A Dual-Targeting Upconversion Nanoplatform for Two-Color

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Letter pubs.acs.org/ac

A Dual-Targeting Upconversion Nanoplatform for Two-Color Fluorescence Imaging-Guided Photodynamic Therapy Xu Wang,† Cheng-Xiong Yang,† Jia-Tong Chen,‡ and Xiu-Ping Yan*,† †

State Key Laboratory of Medicinal Chemical Biology (Nankai University), Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), and Research Center for Analytical Sciences, College of Chemistry, Nankai University, Tianjin 300071, China ‡ Department of Biochemistry and Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071, China S Supporting Information *

ABSTRACT: The targetability of a theranostic probe is one of the keys to assuring its theranostic efficiency. Here we show the design and fabrication of a dual-targeting upconversion nanoplatform for two-color fluorescence imaging-guided photodynamic therapy (PDT). The nanoplatform was prepared from 3-aminophenylboronic acid functionalized upconversion nanocrystals (APBA-UCNPs) and hyaluronated fullerene (HAC60) via a specific diol-borate condensation. The two specific ligands of aminophenylboronic acid and hyaluronic acid provide synergistic targeting effects, high targetability, and hence a dramatically elevated uptake of the nanoplatform by cancer cells. The high generation yield of 1O2 due to multiplexed Förster resonance energy transfer between APBA-UCNPs (donor) and HAC60 (acceptor) allows effective therapy. The present nanoplatform shows great potential for highly selective tumor-targeted imaging-guided PDT.

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strategy. Thus, it is of great significance to integrate such dualtargeting strategy into the UCNPs-C60 nanoplatform to enhance the tumor-targeted selectivity. To our knowledge, however, no work on UCNP-based PDT nanoplatform based on the dual-targeting strategy has been reported to improve tumor-targeted imaging and PDT efficiency so far. Herein, we show the design and fabrication of a two-color fluorescence imaging-guided dual-targeting nanoplatform for PDT (Scheme 1). The nanoplatform was constructed from aminophenylboronic acid (APBA) functionalized NaYF4:Yb3+, Gd3+, Tm3+ UCNPs (APBA-UCNPs) and hyaluronated fullerene (HAC60) via the specific diol-borate condensation.24−26 Efficient generation of 1O2 for PDT resulted from multiplexed Förster resonance energy transfer (FRET) between APBA-UCNPs (donor) and HAC60 (acceptor) due to the overlap of the upconversion luminescence emission spectra of the UCNPs around 475, 650, and 700 nm and the broad absorption spectra of HAC60. We incorporated two types of targeting ligands APBA and hyaluronic acid (HA), which have specificity toward polysialic acid (PSA) and cluster determinant 44 (CD44) receptors overexpressed in cancer cells, respectively,27,28 into the present nanoplatform to enhance the selectivity toward cancer cells, and to improve the therapy efficiency. The ligands HA and APBA along with the NIR upconversion luminescence emission at 800 nm from the UCNPs and the red emission at 640 nm from HAC60 allowed dual-targeting and two color fluorescence imaging. The

heranostic probes are a class of agents that can simultaneously deliver diagnostic and therapeutic functions, enabling detection and treatment of diseases in a single procedure.1−3 Photodynamic therapy (PDT), combined utilization of light and a photosensitizer (PS), has emerged as a viable treatment option for early stage cancer and an adjuvant for surgery in late-stage cancer because of the important role of oxygen in the process of light-induced therapy.4,5 Fullerenes, with almost 100% 1 O 2 generation yield, have gained considerable attention as a PS in antitumor application.6−10 Therefore, much effort has been made to design and develop novel fullerenes with good water solubility, excellent photoluminescent properties, and active tumor targetability for bioimaging and PDT.11−15 However, as most of developed fullerenes for PDT are mainly excited by UV/visible light, the limited depth of light penetration and the high autofluorescence have severely hindered the fullerene-involved PDT efficiency. Recent developed upconversion nanoparticle (UCNP)-based PDT is capable of converting NIR light to UV−vis light, facilitates the delivery of light to deep lesions, and allows PDT by means of 1O2 generation via energy transfer from UCNPs to the PS upon NIR excitation.16−19 Lately, a UCNPs-C60 nanoplatform conjugated with folic acid as the sole targeting ligand for monochrome fluorescence imaging-guided photodynamic therapy in vitro was reported.20 Dual-targeting strategy based on the fact that tumor cells typically overexpress multiple types of surface receptors has been exploited mainly in the field of diagnostics and drug delivery.21−23 Enhanced cellular association effects of imaging agent and magnified cytotoxicity of delivered drug have verified the availability and reliability of this fascinating dual-targeting © 2014 American Chemical Society

Received: January 6, 2014 Accepted: March 13, 2014 Published: March 13, 2014 3263

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Scheme 1. Schematic Illustration of the Design and Cell Internalization of the Dual-Targeting Nanoplatform for Two-Color Fluorescence Imaging-Guided PDT

Figure 1. (A) TEM image of the prepared APBA-UCNPs (Insert: HRTEM showing lattice fringes). (B) XRD patterns of APBA-UCNPs powders in contrast to the standard hexagonal structure card of NaYF4 (JCPDS 028−1192). (C) Comparison of the upconversion luminescence spectra of APBA-UCNPs (3.0 mg mL−1) and UCNPsC60 (3.0 mg mL−1, equivalently containing 120 μg mL−1 HAC60) in PBS buffer (pH 7.4, 10 mM). (D) HRTEM images of HAC60 prepared with the feeding concentration of C60 at 1.0 mg mL−1. (E) UV−vis absorbance spectra and fluorescence spectra (420 nm excitation) of HAC60 (0.01 wt %) in PBS buffer (pH 7.4, 10 mM). (F) HRTEM image of UCNPs-C60. Inset: photograph of UCNPs-C60 colloid solution (0.05 wt %) under ambient light.

developed theranostic nanoprobe not only has the potential to treat lesions or tumors in deep tissue via NIR excitation but also shows higher affinity and selectivity over sole-targeting probes. The APBA-UCNPs involved in the developed nanoplatform were prepared via a facile one-step solvothermal approach (Supporting Information). APBA was used as both a stabilizer in the synthesis and a functional ligand for targeting bioimaging. The synthesized APBA-UCNPs were spherical with an average size of 15.8 ± 3.8 nm (Figure 1A), showing clear lattice fringes with a d spacing of 0.258 nm lattice spacing in accordance with (200) planes of hexagonal NaYF4. Further evidence of the hexagonal crystal phase of APBA-UCNPs were also given by the XRD analysis and the selected area electron diffraction (SAED) analysis (Figure 1B and Figure S1 of the Supporting Information). The presence of APBA on the UCNPs was confirmed by Fourier-transform infrared (FT-IR), energy-dispersive X-ray analysis (EDXA), X-ray photoluminescence spectroscopy (XPS), and thermogravimetric analysis (TGA) (Figure S2−S6 of the Supporting Information). The prepared APBA-UCNPs gave strong multicolour upconversion luminescence at 475, 650, 700, and 800 nm (Figure 1C) with the upconversion luminescence absolute quantum yield of 5.38% (powders). No change in all emissions of APBA-UCNPs colloid solution (0.05 wt %) was observed upon continuous 980 nm NIR light irradiation (500 mW) for 180 min (Figure S7 of the Supporting Information), which allows the further use of APBA-UCNPs for PDT under long exposure time and high-dose irradiation in vitro. HAC60 was synthesized according to Jeong et al.11 but with the use of HA instead of tetraethylene glycol (TEG) as the ligand for targeting tumor cells. The preparation of HAC60 was achieved by optimizing the concentration of C60 while fixing that of HA. A feeding HA:C60 mass ratio of 10:1 was used for the fabrication of UCNPs-C60 nanoplatform to give the prepared HAC60 with relatively high UV−vis absorbance and broad absorption spectra from 200 to 700 nm (Figure S8 of the Supporting Information). The prepared HAC60 was spherical with an average size of 10.0 ± 1.5 nm (Figure 1D). The

covalent bonding of HA to C60 via a nucleophilic addition was confirmed by FT-IR, XPS, and 1H NMR (Figure S2 and S9− S11 of the Supporting Information). TGA data of HAC60 reveal that 0.25 C60 molecules were attached to one sugar unit of HA (Figure S6 of the Supporting Information). The HAC60 gave strong emission at 640 nm (Figure 1E and Figure S12 of the Supporting Information), providing another fluorescence signal for bioimaging except for upconversion luminescence from UCNPs. The multifunctional UCNPs-C60 nanoplatform was fabricated via the diol-borate condensation of APBA-UCNPs and HAC60 (Scheme 1). To obtain the highest PDT efficiency, the mass ratio of HAC60 to APBA-UCNPs was optimized to be 1:25 via steady-state upconversion luminescence determination (Figure S13 of the Supporting Information). The prepared UCNPs-C60 was dumbbell-like, presenting obvious lattice fringes of APBA-UCNPs and the amorphous structure of HAC60 (Figure 1F). Dynamic light scattering experiments showed narrow size distribution of fresh UCNPs-C60 aqueous colloids in an average hydrodynamic diameter of 152.6 nm and no significant change of the size distribution after 24 h standing (Figure S14 of the Supporting Information). The results indicate good water dispersibility and stability of UCNPs-C60. The bioconjugation of HAC60 with APBA-UCNP was verified by a shift in the B−O stretching mode from 1349.6 cm−1 of the boronic acid of APBA-UCNPs to 1403.8 cm−1 of the 3264

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phenylboronate ester mode of UCNPs-C60 in the FT-IR spectra (Figure S2 of the Supporting Information).29 Conjugation of HAC60 to APBA-UCNPs led to a significant quenching of the upconversion luminescence at 475, 650, and 700 nm bands with the FRET efficiency of 50% at 475 nm, 30% at 650 nm, and 19% at 700 nm but no obvious quenching of the upconversion luminescence at 800 nm (Figure 1C). Therefore, the 800 nm emission can be used for high-contrast NIR luminescence imaging while the adequate energy transfer from APBA-UCNPs to HAC60 is the prerequisite for PDT. In a further set of experiments, we found that the energy transfer efficiency from APBA-UCNPs to HAC60 involved in UCNPsC60 were independent of ion strength (0.5−500 mM NaCl), conventional amine acids (10 mM) and other biomolecules and ions (10 mM for glucose and 30 μM for others) (Figure S15 of the Supporting Information). 1,4-Diphenyl-2,3-benzofuran (DPBF), a singlet-oxygen chemical probe, was utilized to evaluate the capability of the UCNPs-C60 nanoplatform to generate 1O2 for PDT. DPBF reacts irreversibly with 1O2, and the reaction can be followed by recording the decrease in the intensity of the DPBF absorption around 420 nm.30,31 To ensure the high 1O2 generation yield of activated UCNPs-C60 and to avoid any photothermal-induced cell damage in further bioapplication, an irradiation time of 15 min was used (Figure S16 and S17 of the Supporting Information). A rapid decrease (∼45%) of DPBF absorption was observed in the presence of UCNPs-C60 after 980 nm laser irradiation for 15 min (Figure S18 of the Supporting Information). On the contrary, the DPBF absorbance showed a negligible change with time in the absence of UCNPs-C60 or without 980 nm light illumination. No significant effect of pH on the 1O2 generation of UCNPs-C60 was observed under the extracellular microenvironment in the pH range of 6.5−7.4 (Figure S19 of the Supporting Information). The effective 1O2generating capability and high pH stability of UCNPs-C60 make it possible to be applied in fluorescence-induced PDT. The UCNPs-C60 dual-targeting nanoplatform showed significantly improved tumor-targeted capacity as compared to the single-targeting probes of APBA-UCNPs and HAC60. To address this issue, PC12 cells with overexpressed PSA and CD44 receptors were incubated with UCNPs-C60, APBAUCNPs, and HAC60, respectively, and the corresponding cell images were collected (Figure 2A). After 24 h incubation, the localization of the HAC60 (640 nm, color-coded as green) (Figure 2A-a, a′) or APBA-UCNPs (800 nm, color-coded as red) (Figure 2A-b, b′) in PC12 cells were observed in the cytoplasm region. To further reveal the internalization mechanism of the single-targeting probes, the ligand blocking assay using free APBA (1.0 mg mL−1) or free HA (1.0 mg mL−1) was performed (Figure S20 of the Supporting Information). The results show that few APBA-UCNPs were stained in the cancer cells after excess free APBA treatment due to the blocking of PSA receptors. In contrast, PC12 cells preincubated with free HA before incubating with APBAUCNPs exhibited similar fluorescence intensity to nonblocking cells. Upconversion luminescence analysis in cells showed a significant difference (P < 0.05) in the cellular uptake of APBAUCNPs between unblocked cells and APBA-blocked cells (Figure S20B of the Supporting Information). All the results demonstrate that the endocytosis of APBA-UCNPs was mainly mediated by PSA receptors. PC12 cells incubated with HAC60 after blocking with free HA or APBA resulted in an opposite outcome (Figure S20, panels A and C, of the Supporting

Figure 2. (A) Confocal microscopic images of PC12 cells incubated with APBA-UCNPs (100 μg mL−1), HAC60 (4 μg mL−1), and UCNPs-C60 (100 μg mL−1) for 24 h: (a) Fluorescence images of PC12 cells treated with HAC60 collected by 610−670 nm detection channels. (b) Upconversion luminescence images of PC12 cells treated with APBA-UCNPs collected by 740−800 nm detection channels. (c, c′, and c″) Luminescence images of PC12 cells treated with UCNPs-C60 collected by 610−670 nm detection channels (c), 740−800 nm detection channels (c′), and the overlay of two detection channels (c″). (a′, b′, and c‴) The corresponding bright field cell images. All scale bars are 50 μm. (B) Upconversion luminescence analysis of APBA-UCNPs and UCNPs-C60 in cells. (C) Downconversion fluorescence analysis of HAC60 and UCNPs-C60 in cells. *P < 0.05; **P < 0.01. Error bars indicate one standard deviation (n = 3).

Information), revealing a CD44 receptor-dependent endocytosis for the intracellular delivery of HAC60. The intracellular uptake of UCNPs-C60 was monitored by two excitation pathways imaging, upconversion luminescence (800 nm) from APBA-UCNPs and downconversion fluorescence (640 nm) from HAC60. UCNPs-C60 gave much brighter upconversion and downconversion fluorescence images of PC12 cells than APBA-UCNPs or HAC60 (Figure 2A). The mean fluorescence intensity of UCNPs-C60 incubated PC12 cells was about two times that of APBA-UCNPs or HAC60 incubated PC12 cells, confirming the higher uptake of UCNPsC60 (Figure 2, panels B and C). To find out the subcellular distribution of UCNPs-C60 nanoplatform, the UCNPs-C60treated PC12 cells were stained with Giemsa dye. The results demonstrated that UCNPs-C60 bound specifically to the membrane of PC12 cells and then internalized into the cytoplasm through dual-receptor-mediated endocytosis up to 24 h (Figure S21 and S22 of the Supporting Information). The present dual-targeting approach significantly promoted the cellular uptake of UCNPs-C60 due to the joint efforts of active targeting mediated by the high affinity of APBA-UCNPs to PSA receptors, HAC60 to CD44 receptors, and a passive targeting based on the EPR effect. The involvement of PSA and CD44 receptors in the UCNPsC60 internalization was also revealed by blocking assays. To address this point, PSA or CD44 receptors in PC12 cells were selectively blocked by exploiting free APBA or HA as the blocking agent (Figure S23 of the Supporting Information). The amount of UCNPs-C60 uptaken by PC12 cells was remarkably reduced by 34.8% after APBA blocking and 31.6% after HA blocking via upconversion luminescence determi3265

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nation. The above results further verify the synergistic targeting effects of the incorporated APBA and HA dual-targeting ligands on the cell uptake of UCNPs-C60. The cytotoxicity of APBA-UCNPs, HAC60, and UCNPs-C60 was assessed to further study the potential utility of UCNPs-C60 for PDT. NIH-3T3 cells and PC12 cancer cells were incubated with APBA-UCNPs (0−120 μg mL−1), HAC60 (0−50 μg mL−1), and UCNPs-C60 (0−120 μg mL−1) in the dark, and cell viability was then examined by the 3-(4,5-dimethythiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay. After 24 h incubation, no significant decrease in cell viability was observed (Figure S24 of the Supporting Information). The above cytotoxicity study shows that APBA-UCNPs, HAC60, and UCNPs-C60 themselves possesses low cytotoxicity without light irradiation. The potential of UCNPs-C60 for PDT upon near-infrared laser irradiation was also examined by the MTT assay. The treatment of UCNPs-C60 (50 or 100 μg mL−1) under irradiation led to a concentration-dependent decrease by 32− 55% in the viability of PC12 cells due to the PDT effect of excited UCNPs-C60 (Figure S25 of the Supporting Information). APBA-UCNPs (50 or 100 μg mL−1) resulted in little decrease in the viability of PC12 cells, whereas HAC60 (2 or 4 μg mL−1) led to a slight decrease of cell viability probably due to a little phototoxicity arising from light contact during experiments. Laser irradiation alone or incubation with UCNPs-C60 in the dark caused negligible decrease in cell viability, verifying that obvious cytotoxicity was produced only when NIR light irradiation and UCNPs-C60 are simultaneously available. To further evaluate the therapeutic efficacy of the UCNPsC60 PDT system, reactive oxygen species (ROSs) produced from UCNPs-C60 in cancer cells were detected by using the 2′,7′-dichlorofluorescin diacetate (DCFH-DA) probe.32,33 The fluorescence emission of 1O2 activated DCFH-DA under excitation at 488 nm was recorded in the range of 500−550 nm. UCNPs-C60 incubated PC12 cells were labeled with DCFH-DA for 30 min, and the ROSs produced upon NIR light irradiation was determined by flow cytometry. The control group, APBA-UCNPs (Figure S26A of the Supporting Information) and HAC60 (Figure S26C of the Supporting Information) with 980 nm laser irradiation and UCNPs-C60 without irradiation (Figure S26B of the Supporting Information) gave little fluorescence change. In contrast, a significant increase in fluorescence at 540 nm was detected for UCNPsC60 irradiated by the 980 nm laser (Figure S26D of the Supporting Information), indicating an intracellular generation of ROSs. In conclusion, we have fabricated a dual-targeting UCNPsC60 nanoplatform for two-color fluorescence imaging-guided PDT. A dual-targeting strategy was first applied in the UCNPbased PDT nanoplatform to promote the selectivity of the nanoplatform toward tumor cells and to enhance the PDT efficiency. We have shown the unambiguously synergistic targeting effect of dual-targeting ligands on a dramatically elevated cell uptake of UCNPs-C60 in comparison to the singletargeting probes of APBA-UCNPs and HAC60. The favorable characteristic of UCNPs-C60 such as convenient synthesis, high specific activity, two-color fluorescence signals, and improved PDT efficiency, warrants its further investigation for in vivo therapeutic and imaging application.

Letter

ASSOCIATED CONTENT

S Supporting Information *

Experimental section and other additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel/Fax: (86)-22-23506075. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (Grant 2011CB707703) and the National Natural Science Foundation of China (Grant 21275079). We also thank Prof. Xueyuan Chen and Dr. Haomiao Zhu (Fujian Institute of Research on the Structure of Matter) for help with the measurement of upconversion luminescence absolute quantum yield, and Prof. Xianshun Zeng (Tianjin University of Technology) for help with cell imaging.

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