Article Cite This: ACS Appl. Nano Mater. 2019, 2, 3819−3827
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Self-Assembling Ratiometric Fluorescent Micelle Nanoprobe for Tyrosinase Detection in Living Cells Jiemin Wang,† Jing Qian,† Zhidong Teng,‡ Ting Cao,† Deyan Gong,† Wei Liu,† Yuping Cao,† Wenwu Qin,*,† Huichen Guo,*,‡ and Anam Iqbal*,§
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Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, Key Laboratory of Special Function Materials and Structure Design (MOE), and State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, 730000, P. R. China ‡ State Key Laboratory of Veterinary Etiological Biology and Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 1, Lanzhou, Gansu Province 730046, P. R. China § Department of Chemistry, University of Baluchistan, Quetta, 87300, Pakistan S Supporting Information *
ABSTRACT: Herein, a novel self-assembling ratiometric fluorescent micelle nanoprobe (NanoDPA-NMP-tyr) based on Fö r ster Resonance Energy Transfer (FRET) was developed and was used to respond to tyrosinase (TYR) activity in B16 cells. The absorption band of the small organic molecular probe NMP-tyr (λabs = ∼450 nm) composed of fluorophore naphthalimide derivative and detecting group tyramine, and the emission band of 9,10-diphenylfluorene DPA (λem = ∼445 nm) overlap. NMP-tyr acts as energy acceptors, and DPA acts as donors to supplement energy. Both of these substances were encapsulated in the hydrophobic interior of the amphiphilic copolymer mPEG-DSPE. The FRET mechanism was formed when NanoDPA-NMP-tyr was excited at 377 nm. When TYR and oxygen were present at the same time, the maximum absorption peak of NMP-tyr blue-shifted from ∼450 nm to ∼350 nm (NMP-tyro), which blocked the energy transfer from DPA to NMP-tyr and effectively turned off FRET. The blue fluorescence of donor dye DPA was restored. NanoDPA-NMP-tyr is able to respond quickly to TYR in 7.5 min and exhibits excellent specificity and sensitivity toward TYR. Fluorescence imaging applied to B16 cells shows that NanoDPA-NMP-tyr is highly responsive to TYR in vivo. KEYWORDS: tyrosinase, micelle, fluorescent nanoprobe, ratiometric, FRET, B16 cell
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INTRODUCTION Melanoma is a malignant cancer that is highly specific to skin.1 Skin malignant melanoma, which is difficult to diagnose, is a major cause of malignant invasive skin cancer and death caused by skin diseases.2−4 According to statistics, cases of cutaneous melanoma have been increasing over the past two decades and it has become one of the fastest growing cancers.5−7 Tyrosinase (TYR), a copper containing enzyme is a biomarker of melanoma cancer cells and a key enzyme for the biosynthesis of melanin, which plays an important role in the formation and expression of melanin in melanocytes and malignant melanoma cells.8−10 TYR and its mRNA can be detected in the serum of patients with melanoma.10−13 Therefore, if the function of TYR expression is impaired, it may cause the disorder of melanin formation, which may cause many diseases, such as albinism or vitiligo;14,15 therefore, the development of a highly selective and sensitive assay of TYR activity is in urgent demand for fundamental research and practical applications in clinical diagnosis. © 2019 American Chemical Society
Many scientists have explored some methods for TYR detection,16−18 such as the colorimetric method,19 electrochemical method,20 and radiation method.21 Compared with these methods, fluorescent detection has the advantages of good specificity, high sensitivity, and low cost. As reported in the literature, many fluorescent methods were used to detect TYR, such as fluorescent quantum dots,22−24 noble metal nanoclusters,25,26 polymer nanoparticles,27−29 and small molecule dyes, etc.30−33 By comparison, it is found that the small organic molecule fluorescent probe has the advantages of good penetrating power and simple operation, and it offers more advantages in clinical and biological applications. Recently, Zhan et al.1 reported a near-infrared organic small molecule turn-on fluorescent probe for imaging in vitro and in vivo by attaching a hydroxyphenyl urea used as a TYR Received: April 14, 2019 Accepted: May 21, 2019 Published: May 22, 2019 3819
DOI: 10.1021/acsanm.9b00689 ACS Appl. Nano Mater. 2019, 2, 3819−3827
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ACS Applied Nano Materials Scheme 1. (a) Schematic Diagram of the FRET Reaction. (b) Synthesis of NMP-tyr and Its Response to TYR
absorption peak of the fluorescent probe NMP-tyr was blueshifted by about 100 nm. The fluorescence emission spectrum of DPA no longer overlapped with the absorption spectrum of NMP-tyr, and energy transfer between the two was interrupted. So FRET was blocked leading the change in fluorescence from NMP-tyr green to DPA blue, thus achieving a fast ratiometric response to TYR. NanoDPA-NMP-tyr has a response time of 7.5 min for TYR, which is shorter than that previously reported. In addition, NanoDPA-NMP-tyr micellar fluorescent nanoprobes have the advantages of high water solubility and low cytotoxicity, which can promote our further real-time fluorescence imaging of TYR related biological processes (such as in tissues and living organisms).
responsive group to a fluorescent dye phenoxazine derivative. Li et al.34 published a small molecule turn-off fluorescent probe which was converted to the corresponding o-benzoquinone by TYR by using a monophenol derivative of tyramine as a detection group. Hence ratiometric fluorescent probes can enhance the accuracy of quantitative detection and reduce the error caused by the single emission compared to many turn-off or turn-on responding probes. However, until now, only a few probes provided selective ratiometric fluorescent detection for TYR. Therefore, to obtain a ratiometric fluorescent probe that is fast and sensitive to TYR detection, the micelle nanoprobe NanoDPA-NMP-tyr was designed, which uses the FRET mechanism to detect TYR.35 The small organic molecule NMP-tyr composed of a naphthalimide derivative and a detection group tyramine18,23−25,29,31,36−40 and the dye 9,10diphenylanthracene (DPA) were encapsulated in the hydrophobic interior of amphiphilic copolymer 1,2-dimyristoyl-snglycero-3-phospho-ethanolamine-N-(methoxy(polyethylene glycol)-2000) (mPEG-DSPE). The emission band of DPA (λem ∼ 445 nm) overlaps with the absorption band of NMP-tyr (λabs ∼ 450 nm), which satisfies the FRET mechanism. NMPtyr is used as an energy acceptor in NanoDPA-NMP-tyr, and DPA is used as a donor of energy complementation. In addition, the mPEG-DSPE allows the two chromophores to be more tightly connected, thus facilitating energy transfer. Therefore, a FRET system has formed (Scheme 1a). When TYR and oxygen were present at the same time, the monophenol derivative of the tyrosine in response to the group was oxidized by TYR to get the corresponding phenylhydrazine NMP-tyro (Scheme 1b). The maximum
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EXPERIMENTAL SECTION
1. Materials and Instruments. All drugs used in chemical experiments were purchased using the market agreement and were used without further purification. 1H and 13C NMR spectra were obtained on a Bruker DRX-400 and DRX-400/4 spectrometer. The mass spectrum is received in an excitation source with EI. Fourier transform infrared spectroscopy (FTIR) in the range of 4000−400 cm−1 was recorded on a Mattson Alpha-Centauri spectrometer (KBr). Transmission electron microscopy (TEM) was measured by the instrument JEOL JEM2100 TEM; model BI-200SM was used to measure the dynamic scatter diameter (DLS) hydrodynamic diameter and size distribution. The pH was measured by using a pH-10C digital pH meter. Selective experiments were performed in 20 mM PBS buffer (pH 7.4, 37 °C). 2. Experimental Section. Synthesis of Compound 2. 1 (1.662 g, 6 mmol) was dissolved in ethanol (14 mL). After this N(ethylaminoethyl)-morpholine (2.400 mL, 3 mmol) was added, and the mixture was refluxed and stirred for 3 h. Thin-layer chromatography (TCL) was used to monitor the progress of the 3820
DOI: 10.1021/acsanm.9b00689 ACS Appl. Nano Mater. 2019, 2, 3819−3827
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ACS Applied Nano Materials reaction. After the completion of the reaction, the mixture was then cooled to room temperature and filtered. The filter cake was washed with ethanol to give white 2, 0.829 g (yield 50%) (Figure S9). 1H NMR (CDCl3, 400 MHz): δ 8.64 (d, H-e, J = 8.0 Hz, 1H), δ 8.57 (d, H-g, J = 8.0 Hz, 1H), δ 8.40 (d, H-h, J = 8.0 Hz, 1H), δ 8.04 (d, H-i, J = 8.0 Hz, 1H), δ 7.86 (t, H-f, J = 8.0 Hz, J = 8.0 Hz, 1H), δ 4.34 (t, Hd, J = 6.8 Hz, 2H), δ 3.67 (t, H-a, J = 4.40 Hz, 4H), δ 2.70 (t, H-c, J = 6.8 Hz, 2H), δ 2.57 (s, H-b, 4H). Synthesis of NMP-tyr. 2 (776 mg, 1.5 mmol) and 4hydroxyphenethylamine (288 mg, 0.5 mmol) were added to a 100 mL round-bottom flask, and the mixture was dissolved in DMF (15 mL) and stirred at 50 °C overnight. After the TCL monitoring reaction was completed, the mixture was cooled to room temperature and the solvent was evaporated under vacuum. The crude product was purified by silica gel column chromatography eluting with dichloromethane/methanol (50:1, v/v) to obtain compound NMPtyr, 810 mg (yield 80%) (Figure S10−S12). 1H NMR (CDCl3, 400 MHz): δ 8.50 (d, H-e, J = 7.2 Hz, 1H), δ 8.41 (d, H-g, J = 8.0 Hz, 1H), δ 7.87 (d, H-h, J = 8.4 Hz, 1H), δ 7.53 (t, H-f, J = 8.0 Hz, J = 7.2 Hz, 1H), δ 7.09 (d, H-m, J = 7.6 Hz, 2H), δ 6.79 (d, H-n, J = 7.6 Hz, 2H), δ 6.72 (d, H-i, J = 8.4 Hz, 1H), δ 5.28 (s, H-o or H-j, 1H), δ 4.34 (t, H-d, J = 6.0 Hz, 2H), δ 3.70 (s, H-a, 4H), δ 3.61 (d, H-k, J = 6.0 Hz, 2H), δ 3.00 (t, H-l, J = 6.0 Hz,2H), δ 2.74 (t, H-c, J = 6.0 Hz, 2H), δ 2.64 (s, H-b, 4H). 13C NMR (DMSO-d 6, 400/4 MHz (100 MHz)): δ 164.25, 163.37, 161.15, 156.34, 150.93, 134.75, 131.11, 129.97, 129.03, 124.68, 122.30, 120.61, 115.73, 108.12, 104.37, 66.72, 56.31, 53.93, 45.36, 36.88, 34.76. MS calcd for NMP-tyr (C26H27N3O4), 445.20; found, 446.28 (M + 1). IR (KBr) ν max/ cm−1 833 (s), 1115 (m), 1246 (m), 1548 (h), 1580 (m), 1612 (h), 1635 (m), 1680 (s), 3356 (s). Preparation of NanoDPA-NMP-tyr. Distilled deionized (9 mL) water was added to the centrifuge tube and sonicated for about 20 min. mPEG-DSPE (5.70 mg) was quickly poured into the water under vigorous ultrasound and maintained for about 20 min. NMP-tyr (2.7 × 10−3 μmol) and DPA (3.1 × 10−5 μmol) were quickly poured into the mixture for 20 min under continuous sonication. Then, the polymer was filtered through 0.22 μm polyvinylidene fluoride (PVDF) syringe, and the free dye was removed by MWCO 300 D dialysis bag to obtain a stock solution of NanoDPA-NMP-tyr. Preparation of NanoDPA and NanoNMP-tyr. NanoDPA was synthesized by separately encapsulating DPA (3.1 × 10−5 μmol) in mPEG-DSPE (5.70 mg) under the same synthetic method as above. NanoNMP-tyr was synthesized by encapsulating NMP-tyr (2.7 × 10−3 μmol) in mPEG-DSPE (5.70 mg). Detection of TYR by NanoDPA-NMP-tyr in PBS Buffer Solution. Three milliliters of PBS buffer (pH 7.4) with NanoDPA-NMP-tyr solution was added to the centrifuge tube, then 0−20 U/mL of TYR was added. Finally, the entire solution was transferred to the quartz cuvette after incubation at 37 °C for 5−30 min.
Figure 1. (a) Average hydrodynamic size of NanoDPA-NMP-tyr as measured by DLS; (b) representative TEM image of NanoDPANMP-tyr. The scale bar is 100 nm.
broad band at around 450 nm, respectively. The absorption spectrum of NanoDPA-NMP-tyr contains a sharp absorption band around 322, 377, and 400 nm, and another strong and broad absorption band can be discovered at about 450 nm. Therefore, the absorption spectra of NanoDPA-NMP-tyr contain the typical absorption spectra of both NanoDPA and NanoNMP-tyr. The emission spectra of NanoDPA show the main band which is at a maximum at ∼445 nm. NanoNMP-tyr shows dual fluorescence, the band at the higher energy of ∼436 nm and another emission band at ∼544 nm. The fluorescence emission spectra of NanoDPA-NMP-tyr are quite similar to that of NanoNMP-tyr in the same PBS buffer solution. Interestingly, it can be seen from the spectrogram that the emission band of NanoDPA (λem ∼ 445 nm) overlaps well with the absorption band of NanoNMP-tyr (λabs ∼ 450 nm). It shows that both the donor DPA and receptor NMP-tyr were encapsulated together in the same micelle aggregate NanoDPA-NMP-tyr, the transfer of effective energy in NanoDPANMP-tyr was found. Then NanoDPA-NMP-tyr shows green fluorescence emission (∼544 nm). The efficiency of FRET was calculated by eq 1 (see Supporting Information), and it was confirmed that FRET has an energy conversion rate of 86.3%. We have calculated the fluorescence quantum yields of NanoDPA, NanoNMP-tyr, and NanoDPA-NMP-tyr in PBS buffer (Table S1). The fluorescence quantum yield of NanoDPA-NMP-tyr has been calculated as 0.11 ± 0.009.
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RESULTS AND DISCUSSION DLS and TEM Characterizations for NanoDPA-NMPtyr. The amphiphilic polymer mPEG-DSPE was dissolved in water. When NMP-tyr and DPA are added, the hydrophobic portion of the hydrophilic micelle and the organic matter attract each other, and the organic portions are associated together to form self-assembled micelle NanoDPA-NMP-tyr. The shape and appearance of NanoDPA-NMP-tyr were measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM) images (Figure 1). Also the average hydrodynamic size of this nanoparticle was obtained and is about 40 ± 10 nm. Spectroscopic Properties of NanoDPA-NMP-tyr. Figure 2 shows the spectra of NanoDPA, NanoNMP-tyr, and NanoDPA-NMP-tyr in PBS buffer. The absorption of NanoDPA shows a typical anthracene absorption spectrum which has three sharp absorption peaks around 322, 377, and 400 nm. The absorption spectra of NanoNMP-tyr show one 3821
DOI: 10.1021/acsanm.9b00689 ACS Appl. Nano Mater. 2019, 2, 3819−3827
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Figure 2. (a) Absorption spectra; (b) emission spectra (λex = 377 nm) of NanoDPA (6.2 × 10−5 μmol), NanoNMP-tyr (5.4× 10−3 μmol), and NanoDPA-NMP-tyr (0.333 mL of aqueous solution of NanoDPA-NMP-tyr was diluted into PBS buffer to volume of 3 mL) in buffer PBS, pH 7.4, 37 °C.
Moreover, Figure S1a demonstrates that the optimum value of NMP-tyr/DPA for forming NanoDPA-NMP-tyr is 87:1. The loading efficiency of DPA and NMP-tyr in NanoDPA-NMP-tyr was calculated as 0.5366 (Figure S1b). Therefore, the ideal amounts of DPA and NMP-tyr in NanoDPA-NMP-tyr are 1.66 × 10−5 μmol and 1.45 × 10−3 μmol, respectively. Spectral Performance Analysis of NanoDPA-NMP-tyr for TYR Ratiometric Fluorescence Detection. Figure 3a, and Figure 3b show the absorption and emission spectra of incubation without and with TYR for 7.5 min incubation in a 37 °C water bath. NanoDPA-NMP-tyr shows a maximum absorption peak at ∼450 nm. The lowest-energy absorption band of NanoDPA-NMP-tyr will shift hypsochromically from ∼450 nm to ∼340 nm when TYR is added to the PBS solution. The relative contributions of the 340/450 nm signals change with varying TYR and show an isosbestic point at ∼400 nm. Figure S2a and Figure S2b also show a similar change of absorption spectra of organic probe NMP-tyr without and with TYR. As seen in Figure S2c, the fluorescence response of NMP-tyr to TYR was measured in oxygen and argon atmosphere, respectively. It was demonstrated that the fluorescence response only occurs in the presence of oxygen.
Figure 3. (a) UV absorption and (b) fluorescence intensity. NanoDPA-NMP-tyr varies with different concentrations of TYR in buffer PBS, pH 7.4, 37 °C. (c) A linear function of TYR concentration with I436 nm/I544 nm.
The probe NanoDPA-NMP-tyr shows a weak emission peak at ∼436 nm and strong fluorescence intensity at ∼544 nm. With the continuous addition of TYR, the emission peak at ∼544 nm decreases while that at ∼436 nm increases in the fluorescence spectrum of NanoDPA-NMP-tyr. As mentioned before, the emission of DPA and the well-absorbed spectrum of NMP-tyr satisfies the FRET mechanism for which the dye 3822
DOI: 10.1021/acsanm.9b00689 ACS Appl. Nano Mater. 2019, 2, 3819−3827
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After the addition of TYR, the fluorescence intensity ratio (I436 nm/I544 nm) of NanoDPA-NMP-tyr showed an obviously different value, but remained stable in the physiological pH range 6−7. To prove the reaction mechanism of NanoDPA-NMP-tyr to TYR, the products of the reaction of a small molecule probe NMP-tyr with TYR was obtained by separation and purification. The structure of the purified product was confirmed by LG−MS, HRMS (NMP-tyr (m/z 445.20; M +1, 446.21; C26H27N3O4 measured value, 446.21) and NMPtyro (m/z 459.18; M+1, 460.19; C26H27N3O5 measured value, 460.19)) and infrared spectra (Figures S13−S15), which is almost identical with that of the hypothetical NMP-tyro. A comparison of the infrared spectra of NMP-tyro and NMP-tyr shows that the IR spectrum of NMP-tyro has a new functional group to generate the CC (1510 cm−1) double bond and the CO (1646 cm−1). Also the UV absorption spectra of NMPtyro and NMP-tyr + TYR were found to be very similar (Figure S16). Specificity and Selectivity of NanoDPA-NMP-tyr. To investigate the specificity and selectivity of NanoDPA-NMPtyr for TYR, NanoDPA-NMP-tyr was used for fluorescence detection of a series of substances (Figure 4). Some
DPA is used as a donor for energy transfer and the fluorescent probe NMP-tyr acts as a receptor. Also the amphiphilic copolymer mPEG-DSPE allows for closer linkage within the chromophore. So the FRET system is formed. When TYR and oxygen are present at the same time, the monophenolic derivatives of the response group tyramine are converted to the corresponding phenylquinones NMP-tyro by TYR. Chromophore naphthalimide derivatives are used as precursors, while the hydroxyl groups act as an auxochrome to shift the overall absorption wavelength to a long wavelength. After being converted to NMP-tyro, the hydroxyl group in the product is converted into the structure of two ketones, the blue group, which makes the absorption peak move to in the short-wave direction. Therefore, the maximum absorption peak shifted from ∼450 nm of NMP-tyr to ∼350 nm of NMP-tyro. As a result, the fluorescence emission spectra of DPA no longer overlap with the absorption of NMP-tyr, and the energy transfer between them is cut off. Then the effect of FRET no longer exists and fluorescence changes from the green of NMPtyr to the blue of DPA, so the fluorescence ratio (I436 nm/ I544 nm) is shown. Under normal physiological conditions (37 °C, pH 7.4), the ratiometric fluorescence probe NanoDPA-NMP-tyr (I436 nm/ I544 nm) shows a good linear relationship to TYR, and the detection range of TYR is 0 to 20 U/mL (Figure 3c). The fluorescence spectral titration of NanoDPA-NMP-tyr was carried out under the premise of different concentrations of TYR. From the linear equation in Figure 3c, the detection limit of NanoDPA-NMP-tyr for TYR is as low as 0.057 U/mL by formula calculation. As shown in Figure S3, the timedependent fluorescence response of NanoDPA-NMP-tyr under the presence of different equivalents of TYR (0, 5, 15, and 20 U/mL) was investigated. The detection reaction of NanoDPA-NMP-tyr for TYR was found to be time-dependent, the reaction time of adding TYR 5 U/mL was 30 min and the time of adding 20 U/mL was 7.5 min. According to the table statistics (Table S2), we found that the reaction time of NanoDPA-NMP-tyr for TYR detection is shorter than that of fluorescent probes previously used for TYR detection. According to the literature41−43 reported, the kinetic properties were used to study the detection of TYR by the probe NanoDPA-NMP-tyr in 60 min (Figure S4). In the PBS buffer solution at 25 °C, the fluorescent probe NanoDPANMP-tyr (0.333 mL of aqueous solution of NanoDPA-NMPtyr was diluted into PBS buffer to volume of 3 mL) in the presence of TYR (10 U/mL) was used to calculate the pseudofirst-order rate in the kinetics (kobs = 0.16 ± 0.24). NanoDPANMP-tyr shows good photostability after being exposed to Xe lamps for 20 min (Figure S5a). By testing the absorption and emission spectra, it was found that the micellar nanoprobe NanoDPA-NMP-tyr remained stable after being left at room temperature for 1 week (Figure S5b and Figure S5c). Under normal physiological conditions, the in vivo imaging optical stability in RPMI-1640 and DMEM media of NanoDPANMP-tyr was studied. When NanoDPA-NMP-tyr was incubated in the medium for 1 h, its fluorescence intensity ratio (I436 nm/I544 nm) remained stable (Figure S6). To investigate that whether NanoDPA-NMP-tyr is pH dependent, we tested the response of NanoDPA-NMP-tyr and NanoDPANMP-tyr + TYR at different pH values. As shown in Figure S7, their fluorescence intensity ratios (I436 nm/I544 nm) were measured in the range of pH 4−9. It was found that NanoDPA-NMP-tyr remains stable in the range of PH 4−9.
Figure 4. Fluorescence responses (λex = 377 nm) of NanoDPA-NMPtyr toward 20 U/mL TYR and other biorelated analytes in 20 mM PBS (PH 7.4); each data were acquired 10 min after addition of the analytes: 0, free; 1, TYR; 2, CaCl2 (100 μM); 3, ClO− (100 μM); 4, Cys (100 μM); 5, peroxidase (100 mU·mL−1); 6, GSH (100 μM); 7, H2O2 (100 μM); 8, Hcy (100 μM); 9, KCl (100 μM); 10, Tyr (100 μM); 11, leucine aminopeptidase (100 mU·mL−1); 12, MgCl2 (100 μM); 13, chymotrypsin (100 mU·mL−1); 14, glucose oxidase (100 mU·mL−1); 15, glucose (100 μM); 16, vitamin B1 (100 μM); 17, vitamin C (100 μM); 18, esterase (100 mU·mL−1).
representative substances in cells such as, reactive oxygen species (ROS), amino acids, inorganic salts, vitamins, glucose, and other different enzymes were measured under the same conditions (PBS, pH = 7.4, 37 °C). The fluorescence intensity ratio of NanoDPA-NMP-tyr + TYR at 436 and 544 nm (I436 nm/I544 nm) was about 6 times that of the other substances. It shows that NanoDPA-NMP-tyr has good selectivity for TYR. Laser Confocal Imaging of TYR by NanoDPA-NMPtyr. The cytotoxicity of the fluorescent micellar nanoprobe NanoDPA-NMP-tyr was determined by the MTS method before performing cell laser confocal biological imaging.44,45 A series of NanoDPA-NMP-tyr aqueous solutions (0, 5.6, 11.2, 3823
DOI: 10.1021/acsanm.9b00689 ACS Appl. Nano Mater. 2019, 2, 3819−3827
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Figure 5. Fluorescence imaging of TYR B16 cells by NanoDPA-NMP-tyr: (a) Confocal imaging of cells after incubation for 2 h with NanoDPANMP-tyr. (b) B16 cells were treated with kojic acid (100 μM) for 2 h, then NanoDPA-NMP-tyr was added with 2 h incubation. (c) Fluorescence images of TYR added for another 2 h after incubation with NanoDPA-NMP-tyr for 30 min. (d) Ratiometric value calculation results, from blue to green channel in fluorescence images. Data represent mean standard error, n = 3.
22.2, 55.6, and 111.1 μL) were diluted into the cell culture medium containing 100 μL of culture medium (Figure S8), and the number of viable cells exceeded 95% after 24 h of culture, indicating that NanoDPA-NMP-tyr is less cytotoxic. According to the literature,46,47 the content of TYR in melanoma cells of mouse (B16) is higher than that in human cervical cancer cells (Hela). Therefore, B16 and Hela cells were used for laser confocal imaging. Figure 5 and Figure 6 show fluorescence-response confocal imaging of the endogenous and exogenous of the two cells to the probe. For B16 cells, when NanoDPA-NMP-tyr was added for 2 h, the fluorescence of the green channel in the cells was attenuated to almost none, while the fluorescence of the blue channel was enhanced (Figure 5a). In Figure 5b, the strong green fluorescence of NanoDPA-NMP-tyr was stable in the B16 cells pretreated with kojic acid (the agent that inhibits TYR production). In Figure 5c, the exogenous TYR (10 μM) was added, and the NanoDPA-NMP-tyr showed a similar response toward TYR; the fluorescence changed in endogenous TYR detection (Figure 5a). Moreover, to fully describe the
difference of fluorescence intensity between blue and green channel, the fluorescence ratios of the two channels were calculated. In Figure 5d, this value (I436 nm/I544 nm) shows the changes in blue and green fluorescence after the addition of NanoDPA-NMP-tyr to B16 cells pretreated with and without kojic acid (R = 1.22, 0.22, 1.50). In Figure 6a, when Hela cells were used for endogenous detection of TYR, the fluorescence of green channel was stronger than that of blue channel, which is opposite to that of the B16 cells (Figure 5a). However, in Figure 6c, when TYR was added to the Hela cells for incubation for a period of time, it showed a similar response toward TYR as the fluorescence change in B16 cells pretreated with NanoDPA-NMP-tyr only. In short, the results demonstrated that the change of intracellular fluorescence is caused by the response of NanoDPA-NMP-tyr to TYR. Thus, the selfassembling ratiometric fluorescent micelle nanoprobe NanoDPA-NMP-tyr can be used for imaging TYR in cells. 3824
DOI: 10.1021/acsanm.9b00689 ACS Appl. Nano Mater. 2019, 2, 3819−3827
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Figure 6. Fluorescence imaging of TYR Hela cells by NanoDPA-NMP-tyr. (a) Confocal imaging of cells after incubation with NanoDPA-NMP-tyr for 2 h. (b) Hela cells were treated with kojic acid (100 μM) for 2 h, and then NanoDPA-NMP-tyr was added for 2 h. (c) Fluorescence images of TYR were added for another 2 h after incubation with NanoDPA-NMP-tyr for 30 min. (d) Ratiometric value calculation results, from blue to green channel in fluorescence images.
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CONCLUSIONS
In summary, we developed a new self-assembled FRET-based ratiometric fluorescent micelle nanoprobe (NanoDPA-NMPtyr) to respond to TYR activity in B16 cells. The organic small molecule probe NMP-tyr used as an energy acceptor is composed of naphthalimide as a fluorophore and tyramine as a detecting group, and a complementary energy donor DPA. These compounds are encapsulated in the hydrophobic end of mPEG-DSPE. NanoDPA-NMP-tyr is highly selective and sensitive toward TYR with a rapid fluorescence response, and the detection limit of TYR is 0.057 U/mL. This is also a new detection method which is used to detect TYR. This fluorescent micelle nanoprobe is expected to be a powerful tool for clinical diagnosis and monitoring of TYR in cells.
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General information and methods; calculations; 1H and C NMR and MS spectra (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*Fax: +86-931-8912582. E-mail:
[email protected]. *E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Wenwu Qin: 0000-0002-9782-6647 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Ministry of Science and Technology of China (2014DFA31890). The authors would like to thank the Natural Science Foundation of China (no. 21771092). This work was also supported by the State Key Laboratory of Veterinary Etiological Biology, Lanzhou
ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.9b00689. 3825
DOI: 10.1021/acsanm.9b00689 ACS Appl. Nano Mater. 2019, 2, 3819−3827
Article
ACS Applied Nano Materials
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Veterinary Research Institute, Chinese Academy of Agricultural Sciences, China,
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