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Nov 15, 2017 - cence resonance energy transfer (FRET),12 contact quenching,13 ..... The pH responsive study materials were prepared by dialysis at dif...
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Determination of Cancer Cell-Based pH-Sensitive Fluorescent Carbon Nanoparticles of Cross-Linked Polydopamine by Fluorescence Sensing of Alkaline Phosphatase Activity on Coated Surfaces and Aqueous Solution Eun Bi Kang,† Cheong A. Choi,† Zihnil Adha Islamy Mazrad,‡ Sung Han Kim,‡ Insik In,*,‡,§ and Sung Young Park*,†,‡ †

Department of Chemical and Biological Engineering, ‡Department of IT Convergence, and §Department of Polymer Science and Engineering, Korea National University of Transportation, Chungju 380-702, Republic of Korea S Supporting Information *

ABSTRACT: The tumor-specific sensitive fluorescence sensing of cellular alkaline phosphatase (ALP) activity on the basis of host−guest specific and pH sensitivity was conducted on coated surfaces and aqueous states. Cross-linked fluorescent nanoparticles (C-FNP) consisting of β-cyclodextrin (β-CD)/ boronic acid (BA) and fluorescent hyaluronic acid [FNP(HA)] were conjugated to fluorescent polydopamine [FNP(pDA)]. To determine the quenching effect of this system, hydrolysis of 4-nitrophenyl phosphate (NPP) to 4-nitrophenol (NP) was performed in the cavity of β-CD in the presence of ALP activated photoinduced electron transfer (PET) between NP and C-FNP. At an ALP level of 30−1000 U/L, NP caused offemission of C-FNP because of their specific host−guest recognition. Fluorescence can be recovered under pH shock due to cleavage of the diol bond between β-CD and BA, resulting in release of NP from the fluorescent system. Sensitivity of the assays was assessed by confocal imaging not only in aqueous states, but also for the first time on coated surfaces in MDAMB-231 and MDCK cells. This novel system demonstrated high sensitivity to ALP through generation of good electron donor/acceptor pair during the PET process. Therefore, this fluorescence sensor system can be used to enhance ALP monitoring and cancer diagnosis on both coated surfaces and in aqueous states in clinical settings.

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techniques for monitoring ALP activity as a targeted tumor assay has great potential due to their intrinsic advantages such as noninvasiveness, sensitivity, and high solubility. Various mechanisms, such as assembly of nanoparticles, host−guest interactions, and change in solubility, can guide changes in the fluorescent behavior of bioreactive ALPs that can then be visualized both in vitro and in vivo.8 One of these mechanisms, the hydrolysis of 4-nitrophenyl phosphate (NPP) to 4-nitrophenol (NP), has been widely used to detect ALP activity in biological studies. Among various biosubstances, hyaluronic acid (HA), which interacts with the CD44 receptor of cancer cells, can be used to achieve high selectivity in cancer diagnosis. However, its detection requires the ability to track fluorescently labeled HA within cells. Currently, fluorescent nanoparticles (FNP) have replaced conventional dyes, noble metal clusters, and semiconductor quantum dots in various applications. Previously, our group has successfully developed FNPs derived from carbonized

lkaline phosphatase (ALP) mediates important functions in the biological system. It can hydrolyze a variety of phosporylated substrates in signal transduction pathways, and correlations have been found between level of ALP and disease states.1 ALP abnormalities have been used as an important indicator for various diseases including breast cancer, prostate cancer, bone diseases, liver dysfunction, and diabetes.2,3 Currently, much focus has been placed on developing more sensitive methods of monitoring ALP activity for cancer diagnosis to overcome the many limitations of conventional diagnostics such as magnetic resonance imaging (MRI), enzymelinked immunosorbent assay (ELISA), hematoxylin and eosin staining, computed tomography (CT), positron emission tomography (PET), bone scan, thyroid scanning, and tumor marker test.4 Measurements of ALP have previously been used in biosensors,5 DNA sensors,6 and ion sensors.7 However, the existing methods, which use aqueous states for ALP detection, are complicated. In addition, they generally require a long detection time and are subject to numerous limitations. Therefore, there is still a need to develop an ALP detection method with high sensitivity, selectivity, and simplicity that requires fewer samples and has high cost-effectiveness. The use of fluorescent © 2017 American Chemical Society

Received: September 21, 2017 Accepted: November 15, 2017 Published: November 15, 2017 13508

DOI: 10.1021/acs.analchem.7b03853 Anal. Chem. 2017, 89, 13508−13517

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Analytical Chemistry HA, which show good solubility and biocompatibility.9 We have also successfully designed FNPs from polydopamine (pDA) through dehydration by acidic catalyst followed by conjugation with boronic acid (BA). Formation of the boronic ester bond was achieved via binding of catechol-containing molecules of FNPpDA with BA.10 It has been shown that the remaining catechol moieties can act as a coating for FNP-pDA on various surfaces.9 Recently, studies on biosensors using the fluorescence on/off system have been actively conducted. For example, biosensor using β-CD, cyclic receptors consisting of seven glucose units linked together, can detect guest molecule complexes within the β-CD cavity by introducing fluorescent compounds into β-CD.11 The chromophore-modified β-CD has been utilized as fluorescence sensors in various quenching methods, such as fluorescence resonance energy transfer (FRET),12 contact quenching,13 and collision quenching.14 Here, we designed a FNP by cross-linking BA/β-CD-functionalized FNP (HA) and FNP (pDA) via a boronic ester complex. This leads to the quenching effect in the presence of ALP by photoinduced electron transfer (PET) due to host− guest recognition between β-CD and 4-nitrophenol. To enhance sensitivity for cancer detection, HA of cross-linked FNP can be used as a significant moiety in traditional ALP-based hydrolysis of 4-nitrophenyl phosphate (NPP) to 4-nitrophenol (NP). The adhesive catechol in cross-linked FNP in our novel design allows the detection of ALP on coated surfaces that can be visualized in MDAMB-231 and MDCK cells. Currently, ALP detection is limited to the solution state, and no specific reports have been published regarding the detection of ALP on coated surfaces. This unique FNP has great potential for tumor-triggered detection assays for monitoring cellular alkaline phosphatases in different mediums.

(HA) and boronic acid (BA), FNP (HA) (0.25 g) was dissolved in 30 mL of degassed deionized water, and the solution was kept under nitrogen. EDC (0.02 g) and NHS (0.0121 g) were slowly added, and the resulting solution was mixed for 20 min. This was followed by addition of 3-amino phenyl boronic acid (BA) (0.0162 g) to the mixture. Solution pH was monitored and adjusted to be 3−4 for 12 h. Subsequently, the solution was allowed to react overnight at room temperature. The solution was then purified by dialysis (MWCO = 3500 Da) and freezedried. The percentage yield was 9.8%. Synthesis of β-Cyclodextrin Grafted FNP (HA-BA) [FNP(HA-CD)]. FNP (HA-BA) (0.1 g) and β-CD (0.0324 g) were dissolved in 20 mL of phosphate-buffered saline (PBS, pH 12) in a 150 mL flask. The mixture was stirred for 24 h at room temperature, and the reaction solvent was dialyzed (MWCO: 3500) for 24 h after freeze-drying. Synthesis of Cross-Linked Fluorescent Nanoparticles (C-FNP) Using Carbonized Polydopamine [FNP (pDA)] and FNP (HA-CD). FNP (pDA) was synthesized according to previously published methods.15 FNP (HA-CD) (0.1 g) and FNP (pDA) (0.1 g) were dissolved in PBS buffer at pH of 12.0. The solution was allowed to react overnight at room temperature to obtain C-FNP. The reaction solvent was dialyzed (MWCO: 3500) for 24 h after freeze-drying. The final product yield was 70.7%. Preparation of 4-Nitrophenol (NP) Complexed C-FNP Using ALP Activity with NPP [C-FNP/NP]. A solution (1 mL) containing C-FNP (1 mg/mL), 10 mM NPP, and MgSO4 (0.1 μM) was placed in a cuvette. The 4-nitrophenol (NP) complexed C-FNP (TBS, pH 7.4) was assessed with continuous addition of ALP (10 μL), where the activity of ALP was 500 U/L. This was followed by a 15 min incubation period. The 4-nitrophenol (NP) complexed C-FNP was then purified through dialysis (MWCO = 1000) and freeze-dried. Fluorescent Quenching of C-FNP by NP. To optimize the concentration of NP required for fluorescent quenching, solutions (1 mL) containing C-FNP (1 mg/mL) and various concentrations of NP (0−10 mM) in Tris−HCl (10 mM, pH 7.4) were monitored by fluorescence spectroscopy at the optimal excitation wavelength. Briefly, 45 μL of NP solution (0−10 mM) was added to 1 mL of C-FNP (1 mg/mL) buffer solution (TBS, pH 7.4). The solution was shaken for 5 min following addition of NP. The fluorescence of mixed solution was recorded after 15 min of incubation. The emission spectra of solutions with different concentrations of NP were recorded at an excitation wavelength of 420 nm. All experiments were conducted at 37 °C. Fluorescence Assay of ALP Activity in Aqueous and Solid States. A solution (1 mL) containing C-FNP (1 mg/mL) in the aqueous state or C-FNP coated PP films was mixed with 10 mM NPP and MgSO4 (0.1 μM). Fluorescence quenching of C-FNP (TBS, pH 7.4) or C-FNP coated PP films was assessed with continuous addition of ALP (10 μL), where the activity of ALP ranged between 0−500 U/L. Following addition of ALP, the solution was incubated for 15 min. The emission spectra of solutions containing different concentrations of ALP were recorded at an excitation wavelength of 420 nm. Temperature was maintained at 37 °C throughout the experiment. Surface Coating of PP with C-FNP or C-FNP/NP. Solid substrate (PP) was prepared through dip-coating methods. In detail, PP films were cleaned using acetone or ethanol for 5 min via sonication. Surface coating with C-FNP or C-FNP/NP was performed by immersing substrates in a buffer solution (10 mM Tris, pH 8.5) containing 10 mg/mL of C-FNP or

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EXPERIMENTAL SECTION Materials and Characterization. β-Cyclodextrin (CD, β-CD), dopamine hydrochloride (DA), Trizma base, Trizma HCl, [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT), N-hydroxy succinimide (NHS), 1-ethyl-3-(3(dimethylamino)propyl)carbodiimide (EDC), 3-amino phenyl boronic acid, Triton X-100, H2SO4, alkaline phosphatase (ALP), and 4-nitrophenyl phosphate (NPP) were purchased from SigmaAldrich, Korea. Hyaluronic acid (Mw 7000 kDa) was obtained from Shinpoong Pharm (Korea) as a kind donation. Penicillinstreptomycin, fetal bovine serum (FBS), 0.25% (w/v) Trypsin, 0.05% (w/v) EDTA solution, and RPMI-1640 medium were purchased from Gibco BRL (Carlsbad, CA). 1 H NMR spectra were recorded using a Bruker Advance 400 MHz spectrometer with deuterium oxide (D2O) and deuterium dimethyl sulfoxide (DMSO-d6) as the solvent. The UV−vis spectra were recorded using an Optizen 2020UV (Mecasys Co.). Particle size was measured using dynamic light scattering (DLS) (Zetasizer Nano, Malvern-Germany). Photoluminescence (PL) spectra were obtained using a PerkinElmer L550B luminescence spectrometer. Fluorescence lifetimes were measured using a NanoLED laser light source (Horiba Jobin Yvon NanoLog spectrophotometer) at 375 nm excitation, and the data were fitted onto a multiexponential decay model. The multimode microplate reader Filter MaxF3 (Molecular Devices, LLC) was used for the MTT assay and quantitative cellular accumulation. Synthesis of Boronic Acid (BA) Conjugated to Partially Carbonized Hyaluronic Acid (FNP(HA)) [FNP(HA-BA)]. Protocol for FNP (HA) synthesis was adopted from methods described in the literature.9 To perform the conjugation of FNP 13509

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Scheme 1. Schematic Illustration of the Fluorescent Assay for Alkaline Phosphatase Based on β-CD-Functionalized C-FNP in Aqueous Solutions and on Solid Surfaces

C-FNP at 0.5 mg/mL for 30 min in fresh medium. The cells were then washed with PBS several times to remove any unbound C-FNP, and were stained with LysoTracker Red with the addition of NPP (10 mM) and MgSO4 (0.1 μM). Finally, the cells were examined at 20× magnification using an LSM510 confocal laser scanning microscope (Carl Zeiss, Germany) for 4 h. For solid-state confocal imaging, MDAMB and MDCK cells were grown over a coated film in a six-well plate at a density of 2 × 105 cells/mL per well, and were incubated for 24 h at 37 °C in humidified atmosphere containing 5% CO2. The cells were then washed with PBS several times, and were stained with LysoTracker Red with the addition of NPP (10 mM) and MgSO4 (0.1 μM). Finally, the cells were examined at 20× magnification using a LSM510 confocal laser scanning microscope (Carl Zeiss, Germany). Cellular uptake of C-FNP on coated film was analyzed via confocal imaging. In addition, we used EDTA to release the cell grown on the coated film. Next, we centrifuged the EDTA solution and then collected the cell on solid base. The cells were examined using 20× magnification via a LSM510 confocal laser scanning microscope (Carl Zeiss, Germany).

C-FNP/NP at room temperature. After 24 h, coated substrates were rinsed extensively with deionized water. MTT Assay. Cytotoxicity was measured using the [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] MTT assay method. Here, 200-μL aliquots of MDAMB (MDAMB231) and MDCK cells, at a density of 2 × 105 cells/mL, were seeded in 96-well plates. The cells were then incubated for 24 h at 37 °C in humidified 5% CO2 atmosphere. To determine cellular viability, a stock solution of C-FNP was dissolved in RPMI medium at a concentration of 1 mg/mL, after which the stock solution was diluted to 0.01 mg/mL. The medium was removed, and the cells were treated with different concentrations of the C-FNP composite material. Cells were then incubated as described above for another 24 h. The medium containing C-FNP was then replaced with 180 μL of fresh medium and 20 μL of a stock solution containing 15 mg of MTT in 3 mL of PBS, after which the cells were incubated for another 4 h. Finally, the medium was removed, and 200 μL of MTT solubilizing agent was added to the cells. Cells were shaken for 15 min, and absorbance was measured at 570 nm using a microplate reader (Varioskan Flash, Thermo Electron Corp.). Relative cell viability was measured by comparison with control wells without C-FNP. Confocal Imaging of ALP Sensitive C-FNP in Aqueous and Solid States. Cellular uptakes of ALP sensitive C-FNP were analyzed through confocal imaging. MDAMB and MDCK cells were plated on a cover slide in an eight-well plate at a density of 2 × 105 cells/mL per well, and were incubated for 24 h at 37 °C in humidified atmosphere with 5% CO2. Cells were treated with

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RESULTS AND DISCUSSION Synthesis of Functionalized Cross-Linked Fluorescent Nanoparticles (C-FNP). Tumor-specific fluorescent nanoparticles (FNPs) were used as ALP biosensors, and were designed on the basis of a fluorescent on/off system, where ALP catalyzes the hydrolysis of 4-nitrophenyl phosphate (NPP) to 4-nitrophenol (NP) due to association between NP and the cavity of 13510

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using the BA-diol complex to prepare a smart nanoprobe for ALP detection. The catechol moieties on the surface of crosslinked FNP with host−guest recognition can support solid-phase assays, which are superior to traditional aqueous media-based ALP sensors. This study has shown that this novel nanoprobe can detect cancer cells through cross-linked FNP that has alkaline phosphatase (ALP) sensitivity in both aqueous states and on coated surfaces. Material Characterization. Figure 1 shows the structural analysis by 1H NMR during each synthesis step. As shown in Figure 1a, the β-CD and HA peak were confirmed at 3−4 ppm. In Figure 1b, the aromatic structure of boronic acid could be observed at 7.2 ppm using d-DMSO. The peaks of H4 at 3.45 ppm, H2 at 3.50 ppm, H5 at 3.75 ppm, and H3 at 3.85 ppm of β-CD were detected following conjugation of β-CD onto FNP (HA). As shown in Figure 1, the NMR peaks for H3 and H5 of CD were shifted to 3.75 and 3.65 ppm, respectively, after 4-nitrophenol complex was formed during the ALP reaction.16 The optical properties of the prepared materials in each synthesis step were observed by UV−vis−NIR absorption spectra, as shown in Figure 2a. The absorbance of FNP (pDA) could be observed in the NIR region.15 The weak BA peak was detected at 294 nm when BA was conjugated to FNP (HA),16 confirming the presence of the π−π* bond in carbonized polydopamine.15 The peak in the NIR region was also found following reaction of C-FNP with FNP (pDA). We also studied the fluorescent profiles of FNP (pDA) and FNP (HA-CD) prior

Figure 1. (a) 1H NMR spectra (400 MHz, D2O) of FNP(HA-BA), FNP(HA-CD), C-FNP, and C-FNP/NP. (b) 1H NMR spectra (400 MHz, d-DMSO) of FNP(HA-BA), FNP(HA-CD), and C-FNP.

β-CD. The design and preparation of this tumor-triggered fluorescent assay to monitor cellular ALP are shown in Scheme 1. As shown in Scheme 1, FNP (pDA) was synthesized through polymerization of dopamine followed by its carbonization using strong sulfuric acid (H2SO4). Hyaluronic acid was carbonized using sulfuric acid (H2SO4) to synthesize FNP (HA). FNP (HA-BA) was then prepared by conjugation with boronic acid. Meanwhile, β-CD was allowed to react with PBS at pH 12 to synthesize FNP (HA-CD). These two FNPs were cross-linked

Figure 2. (a) UV−vis−NIR absorption spectra of FNP (pDA), FNP (HA-BA), FNP (HA-CD), and C-FNP. (b) Fluorescence emission intensity of C-FNP at different excitation (340−500 nm) wavelengths at pH 7.4. (c) DLS measurements of FNP (pDA), FNP (HA-CD), and C-FNP for changes in the distribution of hydrodynamic radius in aqueous solution. (d) TEM image of C-FNP of multiple and a single nanoparticle with HR-TEM images of C-FNP. 13511

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Figure 3. (a) Fluorescence spectra of C-FNP in the presence of different concentrations of p-nitrophenol. (b) Changes in the fluorescence spectra of C-FNP (1 mg/mL) in the presence of NPP (10 mM) with different concentrations of ALP. (c) Fluorescent emission intensity of C-FNP/NP (1 mg/mL) at different pH values. Excitation wavelength was set at 420 nm. The pH responsive study materials were prepared by dialysis at different pH values followed by freeze-drying of the remaining materials. (d) DLS measurements of C-FNP to monitor changes in the hydrodynamic radius distribution in response to changing pH (7.4 to 5.0).

wavelength of 365 nm, where intensity of the blue color should be reduced at higher concentrations. As shown in Figure 3b, fluorescence of C-FNP was assessed at various concentrations of ALP in the presence of 10 mM NPP and 1 mg/mL C-FNP. Results indicated that greater quenching of C-FNP fluorescence due to hydrolysis of NPP to NP was achieved at higher ALP concentrations.19 On the basis of the previous data, complete quenching could be achieved at an ALP concentration of 500 U/L. Figure 3c demonstrates pH-dependent changes in fluorescence of C-FNP/NP upon reaction of ALP (500 U/L) with NPP (10 mM) in C-FNP. In Figure 3c, fluorescence of C-FNP/NP was quenched at pH 7.4, while under acidic conditions (pH 5.0 and 6.0), fluorescence intensity was increased. This was due to cleavage of the boronate ester bond of C-FNP, thereby releasing β-CD and NP from C-FNP.16 Figure 3d also shows the average size of C-FNP at different pH, as measured by DLS. The size of C-FNP was confirmed to be 64.7 nm at pH 5.0, 79.5 nm at pH 6.0, and 167.6 nm at pH 7.4. In addition, the particle size in acidic condition over time demonstrated that the boronate ester in C-FNP hydrolyzes after 30 min incubation and is totally stable after 1 h incubation time (Figure S2). The reduction in particle diameters dependent on times suggests that cleavage of the boronate ester linker is pH-sensitive, and is more effective in an acidic environment.16 Furthermore, at fixed ALP concentration, we observed the time dependence of the PL intensity and particle size of C-FNP in response to ALP concentration in Figure S3. The C-FNP starts to quench at 10 min

to cross-linking (Figure S1). FNP (pDA) and FNP (HA-CD) showed the highest emission at 420 and 340 nm, respectively. As depicted in Figure 2b, the fluorescence emission spectra of the synthesized C-FNP displayed an excitation-dependent red shift from 340 to 500 nm. The wavelength exhibiting the highest fluorescence intensity was 420 nm. Figure 2c shows the average size distribution of FNP (pDA), FNP (HA-CD), and C-FNP measured by DLS in the aqueous state. The sizes were confirmed to be 19.59 nm for FNP (pDA), 64.39 nm for FNP (HA-CD), and 167.6 nm for C-FNP. The large size of C-FNP was confirmed to be due to cross-linking of FNP via the BA-diol complex. The size and shape morphology of C-FNP were observed through transmission electron microscopy (TEM). As can be seen from the TEM image in Figure 2d, the morphology of single C-FNP shows spherical shapes in which it was observed that the C-FNP showed regular dots shapes on multiple particles images with the particle size of approximately 80−100 nm, and graphene-type lattice structures of 0.308−0.329 nm were found by HR-TEM analysis.17 Fluorescent Assay of Alkaline Phosphatase in Aqueous Solution. The quenching effect of C-FNP as a function of NP concentration is shown in Figure 3a. It was confirmed that at the concentration range between 0−10 mM, the quenching effect became significant at higher concentrations. This suggests that photoinduced electron transfer occurs when NP can effectively act as an electron acceptor for C-FNP.18 Quenching at various concentrations could be visually confirmed using a UV lamp at a 13512

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Figure 4. Confocal laser scanning microscope (CLSM) images of MDAMB-231 and MDCK cells incubated with C-FNP (1 mg/mL) in the presence of NPP (10 mM) for 0.5, 1, 2, 3, and 4 h. The scale bars represent a distance of 50 μm.

Figure S4b,c, cell viability of greater than 90% was confirmed even after we treated the NPP with cell lines. The normal level of ALP in cells ranges between 40−150 mU/mL; abnormally high levels of ALP have been linked to several diseases, including cancer (Ozer et al., 2015). Endogenic monitoring of ALP activity in cancer (MDAMB-231) and normal (MDCK) cells was carried out through time-dependent confocal imaging as a method for tumor detection (Figure 4). The cells were incubated in C-FNP (1 mg/mL) for 30 min, and NPP (10 mM) was added. Because of the high ALP concentration in MDAMB-231 cells, fluorescence of the C-FNP nanoprobe in the cancer cell MDAMB-231 started to disappear after 30 min. ALP localized to cancer cells can hydrolyze NPP to NP, which complexes with β-CD because of host−guest recognition.19 This then leads to changes in the fluorescent profile of C-FNP by photoinduced electron transfer (PET).23 However, no significant fluorescent changes were observed in cells without addition of NPP, confirming the absence of NP/CD complex formation. Furthermore, as the boronate ester bond dissociated due to low pH in lysosomes, emission color was detected in lysosomes, as shown in the merged confocal images confirmed by lysotracker.24 In the case of MDCK cells, no significant changes were observed in the overall fluorescence within the cells due to the absence of ALP activity. In addition, Figure S6 shows confocal laser scanning microscope images of MDAMB-231 cells using quenched C-FNP/NP. It was observed that fluorescence

and totally at 30 min incubation, which correspond to decreasing particle size simultaneously. This phenomenon might be applied as an ALP monitoring platform in the cell because the ALP was dominantly localized in intracellular fluid, which induces C-FNP response to ALP before reaching the lysosome environment, indicating that hydrolysis of C-FNP will not influence the ALP responsibility. Cellular uptake was carried out to verify the targeting effect of C-FNP on normal (MDCK) and cancer (MDAMB-231) cells prior to time-dependent cell imaging. As can be observed in Figure S4a, the cellular uptake of C-FNP in MDAMB-231 cells was 38%, while that in MDCK cells was 19%, confirming the selective targeting of C-FNP to cancer cells in the presence of HA. The surface electrical charge of C-FNP under different pH values was measured to support this analysis. The C-FNP showed a negative charge (−mV) irrespective of the pH (ranged from 4.0 to 7.4), implicating negatively charged carboxyl groups resulted from carbonization treatment (Figure S5). Because of the negative charge of the cell membrane, it clearly demonstrated that there are no effects of electrical charge of C-FNP surfaces toward cellular uptake. However, the mechanism of cellular uptake was confirmed by cellular internalization of HA regulated by CD44 receptors on the tumor cell.9 The cells (MDAMB-231 and MDCK) and NPP treated cells were cultured with C-FNP at different concentrations for 24 h for further cytotoxic observation via MTT assays.20,21 On the basis of results shown in 13513

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Figure 5. (a) Confocal laser scan microscopy images of (a) C-FNP coated PP substrate in the presence of NPP (10 mM) at different concentrations of ALP, (b) C-FNP coated PP substrate, C-FNP coated PP substrate treated with ALP in the presence NPP, and after pH shock (pH 6.0) for C-FNP coated PP substrate treated with ALP in the presence NPP. Inset of (a) is the fluorescent intensity graph of C-FNP coated PP substrate.

confirmed that fluorescence was emitted from the same position as the lysotracker.25 ALP level in MDAMB-231 is higher than in MDCK, which could be able act as parameters to determine

began to appear over a period of 0.5−1 h, as the cross-link became loose due to the low pH in lysosomes. Fluorescence intensity gradually increased over a period of 2−4 h. It was 13514

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Figure 6. Confocal laser scanning microscope (CLSM) images of MDAMB-231 and MDCK cells grown on coated PP in the presence of NPP (10 mM) for 0.5, 1, 2, 3, and 4 h. The scale bars represent a distance of 50 μm.

cancer cell.22 When the particles entering the cells via the endocytic pathway encounter ALP in intracellular, the NPP capped in β-CD cavity changes to the NP form, resulting in fluorescent quenching of C-FNP during 30 min. They will then be entrapped in endosome, due to the acidic condition inside the pH responsive linker boronate ester bond of C-FNP being broken down after 30 min to release the NP to recover the emission and eventually end up with lysosome escaping to cytoplasm. No apparent fluorescence could be observed besides lysosome positions in CLSM images of MDAMB-231 on Figure 4, indicating the existence of high ALP level in cytoplasm generating the fluorescent quenching. However, the C-FNP keeps the fluorescence in the normal MDCK due to low level ALP. It does not mean that the C-FNP appears from lysosome escape, but that the fluorescent quenching might be representative of another certain C-FNP system, which did not go to lysosome and remained at cytoplasm. To investigate the efficiency of ALP performance in mammalian cell, we have also done cellular uptake of C-FNP by adding the enzyme inhibitor. Na3VO4 commonly used as ALP inhibitor was employed in MDAMB-231 and MDCK cells incubation. As shown in Figure S7, the fluorescence intensity of C-FNP in the MDAMB-231 was stable in the lysosome environment after the addition of the inhibitor, implicating that the fluorescent quenching has been inhibited by blocking the ALP level in the cell, which equals the result with MDCK cell treated Na3VO4 ALP inhibitor, confirming no ALP activity.

Fluorescent Assay of Alkaline Phosphatase on Solid Surface. Currently, there has been no report that includes ALP monitoring on coated surface materials using fluorescent on/off assays. In our study, we coated PP surface to understand the adhesive properties of the catechol compartment in the FNP (pDA) site. The contact angle of bare PP as shown in Figure S8 was 91.6° prior to coating, and was 76.1° following C-FNP coating. It then increased to 93.5° after NP complex formation.26,27 Accordingly, the value was decreased to 72.3° by pH shock. To advance our study, we measured the XPS spectra (Figure S9) to confirm chemical changes to the coating surface. Results from C 1s analysis confirmed formation of the NP complex with an increase in the CC binding energy. Following pH shock, the binding energy intensity of CC, C−O−C, and O−CO was reduced due to release of HA, CD, and NP, which suggested that the coating surface was affected by pH changes. After PP film was coated with C-FNP, ALP activity was assessed by changes in fluorescence patterns, which was analyzed by confocal microscopy.26 We saw that uncoated PP film did not emit fluorescence. However, following C-FNP coating, fluorescent signal was detected. As shown in Figure 5a, blue, green, and red fluorescence were observed at 405, 488, and 543 nm, respectively. When 500 U/L of ALP and NPP was added, NPP was hydrolyzed to NP. This resulted in fluorescence quenching based on host and guest complexity of CD and NP.28 This effect was enhanced by increased concentration of ALP. Furthermore, 13515

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Analytical Chemistry

β-CD and NP leads to fluorescent quenching through the PET mechanism. Because of cleavage of the diol bond between CD and BA, fluorescence can be recovered in the lysosome due to its acidic environment. In this study, we showed that our C-FNP can be used to monitor ALP activity in MDAMB-231 and MDCK cells via confocal imaging. To conclude, this biosensor has the potential to be a simple and sensitive cancer diagnostic tool that can monitor cellular ALP activity on coated surfaces and in aqueous states.

we investigated the effect of fluorescence on C-FNP/NP coated PP surface (Figure 5b). We observed loss of emission with C-FNP/NP, which recovered as a function of pH.16 Thus, we have demonstrated that the present system can be used to effectively monitor ALP activity within cells through fluorescence imaging with diverse substrates. We further tested the ability of polypropylene (PP) coated C-FNP to detect ALP activity by growing cancer cell on the surface PP. ALP activity was analyzed using confocal images to determine the fluorescence quenching response. Figure 6 shows the changes in fluorescence over time following addition of NPP to cultured cells on the coated surface for 0−4 h. As in the previous experiment under aqueous state, ALP could be detected through the quenching effect of C-FNP after 0.5 h due to high concentration of ALP in MDAMB-231 cells. During the growth of cell on the coated film, we hypothesized that pH conditions decline due to the acid condition inducing the hydrolysis of boronate ester formation of C-FNP to generate FNP(pDA) and FNP(HA-CD). Therefore, only the FNP(HA-CD) can internalize to the cell line due to hyaluron-mediated motility receptor of CD44, whereas the FNP(pDA) might remain deposited on the film. To realize the cellular uptake of them on the coated surface toward MDAMB-231 and MDCK, we further investigated confocal images of the cell grown released by EDTA from coated surfaces after and before pH treatment (Figure S10). The cellular uptake in MDAMB-231 cells (35%) was higher than in MDCK (15%), confirming only FNP(HA-CD) uptake to the cell after release by acid condition. When FNP(HA-CD) has dominantly localized in intracellular MDAMB-231 fluid before forming the endosome, the FNP(HA-CD) fluorescent will turn off realizing the high ALP level as shown in confocal images, while the brighter emission on the MDCK cell than on MDAMB-231 was due to the different level of ALP. In addition, the remaining emission of C-FNP was observed in lysosomes due to breaking down of the pH responsive linker, which resulted in nonspecific quenching. On the other hand, no significant change in fluorescence was detected in MDCK cells. As compared to previous methods, which regularly using synthetic material, organic dye, and metal involving complex preparation, our developed approach shows much better sensitivity and selectivity efficiency for ALP detection due to rapid detection process by fluorescent changes upon 30 min.29−31 Without typical quenching by other particles, ALP can directly quench our C-FNP system and simultaneously play as detector for their existence. This developed strategy shows remarkable advantages on coated surfaces as a smart material to monitor ALP activity in tumor-triggered fluorescent assay probes through sensitive, rapid, simple, convenient, and low-cost techniques. In addition, this approach can be suggested as a great potential method for early diagnosis of various severe diseases such as bone cancer and Alzheimer based on the ALP level.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b03853. Fluorescence emission intensity of FNP (pDA) and FNP (HA-CD) at different excitation wavelengths at pH 7.4; DLS histogram of C-FNP at various pH values (7.4, 6.0, and 5.0) in varied time; changes in the fluorescence spectra of C-FNP in the presence of NPP at fixed ALP concentration; quantitative cellular accumulation of C-FNP nanoparticles in MDAMB-231 and MDCK cells; in vitro biocompatibility of MDCK and MDAMB-231 cells of C-FNP; zeta potential of C-FNP under pH condition; confocal laser scanning microscope (CLSM) images of MDAMB-231 cells incubated with C-FNP/NP; confocal laser scanning microscope (CLSM) images of MDAMB-231 cells and MDCK incubated with C-FNP/NP and inhibitor-treated cells; contact angle investigation on C-FNP coated PP substrates; XPS spectra of coated PP surface; and quantitative cellular accumulation (%) of C-FNP nanoparticles in MDAMB-231 and MDCK cells grown on coated surfaces (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *Fax: 043-841-5220. E-mail: [email protected]. ORCID

Insik In: 0000-0001-7852-1162 Sung Young Park: 0000-0002-0358-6946 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by grant nos. 10062079 and R0005237 from the Ministry of Trade, Industry & Energy (MOTIE), by the Korea Institute for Advancement of Technology (KIAT) through Research and Development for Regional Industry (no. R0005303), and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF2017R1A2B2002365).

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CONCLUSIONS In summary, we prepared a C-FNP for tumor-specific fluorescent sensing-based intracellular ALP activity by conjugating crosslinked β-CD in FNP (HA) with boronic acid (BA) and FNP (pDA) via pH cleavable boronate ester bonds. The adhesive catechol in C-FNP provides a novel aspect to our design, which allows detection of ALP on coated surfaces. The prepared tumor sensing nanoprobe was developed as a fluorescent assay that can be used for cancer diagnosis in both aqueous states and on coated surfaces through fluorescence quenching by ALP. ALP can hydrolyze NPP to NP, and host−guest recognition between

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