Enhanced Afterglow Performance of Persistent Luminescence

May 24, 2017 - More importantly, the intratumorally fixed ZGC PL implants can serve as a built-in excitation source for repeatable light emitting diod...
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Enhanced Afterglow Performance of Persistent Luminescence Implants for Efficient Repeatable Photodynamic Therapy Wenpei Fan,†,‡,§ Nan Lu,§ Can Xu,§ Yijing Liu,§ Jing Lin,*,† Sheng Wang,†,§ Zheyu Shen,§ Zhen Yang,§ Junle Qu,‡ Tianfu Wang,† Siping Chen,† Peng Huang,*,† and Xiaoyuan Chen*,§ †

Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging, School of Biomedical Engineering, Health Science Center, Shenzhen University, Shenzhen 518060, China ‡ Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China § Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, United States S Supporting Information *

ABSTRACT: Persistent luminescence nanoparticles (PLNPs) have been used for bioimaging without autofluorescence background interference, but the poor afterglow performance impedes their further applications in cancer therapy. To overcome the Achilles’ heel of PLNPs, herein we report the construction of injectable persistent luminescence implants (denoted as PL implants) as a builtin excitation source for efficient repeatable photodynamic therapy (PDT). The injectable ZGC (ZnGa1.996O4:Cr0.004) PL implants were prepared by dissolving ZGC PLNPs in poly(lactic-co-glycolic acid)/N-methylpyrrolidone oleosol, which demonstrated much stronger persistent luminescence (PersL) intensity and longer PersL lifetime than that of ZGC PLNPs both in vitro and in vivo. More importantly, the intratumorally fixed ZGC PL implants can serve as a built-in excitation source for repeatable light emitting diode (LED) and PersL-excited PDT upon and after periodic LED irradiation, which leads to the overall improvement of therapeutic effectiveness for efficient tumor growth suppression. This work represents efficient repeatable PDT based on the injectable yet periodically rechargeable ZGC PL implants. KEYWORDS: persistent luminescence implants, afterglow, phase transformation, built-in, photodynamic therapy lasting phosphorescence,23−25 have been widely used in a variety of fields, including but not limited to optoelectronic devices, night illumination, and military forces.26−28 In particular, the past few years have witnessed a growing number of nanoscale afterglow materials, the most representative of which are ZnGa1.996O4:Cr0.004 persistent luminescence nanoparticles (denoted as ZGC PLNPs) featured with NIR (λ = 650−750 nm) persistent luminescence (PersL) emission.29−34 These ZGC PLNPs can be excited by a white light emitting diode (LED, emission spectral range in 400−750 nm)35 and applied for optical imaging with negligible autofluorescence background interference.36−38 Both the NIR fluorescence

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ollowing the growing demands for noninvasive cancer treatment techniques, photodynamic therapy (PDT), recognized as a “magic bullet” to destroy tumors with specific spatiotemporal controllability and minimal invasiveness,1−5 has been rapidly developed and greatly improved during the past decades. In particular, nanotechnology has made it possible to shift the excitation sources of PDT from ultraviolet/visible (UV/vis) light6−10 to near-infrared (NIR) light11−14 and X-ray radiation,15−17 which significantly increases the tissue penetration depth of PDT.18,19 However, the optimized PDT output is strongly dependent on the prolonged and repeated use of NIR light or X-ray irradiation, which inevitably causes severe damage to normal tissues.20−22 It is rational to explore “clean” excitation sources for nonhazardous repeatable PDT with minimal side effects. Persistent luminescence materials, analogous to luminous pearls capable of storing excitation energy and emitting long© 2017 American Chemical Society

Received: March 2, 2017 Accepted: May 24, 2017 Published: May 24, 2017 5864

DOI: 10.1021/acsnano.7b01505 ACS Nano 2017, 11, 5864−5872

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Figure 1. Schematic illustration of the construction of ZGC PL implants for in vivo LED/PersL−PDT upon LED irradiation.

Figure 2. (a, b) TEM images of ZnGa1.996O4:Cr0.004 nanoparticles (ZGC PLNPs). (c) SEM image of ZGC PL implants. (d−h) Elemental mapping images of ZGC PL implants: (d) Zn; (e) Ga; (f) O; (g) C; (h) merged. (i−l) Observation of the liquid−solid-phase transformation process of ZGC PL implants. Digital photos of injectable ZGC PL implants (i) before, (j) during, and (k, l) after water contact.

ZGC PL implants by dissolving ZGC PLNPs in the PLGA/ NMP oleosol (Figure 1). The intratumorally injected liquid oleosol can quickly turn into solid ZGC PL implants, thus favoring prolonged retention in the tumor region. Due to the decreased surface defects, the solid ZGC PL implants exhibit enhanced afterglow intensity and extended PersL lifetime. This study presents a fresh concept of PersL−PDT by using the NIR afterglow to trigger a tumor-sensitive PS, 2-(1-hexyloxyethyl)-2devinyl pyropheophorbide-α (HPPH, Photochlor),42,43 for ROS generation. More significantly, the firmly fixed ZGC PL implants may represent an excellent built-in excitation source for repeatable LED plus NIR PersL-excited PDT upon periodic environmentally friendly LED irradiation.

emission upon LED irradiation and NIR PersL emission after LED irradiation of ZGC PLNPs can be used to activate the adjacent photosensitizers (PSs) for reactive oxygen species (ROS) generation, which gives rise to LED−PDT and PersL− PDT for enhanced PDT efficacy. However, to achieve longtime repeatable LED/PersL−PDT upon periodical LED irradiation, a lingering problem remains in maintaining sufficiently high concentration of PLNPs within the tumor, which again underscores the need for tailor-designed built-in PL implants allowing for firmly fixing PLNPs within tumors. Inspired by the fascinating liquid−solid-phase transformation behavior of poly(lactic-co-glycolic acid) (PLGA)/N-methylpyrrolidone (NMP) oleosol,39−41 herein we designed injectable 5865

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Figure 3. (a−c) Comparison of the PersL performance of ZGC PLNPs after LED excitation for different periods of time: (a) PersL spectra, (b) PersL images, and (c) PersL decay curves of ZGC PLNPs after 2 and 5 min LED excitation, respectively. (d−f) Comparison of the PersL performance of ZGC PLNPs at different mass concentrations (3 and 6 mg/mL): (d) PersL intensities, (e) PersL images, and (f) PersL decay curves of 3 and 6 mg/mL ZGC PLNPs after 2 min LED excitation, respectively. (g−i) Comparison of the PersL performance of ZGC PLNPs and ZGC PL implants: (g) PersL intensities, (h) PersL images, and (i) PersL decay curves of ZGC PLNPs and ZGC PL implants. Acquisition time: 0.5 min. **P < 0.01.

RESULTS AND DISCUSSION ZGC PLNPs were synthesized by a facile one-pot hydrothermal method without sintering.29 In order to obtain sub-20 nm ZGC nanoparticles (Figure 2a,b), the molar ratio of Zn(NO3)2/ Ga(NO3)2 precursors should be maintained at 1:1 rather than the stoichiometric ratio of 1:2 because the latter usually yields big particles with size over 30 nm (Figure S1). ZGC PLNPs are composed of Zn, Ga, and O elements (Figure S2) and display typical cubic crystals (Figure S3). Similar results were also confirmed by the elemental linear scanning images (Figure S4). The obtained ZGC PLNPs have a narrow hydrodynamic size distribution centered at 35.2 nm (Figure S5), indicating their good dispersity in water. As shown in Figure 2c, the solid state of ZGC PL implants was clearly presented. As seen from the corresponding elemental mapping images in Figure 2d−h, the Zn and Ga elements of ZGC PLNPs fill in the C−O framework of PLGA, which indicates the uniform distribution of ZGC PLNPs within the solid implants. For the construction of ZGC PL implants, the dried ZGC PLNP powder and PLGA were dissolved in NMP and stirred for 24 h to form a homogeneous ZGC

PLNPs/PLGA/NMP oleosol with high syringeability (Figure 2i). Upon water contact, NMP is quickly diffused to form an insoluble hydrophobic ZGC PLNPs/PLGA precipitate (Figure 2j,k) according to a fast solvent-removal precipitation mechanism, which yields solid ZGC PL implants (Figure 2l) via a quick liquid−solid-phase transformation process. Besides water, the liquid ZGC PLNPs/PLGA/NMP oleosol can also quickly turn into solid ZGC PL implants in a physiological environment (e.g., PBS (pH = 7.4), DMEM media (with 10% FBS) media, etc.), as shown in Figure S6. It is well-known that the optical excitation energy can be trapped at the specific defects of ZGC PLNPs during excitation and slowly released by afterglow in the case of photostimulated phosphors after excitation.36 Herein, a commercial white LED was used to excite ZGC PLNPs, and the PersL signals were recorded on an IVIS imaging system. As shown in Figure S7, by comparison with PBS as a control, ZGC PLNPs indeed emitted PersL after LED excitation was ceased. The corresponding PersL spectrum shows that the afterglow lies in the NIR region centered at 695 nm (Figure S8), which demonstrates the ability of ZGC PLNPs to emit NIR PersL after LED excitation. The 5866

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Figure 4. (a) Vis absorption and fluorescence emission (695 nm light excitation) spectra of HPPH and vis absorption and NIR PersL emission (after 2 min LED excitation) spectra of ZGC PLNPs. (b) Fluorescence emission spectra of SOSG for HPPH, HPPH (with 2 min LED irradiation), HPPH plus ZGC PLNPs (without LED excitation), and HPPH plus ZGC PLNPs (after 2 min LED excitation).

Figure 5. (a) Flow cytometry for intracellular ROS generation in U87MG cells treated with HPPH, HPPH + ZGC PLNPs (without LED excitation), and HPPH + ZGC PLNPs (after 2 min LED excitation), respectively. (b) Corresponding quantitative analysis of intracellular ROS generation by measuring the mean green fluorescence intensity of DCF. (c) Viabilities of U87MG cells treated with HPPH plus different concentrations of ZGC PLNPs (after 2 min LED excitation). (d) Viabilities of U87MG cells treated with HPPH (1 μg/mL) and HPPH (1 μg/ mL) plus ZGC PLNPs (50 μg/mL) upon one, two, and three cycles of 2 min LED irradiation, respectively. (e−g) Fluorescence images of calcein AM (green fluorescence for live cells) and PI (red fluorescence for dead cells) costained U87MG cells treated with 1 μg/mL HPPH plus 50 μg/mL ZGC PLNPs upon (e) one, (f) two, and (g) three cycles of 2 min LED irradiation, respectively. **P < 0.01, ***P < 0.001.

the PersL emission of persistent luminescence materials (Figure S10).44 Very interestingly, a 3-fold stronger PersL emission is observed for ZGC PL implants due to the largely suppressed nonradiative transitions of ZGC PLNPs in NMP rather than water (Figure 3g,h). Furthermore, the PersL lifetime of ZGC PL implants is remarkably extended to over 30 min (Figure 3i). Thanks to the spectral overlap between the absorption of HPPH and the PersL emission of ZGC PLNPs (Figure 4a), it is reasonable to apply the NIR afterglow of ZGC PLNPs to activate HPPH to generate 1O2 for PersL−PDT. The generated 1 O2 in PersL−PDT was measured chemically using singlet oxygen sensor green (SOSG) as a detector. As shown in Figure 4b, HPPH or HPPH plus ZGC PLNPs (without LED

PersL intensity and decay of ZGC PLNPs were quantified by region of interest (ROI) analysis. As shown in Figure 3a,b, after either 2 or 5 min LED excitation, the PersL intensities of ZGC PLNPs were almost the same. The PersL decay times of both cases were about 10 min (Figure 3c), which suggests that ZGC PLNPs are fully “charged” by 2 min LED excitation. After each cycle of 2 min LED excitation, ZGC PLNPs exhibit the similar PersL decay curve (Figure S9), indicating their excellent PersL stability after periodic excitation. Although the elevated mass concentration leads to an increase in PersL intensity (Figure 3d,e), the PersL lifetime is still suboptimal (Figure 3f) because the nonradiative luminescence decay and quenching processes most frequently take place in the water environment to weaken 5867

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Figure 6. (a) PersL images and (b) PersL decay curves of U87MG tumor-bearing mice injected with ZGC PLNPs and ZGC PL implants after 2 min LED excitation. (c) PersL intensities of U87MG tumor-bearing mice at different time points (1, 3, 5, 7 d) of postinjection of ZGC PLNPs and ZGC PL implants after 2 min LED excitation. Acquisition time: 1.5 min. (d) Fluorescence imaging of PBS and HPPH. (e) In vivo fluorescence imaging of U87MG tumor-bearing mice at different time points (5 min, 1, 2, 4, 24, 48 h) postinjection of HPPH.

excitation) generates a negligible amount of 1O2. On the contrary, 1O2 can be effectively produced by HPPH plus ZGC PLNPs (after 2 min LED excitation), which confirms the feasibility of ZGC PLNPs-triggered PersL−PDT. The in vitro biosafety and cellular uptake of ZGC PLNPs and HPPH were evaluated by MTT assay and confocal laser scanning microscopic (CLSM) imaging, respectively. Figure S11 shows that over 95% of U87MG cells remain alive after 24 h of incubation with ZGC PLNPs. The perinuclear region of the U87MG cells was lit up by red fluorescence (Figure S12), which suggests that ZGC PLNPs can be efficiently uptaken into the cytoplasm via an endocytosis mechanism. As a typical PS, HPPH has virtually no toxicity in the dark (Figure S13) and can diffuse into the cytoplasm (Figure S14), allowing for intracellular photoactivated ROS generation. Afterward, the in vitro PersL−PDT was validated by detecting the ROS generation in U87MG cells treated with HPPH and ZGC PLNPs (after LED excitation). 2,7-Dichlorofluorescin diacetate (DCFH-DA) was used as a fluorogenic probe for intracellular ROS detection. As seen from Figure 5a,b, there is little ROS generation in U87MG cells treated with HPPH or HPPH plus ZGC PLNPs (without LED excitation). Interestingly, after being treated with HPPH plus ZGC PLNPs (after 2 min LED excitation), intracellular ROS was generated (Figure S15), which suggests that the NIR afterglow of ZGC PLNPs after LED excitation can be used for HPPH activation in vitro. Following the validation of NIR PersL-induced intracellular ROS production, the in vitro PersL−PDT effect was evaluated using both MTT and live/dead cell staining assays. As shown in Figure 5c, only a small percentage of U87MG cells were killed by only HPPH or HPPH plus ZGC PLNPs (without LED excitation). Conversely, HPPH plus ZGC PLNPs (after 2 min LED excitation) caused a considerable cell-killing effect, as also evidenced by the calcein AM/PI-stained fluorescent images in Figure S16. Moreover, the increased concentrations of ZGC PLNPs (after 2 min LED excitation) result in decreased cell viability (Figure 5c). However, the PersL−PDT effect was not significantly improved by a longer period of LED excitation, as shown by the negligible difference in U87MG cell viability

using ZGC PLNPs (after 2−6 min LED excitation) (Figure S17). By taking into consideration that ZGC PLNPs can be repeatedly “charged” by LED excitation, the repeated LED− PDT and PersL−PDT may be simultaneously achieved during and after periodic LED irradiation, respectively. As shown in Figure 5d, a limited number of U87MG cells were killed by HPPH plus three cycles of 2 min LED illustration. In contrast, the combination of HPPH and ZGC PLNPs remarkably reduced cell viability to about 20% after three cycles of 2 min LED irradiation, mainly attributed to both the NIR fluorescence (upon LED irradiation) and NIR PersL (after LED irradiation) of ZGC PLNPs to activate HPPH and continuously generate 1O2. ZGC PLNPs may play an “energy reservoir” role in repeated LED/PersL−PDT via periodic “charging” (with LED excitation) and “discharging” (after LED excitation) processes, as confirmed by increasingly more dead cells after each cycle of 2 min LED irradiation (Figure 5e−g). One main key to in vivo application of ZGC PLNPs is their tumor accumulation, which is highly dependent on the suitable surface modification as well as ligand−receptor targeting. Here, we propose a simple yet convenient method to fix ZGC PLNPs within a tumor by injecting a ZGC PLNPs/PLGA/NMP oleosol. The fast liquid−solid-phase transformation prolongs intratumoral retention of ZGC PL implants (Figure S18) as a built-in light source. To further confirm the superiority of ZGC PL implants over ZGC PLNPs in vivo, a comparative PersL imaging experiment was performed on U87MG tumor-bearing mice. As seen from Figure 6a, after 2 min LED irradiation of U87MG tumors, ZGC PL implants exhibit about 5-fold stronger PersL intensity over ZGC PLNPs. A much longer PersL lifetime was also observed for solid ZGC PL implants than liquid ZGC PLNPs (Figure 6b) thanks to the decreased surface defects and suppressed luminescence quenching of ZGC PLNPs within solid plants. Moreover, the PersL intensity of ZGC PL implants nearly remained at the same level within 1 week (Figure S19), while that of ZGC PLNPs gradually decreased (Figure S20), most likely due to the diffusion of liquid ZGC PLNPs away from tumor over time while the solid ZGC PL implants are intratumorally fixed to prevent the loss of 5868

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Figure 7. (a) Volumes of U87MG tumors subjected to different treatments. (b−e) H&E-stained tumor sections from U87MG tumor-bearing mice after treatment with (b) HPPH, (c) HPPH + ZGC PL implants, (d) HPPH + LED, and (e) HPPH + ZGC PL implants + LED, respectively. The treatment efficacy is reflected by the degree of the tumor cell apoptosis/necrosis. ***P < 0.001.

ZGC PLNPs.39,40,45 In view of the much longer intratumoral retention, stronger PersL performance, and greater PersL stability of ZGC PL implants than ZGC PLNPs (Figure 6c), ZGC PL implants show great potential for in vivo PDT treatment. For in vivo PDT, HPPH was intravenously injected into the mice via tail vein to “find” tumor by its fluorescence signals (Figure 6d,e). At ∼24 h postinjection, the tumor area had the strongest fluorescence of HPPH, which guided the intratumoral injection of ZGC PLNPs/PLGA/NMP oleosol that quickly turned into solid ZGC PL implants upon contact with the physiological environment of tumor. Considering the homogeneous distribution of HPPH within the whole tumor, both the NIR fluorescence (upon LED irradiation) and NIR PersL (after LED irradiation) of the intratumorally fixed ZGC PL implants could activate HPPH to generate 1O2 for PDT treatment of tumors. As shown in Figure 7a and Figure S21, both HPPH and the combination of HPPH and ZGC PL implants without LED irradiation resulted in little to no tumor growth delay by comparison with the control group. HPPH plus LED irradiation caused limited inhibitory effect on tumor progression. The tumors administered with HPPH and ZGC PL implants plus LED irradiation, on the other hand, shrunk remarkably within 1 week and exhibited a large scale of tissue apoptosis/necrosis (Figure 7b−e), mainly attributed to the significant ROS-mediated damage arising from the LED−PDT during LED irradiation as well as PersL−PDT after LED irradiation. In order to further inhibit tumor regression after 1 week, LED irradiation was performed again at 24 h postinjection of a second dose of HPPH at day 7, and then the tumor growth was completely suppressed in about half a month, which demonstrates the effectiveness of repeatable LED/PersL−PDT in vivo for an optimized therapeutic outcome. It is of note that the fixed ZGC PL implants can be repeatedly “charged”, and although the intravenously injected HPPH may be excreted within 48 h, the ZGC PL implants may be used for repeatable LED/PersL−PDT by only periodic HPPH injection and LED irradiation, thus leading to complete inhibition or even elimination of malignant tumors. No significant body weight variation was noticed in Figure S22, which indicated the biosafety of these treatments. The relatively high long-term biocompatibility of ZGC PL implants and HPPH in vivo was confirmed by the hematoxylin and eosin (H&E) staining results. As shown in Figure S23, HPPH and ZGC PL implants caused no noticeable abnormality to the major organs (heart, liver, spleen, lung, kidney) on day 30 after

injection, which also indicates that the administrated dosage of NMP causes negligible side effects on the mice’s normal tissues and health status.

CONCLUSION In summary, an injectable ZGC PL implant has been successfully constructed with much stronger PersL intensity and longer PersL lifetime than ZGC PLNPs. More importantly, through the fast liquid−solid phase transformation upon contacting water both in vitro and in vivo, the ZGC PL implants firmly fixed within tumors are featured with great PersL stability in vivo, enabling repeated “charging” (by LED excitation) processes. By validating the feasibility of PersL− PDT based on the NIR PersL-activated HPPH in vitro, both LED−PDT and PersL−PDT can be achieved during and after LED irradiation, respectively, which contributes greatly to the remarkably improved therapeutic effects. In addition to repeatable PersL imaging and LED/PersL−PDT, the wellestablished biocompatible ZGC PL implants (Figures S23 and S24) may represent a significant step toward breaking through the short PersL lifetime and poor tumor retention of nanoscale persistent luminescence materials, thus broadening their biomedical applications. Despite the negligible side effects on the cell proliferation and skin tissue (Figures S25 and S26), LED irradiation bears limited tissue penetration (Figures S27 and S28), so further studies will focus on the design of superior PL implants “charged” by highly penetrating excitation sources for repeatable deep tissue PersL imaging and PDT treatment. MATERIALS AND METHODS Materials. Ga(NO3)3·xH2O, Zn(NO3)2·6H2O, Cr(NO3)3·9H2O, ammonium hydroxide, Poly(D,L-lactide-co-glycolide) (PLGA, lactic acid/glycolic acid ratio of 50:50), N-methylpyrrolidone (NMP), HCl (36.5−38 wt %), and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma-Aldrich. All reagents were of analytical grade and used without any purification. Characterization. Transmission electron microscopy (TEM) images were recorded on a Tecnai TF30 transmission electron microscope (FEI, Hillsboro, OR). SEM images were obtained on a Hitachi SU-70 Schottky field emission gun scanning electron microscope (FEG-SEM). A scientific nanoparticle analyzer (SZ-100, Horiba) was used for dynamic light scattering (DLS) measurements. UV−vis spectra were recorded on a Genesys 10S UV−vis spectrophotometer (Thermo Scientific, Waltham, MA). Powder Xray diffraction pattern were measured on a Rigaku D/MAX-2250 V diffractometer with graphite-monochromatized Cu Kα radiation. Persistent luminescence imaging experiments were performed on an 5869

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μg/mL) into each well. After 6 h of co-incubation, 50 μg/mL ZGC PLNPs were added into the wells. After one, two, and three cycles of 2 min LED irradiation and subsequent co-incubation for 24 h, the old MEM media were discarded followed by addition of 5 mg/mL MTT (in 100 μL MEM media) into each well. After co-incubation for another 4 h, the old MEM media were replaced by 100 μL of DMSO per well, and the absorbance was monitored by a microplate reader at the wavelength of 570 nm. The cytotoxicity was finally expressed as the viability percentage of the treated cells in contrast to the untreated control cells. Measurement and Observation of Intracellular Reactive Oxygen Species Arising from PersL−PDT. 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) was used as a fluorogenic probe for detecting intracellular ROS. DCFH-DA could be deacetylated by nonspecific esterase to form DCFH, which was further oxidized by ROS to yield a stable fluorescent ROS-sensitive compound, 2,7dichlorofluorescein (DCF). In this study, U87MG cells were seeded in a 6-well plate at a density of 105/well and then cultured at 37 °C for 24 h. The cells were incubated with 30 μM DCFH-DA for 20 min, and then 1 μg/mL HPPH and/or 100 μg/mL ZGC PLNPs (after 2 min LED excitation) were added into the wells. During 30 min, the cells were harvested, washed with serum-free MEM media three times, and resuspended in the fresh serum-free MEM media. Finally, the fluorescent density of 10000 events for each sample was detected by flow cytometry (Backman USA). ROS (treated cells) = I (treated cells)/I (control cells) × 100, where I refers to the mean green fluorescent intensity measured by flow cytometry. In addition, fluorescent images of intracellular ROS were captured by excitation at 488 nm on an Olympus FV1000 fluorescent microscope. In Vitro Observation of Live/Dead Cells. U87MG cells were seeded in several 96-well plates at a density of 104 cells per well and then cultured at 37 °C for 24 h. After different treatments, the cells were costained by calcein AM and PI and then imaged using an Olympus FV1000 fluorescent microscope. In Vivo Toxicity Assessment of HPPH and ZGC PL Implants. Healthy female Balb/c nude mice (∼20 g) were obtained from Harlan Laboratories (Frederick, MD) and raised under a National Institutes of Health Animal Care and Use Committee (NIHACUC) approved protocol. Some mice were intravenously injected with HPPH (1 mg/ mL, 150 μL), and others were intratumorally injected with ZGC PL implants (10 mg/mL, 20 μL). The rest of the untreated mice were used as the control. The mice were anesthetized and dissected 30 days postinjection. The major organs (heart, liver, spleen, lung and kidney) were dissected, fixed, and H&E stained for histological analysis. In Vivo PersL Imaging Observation of ZGC PLNPs and ZGC PL Implants. U87MG tumor model was established by subcutaneously injecting U87MG cells (2 × 106 cell/site) in female balb/c nude mice. PersL imaging experiments were performed on the IVIS imaging instrument. ZGC PLNPs or ZGC PL implants (10 mg/mL) were injected into the U87MG tumors. After 2 min, LED irradiation (excitation spectral range in 400−750 nm), the PersL (emission spectral range in 650−750 nm) signals were acquired at an interval of 1.5 min. Then the corresponding in vivo PersL decay curves were obtained by depicting the change curve of the PersL photon counts as a function of time. This similar experiment was repeated at days 3, 5, and 7 to compare the in vivo PersL imaging performance between ZGC PLNPs and ZGC PL implants. In Vivo PDT Evaluation of ZGC PL Implants. U87MG cells (2 × 106 cell/site) were implanted subcutaneously in female balb/c nude mice (∼20 g). In vivo PDT experiment was performed when the tumor reached 6−8 mm in average diameter (10 days after implant). The mice were divided into five groups. The first group of mice received PBS, as control group; the second group was intravenously injected with HPPH (1 mg/mL, 150 μL), as HPPH group; the third group was intravenously injected with HPPH (1 mg/mL, 150 μL) and then intratumorally injected with ZGC PL implants (10 mg/mL, 20 μL), as HPPH + ZGC PL implants group; the forth group was intravenously injected with HPPH (1 mg/mL, 150 μL) and subjected to 15 min LED irradiation at 1 day and 8 day, respectively, as HPPH + LED group; the fifth group was intravenously injected with HPPH (1 mg/

IVIS imaging system. An Utilitech 1000-lm LED hand-held battery flashlight (FT-LOW1415, emission spectral range in 400−750 nm) was used to charge ZGC PLNPs and ZGC PL implants. Statistical analysis was performed by using the student’s two-tailed t test. Synthesis of ZGC PLNPs. Cubic-phase ZnGa1.996O4:Cr0.004 persistent luminescence nanoparticles (ZGC PLNPs) were synthesized via a hydrothermal method. Zn(NO3)2·6H2O (594.96 mg, 2 mmol), Ga(NO3)3·xH2O (511.48 mg, 2 mmol), and Cr(NO3)3·9H2O (1.6 mg, 0.004 mmol) in 15 mL deionized water were added to a 50 mL flask. One milliliter of ammonium hydroxide (28%) solution was quickly added into the flask. After 30 min of stirring, the mixture was transferred to a Teflon-lined autoclave (25 mL). After 10 h of reaction at 220 °C, the system was cooled to room temperature. The products were obtained after centrifugation and dispersed in 0.01 mol/L HCl to remove the ZnO impurities. The resulting ZGC PLNPs were washed with 2-propanol several times and freeze-dried for further use. Synthesis of ZGC PL Implants. PLGA (2.5 mg) was dissolved in 1 mL of NMP under ultrasonication. The PLGA/NMP oleosol was magnetically stirred for 12 h, followed by addition of 10 mg ZGC PLNPs. After the mixture was stirred for another 12 h, such a homogeneous ZGC PLNPs/PLGA/NMP oleosol was obtained as the injectable ZGC PL implants. In Vitro PersL Imaging Observation of ZGC PLNPs and ZGC PL Implants. ZGC PLNPs (in water solution) or ZGC PL implants (ZGC PLNPs in PLGA/NMP oleosol) were put in an IVIS imaging system. After 2 or 5 min LED excitation (excitation spectral range in 400−750 nm), the PersL (emission spectral range in 650−750 nm) signals of ZGC PLNPs or ZGC PL implants were acquired at intervals of 0.5 min. Then the PersL decay curves were obtained by depicting the change curve of the PersL photon counts as a function of time. In Vitro Toxicity Assessment of ZGC PLNPs and HPPH against U87MG Cells. U87MG cells were seeded in two 96-well plates at 104/well and then cultured at 37 °C for 24 h. Different concentrations of ZGC PLNPs (7.5, 15.5, 31.5, 62.5, 125, 250, and 500 μg/mL) and HPPH (0.75, 1.55, 3.15, and 6.25 μg/mL) in the MEM media were added into the wells for 24 h of co-incubation. Then the old MEM media were discarded, followed by addition of 5 mg/mL MTT (in 100 μL MEM media) into each well. After co-incubation for another 4 h, the old MEM media were replaced by 100 μL DMSO per well, and the absorbance was monitored by a microplate reader at the wavelength of 570 nm. The cytotoxicity was finally expressed as the viability percentage of ZGC PLNPs or HPPH-treated cells in contrast to the untreated control cells. In Vitro Observation of Cellular uptake of ZGC PLNPs and HPPH by Confocal Laser Scanning Microscopic Imaging. U87MG cells were seeded in an eight-well CLSM-special plate at 104/well and then cultured at 37 °C for 24 h. MEM solutions (200 μL) of ZGC PLNPs (100 μg/mL) or HPPH (1 μg/mL) were added into the wells. After 6 h of co-incubation, the cells were washed with PBS three times to remove free ZGC PLNPs or HPPH followed by nuclei staining using DAPI. Confocal luminescent imaging experiments were carried out on an Olympus FV1000 laser-scanning microscope using a 60 × oil immersion objective lens. In Vitro PersL−PDT Evaluation of ZGC PLNPs. U87MG cells were seeded in several 96-well plates at a density of 104 cells per well and then cultured at 37 °C for 24 h, followed by addition of HPPH (1 μg/mL) into each well. After 6 h of co-incubation, different concentrations (25, 50 100 μg/mL) of ZGC PLNPs (after 2 min LED excitation) or 100 μg/mL ZGC PLNPs (after 2/4/6 min of LED excitation) were added into the wells. After 24 h of co-incubation, the old MEM media were discarded, followed by addition of 5 mg/mL MTT (in 100 μL MEM media) into each well. After co-incubation for another 4 h, the old MEM media were replaced by 100 μL DMSO per well, and the absorbance was monitored by a microplate reader at the wavelength of 570 nm. The cytotoxicity was finally expressed as the viability percentage of the treated cells in contrast to the untreated control cells. In Vitro Repeatable PDT Evaluation of ZGC PLNPs. U87MG cells were seeded in a 96-well plate at a density of 104 cells per well and then cultured at 37 °C for 24 h, followed by addition of HPPH (1 5870

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ACS Nano mL, 150 μL) and intratumorally injected with ZGC PL implants (10 mg/mL, 20 μL), and then subjected to 15 min LED irradiation at 1 day and 8 day, respectively, as HPPH + ZGC PL implants + LED group. During half a month after the corresponding treatments, the volume of tumors was measured every other day and calculated by the following equation: V = L × W2/2. Finally, the tumors were sectioned into slices and H&E stained for histological analysis.

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ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b01505. TEM image, XRD pattern, EDS spectrum, DLS, in vitro PersL imaging and PDT evaluation of ZGC PLNPs, in vivo PersL imaging and PDT evaluation of ZGC PL implants, and other related experimental data (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Zhen Yang: 0000-0003-4056-0347 Peng Huang: 0000-0003-3651-7813 Xiaoyuan Chen: 0000-0002-9622-0870 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This project is financially supported by the startup fund from Shenzhen University, the National Science Foundation of China (51602203, 81401465, 51573096), the Postdoctoral Science Foundation of China (2016M590808), and the Intramural Research Program (IRP) of the NIBIB, NIH. REFERENCES (1) Chatterjee, D. K.; Fong, L. S.; Zhang, Y. Nanoparticles in Photodynamic Therapy: an Emerging Paradigm. Adv. Drug Delivery Rev. 2008, 60, 1627−1637. (2) Nishiyama, N.; Morimoto, Y.; Jang, W.-D.; Kataoka, K. Design and Development of Dendrimer Photosensitizer-Incorporated Polymeric Micelles for Enhanced Photodynamic Therapy. Adv. Drug Delivery Rev. 2009, 61, 327−338. (3) Huang, P.; Lin, J.; Wang, X.; Wang, Z.; Zhang, C.; He, M.; Wang, K.; Chen, F.; Li, Z.; Shen, G.; Cui, D.; Chen, X. Light-Triggered Theranostics Based on Photosensitizer-Conjugated Carbon Dots for Simultaneous Enhanced-Fluorescence Imaging and Photodynamic Therapy. Adv. Mater. 2012, 24, 5104−5110. (4) Zhen, Z.; Tang, W.; Chuang, Y.-J.; Todd, T.; Zhang, W.; Lin, X.; Niu, G.; Liu, G.; Wang, L.; Pan, Z.; Chen, X.; Xie, J. Tumor Vasculature Targeted Photodynamic Therapy for Enhanced Delivery of Nanoparticles. ACS Nano 2014, 8, 6004−6013. (5) Liu, K.; Xing, R.; Zou, Q.; Ma, G.; Möhwald, H.; Yan, X. Simple Peptide-Tuned Self-Assembly of Photosensitizers towards Anticancer Photodynamic Therapy. Angew. Chem., Int. Ed. 2016, 55, 3036−3039. (6) Lovell, J. F.; Liu, T. W. B.; Chen, J.; Zheng, G. Activatable Photosensitizers for Imaging and Therapy. Chem. Rev. 2010, 110, 2839−2857. (7) Yaghini, E.; Seifalian, A. M.; MacRobert, A. J. Quantum Qots and Their Potential Biomedical Applications in Photosensitization for Photodynamic Therapy. Nanomedicine 2009, 4, 353−363. (8) O’Connor, A. E.; Gallagher, W. M.; Byrne, A. T. Porphyrin and Nonporphyrin Photosensitizers in Oncology: Preclinical and Clinical 5871

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DOI: 10.1021/acsnano.7b01505 ACS Nano 2017, 11, 5864−5872