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Biomaterials

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Turning solid into gel for high-efficient persistent luminescence-sensitizedphotodynamic therapy

Shao-Kai Suna,*, Jian-Cheng Wua, Haoyu Wanga, Li Zhoua, Cai Zhangc, Ran Chenga, Di Kana,Xuejun Zhanga, Chunshui Yub,**

a School of Medical Imaging, Tianjin Medical University, Tianjin, 300203, ChinabDepartment of Radiology, Tianjin Key Laboratory of Functional Imaging, Tianjin Medical University General Hospital, Tianjin, 300052, Chinac College of Chemistry, Research Center for Analytical Sciences, State Key Laboratory of Medicinal Chemical Biology, and Tianjin Key Laboratory of Molecular Recognitionand Biosensing, Nankai University, Tianjin, 300071, China

A R T I C L E I N F O

Keywords:Persistent luminescenceTurning solid into gelPhotodynamic therapy

A B S T R A C T

Bioavailable persistent luminescence material is an ideal internal light source for long-term photodynamictherapy, but inevitably suffers from low utilization efficiency and weak persistent luminescence due to corrosionand screening processes. Herein, we show a facile and smart “turning solid into gel” strategy to fabricate per-sistent luminescence hydrogel for high-efficient persistent luminescence-sensitized photodynamic therapy. Thehomogeneous persistent luminescence hydrogel was synthesized via dispersing high-temperature calcined per-sistent luminescence material without corrosion and screening into a biocompatible alginate-Ca2+ hydrogel. Thesimple synthesis strategy allows 100% of utilization efficiency and intact persistent luminescence of persistentluminescence material. The persistent luminescence hydrogel possesses favorable biocompatibility, bright per-sistent luminescence, red light renewability, good syringeability, and strong fixing ability in tumors. The per-sistent luminescence hydrogel can be easily injected in vivo as a powerful localized light source for superiorpersistent luminescence-sensitized photodynamic therapy of tumors. The “turning solid into gel” strategy en-ables taking full advantages of persistent luminescence for biological applications, and shows great potential inutilizing diverse theranostic agents regardless of hydrophilicity and hydrophobicity.

1. Introduction

Photodynamic therapy (PDT) with specific spatiotemporal se-lectivity and minimal invasiveness has become an emerging solution forcancer therapy from fundamental research to clinic [1,2]. Revolu-tionary technologies that shift the excitation sources of PDT from ul-traviolet/visible (UV/vis) light to near-infrared (NIR) light [2], up-conversion fluorescence [3,4], NIR two-photon excitation [5,6], X-rayradiation [7], self-illumination (chemiluminescence [8,9], biolumines-cence [9,10], and Cerenkov luminescence [11]) greatly promote theflourishing development of PDT with superior therapeutic efficacy [2].However, these technologies suffer from the risks of ionizing radiationinjury, tissue ablation induced by long-term irradiation and insufficientself-illumination efficiency [1,2]. Persistent luminescence (PersLum)material is a kind of optical material that can emit luminescence afterthe ceasing of exciting light [12–17]. The optical excitation energy canbe trapped at the specific defects of PersLum material during excitation

and slowly released as afterglow after excitation. The unique NIR per-sistent luminescence and the renewability of PersLum not only permitbiosensing [18–24] and rechargeable bioimaging without the need of insitu excitation [25–47], but also can serve as an internal light source forrepeated long-term photodynamic therapy (PDT) in deep tissues[48–54] and other theranostic applications [55–65]. Excellent PersLumproperties play a crucial role in biological applications of persistentluminescence material [12–15]. Traditional PersLum-sensitized PDTrequired PersLum material with small size and favorable water solu-bility, which can be easily dispersed in water for intravenous andsubcutaneous injections. However, there is a conflict between uniformmorphology/small size and intense and long-lasting afterglow of Per-sLum material, and this troublesome problem has not been solvedcompletely up to now [12–15]. For instance, high temperature sinteringis the most efficient method for improving the PersLum performance,but results in agglomerated products with irregular morphologies (hightemperature sintering PersLum material was defined as “PLM”).

https://doi.org/10.1016/j.biomaterials.2019.119328Received 15 March 2019; Received in revised form 29 June 2019; Accepted 30 June 2019

* Corresponding author.** Corresponding author.E-mail addresses: shaokaisun@tmu.edu.cn (S.-K. Sun), chunshuiyu@tmu.edu.cn (C. Yu).

Biomaterials 218 (2019) 119328

Available online 02 July 20190142-9612/ © 2019 Elsevier Ltd. All rights reserved.

T

Subsequent corrosion under alkali condition and screening are neces-sary to obtain small-size and water-soluble persistent luminescencenanoparticles (PLNPs), but lead to 90% mass loss of PLM and remark-able decrease of luminescence performance due to the damage ofcrystal lattices [12–15]. While hydrothermal method without hightemperature calcining is employed to produce with small size and goodwater solubility (H-PLNPs), but leads to a much lower PersLum per-formance [12–15]. Besides, multiple surface modifications, such asamination and PEGylation, are always necessary to improve water so-lubility, biocompatibility and targeting ability for PersLum-sensitizedPDT [12–15]. Recently, a novel solid PLM implant has been developedfor irradiation-free PDT, but the invasive transplantation of solid im-plants requires complicated and professional operation, brings potentialside effects induced by invasive administration, and has difficulty in thetreatment of deep tumors [51]. Therefore, it is highly desired to developa smart strategy to take full advantages of PersLum for PDT, unifyingthe high utilization efficiency of PLM, high-performance PersLum, andfacile synthesis and administration processes.

Herein, we show a facile and smart “turning solid into gel” strategyto fabricate PersLum material hydrogel (PLM-hydrogel) for high-effi-cient PersLum-sensitized PDT (Scheme 1). The homogeneous PLM-hy-drogel was prepared by simply dispersing the high temperature cal-cined PLM without corrosion under alkali condition and screening intoalginate-Ca2+ hydrogel, which is formed by mixing FDA approved al-ginate solution and Ca2+ [65–67]. The intense PersLum and longafterglow of PLM synthesized under high temperature was retained tothe maximum degree, and the utilization efficiency of PLM reached100%. The as-prepared PLM-hydrogel with good syringeability can beeffortlessly injected in tumor sites of mice in vivo without the leakagefrom the path of the needle. The intelligent introduction of PLM with abig size into tumors via hydrogel injection facilitates the accumulationof PLM in tumors and minimizes the side effects induced by the pene-tration into bloodstream and other tissues of PLM. The PLM-hydrogelwith intact PersLum of PLM and red light renewability as a robust in-ternal light source permits superior repeated PersLum-sensitized PDT oftumors. The proposed PLM-hydrogel provides a promising way to takefull advantages of the PersLum for PDT, fusing 100% utilization effi-ciency of PLM, intact PersLum, and facile synthesis and administrationprocesses. More importantly, the “turning solid into gel” strategy showsgreat potential in the utilization of diverse theranostic agents regardless

of their hydrophilic and hydrophobic features. To the best of ourknowledge, it is the first time that PLM-hydrogel was developed via“turning solid into gel” strategy for superior PDT of tumors.

2. Experimental section

2.1. Chemicals

All chemicals were at least of analytical grade and used withoutfurther purification. Ultrapure water was provided by HangzhouWahaha Group Co. Ltd (Hangzhou, China). Zinc nitrate, gallium oxide,germanium oxide, chromium nitrate, ytterbium nitrate, erbium nitrate,Na2HPO4·12H2O, NaH2PO4·2H2O, nitric acid, cetyl-trimethylammoniumbromide (CTAB), ammonia, sodium alginate, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),Indocyanine Green (ICG), 2′,7′-Dichlorodihydrofluorescein diacetate(DCFH-DA) and 1,3-Diphenylisobenzofuran (DPBF) were bought fromAladdin Reagent Co. Ltd. (Shanghai, China). Ce6 was obtained from J&K Scientific (Beijing, China). DMSO was supplied by ConcordTechnology (Tianjin, China).

2.2. Characterization

X-ray diffraction (XRD) patterns were measured on a D/max-2500X-ray diffractometer (Rigaku, Japan). The scanning electron micro-scope (SEM) images of PLM and PLM-hydrogel were characterized by aMerlin Compact Field-Emission-Scanning Electron Microscope (CarlZeiss, Germany). Rheological tests were performed at 25 °C using aDHR-2 rheometer (TA Instruments, U. S. A). The absorption spectrawere obtained on a UV-3600 UV-vis-NIR spectrophotometer (Shimadzu,Japan). Photoluminescence spectra and time decay curves were col-lected on an F-4500 spectrofluorometer (Hitachi, Japan). The persistentluminescent imaging of PLM, PLNPs, H-PLNPs, PLM-hydrogel, PLM-hydrogel pattern, PLNPs-hydrogel, and H-PLNPs-hydrogel were carriedout on an IVIS imaging system (Caliper Co.) under bioluminescenceimaging mode. Red light illuminations (0.7W/cm2) were used for there-activation of solid persistent phosphors and persistent phosphorhydrogel, respectively. The absorbance for the MTT assay was recordedon a microplate reader (Bio-tek, USA Park, CA).

Scheme 1. Schematic illustration of fabrication of PLM-hydrogel via turning solid into gel for high-efficient PersLum-sensitized photodynamic therapy.

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2.3. Preparation of PLM, H-PLNPs and PLNPs

PLM (Zn1.25Ga1.5Ge0.25O4: 0.5%Cr3+, 2.5%Yb3+, 0.25%Er3+,ZGGO: Cr3+,Yb3+,Er3+) was synthesized by a hydrothermal method incombination with sintering in air. Firstly, Ga2O3 was dissolved in nitricacid under hydrothermal conditions at 120 °C, and GeO2 was dissolvedin dilute ammonia solution (3%). 0.375mL of chromium nitrate(0.1 M), 12.5 mL of zinc nitrate (0.5M), 1.875mL of ytterbium nitrate(0.1 M), 1.875mL of erbium nitrate (0.01M) and 6.25mL ammoniumgermanate (0.2M) were added to 7.5mL of gallium nitrate solution(1M) in order according to the chemical formula ofZn1.25Ga1.5Ge0.25O4:0.5%Cr3+, 2.5%Yb3+, 0.25%Er3+ under vigorousstirring. After the addition of 80mg cetyltrimethyl ammonium bromide(CTAB), the resulting solution was adjusted to pH=8.0. The reactionsolution was then ultrasonicated for 30min, stirred for 1 h, and sealedinto a 30mL teflon-lined autoclave at 120 °C for 15 h. The resultingproducts was washed orderly with ultrapure water and ethanol, andthen lyophilized to obtain H-PLNPs. The dried H-PLNPs was sintered inair at 1000 °C for 1.5 h and grinded in quartz to obtain PLM solid powerfor further use.

To obtain PLNPs, the PLM powder was wet grinded in minimumethanol, dispersed in 5mM NaOH and vigorously stirred for 24 h, andthe resulting colloid solution was centrifuged at 10000 rpm for 5min toremove NaOH. The collected precipitation was redispersed in waterunder ultrasonication, and centrifugated at 3500 rpm for 5min to re-move large size particles. The PLNPs in resulting supernatant werecollected by centrifugation at 10000 rpm for 5min and washed withwater, and the obtained PLNPs were freeze-dried for further study.

2.4. Preparation of PLM-hydrogel, H-PLNPs-hydrogel and PLNPs-hydrogel

PLM, PLNPs or H-PLNPs solid (8 mg) was dispersed in ALG solution(10mg/mL, 0.9 mL) respectively and kept magnetically stirring for2 h at room temperature. Then CaCl2 solution (0.1 mL, 10mg/mL) wasdropwise added into mixture to fabricate PLM-hydrogel, PLNPs-hy-drogel and H-PLNPs-hydrogel.

2.5. The stability assessment of PLM-hydrogel in different media

PLM-hydrogel (8 mg/mL) was incubated with various media in-cluding normal saline, phosphate buffer solution (PBS), and DMEMculture medium, and the stability of PLM-hydrogel was monitored bytaking photos for 25 days.

2.6. In vitro singlet oxygen (1O2) detection

The generation of 1O2 was measured by the absorption decrease ofDPBF as a classic probe. The photostability of free DPBF (25 μg/mL) orCe6 (2 μg/mL) was firstly evaluated after red irradiation for differenttimes, and the absorption of DPBF and Ce6 mixture in ethanol wererecorded as control group. Pre-excited PLM, PLNPs and H-PLNPs(15mg) were added to 3mL DPBF-Ce6 solution (DPBF: 25 μg/mL; Ce6:2 μg/mL), and the supernatant was collected by centrifugation at dif-ferent time points (5 and 10min respectively). The absorbance of thesupernatant was measured to evaluate PersLum sensitized generationability of 1O2.

To further investigate the renewable 1O2 generation ability of per-sistent phosphors, Pre-excitation PLM, PLNPs and H-PLNPs (5mg PLM/mL) suspended in water were added to the DPBF-Ce6 ethanol solution.The supernatant and persistent phosphor precipitate were collected bycentrifugation respectively at 10min. The absorbance of the super-natant was recorded to evaluate the generation of 1O2, and persistentphosphor precipitate was irradiated with red light (0.7W/cm2, 5min)to re-activate their PersLum. The above-mentioned corresponding su-pernatant was then mixed with re-activated PLM, PLNPs and H-PLNPsfor 5min, and the new supernatant was collected for the measurement

of its absorbance to evaluate the renewable 1O2 generation ability ofdifferent persistent phosphors.

2.7. In vitro toxicity assessment of PLM-hydrogel and Ce6

The cytotoxicities of PLM-hydrogel and Ce6 were investigated via astandard MTT assay using 4T1 cells. 4T1 cells were seeded in 96-wellplates at a density of 1×104/well and cultured in DMEM supplementedwith 10% FBS at 37 °C in 5% CO2 for 24 h. Then the wells were washedwith PBS and incubated with fresh media containing different con-centrations of PLM-hydrogel (2, 4, 6, 8 and 10mg PLM/mL) or Ce6(0.05, 0.1, 0.2, 0.4 and 0.8mg/mL). After cultured for 24 h, stale mediawere replaced by fresh culture media containing MTT (10 μL, 5mg/mL)and incubated at 37 °C for another 4 h. Then the old media were dis-carded, followed by the addition of 120 μL DMSO to dissolve the formedformazan crystals. The absorbance of each well was determined by amicroplate reader at the wavelength of 490 nm. The cell viability wascalculated using the following formula: Cell viability (%) = (Mean ofabsorption of treatment group/mean absorption of controlgroup)× 100%.

2.8. Singlet oxygen (1O2) generation at cellular level

4T1 cells were seeded in 96-well plates at a density of 1×104/welland cultured in DMEM supplemented with 10% FBS at 37 °C in 5% CO2

for 24 h. The cells were then exposed to different treatments: PBS(control group); pre-excitation PLM-hydrogel; Ce6 for 6 h; Ce6 for6 h + pre-excitation PLM-hydrogel; Ce6 for 6 h + red light irradiation(0.7 W/cm2, 3 min); Ce6 for 6 h + red light irradiation (0.7 W/cm2,3 min); and Ce6 for 6 h + pre-excitation PLM-hydrogel + red lightirradiation (0.7 W/cm2, 1min three times with an interval of 1 h). Theconcentrations of Ce6 and PLM-hydrogel used in each group were fixedto 0.2 mg/mL and 0.8mg PLM/mL, respectively. The stale media werereplaced by the fresh culture media containing DCFH-DA (1 μM) for20min, and then cellular fluorescent imaging was carried out to eval-uate 1O2 generation via an inverted fluorescence microscope.

2.9. In vitro PDT evaluation

The PersLum sensitized repeated PDT effect in vitro of PLM-hy-drogel was assessed via a MTT assay using 4T1 cells. 4T1 cells wereseeded in 96-well plates at a density of 104/well and cultured in DMEMsupplemented with 10% FBS at 37 °C in 5% CO2 for 24 h. The cells werethen exposed to different treatments: PBS (control group); pre-excita-tion PLM-hydrogel; Ce6 for 6 h; Ce6 for 6 h + pre-excitation PLM-hydrogel; Ce6 for 6 h + red light irradiation (0.7 W/cm2, 3 min); Ce6for 6 h + red light irradiation (0.7 W/cm2, 3 min); and Ce6 for6 h + pre-excitation PLM-hydrogel + red light irradiation (0.7 W/cm2,1min three times with an interval of 1 h). The concentrations of Ce6and PLM-hydrogel used in each group were fixed to 2 μg/mL and 0.8mgPLM/mL respectively. After the incubation for 18 h, the stale mediawere replaced by the fresh culture media containing 5mg/mL MTT andcultured at 37 °C for 4 h. Thereafter, 120 μL DMSO per well was addedto dissolve the formazan crystals, and the absorbance at 490 nm of eachwell was recorded by a microplate reader. The cell viability was de-termined to evaluate PersLum sensitized repeated PDT effect of PLM-hydrogel.

2.10. Animal models

All small animal experiments were approved by the Tianjin MedicalUniversity Animal Care and Use Committee. The tumor models wereestablished by the subcutaneous injection of 4T1 cells into the backs ofthe Balb/c mice (Beijing HFK Bioscience Co. Ltd., China). In vivoimaging and therapy were preformed when the tumor size reached to0.5–0.8 cm3.

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2.11. In vivo PersLum imaging observation of PLM-hydrogel

The mice bearing 4T1 tumors were intratumorally injected with200 μL pre-formed PLM-hydrogel (8 mg PLM/mL) with a syringe, whichwas pre-excited by a 254 UV lamp for 5min. The PersLum imagingbefore and after injection was performed on an IVIS imaging system atdifferent time points (5, 30 and 60min). Red light illumination (1W/cm2) was conducted on the tumors of mice for 10min before re-acti-vated PersLum imaging at 1 h, 1 d, 3 d, 5 d and 7 d after PLM-hydrogelinjection.

2.12. In vivo PersLum sensitized PDT of tumors using PLM-hydrogel

To investigate in vivo PDT, the mice bearing 4T1 tumors wererandomly divided into five groups (n=3 in each group) for differenttreatments as follows:

Group A: Control group without the administration of any agentsand light illumination;Group B: Intravenous injection of Ce6 (200 μg/mL);Group C: Intravenous injection of Ce6 (200 μg/mL) and red lightillumination (1W/cm2, 10min) after 2 h;Group D: Intravenous injection of Ce6 (200 μg/mL) and in-tratumoral administration of 254 UV lamp pre-excitation PLM-hy-drogel (200 μL, 8mg PLM/mL) with for 5min after 2 h;Group E: Intravenous injection of Ce6 (200 μg/mL), intratumoraladministration of 254 UV lamp pre-excited PLM-hydrogel (200 μL,8mg PLM/mL) with for 5min after 2 h, and red light illumination(1W/cm2, 10min) after 3 h.

The images of mice were taken by digital camera to monitor thetumor change. The tumors volumes of mice with various treatmentswere measured by a caliper according to the following formula: tumorvolume (V) = (length)× (width)2/2. Relative tumor volume was cal-culated as V/V0, where V means tumor volume after treatment and V0

refers to tumor volume before treatment.During the tumor monitoring, the body weights of mice in each

group were also recorded. Besides, Kunming mice were treated with orwithout intravenous injection of Ce6 (200 μg/mL) and subcutaneouslyinjection of PLM-hydrogel (200 μL, 8mg PLM/mL), the major organs(heart, liver, spleen, lung and kidney) of mice in the control andtreatment groups were collected and fixed by 4% paraformaldehyde at1, 7, 15 day. The histopathological study was performed by the he-matoxylin−esin (H&E) staining and analyzed by optical microscope.

3. Results and discussion

3.1. Synthesis and characterization of PLM, PLNPs and H-PLNPs

PLM with bright PersLum, ZGGO: Cr3+,Yb3+,Er3+, was synthesizedby a classic hydrothermal process in combination with high tempera-ture calcining [35]. The synthesized PLM exhibited a spinel cubic phasestructure (JCPDS 25–1240) without other phases of impurities such asZnO, GeO2, and Ga2O3 by X-ray diffraction (XRD) pattern analysis(Fig. 1a) [35]. Scanning electron microscope (SEM) images showed thePLM had an agglomerated irregular morphology due to the high tem-perature sintering (Figs. S1a and S1b). The excitation spectrum of PLMgave four bands derived from the characteristic peaks of Er3+, Cr3+ andYb3+ in a wide range of spectral regions from 200 nm to 600 nm(Fig. 1b) [35]. The excitation band at 254 nm was attributed to thecombination of the ZGGO host excitation band and the O-Cr chargetransfer band [35]. The other three bands at 410 nm, 463 nm, and570 nm resulted from the 3d intrashell transitions of Cr3+, i.e. 4A2→

4T1

(te2), 4A2→4T1 (t2e), and 4A2→

4T2 transition, respectively [35]. ThePLM gave a NIR emission band ranging from 600 to 800 nm with anemission peak at 695 nm under the excitation at 254 nm (Fig. 1b) [35].

The PLM had a brighter NIR fluorescence compared to PLNPs and H-PLNPs, and the much stronger PersLum of PLM can be clearly observedby naked eye (Fig. 1c). Quantitative analysis further revealed the Per-sLum intensity of PLM was at least 2 and 300 times higher than PLNPsand H-PLNPs respectively after 7min past since excitation stopped(Fig. 1d). In addition, PersLum images revealed all of these three ma-terials can be reactivated by mild red light illumination repeatedly(Fig. 1e), and recharged PLM still exhibited much stronger PersLumthan PLNPs and H-PLNPs (Fig. 1e). The superior PersLum makes PLMan ideal light source for PDT application instead of PLNPs and H-PLNPs.

3.2. Synthesis and characterization of PLM-hydrogel, PLNPs-hydrogel andH-PLNPs-hydrogel

Despite appealing afterglow of PLM, the agglomerated irregularmorphology makes it impossible for biological applications. Thus cur-rent PersLum sensitized PDT was limited to the usage of PLNPs and H-PLNPs with much weaker PersLum. The introduction of hydrogel totransform PLM from solid state to injectable hydrogel provides a pro-mising way for taking full advantages of PersLum. Sodium alginatehydrogel (ALG) with excellent biocompatibility, which is formed bysimply mixing Ca2+ and FDA approved alginate in aqueous solution, isextensively used in various biological applications, such as drug de-livery, cell transplantation, and biomedical engineering. The PLM-hy-drogel with good stability was fabricated by simply dispersing PLM inALG hydrogel (Scheme 1 and Fig. S2), and the syringeability, hardnessand viscosity of PLM-hydrogel can be readily tuned by changing theamounts of Ca2+ and PLM. Compared to the 90% mass loss in corrosionand screening processes in the synthesis of widely used PLNPs, the PLM-hydrogel with 100% of utilization efficiency of PLM showed great su-periority in reducing time, cost, and energy consuming. Higher hard-ness and viscosity favored the PLM loading efficiency, but was adverseto syringeability of the PLM-hydrogel. To compromise high hardness/viscosity and syringeability, the optimal amounts of Ca2+ (1mg/mL)and PLM (8mg/mL) was used for the following studies (Fig. 2a). Therheology of PLM-hydrogel showed that G′ is higher than G″, whichrevealed the proposed PLM-hydrogel is indeed a gel (Fig. S3) [68].

SEM images indicated the aggregate PLM became uniform nano-particles with an average size of approximately 200 nm, and werehomogeneously dispersed in the PLM-hydrogel (Fig. 2b and Figs. S1cand S1d). This morphology transformation was attributed to the strongcoordination interaction between PLM and ALG, greatly facilitating thehomogeneous distribution of PLM in PLM-hydrogel for effective PDT.The stability of PLM-hydrogel incubated with various media includingnormal saline, PBS, and DMEM was evaluated for 25 days (Fig. S4). Thephotos revealed the hydrogel only gradually swelled along with thetime and enabled maintaining their homogeneous structure for a longtime due to its good stability.

Moreover, the good syringeability of as-prepared PLM-hydrogelenabled the formation of PLM-hydrogel pattern (TMU) easily, and thefluorescent and PersLum images of PLM-hydrogel both proved theirattracting afterglow property, favorable syringeability and homo-geneity (Fig. 2c–f). Furthermore, various volumes (20–100 μL) of PLM-hydrogel with excellent reproducibility and homogenous luminescencecan be readily extruded (Fig. 2g and h), facilitating the administrationin biological application in vivo.

3.3. Optical properties of PLM-hydrogel

PLM-hydrogel can inherit the PersLum of PLM to the maximumextent due to the retainment of intact crystal structure during the mildmixing process without the need for corrosion and screening. The muchstronger fluorescence of PLM-hydrogel can be observed under UV lampcompared to PLNPs-hydrogel and H-PLNPs-hydrogel (Fig. 2i-n). Theexcitation and emission spectra of PLM-hydrogel were similar to PLMowing to the minimized influence on the luminescent features of PLM

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during the formation of PLM-hydrogel (Fig. 3a). The synthesized PLM-hydrogel exhibited bright PersLum in NIR region, and the PersLumintensity of PLM-hydrogel was at least 1.7 times higher than PLNPs-hydrogel and 200 times higher than H-PLNPs-hydrogel after 25 s pastsince excitation stopped (Fig. 3b). The long-lasting afterglow of PLM-hydrogel can be detected even after 24 h in PersLum imaging (Fig. 3c).The PersLum of PLM-hydrogel can be repeatedly reactivated by mildred light illumination (Fig. 3c and d), and the reactivated PersLum ofPLM-hydrogel was still much brighter than PLNPs-hydrogel and H-PLNPs-hydrogel (Fig. 3c). These results clearly testified the appealingsyringeability and superior PersLum of PLM-hydrogel, showing greatpotential in PersLum sensitized PDT.

3.4. In vitro generation of 1O2

Photosensitizers play a crucial role in PDT, which can convert O2 tocytotoxic singlet oxygen (1O2) under light irradiation. In our study,chlorin e6 (Ce6) with high PDT efficiency was chosen as a modelphotosensitizer. The overlap of absorption of Ce6 as a model photo-sensitizer and luminescence of PLM permits NIR PersLum sensitizedPDT without the need for continuous light irradiation (Fig. 4a). 1, 3-Diphenylisobenzofuran (DPBF) as a classic probe was used for the de-termination of the generation of 1O2, which can destroy the structure ofDPBF and lead to a decrease of its absorption at 410 nm [69]. Free Ce6did not give rise to the absorption decrease of DBPF unless red lightirradiation was applied (Figs. S5–7). In contrast, pre-excitation PLM ledto remarkable decrease of absorption of DBPF in the presence of Ce6without the need for red light illumination, indicating the effectivegeneration of 1O2 triggered by PersLum instead of continuous light ir-radiation (Fig. 4b and Fig. S8). In addition, PLM-induced production of1O2 was renewable after mild red light illumination, guaranteeing re-peated PDT capability to realize high therapeutic efficacy. The

generation ability of 1O2 induced by PersLum highly depended on thePersLum intensity of PLM, and PLNPs and H-PLNPs with much weakerPersLum only triggered the production of less 1O2 (Fig. 4b, Figs. S9 andS10).

3.5. Cytotoxicity and PersLum sensitized PDT in vitro

The cytotoxicity of PLM and PLM-hydrogel was evaluated by astandard MTT assay using 4T1 cells. Over 90% cancer cells remainedalive even after the incubation with 0.8 mg/mL of PLM and PLM-hy-drogel for 24 h, demonstrated their excellent biocompatibility (Fig. 4c).PersLum-sensitized generation of 1O2 in cellular level was then eval-uated by cellular fluorescent imaging using a DCFH-DA probe (Fig.S12). The results revealed pre-excited PLH or red light both can lead toa remarkable generation of 1O2 in Ce6-treated cancer cells, and thecombination treatment of pre-excited PLH and red light is capable ofinducing the maximum amounts of 1O2 in Ce6-treated cancer cells. ThePLM with good biocompatibility and high generation capability of 1O2

was then employed for PDT in vitro. 4T1 cells were exposed to control,Ce6, Ce6 + red light, Ce6 + pre-excitation PLM-hydrogel, andCe6 + pre-excitation PLM-hydrogel + red light, and the cell viabilitieswere determined by a MTT assay (Fig. 4d and Fig. S11). Fig. 4d re-vealed only pre-excitation PLM-hydrogel or Ce6 can't lead to obviouscell death, and 53% of cancer cells were damaged after the treatmentsof Ce6 and red light based on traditional PDT. In contrast, the pre-excitation PLM-hydrogel plus Ce6 led to 40% of cell death without theneed for external light source, and further introduction of red light re-activation (0.7W/cm2, 3min) remarkably improved the therapeuticefficacy and 82% of cells were destroyed due to the renewability ofPersLum sensitized PDT. Moreover, giving an 1-min red light illumi-nation every hour for 3 times also resulted in a similar cell-killing effectvia PersLum-sensitized PDT, which is an alternative recharging

Fig. 1. Characterization of PLM, PLNPs and H-PLNPs. (a) The XRD patterns of PLM, PLNPs and H-PLNPs solid powder. (b) The excitation and emission spectra ofPLM: excitation (Ex.) at 254 nm, 410 nm and 570 nm; emission (Em.) at 696 nm. (c) The photos of H-PLNPs, PLNPs and PLM under natural light, UV light, and in thedark just after the stoppage of UV light irradiation. (d) PersLum decay curves of H-PLNPs, PLNPs and PLM (0.5 g solid), monitored at 695 nm after 5min 254 nm UVlamp irradiation. (e) In vitro PersLum images of pre-excitation PLM, PLNPs and H-PLNPs, re-activated by red light (0.7W/cm2, 5min) after 480 h. (For interpretationof the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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approach to 3-min red light re-activation, and the flexible choice ofrepeated PDT strategy facilitates the potential clinical applications. Theabove results prove the low cytotoxicity of PLM and PLM-hydrogel, andeffective PersLum-sensitized PDT.

3.6. PersLum imaging, PersLumPersLum-sensitized PDT and toxicityevaluation in vivo

We then investigated the feasibility of PersLum sensitized PDT in

vivo using PLM-hydrogel. High accumulation of theranostic agents intumors and low leakage to surrounding tissues play a crucial role inhigh-efficient tumor theranostics with minimal side effects. The smartdesign of turning solid PLM into injectable PLM-hydrogel not only al-lowed the injection without leakage of PLM from the path of needle, butalso permitted the introduction of PLM with big size into tumors (Fig.S13). In contrast, the injected ICG solution leaked out from path ofneedle obviously. The strong fixing ability in tumors of PLM with bigsize greatly benefited the high PDT efficacy, and minimized the

Fig. 2. Synthesis and characterization of PLM-hydrogel. (a) Optimization of the synthesis of PLM-hydrogel via varying the amounts of Ca2+ and PLM. (b) SEM imageof PLM-hydrogel. Photographs of PLM-hydrogel pattern (TMU) under (c) natural light and (d) UV light. (e) Photograph and (f) Persistent luminescent image of PLM-hydrogel pattern on an IVIS imaging system. Photographs of Different volumes of PLM-hydrogel under (g) natural light and (h) UV light. (i)–(n) Various persistentphosphors dispersed in ALG-Ca2+ hydrogel under natural light (up) and UV light (down), i and l: H-PLNPs-hydrogel, j and m: PLNPs-hydrogel, k and n: PLM-hydrogel.

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potential safety threats induced by the penetration into bloodstreamand other tissues. For in vivo application, the pre-excited PLM-hydrogelwas injected to the tumors of the mice, and the strong PersLum of PLM-hydrogel can be observed from optical imaging clearly (Fig. 5a). ThePersLum can be readily reactivated by red light illumination at 1 h, andeven 7 days after the administration of PLM-hydrogel. The pre-excitedPLH showed intense initial and re-activated PerLum both with highsignal-to-noise ratios indicated the excellent PersLum feature and out-standing renewability (Fig. 5a). Moreover, the long-lasting bright Per-sLum was mainly present in the tumor region for a long time,

demonstrating the outstanding fixing ability in tumors of PLM that canserve as a powerful inert light source for PDT (Fig. 5a). The micebearing tumors were intravenously injected with Ce6, and fluorescentimaging indicated the Ce6 was mainly accumulated in tumors at 2 h(Fig. S14). The intratumoral injection of pre-excitation PLM-hydrogeland red light irradiation were then employed to trigger PDT at 2 h afterthe administration of Ce6 (Fig. 5b and c and Fig. S15). PLM-stimulatedPDT exhibited a similar tumor suppression effect as external lightsource, revealing that PLM can serve as an excellent internal light forPDT instead of continuous light irradiation. Furthermore, the mild red

Fig. 3. Optical properties of PLM-hydrogel. (a) Theexcitation and emission spectra of PLM-hydrogel:emission at 254 nm, 410 nm and 570 nm and excita-tion at 696 nm. (b) NIR PersLum decay curves of H-PLNPs-hydrogel, PLNPs-hydrogel, and PLM-hydrogelunder the excitation of 254 nm UV lamp. (c) NIRPersLum and red light-reactivated PersLum images invitro of H-PLNPs-hydrogel, PLNPs-hydrogel, andPLM-hydrogel (8 mg/mL). (d) The renewablePersLum decay curves under the excitation of a254 nm UV lamp and re-activation of red light. (Forinterpretation of the references to colour in thisfigure legend, the reader is referred to the Web ver-sion of this article.)

Fig. 4. PersLum sensitized generation of 1O2 andphotodynamic therapy in vitro. (a) The overlap ofabsorption spectrum of Ce6 and luminescent spec-trum of PLM-hydrogel. (b) PersLum sensitized gen-eration capability of 1O2 of PLM-hydrogel, PLNPs-hydrogel and H-PLNPs-hydrogel. (c) The cytotoxicityof PLM-hydrogel and PLM via an MTT assay. (d) PDTin vitro using PLM (group 1: Control; group 2: 0.8 mgPLM/mL pre-excitation PLM-hydrogel; group 3: 2 μg/mL Ce6; group 4: 2 μg/mL Ce6 + 0.8 mg PLM/mLpre-excitation PLM-hydrogel; group 5: 2 μg/mLCe6 + 3 min red light (L1) irradiation; group 6: 2 μg/mL Ce6 + 0.8 mg PLM/mL pre-excitation PLM-hy-drogel + 3 min red light (L1) irradiation; group 7:2 μg/mL Ce6 + 0.8 mg PLM/mL pre-excitation PLM-hydrogel + 1 min red light (L2) irradiation; group 8:2 μg/mL Ce6 + 0.8 mg PLM/mL pre-excitation PLM-hydrogel + 1 min red light (L2) irradiation twicewith an interval of 1 h; group 9: 2 μg/mLCe6 + 0.8 mg/mL pre-excitation PLM-hy-drogel + 1 min red light (L2) irradiation three timeswith an interval of 1 h). ns: no significant difference,*p < 0.05, **p < 0.01, ***p < 0.001. (For inter-pretation of the references to colour in this figurelegend, the reader is referred to the Web version ofthis article.)

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right irradiation (1W/cm2, 5min) enables the renewability of PLM andfurther improves the tumor therapeutic efficacy remarkably. In con-trast, the tumors of mice in the control group kept growing quickly allthe time. The sizes of tumors in mice treated with PLM-PDT with redlight re-activation were 16 times smaller than those in the controlgroup, proving the appealing PDT efficacy of PLM (Fig. 5c).

To evaluate in vivo toxicity, body weight change monitoring andhistopathological analysis of major organs were preformed after various

treatments. The body weight change of mice after PDT did not exhibitobvious difference from the control group, revealed the low toxicity invivo of PLM and PLM-hydrogel (Fig. 5d). Hematoxylin and eosin (H&E)staining results indicated the major organs of mice (including heart,liver, spleen, lung, and kidney) did not show obvious histopathologicalchange compared to the normal mice, confirming the excellent bio-compatibility of PLM and PLM-hydrogel and biosafety of PLM-hydrogeltriggered PDT (Fig. S16).

Fig. 5. In vivo PersLum imaging and PLM-hydrogel triggered PDT. (a) Persistent and renewable luminescent imaging in vivo. (b) The photos of tumors in mice withdifferent treatments (group A: Control; group B: Ce6; group C: Ce6 + red light; group D: Ce6 + PLM; group E: Ce6 + PLM + red light). (c) The relative volumes oftumors in mice with different treatments (**p < 0.01, ***p < 0.001). (d) The body weight monitoring of mice with different treatments. (For interpretation of thereferences to colour in this figure legend, the reader is referred to the Web version of this article.)

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4. Conclusion

In summary, we developed a facile and smart “turning solid intohydrogel” strategy to synthesize PLM-hydrogel to take full advantagesof PersLum for high-efficient PDT. The homogeneous PLM-hydrogelwas synthesized by simply dispersing high temperature sintering PLMinto ALG-Ca2+ hydrogel. Compared to traditional PLNPs and H-PLNPswith 90% loss of mass and weaken PersLum, the PLM-hydrogel not onlyhad 100% of utilization efficiency of PLM, but also maintained the in-tact PersLum of PLM. The proposed PLM-hydrogel possessed goodsyringeability, appealing biocompatibility, intense NIR PersLum, andred light renewability, which can trigger the continuous and renewableproduction of 1O2. The PLM-hydrogel can be noninvasively injectedinto body without the leakage from the path of needle, and the in-troduction of PLM with a big size exhibited strong fixing ability in tu-mors, favoring therapeutic efficacy and biosafety. Finally, the PLM-hydrogel with bright PL and red light renewability was applied inPersLum-sensitized repeated PDT in vitro and in vivo successfully. Webelieve the proposed strategy of turning solid to hydrogel not only takesfull advantage of PersLum for superior PDT, but also lays down a novelway to make theranostic agents become bioavailable regardless of theirhydrophilic and hydrophobic features.

5. Data availability

The raw/processed data required to reproduce these findings cannotbe shared at this time as the data also forms part of an ongoing study.

6. Competing financial interests

The authors declare no competing financial interests.

Acknowledgements

This work was supported by the National Natural ScienceFoundation of China (Grants 81671676, 21435001), Natural ScienceFoundation of Tianjin City (No. 18JCYBJC20800).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.biomaterials.2019.119328.

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