Superior temperature sensing of small-sized upconversion nanocrystals for simultaneous bioimaging and enhanced synergetic therapy

Journal Pre-proof Superior temperature sensing of small-sized Upconversion Nanocrystals for simultaneous bioimaging and enhanced synergetic therapy

Guofeng Liu, Fan Jiang, Yeqing Chen, Chang Yu, Binbin Ding, Shuai Shao, Mochen Jia, Ping'an Ma, Zuoling Fu, Jun Lin PII:

S1549-9634(19)30219-9

DOI:

https://doi.org/10.1016/j.nano.2019.102135

Reference:

NANO 102135

To appear in:

Nanomedicine: Nanotechnology, Biology, and Medicine

Revised date:

12 November 2019

Please cite this article as: G. Liu, F. Jiang, Y. Chen, et al., Superior temperature sensing of small-sized Upconversion Nanocrystals for simultaneous bioimaging and enhanced synergetic therapy, Nanomedicine: Nanotechnology, Biology, and Medicine(2019), https://doi.org/10.1016/j.nano.2019.102135

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© 2019 Published by Elsevier.

Journal Pre-proof Article type: original article Superior Temperature Sensing of Small-sized Upconversion Nanocrystals for Simultaneous Bioimaging and Enhanced Synergetic Therapy

Guofeng Liu Doctora,b, Fan Jiang Bachelor a, Yeqing Chen Doctor c, Chang Yu Doctor a, Binbin Ding Bachelor a, Shuai Shao Master a, Mochen Jia Bachelor b, Ping’an Ma a,

b,

a,

Doctor *, Zuoling Fu Doctor *, Jun Lin Doctor *

State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of

of

a

b

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Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, China. Coherent Light and Atomic and Molecular Spectroscopy Laboratory, Key Laboratory

School of Applied Physics and Materials, Wuyi University, Jiangmen, Guangdong

529020, China.

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*Corresponding Author:

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c

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University, Changchun 130012, China.

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of Physics and Technology for Advanced Batteries, College of Physics, Jilin

Dr. Ma, [email protected], Changchun Institute of Applied Chemistry, Chinese

Dr.

Fu,

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Academy of Sciences, Changchun, 130022, China. +86-431-85262614. [email protected],

Jilin

University,

Changchun

130012,

China.

+86-431-85167966.

Dr. Lin, [email protected], Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, China. +86-431-85262031.

Conflicts of interest: the authors declare no conflicts of interest

Word count for abstract: 147 Word count for manuscript: 4589 Number of references: 59 Number of figures: 7 Number of tables: 0 1

Journal Pre-proof Number of Supplementary online-only files, if any: 1 ABSTRACT The upconversion nanoparticles (UCNPs) exhibit versatility applications aiming at biological domains for decades on account of superior optical characteristics. Nevertheless, the UCNPs are confronted with tremendous difficulties in biological field owing to large grain size, low fluorescence efficiency, and single function.

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Herein, the small-sized CaF2: Yb3+/Er3+ UCNPs coated with NaGdF4 shells (activator

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and inert, UCNPs-RBHA-Pt-PEG) not only burst out strong fluorescence, but also

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provide prominent diagnosability by taking advantage of magnetic resonance (MR)

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imaging as well as temperature sensing and inhibiting capability for CT26 tumor

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tissues based on synergetic therapy modality of photodynamic therapy (PDT) and

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chemotherapy. Ultimately, the tumor sizes decrease visibly after injected with UCNPs-RBHA-Pt-PEG and simultaneously irradiated with near infrared (NIR) light

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at low power density (0.35 W/cm2, 6 min). In summary, the small-sized and strong-fluorescent single nanoparticles with multi-functions may provide a valuable enlightenment for diagnosis and treatment of cancer in the future. Keywords: Calcium fluoride; Upconversion luminescence; Temperature sensing; Synergetic therapy

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BACKGROUND Nowadays, many methods have been developed for antitumor therapy based on the superior loading capacity of nanoparticles, including magnetic resonance (MR) imaging, photoacoustic (PA) imaging, temperature sensing, computed tomography

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(CT) and fluorescence imaging (FI) for diagnosis; photodynamic therapy (PDT), chemotherapy, photothermal therapy (PTT) and radiotherapy for treatment,

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respectively.1-7 These techniques have a great prospects in medical applications and

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some of them have been widely used in to the clinic, but each of them is not perfect

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on account of their own limitations. For example, the PA imaging sensitivity is higher

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compared to the MR and CT ones while the penetration depth of it is shallow.8,9 The

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efficacy of a single mode of treatment is not obvious while the tumor size can be inhibited effectively by synergetic therapy.3,10 On the other hand, the nanoparticles

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with large particle size, appearing as carriers of photosensitizers (PS), photothermal agent, and chemotherapy drug, are hard to clean out by living body.11 Therefore, the small sized and multifunctional single nanoparticles are needed urgently for diagnosis and treatment of cancer. The PDT, emerging as a new noninvasive method for anticancer therapy, has high inhibition capability for tumor proliferation since the cancer cells could be killed effectively by reactive oxygen species (ROS) generated from PS under light irradiation.12 However, most of PS only can be excited by ultraviolet visible (UV-Vis) light, the penetration depth of whose is superficial, impeding their efficacy for cancer 3

Journal Pre-proof treatment.13 The problem can be solved well after attaching the PS to the surface of upconversion nanoparticles (UCNPs), the emission light from latter can be used to excite the former while the excitation source is NIR light. Among of many chemotherapy drugs, the platinum (Pt (II)) possesses a long research history owing to superior effect of anticancer.14 Despite this, its development is not ideal due to the high cytotoxicity for normal cells.15,16 A lot of works have been done for it, one of

of

feasible methods is the change of valence state of platinum.17,18 In particular,

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cis,cis,trans-diamminedichlorodisuccinato-platinum(IV)(c,c,t-Pt(NH3)2Cl2(OOCCH2C

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H2COOH)2 (DSP)) has low cytotoxicity and can be taken easily by cells. In addition,

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the DSP can be translated into Pt (II) through intracellular reduction of glutathione

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(GSH), enhancing the anticancer efficacy of platinum drug.19,20

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Calcium fluoride (CaF2) nanoparticles have been paid more attention in recent years due to versatility merits as diverse as uniform particle size, good thermal stability, low

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toxicity, nonhygroscopic, low phonon energy, high crystallizability, ideal optical transparence and etc.3,21 In particular, strong fluorescence can be obtained after doped with rare earth (RE) ions based on alkaline-earth fluorides (MF2) structure, which is related to low phonon energy and charge compensation effects. The former decreases the multi-phonon relaxation while the latter introduces more fluorinion to form lattice distortion, resulting in enhanced luminescence emission.21,22 In general, the near infrared (NIR) light can be absorbed and emitted availably by UCNPs, giving rise to deeper penetration depth compared with UV-Vis light.23 Contrary to X-ray, the NIR light has less harm for organism on account of low energy.24 Most important of all, the 4

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thermometers based on thermal coupled levels (TCLs) of UCNPs can be made into nanostructures with low cytotoxicity, which provide a promising choice for temperature measurement in living body. In addition, the fluorescence intensity ratio (FIR) strategy, which comes from TCLs of lanthanide ions, is minimally affected by external environment and brings out higher resolution as well as minimal or

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noninvasive compromise for living body.25,26 Briefly, the temperature of nanoparticles can be calculated by measuring upconversion (UC) emission spectrum to avoid direct

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contact. In addition, the irreversible damage of normal tissues caused by overheating

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in photothermal process also can be avoided through monitoring the emission spectra

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of UCNPs.

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In this paper, the small-sized and strong-fluorescent CaF2: Yb3+/Er3+ nanoparticles are

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prepared by solvothermal way, the fluorescence intensity is further enhanced by coating with NaGdF4 shells (activator and inert). The small sized nanoparticles are

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purged easily by living body while the strong fluorescence is contributed to FI in vitro and in vivo.27,28 Rose-bengal hexanic acid (RBHA) and DSP are attached to them by dehydration condensation (UCNPs-RBHA-Pt-PEG). The result demonstrates that tumor sizes are inhibited effectively by synergetic therapy of PDT and chemotherapy. Moreover, the MR imaging, upconversion luminescence (UCL) imaging and temperature sensing have also come true on the single nanoparticles. In conclusion, the UCNPs-RBHA-Pt-PEG nanoparticles exhibit an excellent prospect in diagnosis and treatment of cancer.

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METHODS Cells and animals CT26 cells (murine colon carcinoma cell line) were used to test, which bought from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cells were cultured in RPMI-1640 medium (1640, GIBCO), which including 10%

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fetal bovine serum (FBS, Kang Yuan Biology), 100 units mL–1 penicillin, and 100 μg

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mL–1 streptomycin (Sigma) in an atmosphere of 5% CO2 at 37 oC. Balb/c mice

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(25-30 g) were purchased from Center for Experimental Animals, Jilin University

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(Changchun, China). All mice were managed using the agreement ratified by the

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Institutional Animal Care and Use Committee of Jilin University.

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Preparation of core/core-shell/core-shell-shell nanoparticles The CaF2: Yb3+/Er3+ (CaF2) nanoparticles were prepared by solvothermal method.29

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Briefly, Ca(CH3COO)2·2H2O (1.78 mmol, 89%), YbCl3·6H2O (0.2 mmol, 10%), ErCl3·6H2O (0.02 mmol, 1%) were added into flask with three necks, then OA (6 mL) and ODE (15 mL) were poured into it, and the flask was heated to 150 °C and keeping for 30 min to form light yellow transparent solution under nitrogen airflow environment. Subsequently, the solution was cooled naturally to room temperature and methanol solution containing NH4F (4 mmol) was added into it. Next, the mixed solution was heated to 70 °C and keeping for 0.5 h volatilizing methanol. Then the mixed solution was heated to 300 °C and keeping for 1 h under nitrogen airflow environment. Then excess ethanol was added into solution at room temperature. The 6

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nanoparticles were obtained by centrifugation, and washed once with ethanol. Finally, the CaF2 nanoparticles were dispersed in cyclohexane (10 mL). Next, the CaF2: Yb3+/Er3+@NaGdF4: Yb3+ (CaF2@NaGdF4) nanoparticles were prepared by a similar process except adding CaF2: Yb3+/Er3+ as core. The CaF2: Yb3+/Er3+@NaGdF4:

Yb3+@NaGdF4

(CaF2@NaGdF4@NaGdF4,

UCNPs)

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nanoparticles were prepared by a similar process except that the CaF2 naoparticles

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were replaced by CaF2@NaGdF4 ones.

Synthesis of RBHA and DSP conjugated with UCNPs

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NOBF4 (100 mg) was dissolved in a mixed solution (DMF 5 mL, cyclohexane 3 mL),

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cyclohexane (5 mL) containing CaF2@NaGdF4@NaGdF4 nanoparticles was added

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into the solution and stirred for 10 min. And the nanoparticles removed OA were

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obtained by centrifugation. Then the nanoparticles were dispersed DMF (5 mL) containing PEI (200 mg) and stirred for overnight. The solution was washed with

mL).

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water for two times, and the CaF2@NaGdF4@NaGdF4-PEI was dispersed in water (5

HA (20 mg) and RB (100 mg) were added into mixed solution (Vacetone: Vwater = 7:3) and stirred for 24 h at 75 °C. Then the acetone was removed by rotary evaporator. And extraction method was used to RBHA in water and ethyl acetate mixed solution. Finally, the sample was obtained by lyophilization. RBHA (4 mg), EDC (20 mg) and NHS (10 mg) were added into water (10 mL) and stirred for 2 h. 5 mL water containing CaF2@NaGdF4@NaGdF4-PEI was added into it and stirred for 24 h. The precipitate was obtained by centrifugation. Then the 7

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CaF2@NaGdF4@NaGdF4-RBHA (UCNPs-RBHA) nanoparticles were dispersed in water (5 mL). DSP was obtained according to reported paper.18 Thereafter, DSP (20 mg), EDC (20 mg) and NHS (10 mg) were dissolved into water (10 mL) and stirred for 2 h. Then UCNPs-RBHA (5 mL) and polyethylene glycol 2000 (PEG2000-NH2, 5 mg/mL, 10 mL) added

into

the

solution

and

stirred

for

overnight.

Finally,

the

of

were

CaF2@NaGdF4@NaGdF4-RBHA-Pt-PEG (UCNPs-RBHA-Pt-PEG) can be obtained

ro

by centrifugation.

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Temperature sensing

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The UCNPs-RBHA-Pt-PEG nanoparticles (2 mL, 0.8 mg/mL) were dispersed in PBS

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with pH=7.4 (1.5 mL), a temperature change device (QNW luma 40) was used to heat

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the solution from 299-341 K, and the spectrum signals were collected by Andor

irradiation.

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SR-500i spectrometer (Andor Technology Co, Belfast, U.K.) upon 980 nm laser

In vitro and in vivo T1-weighted MR imaging The UCNPs-RBHA-Pt-PEG nanoparticles with different Gd3+ concentrations were placed into tubes (0, 0.1875, 0.375, 0.75, 1.5, 3 mM). The signal was collected by Huantong 1.5 T MR scanner for small animal imaging system (Shanghai, China) with the following parameters: TR = 2500 ms, TE = 19.2 ms. The exact sequence of T1 imaging was gradient recalled echo, which gradient strength was 11 Gs/cm. And the evolution of 1/T1 value with Gd3+ concentration can be fitting by a line, which the slope was r1 value. 8

Journal Pre-proof 150 μL PBS (PH=7.4) solution containing UCNPs-RBHA-Pt-PEG (Gd3+: 2 mM) was injected into Balb/c mouse in situ. Then the Huantong 1.5 T MR scanner for small animal imaging system was used to detect the MR imaging of mice. Cell uptake The CT26 cells were incubated in an atmosphere of 5% CO2 at 37 °C. They were seeded in 6-well plate with cell density of 105 cells/well and incubated for overnight.

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Then the cells were incubated with UCNPs-RBHA (10 μM) for different times (0, 10

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min, 0.5, 2, 6 h), respectively. Subsequently, the 4% formaldehyde was used to fix the

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cells and the DAPI was used to dye nucleus. The inverted fluorescence microscope

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(Nikon Ti-S) was used to detect DAPI and UCL imaging.

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In addition, the amount of platinum taken by cells also was detected. The CT26 cells

overnight.

Then

the

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were seeded in 6-well plate with cell density of 105 cells/well and incubated for cells

were

incubated

with

cisplatin,

DSP

and

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UCNPs-RBHA-Pt-PEG at same concentration (Pt: 0.72 μM) for 0.5, 6 h, respectively (n=3). Then cell lysis solution was added into wells and the amounts of platinum were detected by inductively coupled plasma-mass spectrometer (ICP-MS). Singlet oxygen detection The ROS was measured by 1,3-diphenylisobenzofuran (DPBF) probe. Generally, 20 μL dimethyl sulfoxide (DMSO) solution containing DPBF (10 mg/mL) was placed into UCNPs-RBHA-Pt-PEG nanoparticles solution (2 mL, 25 mM). Then the mixed solution was sonicated for 10 min in the dark. Finally, the solution was irradiated under 980 nm laser (0.5 W/cm-2) for different times (0, 3, 6, 10, 15, 20 min), and the 9

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related absorption curves were tested for a comparison of the DPBF absorption intensity at 417 nm. 2,7-dichlorofluorescein diacetate (DCFH-DA) was an effective probe for the detection of ROS in CT26 cells. The CT26 cells were incubated in an atmosphere of 5% CO2 at 37 °C. In detail, the cells were placed to 6-well plate with density of 105 cells/well for

of

overnight. Simply, the irradiated with or without 980 nm laser were written as 980+ and 980-, respectively. The cells were incubated with PBS-980+ (pH=7.4, 0.35 6

min),

UCNPs-RBHA-Pt-PEG-980-

ro

W/cm-2,

(0.16

μM),

and

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UCNPs-RBHA-Pt-PEG-980+ (0.16 μM, 0.35 W/cm-2, 6 min) nanoparticles for 4 h,

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respectively. Then DCFH-DA (10 μM) was added into each well and incubated for 20

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min in the dark. Then the cells were washed with PBS for three times. Finally, the

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existence of ROS was characterized by green luminescence signal using an inverted fluorescence microscope (Nikon Ti-S).

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In vitro tumor inhibition

In vitro tumor inhibition of UCNPs-RBHA-Pt-PEG was assayed against CT26 cells. Briefly, the CT26 cells were incubated in an atmosphere of 5% CO2 at 37 °C. They were seeded in 96-well plate with density of 3500 cells/well and incubated for overnight. Then the cells were incubated with 980+, UCNPs, cisplatin, DSP, UCNPs-RBHA-Pt-PEG-980-, UCNPs-RBHA-Pt-PEG-980+ (0.35 W/cm-2, 6 min) at different concentrations for 48 h, respectively. Finally, the cell viabilities were evaluated by MTT method. Cell apoptosis 10

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The CT26 cells were incubated in an atmosphere of 5% CO2 at 37 °C. They were placed into 6-well plate with density of 2×105 cells/well and treated with (a) PBS, 980+ (0.35 W/cm-2, 6 min), UCNPs; (b) DSP, cisplatin (0.62 μM of Pt); (c) UCNPs-RBHA-980+ (15.6 μM, 0.35 W/cm-2, 6 min, )；(d) UCNPs-RBHA-Pt-PEG 980-, UCNPs-RBHA-Pt-PEG 980+ (0.08 μM of Pt) for 24 h. Then the CT26 cells

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were treated by trypsinization and PBS. Subsequently, the cells were stained by Annexin V-FITC and PI staining kit. Finally, the result of cell apoptosis was observed

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by fluorescence signal using an inverted fluorescence microscope (Nikon Ti-S).

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In vivo tumor inhibition

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The mice were injected with CT26 cells (100 μL, 107 cells/mL) in the left axilla. After

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one week, the tumor sizes reached to about 100-150 mm3. The mice (6-8 weeks old)

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are used for in vivo tumor inhibition. In situ injection is used for in vivo tumor inhibition study, the location of the tumor is irradiated with 980 nm laser after

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injection for 4 h. Then the mice were randomly separated into six groups (4 mice/group), which were injected with

(a) PBS, 980+ (0.35 W/cm-2, 6 min); (b)

cisplatin (1.28 mM of Pt, 100 μL); (c) UCNPs-RBHA-980+ (2.5 mM, 0.35 W/cm-2, 6 min); (d) UCNPs-RBHA-Pt-PEG-980-, UCNPs-RBHA-Pt-PEG-980+ (0.35 W/cm-2, 6 min, 1.28 mM of Pt, 100 μL). The solution injected into mice is PBS (pH=7.4), which is filtered firstly, then sterilized by high-temperature and high-pressure sterilization, and then irradiated by ultraviolet (UV) light. The nanoparticles for injection are sterilized by UV light irradiation, and finally disperse in PBS (pH=7.4) solution and inject into mice. The tumor sizes and body weights of mice were detected every 2 11

Journal Pre-proof days, and the former was calculated by the equation: V=L×W2/2. The body weights of mice are weighed after injection and irradiated. After two weeks, the major organs (heart, liver, spleen, lung, and kidney) of different groups were used to histological analysis. The tissues were fixed in 10% neutral buffered formalin, and packaged in paraffin. The hematoxylin-eosin (H&E) was used for staining thin slices of tissues (4

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μm). Materials and characterization were found in the supplementary information (SI)

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section.

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RESULTS

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Sample preparation and characterization

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CaF2, CaF2@NaGdF4 and CaF2@NaGdF4@NaGdF4 UCNPs are obtained by

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solvothermal process.30 The TEM images of them are shown in Figure 1a, b and c, it can be found that the UCNPs are homogeneous and the average sizes are 8.6, 10.0,

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and 13.6 nm, respectively. The size of sample is smaller than common rare earth fluoride by same method (same ratio between OA and ODE), such as NaYF4.31,32 In addition, the particle size is related to charge compensation effects while calcium ions are replaced by rare earth ions, excess fluorinion on the surface of crystal will form transient electric dipoles by negative poles outward, which limits the diffusion of fluorinion. Furthermore, the particle size of nanoparticles is limited.32 Dynamic light scattering (DLS) measurement indicates that the average sizes are 61.33, 72.09, and 81.51 nm, respectively (Figure 1d, e and f). The diameters of DLS are bigger than TEM ones due to the effect of hydrodynamic size.33 The X-ray diffraction (XRD) 12

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patterns are shown in Figure 1g, which are consistent with standard card, respectively. As we all know, core-shell structure is a good strategy to enhance upconversion emission.34 The emission intensities at 525 (2H11/2 → 4I15/2), 545 (4S3/2 → 4I15/2), and 654 (4F9/2 → 4I15/2) nm become stronger after coating shell layer under the same power (Figure 1h). Compared with the core (CaF2), the intensity of core-shell

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(CaF2@NaGdF4) is 187, 91, and 24 times higher than it. Moreover, the intensity of core-shell-shell (CaF2@NaGdF4@NaGdF4) is 904, 457, and 47 times higher than it.

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In the Figure S1, we can find that the emission intensity of core coated by an activator

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UCNPs are simply written as UCNPs.

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shell is stronger than the coated by an inert shell, and the CaF2@NaGdF4@NaGdF4

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Fourier transform infrared spectroscopy (FTIR) spectrum of UCNPs-PEI is shown in

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Figure 1i. The peaks at 1468 and 1556 cm-1 are pointed to the stretching vibration of N-H (amine group) in the PEI (polyethyleneimine). The peaks of 2848 and 2924 cm -1

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could be attributed to asymmetric and symmetric stretching vibrations of –CH2 (methylene group) in the PEI. The above results illustrate that the UCNPs have been coated by PEI.

Temperature sensing Thermometers have been widely used in many fields, but most of them are carried out in the solid, rarely involving the temperature measurement in biological field, especially in living body.7,35,36 Moreover, ambient normal tissues are easily damaged by overheating in the course of PTT since nanoparticles are hotter than their surroundings under laser irradiation.26 However, the outstanding PTT curative 13

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efficacy means that tumor tissues are inhibited effectively while normal tissues are damaged slightly. Thus it is especially important to accurately measure the temperature of nanoparticles during PTT process to avoid large damage for ambient normal tissues. Figure 2a shows the green light area spectra of nanoparticles at different temperatures (biological temperature) in the PBS (pH=7.4). The changes of fluorescence intensities

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of 2H11/2 → 4I15/2 and 4S3/2 → 4I15/2 transitions are different with temperature rising

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(Figure 2a, Figure S2). The former rises while the latter declines owing to thermal

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process, resulting that the FIR between I525 and I545 ascends, as shown in Figure 2b.

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According to previous reports, we obtain lnB (2.31) and △E/kB (1079.31) by

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calculating ln(FIR) (Figure 2c, Figure S8). Furthermore, the values of sensitivity with

S8).

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UCL and MR imaging

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different temperatures are calculated according to previous reports (Figure 2d, Figure

Strong fluorescence from UCNPs-RBHA-Pt-PEG has potential application in bioimaging. In order to explore it, the cells are incubated with UCNPs-RBHA-Pt-PEG for 0, 10 min, 0.5, 2, 6 h. As shown in Figure 3b, green emission can be found at a short time with 980 nm irradiation, and the UCL signal is stronger after incubated for 6 h than 2 h, demonstrating that some time is needed for cell uptake. In addition, the amount of materials entering cells is detected by ICP-MS method. As shown in Figure 3c, the endocytosis content of 6 h is higher than 0.5 h one, which is consistent with Figure 3b. Interestingly, the endocytosis content of DSP is more than cisplatin one 14

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from 0.5 to 6 h, showing that DSP is easily uptaken by CT26 cells. In particular, the amount of platinum from UCNPs-RBHA-Pt-PEG in cells is 0.422 and 0.645 μg/104 cells from 0.5 to 6 h, respectively, which is about 10 times higher than cisplatin and DSP

at

the

corresponding

time.

We

can

find

that

the

amount

of

UCNPs-RBHA-Pt-PEG taken by cells is higher than other materials at the same time, causing that more tumor cells can be killed by platinum to realize tumor suppression.

of

The Gd3+ exists both in active and inert shells of synthesized UCNPs. The

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UCNPs-RBHA-Pt-PEG can be regard as T1-weighted MR agents on account of the

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strong positive signal properties of Gd3+ ions.17,37,38 As shown in Figure 3d,

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T1-weighted MR signal intensity decreases with the decrease of Gd3+ concentrations.

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The evolution of 1/T1 values with Gd3+ concentrations could be well fitted by a

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straight line in Figure 3e, and the slope (2.177 mM-1 s-1) can be obtained, which represents the longitudinal relaxivity (r1).

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Moreover, the MR imaging in vivo is also detected. The mouse bearing with tumor is injected with UCNPs-RBHA-Pt-PEG, whose T1-weighted MR signal of the mouse is stronger than the pre-injected (Figure 3f and g). The result indicates that the UCNPs-RBHA-Pt-PEG can be regard as an excellent contrast agent for T 1-weighted MR imaging. Detection of ROS generation In general, the absorption peaks of PS locate in UV-Vis light region.39 However, they have low penetration depth in biological tissues while UV light has strong damage for organism.40 The problem could be solved perfectly after attaching PS to UCNPs. As 15

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shown in Figure 4b, the red line denotes absorption spectrum of RBHA and the blue line represents emission spectrum of UCNPs. Fluorescence resonance energy transfer (FRET) can be come true on account of the large overlap between them from 510 to 565 nm, boosting the PDT function triggering by 980 nm laser. As shown in Figure S3, the existence of RBHA absorption peak with a slight red-shift

of

suggests that it has been coupled to the surface of UCNPs.41 The absorption curves of different RBHA concentrations are shown in Figure 4c, and the evolution of

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absorption intensity with concentration can be fitted by a line (slope 3.26, Figure 4d).

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Furthermore, the concentration of RBHA in the UCNPs-RBHA-Pt-PEG can be

re

calculated based on it, and the drug loading of RBHA is about 8.04%. Moreover,

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ICP-MS result suggests that DSP also has been loaded on the surface of UCNPs, and

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the concentration is 1.5 mM with the drug loading 1.87% (Table S1). The ROS, playing a significant role in PDT, can be generated based on FRET between

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UCNPs and RBHA upon 980 nm laser irradiation. In fact, the PDT efficacy is directly related to ROS production.42,43 DPBF probe is introduced in order to detect the generation of ROS. The characteristic absorption peak (about 425 nm) of DPBF declines owing to the irreversible reaction between DPBF and ROS.44,45 As shown in Figure 4e, the absorption intensities keep declining with different times (0-20 min) under 980 nm excitation (0.25 W/cm2), proving that the generation of ROS is continuous upon 980 nm laser irradiation. Furthermore, the production of ROS by UCNPs-RBHA-Pt-PEG in living cells is detected by DCFH-DA probe.46,47 In fact, DCFH-DA can’t emit fluorescence, which 16

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can be transformed into DCFH (no fluorescence), and then the latter can react with ROS to become 2,7-dichlorofluorescein (DCF). Finally, intense green fluorescence emitted by DCF can be seen. As shown in Figure 4f, there is no obvious fluorescence signal can be found, suggesting that no ROS produced after incubated with PBS under irradiation

or

UCNPs-RBHA-Pt-PEG

without

irradiation

of

(UCNPs-RBHA-Pt-PEG-980-). Oppositely, the clear green fluorescence is observed, indicating the generation of ROS only both UCNPs-RBHA-Pt-PEG and irradiation

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In vitro PDT and chemotherapy

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coexist (UCNPs-RBHA-Pt-PEG-980+).

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The biocompatibility of material is an important standard for its actual biological

lP

application, which can be detected by MTT assay. PEI has benign biocompatibility in

viabilities

after

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vitro and in vivo, proved by reported papers.18,48 Figure 5a shows the CT26 cell incubated

with

UCNPs-RBHA-Pt-PEG-980-,

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UCNPs-RBHA-Pt-PEG-980+, cisplatin, and DSP for 48 h. For comparison, the cell viability of UCNPs-RBHA is higher than other materials at the same concentration, indicating that it possesses better biocompatibility and lower cytotoxicity for CT26 cells (Figure S4). The DSP can be translated into Pt (II) after taken by cells, and then linking with DNA to form platinated DNA crosslinks, which is larger harmfulness for cell.49,50 However, pure cisplatin has higher cytotoxicity for organism than DSP at the same concentration.50-52 In fact, DSP can get in and out of the cell by active transport when the amount is constant, but there is little DSP can be translated into Pt (II), giving rise to little toxicity for cell.53 Compared with it, the cell viability is lower 17

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when

CT26

cells

incubated

with

UCNPs-RBHA-Pt-PEG-980-

or

UCNPs-RBHA-Pt-PEG-980+, demonstrating that more DSP is taken and translated into Pt (II) by cells after loaded on the surface of UCNPs-RBHA (Figure 5a). In addition,

we

can

find

that

the

cell

viabilities

are

different

between

UCNPs-RBHA-Pt-PEG-980- and UCNPs-RBHA-Pt-PEG-980+ at same concentration

of

since the enhanced PDT of latter exhibits higher inhibition effect for CT26 cells. The most suitable power density and irradiation time have been explored in order to

ro

eliminate the effect of laser irradiation (Figure S5). As shown in Figure S6, the cell

-p

viability is high (over 90%) after irradiation (0.35 W/cm2, 6 min). The value of 50%

shown

in

Figure

5b,

the

lP

As

re

inhibitory concentrations (IC50) is a significant indicator for tumor cells apoptosis.54 IC50

values

of

cisplatin,

DSP,

0.718,

and

0.496

na

UCNPs-RBHA-Pt-PEG-980-, and UCNPs-RBHA-Pt-PEG-980+ are 1.324, 8.427, μM,

respectively.

Furthermore,

the

cell

lethality

of

Jo ur

UCNPs-RBHA-Pt-PEG-980+ is 17, 2.7, and 1.45 times higher than DSP, cisplatin, and CaF2-RBHA-Pt-980-, respectively.55 Moreover, the apoptosis experiment also can explain it. The early and late apoptosis are indicated by green luminescence from Annexin V and red luminescence from PI, respectively. As shown in Figure 5c, the data indicate that CT26 cells can be killed by cisplatin, UCNPs-RBHA-980+, UCNPs-RBHA-Pt-PEG-980-, and UCNPs-RBHA-Pt-PEG-980+ groups (Figure 5c). In particular, the mortality of UCNPs-RBHA-Pt-PEG-980+ group is highest. In vivo PDT and chemotherapy The anticancer effect of UCNPs-RBHA-Pt-PEG-980+ is further studied by in vivo 18

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experiment. As shown in Figure 6a, the body weights of mice keep growing in all groups. In addition, the histological analysis of organs is shown in Figure S7, and there is no difference between control and experimental groups, suggesting that all the treatments have low toxicity for the mice.56 In the Figure 6b and c, the tumor sizes of PBS and 980+ groups (control groups) grow rapidly. On the contrary, the tumor sizes

of

of cisplatin, UCNPs-RBHA-980+, and UCNPs-RBHA-Pt-PEG-980- groups are smaller than control groups ones, proving that individual PDT or chemotherapy has effect

in

some

extent.

Interestingly,

ro

inhibition

the

effect

of

na

DISCUSSION

lP

re

agreement with the in vitro experiment.

-p

UCNPs-RBHA-Pt-PEG-980- group is more significant than cisplatin one, which is in

In this study, the small-sized and abundant-fluorescent CaF2: Yb3+/Er3+ UCNPs are

Jo ur

achieved by solvothermal route. In particular, the charge compensation effects and core-shell structure have made a great contribution for it, respectively. After coated by PEI, the RBHA and DSP can be combined with UCNPs by the dehydration condensation between amine and carboxyl group, which lays the foundation for the realization of dual functions of PDT and chemotherapy. In addition, the temperature test in water has been accomplished using FIR technique, which will provide a good guidance for temperature measurement and photothermal therapy in living body. Furthermore, the UCNPs-RBHA-Pt-PEG also reveals excellent capability in UCL and MR imaging for biological field based on enhanced fluorescence and prominent 19

Journal Pre-proof contrast effect of Gd3+. Besides, the in vitro experiment demonstrates that CT26 cells can be killed effectively after incubated with UCNPs-RBHA-Pt-PEG-980+ based on synergetic therapy of PDT and chemotherapy while the UCNPs-RBHA displays benign biocompatibility without laser irradiation. Moreover, the CT26 tumors are inhibited markedly in the

of

UCNPs-RBHA-Pt-PEG-980+ group based on the synergetic therapy of chemotherapy and PDT. All the results indicating that UCNPs-RBHA-Pt-PEG-980+ can be regard as

ro

an excellent therapeutic agent for anticancer therapy. There are big differences

-p

between our study and ethanol injection. Firstly, after intratumoral ethanol injection,

re

more cells die in the center of the injection area while many cells survive in the

lP

marginal area because they can’t be infiltrated effectively by ethanol.57,58 Compared to

na

this, the curative effect of experimental group (UCNPs-RBHA-Pt-PEG-980+) is satisfactory, tumor volume decreases a lot, some even disappears. Secondly, the

Jo ur

ethanol is injected many times for killing cells in the marginal area.59 The materials and 980 nm laser irradiation used in our experiment only need to be injected once. Thirdly, if the tumor recurred after the injection of ethanol, which will cause rapid proliferation of cancer cells.59 In our experiment, the synergetic therapy modality of PDT and chemotherapy shows good curative effect, the volume of all tumors decreases with time in the experimental group (UCNPs-RBHA-Pt-PEG-980+), and there is no increase in recurrence. In summary, the strong fluorescence and small size promotes the imaging capability and reduces the physical burden, respectively. In addition, this work also has proved 20

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the capability of UCNPs-RBHA-Pt-PEG in UCL and MR imaging, temperature sensing, and synergetic therapy of PDT and chemotherapy, constructing a composite nanoparticle platform for its application in biological field, which may provide a valuable enlightenment for diagnosis and treatment of cancer in the future.

ACKNOWLEDGEMENTS

of

This project is financially supported by the National Natural Science Foundation of

ro

China (Grant Nos. NSFC 51720105015, 11874182，51672269, 51772124, 21521092, and 51872282), the National Basic Research Program of China (Grant No.

-p

2014CB643803), Science and Technology Development Planning Project of Jilin

re

Province (Grant 20170101188JC, and 20180520163JH), Youth Innovation Promotion Association of CAS (Grant No. 2017273), Overseas, Hong Kong & Macao Scholars

lP

Collaborated Researching Fund (Grant No. 21728101), Science and Technology Project of the 13th Five-Year Plan of Jilin Provincial Department of Education (No.

Jo ur

2018C043-4).

na

JJKH20190179KJ) and provincial industrial innovation in Jilin Province (No.

21

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Figure legends Scheme 1. Schematic illustration of the synthesis and antitumor therapy mechanism of UCNPs-RBHA-Pt-PEG. The small-sized CaF2 UCNPs prepared by solvothermal method emit strong fluorescence after coated with NaGdF4 shells (activator and inert). The RBHA and DSP are attached to the surface of UCNPs on account of dehydration

of

condensation between amino and carboxyl groups (UCNPs-RBHA-Pt-PEG). The

synergetic therapy of PDT and chemotherapy.

ro

compound material possesses strong antitumor effect in vitro and in vivo based on the

-p

Figure 1. Synthesis and characterization of CaF2@NaGdF4@NaGdF4 UCNPs. (a)-(c)

re

TEM images; (d)-(f) size distributions; (g) XRD patterns; (h) emission spectra of

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of UCNPs-PEI.

lP

CaF2, CaF2@NaGdF4 and CaF2@NaGdF4@NaGdF4 UCNPs, respectively; (i) FTIR

Figure 2. Temperature sensing profile of UCNPs-RBHA-Pt-PEG in PBS (pH=7.4).

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The change of (a) fluorescence intensity; (b) FIR; (c) ln(FIR); (d) sensitivity with different temperatures.

Figure 3. UCL and MR imaging of UCNPs-RBHA-Pt-PEG. (a) Schematic illustration of endocytosis with different times; (b) The UCL imaging under different times; (c) The endocytosis amount of platinum drug for different types under different times; (d) In vitro T1-weighted MR imaging of UCNPs at different concentrations of Gd3+; (e) Relaxation rate r1 versus different molar concentrations; (f) pre-injection and (g) after injection of UCNPs-RBHA-Pt-PEG in situ. ANOVA was used to assess statistical significance.*p< 0.05, **p< 0.01, ***p< 0.001. 31

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Figure 4. Detection of ROS generation. (a) Schematic illustration of ROS generation; (b) Spectral overlap between upconversion emission spectrum of UCNPs (blue curve) and absorption spectrum of RBHA (red curve); (c) The changes of absorption intensity of RBHA at different concentrations; (d) The absorption intensity of RBHA fitted with a line; (e) The changes of DPBF absorption intensity when the

of

UCNPs-RBHA excited with 980 nm laser for different times; (f) The detection of ROS generation for different treatments: PBS-980+, UCNPs-RBHA-Pt-PEG-980- and

ro

UCNPs-RBHA-Pt-PEG-980+.

-p

Figure 5. In vitro PDT and chemotherapy. (a) CT26 cell viabilities after incubated

re

with UCNPs-RBHA-Pt-PEG-980+, UCNPs-RBHA-Pt-PEG-980-, cisplatin, and DSP

Pt)

of

different

treatments:

UCNPs-RBHA-Pt-PEG-980+,

na

(IC50,

lP

for 48 h at 37 °C in an atmosphere of 5% CO2; (b) The 50% inhibitory concentration

UCNPs-RBHA-Pt-PEG-980-, cisplatin and DSP; (c) Apoptosis of CT26 cells by

cisplatin,

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staining with Annexin V-FITC and PI after different treatments: PBS, 980+, DSP, UCNPs,

UCNPs-RBHA-980+,

UCNPs-RBHA-Pt-PEG-980-,

UCNPs-RBHA-Pt-PEG-980+. ANOVA was used to assess statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001. Figure 6. In vivo PDT and chemotherapy. (a) The evolution of body weights with different times and treatments; (b) The evolution of tumor volumes with different times and treatments; (c) Digital photographs of excised tumors for different treatments: PBS, 980+, cisplatin, UCNPs-RBHA-980+, UCNPs-RBHA-Pt-PEG-980-, UCNPs-RBHA-Pt-PEG-980+. 32

Journal Pre-proof Author contribution Statement Guofeng Liu: Data curation, Writing- Original draft preparation, Visualization, Investigation, Supervision Software, Validation, Writing- Reviewing and Editing, Fan Jiang: Writing- Reviewing and Editing, Yeqing Chen: Visualization, Investigation. Chang Yu: Software, Validation.: Binbin Ding: Conceptualization, Methodology Shuai Shao: Writing- Original draft preparation

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Mochen Jia: Application of statistical, mathematical, computational

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Ping’an Ma: Development or design of methodology Zuoling Fu: Application of statistical, mathematical

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na

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Jun Lin: Preparation, creation and/or presentation of the published work

33

Journal Pre-proof Graphical abstract The small-sized and enhanced-fluorescent CaF2 UCNPs emit strong fluorescence after coated with NaGdF4 shells (activator and inert). The ample PDT efficacy is achieved relied on the fluorescence resonance energy transfer owing to a large overlap between upconversion emission and rose-bengal hexanic acid absorption (UCNPs-RBHA). At the same time, the UCNPs-RBHA conjugated with platinum (IV) and polyethylene glycol possesses high cell apoptosis for the CT26 cells due to high endocytosis. The UCL and MR imaging, temperature sensing, and strong antitumor effect based on the

of

synergetic therapy of PDT and chemotherapy are achieved on the single nanoparticles.

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na

lP

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-p

ro

(Graphical Abstract: Scheme 1)

34

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6