Large Hollow Cavity Luminous Nanoparticles with Near-Infrared

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Large Hollow Cavity Luminous Nanoparticles with NearInfrared Persistent Luminescence and Tunable Sizes for Tumor Afterglow Imaging and Chemo/Photodynamic Therapies Jun Wang, Jinlei Li, Jiani Yu, Hongwu Zhang, and Bingbo Zhang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b07606 • Publication Date (Web): 20 Apr 2018 Downloaded from http://pubs.acs.org on April 20, 2018

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Large Hollow Cavity Luminous Nanoparticles with Near-Infrared Persistent Luminescence and Tunable Sizes for Tumor Afterglow Imaging and Chemo/Photodynamic Therapies Jun Wang,† Jinlei Li,‡ Jiani Yu,† Hongwu Zhang,‡ and Bingbo Zhang*† †

Institute of Photomedicine, Shanghai Skin Disease Hospital; The Institute for Biomedical

Engineering & Nano Science, Tongji University School of Medicine, Shanghai 200443, China. ‡

Key Lab of Urban Pollutant Conversion, Institute of Urban Environment, Chinese Academy of

Sciences, Xiamen, Fujian 361021, China.

Corresponding Author Bingbo Zhang, Email: [email protected].

Tel: +86-21- 65988029; Fax: +86 21

65987071

ABSTRACT Persistent luminous nanoparticles (PLNPs) have been capturing increasing attention in biomedical imaging because of their long-life emission and concomitant benefits (e.g., zero-autofluorescence background, high signal-to-noise ratio). Although there are quite some synthetic methodologies to synthesize PLNPs, those for constructing functional structured PLNPs remain largely unexplored. Herein we report the design principle, synthesis route, and proof-of-concept applications of hollow structured PLNPs with near-infrared (NIR) persistent luminescence, namely afterglow, and tunable sizes for tumor afterglow imaging and chemical/photodynamic therapies. The design

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principle leverages on the crystallization of the immobilized parent ions on the purgeable carbon spheres. This strategy provides large and size-tunable hollow cavities to PLNPs after calcination. Building on the hollow cavity of PLNPs, high chemical drug (DOX) or photosensitizer (Si-Pc) loading can be achieved. The DOX/Si-Pc-loaded hollow PLNPs exhibit efficient tumor suppression based on the features of large cavity and afterglow of PLNPs. These hollow structured PLNPs, like traditional solid PLNPs, are quite stable and can be repeatedly activated, and particularly can selectively target tumor lesion, permitting rechargeable afterglow imaging in living mice. Our research supplies a strategy to synthesize hollow structured PLNPs, and hopefully it could inspire other innovative structures for cancer theranostics. KEYWORDS: carbon spheres, hollow structure, drug loading, afterglow imaging, tumor therapy


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Persistent luminous nanoparticles (PLNPs), a kind of material that can be luminescent from a few minutes to a few days after the cessation of the excitation light, are superior to conventional luminous materials in elimination of autofluorescence interference for biomedical imaging.1 Actually the use of bulk persistent luminous materials, namely the Night Pearl, can be traced back to almost a millennium ago. The study of nano-sized PLNPs, however, just started in the last decade, with their focuses on biomedicine, such as in vitro biosensing,2-6 in vivo bioimaging,7-11 and imaging-guided therapy.12-17 The first case of PLNPs in biomedical imaging was reported by Scherman group, who synthesized nano-sized NIR PLNPs by sol-gel method and used them for in vivo afterglow imaging over 1 h.18 Since then, various types of PLNPs have been synthesized.19,20 It is clear from the literature that most of persistent luminous materials are fabricated by solid-state annealing reaction method at high temperature, even with good luminance but resulting in agglomerations. Despite they can be grinded into nanoparticles, their sizes and morphologies are hard to be controlled. Hydrothermal method is reported competent in fabricating small sized and monodisperse PLNPs, but the their luminance are usually dissatisfactory because of fabrication at low reaction temperatures.6,21,22 High temperature environment (e.g., solid-state annealing reaction) is of great importance for crystallization and luminance enhancement of PLNPs. Recently, the concern of agglomeration in solid-state annealing reaction is tactfully solved by putting the reaction on the surfaces or the pores of porous silicon nanoparticles.20,23-27 Silica-templated strategy can perform over 600 °C, favouring PLNPs crystallization and improving luminance of PLNPs. Also, the templated structure of silica spheres can regulate the size and morphology of the resultant PLNPs.


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However, the resultant PLNPs lack of multifunctionality, since the pore channels of the silica template are often occupied by the tiny PLNPs which blocks other functional loadings. In this study, we propose a synthetic strategy to synthesize large hollow cavity PLNPs with near-infrared (NIR) persistent luminescence and tunable sizes for tumor afterglow imaging and therapy. By using of purgeable carbon spheres as the templates, metal ions are first immobilized on the surface of the carbon spheres and then in situ crystalized into nanoparticles at high temperature. Upon calcination, a large hollow cavity can be formed as the templated carbon spheres are burned away. This method is found highly efficient and reliable. The obtained hollow PLNPs not only possess good morphology and cavity size tunability, but also show potent afterglow. By leveraging on these features, the hollow PLNPs were used to largely load a chemical drug (DOX) and a photosensitizer (Si-Pc) for proof-of-concept applications in tumor imaging and therapies. To the best of our knowledge, hollow structured PLNPs have never been reported. The specific synthesizing procedure and applications of hollow PLNPs is outlined in Scheme 1.


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Scheme 1. Schematic illustrations of the synthesis, functionalization and applications of the hollow NIR PLNPs. The large cavity of hollow PLNPs can accommodate a large amount of DOX for chemotherapy. In addition, afterglow-assisted imaging and PDT are also demonstrated by leveraging on the potent persistence luminescence of hollow PLNPs on tumor-bearing mice.


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Figure 1. TEM images of the original carbon spheres with a size of ca.150 nm (A); carbon spheres labelled by precursor ions (B); the NIR hollow PLNPs (C); the BSA-modified hollow PLNPs (D); Hydrodynamic diameters of carbon spheres (E), precursor ions labelled carbon spheres (F), NIR hollow PLNPs (G) and the BSA-modified hollow PLNPs (H); SEM images of the resultant hollow PLNPs (I); High resolution TEM images (J); SAED pattern (K); Element mapping images (L-Q); and EDS (R, S) of the resultant hollow PLNPs.

RESULTS AND DISCUSSION


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Synthesis and Characterizations of the Hollow NIR PLNPs. The hollow NIR PLNPs were fabricated using a controllable template method. First, highly dispersed and uniform carbon spheres with a mean diameter of 150 nm were synthesized via a hydrothermal method as the consumable template (Figure 1A, 1E). Leveraging on the chelate effect of metal ions with reactive oxygen functional groups, precursor ions can be easily absorbed onto the surface of carbon spheres and further form a shell of M(OH)CO3 (M = Zn, Ga, Cr) upon the OH- and CO32ions are released from urea under a high temperature condition.28 Therefore a rough surface of carbon spheres is observed and the size is slightly increased (Figure 1B, 1F). Importantly, the shell of M(OH)CO3 can gradually decompose and crystalize into PLNPs during calcination at 800 0

C. In this transfer process, the original carbon sphere templates are burned away, and the hollow

structured PLNPs is accordingly obtained. Transmission electron microscopy (TEM) and scanning electron microscope (SEM) images in Figure 1C, 1D and 1I show that the resultant PLNPs are hollow with a large cavity, and interestingly the size is magically shrunk to ca. 50 nm from the original 150 nm. The decrease in size is mainly due to the dehydration and contraction effects of the organic template during the calcination.29 After BSA modification, the hydrodynamic diameter has a slight increase (Figure 1G, 1H). We also used three dimensional (3D) TEM technique to analyse the topological structure of PLNPs and the reconstruction image and movie clearly show an inner hollow structure of the prepared PLNPs (Figure S1 and Movie S1). High resolution TEM further shows high crystallinity of the as-synthesized hollow PLNPs and the distance between the two adjacent lattice fringes was calculated to be 0.15 nm, which corresponds to the spacing for the (440) lattice planes (Figure 1J). The selected area electron


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diffraction (SADE) also reveals good crystallinity of the hollow PLNPs (Figure 1K). Results from the X-ray diffraction (XRD) pattern support the cubic-phase structure of the obtained hollow PLNPs (Figure S2). We further used high-angle annular dark-field scanning TEM (HAADF-STEM) imaging combined with energy dispersive spectroscopy (EDS) technique to confirm the presence of Zn, Ga, O and Cr elements in the hollow structured PLNPs (Figure1L-1S). Further, N2 adsorption-desorption analysis was conducted to study the specific surface area and pore structure of the hollow PLNPs. The specific surface area of the prepared PLNPs was measured to be 23.01 m2/g (Figure S3A). The existence of hysteresis in the adsorption-desorption curve reveals the presence of mesopores in the shell. The pore size is about 10.67 nm calculated using the Barrett-Joyner-Halenda (BJH) distribution (Figure S3B). The mesopores, most likely resulted from the CO2 gas releasing during the calcination of carbon spheres at high temperature, should locate on the shell layer of PLNPs. These mesopores and large cavity obviously favour drug loading and release. In order to obtain high quality PLNPs, some key parameters of synthesis were studied. Initially, we investigated the effect of different amounts of precursors but keeping an identical molar ratio of Zn:Ga:Cr at 1:2:0.01 on the formation of shell structure by using of 150 nm carbon spheres as the template. TEM shows that hollow structure shell is gradually formed along with the increase of the amounts of Zn2+, Ga3+, and Cr3+ precursor ions, and it achieves the best morphology in the case of 8 X initial amount (Figure S4 and Table S1). Besides, we investigated the effects of the doped Cr3+ content (Figure S5 and Table S2), calcination temperature (Figure S6 and Table S3) and calcination time (Figure S7 and Table S4) on the luminescent performance


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and the morphology of the resultant PLNPs. The results indicate that the optimal doped Cr3+ content is determined at 0.024 mmol and the final optimized calcination time and temperature are 2 h and 800 0C, respectively. The doping content of Cr3+ hardly changes the morphology of PLNPs, however, higher calcination temperature can destroy the shell structure and decrease the luminescent intensity. Longer calcination time also can destroy the shell structure, but it has no significant influence on the luminescent intensity.

Figure 2. Synthesizing hollow PLNPs on 300 nm-sized carbon spheres. TEM images of the carbon spheres with a size of 300 nm (A), precursor ions labelled carbon spheres (B), the hollow NIR PLNPs (C); SEM image of the hollow NIR PLNPs (D); High resolution TEM image of the hollow NIR PLNPs (E); SAED pattern of the hollow NIR PLNPs (F); Element mapping (G-M) and EDS of the hollow NIR PLNPs (N).


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Figure 3. Synthesizing hollow PLNPs on 500 nm-sized carbon spheres. TEM images of the carbon spheres with a size of 500 nm (A), precursor ions labelled carbon spheres (B), the hollow NIR PLNPs (C); SEM image of the hollow NIR PLNPs (D); High resolution TEM image of the hollow NIR PLNPs (E); SAED pattern of the hollow NIR PLNPs (F); Element mapping (G-M) and EDS of the hollow NIR PLNPs (N).

To demonstrate the universal applicability of the proposed carbon spheres-templated approach in fabricating hollow structured PLNPs, another two sizes (300 nm and 500 nm) of carbon spheres were used as the purgeable template, and we found both can serve well in fabrication of hollow structured PLNPs (Figure 2, Figure 3, Figure S2 and Table S5). Likewise, the products synthesized on the 300 nm and 500 nm of carbon spheres are also shrunk to 100 nm and 250 nm, respectively. Surface Modification of Hollow NIR PLNPs. Surface engineering was done to improve water-dispersion and biocompatibility of PLNPs before biomedical applications. Bovine serum albumin (BSA), a highly hydrophilic, stable and cheap protein, was used to modify PLNPs. Our


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previous work have demonstrated that BSA protein can effectively transfer hydrophobic nanoparticles (e.g. quantum dots,30 magnetic nanoparticles,31,32 upconversion nanoparticles33) to their hydrophilic counterparts, ensuring enhanced stability in aqueous phase. FT-IR spectra in Figure S8A reveal BSA successfully encapsulates on the surface of PLNPs. The characteristic peaks of BSA at 1655 cm-1 and 1537 cm-1 which are ascribed to C=O stretching vibration and N-H bending vibration at 2950 cm-1 and 2924 cm-1 which are assigned to the vibration of -CH3 are also present on the spectrum of PLNPs, indicating the immobilization of BSA on the surface of PLNPs. Consistently, the results of zeta potential measurements also prove BSA successfully absorbed onto the surface of PLNPs. The initial potential of PLNPs was +4.2 mV and changed to -29.7 mV after treating with NaOH solution due to the introduction of hydroxyl groups. After amination, the zeta potential was shifted to +36.5 mV. However, it became negatively charged again with a zeta potential of -24.2 mV upon BSA anchoring (Figure S8B). Furthermore, the normalized fluorescence emission spectra and their size distribution were monitored after each surface modification process. It shows that the maximum fluorescence emission peak nearly remains while the size of BSA-modified PLNPs slightly increases due to the macromolecular BSA capping (Figure S8C and S8D).


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Figure 4. Excitation and emission spectra of the hollow NIR PLNPs (A); NIR afterglow decay curve of the hollow NIR PLNPs powder after 3 min of excitation at 254 nm, and inset image refers to the powder of as-prepared PLNPs (B); NIR afterglow decay curve of the BSA-modified hollow NIR PLNPs powder after repeated excitation at 650 nm (C); NIR afterglow decay curve of the hollow NIR PLNPs aqueous solution (1 mg/mL) after 5 min of excitation at 254 nm (D); The NIR afterglow images at 5 min, 10 min, 15 min, 30 min, 1 h and 1.5 h of the hollow NIR PLNPs powder after 10 min irradiation by a 254 nm UV lamp, and two afterglow decay images at 5 min and 10 min were obtained after 5 min of re-activation by a white LED lamp (1100 lm) (E); The NIR afterglow images at 5 min, 10 min, 15 min, 30 min, 45 min and 1 h of the BSA-modified hollow NIR PLNPs aqueous solution (2 mg/mL) after 10 min irradiation by a 254 nm UV lamp, and two afterglow decay images at 1 min and 5 min were obtained after 5 min of re-activation by a white LED lamp (1100 lm) (F).


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The size of the hollow NIR PLNPs in this study was 50 nm.

Persistent Luminescence Properties of Hollow NIR PLNPs. The excitation and photoluminescence spectra of the hollow PLNPs are given in Figure 4A, showing an intensive NIR emission at 696 nm under 254 nm excitation due to 2E→4A2 transition of Cr3+ ions in the zinc gallate host.34 It can be found there are three main excitation bands from the excitation spectrum, namely 256 nm, 467 nm and 590 nm, which are ascribed to CrO+→O2-, 4A2→4T1 and 4

A2→4T2, respectively.1,24,35,36 The emission spectrum of the hollow NIR PLNPs in aqueous

solution (1 mg/mL) was measured under irradiation of 254 nm, as shown in Figure S9. The emission spectral profile is almost symmetric, featuring the major emission peak at ca. 696 nm with a tail more extending to the near-infrared region. Figure 4B and 4D show the NIR afterglow decay curves of the prepared hollow PLNPs powder and its aqueous solution monitored at 696 nm for 30 min after 3 min and 5 min irradiation with a 254 nm UV lamp, respectively. Both have a very similar decay pattern showing of a fast decay in the beginning and followed by a mitigation, and particularly their afterglow can last over 30 min at least. More importantly, the prepared hollow NIR PLNPs can be repeatedly activated by a 650 nm NIR light with no obvious loss of intensity (Figure 4C), showing the good optical stability and signal consistency of hollow NIR PLNPs. This rechargeable luminescence, particularly renewable by a red lamp, should be of great significance for longitudinal detection and imaging. For a more intuitive experience, the afterglow images of the power and the water solution samples of hollow NIR PLNPs were photographed by a CCD camera at different time points after ceasing the excitation lamp. Both the powder and aqueous products can be luminescent over one hour. And particularly the extinguishing PLNPs can be re-activated by a white LED lamp irradiation and


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continue to emit (Figure 4E and 4F). This luminescent feature favours its utility in in vivo imaging, particularly for those cases that long-term or multiple imaging is needed. The emission spectra of the other two sizes (namely, 100 nm and 250 nm) of BSA-modified hollow NIR PLNPs were also measured. They both show a NIR luminescence emission at 696 nm under the 254-nm excitation (Figure S10 and S11). The fluorescence quantum yields of 50 nm, 100 nm and 250 nm hollow PLNPs were measured to be 4.35 %, 4.60 % and 5.55 %. Moreover, their afterglow images in powder and aqueous solution state at varying time points are provided in Figure S12, showing quite similarity with that of 50 nm-sized product. The hollow NIR PLNPs Stability Study. For in vivo application, the stability of nanomaterials is required due to the challenges of complicated biological environment, such as pH, and ionic strength.37-40 Different pH PBS solution and different ionic strength NaCl solution against the fluorescence intensity and the persistent luminescence intensity of the hollow PLNPs were investigated, respectively. The results show that different pH PBS solution and ionic strength NaCl solution have no obvious influence on the fluorescence intensity and the persistent luminescence intensity of the hollow PLNPs (Figure S13). Meanwhile, the emission stability of 50 nm-sized BSA-modified hollow NIR PLNPs was also investigated and found to be quite stable both in deionized (DI) water and phosphate-buffered saline (PBS) (pH 7.4, 10 mM) for over 5 days (Figure S14), which is attributed to the superior anti-hydrolysis capability of zinc gallate composed PLNPs over the aluminate PLNPs.1 The good stability of the hollow NIR PLNPs favors in vivo application.


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Figure 5. Cumulative drug release profiles of the DOX-hollow NIR PLNPs in PBS with two pH values of 7.4 and 5.0 (A); Cell viabilities of free DOX and DOX-hollow NIR PLNPs against 4T1 cells at different DOX concentrations for 24 h (B); Confocal images of 4T1 cells incubated with DOX-hollow PLNPs for 4 h at 37 0C (DOX concentration = 2 µg/mL). The nuclei was stained by DAPI. The green and red fluorescence signal was from the hollow PLNPs and DOX, respectively. Scale bar is 25 µm. Notes: Due to the coincidence of the fluorescence color of DOX and hollow PLNPs, the fluorescence signal of the hollow PLNPs nanoparticles is labelled with green color for a better distinction (C); Bio-TEM images of the 4T1 cells incubated with


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DOX-hollow PLNPs. The red arrow shows the local magnification image of the red frame nanoparticles (D). The hollow NIR PLNPs with a size of 50 nm was used in this study.

In Vitro Drug Loading and Release Study of Hollow NIR PLNPs. Doxorubicin (DOX) was chosen as a model drug to investigate drug loading, release and its anti-tumor capability of the prepared hollow NIR PLNPs. The UV-vis absorption and fluorescence spectra collectively verify the successful incorporation of DOX into the hollow cavity of NIR PLNPs (Figure S15). The loading content of DOX in hollow PLNPs was measured to be 181 mg/g through calculating the absorbance of the supernatant according to the DOX standard curve (Figure S16). The high drug loading content may be attributed to the large inner cavity and mesoporous holes of the hollow structured PLNPs. Cumulative drug release study discloses a pH-dependent release behaviour of the DOX-loaded hollow PLNPs (Figure 5A), showing after 48 h, 16.1 % of DOX was released in the pH 7.4 of PBS solution, while this amount is speeded up to 29.1 % in the pH 5.0 of PBS solution. This increased release in a lower pH solution is mainly attributed to the protonation of DOX in acidic solution which facilitates the release of DOX.41 Before cancer cell killing studies, the cytotoxicity of BSA-modified hollow NIR PLNPs without DOX was evaluated by CCK-8 assays. All sizes of the prepared PLNPs exhibit over 90 % of cell viability after incubation with 4T1 cells for 24 h, even at a high dose of 250 µg/mL, indicating good cellular biocompatibility of the prepared PLNPs in this study (Figure S17-S19). Upon loading DOX, the hollow PLNPs show good performance in tumor cell killing (Figure 5B). Along with the increase of concentration, DOX-loaded PLNPs exert increasing lethality on cancer cells as intended. It is worth noting that free DOX treatment achieved higher cell mortality rate. The slight discount in therapeutic effect of hollow PLNPs is mainly due to the size effect of nanoparticles which could slow down their endocytosis as compared to the free DOX drug. But 16 ACS Paragon Plus Environment

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the free DOX non-selectively intercalate into the structure of DNA in the cell nucleus and finally kill them regardless of normal or cancerous cells. This indiscriminate killing however can be circumvented by nano-encapsulation and particularly further binding with a targeting ligand. The cellular uptake and drug release of DOX-hollow PLNPs by 4T1 cells was further investigated by tracking the fluorescence signals of hollow PLNPs and DOX, respectively. The cellular nucleus was stained blue by DAPI. As shown in Figure 5C, after 4 h of incubation, a green fluorescence signal (pseudo color) is observed in the cytoplasm, suggesting the successful intracellular accumulation of the hollow PLNPs in tumor cells via an endocytosis mechanism. For the hollow PLNPs loading with DOX, red fluorescence signal is observed in the nucleus after 4 h incubation, which indicates that DOX could be released from DOX-hollow PLNPs into the nucleus. In addition, biological transmission electron microscopy (bio-TEM) also proves the hollow PLNPs are effectively phagocytized by 4T1 cells and mainly locate in the cytosolic vesicles (Figure 5D).


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Figure 6. Overview of persistent luminescent (PL) or afterglow imaging experiments in vivo (A); In vivo afterglow images of normal mice with intravenous injection of BSA-modified hollow NIR PLNPs (dosage: 2 mg/mL, 200 µL) (B); In vivo recharged afterglow images by a white LED light (1100 lm) for 5 min per each cycle (C); Ex vivo afterglow images of heart (a), liver (b), spleen (c), lung (d), kidneys (e) and intestine (f) from the injected mouse at 24 h post intravenous injection (D); and their quantified pattern (E).

In Vivo Afterglow Imaging of Hollow NIR PLNPs. The ability of hollow NIR PLNPs for in vivo imaging was validated both in normal mice and 4T1 tumor-bearing mice. BSA-modified hollow NIR PLNPs (200 µL, 2 mg/mL) were charged by an external 254 nm UV lamp for 10 min, and then intravenously injected into the normal mice (Figure 6A). As shown in Figure 6B, a 18 ACS Paragon Plus Environment

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strong luminous signal is observed in the liver area without any excitation source under a bioluminescence mode. The afterglow signal is found gradually decreased along with the time increases. As discussed above, the afterglow of PLNPs decays rapidly in the earlier stage, and possibly the annihilated signal cannot be collected for a specific diagnosis. Fortunately, PLNPs can be renewed repetitively by a white LED light source, as demonstrated above. This characteristic allows us to recharge the failed PLNPs at a desired time to provide persistent images, which is of great advantage for long-time in vivo imaging. Very much Like the in vitro observation, the prepared hollow NIR PLNPs can be reactivated in vivo at least three cycles and the quality of the decayed images remains nearly unchanged in each cycle (Figure 6C), showing its capability for longitudinal imaging. The biodistribution of BSA-modified hollow NIR PLNPs was investigated by collecting the main organs (heart, liver, spleen, lung, kidneys and intestines) 24 h post intravenous injection. The ex vivo imaging result demonstrates the luminescent signal is mainly from liver, indicating the biggest uptake of PLNPs there. This finding agrees well with the published results.23,42 Lung and spleen are also found luminous but much weaker as compared to liver area (Figure 6D). Furthermore, the quantified luminous intensity in each tissue is also provided in Figure 6E.


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Figure 7. In vivo afterglow images at various time points of a 4T1-tumor-bearing mouse with intravenous injection of BSA-modified hollow NIR PLNPs (dosage: 2 mg/mL, 100 µL) (A); Ex vivo afterglow images of heart (a), liver (b), spleen (c), lung (d), kidneys (e), intestine (f) and tumor (g) from an injected mouse at 2 h post intravenous injection (B); and their quantified pattern (C); Body weight of mice (0-14 days) (D); Tumor growth curves after various treatments (E); Representative photographs of mice after different treatments (F); Hematoxylin/eosin staining of tumor tissues from different treatment groups (G). Scale bar is 100 µm.

To study the potential of the hollow NIR PLNPs for in vivo tumor imaging, BSA modified hollow NIR PLNPs (100 µL, 2 mg/mL) were charged by an external 254 nm UV lamp for 10 min, and then intravenously injected into 4T1 tumor-bearing mice. The luminescence signal in the tumor area was monitored at 10 min, 15 min, 20 min, 25 min and 30 min post-injection. The dynamic afterglow imaging shows that the hollow PLNPs can selectively target the tumor site. This clear tumor enhancement by PLNPs is most probably through enhanced permeability and retention (EPR) effect (Figure 7A). Moreover, the ex-vivo luminescent signals of the isolated organs and tumor are provided and quantified (Figure 7B and 7C). In Vivo Chemotherapy. To investigate the chemotherapy efficacy of DOX-hollow PLNPs, 4T1 tumor-bearing mice were treated by PBS, free DOX and DOX-hollow PLNPs, respectively. Compared with PBS and free DOX group, changes in tumor volume of the mice indicate the tumor growth is significantly inhibited by DOX-hollow PLNPs (Figure 7E and 7F). The body weight of the mice treated with free DOX and DOX-hollow PLNPs has a slight decrease in the whole experiment due to the side effect of chemotherapy to the mice (Figure 7D). H&E staining was used to further evaluate the therapeutic effect in the tumor tissues. Compared to PBS and free DOX group, the DOX-hollow PLNPs exhibit a large scale of tissue apoptosis, suggesting the


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hollow PLNPs could be as a promising drug carrier for tumor chemotherapy in vivo (Figure 7G).

Figure 8. Fluorescence emission spectrum of the hollow PLNPs excited under 254 nm and UV-vis absorption spectrum of Si-Pc (A); Monitoring of persistent luminescence sensitized generation of 1O2. The Si-Pc loaded hollow PLNPs (1 mL, 9.25 mg/mL) were excited with a white LED lamp (1100 lm) for 10 min before adding


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DPBF. The continuous quenching of DPBF was recorded for 9 min after the cessation of the white LED light irradiation (B); Body weight of mice (0-14 days) (C); Tumor growth curves after various treatments (D); Representative photographs of mice after different treatments (E); Hematoxylin/eosin staining of tumor tissues from different treatment groups (F). Scale bar is 100 µm.

In Vivo Photodynamic Therapy (PDT). In addition to chemotherapy, the NIR hollow PLNPs was also used to load a photosensitizer, Si-Pc, for tumor photodynamic therapy, by leveraging on the afterglow of PLNPs. The loading content of Si-Pc in hollow PLNPs was measured to be 850 mg/g through calculating the absorbance of the supernatant according to the Si-Pc standard curve (Figure S20). As shown in Figure 8A, the spectral overlap of the emission spectrum of hollow PLNPs and the absorption spectrum of Si-Pc provides a possibility to apply the afterglow of the hollow PLNPs to activate the photosensitizer. Singlet oxygen (1O2) generation was monitored by DPBF whose fluorescence can be quenched by the generated 1O2. It shows that Si-Pc with 1 min LED irradiation can cause a slight fluorescence quenching of DPBF. However, Si-Pc loaded hollow PLNPs exhibit heavier fluorescence quenching of DPBF, which indicates the afterglow of the NIR hollow PLNPs could collectively excite the photosensitizer (Figure S21). The fluorescence intensity of the DPBF was gradually decreased along with the time after the cessation of the white LED light irradiation and this could last for 9 min, which indicates the continuous generation of 1O2 triggered by the afterglow of PLNPs (Figure 8B). In vivo PDT was further conducted on 4T1 tumor-bearing mice. The mice were randomly divided into five groups, namely control, hollow PLNPs, free Si-Pc (without LED irradiation), free Si-Pc (with LED irradiation) and Si-Pc loaded hollow PLNPs (with LED irradiation). Compared to the other groups, the tumor growth was dramatically suppressed by Si-Pc loaded


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hollow PLNPs (with LED irradiation). This result combined with the above in vitro 1O2 generation data collectively indicates that the afterglow of PLNPs can contiguously sensitize the photosensitizer to produce 1O2, therefore enhancing the treatment efficacy on tumors (Figure 8D and 8E). H&E staining image from Si-Pc loaded hollow PLNPs (with LED irradiation) shows significant tissue apoptosis, which demonstrates good antitumor efficacy of Si-Pc loaded hollow PLNPs (with LED irradiation) (Figure 8F). Meanwhile, the relative body weight was monitored during the treatment period. There is no significant difference in the body weights of the mice receiving various treatments in the whole experiment (Figure 8C). Finally, histological assessments were performed to evaluate the potential in vivo toxicity of BSA-modified hollow NIR PLNPs without loading drugs. We found no obvious damage in the main organ tissues including heart, liver, spleen, lungs, kidney and intestines by hematoxylin and eosin (H&E) staining (Figure S22). However, must approbatory is, this new structured PLNPs deserve further in-depth investigations on their long-term in vivo toxicity before its further use in cancer imaging and therapy. CONCLUSIONS In sum, a large hollow cavity and size-tunable NIR PLNPs are fabricated using consumable carbon spheres as the template. Not only do the synthesized PLNPs have a uniform size and regular morphology, but they also possess intense persistent luminescence and particularly can be repeatedly renewed by a white LED for rechargeable afterglow imaging in vivo. This PLNPs-mediated afterglow imaging is advantageous over bioluminescence or radionuclide imaging that is also free of background interferences but it fails in signal emission once consumption of the injected substrates or disintegration of radioactive isotopes, accentuating the


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prospects of PLNPs for longitudinal imaging. More importantly, the resultant particles receive large cavity volume after calcination, therefore allowing high drug loading. To show the talents of this kind of hollow-structured PLNPs, a chemical drug, DOX, and a photosensitiser, Si-Pc, were largely loaded for chemotherapy and afterglow-assisted PDT. As intended, these two therapeutical modes show good antitumor capability, mainly attributed to the large cavity structure and potent afterglow of PLNPs. To the best of our knowledge, hollow structured PLNPs for afterglow imaging, drug loading and tumor therapy have not been reported. Admittedly, further in-depth study is needed, but we hope this proof-of-concept study of hollow structured PLNPs could inspire other functional structures of PLNPs and further extend their applications in biomedicine.

EXPERIMENTAL SECTION Materials. Zinc nitrate hexahydrate (Zn(NO3)2·6H2O), Chromium(III) nitrate nonahydrate (Cr(NO3)3·9H2O), dimethyl sulfoxide (DMSO) and bovine serum albumin (BSA) were purchased from Alfa Aesar. 1, 3-Diphenylisobenzofuran (DPBF), Silicon phthalocyanine (Si-Pc), urea and 3-aminopropyl-trimethoxysilane (APTMS) were purchased from Sigma-Aldrich. Ethanol absolute and N, N-Dimethylformamide were acquired from Sinopharm Chemical Reagent Co., Ltd. Sodium hydroxide, acetonitrile, glucose monohydrate, 4',6-diamidino-2-phenylindole (DAPI) and gallium nitrate hydrate (Ga(NO3)3·xH2O) were purchased from Aladdin (Shanghai, China). Millipore water with 18.2 MΩ was used in the experiment. Synthesis of Different Size of Carbon Spheres. 150 nm Carbon Spheres. The carbon spheres with 150 nm were prepared according the reported literature previously.29 Glucose monohydrate (6.0 g) were added to 35 mL of deionized water to form transparent solution. The solution was transferred into a 100 mL Teflon-lined


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autoclave, which maintained at 180 °C for 5.3 h. The solution was naturally cooled to room temperature. The carbon spheres were collected by centrifugation (10,000 rpm for 10 min) and washed with ethanol absolute and deionized water several times. The resultant product was dried in a vacuum oven at 60 °C for 8 h. 300 nm Carbon Spheres. The carbon spheres with 300 nm were prepared according the reported literature previously with a slight modification.28 Glucose monohydrate (4.0 g) were added to 60 mL of deionized water to form transparent solution. The solution was transferred into a 100 mL Teflon-lined autoclave, which maintained at 200 °C for 5.0 h. The solution was naturally cooled to room temperature. The carbon spheres were collected by centrifugation (10,000 rpm for 10 min) and washed with ethanol absolute and deionized water several times. The resultant product was dried in a vacuum oven at 60 °C for 8 h. 500 nm Carbon Spheres. Glucose monohydrate (17.8 g) were added to 50 mL of deionized water to form transparent solution. The solution was transferred into a 100 mL Teflon-lined autoclave, which maintained at 180 °C for 5.5 h. The solution was naturally cooled to room temperature. The carbon spheres were collected by centrifugation (10,000 rpm for 10 min) and washed with ethanol absolute and deionized water several times. The resultant product was dried in a vacuum oven at 60 °C for 8 h. Synthesis of Hollow Near Infrared Persistent Luminous Nanoparticles (Hollow NIR PLPNs) at Different Amount of Zn2+, Ga3+, Cr3+. Different amount of Zn(NO3)2·6H2O, Ga(NO3)3·xH2O, Cr(NO3)3·9H2O were mixed with 10 mL of ethanol absolute and 10 mL of deionized water (Table S1). Then urea (0.6 g) was added to the aboved mixture solution under vigorous stirring. After 5 min of stirring, the carbon spheres (8.0 mg, 150 nm) were added with


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sonication for 15 min. Subsequently, the solution was placed in a oil bath and heated at 90 °C for 6 h under vigorous stirring. The precursor was collected by centrifugation and washed with ethanol absolute and deionized water several times, and then dried at 60 °C in a oven. The sample was calcined at 800 °C for 2 h with a heating rate of 2 °C/min in air atmosphere to obtain the hollow NIR PLNPs. Optimization of Cr3+ Doping Content. Zn(NO3)2·6H2O (8 mmol), Ga(NO3)3·xH2O (16 mmol) and different amount of Cr(NO3)3·9H2O were mixed with 10 mL of ethanol absolute and 10 mL of deionized water (Table S2). Then urea (0.6 g) was added to the aboved mixture solution under vigorous stirring. After 5 min of stirring, the carbon spheres (8.0 mg, 150 nm) were added with sonication for 15 min. Subsequently, the solution was placed in a oil bath and heated at 90 °C for 6 h under vigorous stirring. The precursor was collected by centrifugation and washed with ethanol absolute and deionized water several times, and then dried at 60 °C in a oven. The sample was calcined at 800 °C for 2 h with a heating rate of 2 °C/min in air atmosphere to obtain the hollow NIR PLNPs. Synthesis

of the Hollow

NIR PLNPs at Different Calcination Temperature.

Zn(NO3)2·6H2O (8 mmol), Ga(NO3)3·xH2O (16 mmol) and Cr(NO3)3·9H2O (0.024 mmol) were mixed with 10 mL of ethanol absolute and 10 mL of deionized water. Then urea (0.6 g) was added to the aboved mixture solution under vigorous stirring. After 5 min of stirring, the different size of carbon spheres were added with sonication for 15 min. Subsequently, the solution was placed in a oil bath and heated at 90 °C for 6 h under vigorous stirring. The precursor was collected by centrifugation and washed with ethanol absolute and deionized water several times, and then dried at 60 °C in a oven. The sample was calcined at different calcination temperature for 2 h with a 27 ACS Paragon Plus Environment

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heating rate of 2 °C/min in air atmosphere to obtain the hollow NIR PLNPs (Table S3). Synthesis of the Hollow NIR PLNPs at Different Calcination Time. Zn(NO3)2·6H2O (8 mmol), Ga(NO3)3·xH2O (16 mmol), Cr(NO3)3·9H2O (0.024 mmol) were mixed with 10 mL of ethanol absolute and 10 mL of deionized water. Then urea (0.6 g) was added to the aboved mixture solution under vigorous stirring. After 5 min of stirring, the carbon spheres (8.0 mg, 150 nm) were added with sonication for 15 min. Subsequently, the solution was placed in a oil bath and heated at 90 °C for 6 h under vigorous stirring. The precursor was collected by centrifugation and washed with ethanol absolute and deionized water several times, and then dried at 60 °C in an oven. The sample was calcined at 800 °C under different time with a heating rate of 2 °C/min in air atmosphere to obtain the hollow NIR PLNPs (Table S4). Synthesis of Different Size of the NIR Hollow PLNPs. Zn(NO3)2·6H2O (8 mmol), Ga(NO3)3·xH2O (16 mmol) and Cr(NO3)3·9H2O (0.024 mmol) were mixed with 10 mL of ethanol absolute and 10 mL of deionized water. Then urea (0.6 g) was added to the above mixture solution under vigorous stirring. After 5 min of stirring, 150 nm size of carbon spheres (8.0 mg) were added with sonication for 15 min (Table S5). Subsequently, the solution was placed in an oil bath and heated at 90 °C for another 6 h vigorous stirring. The precursor-labelled carbon spheres were then collected by centrifugation and washed with ethanol absolute and deionized water several times, and then dried at 60 °C in an oven. The sample was finally calcined at 800 °C for 2 h with a heating rate of 2 °C/min in air atmosphere to obtain hollow NIR PLNPs. The preparation of the other two sizes of hollow PLNPs on 300 nm and 500 nm sizes of carbon spheres is just like the above protocol. Surface Modification of the Hollow NIR PLNPs. The amino-modified hollow NIR PLNPs 28 ACS Paragon Plus Environment

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was synthesized by a previously reported protocal with a slight modification.8,9 The hollow NIR PLNPs (2.0 mg) were dispersed in 5 mM NaOH solution under vigorous stirring for 24 h. The product were collected by centrifugation. The hydroxylated nanoparticles were dispersed in 2 mL of DMF. Then, 20 µL APTMS was added and reacted at 80 °C under vigorous stirring for 24 h. The resultant product was centrifuged and washed with DMF and deionized water several times to remove excess APTMS. To absorb BSA to the surface of the hollow NIR PLNPs-NH2, BSA (200 µL, 1 mg/mL) was added into the hollow NIR PLNPs-NH2 solution. The mixture was stirred at room temperature for 30 min. The BSA-modified hollow NIR PLNPs (BSA-hollow PLNPs) were collected by centrifugation and washed by deionized water. The final nanoparticles were dispersed in deionized water. Loading of Doxorubicin Hydrochloride (DOX·HCl). The hollow NIR PLNPs (2.6 mg) were added to DOX·HCl (1 mg/mL, 3.0 mL) aqueous solution and stirred in dark at the room temperature for 24 h. The product was acquired by centrifugation and washed with deionized water until the supernatant became colorless. The loading content of DOX was analyzed by UV-vis absorption specrum. The loading content (LC) was calculated as follows: LC% = (weight of loaded drug/weight of drug carrier). In Vitro Drug Release. DOX-hollow NIR PLNPs were suspended in 2 mL of buffer solution. The dispersion solution was then transferred into a dialysis bag (molecular weight cut off =8 000-14 000 kDa). The dialysis bag was placed in 100 mL of PBS buffer solutions (pH 5.0 and 7.4) at 37 °C and shaked at 180 rpm. At timed intervals, 3 mL of solution was withdrawn from the solution. The released DOX was analyzed by UV-vis. The volume of the release medium kept constant by adding 3 mL fresh medium after each sampling.


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Loading of Si-Pc. The hollow NIR PLNPs (20 mg) were added to Si-Pc (23.5 mg) DMSO solution and stirred in dark at the room temperature for 24 h. The product was acquired by centrifugation and washed with DMSO several times. The loading content of Si-Pc was analyzed by UV-vis absorption spectrum. The loading content (LC) was calculated as follows: LC% = (weight of loaded drug/weight of drug carrier). Singlet Oxygen Detection. The detection of singlet oxygen was measured by monitoring the fluorescence quenching of DPBF. The Si-Pc loaded hollow PLNPs (9.25 mg) were dispersed in anhydrous acetonitrile (1 mL), and was then irradiated by a white LED lamp for 10 min. DPBF (34 µM, 100 µL) was added into the Si-Pc loaded hollow PLNPs solution. The fluorescence of DPBF was recorded at different time points. In Vitro Cytotoxicity. The cell viability was determined by CCK-8 assay. 4T1 cells was seeded into a 96-well plate at a density of 1 × 104 cells and cultured at 5% CO2 and 37 °C for 24 h. Different concentrations of BSA modified hollow NIR PLNPs, free DOX and DOX-hollow NIR PLNPs were added to the medium, and the cells were incubated at 5% CO2 and 37 °C for 24 h. After 24 h, the culture media was replaced by fresh media containing 10 % of CCK-8 medium solution, and cultured for an another 2 h. The absorbance value at the wavelength of 450 nm was determined by microplate reader. In Vitro Cell Imaging. 4T1 cells at a density of 1 × 104 cells were seeded into confocal dish and cultured 12 h at 5% CO2 and 37 °C. The cells were treated with DOX-hollow PLNPs for 4 h (DOX concentration = 2 µg/mL). Then, the cells were washed three times with pH 7.4 PBS solution and fixed with 4% formaldehyde for 10 min, stained with DAPI before cell imaging. The bio-TEM sample was prepared according to the standard procedure for observation.


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In Vivo Biosafety Analysis. Hematoxylin and eosin (H&E) staining was adopted to evaluate in vivo toxicity of the BSA-hollow PLNPs with a size of 50 nm. All the animal procedures conformed to the guidelines of the Institutional Animal Care and Use Committee of Tongji University. Four weeks old healthy female BALB/c mice were treated with the BSA-hollow PLNPs (2 mg/mL, 200 µL) via the tail veins injection as experiment group. The control group was injected with PBS 7.4 solution at the same dosage. The main organs, including heart, liver, spleen, lung, kidney and intestines were collected at 15 d after the mice were anaesthetized. The different organs were fixed by 4% paraformaldehyde, sliced and dyed with H&E. The tissue samples were observed by using optical microscope. In Vivo Luminescence Imaging. Four weeks old healthy female BALB/c mice was treated with the BSA-hollow PLNPs (2 mg/mL, 200 µL) by intravenous injection. The BSA-hollow PLNPs were excited for 10 min with a 254 nm UV lamp (6 W) before injection. The luminescence signal was collected by Indigo imaging system with an exposure time of 60 s. In vivo re-excited imaging was conducted by white LED lamp (1100 lm) irradiation for 5 min. The exposure time was set as 60 s. Ex Vivo Biodistribution Analysis. The mice were sacrificed at 24 h after mice were injected the BSA-hollow PLNPs (2 mg/mL, 200 µL) through tail vein. The main organs (including heart, liver, spleen, lung, kidney and intestine) were taken out and irradiated by 254 nm UV lamp for 5 min. The luminescence signal was collected by Indigo imaging system with an exposure time of 60 s. Stability Study. For fluorescence stability study, The prepared BSA-hollow PLNPs with a size of 50 nm were dispersed in PBS buffer (pH 7.4) and deionized water. Subsequently, the


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fluorescence intensity of BSA-hollow PLNPs was monitored by fluorescence analyzer for 5 days at room temperature. For the afterglow and fluorescence stability in various concentrations of NaCl and different pH PBS aqueous solution, the prepared BSA modified hollow NIR PLNPs with a size of 50 nm were dispersed in different pH PBS aqueous solution (pH 4.0, 7.4 and 9.0) and various concentrations of NaCl (0 mM, 25 mM, 50 mM and 100 mM). The fluorescence intensity of BSA-hollow PLNPs was monitored by fluorescence analyser. The luminescence signal was collected by Indigo imaging system with an exposure time of 60 s. In Vivo Luminescence Imaging of 4T1 Tumor bearing mice. The BSA-hollow PLNPs (2 mg/mL, 100 µL) were injected into 4T1-tumor-bearing mice by intravenous injection. The BSA-hollow PLNPs were excited for 10 min with a 254 nm UV lamp (6 W) before injection. The luminescence signal was collected by Indigo imaging system with an exposure time of 60 s. The mice were sacrificed at 2 h after mice were injected the BSA-hollow PLNPs (2 mg/mL, 100 µL) intravenously. The main organs (including heart, liver, spleen, lung, kidney and intestine) and tumor were taken out and irradiated by 254 nm UV lamp for 5 min. The luminescence signal was collected by Indigo imaging system with an exposure time of 60 s. In Vivo Chemotherapy. Female BALB/c mice (4 weeks and weight ∼20 g) were acquired from Shanghai Slac laboratory animal Co, Ltd. 4T1 cells (1*106 cells) were implanted by subcutaneous injection into the flank region. When the volume of the tumor grew to approximately 80 mm3, the tumor-bearing mice were randomized into three groups (n=3 per group): PBS, free DOX and DOX-hollow PLNPs. Free DOX and DOX-hollow PLNPs (200 µL, 0.5 mg/mL as DOX) were injected into the mice through the tail vein once every two days for three times in the first six days. The tumor volume was measured by a digital cliper every two


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days. The tumor volume was calculated according to the follow formula: the tumor volume = length*width2/2 In Vivo Photodynamic Therapy. Female BALB/c mice (4 weeks and weight ∼20 g) were acquired from Shanghai Slac laboratory animal Co, Ltd. 4T1 cells (1*106 cells) were implanted by subcutaneous injection into the flank region. When the volume of the tumor grew to approximately 80 mm3, the tumor-bearing mice were randomized into five groups (n=3 per group): PBS (control), hollow PLNPs, Si-Pc, Si-Pc + LED and Si-Pc loaded-hollow PLNPs + LED. PBS (50 µL), hollow PLNPs (5 mg/mL, 50 µL), Si-Pc ( 4.25 mg/mL, 50 µL) and Si-Pc loaded hollow PLNPs (50 µL, 4.25 mg/mL as Si-Pc and 5 mg/mL as hollow PLNPs) were intratumorally injected into the mice at 1 day and 7day, and then subjected to 10 min irradiation by white LED lamp. The tumor volume was measured by a digital cliper every two days. The tumor volume was calculated according to the follow formula: the tumor volume = length*width2/2. Characterizations. The fluorescence spectrum of the hollow NIR PLNPs was conducted using Pekin-Elmer fluorescence spectrometer. High resolution-transmission electron microscopy (HR-TEM) was conducted on a JEM-2100 operated at 200 kV. The 3D TEM was conducted on a FEI Tecnai G2 F30 and Inspect 3D software was adopted to reconstruct the 3D TEM morphology of the hollow NIR PLNPs. The hydrodynamic size and zeta potential were measured on a Zetasizer Nano-ZS90 (Malvern, UK). The surface area, pore volume and pore size were measured on by N2 adsorption–desorption isotherms obtained at 77 K on a Quantachrome Autosorb-1, USA. The sample was outgassed at 10-3 Torr and 200 0C for approximately 6 h prior to the adsorption experiment. Fourier Transform Infrared (FT-IR) was measured on a SHIMADZU IRprestige-21 spectrometer. Powder X-ray diffraction (XRD) analysis was measured on a XD-3 X-ray


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diffractometer using Cu (36 kV, 20 mA) radiation in the 2θ range from 10° to 70°. The excitation, emission and afterglow spectra were acquired from FLS920 spectrometer (Edinburgh, UK). The fluorescence quantum yield of the hollow PLNPs was measured from FLS1000 (Edinburgh, UK). Cell imaging was conducted on TCS SP5 confocal laser scanning microscope (Leica, Germany). The images in vivo were observed on a NightOWL LB 983 imaging system. ASSOCIATION CONTENT Supporting Information 3 Dimensional TEM image and video of the hollow PLNPs; XRD; BET data; Fluorescence spectrum of different size of hollow PLNPs; TEM images of the hollow PLNPs at different concentration of Zn2+, Ga3+, Cr3+; TEM images and fluorescence spectrum of the hollow PLNPs at different Cr3+ content; TEM images and fluorescence spectrum of the hollow PLNPs at different calcination temperature; TEM images and fluorescence spectrum of the hollow PLNPs at different calcination time; stability study; FT-IR, zeta potential, size changes and fluorescence spectrum under each modification; NIR persistent luminescent afterglow decay images of other two size of hollow NIR PLNPs powder and aqueous solution; cytotoxicity assay; The hollow NIR PLNPs stability study; In vivo biosafety study; DOX and Si-Pc standard curve and tables as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail:[email protected] ACKNOWLEDGMENTS This research was support by the National Natural Science Foundation of China (Grants number:


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81571742, 81371618) and the Fundamental Research Funds for the Central Universities.


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The graphic used in the table of contents and abstract:


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Large Hollow Cavity Luminous Nanoparticles with Near-Infrared

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