CoreâShellâShell Multifunctional Nanoplatform for Intracellular Tumor

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A Core-Shell-Shell Multifunctional Nanoplatform for Intracellular Tumor-Related mRNAs Imaging and Near-Infrared Light Triggered Photodynamic-Photothermal Synergistic Therapy Yao Cen, Wen-Jing Deng, Yuan Yang, Ru-Qin Yu, and Xia Chu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02081 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 6, 2017

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Analytical Chemistry

A Core-Shell-Shell Multifunctional Nanoplatform for Intracellular Tumor-Related mRNAs Imaging and Near-Infrared Light Triggered Photodynamic-Photothermal Synergistic Therapy Yao Cen†,‡, Wen-Jing Deng†, Yuan Yang†, Ru-Qin Yu† and Xia Chu†,* † State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China ‡ School of Pharmacy, Nanjing Medical University, Nanjing, 211166, P. R.China ABSTRACT: A multifunctional nanoplatform, which generally integrates biosensing, imaging diagnosis, and therapeutic functions into a single nanoconstruct, has great important significance of biomedicine and nanoscience. Here, we developed a core-shell-shell multifunctional polydopamine (PDA) modified upconversion nanoplatform for intracellular tumor-related mRNAs detection and near-infrared light triggered photodynamic and photothermal synergistic therapy (PDT-PTT). The nanoplatform was constructed by loading a silica shell on the hydrophobic upconversion nanoparticles (UCNPs) with hydrophilic photosensitizer methylene blue (MB) entrapped in it, and then modifying PDA shells through an in situ self-polymerization process, thus yielding a core-shell-shell nanoconstruct UCNP@SiO2-MB@PDA. By taking advantages of preferential binding properties of PDA for single-stranded DNA over double-stranded DNA and the excellent quenching property of PDA, a UCNP@SiO2-MB@PDA-hairpin DNA (hpDNA) nanoprobe was developed through adsorption of fluorescently labeled hpDNA on PDA shells for sensing intracellular tumor-related mRNAs and discriminating cancer cells from normal cells. In addition, the fluorescence resonance energy transfer from the upconversion fluorescence (UCF) emission at 655 nm of the UCNPs to the photosensitizer MB molecules could be employed for PDT. Moreover, due to the strong NIR absorption and high photothermal conversion efficiency of PDA, the UCF emission at 800 nm of the UCNPs could be used for PTT. We demonstrated that the UCNP@SiO2-MB@PDA irradiated with NIR light had considerable PDT-PTT effect. These results revealed that the developed multifunctional nanoplatform provided promising applications in future oncotherapy by integrating cancer diagnosis and synergistic therapy.

A multifunctional nanoplatform, which generally integrates biosensing, imaging diagnosis, and therapeutic functions into a single nanoconstruct, has attracted increasing attention due to the rapid development of nanoscience and biomedicine.1-6 The combination of multiple imaging techniques (such as computer tomography, magnetic resonance imaging, ultrasound imaging, and fluorescence imaging) with multimodality therapy (such as photodynamic therapy (PDT), photothermal therapy (PTT) and chemotherapy) is of great interest for higher diagnosis requirement and enhanced therapeutic efficacy.7-11 Lanthanide-doped upconversion nanoparticles (UCNPs) have recently gained tremendous attention in biosensing of complex biological samples, medical diagnosis and cancer therapy due to their outstanding properties, for instance tunable multicolor sharp emission, superior photostability, excellent light penetration depth, and autofluorescence-free nature.12-20 Up to now, several works have developed UCNPs-based biosensors with both biosensing and imaging functions.21-23 Moreover, some UCNPs-based nanoconstructs have combined imaging and cancer therapy.24-27 However, to our knowledge, the UCNPsbased integration of intracellular biomolecules sensing, imaging diagnosis, and multimodality therapy remains a great challenge in biomedical applications. Dopamine (DA) could be easily self-polymerized through spontaneous oxidation and form polydopamine (PDA) shell on a wide variety of substrate surfaces.28 PDA surface has abun-

dant catechol and amino groups, which enables further surface various biomolecules modification.29 In addition, the excellent biocompatibility and biodegradability of PDA makes it an ideal choice in biomedical applications.30,31 Furthermore, PDA could also act as a PTT agent because of its pronounced adsorption and photothermal energy conversion.32 Recently, tumor-related mRNAs can be widely utilized as specific markers to assess the stage of the tumor. 33-35 The abnormalities in tumor-related mRNAs expression are commonly associated with tumor progression.36 Currently, there have been several methods for detection of tumor-related mRNAs, including northern blot analysis,37 electrochemical analysis,38 and fluorescence assay.39 However, these methods are incapable of imaging the expression levels of mRNAs in living cells. Therefore, there is still a high demand to develop methods for detecting multiple tumor-related mRNAs in living cells. In the present study, we developed a multifunctional PDA modified upconversion nanoplatform for intracellular tumorrelated mRNAs detection and near-infrared (NIR) light triggered photodynamic and photothermal synergistic therapy (PDT-PTT). As shown in Scheme 1, the nanoplatform was constructed by loading a silica shell onto the hydrophobic UCNPs surface with hydrophilic photosensitizer methylene blue (MB) molecules entrapped in it, forming core-shell UCNP@SiO2-MB nanoparticles. The silica coating endowed the UCNPs with water-dispersibility and biocompatibility,

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whereas the MB molecules afforded the PDT function. Then, the UCNP@SiO2-MB nanoparticles were further modified with a PDA shell through an in situ self-polymerization process, yielding a core-shell-shell nanoconstruct UCNP@SiO2MB@PDA. The PDA shell could adsorb single-stranded DNA (ssDNA) through hydrogen bonding or π–π stacking between the aromatic groups of PDA and nucleobases of ssDNA. When forming the duplex structure with its target, the nucleobases of ssDNA formed hydrogen bonding with the corresponding nucleobases of target through complementary base pairing, which weakened the interaction between the nucleobases of ssDNA and the aromatic groups of PDA, resulting in the dissociation of the double-stranded DNA (dsDNA) from the PDA shell.31,40 Based on the preferential binding properties of PDA for ssDNA over dsDNA and the excellent fluorescence quenching property of PDA due to energy transfer and/or electron transfer, we could develop an UCNP@SiO2-MB@PDAhairpin DNA (hpDNA) nanoprobe by adsorbing fluorescently labeled hpDNA on PDA shell for sensing intracellular tumorrelated mRNAs and discriminating cancer cells from normal cells. In addition, the fluorescence resonance energy transfer (FRET) from the upconversion fluorescence (UCF) emission at 655 nm of the UCNPs to the photosensitizer molecules MB could be employed for PDT. Moreover, due to the strong NIR absorption and high photothermal conversion efficiency of PDA, the FRET from the UCF emission at 800 nm of the UCNPs to the PDA shell could be utilized for PTT. The results indicated that the developed multifunctional nanoplatform could integrate biosensing, imaging diagnostics and synergistic therapy, and hold great potential for the early cancer diagnosis and therapy.

Scheme 1. Schematic Illustration of the Principle of CoreShell-Shell Mulitifunctional UCNP@SiO2-MB@PDA Nanoplatform and Its Application for Intracellular TumorRelated mRNAs Imaging and Near-Infrared Light Triggered Photodynamic-Photothermal Synergistic Therapy.

EXPERIMENTAL SECTION Materials. YCl3·6H2O, YbCl3·6H2O, TmCl3·6H2O, ErCl3·6H2O,oleic acid (OA, 90%), 1-octadecene (ODE, 90%), ammonium fluoride (NH4F), sodium hydroxide (NaOH),

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tetraethyl orthosilicate (TEOS), Igepal CO-520, methylene blue (MB), dopamine hydrochloride, and 1,3diphenylisobenzofuran (DPBF) were obtained from SigmaAldrich. DNA and RNA sequences listed in Table S1 were provided by Sangon Biotechnology Co., Ltd. (Shanghai, China) and TaKaRa Biotechnology Co., Ltd. (Dalian, China). Singlet oxygen sensor green (SOSG) was purchased from Molecular Probes. CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit was purchased from Promega (Madison, USA). Instruments. The particle size and crystal phase were measured by transimission electron microscopy (TEM) and Xray powder diffraction (XRD). UV-vis absorption spectra, dynamic light scattering (DLS) measurements, Fourier transform infrared (FT-IR) spectra, Energy dispersive X-ray spectroscope (EDS) analysis, X-ray photoelectron spectroscopy (XPS) analysis and upconversion fluorescence spectra were all performed similarly with our previously reported methods.21 The fluorescence spectra of organic dye were recorded on FluoroMax-4 Spectrofluorometer with the internal excitation source. Synthesis of OA-Capped NaYF4: 20%Yb, 2%Er, 1%Tm UCNPs (OA-UCNPs). OA-UCNPs were prepared using a modified solvothermal method according to our previous literature.21 6 mL OA and 15 mL ODE were mixed with YCl3·6H2O (0.77 mmol), YbCl3·6H2O (0.20 mmol), ErCl3·6H2O (0.02 mmol), and TmCl3·6H2O (0.01 mmol) in a 50 mL three-necked flask. The procedures for subsequent synthetic process were similar to those used in our previous literature.21 The resulting OA-UCNPs were finally redispersed in cyclohexane for silica coating. Synthesis of Silica-Coated UCNPs Loaded with MB (UCNP@SiO2-MB). UCNP@SiO2-MB was synthesized according to the literature method with modified reverse microemulsion method.41 Igepal CO-520 (1 mL) was mixed with cyclohexane (20 mL) in a 50 mL flask and stirred for 1 h at 22 °C. OA-UCNPs in cyclohexane (8 mg) were added into the flask, and the mixture was stirred for 3 h. MB aqueous solution (30 µL) was added and stirred for 1 h. After that, 150 µL NH3·H2O was slowly added into the flask and stirred for 2 h. Subsequently, the system was sealed and stirred for 24 h at 22 °C after slowly adding 200 µL TEOS. The products were precipitated with methanol and washed with ethanol for several times. The obtained UCNP@SiO2-MB was finally suspended in 1 mL of ethanol. The concentration of UCNP@SiO2-MB was calculated as 8.0 mg mL-1. For the preparation of silicacoated UCNPs without loaded MB (denoted as UCNP@SiO2), there were the same procedure except for MB addition. Preparation of PDA-Modified UCNP@SiO2-MB (UCNP@SiO2-MB@PDA). Briefly, UCNP@SiO2-MB (125 µL, 8.0 mg mL-1) was collected by centrifugation and discarded the supernatants. To decorate UCNP@SiO2-MB with 2 nm thickness of PDA shell, 30 µL of dopamine hydrochloride solution (10 mg mL-1, dissloved in 10 mM Tris-HCl at pH 8.5) was mixed with 2.97 mL of 10 mM Tris-HCl (pH 8.5) containing 1 mg of UCNP@SiO2-MB under continuous sonication for 90 min at room temperature. After that, UCNP@SiO2-MB@PDA was obtained by centrifugation and washing. The prepared UCNP@SiO2-MB@PDA was finally dispersed in 250 µL of ultrapure water. The concentration of UCNP@SiO2-MB@PDA was calculated as 4.0 mg mL-1. Preparation of UCNP@SiO2-MB@PDA-hpDNA Nanoprobe. UCNP@SiO2-MB@PDA-hpDNA nanoprobe was pre-


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pared through adsorping fluorescently labeled hpDNA on PDA shells. 500 nM FAM-hpDNA was mixed with 2.0 mg mL-1 of UCNP@SiO2-MB@PDA and the mixture was incubated for 1 h. Excess hpDNAs were removed by centrifugation. The as-prepared UCNP@SiO2-MB@PDA-hpDNA (FAM) nanoprobe was redispersed and stored at 4 °C. The multiplexed nanoprobe was synthesized using the similar procedure with FAM-hpDNA and Cy3-hpDNA. The FAM-hpDNA was used to quantify the amounts of hpDNA on the surface of the UCNP@SiO2-MB@PDA. Determination of mRNA Target Based on UCNP@SiO2MB@PDA-hpDNA Nanoprobe in Buffer Solution. In a typical assay, 0.2 mg mL-1 of UCNP@SiO2-MB@PDAhpDNA (FAM) was incubated with different concentrations of mRNA target (0, 1, 2.5, 5, 10, 25, 50, 75, 100, 150, and 200 nM) at 37 °C for 1 h, and the fluorescence intensity was measured on FluoroMax-4 Spectrofluorometer. The reaction buffer contained 20 mM Tris–HCl buffer (pH 7.4), 50 mM NaCl and5mM MgCl2. Non-complementary sequence was used to verify the specificity of the nanoprobe toward target mRNA. Confocal Fluorescence Imaging of mRNA Target Based on UCNP@SiO2-MB@PDA-hpDNA Nanoprobe in Living Cells. MCF-7 and MCF-10A cells were seeded on a 35-mm Petri dish with culture medium for 24 h, respectively. After washing three times with PBS buffer (pH 7.4), the cells were incubated in the culture medium containing 0.2 mg mL-1 UCNP@SiO2-MB@PDA-hpDNA nanoprobe for 8 h at 37°C, then imaged by confocal fluorescence microscope (Olympus FV1000) after washing the cells with PBS. UCF images were collected by using a 980 nm excitation laser. Cytotoxicity Assay. MCF-7, HeLa, HepG2 and MCF-10A cells were seeded in 96-well plates at 1.5×104 cells per well and incubated for 24 h. After incubating with different concentrations of UCNP@SiO2-MB@PDA for another 24 h, the cell viability was measured by a CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit. Detection of Singlet Oxygen (1O2) Generation Based on UCNP@SiO2-MB@PDA. DPBF, which reacted with 1O2 to cause an absorption decrease at about 410 nm, was employed for detection of 1O2 generation. 10 µL of a DPBF/ethanol solution (5 mM) was mixed with 1 mL of UCNP@SiO2MB@PDA or UCNP@SiO2@PDA (as control experiment) solution. The mixture was then irradiated by a 980 nm laser (1.5 W cm-2), and the UV-vis absorption spectra were collected every 5 min. Intracellular 1O2 Measurements. 1O2 generation inside cells was detected using SOSG. MCF-7 cells were seeded on a 35-mm Petri dish with culture medium for 24 h. After treated with UCNP@SiO2-MB@PDA for 8 h, SOSG was incubated with the cells for 1 h. Cells were washed three times with PBS and exposed to 980 nm laser (1.5 W cm-2) for 5 min, then fluorescence images were captured. Measurement of Photothermal Performance. UCNP@SiO2-MB@PDA with different concentrations (0, 0.05, 0.1, 0.15, and 0.2 mg mL-1) was irradiated by a 980 nm laser (1.5 W cm-2) for 10 min. The solution temperature was measured by a thermometer with thermocouple probe. Photodynamic and Photothermal Synergistic Therapy. MCF-7 cells were plated on a 35-mm Petri dish with culture medium for 24 h. After washing three times with PBS buffer (pH 7.4), the cells were incubated in the culture medium con-

taining 0.2 mg mL-1 UCNP@SiO2-MB@PDA for 8 h at 37°C. After irradiated by a 980 nm laser (1.5 W cm-2), the cells were dyed with propidiumiodide (PI) and for confocal fluorescence image. To evaluate the PDT-PTT cytotoxicity of UCNP@SiO2MB@PDA, MCF-7 cells were seeded in 96-well plates at 1.5×104 cells per well and incubated for 24 h. The cells were incubated with different concentrations of UCNP@SiO2MB@PDA for 8 h. After irradiated by a 980 nm laser (1.5 W cm-2), the cells were incubated for an additional 24 h. The cell viability was measured by a CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit.

RESULTS AND DISCUSSION Synthesis and Characterization of UCNP@SiO2MB@PDA Nanoconstruct. The OA-capped NaYF4: 20%Yb, 2%Er, 1%Tm nanoparticles (OA-UCNPs) were prepared using a modified solvothermal method according to our previous literature.21 OA-UCNPs showed uniform spherical-like morphology with an average diameter of 20 nm (Figure 1A). The XRD analysis (Figure 1D) confirmed the pure hexagonalphase of these nanoparticles (JCPDS no. 28-1192). A thin silica shell loading with photosensitizer MB was coated on the surface of OA-UCNPs, resulting in core-shell structure UCNP@SiO2-MB. The TEM image revealed that approximate 2.7 nm thick silica shell was coated on the surface of OAUCNPs (Figure 1B). DA could easily form PDA shell on the substrate through in situ spontaneous polymerization in an alkaline solution and the presence of oxygen.28 The asprepared core-shell-shell nanostructural UCNP@SiO2MB@PDA showed monodispersed morphology with a 2 nmthickness PDA shell on the surface of UCNP@SiO2-MB (Figure 1C). The DLS measurement results showed that the UCNP@SiO2-MB@PDA was well-dispersed in water and the hydrodynamic diameter of the OA-UCNPs increased from 23 nm to 38 nm after coating the silica and PDA shell (Figure S1), which was consistent with the TEM results. In addition, the thickness of the PDA shell could be tuned to 4 nm by prolonging the polymerization time to 3 h (Figure S2). Furthermore, FT-IR spectra of the UCNP@SiO2-MB@PDA nanoparticles exhibited new peaks at 3410 cm-1 (phenolic N-H and O-H stretching vibration), 1605 cm-1 (aromatic ring stretching vibration and N-H bending vibration), and 1296 cm-1 (phenolic C-O stretching vibration) compared with that of UCNP@SiO2MB, confirming the successful polymerization of PDA shell(Figure S3). EDS measurements clearly showed the elements of N, Si, and S in UCNP@SiO2-MB@PDA (Figure S4). XPS measurements further verified the presence of PDA on the surface of OA-UCNPs (Figure S5). All of results strongly demonstrated the successful coating of SiO2 shell and polymerization of PDA shell on the OA-UCNPs surface.



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Figure1. TEM images of (A) the as-prepared OA-UCNPs, (B) UCNP@SiO2-MB, and (C) UCNP@SiO2-MB@PDA. (D) XRD pattern of OA-UCNPs compared to the standard pattern of NaYF4 (JCPDS no. 28-1192). Compared with UCNP@SiO2, there was a remarkable MB absorption peak at about 620 nm in UV-vis spectrum of UCNP@SiO2-MB, indicating the successful loading of MB within the silica matrix (Figure 2A). In addition, UCNP@SiO2-MB@PDA exhibited higher adsorption due to the presence of PDA shell, which was favorable for PTT (Figure 2A). Furthermore, as shown in Figure 2B, the upconversion fluorescence spectrum of OA-UCNPs in cyclohexane showed three major UCF emissions at 540 nm, 655 nm and 800 nm, respectively. Since the absorbance peak of MB obviously matched the red emission at 655 nm of OA-UCNPs, the red emission of UCNP@SiO2-MB was quenched remarkably after loading MB in the silica matrix through FRET process (Figure 2B). In addition, the lifetime of the UCNPs was significantly reduced by MB, confirming the efficient energy transfer between the UCNP and MB (Figure S6). Compared with that of UCNP@SiO2-MB, the upconversion fluorescence intensity of UCNP@SiO2-MB@PDA decreased obviously due to the absorption of PDA shell (Figure 2B).

Figure 2. (A) UV-vis absorbance spectra of UCNP@SiO2 (black line), UCNP@SiO2-MB (red line), and UCNP@SiO2MB@PDA (blue line). Inset: photos of UCNP@SiO2 (left), UCNP@SiO2-MB (middle), and UCNP@SiO2-MB@PDA (right). (B) Upconversion fluorescence spectra of OA-UCNPs in cyclohexane (black line), UCNP@SiO2-MB in water (red line), and UCNP@SiO2-MB@PDA in water (blue line). Construction of UCNP@SiO2-MB@PDA-hpDNA Nanoprobe and Sensing of mRNA Targets in Buffer. PDA has abundant catechol and amino groups, which facilitate the direct binding of ssDNA via non-covalent interactions and lead

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to the fluorescence quenching of dye-labeled ssDNA. However, the interactions would be weakened upon the reaction with target sequences, leading to the release of the binding DNA probes and recovery of the fluorescence.40 The Zeta potential changes indicated that hpDNA was successfully conjugated onto the surface of UCNP@SiO2-MB@PDA (Figure S7). Using FAM-hpDNA, the average amount of hpDNA on UCNP@SiO2-MB@PDA was estimated to be about 0.23 µmol g-1 nanosystem. FAM-hpDNA, which specifically recognized c-myc mRNA, mixed with prepared UCNP@SiO2-MB@PDA nanoparticles to form UCNP@SiO2-MB@PDA-hpDNA (FAM) nanoprobe. As shown in Figure 3A, the fluorescence of the FAM-hpDNA decreased gradually with increased UCNP@SiO2-MB@PDA concentration, and significant quenching (96%) was observed in the presence of 0.2 mg mL-1 UCNP@SiO2-MB@PDA, indicating the successful adsorption of FAM-hpDNA onto the surface of UCNP@SiO2-MB@PDA and the high fluorescence quenching efficiency of UCNP@SiO2-MB@PDA. The performance of UCNP@SiO2MB@PDA-hpDNA (FAM) nanoprobe for mRNA target detection was then investigated. As shown in Figure 3B, the UCNP@SiO2-MB@PDA-hpDNA (FAM) nanoprobe showed no visible fluorescence intensity. However, upon incubation with 200 nM complementary c-myc mRNA target, the nanosystem showed an intense fluorescence signal and 21-fold fluorescence enhancement. In contrast, the addition of noncomplementary sequence would not result in obvious fluorescence increase. Under the optimal reaction buffer containing 20 mM Tris–HCl buffer (pH 7.4), 50 mM NaCl and 5mM MgCl2, the fluorescence intensity of UCNP@SiO2MB@PDA-hpDNA (FAM) nanosystem gradually increased with increasing c-myc mRNA target concentrations (Figure 3C). The peak intensity gradually recovered with c-myc mRNA concentration in the range of 0−200 nM (Figure 3D). The detection limit of c-myc mRNA was estimated to be 0.40 nM according to the 3σ rule. In addition, the fluorescence images at the different concentrations of target of mRNA were given in Figure S8. It can be seen that the fluorescence intensity gradually increased with increasing c-myc mRNA target concentrations. The UV-vis absorption spectrum of UCNP@SiO2-MB@PDA showed strong wide-band absorption, which made it a potential dark quencher to a wide variety of fluorescence donors. Cy3-hpDNA, which specifically recognized TK1 mRNA, mixed with prepared UCNP@SiO2MB@PDA to form UCNP@SiO2-MB@PDA-hpDNA (Cy3) nanoprobe. The fluorescence intensity of UCNP@SiO2MB@PDA-hpDNA (Cy3) nanosystem gradually increased with increasing TK1 mRNA target concentrations (Figure S9A). The peak intensity gradually recovered with TK1 mRNA concentration in the range of 0−200 nM (Figure S9B). The detection limit of TK1 mRNA was estimated to be 0.50 nM according to the 3σ rule. These results indicated that the proposed nanoprobes were capable of sensing multiple mRNA targets through specific hybridization with their corresponding mRNA targets.


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Figure 3. (A) Fluorescence spectra of 50 nM FAM-hpDNA with different concentrations of UCNP@SiO2-MB@PDA (0, 0.02, 0.04, 0.08, 0.12, 0.16, 0.20 mg mL-1). Inset: fluorescence quenching efficiency versus UCNP@SiO2-MB@PDA concentrations. (B) Fluorescence spectra of UCNP@SiO2MB@PDA-hpDNA (FAM) before (blue line) and after incubation with 200 nM target sequence (black line), and 200 nM non-complementary sequence (red line). (C) Fluorescence spectra of UCNP@SiO2-MB@PDA-hpDNA (FAM) upon incubation with different concentrations of c-myc mRNA. (D) Fluorescence intensity as a function of c-myc mRNA concentration. Confocal Fluorescence Imaging of mRNA Targets in Living Cells. For the application of UCNP@SiO2-MB@PDAhpDNA nanoprobe in monitoring intracellular tumor-related mRNAs, confocal fluorescence microscopy experiments were perfomred. C-myc mRNA is involved in tumorigenesis and progression, and there are deregulation in c-myc mRNA expression level in many cancers.42 TK1 mRNA is implicated in cell division and a potent marker of tumor growth.43 C-myc and TK1 mRNA were all overexpressed in human breast cancer cell line MCF-7, whereas normally expressed in normal human mammary cell line MCF-10A.42,43 As shown in Figure 4A, MCF-10A cells incubated with UCNP@SiO2-MB@PDAhpDNA (FAM) nanoprobe displayed a weak green fluorescence, while MCF-7 cells showed a strong green fluorescence signal, indicating that the proposed nanoprobe could be used for monitoring intracellular tumor-related mRNA. Moreover, we have also prepared multiplexed nanoprobe for simultaneous imaging both c-myc and TK1 mRNA in living cells. As shown in Figure 4B, after being treated with the multiplexed nanoprobes, intensive green fluorescence for c-myc mRNA and red fluorescence for TK1 mRNA were observed in MCF-7 cells, whereas no obvious fluorescence could be obtained in MCF-10A cells under the same conditions. In addition, the UCF images of MCF-7 cells were also performed by incubating with UCNP@SiO2-MB@PDA-hpDNA nanoprobe. The bright green and red upconversion fluorescence can be observed in the cells, demonstrating the efficient cellular uptake of UCNP@SiO2-MB@PDA-hpDNA nanoprobe (Figure S10). These results suggested that the UCNP@SiO2-MB@PDAbased multiplexed nanoprobes enabled simultaneous imaging of multiple mRNAs in living cells, which could be used for discriminating cancer cells from normal cells.

Figure 4. Intracellular imaging of mRNAs based on UCNP@SiO2-MB@PDA-hpDNA nanoprobe. (A) Confocal fluorescence images of MCF-7 and MCF-10A cells incubated with nanoprobe targeting c-myc mRNA. Left panels represented FAM channel, middle panels represented bright filed mode, and right panels represented the overlay of bright filed and FAM channel. (B) Multiplexed imaging of c-myc mRNA and TK1 mRNA. Left panels represented FAM channel, middle panels represented Cy3 channel, and right panels represented the overlay of FAM channel, Cy3 channel, and bright filed images. Scale bar: 50 µm. NIR Light-Triggered 1O2 Generation. Since the absorption peak of MB obviously matched the red emission at 655 nm of OA-UCNPs, the red UCF emission could activate the photosensitizer MB through FRET process to produce 1O2 for PDT upon 980 nm excitation. To evaluate the generation of 1 O2 in the UCNP@SiO2-MB@PDA-based PDT system, DPBF was employed for detection of 1O2. As shown in Figure 5A, in control experiments of UCNP@SiO2@PDA upon 980 nm irradiation or UCNP@SiO2-MB@PDA without 980 nm irradiation, no obvious bleaching of DBPF absorption at 410 nm was detected, suggesting that there was no 1O2 generation under these two conditions. In contrast, the time-dependent decrease in DBPF absorption at 410 nm was observed in UCNP@SiO2-MB@PDA system with 980 nm irradiation, verifying the 1O2 generation. The 1O2 generation within cells when incubated with UCNP@SiO2-MB@PDA was also demonstrated by using SOSG probe through confocal fluorescence imaging. There was negligible SOSG fluorescence in itself (Figure 5B) or in UCNP@SiO2-MB@PDA incubated cells without 980 nm irradiation (Figure 5C), whereas strong SOSG fluorescence was observed after 980 nm irradiation in UCNP@SiO2-MB@PDA incubated cells, confirming the 1O2 generation in cells (Figure 5D).



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Figure 5. (A) Time-dependent bleaching curve of DBPF absorbance at 410 nm for UCNP@SiO2@PDA under 980 nm irradiation (black line), UCNP@SiO2-MB@PDA without 980 nm irradiation (red line), and UCNP@SiO2-MB@PDA under 980 nm irradiation (blue line). Confocal fluorescence images of (B) SOSG only, (C) SOSG and UCNP@SiO2-MB@PDA without 980 nm irradiation, and (D) SOSG and UCNP@SiO2MB@PDA with 980 nm irradiation. Scale bar: 50 µm. Photothermal Performance of UCNP@SiO2-MB@PDA. Since UCNP@SiO2-MB@PDA exhibited high absorption due to the presence of PDA shell, the UCF emission at 800 nm of OA-UCNPs could activate the photothermal agent PDA through FRET process to induce PTT upon 980 nm irradiation. The photothermal conversion performance of UCNP@SiO2MB@PDA was evaluated by monitoring the temperature variation curve of different nanoparticles irradiating with a 980 nm laser at 1.5 W cm-2 for 10 min. As shown in Figure 6, a slight increase could be observed in the time-dependent temperature curves for water, UCNP@SiO2, and UCNP@SiO2MB under 980 nm laser irradiation. The overheating issue induced by 980 nm excitation could be largely solved by using Nd3+-based 800 nm laser excitation.44,45 However, the temperature was remarkably increased with increasing irradiation time and increasing UCNP@SiO2-MB@PDA nanoparticle concentrations. In the presence of 0.2 mg mL-1 UCNP@SiO2MB@PDA, the solution temperature was increased to 52.2 °C after 10 min irradiation, suggesting that UCNP@SiO2MB@PDA had high photothermal conversion capability and could be used as an effective candidate for cancer PTT.

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(red line), 0.10 mg mL-1 (green line), 0.15 mg mL-1 (purple line) and 0.20 mg mL-1 (pink line) with 980 nm irradiation. Photodynamic-Photothermal Synergistic Therapy. The cytotoxicity of UCNP@SiO2-MB@PDA nanoparticles on MCF-7, HeLa, HepG2 and MCF-10A cells was first evaluated using CellTiter 96® Aqueous One Solution Cell Proliferation Assay. The results confirmed that UCNP@SiO2-MB@PDA nanoparticles had low cytotoxicity and excellent biocompatibility (Figure 7A). To investigate the PDT and PTT synergistic effect of UCNP@SiO2-MB@PDA on cancer cells, UCNP@SiO2-MB@PDA incubated MCF-7 cells with or without 980 nm irradiation was stained with PI to visualize dead cells and for confocal fluorescence image. Cells treated by either laser irradiation alone or incubating with UCNP@SiO2-MB@PDA only (Figure 7B1 & 7B2) showed weak red PI signal. In contrast, UCNP@SiO2-MB@PDA incubated MCF-7 cells combined with 980 nm irradiation exhibited strong red PI signal, indicating obvious cell death (Figure 7B3 & 7B4). The confocal fluorescence images of MCF-7 cells co-stained with calcein AM and PI revealed the similar results. For cells incubated with UCNP@SiO2-MB@PDA nanoparticles and then with 980 nm irradiation, almost all cells showed red fluorescence (Figure S11), indicating that the UCNP@SiO2-MB@PDA nanoparticles could effectively induce the cancer cells apoptosis due to the PDT-PTT synergistic effects. We further evaluated the PDT-PTT cytotoxicity of UCNP@SiO2-MB@PDA nanoparticles on MCF-7 cells. As shown in Figure 7C, the cell viabilities of UCNP@SiO2-MB, UCNP@SiO2@PDA or UCNP@SiO2-MB@PDA incubated MCF-7 cells without laser irradiation remained above 92% at a high concentration of 0.2 mg mL-1. However, UCNP@SiO2MB or UCNP@SiO2@PDA incubated MCF-7 cells with laser irradiation showed apparent cytotoxicity due to the 1O2 generation of PDT effect or hyperthermia of PTT effect, respectively. Likewise, the cell viability of UCNP@SiO2- MB@PDA incubated MCF-7 cells was significantly decreased upon 980 nm irradiation due to the considerable PDT-PTT synergistic effect. The synergistic effect between PDT and PTT effect was further demonstrated by using student’s two-tail t test (Figure S12).46 It showed that the cancer cells after incubation with UCNP@SiO2-MB@PDA and 980 nm irradiation had a lower cell viability than the projected additive value (37.1%, *P< 0.05), strongly confirming the considerable synergistic effect.

Figure 6. Temperature elevation of water (black line), UCNP@SiO2 (cyan line), UCNP@SiO2-MB (blue line), UCNP@SiO2-MB@PDA at a concentration of 0.05 mg mL-1


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Figure 7. (A) Cytotoxicity investigation of UCNP@SiO2MB@PDA nanoparticles tested on MCF-7, HeLa, HepG2 and MCF-10A cells. (B) Confocal fluorescence images of MCF-7 cells with different treatments after dyed with PI: (B1) with 980 nm irradiation for 6 min, (B2) incubation with UCNP@SiO2-MB@PDA nanoparticles, and (B3 and B4) incubation with UCNP@SiO2-MB@PDA nanoparticles then with 980 nm irradiation for 3 min and 6 min, respectively. Left panels represented PI channel and right panels represented the overlay of bright filed and PI channel. Scale bar: 100 µm. (C) Cell viability of MCF-7 cells incubated with various concentrations of UCNP@SiO2-MB, UCNP@SiO2@PDA and UCNP@SiO2-MB@PDA nanoparticles with or without 980 nm irradiation.

CONCLUSIONS In summary, we have demonstrated a core-shell-shell multifunctional UCNP@SiO2-MB@PDA nanoplatform for intracellular tumor-related mRNAs imaging and NIR light triggered PDT-PTT. The nanoplatform could be prepared by coating a silica shell on the surface of OA-UCNPs with hydrophilic photosensitizer MB entrapped in it, and then modifying PDA shell through an in situ self-polymerization process. By taking advantage of preferential binding capability for ssDNA and excellent quenching property of PDA, the developed UCNP@SiO2-MB@PDA-hpDNA nanoprobe could realize the simultaneous intracellular imaging of multiple tumor-related mRNAs and discriminating cancer cells from normal cells. Importantly, the NIR light triggered UCNP@SiO2-MB@PDA nanoplatform had considerable PDT-PTT synergistic effect due to the FRET process from the UCF emissions of the UCNPs to the absorbance of the MB and PDA. These results revealed that the developed multifunctional nanoplatform provided the proof of concept for the integration of imaging diagnosis and synergistic therapy. Meanwhile, our successful multifunctional nanoplatform provides a new design strategy for further explorations of this integration in biomedical fields.

ASSOCIATED CONTENT Supporting Information Experimental details and additional figures as noted in the text. This material is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author * Phone/Fax: +86-731-88821916. E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 21525522) and the Foundation for Innovative Research Groups of NSFC (Grant 21521063).

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