Upconversion Composite Nanoparticles for Tumor Hypoxia

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Biological and Medical Applications of Materials and Interfaces

Upconversion Composite Nanoparticles for Tumor Hypoxia Modulation and Enhanced NIR-triggered Photodynamic Therapy Tongxu Gu, Liang Cheng, Fei Gong, Jun Xu, Xiang Li, Gaorong Han, and Zhuang Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b03238 • Publication Date (Web): 23 Apr 2018 Downloaded from http://pubs.acs.org on April 23, 2018

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ACS Applied Materials & Interfaces

Upconversion Composite Nanoparticles for Tumor Hypoxia Modulation and Enhanced NIR-triggered Photodynamic Therapy Tongxu Gu,† Liang Cheng,‡ Fei Gong,‡ Jun Xu,‡ Xiang Li,*,† Gaorong Han,† Zhuang Liu*,‡ †

State Key Laboratory of Silicon Materials, School of Materials Science and Engineering,

Zhejiang University, Hangzhou, Zhejiang 310027, P. R. China ‡

Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-

Based Functional Materials & Devices, Soochow University, Suzhou 215123, P. R. China KEYWORDS. mesoporous silica nanoparticles, upconversion nanoparticles, manganese oxide photodynamic therapy, tumor hypoxia,

ABSTRACT. The efficacy of conventional photodynamic therapy (PDT) is markedly suppressed by limited penetration depth of light in biological tissues and oxygen depletion in the hypoxic tumor microenvironment. Herein, mesoporous silica nanospheres with fine CaF2: Yb, Er upconversion nanocrystals entrapped in their porous structure are synthesized via a thermal decomposition method. After subsequently coating with a thin MnO2 layer and loading with a photosensitizer, Chlorin e6 (Ce6), a new type of nanoscale PDT platform is obtained. Within such composite nanoparticles, Mn2+ ions doped into the lattice of CaF2 crystals effectively enhance the near-infrared (NIR)-triggered red-light upconversion photoluminescence for exciting

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the adsorbed Ce6 via resonance energy transfer, enabling improved photodynamic phenomenon. Meanwhile, the MnO2 coating modulates the hypoxic tumor microenvironment by in-situ generating O2 through the reaction with tumor endogenous H2O2. Both mechanisms acting synchronously lead to the superior therapeutic outcome in NIR-triggered photodynamic tumor therapy.

1. INTRODUCTION Photodynamic therapy (PDT) as an effective therapeutic approach with great spatiotemporal accuracy and minimal invasiveness, has aroused tremendous interests in tumor therapy.1-4 PDT causes cell oxidative photodamage by photosensitizers that produce reactive oxygen species (ROS) under light irradiation such as singlet oxygen (1O2).5 However, there still remains two challenges in conventional PDT. First, the majority of current photosensitizing agents are often excited by visible light which has poor tissue penetration.6-7 The other issue that limits direct cancer cell damage is the availability of oxygen within the tumor tissue.8 It is well known that low oxygen concentration regions are contained in most solid tumors due to the poor tumor vasculature. As PDT is an oxygen depletion process, the hypoxia within solid tumors remarkably suppresses the efficacy of PDT cancer treatment.9-12 Compared to visible light, near-infrared (NIR) light in the range of 700-1000 nm displays lower tissue absorbance, reduced scattering, and minimum photodamage to cells, and thus has received much attention for deep PDT treatment of tumors.13-15 One strategy to achieve NIRactivated PDT is based on lanthanide ions doped upconversion nanoparticles (UCNPs),16-17 which can convert NIR light into ultraviolet-visible light through two-photon or multiphoton processes, so as to excite nearby photosensitizers via resonance energy transfer.18-20 Thus, there


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is a burgeoning interest in the development of UCNPs-based deep-tissue PDT.21-24 As a typical strategy to contrast UCNP-based PDT platform, generally, UCNPs are synthesized by hydrothermal method, and subsequently coated with a silica shell by sol-gel method to form core-shell structures, into which photosensitizers are loaded.25-27 This approach involves cumbersome procedures in surface modification, and induces the pores with low dimensions in the silica shell which often shows restricted storage of functional molecules. On the other hand, aiming at improving the therapeutic responses of PDT to hypoxic tumors, there have been a number of groups reporting that MnO2 nanostructures could effectively relieve tumor hypoxia by their unique reactivity with tumor endogenous H2O2 to produce O2.21, 28-31 By incorporating UCNPs on the surface of MnO2 nanosheets, NIR-triggered PDT with tumor hypoxia relief has also been demonstrated in a recent work.32 However, in such system, the flaky MnO2 nanosheets would impact the dispersity of UCNPs and the upconversion luminescence (UCL) signals would be partially quenched by the black MnO2 nanosheets. Recently, a series of new methods have been proposed to incorporate functional factors within the mesopores in a more effective way.33-35 For instance, Chen et al. demonstrated that mesoporous silica nanoparticles (MSNs) can serve as a multifunctional nanoarchitecture by embedding iron oxide nanoparticles and seeding multi-gold nanorods within the pores.33 Guest factors that feature functional properties can be effectively maintained within the mesostructure, inspiring a promising methodology to create multifunctional composite structures. Herein, a new type of multifunctional UCNP-based nanocomposites was fabricated by utilizing MSNs with large pore sizes at the template. In our system, CaF2: Yb, Er nanocrystals were incorporated within the highly radial pores of MSN templates with the average pore size at ~8 nm, through the capillary effect and the thermal decomposition method (Scheme 1). Such upconversion


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composite nanoparticles maintained favorable dispersibility and high specific surface area. Afterwards, Mn2+ ions were doped into the lattices of CaF2: Yb, Er crystals during MnO2 coating on the surface of CaF2: Yb, Er@Silica (C@S) nanoparticles. Interestingly, the lattice distortion and exchange-energy transfer process with Er3+ caused by Mn2+ doping could lead to the enhanced UCL emission of red light under NIR irradiation, favorable for more efficient PDT with loading of Ce6 as the photosensitizer. Meanwhile, the thin MnO2 shell in the final CaF2: Yb, Er@Silica/MnO2-Ce6 (C@SMn-Ce6) nanocomposites showed the capability to effectively ameliorate tumor hypoxia by triggering the decomposition of tumor endogenous H2O2, further promoting the efficacy of PDT in destructing hypoxic tumors. Therefore, multifunctional upconversion composite nanoparticles that allow NIR-triggered effective PDT are fabricated in this work via a unique mesopore-based nano-templating method, which may be extended to the design of other types of nanocomposites for various interesting applications. 2. EXPERIMENTAL SECTION 2.1. Chemical and Reagents. Tetraethoxysilane (TEOS), cetyltrimethylammonium bromide (CTAB), ethanol, calcium acetate monohydrate (99%), Manganese(II)chloride, anhydrous (98%), Sodium hydroxide were acquired from Sinopharm Chemical Reagent Co., Ltd. Lysine, noctane (99%), 2, 2'-azobis(2-amidinopropane) dihydrochloride (AIBA), styrene (99%, contains 4-tert-butylcatechol as stabilizer), N-hydroxysulfosuccinimide (NHS), trifluoroacetic acid (TFA, 99.5%), 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC), PAA (Mw ≈ 1800),

3-aminopropyltriethoxysilane

(APTES),

3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aladdin Industrial Co., Ltd. Ytterbium(III) acetate hydrate (99.9%) and Erbium(III) acetate tetrahydrate (99.9%) were obtained from Thermo Fisher Scientific. Amino-terminated PEG (PEG-NH2, Mw ≈ 5000) was


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purchased from Biomatrik Inc. Hydrogen peroxide (H2O2, 30 wt%) was acquired from J&K chemical Co., Ltd. All chemicals were used as received. 2.2. Characterization. The morphology of nanoparticles were characterized by field-emission scanning electron microscopy (FESEM, Hitachi SU-70, Japan) operated at 3 kV and transmission electron microscopy (TEM, Tecnai F20, FEI, USA) operated at 200 kV. The phase identiﬁcation was done by X-ray diffraction (XRD, X’pert PRO MPD, Netherlands) for powder samples. The scan range was set from 10° to 80° and the step size was 0.167°. The zeta potential measurements

were

carried

out

with

Zetasizer

(ZEN3690,

Malvern,

UK).

N2

adsorption/desorption analysis (OMNISORP-100 apparatus) were carried out to evaluate the specific surface area, pore volumes and pore size distribution at liquid nitrogen temperature (77 K). The FTIR spectra (Tensor 27, Bruker, Germany) were measured by using a PerkinElmer 580B infrared spectrophotometer on KBr pellets. UV/Vis adsorption spectra were recorded by a spectrophotometer of TU-1810. The ﬂuorescence spectrophotometer (PL, FLSP920, Edinburgh) was used to record the upconversion photoluminescence spectra under 980-nm laser excitation. To minimize experimental uncertainties, the sample position and spectra collection were maintained under identical conditions. 2.3. Synthesis of C@S. MSNs were prepared following the standard method reported earlier.36 To incorporate CaF2: Yb, Er nanocrystals within the pore of MSNs, a precursor solution was prepared beforehand. Ca(OAc)2, Yb(OAc)3, and Er(OAc)3 were dissolved in deionized water to prepare a 0.5 M solution, and added with 12 ml TFA under stirring for 24 h. Subsequently, 300 mg MSN and 20 ml precursor solution were mixed and immersed for 24 h under slow stirring at 35 oC. The particles were then centrifugated at 8000 rpm for 5 min, and stood for 4 h at room


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temperature. After being air-dried at 80 oC overnight, the particles were further calcined at 600 o

C for 3 h.37 2.4. Synthesis of C@SMn. MnCl2 and C@S were dissolved in 10 ml deionized water with

different weight ratios (MnCl2 : C@S (w/w) = 1:2, 1:4, 1:8, 1:12, 1:20). After stirring 30 min at room temperature, 5 ml (0.05 M) NaOH was added under stirring for an additional 2 h. The products were collected by centrifugation and washed 3 times with ethanol.38 2.5. Surface Modification of C@SMn. The as-made C@SMn nanoparticles (10 mg) were dispersed in 10 ml ethanol under sonication for 10 min, and then added with 300 µl APTES under stirring at 50 oC. After 24 h, amine-modified nanoparticles were obtained, washed with ethanol once and re-dispersed in 4 ml deionized water, into which 2 ml PAA solution (5 mg/ml, Mw = 1800, pH = 8) was added under ultrasonication. After being stirred vigorously at 1100 rpm for 10 min, the suspension was centrifuged to remove excessive PAA and washed with water for 3 times. The product dissolved in 5 ml ultrapure water was added with 20 mg mPEG-NH2 (Mw = 5000) and 4 mg EDC, and thus stirred for 24 h before being purified by ultracentrifugation. C@S nanoparticles also went through the same surface modification process for subsequent parallel comparison. 2.6. Ce6 Loading. Ce6 was dissovled in DMSO with a concentration of 10 mg/ml first. 5 ml PEGylation C@SMn and 0.2 ml Ce6 were stirred at room temperature in dark with different mass ratios (Ce6 : C@SMn (w/w) = 0.1:1, 0.2:1, 0.5:1, 1:1, 2:1) for 4 h and centrifuged at 8000 rpm for 5 min. Excess Ce6 were removed by washing with deionized water for 3 times. Loading capacities and loading efficiencies were quantified by UV-vis absorption spectra, where the absorption peak of Ce6 was at 404 nm. C@SMn-Ce6 obtained at the feeding mass ratio of 0.2:1


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was used for further experiments. Ce6 were loaded on PEGylation C@S nanoparticles at the same feeding proportion to obtain C@S-Ce6. 2.7. Examination of singlet oxygen production. Singlet oxygen was detected by employing the fluorescence molecular probe Singlet oxygen sensor green (SOSG, USA). Different samples were exposed to 980-nm laser irradiation with the power density of 0.5 W/cm2. SOSG at 2.5 µM was used to evaluate singlet oxygen production by 2.5 mg/ml C@S-Ce6 and C@SMn-Ce6 with or without H2O2 (50 µM). The generation of singlet oxygen was estimated by detecting the recovery of SOSG fluorescence under 494 nm excitation. 2.8. Cellular Experiments. 4T1 (Murine breast cancer) cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin in a 37 °C incubator with 5% CO2. For cell cytotoxicity assay, 4T1 cells seeded in 96-well culture plates were treated with various concentrations of C@SMn-Ce6. After 12 h and 24 h, the cell viabilities were determined by standard methyl thiazolyl tetrazolium (MTT, Sigma Aldrich) assay and living/dead fluorescence staining (Calcein-AM: Ex = 488 nm, Em = 515 nm; propidium iodide: Ex = 543 nm, Em = 617 nm ) compared with untreated cells. To prove the cellular uptake of C@SMn-Ce6, 4T1 cells were incubated with free Ce6 or C@SMn-Ce6 (Ce6: 2 µM) for different time (0.5, 1, 2, and 4 h). After washing with PBS, The samples were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) at room temperature for confocal imaging (Ce6: Ex = 633 nm, Em = 720 nm; DAPI: Ex = 405 nm, Em = 470 nm). Intracellular ROS generation was examined using the 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA, Sigma-Aldrich) probe following the recommended protocol. The DCFH fluorescence intensity was measured using confocal microscopy at 488 excitation and 520 nm emission.


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2.9. In vitro PDT. 4T1 cells were seeded at 1×105 cells/well in 96-well plates and incubated overnight. Then various concentrations of C@S-Ce6 or C@SMn-Ce6 were added. The 96-well plates were incubated in normal environment (37 oC, 5 % CO2, 21 % O2) or hypoxia environment (37 oC, 5 % CO2, 1 % O2). After 4 h, the 96-well plates were exposed to 980-nm laser irradiation for 10 min (1 min interval) at a power density of 0.5 W/cm2. After that, the cells were transferred into fresh RPMI-1640 medium and incubated for additional 24 h. MTT assay was used for assessing the relative cell viabilities. 2.10. Tumor Model. Famale Balb/c mice (6-8 weeks old, 20g) were purchased from Soochow University Laboratory Animal Center. 50 µL PBS solution with 4T1 cells (1×106) were injected subcutaneously into the right side back of each mouse. The mice were used for PDT treatment as tumor volumes reached about 200 mm3. 2.11. In vivo Cancer Treatment. Mice with subcutaneous 4T1 tumors were randomly divided into five groups (five mice per group): (1) Control; (2) NIR; (3) C@SMn-Ce6 alone; (4) C@SCe6 + NIR; (5) C@SMn-Ce6 + NIR. The solution of C@SMn-Ce6 (50 µl, 20 mg/ml) was injected by intratumoral injection in group 3 and 5, while 50 µl of C@S-Ce6 (20 mg/ml) was i.t. injected into group 4. After 2 h, the mice in group 2, 4 and 5 were exposed to the 980-nm laser (0.5 W/cm2) for 30 min (1 min interval after each minute of irradiation to avoid heating damage caused by the laser). The body weights and tumor sizes were monitored in following 2 weeks, 2 days at a time. The following formula was used to calculate tumor volume: width2× length /2. The changes of tumor volume were normalized using the relative tumor volumes, which was calculated as V/V0 (V0 is the tumor volume before PDT treatment). To further confirmed PDT efficiency, all the tumors were sectioned into slices for hematoxylin & eosin (H&E) staining and examined by a microscope.


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2.12. Immunofluorescence staining. To study the hypoxia status in tumors, mice with 4T1 tumors were injected with PBS, C@S-Ce6 or C@SMn-Ce6 (25 µl, 10 mg/ml). The hypoxia specific staining for tumor slices was conducted using pimonidazole (Hypoxiyprobe-1 plus kit, Hypoxyprobe Inc) following the vendor protocol as reported earlier.32 The slices were examined by confocal microscopy (Leica SP5). 3. RESULTS AND DISCUSSION MSN templates were prepared prior to the synthesis of C@SMn nanoparticles. As shown in transmission electron microscope (TEM) and scanning electron microscope (SEM) images (Figure 1a, Supporting Figure S1), our synthesized MSNs present uniform sizes distribution with the average diameter of ~120 nm. And a radial pore structure was exhibited with the pore size at ~8 nm. Afterwards, those MSNs were mixed with the precursor solution containing metal trifluoroacetates including Ca(CF3COO)2, Yb(CF3COO)3 and Er(CF3COO)3 the designated ratio (molar ratio Ca : Yb : Er = 78 : 20 : 2).39 Then the precursor solution was diffused into the pores of MSNs via the capillary effect. After drying and calcination under 600 oC, upconversion CaF2: Yb, Er nanocrystals were formed in-situ within the pores of the MSN template. As shown under TEM imaging (Figure 1b), most of the formed CaF2: Yb, Er nanocrystals with higher contrast were found to be inside mesopores, while the structure of MSN templates was maintained in the obtained CaF2: Yb, Er@Silica (C@S) nanoparticles. For the subsequent growth of the MnO2 shell, C@S nanoparticles were mixed with MnCl2 under vigorous stirring. In this process, Mn2+ ions were adsorbed on C@S nanoparticles with negative surface charges. Afterwards, the solution pH value was adjusted by NaOH. With the following reaction,32 a thin MnO2 layer was formed on the surface of C@S (Figure 1c).


9


2MnCl2 + 4NaOH + O2 → MnO2 + 4NaCl + 4H2O

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(1)

Further characterizations were carried out for our obtained CaF2: Yb, Er@Silica/MnO2 (C@SMn) nanoparticles. In selected area electron diffraction (SAED) pattern (Figure 1c, inset), C@SMn nanoparticles showed distinct diffraction rings, which could assign to the (111), (220), (311) reflections of the CaF2’s cubic phase, implying the crystalline structure of CaF2 was well maintained in the final product. In addition, the element mapping demonstrated the homogenous elemental distribution of Si, Ca, F, O, Yb, Er, and Mn (Figure 1d) for the obtained C@SMn nanoparticles. The chemical valence of Mn element was investigated by X-ray photoelectron spectroscopy (XPS). Two characteristic peaks at 654.2 eV and 642.4 eV, corresponding to the quadrivalence Mn 2p1/2 and 2p3/2 spin-orbit peaks, were observed in the formed C@SMn sample (Figure S2). Furthermore, the X-Ray diffraction (XRD) pattern was in good agreement with the SAED pattern, and evidenced the cubic phase of CaF2 nanocrystals within the obtained C@SMn nanoparticles. The thin layer of MnO2 did not affect the position of XRD peaks, suggesting the amorphous nature of MnO2 (Figure 1e). As revealed by N2 adsorption/desorption isotherms (Figure 1f and Table S1), the specific surface areas were measured to be 747, 454, and 412 m2 g1

, for MSN, C@S and C@SMn samples, respectively, owing to the formulation of upconversion

nanocrytals within the mesopores of MSNs to reduce the measured surface area. Interestingly, the average pore sizes kept at ~8 nm for all three samples without significant variation, even after coating with MnO2. Those results imply that C@SMn nanoparticles remain in a mesoporous nature, paving the way to the subsequent photosensitizers loading. The optical properties of those nanoparticles were then carefully measured. It was found that the intensity ratio between green and red upconversion emission peaks of C@S and C@SMn nanoparticles was highly dependent on the MnO2 coating. As shown in Figure 2a, C@S with or


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without MnO2 exhibited 3 distinct emission bands at 516-533, 537-557 and 640-681 nm, which were assigned to the transition 2H11/2 → 4I15/2, 4S3/2 → 4I15/2, and 4F9/2 → 4I15/2 of Er3+ ions.40-41 As the increase of added MnCl2 during the fabrication of C@SMn, the absolute UCL intensity increased first and then showed decrease, while the intensity ratio of red-to-green peak increased constantly. For the C@SMn nanoparticles prepared with MnCl2 : C@S (Mn : C@S) mass ratio of 1:12, the luminescence intensity reached approximately 3 times higher than that of naked C@S (Figure 2b). To understand the mechanism of UCL tuning by MnO2 coating for our nanoparticles, XRD patterns of C@SMn nanoparticles synthesized with different contents of added Mn source are shown in Figure 2c. All diffraction peaks of the samples show in a good correspondence to the pure cubic CaF2 (PDF#35-0816, space group: Fm3m). However, in the selected region of diffraction peaks magnified on the right, the peaks shifted toward smaller angles gradually as the increase of added MnCl2 concentrations, and then shift toward higher angles. The maximal peak deviation showed up for the C@SMn sample prepared at the Mn : C@S mass ratio of 1:12. Those findings imply the change of lattice constants of CaF2. According to previous research results,42 this phenomenon could be explained as follows. The ionic state Mn2+ were incorporated into CaF2: Yb, Er nanocrystals lattice during the coating of MnO2 layer. The whole process that CaF2: Yb, Er nanocrystals lattice caused by Yb3+ and Mn2+ doping is briefly illustrated in Figure 2d. In C@S, Yb3+ doping would cause the increase of lattice space in the CaF2 crystal. Subsequently, when Mn2+ ions were incorporated into the lattice, Mn2+ ions may possibly act as the interstitial atoms in the lattice of CaF2 nanocrystals at the initial stage when the low amount of Mn2+ ions are diffused, and then occupy Yb3+ sites as the further increase of Mn2+ doping.43-44 The existence of Mn2+ disturbs the transition possibilities between green and red emissions of


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Er3+ and facilitates the dominate occurrence of red emission, resulting in an emission color output from green to red by ratio controlling of Mn2+ doping level (Figure S3). Besides influencing the lattice distortion by Mn2+ doping, quenching of UCL emission by MnO2 would occur with the coating of MnO2 shell. According to the reaction mechanism of MnO2 and H2O2, MnO2 only acts as a catalyst for the disproportionation of H2O2 into O2 in neutral solutions. However, in acidic solutions with H2O2, MnO2 would be decomposed into colorless Mn2+ (Figure S4a). We evaluated the UCL emission of C@SMn (Mn:C@S = 1:12) nanoparticles after reaction with H2O2 in pH 7.4 and pH 5.5 solutions (Figure S4b). The UCL emission of those nanoparticles recovered for the samples in the acidic solution with no obvious change in red to green ratio, suggesting that MnO2 coating caused the partial quenching of UCL emission, although Mn2+ doping at this ratio is favorable for enhancing the red UCL emission. All of these factors above contribute to the changes in photoluminescence performance, and the factor that the maximal red UCL emission could be achieved at an optimal coating ratio of MnO2. To improve the stability of nanoparticles in physiological solutions, surface modification is usually involved.45 In our study (Figure 3a), amino group was first modified on C@SMn nanoparticles through a silane coupling agent, aminopropyltriethoxysilane (APTES). The flourier transform infrared (FITR) spectra of C@S, C@SMn, and C@SMn-NH2 samples are depicted in Figure S5. Comparing with C@SMn nanoparticles, there was a new peak observed at 1560 cm-1, which could be attributed to N-H asymmetric bending vibration, indicating the successful functionalization of our nanoparticles with amino groups.46 Those C@SMn-NH2 nanoparticles were then decorated with anionic polymer polyacrylic acid (PAA) by electrostatic interactions. Subsequently, amino-terminated polyethylene glycol (NH2-PEG, Mw = 5000) was conjugated to carboxyl groups through covalent bonds via 1-(3-(dimethylamino) propyl)-3-ethylcarbodiimide


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hydrochloride (EDC) induced amide formation. Following this, the surface charge was amplified gradually. After such surface modification, the stability and dispersion of PEGylation C@SMn became much better in aqueous solution. The alternating changes of zeta potentials at each step of surface modification (Figure S6) evidenced the successful layer-by-layer polymer coatings on nanoparticles. The PEGylation C@SMn nanoparticles were then loaded with Ce6 photosensitizer following a protocol reported previously.28 The amount of Ce6 load on each sample was evaluated by UV-vis spectra (Figure 3b). The Ce6 loading capacity showed increase as the increase of Ce6 concentrations added. As for the loading efficiency of Ce6 (Figure 3c), reached a maximum level at the Ce6 : C@SMn feeding ratio of 0.2:1 (w/w), and this ratio was used for the following experiments. After Ce6 loading, the whole nanostructures exhibited excellent stability without any agglomeration in physiological solutions including ultrapure water, phosphate buffered saline (PBS), fetal bovine serum (FBS), and 10% serum containing cell culture medium (RPMI-1640) (Figure S7). The hydrodynamic diameters of C@SMn-Ce6 nanoparticles remained constant in different solutions without any agglomeration (Figure S8). In addition, the emission at 660 nm (red) presented a clear decline when more Ce6 was loaded, indicating the effective resonance energy transfer from C@SMn to Ce6 (Figure 3d & Figure S9). The release behavior of Ce6 from C@S-Ce6 and C@SMn-Ce6 nanoparticles in RPMI-1640 cell culture medium (containing 10% serum) was investigated. Within 24 h incubation, most of Ce6 were still loaded on the particles, indicating the good stability of these complexes for following PDT treatment (Figure S10). Singlet oxygen sensor green (SOSG)47 was chosen to determine the production of 1

O2 from different samples under 980-nm laser irradiation. Singlet oxygen level produced by

C@SMn-Ce6 was higher than that of C@S-Ce6 at the same concentration of Ce6, while no


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appreciable singlet oxygen generation was observed for free Ce6 under the 980-nm laser (Figure 3e). It is known that MnO2 shows sound catalytic activity to promote the disproportionation reaction of hydrogen peroxide (H2O2) for producing O2. In this study, the generation of O2 were examined by an oxygen probe in solutions containing 500 µM H2O2 upon addition of C@S-Ce6 or C@SMn-Ce6 nanoparticles at same concentration (Figure 3f). As expected, C@SMn-Ce6 would trigger the rapid generation of O2, while C@S-Ce6 did not increase the dissolved oxygen in water. The production of 1O2 by C@S-Ce6 and C@SMn-Ce6 nanoparticles was also investigated in the presence of 50 µM H2O2. As shown in Figure 3g, obvious increase of 1O2 production was induced by C@SMn-Ce6 with H2O2 under the 980-nm laser irradiation due to the generation of O2 from decomposed H2O2 triggered by MnO2. In contrast, the presence of H2O2 showed no appreciable effect to 1O2 production by C@S-Ce6 under the 980-nm laser. Those findings imply that PDT efficiency of C@SMn-Ce6 nanoparticles can be improved significantly in the presence of H2O2, which is known to exist at the level of 50-100 µM within the tumor microenvironment.48 Afterwards, in vitro studies using mouse 4T1 breast tumor cells were pursued with C@SMnCe6 nanoparticles. No obvious dark toxicity of C@SMn-Ce6 was observed to 4T1 cells in our tested concentration range (Figure 4a). Subsequently, C@SMn-Ce6 and free Ce6 were incubated with 4T1 cells for 4 h to estimate the cellular uptake efficiency. Compared to cells incubated with free Ce6, stronger fluorescence was exhibited inside 4T1 cells which incubated with C@SMnCe6 nanoparticles (Figure 4b). Flow cytometry results also demonstrated the same conclusion (Figure S11). Therefore, using nanoparticles as the delivery system, the cellular uptake of Ce6 was remarkably enhanced, consistent to the previous work reported elsewhere.49


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It is known that the aberrant metabolism of cancer cells inside solid tumors could constitutively produce H2O2, thus the PDT efficiency of C@S-Ce6 and C@SMn-Ce6 were measured by incubating them with 4T1 cells in the presence of exogenous 50 µM H2O2. To certify that the nanoparticles with MnO2 could trigger the decomposition of H2O2 and generate O2 in-situ to improve the efficacy of PDT, 4T1 cells were placed in both normoxia (21% O2) and hypoxia (1% O2) atmosphere throughout the operation. After adding the same concentration of C@S-Ce6 and C@SMn-Ce6 nanoparticles for 4 h, cells were exposed to the 980-nm laser for 10 min (1 min interval) at the power density of 0.5 W/cm2. Their viabilities were measured after further incubation for 24 h. As shown in Figure 4c, strong phototoxicity were shown in both C@S-Ce6 and C@SMn-Ce6 samples within the normoxia environment. Besides, we evaluated the intracellular ROS generation via DCFH-DA fluorescence assay. As expected, the cells treated with C@SMn-Ce6 showed a higher level of ROS generation than those treated with C@S-Ce6 after being irradiated with 980-nm laser for 10 min (Figure S12). These findings imply that the cell killing effect of C@SMn-Ce6 was higher than that of C@S-Ce6. In comparison (Figure 4d), the PDT-induced cell killing was less effective for C@S-Ce6 under hypoxia atmosphere, in which C@SMn-Ce6 still showed a decent level of phototoxicity owing to the decomposition of H2O2 triggered by MnO2. Confocal fluorescence staining of living/dead cells also confirmed the in vitro photodynamic effect by C@SMn-Ce6 nanoparticles (Figure 4e). Next, we ought to demonstrate the performance of C@SMn nanoparticles in ameliorating hypoxia inside tumors. In our experiments, C@S-Ce6 and C@SMn-Ce6 at the same dose were separately injected into 4T1 tumors growing on Balb/C mice. Immunofluorescence assay using a hypoxyprobe, pimonidazole, was then carried out on tumor slices following the standard protocol (Figure 5a).32 Comparing to the control and C@S-Ce6 treated groups, the tumor slices from mice


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treated with C@SMn-Ce6 exhibited obviously reduced hypoxia-specific green fluorescence, which means that the tumor hypoxia was remarkably modulated. The quantitative statistical analysis of hypoxia positive and blood vessel areas further confirmed the obvious hypoxia decrease induced by C@SMn-Ce6 in tumor regions at similar blood vessel densities (Figure 5b). In vivo photodynamic therapeutic effect of C@SMn-Ce6 was then evaluated using 4T1 mouse tumor model. 25 mice bearing 4T1 tumors were randomly separated into 5 groups: Control (Group 1), 980-nm NIR irradiation (Group 2), C@SMn-Ce6 (Group 3), C@S-Ce6 with NIR (Group 4), and C@SMn-Ce6 with NIR (Group 5). For PDT treatment, the dose of nanoparticles was 25 mg kg-1 (Ce6: 3 mg kg-1). At 2 h post injection, mice were exposed to the 980-nm laser (0.5 W/cm2) for 30 min (1 min interval). The mice body weights and tumor sizes were recorded in the following two weeks (Figure 5c and Figure S13). On day 14, all the mice were sacrificed and tumors were collected and weighted (Figure 5d and Figure S14). Compared with untreated tumors, those on mice treated with NIR alone or C@SMn-Ce6 without NIR irradiation showed no obvious delay in their growth. For mice with tumors injected with C@SCe6 and exposed to NIR irradiation, their tumor growth was partially inhibited. More significantly, for those after NIR-triggered PDT with C@SMn-Ce6 nanoparticles, the tumor sizes obviously shrunk after light irradiation and kept in small sizes within 14 days. No appreciable body weight variation was observed for different groups of mice (Figure S8), indicating no obvious acute toxicity of those nanoparticles. Hematoxylin and eosin (H&E) stained microscopy images of tumor slices also manifested that the tumor tissue treated after PDT with C@SMn-Ce6 nanoparticles showed much more serious damages compared PDT by C@S-Ce6, while the cells in control, NIR alone and C@SMn-Ce6 injection alone group basically retained their regular morphology as (Figure 5e). Therefore, our data suggest that due


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to the enhancement of photoluminescence and the amelioration of tumor hypoxia by C@SMnCe6 nanoparticles, the photodynamic therapeutic effect by C@SMn-Ce6 nanoparticles could be remarkably enhanced. 4. CONCLUSION In this work, multifunctional upconversion composite nanoparticles are designed and synthesized via a mesoporous-silica-templated method by in situ growth of CaF2: Yb, Er nanocrystals inside the porous structure of MSNs, the latter of which show retained mesoporous structure after such growth. By further finely tuning the coating of a thin layer of MnO2 on the surface of those nanoparticles, the red-to-green ratio and absolute UCL emission intensity of those nanoparticles could be enhanced, favoring more efficient resonance energy transfer to the loaded photosensitizer Ce6 for better PDT. Moreover, owing to the unique capability of MnO2 in converting endogenous H2O2 produced by cancer cells into O2, such C@SMn-Ce6 nanoparticles have demonstrated outstanding performance in overcoming hypoxia-associated resistance and greatly improved therapeutic efficacy in destructing tumors on mice post NIR irradiation. Compared to UCNP-based PDT nano-systems reported previously, our study presents several unique advantages. 1) Compared to the conventional method by coating UCNPs with mesoporous shells, our method by growing multiple upconversion nanocrytals within the MSN template is a reversed approach. Apparently larger specific surface area in the final nanocomposite could be achieved by our method. 2) Different from growing UCNPs on MnO2 nanosheets, the latter of which may partially quench the UCL luminescence of the former, our fabrication protocol results in marked enhanced UCL emission by fining tune MnO2 growth on top of the upconversion nanocomposite. Our work thus presents a unique materials design strategy to fabricate multifunctional composite nanoparticles, not only promising for cancer


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theranostic as demonstrated herein, but also potentially useful for developing functional nanostructures aiming at other types of applications (e.g. catalysis, energy, etc.).

ASSOCIATED CONTENT Supporting Information. The supporting information is available free of charge on the ACS Publication website at DOI: 10.1021/acsami. xxx. SEM images of MSNs; XPS spectrum of C@SMn nanoparticles; Energy level diagrams of Er3+, Yb3+ and Mn2+ and the relative transitions; FTIR spectra of C@S, C@SMn and C@SMn-NH2 nanoparticles; Zeta potentials of nanoparticles after each step of surface modification; Photograph of C@SMn-Ce6 nanoparticles dissolved in different media; Intensity ratios of red-to green emission before and after Ce6 loading at various feeding Ce6 concentrations; Average body weights of mice in various treatment group as indicated; Tumor weight collected from different groups 14 d after the treatment; Textural properties of MSNs, C@S and C@SMn nanoparticles. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] * E-mail: [email protected] Notes


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The authors declare no competing financial interest. ACKNOWLEDGMENT This work was financially supported by the National Nature Science Foundation of China (51672247, 51525203, 51572180), the 111 program of China (No. B16042), the Major State Research Program of China (2016YFC1101900), and the National Research Programs from Ministry of Science and Technology (MOST) of China (2016YFA0201200)

REFERENCES (1) Wu, W.; Mao, D.; Hu, F.; Xu, S.; Chen, C.; Zhang, C. J.; Cheng, X.; Yuan, Y.; Ding, D.; Kong, D.; Liu, B. A Highly Efficient and Photostable Photosensitizer with Near-Infrared Aggregation-Induced Emission for Image-Guided Photodynamic Anticancer Therapy. Adv. Mater. 2017, 29 (33), 1700548. (2) Han, S.; Hwang, B. W.; Jeon, E. Y.; Jung, D.; Lee, G. H.; Keum, D. H.; Kim, K. S.; Yun, S. H.; Cha, H. J.; Hahn, S. K. Upconversion Nanoparticles/Hyaluronate-Rose Bengal Conjugate Complex for Noninvasive Photochemical Tissue Bonding. ACS Nano 2017, 11 (10), 99799988. (3) Liu, L. H.; Zhang, Y. H.; Qiu, W. X.; Zhang, L.; Gao, F.; Li, B.; Xu, L.; Fan, J. X.; Li, Z. H.; Zhang, X. Z. Dual-Stage Light Amplified Photodynamic Therapy against Hypoxic Tumor Based on an O2 Self-Sufficient Nanoplatform. Small 2017, 13 (37), 1701621. (4) Ju, E.; Dong, K.; Chen, Z.; Liu, Z.; Liu, C.; Huang, Y.; Wang, Z.; Pu, F.; Ren, J.; Qu, X. Copper(II)-Graphitic Carbon Nitride Triggered Synergy: Improved ROS Generation and


19


Page 20 of 33

Reduced Glutathione Levels for Enhanced Photodynamic Therapy. Angew. Chem. Int. Ed. 2016, 55 (38), 11467-11471. (5) Castano, A. P.; Mroz, P.; Hamblin, M. R. Photodynamic Therapy and Anti-tumour Immunity. Nat. Rev. Cancer 2006, 6 (7), 535-545. (6) Deng, K.; Li, C.; Huang, S.; Xing, B.; Jin, D.; Zeng, Q.; Hou, Z.; Lin, J. Recent Progress in Near Infrared Light Triggered Photodynamic Therapy. Small 2017, 13 (44), 1702299. (7) Maria C. DeRosa, R. J. C. Photosensitized Singlet Oxygen and Its Applications. Coord. Chem. Rev. 2002, 233, 351-371. (8) Song, X.; Feng, L.; Liang, C.; Gao, M.; Song, G.; Liu, Z. Liposomes Co-loaded with Metformin and Chlorin e6 Modulate Tumor Hypoxia During Enhanced Photodynamic Therapy. Nano Res. 2016, 10 (4), 1200-1212. (9) Guan, Y.; Lu, H.; Li, W.; Zheng, Y.; Jiang, Z.; Zou, J.; Gao, H. Near-Infrared Triggered Upconversion Polymeric Nanoparticles Based on Aggregation-Induced Emission and Mitochondria Targeting for Photodynamic Cancer Therapy. ACS Appl. Mater. Interfaces 2017, 9 (32), 26731-26739. (10) Xu, J.; Xu, L.; Wang, C.; Yang, R.; Zhuang, Q.; Han, X.; Dong, Z.; Zhu, W.; Peng, R.; Liu, Z. Near-Infrared-Triggered Photodynamic Therapy with Multitasking Upconversion Nanoparticles in Combination with Checkpoint Blockade for Immunotherapy of Colorectal Cancer. ACS Nano 2017, 11 (5), 4463-4474. (11) Cabuy, E. Photodynamic Therapy in Cancer Treatment. Cabuy E. Reliable Cancer Therapies. Energy-based therapies 2012, 3 (2), 54. (12) Wang, P.; Li, X.; Yao, C.; Wang, W.; Zhao, M.; El-Toni, A. M.; Zhang, F. Orthogonal Nearinfrared Upconversion Co-regulated Site-specific O2 Delivery and Photodynamic Therapy


20

Page 21 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


for Hypoxia Tumor by Using Red Blood Cell Microcarriers. Biomaterials 2017, 125, 90-100. (13) Yao, C.; Wang, P.; Li, X.; Hu, X.; Hou, J.; Wang, L.; Zhang, F. Near-Infrared-Triggered Azobenzene-Liposome/Upconversion Nanoparticle Hybrid Vesicles for Remotely Controlled Drug Delivery to Overcome Cancer Multidrug Resistance. Adv. Mater. 2016, 28 (42), 93419348. (14) Smith, A. M.; Mancini, M. C.; Nie, S. Bioimaging: Second Window for In Vivo Imaging. Nat. Nanotechnol. 2009, 4 (11), 710-711. (15) Hu, L.; Wang, P.; Zhao, M.; Liu, L.; Zhou, L.; Li, B.; Albaqami, F. H.; El-Toni, A. M.; Li, X.; Xie, Y.; Sun, X.; Zhang, F. Near-infrared Rechargeable "Optical Battery" Implant for Irradiation-free Photodynamic Therapy. Biomaterials 2018, 163, 154-162. (16) Fan, W.; Bu, W.; Shi, J. On The Latest Three-Stage Development of Nanomedicines Based on Upconversion Nanoparticles. Adv. Mater. 2016, 28 (21), 3987-4011. (17) Gnanasammandhan, M. K.; Idris, N. M.; Bansal, A.; Huang, K.; Zhang, Y. Near-IR Photoactivation

Using

Mesoporous

Silica-coated

NaYF4:Yb,Er/Tm

Upconversion

Nanoparticles. Nat. Protoc. 2016, 11 (4), 688-713. (18) Liu, Y.; Zhang, J.; Zuo, C.; Zhang, Z.; Ni, D.; Zhang, C.; Wang, J.; Zhang, H.; Yao, Z.; Bu, W. Upconversion Nano-photosensitizer Targeting into Mitochondria for Cancer Apoptosis Induction and Cyt c Fluorescence Monitoring. Nano Res. 2016, 9 (11), 3257-3266. (19) Huang, L.; Li, Z.; Zhao, Y.; Yang, J.; Yang, Y.; Pendharkar, A. I.; Zhang, Y.; Kelmar, S.; Chen, L.; Wu, W.; Zhao, J.; Han, G. Enhancing Photodynamic Therapy through Resonance Energy Transfer Constructed Near-Infrared Photosensitized Nanoparticles. Adv. Mater. 2017, 29 (28), 1604789. (20) Chang, Y.; Li, X.; Zhang, L.; Xia, L.; Liu, X.; Li, C.; Zhang, Y.; Tu, L.; Xue, B.; Zhao, H.;


21


Page 22 of 33

Zhang, H.; Kong, X. Precise Photodynamic Therapy of Cancer via Subcellular Dynamic Tracing of Dual-loaded Upconversion Nanophotosensitizers. Sci. Rep. 2017, 7, 45633. (21) Liu, Y.; Liu, Y.; Bu, W.; Cheng, C.; Zuo, C.; Xiao, Q.; Sun, Y.; Ni, D.; Zhang, C.; Liu, J.; Shi, J. Hypoxia Induced by Upconversion-Based Photodynamic Therapy: Towards Highly Effective Synergistic Bioreductive Therapy In Tumors. Angew. Chem. Int. Ed. 2015, 54 (28), 8105-8109. (22) Idris, N. M.; Jayakumar, M. K.; Bansal, A.; Zhang, Y. Upconversion Nanoparticles as Versatile Light Nanotransducers for Photoactivation Applications. Chem. Soc. Rev. 2015, 44 (6), 1449-1478. (23) Cheng, L.; Wang, C.; Feng, L.; Yang, K.; Liu, Z. Functional Nanomaterials for Phototherapies of Cancer. Chem. Rev. 2014, 114 (21), 10869-10939. (24) Wang, F.; Banerjee, D.; Liu, Y.; Chen, X.; Liu, X. Upconversion Nanoparticles in Biological Labeling, Imaging, and Therapy. Analyst 2010, 135 (8), 1839-1854. (25) Yang, D.; Ma, P.; Hou, Z.; Cheng, Z.; Li, C.; Lin, J. Current Advances in Lanthanide Ion (Ln(3+))-based Upconversion Nanomaterials for Drug Delivery. Chem. Soc. Rev. 2015, 44 (6), 1416-1448. (26) Ai, X.; Ho, C. J.; Aw, J.; Attia, A. B.; Mu, J.; Wang, Y.; Wang, X.; Wang, Y.; Liu, X.; Chen, H.; Gao, M.; Chen, X.; Yeow, E. K.; Liu, G.; Olivo, M.; Xing, B. In Vivo Covalent Crosslinking of Photon-converted Rare-earth Nanostructures for Tumour Localization and Theranostics. Nat. Commun. 2016, 7, 10432. (27) Feng, L.; He, F.; Dai, Y.; Gai, S.; Zhong, C.; Li, C.; Yang, P. Multifunctional UCNPs@MnSiO3@g-C3N4 Nanoplatform: Improved ROS Generation and Reduced Glutathione Levels for Highly Efficient Photodynamic Therapy. Biomater. Sci. 2017, 5 (12),


22

Page 23 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2456-2467. (28) Zhu, W.; Dong, Z.; Fu, T.; Liu, J.; Chen, Q.; Li, Y.; Zhu, R.; Xu, L.; Liu, Z. Modulation of Hypoxia in Solid Tumor Microenvironment with MnO2 Nanoparticles to Enhance Photodynamic Therapy. Adv. Funct. Mater. 2016, 26 (30), 5490-5498. (29) Chu, C.; Lin, H.; Liu, H.; Wang, X.; Wang, J.; Zhang, P.; Gao, H.; Huang, C.; Zeng, Y.; Tan, Y.; Liu, G.; Chen, X. Tumor Microenvironment-Triggered Supramolecular System as An In Situ Nanotheranostic Generator for Cancer Phototherapy. Adv. Mater. 2017, 29 (23), 1605928. (30) He, D.; Hai, L.; He, X.; Yang, X.; Li, H. W. Glutathione Activatable and O2/Mn2+ Evolving Nanocomposite for Highly Efficient and Selective Photodynamic and Gene‐Silencing Dual Therapy. Adv. Funct. Mater. 2017, 27 (46), 1704089. (31) Fan, H.; Yan, G.; Zhao, Z.; Hu, X.; Zhang, W.; Liu, H.; Fu, X.; Fu, T.; Zhang, X. B.; Tan, W. A Smart Photosensitizer-Manganese Dioxide Nanosystem for Enhanced Photodynamic Therapy by Reducing Glutathione Levels in Cancer Cells. Angew. Chem. Int. Ed. 2016, 55 (18), 5477-5482. (32) Chen, Q.; Feng, L.; Liu, J.; Zhu, W.; Dong, Z.; Wu, Y.; Liu, Z. Intelligent Albumin-MnO2 Nanoparticles as pH-/H2O2-Responsive Dissociable Nanocarriers to Modulate Tumor Hypoxia for Effective Combination Therapy. Adv. Mater. 2016, 28 (33), 7129-7136. (33) Chen, P. J.; Kang, Y. D.; Lin, C. H.; Chen, S. Y.; Hsieh, C. H.; Chen, Y. Y.; Chiang, C. W.; Lee, W.; Hsu, C. Y.; Liao, L. D.; Fan, C. T.; Li, M. L.; Shyu, W. C. Multitheragnostic MultiGNRs Crystal-Seeded Magnetic Nanoseaurchin for Enhanced In Vivo Mesenchymal-StemCell Homing, Multimodal Imaging, and Stroke Therapy. Adv. Mater. 2015, 27 (41), 64886495.


23


Page 24 of 33

(34) Kong, B.; Tang, J.; Zhang, Y.; Jiang, T.; Gong, X.; Peng, C.; Wei, J.; Yang, J.; Wang, Y.; Wang, X.; Zheng, G.; Selomulya, C.; Zhao, D. Incorporation of Well-dispersed Sub-5-nm Graphitic Pencil Nanodots into Ordered Mesoporous Frameworks. Nat. Chem. 2016, 8 (2), 171-178. (35) Lim, W. Q.; Phua, S. Z.; Xu, H. V.; Sreejith, S.; Zhao, Y. Recent Advances in Multifunctional Silica-based Hybrid Nanocarriers for Bioimaging and Cancer Therapy. Nanoscale 2016, 8 (25), 12510-12519. (36) Nandiyanto, A. B. D.; Kim, S.-G.; Iskandar, F.; Okuyama, K. Synthesis of Spherical Mesoporous Silica Nanoparticles with Nanometer-size Controllable Pores and Outer Diameters. Microporous Mesoporous Mater. 2009, 120 (3), 447-453. (37) Li, Y.; Zhou, Y.; Gu, T.; Wang, G.; Ren, Z.; Weng, W.; Li, X.; Han, G.; Mao, C. A Multifunctional Nanocrystalline CaF2:Tm,Yb@mSiO2 System for Dual-Triggered and Optically Monitored Doxorubicin Delivery. Part. Part. Syst. Charact. 2016, 33 (12), 896905. (38) Ramarajan, D.; Sivagurunathan, P.; Yan, Q. Synthesis and Characterization of Sol-processed α-MnO2 Nanostructures. Mater. Sci. Semicond. Process. 2012, 15 (5), 559-563. (39) Georgescu, S.; Voiculescu, A. M.; Matei, C.; Secu, C. E.; Negrea, R. F.; Secu, M. Ultraviolet and Visible Up-conversion Luminescence of Er3+ /Yb3+ Co-doped CaF2 Nanocrystals in Sol– gel Derived Glass-ceramics. J. Lumin. 2013, 143 (6), 150-156. (40) Zhou, J.; Liu, Z.; Li, F. Upconversion Nanophosphors for Small-animal Imaging. Chem. Soc. Rev. 2012, 41 (3), 1323-1349. (41) Chen, G.; Qiu, H.; Prasad, P. N.; Chen, X. Upconversion Nanoparticles: Design, Nanochemistry, and Applications in Theranostics. Chem. Rev. 2014, 114 (10), 5161-5214.


24

Page 25 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(42) Tian, G.; Gu, Z.; Zhou, L.; Yin, W.; Liu, X.; Yan, L.; Jin, S.; Ren, W.; Xing, G.; Li, S.; Zhao, Y. Mn2+ dopant-controlled Synthesis of NaYF4:Yb/Er Upconversion Nanoparticles for In Vivo Imaging and Drug Delivery. Adv. Mater. 2012, 24 (9), 1226-1231. (43) Tian, D.; Gao, D.; Chong, B.; Liu, X. Upconversion Improvement by The Reduction of Na+vacancies in Mn2+ doped Hexagonal NaYbF4: Er3+ Nanoparticles. Dalton. Trans. 2015, 44 (9), 4133-4140. (44) Wang, J.; Wang, F.; Wang, C.; Liu, Z.; Liu, X. Single-band Upconversion Emission in Lanthanide-doped KMnF3 Nanocrystals. Angew. Chem. Int. Ed. 2011, 50 (44), 10369-10372. (45) Song, G.; Chao, Y.; Chen, Y.; Liang, C.; Yi, X.; Yang, G.; Yang, K.; Cheng, L.; Zhang, Q.; Liu, Z. All-in-One Theranostic Nanoplatform Based on Hollow TaOx for Chelator-Free Labeling Imaging, Drug Delivery, and Synergistically Enhanced Radiotherapy. Adv. Funct. Mater. 2016, 26 (45), 8243-8254. (46) Li, Y.; Chen, X.; Liu, H.; Mou, X.; Ren, Z.; Ahmad, Z.; Li, X.; Han, G. Silica Nanospheres Entrapped with Ultra-small Luminescent Crystals for Protein Delivery. Chem. Eng. J. 2017, 330, 166-174. (47) Gollmer, A.; Arnbjerg, J.; Blaikie, F. H.; Pedersen, B. W.; Breitenbach, T.; Daasbjerg, K.; Glasius, M.; Ogilby, P. R. Singlet Oxygen Sensor Green(R): Photochemical Behavior In Solution and in a Mammalian Cell. Photochem. Photobiol. 2011, 87 (3), 671-679. (48) Chen, Q.; Liang, C.; Sun, X.; Chen, J.; Yang, Z.; Zhao, H.; Feng, L.; Liu, Z. H2O2responsive Liposomal Nanoprobe for Photoacoustic Inflammation Imaging and Tumor Theranostics via In Vivo Chromogenic Assay. Proc. Natl. Acad. Sci. U.S.A. 2017, 114 (21), 5343-5348. (49) Liu, T.; Wang, C.; Cui, W.; Gong, H.; Liang, C.; Shi, X.; Li, Z.; Sun, B.; Liu, Z. Combined


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Photothermal and Photodynamic Therapy Delivered by PEGylated MoS2 Nanosheets. Nanoscale 2014, 6 (19), 11219-11225.


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Scheme 1. Schematic Illustration for the synthesis of C@SMn


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Figure 1. TEM images of (a) MSNs, (b) C@S nanoparticles, (c) C@SMn nanoparticles. Inset: the SAED pattern of C@SMn nanoparticles. (d) EDS element mapping of a C@SMn nanoparticle. The scale bar is 50 nm. (e) XRD patterns of MSNs, C@S and C@SMn nanoparticles. (f) N2 absorption and desorption isotherm curves of MSNs, C@S and C@SMn nanoparticles. Inset: corresponding pore distribution derived from absorption data.


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Figure 2. Tuning of optical properties. (a) Photoluminescence spectrum of C@SMn nanoparticles prepared by adding different mass ratios of Mn source to C@S. (b) The changes of UCL photoluminescence intensity and R/G ratio. (c) Left: XRD patterns of C@SMn samples prepared with different Mn : C@S ratios. Right: Amplified figure in the range of interests. (d) Analog illustration of lattice distortion caused by Yb3+ and Mn2+ doping.


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Figure 3. Ce6 loading on nanoparticles. (a) A scheme showing the surface modification and Ce6 loading procedures. (b) UV-vis-NIR spectra of C@SMn-Ce6 nanoparticles prepared at various feeding Ce6 concentrations. (c) Loading efficiencies of Ce6 on C@SMn nanoparticles. (d) UC emission spectra before and after Ce6 loading at various feeding Ce6 concentrations. (e) The generation of singlet oxygen determined by the increased SOSG fluorescence for C@S, C@SMn, free Ce6, C@S-Ce6 and C@SMn-Ce6 nanoparticles under 980-nm laser irradiation. (f) Simultaneous O2 generation of C@S and C@SMn nanoparticles in H2O2 solutions (500 µM). (g) Changes of SOSG fluorescence for C@S-Ce6 and C@SMn-Ce6 nanoparticles in absence or presence of H2O2 (50 µM) under 980-nm laser irradiation (Power density: 0.5 W/cm2, [Ce6]: 0.3 mg/mL).


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Figure 4. In vitro cell culture study. (a) Relative viability of 4T1 cells after incubation with C@SMn-Ce6 nanoparticles at different concentrations for 12 and 24 h. (b) Confocal images of 4T1 cells incubated with free Ce6 and C@SMn-Ce6 nanoparticles at different time points. Scale bar: 25 µm. (c & d) In vitro PDT treatment of 4T1 cells by PBS, C@S-Ce6 and C@SMn-Ce6 nanoparticles with or without 980-nm laser irradiation (0.5 W/cm2, 10 min, 1 min interval) in normoxia and hypoxia (1% O2) atmosphere. (e) Confocal fluorescence images of 4T1 cells incubated with C@SMn-Ce6 nanoparticles at different concentrations after irradiation using 980nm laser (0.5 W/cm2) and incubated for 24 h. The cells were costained by calcein AM (green, live cells) and PI (red, dead cells) before imaging. (***p < 0.001, **p < 0.01, or *p < 0.05)


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Figure 5. In vivo overcoming tumor hypoxia and cancer treatment. (a) Representative hypoxia staining immunofluorescence images of tumor slices 2.5 h after injecting PBS, C@S-Ce6 and C@SMn-Ce6 nanoparticles. The cell nuclei, hypoxia areas and blood vessels were stained by DAPI (blue), antipimonidazole antibody (green), and anti-CD31 antibody (red), respectively. (b) Quantification of tumor hypoxia and relative blood vessels areas from more than ten micrographs for each group. (c) Tumor volume growth curves of mice after various treatments (five mice for each group). Light irradiation (L+) was conducted by the 980-nm laser at the power density of 0.5 W cm−2 for 30 min (1 min interval). (d) Photograph of tumors collected from different groups of mice 14 days after treatment. (e) H&E stained tumor slices from different groups collected 24 h after laser irradiation. Scaled bar: 100 µm. (***p < 0.001, **p < 0.01, or *p < 0.05)


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Table of Contents (TOC)


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Upconversion Composite Nanoparticles for Tumor Hypoxia

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