Near-Infrared Light-Excited Upconverting Persistent Nanophosphors

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Biological and Medical Applications of Materials and Interfaces

Near-infrared light excited upconverting persistent nanophosphors in vivo for imaging-guided cell therapy Bin Zheng, Yang Bai, Hongbin Chen, Huizhuo Pan, Wanying Ji, Xiaoqun Gong, Xiaoli Wu, Hanjie Wang, and Jin Chang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b05706 • Publication Date (Web): 14 May 2018 Downloaded from http://pubs.acs.org on May 15, 2018

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ACS Applied Materials & Interfaces

Near-infrared light excited upconverting persistent nanophosphors in vivo for imaging-guided cell therapy †

‡

†

†

Bin Zheng, ,# Yang Bai, ,# Hongbin Chen, Huizhuo Pan, Wanying Ji,

†

Xiaoqun Gong,† Xiaoli Wu,† Hanjie Wang,*,† and Jin Chang*,† †

School of Life Sciences, Tianjin University, 92 Weijin Road, Nankai

District, Tianjin 300072, P.R. China. ‡

Department of Stomatology, Tianjin Medical University General

Hospital, 154 Anshan Road, Heping District, Tianjin 300052, P.R. China.

Address correspondence to [email protected] and [email protected].

#

These authors contributed equally to this work.

ACS Paragon Plus Environment

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ABSTRACT: Optical imaging for biological applications is in need of more sensitive tool. Persistent luminescent nanophosphors enable highly sensitive in vivo optical detection and almost completely avoids tissue autofluorescence. Nevertheless, the actual persistent luminescent nanophosphors necessitates ex vivo activation before systemic operation, which severely restricted the use of long-term imaging in vivo. Hence, we introduced a novel generation of optical nanophosphors, based on (Zn2SiO4: Mn): Y3+, Yb3+, Tm3+ upconverting persistent luminescent nanophosphors, these nanophosphors can be excited in vivo through living tissues by highly penetrating near-infrared light. We can trace labeled tumor therapeutic macrophages in vivo after endocytosing these nanophosphors in vitro and follow macrophages biodistribution by a simple whole animal optical detection. These nanophosphors will open novel potentials for cell therapy research and for a variety of diagnosis applications in vivo. Keywords: optical imaging, near infrared light (NIR), upconverting persistent

luminescent nanophosphors

(UPLNs),

macrophages, cell therapy


tumor-associated

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■ INTRODUCTION To increase the imaging accuracy in vivo, various techniques such as magnetic resonance imaging (MRI),1 positron emission tomography (PET),2 computed tomography (CT),3 ultrasound,4 and optical imaging5 have been used for living imaging. Among these methods, optical imaging has been intensively investigated for their potential accurate imaging application due to the stability and convenience in vivo. In particular, luminescent nanoprobes such as semiconductor quantum dots (QDs),6 upconversion luminescent nanoparticles (UCNs),7 carbon nanomaterials8 and so on, have widely used in imaging. However, during in situ excitation of these luminescent nanoprobes it will inevitably cause strong auto-fluorescence from biological tissue, which significantly reduces the signal to noise ratio and even results in false diagnosis.9 So the novel kind of luminescent nanoprobes with high signal to noise ratio is still needed to be developed. Recently, there is an increasing interest toward the application of persistent luminescence nanoparticles (PLNs) to realize in vivo imaging.10 PLNs are a novel group of luminescent nanoprobes which can be used for optical imaging in vivo. As reported, persistent luminescence (long afterglow or long-lasting phosphorescence) could last several hours after optically excited.11,12 On account of the removal of background noise originating from in situ excitation, the signal-to-noise ratio could be remarkably improved.



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Though intended for imaging applications in vivo, PLNs still are subjected to many defects. Especially, conventional PLNs have to be excited ex vivo by low tissue-penetrating ultraviolet light before systemic operations, which severely limited the applications for long-term imaging in vivo. 13-16 In order to overcome these serious defects, efforts to unravel the excitation mechanism of PLNs have resulted in optimized compositions.17-20 Interestingly, near-infrared (NIR) light owing a minimally invasive, deep tissue penetrability and harmless therapeutic modality, has become an ideal excitation light source. Meanwhile, in the lanthanide-doped Y, Yb and Tm upconverting luminescent nanoparticles, Yb3+ can absorb two or more low-energy pump photons from the NIR and Tm3+ transfer them to a higher-energy output photon with a shorter wavelength such as UV or blue light,21-23 which can be used to excite persistent luminescence nanoparticle in vivo. Hence, it is a great potential for

developing

a

novel

NIR-excitation

persistent

luminescence

nanoparticle with super-long afterglow for in vivo imaging. In this paper, the NIR-excited PLNs nanoparticles had been prepared for persistent luminescent imaging-guided cell therapy to tumor in vivo. The system be composed of two parts: 1) Upconverting persistent luminescent nanophosphors (UPLNs) (containing the upconversion parts that absorbs and converts near-infrared light, and green light-emitting Zn2SiO4:Mn which has strong environmental adaptability, good chemical


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stability, easy preparation, and low price) were synthetized with template method of mesoporous silica nanoparticles. They could be excited by extra NIR light and emit persistent luminescence to track therapeutic macrophages for realizing visual-guided cell therapy in vivo. 2) The tumor-associated

macrophages,

which

can

infiltrate

into

tumor

microenvironment in high numbers for inhibiting tumor growth and killing tumor cells by phagocytosis. 24-26 Besides, they can also secrete a wide array of cytokines to activating the anti-tumor immune response, such as TNF α, IL β, etc, 27, 28 served as a targeted cell therapy reagent to destroy cancer cells in vivo. However, conventional macrophages-based immunotherapy was generally difficult to track in vivo without in situ excitation. 29-31 In our system, the highly penetrating near-infrared light excited super-long time persistent luminescence imaging strategy could be used to solve this defect as a novel technique to track the biodistribution of tumor therapeutic macrophages in vivo after endocytosing these nanophosphors in vitro (as shown in Figure 1). This nanophosphor will open novel potentials for cell therapy research and for a variety of diagnosis applications. ■ Results and discussion The Preparation Process and Physicochemical Characterization of the Upconverting Persistent Luminescent Nanophosphors. The synthesis process of the UPLNs nanoparticles could be generally divided



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into three steps (as shown in Figure 2A). Firstly, the mesoporous silica nanoparticles（mSiO2）were synthesized by CTAB template method.32, 33 Secondly, the precursor ions such as Zn2+ and Mn2+（Zn/Mn = 2/1）were absorbed into the nanopores of mesoporous silica layer, and after vacuum drying and 800℃ annealing process the precursor ions formed persistent phosphor Zn2SiO4:Mn crystals inside the mSiO2.34 Then, the precursor ions for Y3+, Yb3+, and Tm3+（Y/Yb/Tm=750/250/3）were absorbed into the nanopores again, and after vacuum drying and 300℃ annealing process the precursor ions formed NIR-excitation upconverting persistent luminescent nanoparticles (UPLNs). The physicochemical characteristics of upconverting persistent luminescent nanoparticles were characterized by TEM, X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDX). The detailed morphological features of mSiO2 and UPLNs were examined by TEM. In Figure

2B,

the

mSiO2

had

many

nanopores

and

the

N2

adsorption–desorption of nanopores was analyzed in Figure S1A, which illustrated that it could be used for loading the precursor ions of Zn2+, Mn2+, Y3+, Yb3+ and Tm3+. The upconverting persistent luminescent nanophosphors of (Zn2SiO4: Mn): Y3+, Yb3+, Tm3+ crystals were obtained after 3 h of sintering process in 800℃ muffle furnace and the second sintering at 300℃, and it could be seen inside the silica nanospheres (Figure 2B, yellow dotted circle). All the nanoparticles were spherical


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and uniform and the particle sizes had been tested by DLS. As seen in Figure 2C and S1B, the diameter of them were all within 120-160 nm, which indicated that they were appropriate for animal experiments as well. In Figure 2D, the XRD measurement was used for confirming crystal texture of as-synthesized UPLNs nanocomposite. All diffraction peaks of the mSiO2 and UPLNs nanoparticles could be indexed according to the literature data (peaks at 12°, 25°, 32°, 34°, 39°, 48°, 66°, JCPDS, 00-008-0492 and 28-1192).34, 35 The clear morphology of the surface on the UPLNs was observed by the high-resolution TEM image in Figure 2E (orange dotted circle). The chemical element for UPLNs was analyzed by EDX and results displayed that Zn2+, Mn2+, Y3+, Yb3+ and Tm3+ had been mingled into the mesoporous silica nanoparticles (Figure 2F1 and 2F2). All data indicated that UPLNs nanoparticles had been successfully generated. The Persistence Luminescent Performance. The luminescence mechanism of upconverting persistent luminescence nanoparticles (UPLNs) contained two steps, upconversion luminescence (UCL) and persistent luminescence (PL), and it could be succinctly explained (Figure 3A.). Firstly, some low-energy incident photons could stimulate the ion system from ground state (G) to high-energy delocalized state (D) via an upconversion excitation channel, followed by filling of electron traps. When the stored excitation energy was gradually released after removal



of excitation light source, a persistent fluorescence could be generated. The trivalent lanthanide ion of Tm3+ would be an ideal activator of ions owing to that it could convert the absorption photons to ultraviolet and blue light for exciting PLNs.36-40 To test persistent luminescence performance of UPLNs, luminescence decay characteristic was tested after exciting the UPLNs by UV light, and it could last 40 min in a very high intensity after removal of the excitation light source. Nevertheless, the persistent emission signal of UPLNs weakened after 40 min, which would also limit the long time monitoring of some physiological processes in vivo (Figure 3B). When the UPLNs were re-irradiated by 980 nm laser, fluorescence was partly recovered and reproduced afterglow, showing that the UPLNs could be repeatedly excited in vivo by deep-tissue-penetrating near-infrared (NIR) light (Figure 3B). Furthermore, we designed a logo on the hard paper and the powder of UPLNs served as ink was printed on it (shown in the inset of Figure 3B). After irradiating by UV light, intense persistent luminescence of UPLNs was monitored by in vivo imaging system. The similar effect was also detected after irradiation by 980 nm NIR light again. These results illustrated that the UPLNs could be regarded as in situ re-excitable persistent luminescent nanophosphors to achieve super-long time in vivo imaging by recharging with highly penetrating near-infrared light. Furthermore, the fluorescence spectrometer of UPLNs was also detected


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under irradiation of 980 NIR light. Excited by 980 NIR light, the UPLNs nanoparticle could generate two intense emission bands at about 450−480 nm and 480−600 nm, and the maximum emission peak was 525 nm (Figure 3C). These results indicated that upconverting persistent luminescence nanoparticles could also be efficaciously excited by deep-tissue-penetrating 980 nm near-infrared light. In order to prove that the UPLNs could be used for persistent luminescence imaging after removal of excitation light source, aqueous solution of these nanocomposites was added into the 96-well plate. The luminescence intensity was monitored by in vivo imaging instrument. As shown in Figure 3D, the UPLNs had intense fluorescence after charging with UV light and 980 nm laser. The fluorescence intensity weakened over time, while the persistent luminescence could still be intensively detected after 40 min. In addition, the persistent luminescence stabilities of UPLNs were tested by recharged with 980 nm NIR light. After recharging, there was still intense luminescence and also the long persistent luminescence (Figure 3D). The powders of UPLNs served as ink were printed on a paper to form an image of “H” letter. After irradiating by 980 nm laser, the persistent luminescence of UPLNs was then monitored by in vivo imaging system. The imaging results showed that bright luminescence lasted up for 40 min after removal of 980 nm NIR light (Figure 3E). The in vivo imaging of mouse was also monitored after the intratumor injection of UPLNs



dissolved in PBS and luminescence sustained to 30 min in vivo after recharging 3 times with 980 nm NIR light (Figure S6). These results indicated that UPLNs was expected to be used for NIR-triggered persistent luminescence imaging for diagnosis applications and tracing cell therapy research in vivo. The Phagocytosis Test of Macrophages. Phagocytosis is a very significant aspect of macrophages. In order to test whether macrophages had strong phagocytosis function, the mouse macrophages (Raw 264.7) were incubated with UPLNs labeled by indocyanine green (ICG) (ICG@UPLNs), as shown in Figure 4A. Almost all the cells were filled with the red from ICG, when the Raw 264.7 cells were incubated with 0.25 mg/ml ICG@UPLNs for 2 hours (Figure S7). This result showed that the upconverting persistent luminescence nanoparticles could been internalized by macrophages. In order to test whether macrophages still had phagocytosis function after uptaking the UPLNs for simulating the processes in the body, the macrophage phagocytosis of cancer cells was designed. Firstly, the Raw 264.7 cells were incubated with UPLNs labeled by indocyanine green (ICG) (ICG@UPLNs) (Figure 4B2). Meanwhile, the B16 tumor cells were labeled with FITC-phalloidin (Green fluorescent probe) in Figure 4B1. Afterwards, the green B16 cells were gently digested and incubated with the Raw 264.7 cells of loading ICG@UPLNs. As seen in Figure 4,


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after macrophages incubated with ICG@UPLNs, the strong red fluorescence was observed in Raw 264.7 cells, which indicated that the ICG@UPLNs had already been uptaked by macrophages (Figure 4B2). Then they were added and incubated with phalloidin-FITC labeled B16 cells for 3 hours. After the culture medium was replaced, the green fluorescence was found in the macrophages (Figure 4B3), which suggested that the B16 cell was devoured into macrophages. And laser confocal fluorescence microscopy was also used for observing the high-resolution phagocytosis and the arrow position was the B16 cell debris after being swallowed into macrophages. (Figure 4C). Meanwhile, the results were also confirmed by flow cytometry studies. These data presented that B16 cells could be taken up by the macrophages. According to this feature, they were expected to be used in persistent luminescence imaging-guided cell therapy to B16 melanoma in vivo. Persistent Luminescence Imaging-Guided Cell Therapy in vivo. To test the fluorescence imaging effect of UPLNs loaded macrophages (UPLNs@M) in vivo, the samples were injected into the B16 subcutaneous tumor model in C57/B6 mice by caudal vein, as shown in Figure 5A. The treatment was carried out when the tumors volume grew to about 350 mm3 and the upconverting persistent luminescence nanoparticles were excited with NIR light before each in vivo imaging. The plentiful UPLNs loaded macrophages (UPLNs@M) had been observed to gather to the tumor area ACS Paragon Plus Environment


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preferably by persistent luminescence imaging after tail vein injection for 24 h (Figure 5B). However, the group of only injectable UPLNs has a more dispersed fluorescence distribution in vivo after tail vein injection by UPLNs for 24 h (Figure 5B and Figure S8). The phenomenon illustrated that macrophages had a strong tumor targeting performance to tumor region and it can serve as a targeted cell therapy reagent to specifically recognize and destroy neoplastic cells in vivo, consistent with the results reported in many literatures.24, 26, 41 After euthanasia, the main internal organs and tumor of mice for different treatment were taken out, and the persistent luminescence were detected by in vivo imaging instrument after 980 nm laser excitation. In Figure 5C and 5D, tumor had a very high persistent luminescence intensity in UPLNs loaded macrophages group compared with UPLNs alone injection. These results manifested that macrophages had a strong tumor targeting performance to tumor region and it can serve as a targeted cell therapy reagent to specifically recognize and destroy neoplastic cells in vivo. Besides, after tail vein injection UPLNs loaded macrophages for 24 h, the tumors were made into ultrathin section

(50-80

nm)

for

detecting

the

upconverting

persistent

luminescence nanoparticles in macrophages by TEM (Figure 6A). It turned out that the macrophages could be targeted to reach the tumor area in vivo and the UPLNs had good stability in the macrophages. These results suggested that repeatedly NIR-excited persistent luminescence


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nanophosphors could be used for super-long time persistent luminescence imaging-guided macrophage-based cell therapy in vivo. Macrophages labeled by UPLNs had better persistent luminescence imaging performance in vivo according to the above mentioned. Then, in order to prove whether it also had a good cure effect for tumor in vivo, the cell therapy experiment of macrophages was also further evaluated. There were four groups of experiment designed, including blank group, received PBS tail vein injection only, received 0.5 mg/ml UPLNs tail vein injection only and experimental group received the 0.5 mg/ml UPLNs loaded macrophages tail vein injection by 106 Raw 264.7 cells. After different treatments for 7 days, there were high concentrations of tumor necrosis factor α (TNF α) and interleukin β (IL β) in the serum of rats for experimental group (n=5), which they were secreted from mainly macrophages and could induce tumor cell death (Figure 6B and 6C).27, 28 After 15 days of treatment, the tumor had a diameter of less than 10 mm, a weight of 0.25 g, and a volume of 300 mm3 in experimental group, which these values were significantly smaller than those in the control group (Figure 6D-6F and S10-S11). These results proved the commendable inhibition tumor growth ability of the macrophages labeled by UPLNs group. Moreover, survival of the mice was significantly prolonged in the macrophages labeled by UPLNs group compared with control groups (Figure 6G). Besides, histological analyses of B16 xenograft tumors were ACS Paragon Plus Environment


also performed using H&E and TUNEL staining. More obvious tumor necrosis with severe structural damage could be observed in tumors of macrophages labeled by UPLNs group more than the other three groups (Figure 6H). These results certified the excellent anti-tumor ability of macrophages labeled by UPLNs. During the animal experiments, macrophages labeled by UPLNs were very good biological tolerance and had no obvious acute toxicity to the examined mice for cell therapy in vivo (Figure S12 and S13). All these results indicated that the tumor targeting macrophages labeled by UPLNs as a super-long time persistent luminescence image-guided cell therapy reagent could be used to specifically recognize and destroy neoplastic cells in vivo. ■ CONCLUSIONS In conclusion, the 980 nm NIR-excited PLNs nanoparticles had been successfully prepared via template method of mesoporous silica nanoparticles, which this method was simple to operate, low in cost, and these products had uniform particle size and morphology. In this composite nanoparticle, the upconversion parts could absorb and convert near-infrared light to UV light and blue light to inspire long afterglow luminescence. The in situ and in vivo repeatable excitation with NIR light not only could provide super-long time in vivo imaging but also could substantially avoid the interference of the auto-fluorescence originating


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from biological tissues. These NIR-rechargeable persistent luminescence nanophosphors could be potentially applied in some situations, where super-long monitoring time was needed in vivo. Based on these features, the novel UPLNs nanocomposites were constructed for recharging persistent luminescence imaging-guided cell therapy against tumor in vivo. After that, the tumor-associated macrophages labeled by UPLNs as a targeted cell therapy reagent to specifically recognize and destroy neoplastic cells by phagocytosis and secreting a wide array of anti-tumor immune cytokines in vivo. The visual treat under 980 nm NIR-triggered persistent luminescence-imaging guidance, tumor targeting and cell therapy efficiency of this system were perfectly integrated. We also elucidated the superior solid tumor inhibitory efficiency of the macrophages labeled by UPLNs targeting cure system in vivo. Hence, such repeatedly NIR-excited persistent luminescence nanophosphors could be commendably used for super-long time persistent luminescence imaging-guided macrophage-based cell therapy in vivo. ■ EXPERIMENTAL SECTION Synthesis of (Zn2SiO4: Mn): Y3+, Yb3+, Tm3+ Upconverting Persistent Luminescent Nanophosphors (UPLNs). The mesoporous silica nanoparticles (mSiO2) were synthesized by CTAB template method.32, 33 The synthesis process of UPLNs could be briefly described



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as follows. Firstly, Zn(NO3)2 and MnCl2 were dissolved in water/ethanol, according to a molar ratio of Zn/Mn (2/1). Then, 400 µL of the precursor solution was mixed with 100 mg mesoporous silica nanoparticles and the mixture was evaporated after centrifugation. Finally, the samples were calcined at 800℃-1000℃ for 3 h.34 Subsequently, according to the same method, the precursor ions of chloride for Y3+, Yb3+, and Tm3+ （Y/Yb/Tm=750/250/3）were absorbed into nanopores of mesoporous silica again, and after vacuum drying and 300℃ annealing process the precursor

ions

formed

NIR-excitation

upconverting

persistent

luminescent nanophosphors (UPLNs). In order to improve the luminescence intensity, the above process could be repeat as required (Figure S2). Persistent Luminescent Imaging and Cell Therapy Efficiency in vitro. In vitro fluorescence imaging was conducted by putting these powder samples into a 96-well plate. The luminescent signal was recorded by a IVIS Spectrum In Vivo Imaging System ( IVIS®Spectrum BL, PerkinElmer) after excited by using ultraviolet lamp or 980 NIR laser (MDL-N-980 (Cnilaser, China) for 10 min. The cells were incubated within the medium containing different samples for evaluating the cell viability by MTT assay and the cytotoxicity by Calcein-AM dying and flow cytometer.


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Persistent Luminescent Imaging and Cell Therapy Efficiency in vivo. For details on animal rearing (female C57/B6) and tumor model construction, please see the Supporting Information and relevant references.16,42 Tumor volume= Length×Width×(Length+Width)/2. The persistent luminescence imaging of the examined mice were acquired on a IVIS Spectrum in vivo Imaging System ( IVIS®Spectrum BL, PerkinElmer) without excitation sources. 42 ■ ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: *****. N2 adsorption–desorption isotherms of mSiO2, SEM of UPLNs, persistent luminescence of UPLNs nanospheres under 980 nm excited, MTT, biocompatibility, hemolytic test of UPLNs, rechargeable persistent luminescence performance of the UPLNs by 980 nm NIR laser excitation in vivo, phagocytosis efficiency of macrophage, targeting efficiency of UPLNs loaded macrophages in vivo, tumor volumes after different treatments, weight change of mice, H&E staining of the main internal organs. (PDF) ■ AUTHOR INFORMATION



Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected]. ORCID Jin Chang: 0000-0002-6752-8526 Hanjie Wang: 0000-0001-9400-814X Author Contributions #

Dr. Zheng and Bai contributed equally to this work.

Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS This work was sponsored by National Key Research and Development Program of China (2017YFA0205104), National Natural Science Foundation of China (51373117, 51573128 and 81771970), Tianjin Natural Science Foundation (15JCQNJC03100).


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Mantovani,


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Figures:

Figure 1. Schematic diagram indicated applications of near infrared (NIR)-excitation upconverting persistent luminescent nanoparticles (UPLNs) loading macrophages for real-time tracking tumor therapeutic macrophages in vivo after endocytosing these nanophosphors in vitro and follow macrophages biodistribution by a simple whole animal optical detection. The UPLNs loading macrophage tracking system consisted of two parts: 1) Upconverting persistent luminescent nanophosphors (UPLNs) (containing Zn, Mn, Y, Yb, Tm) were synthetized with template method of mesoporous silica nanoparticles. They could be excited by extra NIR light and emit persistent luminescence to track therapeutic macrophages for realizing visual-guided cell therapy in vivo. 2) The tumor-associated macrophages, which are a class of common natural immune cells and can specifically recognize and destroy neoplastic cells both in vitro and in vivo without injuring nontumorigenic cells by specific


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phagocytes and release of cytokines, served as a targeted cell therapy reagent to kill cancer cells in vivo. In this system, the highly penetrating near-infrared light excited super-long time persistent luminescence imaging strategy could be used as a novel technique to track the biodistribution of tumor therapeutic macrophages in vivo after endocytosing these nanophosphors in vitro. This nanophosphor will open a novel potentials for cell therapy research and for a variety of diagnosis applications in vivo.



Figure 2. (A) Schematic diagram of preparation of UPLNs; The TEM picture of (B) mesoporous silica nanoparticles (mSiO2) and UPLNs; The dynamic light scattering (DLS) of (C) mSiO2 and UPLNs. X-ray powder diffraction pattern of as-obtained (D) mSiO2 and UPLNs and the numerical value from standard data (JCPDS: 00-008-0492 and 28-1192). (E) The high-resolution TEM image of UPLNs. (F1 and F2) The EDX test for element analysis of UPLNs. Scale bars were 100 nm.


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Figure 3. (A) The schematic diagrams for the luminescence mechanism of the persistent luminescence nanoparticles (PLNs), upconverting persistent luminescence nanophosphors (UPLNs) and upconverting nanoparticles (UCNs), with hypothetical energy level schemes. G, M, E and D represent the ground state, metastable state, emission state, and delocalized state, respectively. Straight-line arrows and curved-line arrows represent optical transitions and electron transfer processes, respectively. (B) The long afterglow decay curve of UPLNs after UV lamp or 980 nm NIR light excitation. The inserts showed imaging of the UPLNs after UV light or 980 nm light exposure for 10 min. (The UPLNs powder as ink were printed on hard paper to form a logo image). (C) The fluorescence spectrum of UPLNs under 980 nm light exposure. The



inserts showed the macrophotograph of UPLNs under 980 nm light exposure. (D) The persistent luminescence imagines of UPLNs by recharging for 3 times was measured within 40 min after UV lamp or 980 nm NIR laser for irradiating 10 min, respectively. (E) The fluorescence imaging of the UPLNs powder as ink were printed on hard paper to form an image of a “H” letter.


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Figure 4. The macrophages phagocytosis activity test of UPLNs and tumor cells. (A) A schematic illustration shows that UPLNs were uptaken by Raw 264.7 cells and then tumors cells were uptaken again. (B1) The cytomembrane of B16 cells was stained by Phalloidin-FITC (Green fluorescent probe); (B2) The Raw 264.7 cells uptaken the UPLNs labeled by indocyanine green (ICG) (ICG@UPLNs); (B3) The green B16 cells were gently digested and added into the Raw 264.7 cells of loading ICG@UPLNs. Each process cells of stained FITC and ICG were quantified with flow cytometry (FCM). Scale bars were 100 µm. (C) The high-resolution laser confocal fluorescence microscopy was used for observing the phagocytosis of B3. The scale bars were 20 µm.



Figure

5.

The

repeatedly

NIR-excited

persistent

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luminescence

nanophosphors used for super-long time persistent luminescence imaging in vivo. (A) The Schematic diagram of NIR-excited persistent luminescence nanophosphors used for super-long time persistent luminescence (PL) imaging in vivo. (B) After tail vein injection different treatment group (including the macrophages group alone, the UPLNs group alone and the UPLNs loaded macrophages (UPLNs@M) group), the persistent luminescence imaging in vivo at different time. (C) After tail vein injection for 24h, the distribution of persistent luminescence nanophosphors in excised organs and tumors for different treatment groups. (D) After tail vein injection for 24h, the quantitative analysis of the fluorescence intensity in excised organs and tumors for different treatment groups. The in vivo imaging was used for detecting the fluorescence intensity after recharging with 980 nm NIR laser.


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Figure 6. The NIR-excited persistent luminescence nanophosphors used for persistent luminescence imaging-guided macrophage-based cell therapy in vivo. (A) After tail vein injection UPLNs loaded macrophages for 24 h, the tumors were made into ultrathin section (50-80 nm) for detecting the upconverting persistent luminescence nanoparticles in macrophages by TEM imaging. Scale bars, 2 um. (B) The concentration of tumor necrosis factor α (TNF α) in the serum of rats for different treatments groups, including blank group, received PBS tail vein injection only, received 0.5 mg/ml UPLNs tail vein injection only and experimental group received the 0.5 mg/ml UPLNs loaded macrophages



tail vein injection by 106 Raw 264.7 cells. (C) The concentration of interleukin β (IL β) in the serum of rats for different treatments groups. (D) Size of excised tumors for different treatments groups after 15 days. (E) Tumors weight for different treatments groups. (F) Volume change of tumors for different treatments groups within 15 days. (G) Kaplan-Meier survival curve of B16 tumor-bearing mice for different treatments groups (Calculated by Survival Curve Comparison: Log-Rank Test). (H) The H&E and TUNEL staining of B16 xenograft tumors with different treatments. The scale bars were 100 µm. Data represent mean ± SD; *P

Near-Infrared Light-Excited Upconverting Persistent Nanophosphors

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