pH-Triggered and Enhanced Simultaneous Photodynamic and

May 31, 2017 - School of Chemical and Biomedical Engineering, Nanyang ... NAB NPs can be activated in weak acidic environment to give high rate of ...
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pH-Triggered and Enhanced Simultaneous Photodynamic and Photothermal Therapy Guided by Photoacoustic and Photothermal Imaging Qianyun Tang,† Wanyue Xiao,† Chuhan Huang,† Weili Si,† Jinjun Shao,† Wei Huang,† Peng Chen,*,§ Qi Zhang,*,‡ and Xiaochen Dong*,† †

Key Laboratory of Flexible Electronics (KLOFE) and Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), Nanjing 211816, P.R. China ‡ School of Pharmaceutical Sciences, Nanjing Tech University (NanjingTech), Nanjing 211816, P.R. China § School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, 637459, Singapore S Supporting Information *

ABSTRACT: Developing smart photosensitizers sensitively responding to tumor-specific signals for reduced side effects and enhanced anticancer efficacy is a major challenge for tumor phototherapy. Herein, a pH-sensitive photosensitizer has been synthesized through introducing a pH-sensitive receptor (dimethylaminophenyl unit) onto the aza-BODIPY core (abbreviated as NAB). Through enveloping hydrophobic NAB with amphiphilic DSPE-mPEG2000, NAB nanoparticles (NPs, diameter ∼ 30 nm) with strong near-infrared absorption (∼792 nm) are obtained. NAB NPs can be activated in weak acidic environment to give high rate of reactive oxygen species (ROS) generation and enhanced photothermal effect. NAB NPs can selectively accumulate in the lysosomes of tumor cells and subsequently activate under the acidic microenvironment of lysosome (pH 5.0) to produce ROS for photodynamic therapy, due to switch-off of the photoinduced electron transfer (PET) pathway. In vivo, pH-enhanced photoacoustic imaging (PAI) and photothermal imaging (PTI) confirm that NAB NPs can selectively aggregate in the tumor, and the tumor growth can be effectively inhibited under xenon lamp irradiation through synergistic phototherapy (photodynamic and photothermal therapy, PDT/PTT). Furthermore, based on PAI signal and terminal elimination half-life (T1/2) obtained by pharmacokinetic experiment, it is concluded that the NAB NPs can be rapidly metabolized. The pH-sensitive NAB NPs offer a new possibility toward PAI and PTI guided synergistic phototherapy. (pH 7.4).20 Therefore, pH-sensitive photosensizers are particularly attractive.21−26 Some organic molecules capable of pH-switchable singlet oxygen generation have been reported for PDT.27−29 Nonetheless, these photosensitizers are hardly practical because they are activated by ultraviolet or visible light which is poor for tissue penetration. As the NIR region (700−1100 nm) is the transparent window for deep tissue penetration because of reduced tissue autofluorescence and photon absorption,30 it is highly desirable to design NIR absorbing pH-sensitive photosensitizers to accomplish deep-tissue and tumor targeting phototherapy. In contrast to the traditional organic photosensitizer (such as, porphyrin derivatives, phthalocyanine derivatives, chlorine derivatives),31−34 aza-boron-dipyrromethene (aza-BODIPY) derivatives exhibit preferable red-shifted absorption. And they possess other desirable photophysical properties, including

1. INTRODUCTION Phototherapy for cancers, including photodynamic therapy (PDT) and photothermal therapy (PTT), relies on the photosensitizers to convert photon energy into reactive oxygen species (ROS, such as singlet oxygen 1O2) or heat to destroy tumor cells.1−6 Considerable efforts have been made to enhance the therapeutic efficacy by improving the photophysical properties, cellular uptake, pharmacokinetic profile, and tumor-targeting performance of photosensitizers.7 Knowing the differences between tumor and normal tissues, “smart” photosensitizers have been designed to reduce the severe side effects and enhance the anticancer efficacy. Such photosensitizers can be devised to be responsive to tumor-specific biomarkers, such as DNAs8−12 and enzymes.13−17 But these biomarkers are often limited in number and tumor type specific.18 Because of the upregulated glycolytic metabolism under the hypoxic conditions,19 tumor tissues, regardless of types and developmental stages, are universally characterized by the acidic extracellular microenvironment (pH 6.5−6.8), in contrast to the neutral condition in blood or normal tissues © 2017 American Chemical Society

Received: March 15, 2017 Revised: May 31, 2017 Published: May 31, 2017 5216

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Scheme 1. Schematic Illustration of pH-Sensitive NAB NPs for PAI and PTI Guided PTT/PDT Synergistic Therapy with Xenon Lamp Irradiation

Figure 1. (a) Expected protonation mechanism of NAB protonation induced by pH change. (b) Color changes of NAB in dichloromethane upon the addition of trifluoroacetic acid (TFA) and triethylamine (TEA). (c) UV−vis absorption spectra of NAB in dichloromethane in the absence and in the presence of TFA.

with good biocompatibility and water solubility are obtained. The NAB NPs show broad absorption range extending to the NIR region (400−1100 nm) and can be switched from “off’ to “on” state in a weak acidic environment to generate singlet oxygen and enhance the photothermal effect. Additionally, both fluorescence (FL) and photoacoustic (PA) signals of NAB NPs are largely increased at pH 5.0. In vitro experiments demonstrate that NAB NPs can be readily uptaken by the tumor cells and preferentially accumulate in lysosomes (pH 4.5−5.0), where PDT is activated due to effective ROS generation. Dual imaging in vivo (PAI and PTI) indicates that NAB NPs selectively accumulate in mice tumor tissues through the enhanced permeability and retention (EPR) effect. PAI and pharmacokinetic studies also suggest that NAB NPs can be rapidly metabolized. Both in vitro and in vivo studies demonstrate the good biosafety and outstanding antitumor

large visible light molar absorption coefficient and good photostability.35,36 We hypothesize that aza-BODIPY can be derived to realize pH-sensitivity and NIR absorption for dual modal cancer phototherapy in deep tumor tissues. Moreover, the photothermal effect of aza-BODIPY can also be utilized for photothermal imaging (PTI) and photoacoustic imaging (PAI) in order to guide localized phototherapy. Although PTI offers good temperature sensitivity and possibility of real-time monitoring, it provides poor spatial resolution. 37,38 In comparison, PAI is an emerging nonionizing and noninvasive modality with high optical contrast, good penetration depth and high spatial resolution.39,40 Herein, two dimethylaminophenyl units are introduced onto the aza-BODIPY core to achieve a pH-sensitive photosensitizer with strong NIR absorption. Through enveloping hydrophobic NAB with amphiphilic DSPE-mPEG2000, nanoparticles (NPs) 5217

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Figure 2. (a) TEM image of NAB NPs. (b) UV−vis absorption spectra of NAB NPs in PBS solution at pH 7.4 and 5.0. (c) Fluorescence spectra of NAB NPs in PBS solution at pH 7.4 and 5.0 (λex = 561 nm). (d) Absorption spectra of DPBF in the presence of NAB NPs at pH 7.4 and xenon lamp irradiation for different durations. (e) Absorption spectra of DPBF in the presence of NAB NPs at pH 5.0 and xenon lamp irradiation for different durations. (f) Absorbance decrease of DPBF with increasing photoirradiation time in the presence of NAB NPs at pH 7.4 and 5.0. (g) Temperature curves of PBS or NAB NP solution at pH 7.4 or 5.0 with xenon lamp irradiation. (h) Relationships between concentration of NAB NPs and PA signal intensity at pH 7.4 or 5.0.

does not oxidize DPBF.42 In contrast to DPBF only solution or DPBF/NAB mixture at neutral condition, the absorption of DPBF/NAB at low pH decrease in an irradiation dependent manner, confirming the acidity activated 1O2 generation by NAB (Figure S3). 2.2. Characterization of NAB NPs. Biocompatible and soluble NAB NPs were prepared through enveloping hydrophobic NAB with amphiphilic 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[methoxy(poly(ethylene glycol))2000] (DSPE-mPEG2000). Transmission electron microscopy (TEM) image showed that the NAB NPs have spherical morphology with uniform particle size of about 30 nm (Figure 2a). And they showed strong and broad absorption centered at ∼830 nm (Figure 2b) in phosphate buffered saline (PBS) at pH 7.4 or pH 5.0, exhibiting a 38 nm red shift comparing to that of NAB. The red shift may be attributable to the strong intramolecular and intermolecular π−π interactions between the aggregated NAB molecules.43,44 The maximum absorption peak of NAB NPs at pH 5.0 (absorption coefficient α = 10.76 L g−1 cm−1) is stronger than that of NAB NPs at pH 7.4 (absorption coefficient α = 9.26 L g−1 cm−1), indicating that more photon energy is absorbed at acidic condition. As shown in Figure 2c, under excitation of 560 nm, NAB NPs at both pH 7.4 and 5.0 gave two emission peaks at ∼624 and ∼850 nm; however, the fluorescence intensity of both peaks was enhanced at pH 5.0, manifesting a pH-sensitive fluorescence characteristic. Similar to individual NAB molecules, NAB NPs under xenon lamp irradiation are also much more active at pH 5.0 than at pH 7.4 in production of singlet oxygen, as reported by the absorption decrease of DPBF (Figure 2d−f). Interestingly, the photothermal effect of NAB NPs is also enhanced to some extent at acidic condition. As shown in Figure 2g, the temperature of NAB NP solution at pH 5.0 increased by 23.2 °C after 20 min of xenon lamp irradiation, which is 1.6 °C higher than that attained at pH 7.4. The photothermal conversion efficiency of the aqueous solution of NAB NPs at 808 nm was calculated to be ∼67.8% (Figure S4), which is

efficiency of NAB NPs in enhanced phototherapy (PDT and PTT) under broad-band xenon lamp irradiation (Scheme 1).

2. RESULTS AND DISCUSSION 2.1. Characterization of NAB. The detailed synthetic route of NAB is shown in Scheme S1 (Supporting Information). In weak acidic condition, one dimethylaminophenyl unit of NAB can be first protonated to form NAB1H (Figure 1a). The purple NAB solution (in dichloromethane) was gradually turned into a light purple with increasing addition of trifluoroacetic acid (CF3COOH, TFA) due to the formation of NAB1H (Figure 1b). With further increase of TFA, NAB2H with two protonated dimethylaminophenyl units results; concurrently, the solution color turned into blue.41 As shown in Figure 1c, NAB displayed a strong absorption peak at ∼792 nm and a weak peak at ∼553 nm. With increasing TFA, both peaks had slight blue-shift, and a new peak at ∼946 nm appeared (Figure S1). With 6.25 mM TFA, the light purple NAB1H solution displayed three absorption peaks at ∼542, 743, and 946 nm, respectively (Figure 1c). With further addition of TFA, a new peak at 670 nm emerged and the absorption at 946 nm elevated (Figure S1). With 125 mM TFA, the blue NAB2H solution showed three peaks at ∼553, 670, and 946 nm, respectively (Figure 1c). Subsequent neutralization by adding 125 mM triethylamine (TEA) into NAB2H solution recovered the solution color and adsorption profile back to nearly the same as NAB, suggesting that the sequential protonation progress of NAB is reversible. At 553 nm excitation, NAB displayed a fluorescence emission peak at ∼838 nm (Figure S2). With increasing addition of TFA (decreasing pH), emission at ∼838 nm was weakened while emissions at ∼608 nm and ∼723 nm were strengthened. And such changes in emission profile were completely rectified after neutralizing TEA. The transition of NAB from “off’ to “on” state of singlet oxygen generation was examined with 1,3-diphenylisobenzofuran (DPBF) as 1O2 probe and TFA as acidity regulator which 5218

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Figure 3. Expected machanism of switch off/on of singlet oxygen generation, enhanced fluorescence, and improved photothermal conversion effect induced by pH.

much higher than those of the previously reported photothermal agents.45 The photothermal stabilities of NAB NPs at pH 7.4 and 5.0 were studied by reversible heating and natural cooling (Figure S5). Almost no temperature decrease of NAB NP solutions after 5 cycles can be observed, confirming the excellent photothermal stability of NAB NPs at both pH 7.4 and 5.0. With the enhanced photothermal performance at pH 5.0, the concentration-dependent PA response is therefore also augmented (Figure 2h). We conceive that the reversible pH-sensitive characteristic of NAB and NAB NPs is originated from the photoinduced electron transfer (PET) mechanism as illustrated in Figure 3.46 The −NMe2 groups serve as the sensitive pH sensor because they can be protonated by weak acidity. According to the Jablonski diagram, the photon-excited electron may return to the ground state through radiative relaxation or vibrational relaxation, which leads to fluorescence emission or the thermal effect, respectively.47 It is also possible for an excited electron to enter the triplet state through intersystem crossing, which in turn triggers photochemical reaction to produce ROS. In neutral condition, the HOMO level of the −NMe2 group is between the HOMO and the LUMO levels of the photosensitizer. Consequently, after excitation, −NMe2 groups transfer an electron to the HOMO of the photosensitizer while the excited electron tends to be shunted to the HOMO of the −NMe2 group, instead of undertaking radiative relaxation, vibrational relaxation, or intersystem crossing. In acidic condition, H+ bonding to the −NMe2 group brings its HOMO lower than that of the photosensitizer and, hence, switches the photosensizer to the “on” state for fluorescence, photothermal, and photodynamic effects. 2.3. In Vitro Cell Experiments. To study the dark toxicity and phototoxicity of NAB NPs to HeLa cells, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was carried out in the presence or absence of xenon lamp irradiation. As demonstrated in Figure S6, NAB NPs exhibited significant phototoxicity in a dose-dependent manner with a half-maximal inhibitory concentration (IC50) of ∼130 μM, whereas the viability of the cells without irradiation remained high. It is known that lysosome has a weak acidic environment (pH 4.5−5.0) compared to the cytoplasm (pH 7.2), which is maintained by pumping protons from the cytosol across the lysosomal membrane via vacuolar-type H+-ATPase.48 As lysome is a cellular compartment in the endocytoic pathways, its acidic environment can be utilized to activate endocytosed pH-sensitive photosensitizers. Figure 4a shows the cellular uptake property of NAB NPs revealed by confocal microscopy at different pH values. Cells at pH 5.0 exhibited a more obvious fluorescence enhancement than those at pH 7.4.

Figure 4. (a) Confocal images of DAPI stained HeLa cells with preincubation with NAB NPs at pH 7.4 or 5.0. (b) Confocal images of LysoTracker Green stained HeLa cells with preincubation with NAB NPs at pH 7.4. (c) Confocal images of DAPI and DCFH-DA stained HeLa cells with or without being preincubated with NAB NPs at pH 7.4.

The fluorescence costaining experiment with a lysosomal indicator (Lyso-Tracker Green) indicated that NAB NPs were segregated in lysosomes (Figure 4b). Then ROS generation of NAB NPs in HeLa cells was reported by 2′,7′dichlorofluorescin diacetate (DCFH-DA), which can react with ROS to form green fluorescent 2′,7′-dichlorofluorescein (DCF) after deacetylation by esterase.49 Figure 4c shows that, after illumination with a xenon lamp, green fluorescence was distributed throughout the HeLa cells treated with NAB NPs and DCFH-DA molecules at pH 7.4. In the control experiment, the same irradiation but without NAB NPs did not cause any green fluorescence, indicating no ROS generation. These observations certify the lysosomal activation of NAB NPs to intracellularly generate ROS. The produced ROS could in turn induce lysosomal membrane permeabilization and subsequent release of lysosimal hydrolases such as cathepsins into cytosol to trigger cell death pathways.48,50 5219

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Figure 5. (a) In vivo PA images of the mouse tumor taken at different time points after tail intravenous injection of NAB NPs. (b) In vivo PT images of HeLa tumor-bearing nude mice after tail intravenous injection of NAB NPs with xenon lamp irradiation. (c) Tumor temperature curves under xenon lamp irradiation.

2.5. Pharmacokinetic Study. To study the pharmacokinetic performance of pH-sensitive NAB NPs, six Wistar rats were injected with saline solution containing NAB NPs through tail vein injection. As shown in Figure S8, the plasma concentration of NAB NPs reduced quickly, and nearly no NAB NPs could be detected after 12 h, indicating the rapid metabolic processes, contrary to the long-term retention of inorganic nanomaterial based photosensitizers. The main pharmacokinetic parameters were calculated by a two compartment model and are presented in Table 1. The terminal

2.4. Dual-Modal in Vivo Imaging. Photoacoustic imaging was employed to monitor in real time the dynamic accumulation and metabolism of NAB NPs in mouse tumor (Figure 5a and Figure S7). Before injection of NAB NPs, PA signals from the blood vessels were observed, resulting from the endogenous light-absorbing hemoglobin molecules in the blood. These PA signals indicate the abundant blood in the tumor region. After tail intravenous injection of NAB NPs, the PA signal increased greatly (particularly near the blood vessels) and reached the maximum after 1 h. Such fast accumulation of NAB NPs was attributed to passive targeting because of the enhanced permeability and retention (EPR) effect and selective target to the acidic extracellular microenvironment (pH 6.5− 6.8) in tumor tissues by their pH-sensitive characteristic.18 Over time, NAB NPs spread into the deep tissue of the tumor, and after the sixth hour, the PA signal started to decrease quickly, suggesting that NAB NPs were metabolized. As shown in Figure S7, 24 h after injection of NAB NPs, the mean PA signal intensity in the tumor was similar to that before injection, indicating NAB NPs were well-metabolized by the body. The photothermal effect of the photosensitizer can be utilized for photothermal imaging (PTI) which can offer good temperature sensitivity and possibility of real-time monitoring. Figure 5b presents the PT images of the nude mice at various time points during the continuous irradiation of the xenon lamp (20 mW cm−2). In contrast to the control group injected with saline, the tumor temperature of the mouse injected with NAB NPs increased steadily and remarkably, indicating the outstanding photothermal effect and photostability (Figure 5b,c). Also evidently, NAB NPs selectively accumulated in the tumor site but not the normal tissues. The tumor temperature gradually escalated to ∼46 °C within 10 min, which is high enough to eliminate tumor cells. Besides, during the heating progress, mild hyperthermia induced by PTT can increase the cell membrane permeability and subsequently improve vascular perfusion whereby relieving the hypoxic condition in tumor and enhancing extravasation of therapeutic nanoparticles into the tumor interstitium.51 Taken together, both PAI and PTI provide important guidance for photosensitizer performance and phototherapy.

Table 1. Pharmacokinetic Parameters of NAB NPs after a Single Intravenous Administration at a Dose of 2.3 mg kg−1 in Healthy Rats (n = 6) pharmacokinetic parameters AUC0−t AUC0−∞ MRT t1/2 CL

values (mean ± SD)

unit −1

μg mL h μg mL−1 h h h mL h−1 kg−1

14.725 15.934 1.908 4.288 148.16

± ± ± ± ±

1.441 1.828 0.123 0.48 18.271

elimination half-life (T1/2) was 4.288 ± 0.48 h. As NAB NPs take 1 h to be most enriched in the tumor (Figure 5a), such T1/2 is long enough to ensure accumulation of NAB NPs. Additionally, the clearance rate (148.16 ± 18.271 mL h−1 kg−1) of NAB NPs is high, indicating a fast elimination progress of NAB NPs in vivo. Taken together, the pharmacokinetic study proves that NAB NPs can be readily metabolized, which ensures the good biosafety of NAB NPs. 2.6. In Vivo Synergistic Phototherapy. To investigate in vivo the synergistic therapeutic effects of NAB NP enabled simultaneous PDT and PTT, HeLa tumor-bearing nude mice were utilized as the animal model. Considering the side effects (such as discomfort, erythema, crusting, blistering, and dyspigmentation) that laser may cause and the narrow bandwidth of lasers which limits their use to specific photosensitizers only,52,53 a broadband xenon lamp was employed to activate the NAB NPs for in vivo phototherapy. These nude mice were randomly divided into 3 groups (5 mice per group): (i) the treatment group with tail intravenous 5220

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Figure 6. (a) Tumor growth curves of 3 differently treated mouse groups (5 mice per group). Error bars represent the standard deviations (no significance or N.S., **p < 0.01). (b) Photographs of tumors collected from the mice treated with saline injection and irradiation (top), the mice treated with NAB NP injection but without irradiation (middle), or the mice treated with both NAB NP injection and irradiation (bottom), respectively. (c) H&E staining and Ki67 immuno-staining of tumor tissues after different treatments.

injection of NAB NPs and xenon lamp exposure; (ii) the comparison group with nanoparticle injection but no irradiation;and (iii) the control group with injection of saline solution and irradiation by lamp. The efficacy of NAB NP mediated phototherapy was assessed by monitoring the tumor volume. As demonstrated in Figure 6a,b, the phototherapy treatment led to significant inhibition to tumor growth (90.7% inhibition as compared to the control group), which is superior to the previously reported methods based on a single phototherapy mode (PDT or PTT).54,55 Comparing with the therapeutic effect of aza-BODIPY nanoparticles lacking pHsensitive property in our recent report, NAB NPs can more effectively inhibit tumor growth.45 In contrast, the tumor volume of both control and comparison groups steadily expanded over time (28 days), and no significant difference was noticed between these two groups. The obvious body weight gain of the mice in the comparison and treatment groups implies no obvious toxicity of NAB NPs (Figure S9). The treatment efficacy was also evaluated by hematoxylin and eosin (H&E) staining as well as Ki67 immuno-staining on tissue sections from the differently treated groups. As shown in Figure 6c, prominent necrosis and apoptosis was observed in histological sections from the treatment group, confirming the successful destruction of the tumor cells by the dual modal phototherapy. In contrast, indiscernible necrosis appeared in both the control and the comparison group, suggesting the low dark toxicity and good biocompatibility of NAB NPs, as well as the low photon toxicity from broadband irradiation by xenon lamp. The Ki67 immuno-staining assay shows that Ki67

proteins (cellular marker for proliferation) expressed at low level in the treatment group and high level in the control and comparison groups, indicating the capability of NAB NPs to potently inhibit the proliferation of tumor cells. Figure S10 presents the H&E staining of the major organs (heart, lung, liver, spleen, and kidney). Compared with the control group, no damage or inflammation appeared in the other two groups injected with NAB NPs, further confirming the in vivo biocompatibility of the nanoparticles.

3. CONCLUSION In summary, a new photosensitizer (NAB) is obtained by incorporating dimethylaminophenyl groups onto the azaBODIPY core. The biocompatible and water-soluble NAB NPs are further assembled by encapsulating the hydrophobic NAB molecules with amphiphilic polymers. The pH-switchable properties of NAB NPs permit their selective activation in intracellular lysosomes and the acidic microenvironment in tumor tissues to give high rate of singlet oxygen generation and enhanced photothermal effect, thus enabling targeted synergistic dual mode (simultaneous photothermal and photodynamic) phototherapy. The strong NIR-absorption property of NAB NPs allows deep tissue phototherapy. The photothermal effect of the nanoparticle is utilized for photothermal and photoacoustic imaging. The dual mode in vivo imaging reveals the preferential localized accumulation of NAB NPs in the tumor site and their rapid metabolic kinetics. Potentially this can also be used for in situ cancer diagnosis in deep tissues. With both in vitro and in vivo experiments, we demonstrated 5221

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the excellent efficacy of NAB NPs as the new multifunctional phototherapy sensitizer.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.7b01075. Synthetic route of NAB, some experimental details, UV− vis absorption spectra and emission spectra of NAB, absorption decrease of DPBF, photothermal conversion efficiency and photothermal stability studies of NAB NPs, cell viability, mean plasma concentration−time curves, body weight of three groups of the mice, and H&E staining images of the major organs (PDF)

4. EXPERIMENTAL SECTION Preparation of Water-Soluble NAB NPs. The solution of NAB in tetrahydrofuran (1.0 mg/mL, 15 mL) was swiftly dropped into the DSPE-mPEG2000 aqueous solution (1.0 mg/mL, 75 mL) under sonication (175 W). After removing tetrahydrofuran, the obtained purple aqueous solution was further filtered with a 0.45 μm filter and rinsed with deionized water, which was freeze-dried to receive the final sphere as a purple powder. Singlet Oxygen Detection of NAB NPs. The singlet oxygen generation of NAB NPs (18 μM) was detected also with DPBF (25 μM) as the 1O2 fluorescent probe in PBS solution at pH 7.4 or pH 5.0. Then the mixed solution was irradiated with a xenon lamp (>510 nm, 20 mW cm−2) for 150 s. The oxidation of DPBF (at 418 nm) vs irradiation time was monitored by UV−vis−NIR spectrophotometer. Photothermal Effect of NAB NPs. The solutions of NAB NPs in PBS solution (1.3 mM) at both pH 7.4 and pH 5.0 were studied with PBS solution at pH 7.4 and pH 5.0 as the control group. These four solutions were irradiated simultaneously with a xenon lamp (20 mW cm−2) for 20 min. The temperature changes were collected by an FLIR thermal camera. Lysosomal Location Study. HeLa cells seeded in glass bottom Petri dish were incubated in media containing NAB NPs (130 μM, 2 mL) for 24 h at 37 °C under 5% CO2. After washing three times with PBS solution, HeLa cells were incubated with LysoTracker Green DND-26 at 37 °C for 30 min in darkness. The cells were rinsed three times with PBS solution (pH 7.4) and refilled with 2 mL of PBS solution at pH 7.4. The fluorescence images of the cells were obtained using confocal laser scanning microscopy (Olympus IX 70 inverted microscope). NAB NP was excitated with a 559 nm laser, and the fluorescence from 570 to 670 nm was collected. LysoTracker Green was excitated with a 488 nm laser, and the fluorescence from 500 to 560 nm was collected. Pharmacokinetic Study. Six Wistar rats (6 males; weight 200 ± 10 g) obtained from the Animal Center of Nanjing Medical University (NJMU, Nanjing, China) were used in pharmacokinetic study. After being fasted for 12 h, six rats were injected with saline solution containing NAB NPs through tail vein injection (dose: 2.3 mg kg−1). Blood samples (500 μL) were collected from the ocular vein of each rat at 3 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 12 h after intravenous administration. Plasma samples were separated by centrifugation at 4000 × g for 10 min at 4 °C and stored at −80 °C until analysis. The concentration of NAB in the plasma samples was determined by a UV−vis−NIR spectrophotometer. The pharmacokinetic parameters including the area under the concentration−time curve (AUC) from zero to the last measurable plasma concentration point (AUC0−t), AUC from 0 to infinity (AUC0−∞), mean residence time (MRT), terminal elimination half-life (T1/2), and clearance (CL) were calculated by a two compartment method using Drug and Statistics (DAS 3.0) software (Mathematical Pharmacology Professional Committee of China, Shanghai, China). In Vivo Combination Therapy. Three groups (5 mice per group) of HeLa tumor-bearing nude mice were employed to study the antitumor effect of NAB NPs. The control group was injected with saline while the other two groups were treated with NAB NPs solution (1.3 mM). At 1 h after injection, both the control and the treatment group were irradiated with a xenon lamp (20 mW cm−2) for 10 min, and the body weight and tumor size were monitored every 2 days. All mice were sacrificed after treatment. Tumors and the major organs (heart, lung, liver, spleen, and kidney) of mice were washed with saline, fixed with 10% formalin, and embedded in paraffin wax for further histological examination (H&E staining) and immunohistochemical (antihuman Ki-67 staining) analysis.

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AUTHOR INFORMATION

Corresponding Authors

*(P.C.) E-mail: [email protected]. *(Q.Z.) E-mail: [email protected]. *(X.D.) E-mail: [email protected]. ORCID

Jinjun Shao: 0000-0001-6446-8073 Wei Huang: 0000-0001-7004-6408 Peng Chen: 0000-0003-3730-1846 Xiaochen Dong: 0000-0003-4837-9059 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was supported by the NNSF of China (61525402, 61604071), Key University Science Research Project of Jiangsu Province (15KJA430006), QingLan Project, Natural Science Foundation of Jiangsu Province (BK20161012), Six Talent Peaks Project (51235018), and AcRF Tier 2 grant (MOE2014T2-1-003) from the Ministry of Education of Singapore.

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REFERENCES

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DOI: 10.1021/acs.chemmater.7b01075 Chem. Mater. 2017, 29, 5216−5224

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DOI: 10.1021/acs.chemmater.7b01075 Chem. Mater. 2017, 29, 5216−5224