COMMUNICATION
www.advmat.de
Temperature- and pH-Sensitive Multicolored Micellar Complexes By Yong-Yong Li, Han Cheng, Jing-Ling Zhu, Lin Yuan, Yu Dai, Si-Xue Cheng, Xian-Zheng Zhang,* and Ren-Xi Zhuo Fluorescence is widely applied in ultrasensitive detection, and the demand for simultaneous detection of multiple signals requires multicolor fluorophores.[1] Various multicolored or desirable optical nanocrystals have been prepared, and the use of semiconductor nanocrystals has become one of the fastest moving and the most exciting areas of nanotechnology.[2,3] However, the fluorescence of nanocrystals is not ‘‘tunable’’, and they usually suffer from cytotoxicity and leakage into the biological environment. Furthermore, nanocrystals are generally synthesized under harsh conditions, and some of the reagents are cytotoxic. Luminescent lanthanide chelates, with unique properties such as high color purity and insensitivity to environmental quenching, were found to have great potential in bioimaging and biolabeling applications.[4] Very recently, we reported the in vitro and in vivo cell tracking of the micelles based on Eu(III) coordination complexes.[5] From an application standpoint, the fluorophores that are needed for single and multisignal biological detection should have some specific properties: i) water solubility, to avoid aggregation effects, ii) tunable fluorescence (sensitive to specific parameters), iii) emission of fluorescence at a significantly longer wavelength than the excitation source, so that observation of the label is not complicated by back-scattering effects, and, iv) low cytotoxicity. In addition, a carrier to load these fluorophores is needed. Micelles were chosen as the carriers due to their good biocompatibility, water solubility, stimulus-responsivity, nanometer-size (entering into the various organisms) and simple preparation.[6] Another advantage of micelles is that they are easily constructed from functional polymers, based on weak interactions such as hydrogen bonding,[7] and host–guest interaction.[8] The amphiphilic polymers poly[N-isopropylacrylamide-co-4(1-ethyl-1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl) phenyl methacrylate]block-poly(vinylphenol) [P(NIPAAm-co-EIPPMMA)-b-PVPhol] and poly(N-isopropylacrylamide-co-hydroxylethyl methacrylate)-blockpoly(vinylpyridine) [P(NIPAAm-co-HEMA)-b-PVP] were synthesized
by reversible addition-fragmentation chain transfer (RAFT) polymerization. The number-average molecular weight of the two polymers, as characterized by gel permeation chromatography (GPC), is shown in Figure S1 in the Supporting Information. The polymers were converted to fluorescent polymers through coordination with tris(dibenzoylmethanato)europium(iii) [Eu(DBM)3] and covalent bonding with fluorescein isothiocyanate (FITC), respectively. The Eu(DBM)3-coordinated P(NIPAAm-co-EIPPMMA)-b-PVPhol and FITC-conjugated P(NIPAAm-co-HEMA)-b-PVP are designated as FC-1 and FC-2, respectively, as illustrated in Scheme 1. The detailed chemical structures of two polymers are shown in Figure S2 in the Supporting Information. The content of Eu(DBM)3 in FC-1 is 0.86 wt% and that of FITC in FC-2 is 3.87 wt%. The critical micelle concentrations (CMCs) of the FC-1 and FC-2 are 55 and 45 mg L1, respectively (see the Supporting Information, Fig. S3). Furthermore, both copolymers show no obvious precipitations up to 5 mg mL1, indicating good solubility in water. Several complexes formed in different solvents, based on the interactions of hydrogen bonds between PVP and PVPhol, have been investigated by other researchers.[7] Here, PVP and PVPhol can induce self-assembly of the two fluorescent polymers in an aqueous environment when above their respective CMCs. The self-assembled core/shell micellar complexes consist of a PVP/PVPhol core and a shell with two types of hydrophilic chains (Scheme 1). To verify the micellar structure of FC-1/FC-2 complexes, the suspension of the complexes was freeze-dried for 1H nuclear magnetic resonance (NMR) spectroscopy in D2O (Fig. 1). The peaks of PVP and PVPhol were found to disappear compared to the 1H NMR spectra of two copolymers in CDCl3, due to the fact that core/shell micellar structures were formed, with isolated hydrophobic inner
[*] Prof. X. Z. Zhang, Y. Y. Li, H. Cheng, J. L. Zhu, Y. Dai, Prof. S. X. Cheng, Prof. R. X. Zhuo Key Laboratory of Biomedical Polymers The Ministry of Education and the Department of Chemistry Wuhan University, Wuhan 430072 (PR China) Fax: þ 86 27 6875 4509; E-mail:
[email protected] Prof. L. Yuan Biomedical Materials and Engineering Research Center Wuhan University of Technology Wuhan 430072 (PR China)
DOI: 10.1002/adma.200803770
2402
Scheme 1. Self-assembly of the two fluorescent polymers in aqueous solution.
ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2009, 21, 2402–2406
www.advmat.de
cores and hydrophilic outer shells. Note that the peaks of PVP reoccurred when the micellar complexes were tuned to be in acidic conditions, since the PVP can be protonated below pH 4, indicating that the P(NIPAAmco-HEMA)-b-PVP would disassemble from the micellar complex below pH 4 (see the Supporting Information, Fig. S4). Transmission electron microscopy (TEM) observations showed that self-assembled complexes at neutral conditions are well dispersed as individual spherical nanoparticles with a mean diameter of around 50 nm (Fig. 2I). PNIPAAm, with a lower critical solution temperature (LCST), is a widely used thermosensitive polymer.[9] The enthalpic changes associated with the transition from the extended to the collapsed state of the polymer were studied by differential scanning calorimetry (DSC, see the Supporting Information, Fig. S5). For FC-1, an endothermic transition is observed at 32.0 8C, while for FC-2 no endothermic transition is observed, indicating that there is no phase change upon temperature changes due to the high HEMA content (15%, from Fig. 1A) in P(NIPAAm-co-HEMA). Light transmittance of the polymer solution at 500 nm as a function of temperature for FC-1 and FC-2 polymer solutions, which also reflect the phase transition of PNIPAAm-based polymers, was also measured. The LCST obtained from this method (see the Supporting Information, Fig. S6) is in accordance with the result obtained from the above DSC measurements.
Adv. Mater. 2009, 21, 2402–2406
COMMUNICATION
Figure 1. 1H NMR spectra of A) P(NIPAAm-co-HEMA)-b-PVP, B) P(NIPAAm-co-EIPPMMA)-b-PVPhol in DMSO-d6, and C) FC-1/FC-2 complexes in D2O. The ratio of the HEMA unit in the backbone chain of P(NIPAAm-co-HEMA)-b-PVP was calculated to be 15 mol% by the formula: mol% of HEMA unit ¼ Sd/4/(Sd/4 þ Sb) 100%, where S is the integral of the corresponding peak.
The disappearance of the LCST for FC-2 indicates that the P(NIPAAm-co-HEMA) segment would be hydrophilic regardless of the alteration of the temperature. Thus, when the temperature changes from below to above 32 8C, the aggregation of the complexes would be largely prevented due to the hydrophilic nature of P(NIPAAm-co-HEMA) when the complexes selfassembled from the FC-1 and FC-2 in aqueous solution. To investigate whether the two polymers above could form complexes in an aqueous environment, the thermal stability of the complexes at 15 and 60 8C was examined; it was found that there was no precipitation at either temperature. If the two polymers form separately, without any association, the micelles formed by FC-1 would precipitate at 60 8C due to their inherent thermal properties, with a LCST at around 32 8C, as mentioned above, while the micelles formed by FC-2 would keep their micellar structure, since they have no thermosensitivity. No aggregation of the complexes at 60 8C indicates that the copolymers have molecularly blended to form the complexes. This is also confirmed by the fact that no endothermic transition is observed for FC-1/FC-2 complexes (see the Supporting Information, Fig. S2). The size versus temperature changes of FC-1, FC-2 and FC-1/ FC-2 complexes across the LCST of FC-1 polymers also demonstrate their thermosensitive properties (see the Supporting
Figure 2. I) TEM image of FC-1/FC-2 micellar complexes with a weight ratio of 1:1 ([FC-1] ¼ 250 mg L1, [FC-2] ¼ 250 mg L1); the inset image is a magnification of the micellar complex indicated. II) Fluorescence spectra of the micellar complexes with a weight ratio of 1:1 ([FC-1] ¼ 250 mg L1, [FC-2] ¼ 250 mg L1) under different pHs excited at 330 nm (the fluorescence measurements were characterized at 25 8C and both excitation and emission bandwidths were 5 nm). III) Photographs of FC-1/FC-2 micellar complexes excited by UV light with different weight ratios under A) different temperatures at pH 7.0 and B) under different pHs at 15 8C. The tubes in each image, from left to right, correspond to the different weight ratios of FC-1/FC-2 in the order 1:0, 3:1, 1:1, 1:3 and 0:1.
ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2403
COMMUNICATION
www.advmat.de
2404
Information, Fig. S7). The size of the micelles formed by polymer FC-1 shows an obvious dependence on temperature. The size, around 260 nm, has no obvious change from 26 to 30 8C, then decreases gradually from 30 to 35 8C to a minimal value of 190 nm due to the dehydration of the micelles across the LCST, and subsequently increases from 36 to 40 8C to 600 nm, due to aggregation of the micelles. In contrast, the size of the micelles formed by FC-2 polymer and FC-1/FC-2 complexes shows no obvious change in the measured temperature range. Considering the large size of micelles in this study, reverse micelles probably exist in the internal structure of the complexes, called compound micelles, as reported by Eisenberg et al.[10] The fluorescence spectra of the micellar complexes formed by the two fluorescent polymers with 1:1 weight ratio were compared with the fluorescence spectra of the micelles formed by FC-1 and FC-2 polymers separately, shown in Figure S8a in the Supporting Information. There are two regions in the fluorescent spectra of the micellar complexes: 500–570 nm, corresponding to the emission of the FITC groups in FC-2, and 600–630 nm, corresponding to the emission of EIPPMMA Eu(DBM)3 in FC-1. It has been reported that FITC is a highly fluorescent molecule and one of the most common acid–base indicators.[11] Its emission intensity is correlated with the proton (of phenolic hydroxyl groups) dissociation equilibrium in aqueous solutions, and is highly pH-dependent. The pH dependence of the fluorescence property of FITC in the pH range 5–8 was examined. It was found that FITC shows stronger green fluorescence in a basic environment than in an acidic environment (see the Supporting Information, Fig. S8b). Simultaneously, polymer FC-1 is thermally responsive. Therefore, it would be expected that the fluorescence properties of the FC-1/FC-2 complexes could be tuned by changing the pH as well as temperature. The fluorescence properties of the complexes at different pH values were investigated (Fig. 2II). The green fluorescence peaks at 520 nm greatly increases with the increasing pH, while the red fluorescence at 620 nm has an almost negligible increase, indicating that the fluorescence depends greatly on pH in the range 5–8, within physiological bounds. Photographs of the complexes at different temperatures and pHs excited by UV light are shown in Figure 2III. At a particular temperature and pH, the fluorescence of the complexes changes from red to green with a change in the weight ratio FC-1/FC-2 from 1:0 to 0:1, and exhibits well-proportioned yellow fluorescence at a weight ratio of 1:1. When the temperature increases from 15 to 45 8C, red fluorescence of the sample reduces; it decreases slightly as the temperature increases from 15 to 35 8C, compared with a large decrease from 35 to 45 8C. The red fluorescence almost disappeared at 45 8C, while the intensity of green fluorescence does not show any obvious change. This phenomenon can be explained as follows: P[NIPAAmco-EIPPMMA Eu(DBM)3] segments switch from being hydrophilic to hydrophobic as the temperature increases, and the EIPPMMA Eu(DBM)3 fluorophores are entrapped into the core of the complexes as the phase transition occurs. The intensity of red fluorescence decreases because the excitation of EIPPMMA Eu(DBM)3 fluorophores is shielded by the shell of the complexes. The green fluorescence does not change because the P(NIPAAm-co-HEMA) segments have no such transition. On the
other hand, the color intensities of the complexes at different pH values exhibit significant differences in green fluorescence. At pH 5.0 and 6.0, the intensity of the green fluorescence is weak, and then it is greatly enhanced when the pH is increased to 7.0 or 8.0. The result is in accordance with the spectra in Figure 2II. Tumor cells (A549) were used as a model to check whether FC-1/FC-2 complexes could emit their peculiar fluorescence under different temperatures and pHs. From Figure 3, it was found that the fluorescence showed obvious pH and temperature dependencies. The complexes accumulate in the cells after 24 h incubation, and are confirmed to be internalized into the cells by a line scan of one single cell on the z-axis. Confocal image slices through the cells (see the Supporting Information, Fig. S9) demonstrated that the signal is present in the cell’s interior rather than on its surface. In addition, as the slices advance from the apical to basal surfaces of the cells, the signal appears primarily in the centermost slices. Green and red fluorescences exist simultaneously, indicating that there is no segregation between
Figure 3. Images of A549 cells with the FC-1/FC-2 complexes after excitation: I) at different temperatures, 26, 32, and 38 8C in A, B and C, respectively, and II) at different pHs, 5.0, 6.5 and 8.0 in D, E and F, respectively, at 25 8C. For each case, image 1 shows only the green fluorescence, image 2 only the red fluorescence and image 3 is under bright field.
ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2009, 21, 2402–2406
www.advmat.de
AIBN (5 mg), vinylpyridine (1.02 g), 600 mg P(NIPAAm-co-HEMA)-b-PVP, 30 mg fluorescein isothiocyanate (FITC), and 0.5 mL triethylamine were dissolved and reacted in dimethyl sulfoxide (DMSO) to obtain the fluorescent block copolymers. The content of FITC in the polymer was quantitatively analyzed by a fluorescence method using the fluorescence intensity at 520 nm; the value was estimated to be 3.87 wt%. Culture with A549 Cells: The human lung adenocarcinoma (A549) cell line, maintained at 37 8C, 5% CO2 in Dulbecco’s modied Eagle’s medium (DMEM), was chosen to assess the fluorescence properties of FC-1/FC-2 complexes. After incubation for 24 h (37 8C, 5% CO2), the culture medium was replaced by 200 mL of DMEM with predetermined pH containing complexes (500 mg L1) and further incubated for 24 h at either 37 8C or 4 8C. Observations were made using an inverted microscope (Leica DM IRE2) with laser confocal system (Leica TCS SP2 AOBS) at 405 nm after the plates had been washed with PBS three times. The XYZ mode was applied in scanning the cells. In order to know the distribution of fluorescent signals, the cells were scanned at different Z distances. In the temperature-sensitivity experiment, the cells were incubated at temperatures from 38 to 26 8C controlled using a thermocontroller (Tempcontrol 37-2). During the experiments, all collection parameters were fixed to be the same, except for the temperature. The fluorescence signals were collected at 500–535 and 603–668 nm by different channels, and the signal intensities were analyzed using the Leica Confocal Software.
Acknowledgements This work was financially supported by the National Natural Science Foundation of China (20774069, 50633020) and the National Key Basic Research Program of China (2005CB623903). Supporting Information is available online from Wiley Inter Science or from the author. Received: December 20, 2008 Revised: January 23, 2009 Published online: March 23, 2009
Experimental Synthesis of P(NIPAAm-co-EIPPMMA) Macro-RAFT: P(NIPAAm-coEIPPMMA) macro-RAFT was synthesized by RAFT polymerization of NIPAAm (2.77 g) and EIPPMMA (12 mg) in 15 mL tetrahydrofuran (THF) using 6 mg azobisisobutyronitrile (AIBN) and 50 mg benzylsulfanylthiocarbonylsulfanylpropyonic acid (BSPA) as initiator and chain transfer agent, respectively. After all the reactants were mixed well in THF, the solution was degassed by bubbling with nitrogen for 30 min. The reaction was performed at 70 8C for 24 h under nitrogen. Upon completion, the product was precipitated out by the addition of diethyl ether. The product was purified by repeated precipitation in diethyl ether from ethanol and then dried in vacuum. Synthesis of P(NIPAAm-co-EIPPMMA)-b-PVPhol: P(NIPAAm-coEIPPMMA) macro-RAFT (1.13 g), AIBN (5 mg) and vinylphenol (1.2 g) were dissolved in 15 mL THF, and the solution was degassed by bubbling with nitrogen for 30 min. The reaction was performed at 70 8C for 24 h under nitrogen. Upon completion, the product was precipitated out by the addition of diethyl ether. The product was purified by repeated precipitation in diethyl ether from dimethylformamide (DMF), and then dried in vacuum. After the synthesis of P(NIPAAm-co-EIPPMMA)-b-PVPhol, it was coordinated with Eu(DBM)3 in ethanol to incorporate the strong fluorescent property to the polymer. The weight ratio of Eu(DBM)3 in the polymer was 0.86%, as determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES). Synthesis of P(NIPAAm-co-HEMA) Macro-RAFT: The procedure for synthesizing P(NIPAAm-co-HEMA) macro-RAFT was the same as for P(NIPAAm-co-EIPPMMA) macro-RAFT except for the reactants: NIPAAm (1.98 g), AIBN (6 mg), hydroxyethylmethacrylate (HEMA; 1.98 g), BSPA (50 mg). Synthesis of P(NIPAAm-co-HEMA)-b-PVP: The procedure for synthesizing P(NIPAAm-co-HEMA)-b-PVP was the same for P(NIPAAmco-EIPPMMA)-b-PVPhol except for the reactants: macro-RAFT (1.13 g),
Adv. Mater. 2009, 21, 2402–2406
COMMUNICATION
FC-1 and FC-2. In addition, the red fluorescence is obviously weakened with an increase in temperature from 26 to 38 8C (see Fig. 3I, boxes A2, B2, and C2). The ratios of green to red fluorescences are 2.08 0.103 at 26 8C and 2.90 0.101 at 32 8C, and exhibit a great increase to 4.04 0.200 at 38 8C. The fluorescence intensity increases with increasing pH: the ratio of green to red fluorescence is 1.77 0.096 at pH 5.0, 1.89 0.102 at pH 6.5, and 2.16 0.108 at pH 8.0. A549 cells with micellar aggregates were cultured at 4 8C to demonstrate whether these micellar aggregates are able to cross the cell membrane through diffusion or endocytosis; at this temperature, endocytosis would be greatly suppressed. Very weak fluorescence was detected inside the cells after the plates were washed with phosphate buffered saline (PBS; see the Supporting Information, Fig. S10). The results show that only a small part of the micellar aggregates could thrill through the cell membrane through diffusion under this condition, indicating that most of the micellar aggregates thrill through the cell membrane through endocytosis at 37 8C. In summary, novel multicolored micellar complexes, selfassembled from two copolymers through hydrophobic interactions and hydrogen bondings, were demonstrated. The fluorescence properties of the resulting micellar complexes can be controlled by temperature and pH changes. These complexes might find great potential in probing the pH or temperature in complicated environments, and have the potential to be used in bioanalysis and the in vivo transfer of small molecules.
[1] I. L. Medintz, H. T. Uyeda, E. R. Goldman, H. Mattoussi, Nat. Mater. 2005, 4, 435. [2] X. G. Peng, L. Manna, W. D. Yang, J. Wickham, E. Scher, A. Kadavanich, A. P. Alivisatos, Nature 2000, 404, 59. [3] a) H. Kobayashi, Y. Hama, Y. Koyama, T. Barrett, C. A. S. Regino, Y. Urano, P. L. Choyke, Nano Lett. 2007, 7, 1711. b) C. Xu, E. Bakker, Anal. Chem. 2007, 79, 3716. c) Y. Wu, S. K. Campos, G. P. Lopez, M. A. Ozbun, L. A. Sklar, T. Buranda, Anal. Biochem. 2007, 364, 180. [4] L. M. Fu, X. F. Wen, X. C. Ai, Y. Sun, Y. S. Wu, J. P. Zhang, Y. Wang, Angew. Chem. Int. Ed. 2005, 44, 747. [5] Y. Y. Li, H. Cheng, Z. G. Zhang, C. Wang, J. L. Zhu, Y. Liang, K. L. Zhang, S. X. Cheng, X. Z. Zhang, R. X. Zhuo, ACS Nano 2008, 2, 125. [6] a) G. Riess, Prog. Polym. Sci. 2003, 28, 1107. b) K. Kataoka, A. Harada, Y. Nagasaki, Adv. Drug Delivery Rev. 2001, 47, 113. c) J. Rodriguez-Hernandez, F. Checot, Y. Gnanou, S. Lecommandoux, Prog. Polym. Sci. 2005, 30, 691. d) E. S. Gil, S. A. Hudson, Prog. Polym. Sci. 2004, 29, 1173. e) J. F. Gohy, Adv. Polym. Sci. 2005, 190, 65. [7] a) W. Q. Zhang, L. Q. Shi, L. C. Gao, Y. L. An, K. Wu, Macromol. Rapid Commun. 2005, 26, 1341. b) M. L. Xiang, M. Jiang, Y. B. Zhang, C. Wu, L. X. Feng, Macromolecules 1997, 30, 2313. c) Y. B. Zhang, M. L. Xiang, M. Jiang, C. Wu, Macromolecules 1997, 30, 6084. d) S. Z. Luo, S. Y. Liu, J. Xu, H. Liu, Z. Y. Zhu, M. Jiang, C. Wu, Macromolecules 2006, 39, 4517. e) J. Kriz, J. Dybal, J. Brus, J. Phys. Chem. B 2006, 110, 18338. f) S. H. Zhang, P. C. Painter, J. Runt, Macromolecules 2004, 37, 2636. g) S. W. Kuo, P. H. Tung, F. C. Chang, Macromolecules 2006, 39, 9388.
ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2405
COMMUNICATION
www.advmat.de
2406
[8] a) L. Zhang, Y. W. Wu, L. Brunsveld, Angew. Chem. Int. Ed. 2007, 46, 1798. b) H. Q. Dong, Y. Y. Li, S. J. Cai, R. X. Zhuo, X. Z. Zhang, L. J. Liu, Angew. Chem. Int. Ed. 2008, 47, 5573. [9] a) Y. Hirokawa, T. Tanaka, J. Chem. Phys. 1984, 81, 6379. b) A. Suzuki, T. Tanaka, Nature 1990, 346, 345.
[10] L. Zhang, A. Eisenberg, J. Am. Chem. Soc. 1996, 118, 3168. [11] a) N. Nakayama-Ratchford, S. Bangsaruntip, X. M. Sun, K. Welsher, H. J. Dai, J. Am. Chem. Soc. 2007, 129, 2448. b) D. Millar, M. Uttamlal, R. Henderson, A. Keeper, Chem. Commun. 1998, 477.
ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2009, 21, 2402–2406
