and PDT simultaneously under the same NIR light irradiation, indicating its great potential in a medical application.

ASSOCIATED CONTENT Supporting Information



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Experimental details and characterization of new compounds, decay curves, ROS generation, stability of these NPs, UCL image and ROS generation in cells. The material is available free of charge on the ACS Publications website at DOI: 10.1021/XXXXX.

AUTHOR INFORMATION Corresponding Author [email protected] or [email protected] Notes The authors declare no competing financial interests. ACKNOWLEDGEMENTS We are grateful to the National Natural Science Foundation of China (21504064, 21674080), the Natural Science Foundation of Tianjin (16JCQNJC02800), the Research Funds for Tianjin Educational Committee (20140501), 131 talents program of Tianjin, the leading talents of Tianjin Educational Committee and the Fundamental Research Funds for the Central Universities (JCKY-QKJC05) for financial support. We are also grateful to Dr. jintao Kong in Fujian Institute of Research on the Structure of Matter for technical support.

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Scheme 1. Schematic illustration of the pH responsive UCNP@PAIE-TPP-PEG NPs as the NIR triggered mitochondria targeting photosensitizer to generate ROS for PDT.


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Scheme 2. Synthetic scheme of the polymer PAIE and PAIE-TPP.



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Figure 1. (A) Normalized UV-Vis absorption spectra and PL spectra (λex = 488 nm) of AIEM, PAIE, UCNPs and UCNP@PAIE. (B) PL spectra of AIEM (200 µM) in DMSO/water mixtures (λex = 488 nm) and dependence of the I/I0 ratios on the solvent composition. (C) UCL spectra of the UCNPs, UCNP@PAIE NPs and UCNP@PAIE-TPP NPs upon excitation at 980 nm. [UCNPs] = 1 mg mL-1. (D) PL spectra of DCF (2 µM) in UCNP@PAIE-TPP NPs (100 µg mL-1) with different 980 nm laser irradiation times (3 W cm-2) and time-dependent DCF fluorescence intensity changes at 523 nm.


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Figure 2. (A) UCL spectra in NIR range of the UCNPs, UCNP@PAIE NPs and UCNP@PAIE-TPP NPs upon excitation at 980 nm. [UCNPs] = 1 mg mL-1. (B) UV-Vis spectra of UCNP@PAIE-TPP NPs (100 µg mL-1) after different times of 980 nm irradiation (3 W cm-2).



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Figure 3. TEM images of UCNPs (A) and UCNP@PAIE-TPP-PEG NPs (B).


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Figure 4. 1H NMR spectra of mPEG-CHO and the pH-sensitivity of the benzoic imine bond generated between UCNP@PAIE-TPP and mPEG-CHO.



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Figure 5. Confocal microscopy images of A549 cells after incubation with UCNP@PAIE NPs (A-D), UCNP@PAIE-TPP NPs (E-H) and UCNP@PAIE-TPP-PEG NPs at pH 6.8 (I-L). The cells were costained with MTG (50 nM). For NPs: λex = 488 nm, λem = 662-737 nm. For MTG: λex = 488 nm, λem = 500-530 nm. The scale bar is 20 µm.


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Figure 6. Cell viability of A549 cells after being incubated with various concentrations of UCNP@PAIE NPs, UCNP@PAIE-TPP NPs and UCNP@PAIE-TPP-PEG NPs for 24 h (A) in the dark, (B) under laser irradiation for 10 min (3 W cm-2, with 1 min break for each 5-min exposure). Control samples are the cells without any treatment.



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Figure 7. Live/dead staining of UCNP@PAIE-TPP NPs (A-C) and UCNP@PAIE-TPP-PEG NPs (D-F) treated A549 cells with 980 nm light irradiation (3 W cm-2) for 0 min (A, D), 5 min (B, E) and 10 min (C, F). The live cells were stained by FDA (green), whereas dead cells were stained by PI (red). The scale bar is 200 µm.


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Table of Contents

Near-Infrared Triggered Upconversion Polymeric Nanoparticles Based on AggregationInduced Emission and Mitochondria Targeting for Photodynamic Cancer Therapy Yue Guan, Hongguang Lu*, Wei Li, Yadan Zheng, Zhu Jiang, Jialing Zou, Hui Gao*



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Scheme 1. Schematic illustration of the pH responsive UCNP@PAIE-TPP-PEG NPs as the NIR triggered mitochondria targeting photosensitizer to generate ROS for PDT. 416x250mm (72 x 72 DPI)

ACS Paragon Plus Environment

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Scheme 2. Synthetic scheme of the polymer PAIE and PAIE-TPP. 409x248mm (72 x 72 DPI)



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Figure 1. (A) Normalized UV-Vis absorption spectra and PL spectra (λex = 488 nm) of AIEM, PAIE, UCNPs and UCNP@PAIE. (B) PL spectra of AIEM (200 µM) in DMSO/water mixtures (λex = 488 nm) and dependence of the I/I0 ratios on the solvent composition. (C) UCL spectra of the UCNPs, UCNP@PAIE NPs and UCNP@PAIE-TPP NPs upon excitation at 980 nm. [UCNPs] = 1 mg mL-1. (D) PL spectra of DCF (2 µM) in UCNP@PAIE-TPP NPs (100 µg mL-1) with different 980 nm laser irradiation times (3 W cm-2) and timedependent DCF fluorescence intensity changes at 523 nm. 160x81mm (300 x 300 DPI)


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Figure 2. (A) UCL spectra in NIR range of the UCNPs, UCNP@PAIE NPs and UCNP@PAIE-TPP NPs upon excitation at 980 nm. [UCNPs] = 1 mg mL-1. (B) UV-Vis spectra of UCNP@PAIE-TPP NPs (100 µg mL-1) after different times of 980 nm irradiation (3 W cm-2). 80x104mm (300 x 300 DPI)



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Figure 3. TEM images of UCNPs (A) and UCNP@PAIE-TPP-PEG NPs (B). 456x188mm (72 x 72 DPI)


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Figure 4. 1H NMR spectra of mPEG-CHO and the pH-sensitivity of the benzoic imine bond generated between UCNP@PAIE-TPP and mPEG-CHO. 38x31mm (300 x 300 DPI)



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Figure 5. Confocal microscopy images of A549 cells after incubation with UCNP@PAIE NPs (A-D), UCNP@PAIE-TPP NPs (E-H) and UCNP@PAIE-TPP-PEG NPs at pH 6.8 (I-L). The cells were co-stained with MTG (50 nM). For NPs: λex = 488 nm, λem = 662-737 nm. For MTG: λex = 488 nm, λem = 500-530 nm. The scale bar is 20 µm. 289x212mm (72 x 72 DPI)


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Figure 6. Cell viability of A549 cells after being incubated with various concentrations of UCNP@PAIE NPs, UCNP@PAIE-TPP NPs and UCNP@PAIE-TPP-PEG NPs for 24 h (A) in the dark, (B) under laser irradiation for 10 min (3 W cm-2, with 1 min break for each 5-min exposure). Control samples are the cells without any treatment. 400x150mm (72 x 72 DPI)



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Figure 7. Live/dead staining of UCNP@PAIE-TPP NPs (A-C) and UCNP@PAIE-TPP-PEG NPs (D-F) treated A549 cells with 980 nm light irradiation (3 W cm-2) for 0 min (A, D), 5 min (B, E) and 10 min (C, F). The live cells were stained by FDA (green), whereas dead cells were stained by PI (red). The scale bar is 200 µm. 370x250mm (72 x 72 DPI)


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