Bioorganic & Medicinal Chemistry 21 (2013) 6292–6302
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Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
A novel pH sensitive water soluble fluorescent nanomicellar sensor for potential biomedical applications Nikolai I. Georgiev a, Rayna Bryaskova b, Rumiana Tzoneva c, Iva Ugrinova d, Christophe Detrembleur e, Stoyan Miloshev b, Abdullah M. Asiri f,g, Abdullah H. Qusti f, Vladimir B. Bojinov a,f,⇑ a
Department of Organic Synthesis, University of Chemical Technology and Metallurgy, 8 Kliment Ohridsky Blvd., 1756 Sofia, Bulgaria Department of Polymer Engineering, University of Chemical Technology and Metallurgy, 8 Kliment Ohridsky Str., 1756 Sofia, Bulgaria Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Science, Acad. G. Bonchev Str., Bl.21, 1113 Sofia, Bulgaria d Institute of Molecular Biology, Bulgarian Academy of Science, Acad. G. Bonchev Str., Bl.21, 1113 Sofia, Bulgaria e Center for Education and Research on Macromolecules (CERM), Chemistry Department, University of Liege (ULg), Sart-Tilman B6a, 4000 Liège, Belgium f Chemistry Department, Faculty of Sciences, King Abdulaziz University, PO Box 80203, Jeddah 21589, Saudi Arabia g Center of Excellence for Advanced Materials Research (CEAMR), King Abdulaziz University, PO Box 80203, Jeddah 21589, Saudi Arabia b c
a r t i c l e
i n f o
Article history: Received 5 July 2013 Revised 27 August 2013 Accepted 29 August 2013 Available online 11 September 2013 Keywords: 1,8-Naphthalimide/rhodamine 6G bichromophore Fluorescent resonance energy transfer (FRET) pH sensor Well-defined PMMA–PMAA block copolymer Fluorescent micelles Living cells
a b s t r a c t Herein we report on the synthesis and sensor activity of a novel pH sensitive probe designed as highly water-soluble fluorescent micelles by grafting of 1,8-naphthalimide–rhodamine bichromophoric FRET system (RNI) to the PMMA block of a well-defined amphiphilic diblock copolymer—poly(methyl methacrylate)–b-poly(methacrylic acid) (PMMA48–b-PMAA27). The RNI-PMMA48–b-PMAA27 adduct is capable of self-assembling into micelles with a hydrophobic PMMA core, containing the anchored fluorescent probe, and a hydrophilic shell composed of PMAA block. Novel fluorescent micelles are able to serve as a highly sensitive pH probe in water and to internalize successfully HeLa and HEK cells. Furthermore, they showed cell specificity and significantly higher photostability than that of a pure organic dye label such as BODIPY. The valuable properties of the newly prepared fluorescent micelles indicate the high potential of the probe for future biological and biomedical applications. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Determination of pH is one of the most important analytical methods in the chemical laboratories and in the industry. The pH is a key parameter in clinical analysis, food production, biotechnological processes, waste water treatment procedures, environmental and life sciences.1–4 Intracellular pH plays a critical role in many cellular events, including cell growth and apoptosis, ion transport and homeostasis and enzymatic activity. Abnormal pH values are associated with inappropriate cell function, growth, and division and are observed in some common disease types such as cancer and Alzheimer’s. Hence the determination of pH has attracted increasing interests.5,6 Although the potentiometric pH sensor is well-established for routine pH measurements, it possesses some limitations as regarding miniaturized and disposable devices, work in a strong
⇑ Corresponding author. Tel.: +359 2 8163206. E-mail address:
[email protected] (V.B. Bojinov). 0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.08.064
electromagnetic field, high throughput screening, presence of organic matter or selectivity in high pH media.7,8 Indeed, in some applications, pH electrode is irreplaceable, but in a number of researches and technological tasks fluorescence probes could be an alternative to overcome the above mentioned limitations. Currently a very intensive research on pH fluorescent chemosensors is focusing on the synthesis of water-soluble fluorophores, that can be miniaturized, are disposable or calibration-free sensors, and are widening the dynamic pH range either by design of new dyes or by combining different sensors in one fluorescence probe.8–19 A large number of organic dyes have been developed for bioimaging purposes to investigate the main processes at the tissue, cellular and molecular level.20,21 Based on their chemical structure, they can be divided into several classes such as cyanine, porphyrin, squaraine, BODIPY, and xanthenes. The main disadvantages of commonly used fluorescent organic dyes are their non-specific to target tissue, instability and toxicity which limit further biomedical application because of potential carcinogenesis, low threshold of photobleaching, and lack of functional groups for further
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modification.22 Moreover, the relatively poor solubility in water significantly restricts their practical applications.23 To overcome these limitations, fluorescent polymeric assemblies of nanoscale dimensions have become a focus of intensive investigations during the past few decades. These systems offer several advantages over the conventional fluorescent dyes such as improved biocompatibility, water dispersibility, stimuli–responsiveness, facile integration into optical detection devices, and the ability of further functionalization.23,24 One approach to prepare such structure consists in the use of amphiphilic diblock copolymers which possess one hydrophilic and one hydrophobic block that can self-assemble in a solution forming different nano-sized morphologies. In many cases, spherical micelles consisting of a core formed by hydrophobic block and a shell arising from the hydrophilic one can be obtained in an aqueous solution.25 On the basis of this consideration different block copolymer micelles have been used to encapsulate fluorophores within micellar core. Small organic dyes can be physically encapsulated into the hydrophobic core of the micelles or chemically coupled to copolymers.23,26–36 Fluorescence resonance energy transfer (FRET) is a distancedependent interaction between the electronic excited states of two different dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon.37,38 The pH-dependent fluorescence properties of the FRET based multifluorophoric systems are very promising because of the long communication wavelengths exhibited by these molecules. Long-wavelength excitation reduces problems of autofluorescence and scattering during fluorescent sensing within many biological and industrial matrices.39 By introduction of fluorescence resonance energy transfer (FRET) processes into polymeric assemblies and nanoparticles (NPs) tracking can be conducted in a ratiometric manner and effectively to exclude background interference. It would be possible to fabricate a FRET based ratiometric probe, in which the ratio of the fluorescent intensities at two different wavelengths provides a built-in correction for environmental effects, and stability under illumination.40 This method allows precise and quantitative analysis and imaging even in complicated systems. Moreover, sensing to the microenvironment can be obtained if the FRET efficiency is responsive to the variation of solution conditions such as pH value, temperature, metal ions, glucose, and tissue-specific enzymes.30 Stimuli-sensitive fluorescent dyes were widely used to modulate FRET processes within polymeric assemblies and NPs, as their spectroscopic properties can be easily switched between two states upon the action of external stimuli as UV irradiation.23,41,42 Multicolor fluorescent polymeric assemblies and NPs which exhibit metal ion-switchable FRET processes by utilizing metal ion-reactive fluorophores as potential FRET acceptors have been developed as well and could find potential practical application for biosensing processes.43 Recently, three-state switching of multicolor thermoresponsive polymer micelles has been demonstrated.44 However, among the fluorescent pH probes reported, most are practically designed for a near-neutral pH range (pH 6.8–7.4). Only a few probes were reported for monitoring pH changes inside lysosomes (pH 4.5–5.5).45–50 Thus, it was of interest to develop a new FRET pH probe based on a 1,8-naphthalimide–rhodamine bichromophoric system and to incorporate it into polymer micelles. The present investigation reports on the preparation of a novel pH sensitive water soluble fluorescence probe obtained by covalent attachment of a Rhodamine 6G/1,8-naphthalimide wavelength-shifting bichromophoric system to amphiphilic copolymer, based on poly(methyl methacrylate)-b-poly(methacrylic acid) (PMMA-b-PMAA), followed by its self-assembly in an aqueous solution (Scheme 1). The possibility to apply the novel fluorescence micelles for intracellular imaging was the other goal of the present investigation.
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2. Materials and methods 2.1. Materials Methyl methacrylate (MMA) (>99%, Aldrich) and tert-butyl methacrylate (t-BMA) (98%, Aldrich) were dried over calcium hydride, degassed by several freeze-thawing cycles before being distilled under reduced pressure. Before use, they were then dried over triethyl aluminium and distilled under vacuum. 1,1-Diphenylethylene (97%, Aldrich) was dried with sec-butyllithium (1.4 M in cyclohexane, Aldrich) and distilled under vacuum. Lithium chloride (anhydrous, >99%, Aldrich) was dried at 200 °C under vacuum. Tetrahydrofuran (anhydrous, >99%, Aldrich) was dried by refluxing over sodium/benzophenone and distilled. Then it was dried over polystyryllithium and distilled under vacuum before use. MMA and t-BMA were dried by distillation over CaH2 and then, over AlEt3. Intermediate compounds 4 (Scheme 3) was synthesized as previously described.51 Rhodamine 6G, hydrazine monohydrate (>98%), ethylenediamine, 4-nitro-1,8-naphthalic anhydride (Aldrich, Merck, Fluka) and DMF (Aldrich, spectroscopic grade) were used as purchased without further purification. NaOH and HCl were supplied by Merck. 2.2. Methods and characterization FT-IR spectra were recorded on a Varian Scimitar 1000 spectrometer. The 1H NMR spectra were recorded on a Bruker DRX250 spectrometer; operating at 250.13 MHz. TLC was performed on silica gel, Fluka F60 254, 20 20, 0.2 mm. Absorption spectra were recorded on Hewlett Packard 8452A spectrophotometer in water. The fluorescent spectra were recorded on a Scinco FS-2 fluorescence spectrophotometer. The excitation source was a 150 W Xenon lamp. Excitation and emission slits width were 5 nm. Fluorescence measurement was carried out in right angle sample geometry. A 1 1 cm quartz cuvette was used for the spectroscopic analysis. Relative fluorescence quantum yield (UF) was determined using Coumarin 6 (Aldrich, UF = 0.78 in ethanol)52 or Rhodamine 6G (UF = 0.95 in ethanol)53 as standards. The spectral data were collected using FluoroMaster Plus 1.3 and further processed by OrginPro 6.1 software. A pH meter Metrohm 704 coupled with combined pH electrode was used for pH measurements. The commercial standard buffers for pH 2, 7 and 10 (Aldrich) were used for calibration. The relative molecular weights (Mn) and polydispersity (Mw/Mn) of the polymer were determined by size-exclusion chromatography (SEC) in tetrahydrofuran (THF) (flow rate: 1 mL min1) at 40 °C with a Waters 600 liquid chromatograph equipped with a 410 refractive index detector and styragel HR columns (four columns HP PL gel 5 lm, 105, 104, 103, 102 Å). Poly(methyl methacrylate) (PMMA) standards were used for calibration. High-performance liquid chromatography (HPLC) analysis was performed on Agilent 1100 Series with UV–vis detector. The column used was Kinetex (Phenomenex) with particle size 2.6 lm and dimensions 100 60 mm. Transmission electron microscopy (TEM) observations were carried out with a JEOL instrument operating at a voltage 100 kV. Samples were prepared by deposition of a droplet of the aqueous micellar solution onto a carbon coated copper TEM grid, which was allowed to evaporate for 2 h. Dynamic light scattering (DLS) measurements were performed on a Brookhaven Instruments Corp. equipped with a He–Ne laser. The temperature was set to 22 °C and the angle of measurements was 90°. The measurements of the hydrodynamic diameter and particle size distribution as well as all other experiments were performed at room temperature after filtration of the aqueous micellar solution through 0.45 lm filter.
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Scheme 1.
Scheme 3.
2.3. Synthesis of a bichromophoric compound based on Rhodamine 6G and 1,8-naphthalimide (RNI)
tal analysis: Calcd for C26H28N4O2 (MW 428.53) C 72.87, H 6.59, N 13.07; Found C 73.23, H 6.38, N 12.82.
2.3.1. Synthesis of Rhodamine 6G hydrazide (2) To a solution of Rhodamine 6G 1 (2.4 g, 5 mmol) in 40 mL of absolute ethyl alcohol, 1.46 mL of hydrazine monohydrate (30 mmol, d = 1.032) was added drop wise at room temperature over a period of 30 min. The resulting solution was stirred at reflux for 5 h. After cooling to room temperature the solid precipitated was filtered off, washed with water and dried to give 1.91 g (89%) of 2 as pale pink crystals (Rf = 0.57 in a solvent system: toluene/ethylacetate/ethanol = 10:2.5:1). IR (KBr) cm1: 3280, 3204 ((mNH and mNH2); 2920, 2810 (mCH); 1662 (mC@O); 1600, 1568 and 1496 (mAr@CH). 1H NMR (CDCl3-d, 250.13 MHz) ppm: 7.96 (m, 1H, 9-Ph H-3); 7.45 (m, 2H, 9-Ph H-4 and 9-Ph H-5); 7.06 (m, 1H, 9-Ph H-6); 6.39 (s, 2H, rhodamine H4 and H-5); 6.26 (s, 2H, rhodamine H-1 and H-8); 3.58 (s, 2H, NH2); 3.52 (br s, 2H, 2 NH); 3.21(q, 4H, J = 3.5 Hz, 2 CH2CH3); 1.92 (s, 6H, 2 Ar-CH3); 1.32 (t, 6H, J = 7.1 Hz, 2 CH2CH3). Elemen-
2.3.2. Synthesis of intermediate (4) A suspension of 4-nitro-1,8-naphthalic anhydride 3 (0.97 g, 4 mmol) and Rhodamine 6G hydrazide 2 (1.71 g, 4 mmol) in 25 mL of glacial acetic acid was stirred at 110 °C for 5 h. After cooling to room temperature, the resulting solution was poured into 40 mL of water and alkalized to pH 8 by addition of 1% sodium hydroxide. The solid product that precipitated was filtered off, washed with water and dried to give 2.30 g (88%) of pure intermediate 4 as a slightly purple solid (Rf = 0.83 in a solvent system: toluene/ethylacetate/ethanol = 10:2.5:1). IR (KBr) cm1: 3312 (mNH); 2918 and 2822 (mCH); 1720 (masN– C@O); 1682 (maN–C@O); 1638 (mC@O); 1515 (masNO2); 1330 (maNO2). 1H NMR (CDCl3-d, 250.13 MHz) ppm: 8.75 (dd, 1H, J = 8.8 Hz, J = 1.3 Hz, naphthalimide H-7); 8.39 (dd, 1H, J = 7.5 Hz, J = 1.2 Hz, naphthalimide H-5); 8.35 (d, 1H, J = 8.0 Hz, naphthalimide H-3); 8.25 (d, 1H, J = 8.0 Hz, naphthalimide H-2); 8.10 (m,
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1H, 9-Ph H-3); 7.84 (dd, 1H, J = 8.8 Hz, J = 7.5 Hz, naphthalimide H6); 7.66 (m, 2H, 9-Ph H-4 and H-5); 7.30 (m, 1H, 9-Ph H-6); 6.55 (s, 2H, rhodamine H-4 and H-5); 6.07 (s, 1H, rhodamine H-1); 6.06 (s, 1H, rhodamine H-8); 3.53 (br s, 2H, 2 NH); 3.08 (q, 4H, J = 7.0 Hz, 2 CH2CH3); 2.01 (s, 6H, 2 Ar-CH3); 1.25 (t, 6H, J = 7.0 Hz, 2 CH2CH3). Elemental analysis: Calcd for C38H31N5O6 (MW 653.68) C 69.82, H 4.78, N 10.71; Found C 70.19, H 4.91, N 10.49.
hydrolysis of the second block with the formation of the corresponding diblock copolymer PMMA48–b-PMAA27: 1H NMR (DMSO-d6, 250.13 MHz) ppm: 12.25 (s, OH); 3.57 (s, OCH3); 2.06–1.64 (m, CH2); 1.12–0.76 (m, CO–C–CH3).
2.3.3. Synthesis of bichromophoric system (5) To a solution of intermediate 4 (1.96 g, 3 mmol) in 30 mL of DMF, 6.2 mL of ethylenediamine was added portion wise at room temperature. After 48 h (TLC control in a solvent system toluene/ ethylacetate/ethanol = 10:2.5:1), the resulting solution was poured into 30 mL of water. The crude precipitated product was filtered off, washed with distilled water and dried at room temperature. Silica gel chromatography (methanol/25% ammonium hydroxide = 2.5:1, v/v) afforded 1.20 g (60%) of the bichromophoric compound 5 as a yellow solid (Rf = 0.45 in a solvent system: toluene/ethylacetate/ethanol = 10:2.5:1). FT-IR (KBr) cm1: 3378 (mNH and mNH2); 2966, 2927 and 2866 (mCH2 and mCH3); 1726 (masN–C@O); 1690 (msN–C@O); 1736 (mN– C@O). 1H NMR (CDCl3-d, 250.13 MHz) ppm: 8.18 (d, 1H, J = 8.4 Hz, naphthalimide H-7); 8.11 (m, 1H, 9-Ph H-3); 7.73 (d, 1H, J = 8.4 Hz, naphthalimide H-2); 7.65 (m, 2H, 9-Ph H-4, 9-Ph H-5); 7.56 (d, 1H, J = 7.1 Hz, naphthalimide H-5); 7.29 (m, 1H, 9Ph H-6); 7.00 (dd, 1H, J = 8.1, Hz, J = 7.5 Hz, naphthalimide H-6); 6.51 (m, 3H, rhodamine H-4, rhodamine H-5, naphthalimide 4NH); 6.21 (d, 1H, J = 8.8 Hz, naphthalimide H-3); 6.05 (s, 1H, rhodamine H-1); 6.00 (s, 1H, rhodamine H-8); 3.41 (br s, 2H, 2 NH); 3.22 (m, 2H, NHCH2CH2NH2); 3.03 (m, 6H, NHCH2CH2NH2, rhodamine 2 CH2CH3); 1.98 (s, 3H, rhodamine CH3); 1.97 (s, 3H, rhodamine CH3); 1.20 (m, 8H, NH2, rhodamine 2 CH2CH3). Elemental analysis: Calcd for C40H38N6O4 (MW 666.77) C 72.05, H 5.74, N 12.60; Found C 71.66, H 5.89, N 12.42.
To a solution of 20 mg PMMA48–b-PMAA27 copolymer (2.8 106 mol) in 10 mL of boiling methanol, a solution of 5 mg of bichromophoric compound RNI (7.5 106 mol) in 5 mL of methanol was added drop-wise under stirring over a period of 2 h. The resulting solution was refluxed for 5 h. The process was controlled by thin-layer chromatography (solvent system toluene/ethylacetate/ethanol = 10:2.5:1) until the complete exhaustion of the starting bichromophore RNI. The crude product was obtained after vacuum evaporation of the solvent used. Then, the product was purified to remove any RNI traces by treatment with acid aqueous solution (pH 2) and analyzed by SEC in THF (Mn = 8900 g/mol; Mw/Mn = 1.09). Micellization of amphiphilic RNI-PMMA48–b-PMAA27 block copolymers in Milli-Q water was performed by dissolution of the copolymer in methanol (20 mg of RNI-PMMA-b-PMAA copolymer in 5 mL of methanol), followed by slow addition of the dissolved copolymer into deionizer water at a controlled rate (8 mL/h). The solution was stirred at room temperature for an additional 24 h before been dialyzed against Milli-Q water for 48 h to remove the methanol. A micellar solution (pH 5.8) consisting of PMMA cores and PMAA shells was thus formed. The carboxylic acid groups of the PMAA block are at least partially deprotonated thus providing polymer shell with a negative charge which stabilized the micelles via electrostatic repulsion.
2.4. Synthesis of poly(methyl methacrylate)-b-poly(methacrylic acid)
One tumor cell line HeLa (ATCC, Manassas, VA, USA) and a nontumorigenic cell line HEK (ATCC, Manassas, VA, USA) were cultivated in DMEM. Cell suspension with a density of 1.5 105 cells/ mL was plated directly on the bottom of 24 well cultivated plates and on glass slides (d = 12 mm, Superior-Marienfeld, Germany), placed on 24 well plates. The cells were incubated during 24 h at 37°C and 5% CO2.
In a 500 mL glass flask is added LiCl (0.992 g, 23.4 mmol) and the reaction flask is gently flamed under vacuum for a few minutes. After cooling, dry argon is injected, followed by 250 mL of THF (dried over calcium hydride and then, distilled over polystyryl lithium). A dried solution of diphenylethylene in THF (13.8 mL; 0.51 M, 7 103 mol) is then added and the reaction medium is cooled to 78 °C. Residual water is then eliminated by slowly adding a solution of sec-butyllithium (0.16 M) until a light pink coloration appears. Then, 1.67 mL of sec-butyllithium (1.4 M, 2.34 mmol) are added (the solution becomes red), followed by 10 mL (93.5 mmol) of dry MMA (dried by distillation over CaH2 and then over AlEt3). After 1 h at 78 °C, a sample is picked out and is precipitated in methanol for analysis by SEC in THF (Mn = 4750 g/mol; Mw/Mn = 1.04). Then dry tert-butyl methacrylate (10.7 mL, 65.8 mmol) is added to the remaining solution. After 1 h of reaction at 78 °C, degassed methanol (5 mL) is added to the solution to deactivate the chains. The polymer is then precipitated two times in a MeOH/H2O (90:10) mixture, dried under vacuum at 80 °C overnight and analyzed by SEC in THF (Mn = 7600 g/mol; Mw/ Mn = 1.04). The composition of the copolymer is determined to P(MMA)48-b-P(t-BMA)27 by 1H NMR spectroscopy: 1H NMR (CDCl3d, 250.13 MHz) ppm: 3.60 (s, OCH3); 1.99–1.74 (m, CH2); 1.44 (s, C(CH3)3); 1.16–0.78 (m, CO–C–CH3). Subsequently, the tert-butyl groups of the P(t-BMA) block were hydrolyzed with concentrated hydrochloric acid in dioxane at 85 °C for 8 h to form a PMAA blocks according to a reported procedure54 and analyzed by SEC in THF (Mn = 7200 g/mol; Mw/Mn = 1.05). 1H NMR analysis evidenced the complete
2.5. Synthesis of RNI labeled PMMA-b-PMAA copolymer and preparation of RNI-PMMA-b-PMAA based micelles
2.6. Cell lines and cultivation conditions
2.7. Cell treatment with RNI-PMMA48–b-PMAA27 micelles and fluorescent imaging After 24 h incubation the cells were treated with 15, 30, 63 and 125 lg/mL solution of RNI-PMMA48–b-PMAA27 micelles and were incubated additionally for 2 and 24 h. The fluorescent images were photographed using an inverted fluorescent microscope (Leica DMI3000 B, Leica Microsystems GmbH, Germany) with the objective lens HI PLAN 20 and 40/0.50. For bleaching experiments the cells were fixed in 3% solution of paraformaldehyde in PBS (phosphate buffer saline, pH 7.4) in order to be photographed with objective lens HCX PL FLUOTAR 63/1.25 oil. 2.8. MTT test for cell survival To analyze the cytotoxic effect of RNI-PMMA48–b-PMAA27 micelles on the breast cancer and non-cancer cells, the MTT test (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Invitrogen, USA) was performed as described by Mosmann,55 with some modifications. Briefly, the adherent cells were treated as described above and incubated additionally for 2 and 24 h. After the incubation period the cell medium was changed with fresh medium (200 lL/well). Then, 50 lL of MTT solution (5 mg/mL in
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PBS) was added. Plates were further incubated for 4 h at 37 °C, and the formazan crystals formed were dissolved by addition of 250 lL solvent (5% formic acid in 2-propanol) per well and mixing. The absorbance was recorded at 570 nm with the 96-well plate reader Tecan Infinite F200 PRO (Tecan Austria GmbH, Salzburg). For each concentration six wells were used. Complete medium (200 lL) and 5% formic acid in 2-propanol (250 lL) were used as a blank solution. 3. Results and discussion 3.1. Design and synthesis of novel RNI-PMMA48–b-PMAA27 fluorescence copolymers The target copolymer was designed as a bichromophoric probe, based on modulating FRET process, comprising a 1,8-naphthalimide donor and a rhodamine acceptor. The 1,8-naphthalimide chromophore was chosen as a fluorescence donor in a view of its chemical stability and high fluorescent efficiency.56 Rhodaminebased dyes are excellent ‘off–on’ fluorescence probes as a result of the spirolactam (nonfluorescent) to ring-open amide (fluorescent) equilibrium of rhodamine.50,57–59 A requirement for efficient energy transfer is the spectral overlap between the emission of the donor dye and the absorbance of the acceptor chromophore. The light absorption properties of the 1,8-naphthalimide derivatives are basically related to the polarization of their chromophoric system. The light absorption in this molecule generates a charge transfer interaction between the substituents at C-4 position and the imide carbonyl groups. In general, the derivatives with alkoxy groups are colorless and have a blue emission, while the amino substituted 1,8-naphthalimides are yellow colored and emit green fluorescence.60–65 Rhodamines are red-orange emitting fluorophores with maximal absorption at the 4-alkylamino-1,8naphthalimides emission region. Consistent with this requirement, 4-alkylamino-1,8-naphthalimides and rhodamine dyes are suitable fluorescence donor–acceptor pair for dyad systems.17,40,50,51 In order to obtain water soluble fluorescence probe, amphiphilic PMMA48–b-PMAA27 block copolymer was chosen. This copolymer self-assembles in aqueous media into micelles consisting of a hydrophobic core arising from the PMMA block and a shell consisting of hydrophilic PMAA block. The covalent attachment of fluorescence RNI probe to the PMMA block of PMMA48–b-PMAA27 copolymer will ensure its incorporation within the hydrophobic core of the PMMA48–b-PMAA27 based micelles and will avoid its leaching out of the micellar system. Due to the pH sensitive character of rhodamine acceptor dye, we expect the fluorescence signal of the novel water soluble fluorescence probe to be a function of pH. In an alkaline solution, Rhodamine 6G derivatives are in the colorless spirolactam closed form and the energy transfer from the donor 1,8-naphthalime to the Rhodamine 6G acceptor is not feasible (Scheme 2). Under these conditions, the target probe will have the typical yellow-green fluorescence for 4-amino-1,8-naphthalimides. In acidic conditions, Rhodamine 6G spirolactam ring is opened, the energy of donor 1,8naphthalimide in the copolymeric probe can therefore be transferred to the acceptor rhodamine and the system will emit a redorange fluorescence signal (Scheme 2). The preparation of the water soluble fluorescence micelles was performed in three steps. First, wavelength-shifting chromophore 5 was synthesized as outlined in Scheme 3. For this purpose, rhodamine hydrazide 2 was obtained by reaction of Rhodamine 6G 1 with hydrazine monohydrate under reflux in absolute ethyl alcohol for 5 h and subsequently converted into the intermediate adduct 4 by condensation with 4-nitro-1,8-naphthalic anhydride 3 in glacial acetic acid at 110 °C for 5 h.
The fluorescent 1,8-naphthalimide moiety in the target bichromophore 5 was prepared by nucleophilically substituting the nitro group in the intermediate 4 with the commercially available ethylenediamine in DMF solution at room temperature for 48 h. The structure and purity of the desired product were confirmed by conventional techniques—TLC (Rf values), elemental analysis data, UV–vis, fluorescence, FT-IR and 1H NMR spectroscopy (see Experimental section). The next step was the synthesis of PMMA48–b-PMAA27 block copolymer. First, a poly(methyl methacrylate)-b-poly(tert-butyl methacrylate) block copolymer was synthesized by sequential living anionic polymerization, followed by hydrolysis of the second block by HCl in dioxane according to a reported procedure.54 The molecular weight and structure were determined by SEC and 1H NMR (see Experimental section). Resonance absorptions in the 1H NMR spectrum of P(MMA)48-b-P(t-BMA)27 at 3.60 and 1.44 are in ratio of 1 to approximately 1.7, which corresponds to the ratio between the two blocks 48 to 27. Further, in the spectrum of the PMMA48–b-PMAA27 block copolymer, the resonance absorption at 1.44 ppm for t-butyl group is absent, confirming the successful hydrolysis of the second block and the final composition of the copolymer. Finally, the PMMA48–b-PMAA27 block copolymer was covalently labeled by amide bonds with the synthesized fluorescent wavelength-shifting chromophore RNI after interaction of the RNI terminal amino groups (–NH2) and the ester groups from PMMA block of PMMA48–b-PMAA27 (Scheme 4). The grafting of organic RNI fluorophore to the PMMA block of PMMA48–b-PMAA27 copolymer was proven by FT-IR spectroscopy (Fig. 1). As can be seen, the FT-IR spectrum of the RNI-PMMA48–b-PMAA27 fully overlaps with that of the PMMA48– b-PMAA27 block copolymer, except the range between 1660 and 1500 cm1, which contains the characteristic adsorption bands of the RNI bichromophore. The new band at 1648 cm1 (carbonyl stretching) demonstrates formation of an aliphatic amide bond between RNI and the PMMA block of a PMMA48–b-PMAA27 copolymer. The attachment of the RNI fluorophore to the polymer was proved by SEC as well. In the UV-detected SEC chromatograms only a single peak was observed for RNI-PMMA48–b-PMAA27 labeled copolymer, PMMA48–b-PMAA27 copolymer and RNI fluorophore, its retention time being different from one another. Furthermore, the elution peak of the covalently labeled PMMA48– b-PMAA27 copolymer with RNI fluorophore clearly shifts to higher molecular weight in respect to the unlabeled copolymer, thus indicating the successful incorporation of the fluorophore to the polymer chain. The covalent attached RNI fluorophore to the PMMA48–bPMAA27 copolymer was further analyzed by HPLC with UV detection. The major HPLC peak (retention time 1.96 min, fraction 80%) gave an UV–vis spectrum possessing several absorption peaks as those with longest-wavelength absorption maxima at 436 and 530 nm belong to the RNI fluorophore (Fig. 2). This is an evidence for the covalent attachment of the starting bichromophoric system RNI to the PMMA48–b-PMAA27 copolymer. Further, the self-assembly of RNI labeled PMMA48–b-PMAA27 amphiphilic block copolymer in water (pH 5.8) afforded micelles possessing PMMA core with embedded RNI fluorophores and shell consisting of PMAA block. The transformation of organic to aqueous solution is illustrated in Figure 3, where a picture of the organic fluorophore in water before encapsulation (left vial) and after incorporation within the micelles (right vial) is shown. The wavelength-shifting RNI bichromophore is insoluble in an aqueous media. However, after its incorporation within the core of the PMMA48–b-PMAA27 micelles, a stable solution is obtained
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Scheme 2.
Scheme 4.
Figure 1. FT-IR spectra of RNI bichromophore, PMMA48–b-PMAA27 block copolymer and RNI labeled PMMA48–b-PMAA27.
without any aggregation. The successful formation of PMMA48–bPMAA27 micelles with embedded fluorophore was proven by DLS and TEM analyses. Micelles with hydrodynamic diameter (Dh) of 73.9 nm and PDI = 0.26 were obtained from fluorophore labeled PMMA48–b-PMAA27 copolymer (Fig. 4A). In comparison, the DLS measurement of pure PMMA48–b-PMAA27 micelles without incorporated fluorophore possesses hydrodynamic diameter (Dh) of 65.9 with a broader polydispersity index (PDI) of 0.35. As expected, the hydrodynamic diameter of the micelles with embedded fluorophores is bigger than the free of fluorophores micelles. TEM picture also reveals the formation of spherical fluorophore labeled PMMA48–b-PMAA27 micelles with an average diameter of 32 nm (Fig. 4B). The different diameter observed by DLS and TEM of the micelles is a result of different degree of stretching of PMAA shell of the micelles. In solution, the PMAA chains are extended to some degree in comparison to their dried state (TEM observation) where the PMAA chains collapsed on the hydrophobic core.
3.2. Photophysical investigation of RNI-PMMA-b-PMAA micelles in aqueous solution Photophysical properties of the novel RNI-PMMA48–b-PMAA27 fluorescence probe were determined in an aqueous solution. The photophysical characteristics of the 1,8-naphthalimide donor dye were recorded at ca. pH 8 and kex = 430 nm, while the rhodamine behavior was observed at ca. pH 2 and kex = 510 nm. The data listed in Table 1 for the donor and acceptor fluorophores are common for the rhodamine and 1,8-naphthalimide derivatives66,67 and do not undergo remarkable changes due to the covalent bonding of RNI to the PMMA48–b-PMAA27 copolymer. The RNI-PMMA48–b-PMAA27 based micelles were then tested as fluorescence sensors for determining pH changes over a wide pH scale. The absorption spectrum of the RNI-PMMA48–b-PMAA27 based probe over a wide pH scale shows typical for 1,8-naphthalimide and rhodamine moieties absorption bands. The 1,8-naphthalimide absorption does not show significant pH-dependent
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Figure 2. UV–vis spectrum of RNI labeled PMMA48–b-PMAA27 copolymer from HPLC analysis.
Figure 4. (A) DLS measurements of RNI labeled PMMA48–b-PMAA27 micelles; (B) TEM image of micelles formed by RNI labeled PMMA48–b-PMAA27 copolymer.
Figure 3. Picture of the RNI fluorophore (left) and RNI labeled PMMA48–b-PMAA27 micelles (right) in aqueous solution.
Table 1 Photophysical characteristics of RNI-PMMA48–b-PMAA27 copolymer in aqueous solution kA (nm) Naphthalimide donor Rhodamine acceptor
changes, since the 1,8-naphthalimide fluorophore does not affect its internal charge transfer (ICT) excited states. Negligible absorption enhancement and slight blue shift were observed only in strong acidic media due to the protonation of the aromatic amine substituent (Fig. 5A). In the pH window 7–12 absorption of the rhodamine unit practically absent due to the spirolactam closed (nonfluorescent) form. However, when the pH decreased from 7 to 2, as a result of spirolactam opening reaction, a novel absorption band corresponding to the acceptor rhodamine in the RNI bichromophoric system is appearing at 532 nm (Fig. 5A, inset of Fig. 5A). From the absorption changes at 532 nm, the titration curve of novel copolymeric pHprobe was obtained. The analysis of the titration plot according to Eq. 139 gives the pKa value of 4.2, which is lower as compared to the other rhodamine hydrazides.68
log½ðAmax AÞ=ðA Amin Þ ¼ pH pK a
ð1Þ
The reason for this observation is a result of (i) the directly attached spirolactam cycle to imide group which possesses strong electron accepting ability, thus resulting in more acid spirolactam properties of rhodamine moiety and (ii) the protective effect of polymer PMAA shell in the micellar architecture.
a b
a
444 532
kF (nm) a
537 550b
mA mF (cm1) a
3871 615b
UF 0.10a 0.39b
Photophysical data recorded at ca. pH 8 and kex = 430 nm. Photophysical data recorded at ca. pH 2 and kex = 510 nm.
In alkaline and neutral media, after excitation at 430 nm (within the spectral region of maximal absorption of the donor 1,8-naphthalimide), the novel fluorescence probe RNI-PMMA48– b-PMAA27 shows emission band in range 480–700 nm with pronounced maximum at 537 nm (Fig. 5B). Under these conditions, the focal acceptor dye is in a spirolactam form, energy transfer from the donor fluorophore to the rhodamine is not possible and the system shows a yellow-green emission that is typical for the 4-amino-1,8-naphthalimide derivatives. Upon acidification from pH 5 to 2, the spirolactam form became opened, which allows the energy transfer to the acceptor moiety. This results in remarkable fluorescence intensity enhancement (FE) in the rhodamine emission region at 550 nm. The fluorescence intensity enhancement (FE = I/I0), calculated using minimal (I0) and maximal (I) fluorescence intensity, recorded in the examined pH interval was more than 20 times (FE = 27). The analysis of the fluorescence changes at 550 nm (Fig. 5B-inset) according to Eq. 2 gives
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cytotoxicity test performed by MTT assay showed function of HeLa and HEK cells cultivated without fluorescent dye and in the presence of different concentrations of RNI-PMMA48–b-PMAA27 micelles. In general HeLa are more stable to the toxic influence of RNI-PMMA48–b-PMAA27 micelles in comparison to HEK cells (Fig. 6). Concentrations of 15 and 30 lg/mL of the fluorescent micelles did not cause any significant reduction in cell viability even after 24 h of incubation. Incubation of HeLa with 63 lg/mL of RNIPMMA48–b-PMAA27 micelles for 24 h decreased the viability compared to the viability of HeLa incubated with the same concentration of fluorescent micelles for 2 h. Concentration of 125 lg/mL of fluorescent micelles in incubation media already caused decreased cell viability even after 2 h of incubation with 30% (1/3 fold) (Fig. 6A). HEK were more sensitive to the action of the fluorescent dye (Fig. 6B). Concentration of 15 lg/mL did not decrease significantly the cell viability after 2 h of incubation. However, with the increasing of RNI-PMMA48–b-PMAA27 micelles concentration, the cell viability gradually decreased and reached 50% when 125 lg/mL of the fluorescent probe are used. Longer incubation (24 h) of the fluorescent micelles with the HEK cells caused significant decrease in cell viability (50%) even at the lowest dye concentration of 15 lg/mL (Fig. 6B).
Figure 5. Absorption (A) and fluorescent (B, kex = 430 nm) changes of RNIPMMA48–b-PMAA27 based micelles in aqueous solutions at different pHs.
the pKa value of 4.1 which is similar to the pKa value calculated from absorption changes.
log½ðIFmax IF Þ=ðIF IFmin Þ ¼ pH pK a
ð2Þ
The results obtained suggested that the spirolactam-opening reaction of the rhodamine acceptor is responsible for the pH sensing properties of the novel probe. This switching process was found to be reversible and no aggregation behavior during the photophysical investigation was observed. The accomplished experiments clearly demonstrate that the synthesized fluorescence micelles are excellent chemosensing materials for determination of pH changes in aqueous solutions in the absence of organic solvent. Furthermore, the pKa value of 4.1–4.2 matches the typical pH values of acidic organelles (pH 4– 6),5 indicating that the probe may be suitable for monitoring pH variations in the acidic environments in bio-samples. 3.3. Cytotoxicity of RNI-PMMA48–b-PMAA27 micelles The cytotoxicity and cellular uptake are the two basic important characteristics for successfully cell labeling or for clinical use. The
Figure 6. Cell cytotoxicity of HeLa (A) and HEK (B) cells. Data are means ± SD of four replicates. The statistics was performed by one-way analysis ANOVA using the Tukey–Kramer post test (⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001).
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3.4. Fluorescence imaging in living cell RNI-PMMA48–b-PMAA27 micelles were applied to HeLa and HEK cells to investigate their imaging ability. The incubation of HeLa cells with concentrations of 15–125 lg/mL of the dye for 2 h (Fig. 7, upper panel) and 24 h (Fig. 7, lower panel) gave intracellular fluorescence as monitored by fluorescence microscopy. The results demonstrated that the fluorescent micelles were cell-permeable and the fluorescence intensity increased in a concentration and time dependent manner. As can be seen from Figure 8, the fluorescence inside the cells is mainly concentrated near the nucleus. In HEK cells the novel RNI-PMMA48–b-PMAA27 micelles was also internalized around the cell nuclei (Fig. 9). In this cell type a high fluorescent intensity of the polymer micelles was visible even at the earlier time of incubation (2 h) and at the lower dye concentration (15–30 lg/mL) (Fig. 9). This result allows us to suppose that the cell membrane of this cell type is more permeable to the dye and its level of saturation in the HEK cell is reached earlier than in HeLa cells. This higher permeability can be responsible for the increased cytotoxicity observed for the RNI-PMMA48–b-PMAA27 micelles to HEK cells (Fig. 6B). 3.5. Photostability of RNI-PMMA48–b-PMAA27 micelles—labeling method To test the photostability of the RNI-PMMA48–b-PMAA27 micelles—labeling method,69 HeLa cells were stained with RNIPMMA48–b-PMAA27 micelles in concentration of 30 lg/mL (nontoxic concentration) and BODIPY558/568 phalloidin (labeling actin filaments), respectively. The concentration of BODIPY dye was usual used —4 U/mL which is equal to approximately 150 lg/mL. BODIPY conjugates were used for photostability experiment because they are more photostable than traditional fluorophores like fluorescein and rhodamine. The samples were excited for 4 min by successive intense irradiation, and fluorescent images were acquired every few seconds. As displayed in Figure 10, the fluorescence on the actin filaments stained by pure BODIPY-labeled actin was bleached very quickly during 30 s, and the red signal of BODIPY was then almost invisible. In contrast, the fluorescence intensity of HeLa cells labeled by RNI-PMMA48–b-PMAA27 micelles decreased very slowly, and the red signals were still clearly visible to the naked eyes after 4 minutes of continuous intensive irradiation. It is demonstrated that the photostability of RNI-PMMA48–bPMAA27 fluorescence copolymer (in which the concentration of the
Figure 8. Cell localization of RNI-PMMA48–b-PMAA27 micelles. HeLa cells were treated with 30 lg/mL dye for 2 h and photographed by fluorescent microscope (merged picture with bright field and fluorescence). Bar scale is 50 lm.
dye is approximately fivefold lower then BODIPY) is much better in comparison with the pure organic dye label BODIPY558/568 phalloidin. The results obtained clearly demonstrate that the main purpose of the study to develop a new highly water soluble self-assembly nanoscale formulation, composed of diblock copolymer and a fluorescent dye through a novel process, is achieved. 4. Conclusions In summary, we have described the synthesis, characterization and intracellular applications of a novel pH sensitive water-soluble fluorescent micellar probe. The new bichromophoric FRET system (RNI) based on 1,8-naphthalimide donor and rhodamine acceptor units was synthesized and then anchored to the novel well defined amphiphilic diblock copolymer—PMMA48–b-PMAA27. This RNIPMMA-b-PMAA adduct self-assembles into micelles with a hydrophobic PMMA core containing the anchored fluorescent probe, and a hydrophilic shell composed of PMAA block. It was demonstrated that the novel fluorescence micelles can serve as a highly sensitive
Figure 7. Internalization of RNI-PMMA48–b-PMAA27 micelles in living HeLa cells. The cells were incubated 2 h (upper panel) or 24 h (lower panel) with fluorescent dye in different concentrations. Bar scale is 40 lm.
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Figure 9. Internalization of RNI-PMMA48–b-PMAA27 micelles in living HEK cells. The cells were incubated 2 h (upper panel) or 24 h (lower panel) with fluorescent dye in different concentrations. Bar is 40 lm.
Figure 10. Photostability comparison of the stained HeLa cells by using (upper panel) pure BODIPY-labeled actin and (lower panel) RNI-PMMA48–b-PMAA27 micelles internalized in HeLa cells. Images were taken after different time intervals of continually intense excitation. Bar is 50 lm.
pH probe in water. They are also internalized in HeLa and HEK cells. It was found that the fluorescence intensity of the cells is concentration and time dependent. The cytotoxicity of the newly prepared fluorescent micelles is cell specific—HeLa cells are more stable to the action of the dye than HEK cells. The RNI-PMMA-bPMAA fluorescence micelles possess much better photostability in comparison to the pure organic dye label such as BODIPY. The results obtained indicated the high potential of the prepared fluorescence micelles for future biomedical applications. Acknowledgments The authors gratefully acknowledge the financial support from the Bulgarian National Science Fund (Project DDVU-02/97), the Science and Research Program of the University of Chemical Technology and Metallurgy, Sofia, Bulgaria, and the Scientific Funds for Scientific Research (F.R.S-FNRS), Bruxelles, Belgium. References and notes 1. Jin, W.; Jiang, J.; Wang, X.; Zhu, X.; Wang, G.; Song, Y.; Ba, C. Respir. Physiol. Neurobiol. 2011, 177, 183.
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