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Near Infrared-Activated Upconversion Nanoprobes for Sensitive Endogenous Zn2+ Detection and Selective On-Demand Photodynamic Therapy Ping Hu, Rui Wang, Lei Zhou, Lei Chen, Qingsheng Wu, MingYong Han, Ahmed Mohamed El-Toni, Dongyuan Zhao, and Fan Zhang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 21, 2017

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Analytical Chemistry

Near Infrared-Activated Upconversion Nanoprobes for Sensitive Endogenous

Zn2+

Detection

and

Selective

On-Demand

Photodynamic Therapy Ping Hu†‡, Rui Wang†, Lei Zhou†, Lei Chen†, Qingsheng Wu#, Ming-Yong Han§, Ahmed Mohamed El-Toni║, Dongyuan Zhao†, and Fan Zhang†* †

Department of Chemistry, Collaborative Innovation Center of Chemistry for Energy

Materials, State Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai 200433, P.R. China ‡

State Key Laboratory of High Performance Ceramics and Superfine Microstructures,

Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050, China #

§

Department of Chemistry, Tongji University, Shanghai 200092, P. R. China Institute of Materials Research and Engineering, 2 Fusionopolis Way, Singapore

138634 ║

King Abdullah Institute for Nanotechnology, King Saud University, Riyadh 11451,

Saudi Arabia Tel: (+86)21-51630322; Fax: (+86)21-5163-0307 E-mail: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT As a light-activated non-invasive cancer treatment paradigm, photodynamic therapy (PDT) has been attracting extensive attentions because of its high treatment efficacy and low side effects. Especially, spatiotemporal control of singlet oxygen (1O2) release is highly desirable for realizing on-demand PDT, which, however, still remains a huge challenge. To address this issue, a novel switchable near infrared (NIR)-responsive upconversion nanoprobe has been firstly designed and successfully applied for controlled PDT that can be optically activated by tumor-associated disruption of labile Zn2+ (denoted as Zn2+ in the following words) homeostasis stimuli. Upon NIR irradiation, this theranostic probe can not only quantitatively detect the intracellular endogenous Zn2+ in situ, but also selectively generate a great deal of cytotoxic reactive oxygen species (ROS) for efficiently killing breast cancer cells under the activation of the excessive endogenous Zn2+, so as to maximally avoid adverse damage to normal cells. This study aims to propose a new tumor-specific PDT paradigm, and more importantly, provide a new avenue of thought for efficient cancer theranostics based on our designed highly sensitive upconversion nanoprobes. KEYWORDS Upconversion nanoprobes, photodynamic therapy, endogenous Zn2+ sensing, singlet oxygen

2


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INTRODUCTION Photodynamic therapy (PDT) has been intensively investigated in recent years as an emerging noninvasive treatment modality for cancerous tumors.1-4 Upon irradiation, 'active' photosensitizers (PS) can directly absorb light at appropriate wavelength, and then transfers the photonic energy to the surrounding 3O2 or H2O for generating cytotoxic reactive oxygen species (ROS) for killing cancer cells.5-7 Conventional PDT is often limited by the extremely low efficiency and selectivity. In previous reports, the photosensitizers based on near infrared (NIR) upconversion nanoparticles (UCNPs) had solved the problem of limited penetration depth and therefore the photodynamic therapy efficiency has been improved.8-16 However, the low selectivity of UCNPs based photosensitizers, which causes the treatment-related toxicity and side effects on adjacent normal tissues, is still a major limitation for clinical PDT against cancer.17 To address this problem, activatable photosensitizers are used instead to achieve real-time controlled release of 1O2 by specific cancer-associated events for controlling the PDT-induced death of cancer cells in an efficient way.18-29 The activatable photosensitizers

were

activated

only

in

cancerous

tissues

by

specific

cancer-associated events to effectively produce 1O2. This permits high therapeutic efficacy against cancer in localized area and thus greatly minimizes the side effects, and thus facilitates the selective destruction of cancerous cells while maintaining the condition of the body’s healthy tissues. Upon interactions with some cancer-related biomarkers such as acids,20-25 thiols,26 proteases,27 H2O228 and nucleic acids,29 the 3



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activatable photosensitizers could be selectively activated in a novel sensing nanostructure to kill cancer cells through generating a large amount of ROS. In addition to the above-mentioned cancer-related biomarkers, the disruption of metal ion homeostasis in biological systems is also an important cancer-related signal. There are a growing number of studies to focus on the changes in the concentration of critical metal ions in cancer cells.30-31 Zn2+ plays important roles in various fundamental biological processes. It has been clear for a longer period of time that the disruption of Zn2+ homeostasis has been linked to several pathologies, including Alzheimer’s disease,32 prostate cancer31 and brain cancer.33 Recently, Kathryn M. Taylor et al.34 identified the protein kinase CK2 as a switch to transport free Zn2+ into breast cancer cells (increased zinc concentration internally), exhibiting that excessive Zn2+ was one of the important tumor-associated biomarkers of breast cancer cells. In the last decades, fluorescence imaging of Zn2+ in living systems has been intensively investigated with various organic sensors including ZP1, ZnAF-1, ZNP1 and Zinbo-5, which absorb UV or visible light without short tissue penetration depth.36 Very recently, Young-Tae Chang et al. reported the semi-qualitative detection of Zn2+ in brain tissue,37 and there is still a great need to develop a method for the quantitative in situ detection of endogenous Zn2+, which has not been reported so far. Develop a method for the quantitative detection of endogenous Zn2+ and achieve Zn2+ guided photodynamic therapy have very important significance. In this study, we report the quantitative detection of endogenous Zn2+ in cancer cells with a switchable upconversion nanoprobe, which is selectively activated by the endogenous Zn2+ under 4


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NIR laser radiation to generate a large amount of ROS for efficiently killing cancer cells (Scheme 1). Our theranostic nanoprobe was based on a design with two successive radiative energy transfer processes under NIR laser irradiation: (i) from NaGdF4:25%Yb,0.5%Tm@NaYF4 UCNPs to Zn2+-sensing molecule L and (ii) from the molecule L to activate photosensitizer Rose Bengal (RB). The activated RB produced 1O2 initiated cell apoptosis via DNA damage as well as lipid and protein peroxidation of mitochondria. Along with the improved tissue penetration depth and reduced autofluorescence background in biological samples, the newly designed UCNPs based theranostic probe has been demonstrated to achieve the intelligent and spatiotemporal control of 1O2 release by endogenous Zn2+, and also used successfully for highly efficient and specific PDT under NIR irradiation to avoid drug overdose. EXPERIMENTAL SECTION Investigation of singlet oxygen with DPBF. The ROS generation (1O2) of UCNPs@β-CD’-RB was detected by commercial probe DPBF. An ethanol solution of 1, 3-diphenylisobenzofuran (DPBF) (20 µL, 10 mM) was added into an aqueous solution of UCNPs@β-CD’-RB/L (10.0 mg, 1 mL) in 3.0 mL of ethanol. The mixed solution was kept in the dark and 1 µmol ZnCl2 was added subsequently followed by 980 nm laser irradiation. Upon NIR laser irradiation in the designated time intervals, the absorption of DPBF (420 nm) was recorded. The absorption of DPBF in the same conditions in the absence of ZnCl2 with or without 980 nm irradiation was also recorded as control. Intracellular exogenous Zn2+ sensing by UCNPs@β-CD’-RB/L in HeLa cells. 5



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HeLa cells (~104 cells per dish) were seeded in glass bottom dishes (20 mm, NEST) for CLSM observations, and then treated with UCNPs@β-CD’-RB/L at the final concentration of 50 µg/mL for 5 h. After removing the medium and the cells were washed for three times with PBS buffer to remove the residual nanoparticles. Then the cells were treated with Zn2+ and pyrithione for two times (1 µM of Zn2+ was added each time). Subsequently, the fluorescence recovery experiments were performed by adding a membrane-permeable Zn2+ chelator TPEN. The cells were further treated with TPEN for two times (1 µM of TPEN was added each time). The cells visualized under a confocal laser scanning microscope (Fluo View FV1000, Olympus). The fluorescence images were taken under 60×oil-immersion objective. Intracellular endogenous Zn2+ sensing by UCNPs@β-CD’-RB/L in MCF-7 cells. MCF-7 cells (~104 cells per dish) were seeded in glass bottom dishes (20 mm, NEST), and then treated with UCNPs@β-CD’-RB/L at the final concentration of 50 µg/mL for 12 h. Then a membrane-permeable Zn2+ chelator (TPEN) was added to recover the fluorescence of the nanoparobes by fluorescence titration via removal of endogenous free Zn2+. We monitored the blue UCL spectra of UCNPs@β-CD’-RB/L continuously by CLSM for 40 min. Blue UCL spectra of UCNPs@β-CD’-RB/L in randomly selected 20 individual cells were recorded by CLSM to obtain the 0 nM reference plots for Zn2+. After the endogenous Zn2+ concentrations was reduced nearly to zero, different concentrations of Zn2+ (100, 200 and 400 nM) and equal molar quantity of pyrithione were added to the cell culture medium. The integral area of blue UCL spectra was obtained as the related reference plot for Zn2+. Finally, MCF-7 cells were 6


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incubated with UCNPs@β-CD’-RB/L directly to detect the endogenous Zn2+ by recording the UCL spectra of UCNPs@β-CD’-RB/L through the similar treatment. Cell apoptosis assay. MCF-7 cells (~3 × 104 cells/well in 6-well plates) were incubated as described for the CCK-8 assay, and then treated with PBS buffer, UCNPs@β-CD’-RB/L (50 µg/mL), UCNPs@β-CD’-RB/L (50 µg/mL), sodium azide (5 µg/mL), UCNPs@β-CD’-RB/L (50 µg/mL) and TPEN (2 µM) at 5% pCO2 for 24 h respectively. Cells in each treatment were collected and divided into two groups, in which one group was treated with 980 nm irradiation (1.5 W/cm2, 10 min) and the other group was not treated further. The cells were re-suspended in 1 mL of binding buffer and incubated for an additional 30 min in the presence of Annexin V-FITC Apoptosis Detection Kit (Beyotime Institute of Biotechnology, Shanghai, China) was used (2.0 µL of annexin V-FITC and 2.0 µL of propidium iodide per mL binding buffer), before analysis by flow cytometry. In vivo PDT experiments. HeLa or MCF-7 cells (~5×106 cell/site) were implanted subcutaneously into female balb/c mice (~20 g). In vivo synergetic therapy experiments were performed when the tumors reached 5-6 mm in average diameter (9 days after implant). The mice were divided into seven groups and each group included five mice. Seven groups of mice were treated with (1) PBS + 980 nm (HeLa), (2) UCNPs@β-CD’-RB/L

(HeLa),

(3)

PBS

+

980

nm

(MCF-7),

(4)

UCNPs@β-CD’-RB/L (MCF-7), (5) UCNPs@β-CD’-RB/L + NaN3 + 980 nm, (6) UCNPs@β-CD’-RB/L + 980 nm (MCF-7). The power density and irradiation time are 1.5 W/cm2 and 30 min (0.5 min interval after each minute of irradiation). In vivo 7



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therapy experiments were performed on HeLa or MCF-7 solid tumors by intratumoral injection of PBS (150 µL) or UCNPs@β-CD’-RB/L (150 µg/mL, 150 µL) by intratumoral injection. Then the tumors were sectioned into slices and H & E stained for histological analysis. RESULTS AND DISCUSSTION Synthesis and characterizations of UCNPs@β-CD’-RB/L theranostic probe. Organic fluorescent ligand (L) (C39H60N4O2) was prepared by condensation reaction of 7-N,N-didodecylaminocoumarin-3-aldehyde and 2-hydrazinopyridine (Scheme S1.). The coordination of Zn2+ with the ligand L was characterized by the XPS experiments (Figure S1). Obvious changes of XPS spectra of Zn2p3, O1s and N1s have taken place in ligand L before and after Zn coordination indicating the obvious coordination of the oxygen and nitrogen atoms to the zinc metal center. The oleic acid (OA)-coated NaGdF4: 25% Yb, 0.5% Tm@NaYF4 core@shell UCNPs (i.e. OA-UCNPs) of ~25 nm in size were synthesized with our modified method according to our previous report (Figure S2a and Figure 1a,).37 The obvious fluorescence enhancement was abtained after NaYF4 shell coating (Figure S2c). Amino-β-CD-NH2 (β-CD’) was covalently bonded to a photosensitizer RB via a condensation reaction to form β-CD’-RB. The as-purified OA-UCNPs in cyclohexane was mixed with organic ligand L in chloroform followed by vaporizing the organic solvents to generate L-embedded OA-UCNPs via hydrophobic interaction. β-CD’-RB in water were mixed, ultrasonicated and purified to load it into hydrophobic interlayer prepare water-soluble UCNPs@β-CD’-RB/L via a simple host-guest self-assembly (Scheme 8


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1). FTIR absorption spectras which indicating L and RB modification successfully show in Figure S5. Upconversion luminescence (UCL) intensity of water-soluble UCNPs@β-CD’-RB/L is weaker than oil soluble OA-UCNPs (Figure S2d). The concentration of L and RB loaded in the UCNPs@β-CD’-RB were calculated using the absorption spectroscopy technique (Figure S3). The concentration of measured UCNPs@β-CD’-RB solution is 10 µg/mL. L and RB content (red point) of UCNPs@β-CD’-RB/L were determined to be 0.75 µg/mL and 2.31 µg/mL respectively. The mass percentages of L and RB in UCNPs@β-CD’-RB were calculated to be 7.5% and 23.1% respectively. A representative TEM image (Figure 1b) shows that the UCNPs@β-CD’-RB/L remained monodisperse without an obvious change in shape and aggregation in comparison with the as-synthesized hydrophobic UCNPs (Figure 1a). Herein, the organic ligand L in the UCNPs@β-CD’-RB/L is a switchable bridge to realize the Zn2+ sensing for guided photodynamic therapy. L is not fluorescent in the absence of Zn2+, inhibiting 1O2 release from RB. The fluorescence of L is turned ON when the Zn2+/L complex is formed in the presence of Zn2+ and a decrease of UCL ranging 400-500 nm occurs by the radiative energy transfer process leading to a significant increase of the green fluorescence at 513 nm of Zn2+/L complex (Figure 1c). The linear relationship between the UCL and Zn2+ concentration is demonstrated in Figure S4. The limit of detection of the probe for Zn2+ had been examined and calculated as 0.18 µM.38-39 The variation in UCL intensity as a function of L concentration allows us to achieve quantitative monitoring of Zn2+ endogenously 9



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generated in living cells. In comparison to metal ions (e.g., Co2+, Cu2+, Ni+, Fe2+, Pb2+, Mn2+, Ca2+, Ba2+, Mg2+, K+ and Na+), Zn2+ induced an obvious signal change of UCNPs because the Zn2+ complex have a large absorption at 475 nm, indicating the good selectivity of sensor (Figure 1d). Cd2+ may interfere with the detection of zinc ions to some extent, but fortunately, there are almost no Cd2+ ions in the human cells. To investigate the mechanism of energy transfer process, UCL lifetimes of the UCNPs before and after the modification of “L” and “RB” had been detected41-42 (Figure S6). No obvious change of lifetimes can be found after “L” and “RB” modification. Thus the energy transfer from UCNPs to the organic fluorescent probes “L” is likely to be radiative energy transfer process. RB is a very efficient photosensitizer in producing singlet oxygen.43 There is a large overlapping between the absorption of RB and the green fluorescence of Zn2+/L complex (Figure 1e), which allows efficient energy transfer from Zn2+/L complex to RB. The activated RB can produce 1O2 to trigger PDT specifically for breast cancer cells.

In

order

to

monitor

the

Zn2+-sensing

guided

PDT

process,

1,

3-diphenylisobenzofuran (DPBF) was used to investigate the generation of 1O2 (Figure 1f). The absorption intensity of DPBF monitored at 420 nm decreased exponentially as a function of time upon 980 nm irradiation. In control experiments with or without 980 nm irradiation in the absence of Zn2+, the absorption of DPBF remains unchanged. This observation illustrates that 1O2 can only be generated from the UCNPs@β-CD’-RB/L in the presence of Zn2+ under 980 nm irradiation. Quantitative detection of intracellular endogenous Zn2+. The standard cell 10


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counting kit (CCK-8) assays of Hela cells exhibited the excellent biocompatibility of UCNPs@β-CD’-RB/L (Figure S7). To assess whether UCNPs@β-CD’-RB/L could be used to in situ quantitative sensing the Zn2+ concentration in living cells, HeLa cells with scarcely endogenous Zn2+ were incubated with the theranostic nanoprobe for 4 h and analyzed under a confocal laser scanning microscopy (CLSM) equipped with a 980 nm laser. Blue UCL emitted by UCNPs@β-CD’-RB/L was clearly observed from the cells, indicating the successful cellular up-taking of the UCNPs@β-CD’-RB/L (Figure

S8).

Moreover,

obvious

decrease

of

blue

UCL

intensity

of

UCNPs@β-CD’-RB/L was observed when the cells were treated alternately with Zn2+ supplied from outside the cells and pyrithione (i.e. 2-mercaptopyridine N-oxide), a reagent that can bring Zn2+ into the cytoplasm through the formation of metal complex (Figure 2a-d). In addition, the blue UCL from UCNPs@β-CD’-RB/L in the presence

of

Zn2+

was

recovered

dramatically

upon

the

addition

of

a

membrane-permeable Zn2+ chelator N,N,N’,N’-tetrakis(2-pyridyl)ethylenediamine (TPEN)35

(Figure

2e-h).

These

results

established

the

capability

of

UCNPs@β-CD’-RB/L for sensing the fluctuations of Zn2+ concentration in living cells for a long period of time with minimum interference from other competing metal ions in cell microenvironment. In

addition

to

UCNPs@β-CD’-RB/L

the

sensing

probes

to

of

exogenous

quantitatively

Zn2+, detect

we

also

spatially

utilized resolved

concentrations of endogenous Zn2+ generated inside living cells with the breast cancer cells (MCF-7) as a typical model (Figure 2i). In order to obtain the reference plots, 11



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MCF-7 cells were pre-treated with TPEN via fluorescence titration to remove endogenous free Zn2+. We monitored the blue UCL spectra in a MCF-7 cell continuously for 40 min until there is no further change/increase in the fluorescence (Figure S9). Twenty MCF-7 cells were randomly selected after removing the free endogenous Zn2+, the UCL spectra in individual cells were recorded by CLSM to obtain the reference plot in the absence of Zn2+. Similarly, after reducing the endogenous Zn2+ concentrations nearly to zero, different concentrations of Zn2+ from 100 to 400 nM were added to obtain the reference plots in the presence of Zn2+ with different

concentrations.

Finally,

MCF-7

cells

were

incubated

with

UCNPs@β-CD’-RB/L nanoparticles directly to detect the endogenous Zn2+ by recording the UCL spectra of UCNPs@β-CD’-RB/L. Comparing the integral area of blue UCL spectra of MCF-7 cells in the presence of endogenous Zn2+ with other reference plots in Figure 2i, the endogenous Zn2+ concentrations in MCF-7 cells falls between 200 to 400 nM. Although the experimental measured Zn2+ concentrations may deviate more or less from the true values due to some systematic errors, the experimental method as well as the measured Zn2+ concentration range is reliable. In the most of previous reports, fluorescent Zn2+ probes were only able to provide the trend change of Zn2+ concentration.35, 36 To our knowledge, this is the first time that local endogenous intracellular Zn2+ levels can be in situ tracked quantitatively. Zn2+-activatable

generation

of

1

O2.

Having

confirmed

that

the

UCNPs@β-CD’-RB/L probe can be used for sensing the endogenous Zn2+ in living cells, we further demonstrated the Zn2+-sensing guided photodynamic therapy upon 12


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980 nm laser irradiation. Firstly, we proceeded to test the intracellular ROS activation in HeLa cells with different concentrations (0, 0.5, 1.0 µM) of the exogenous Zn2+ via CLSM (Figure S10). HeLa cells treated with UCNPs@β-CD’-RB/L were stained with a ROS probe, DCFH-DA (2‘,7‘-dichlorodihydrofluorescein diacetate). This probe is nonfluorescent, but its oxidized product by ROS (2‘,7‘dichlorofluorescin,

DCF)

could emit a green fluorescence.40 Green fluorescent intensity and coverage area in HeLa cells reflect ROS levels. HeLa cells were treated with no exogenous Zn2+ as negative control. After the irradiation by 980 nm laser for 10 min, almost no green fluorescence was found in the control samples. In contrast, the exogenous addition of Zn2+ induced the production of ROS with the UCNPs@β-CD’-RB/L in similar conditions. Moreover, the amounts of 1O2 were dependent on Zn2+ concentrations as indicated by the remarkable green fluorescence enhancement with the increase of the Zn2+ concentration from 0.5 to 1.0 µM. Encouraged by the efficient 1O2 generation with UCNPs@β-CD’-RB/L in HeLa cells upon 980 nm irradiation in the presence of exogenous Zn2+, the feasibility of the theranostic nanoprobe in the endogenous Zn2+ sensing guided antitumor PDT was demonstrated

in

MCF-7

cells.

Firstly,

MCF-7

cells

incubated

with

UCNPs@β-CD’-RB/L before and after 980 nm laser irradiation were characterized by confocal microscopy to track the cell morphology change (Figure 3a-d). As shown in Figure 3a and Figure 3b, after light irradiation for 2 min, cell morphology of MCF-7 cells incubated with UCNPs@β-CD’-RB/L was shrunk, indicating cells were stimulated by 1O2 generation. Moreover, obvious morphological changes of cell 13



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apoptosis occurred when the irradiation time was set for 10 min which indicating highly efficient PDT performance of UCNPs@β-CD’-RB/L theranostic nanoprobes (Figure 3c and 3d). Then, MCF-7 cells were characterized before/after treatment by co-staining with calcein AM and propidium iodide for differentiating living cells from dead ones (green representing the living cells and red indicating the dead ones). After incubated with UCNPs@β-CD’-RB/L without 980 nm laser irradiation, MCF-7 cells exhibited negligible cytotoxicity in dark (Figure 3e). Upon 980 nm irradiation for 10 min, cell viability was decreased to below 20% (Figure 3f), demonstrating high phototoxicity of UCNPs@β-CD’-RB/L to MCF-7 cells due to the presence of abundant endogenous Zn2+. To prove that the cell death was caused by singlet oxygen, we used a ROS quencher sodium azide (NaN3)44 to quench the production of 1O2. In the presence of NaN3, the cell viability remained above 70%, suggesting 1O2 is the key factor that causes the cell death (Figure 3g). In another set of control experiments, a membrane permeable Zn2+ chelator TPEN was used to inhibit the formation of Zn2+/L complex. Figure 3h shows the negligible cytotoxicity was observed upon 980 nm

irradiation

when

MCF-7

cells

were

pre-treated

with

TPEN

and

UCNPs@β-CD’-RB/L. These observations demonstrated that the cause of cell death is ascribed to the endogenous Zn2+-induced PDT of UCNPs@β-CD’-RB/L upon 980 nm irradiation. The ability of UCNPs@β-CD’-RB/L in inducing the death of Hela cells in presence of exogenous Zn2+ which was investigated by CCK-8 assays futher indicating the similar resuilts (Figure S11). The ability of UCNPs@β-CD’-RB/L in inducing the 14


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death of MCF-7 cells was also assessed by flow cytometry analyses (Figure 3i) and CCK-8 cell viability assays (Figure 3j). After the cells were treated with UCNPs@β-CD’-RB/L, most of the cells are viable with a cell mortality rate

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