ARTICLE pubs.acs.org/Langmuir
In Situ Electron-Beam Polymerization Stabilized Quantum Dot Micelles Nathalie Travert-Branger,† Fabien Dubois,† Jean-Philippe Renault,‡ Serge Pin,‡ Benoit Mahler,§ Edmond Gravel,† Benoit Dubertret,*,§ and Eric Doris*,† †
CEA, iBiTecS, Service de Chimie Bioorganique et de Marquage, 91191 Gif-sur-Yvette, France CEA, Service de Chimie Moleculaire/CNRS URA 331, 91191 Gif-sur-Yvette, France § Laboratoire de Physique et d’Etude des Materiaux, UMR8213 du CNRS, ESPCI, 10 rue Vauquelin, 75005 Paris, France ‡
ABSTRACT: A polymerizable amphiphile polymer containing PEG was synthesized and used to encapsulate quantum dots in micelles. The quantum dot micelles were then polymerized using a “clean” electron beam process that did not require any post-irradiation purification. Fluorescence spectroscopy revealed that the polymerized micelles provided an organic coating that preserved the quantum dot fluorescence better than nonpolymerized micelles, even under harsh conditions.
’ INTRODUCTION Colloidal semiconductor quantum dots (QDs) are a promising alternative to organic dyes for fluorescence-based bioapplications.1 The main challenges that need to be addressed for in vitro or in vivo experiments are aqueous solubility, colloidal stability, and biocompatibility. Two main strategies exist for converting initially hydrophobic QDs into hydrophilic nanoparticles: (i) exchange of the original organic layer with hydrophilic ligands2 (however, ligand permutation often deteriorates the fluorescence properties of the QDs3) or (ii) incorporation of the hydrophobic QDs into amphiphilic micelles.4 Methods based on micellar encapsulation of QDs in poly(ethylene glycol) phospholipids (PEG-PL) are attractive processes, since they do not alter the surface of the QDs. In addition, the resulting micelles display a high density of PEG on their surface that prevents nonspecific adsorption and improves biocompatibility.5 However, the supramolecular architecture of the surfactant coating is only maintained by weak, local van der Waals interactions between the interdigitated hydrophobic ligands, initially present on the QD surface, and the hydrophobic tails of the outer phospholipid layer. In harsh environments, denaturation of the micelles and aggregation of the QDs is often observed. Since accumulation of cadmium and other metals is a potential cause of acute toxicity, preservation of the colloidal stability is therefore essential for in vivo imaging applications.6 We conceived that the polymerization of the amphiphilic PEG-phospholipids would improve the stability of a micellar complex surrounding a QD and lead to more robust coating of the latter. Our approach is based on the use of PEG-phospholipids 1 which incorporate a polymerizable acrylate group as linking unit and the use of in situ electron-beam-mediated polymerization of the micelles containing QDs (Scheme 1). The choice of electron beam r 2011 American Chemical Society
Scheme 1
polymerization was governed by the fact that, contrary to chemically induced polymerization, it does not necessitate the use of an initiator that could alter the properties of the QDs. In addition, no purification is needed after the key polymerization step.
’ EXPERIMENTAL SECTION General. All reagents were purchased from Aldrich except for 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine which was purchased from Alexis Biochemicals and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] from Aventi Polar lipids, Inc. CH2Cl2 was dried by distillation over CaH2 and tetrahydrofuran (THF) over Na/benzophenone. NMR spectra were recorded on a Bruker AVANCE DPX 400 spectrometer; chemical shifts Received: October 5, 2010 Published: March 21, 2011 4358
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Langmuir are given in ppm relative to residual CHCl3 (7.26 ppm). Fluorescence spectra and photostability experiments were measured on a Jobin-Yvon Fluoromax-3 instrument. Transmission electron microscopy pictures were obtained with a Phillips CM 12 microscope (120 kV) using carboncoated grids. Nanocrystal Synthesis. CdSe/ZnS nanocrystals were synthesized using modifications of the syntheses first developed by Murray et al.9 and Hines and Guyot-Sionnest.10 Typically, 20 mL of a 0.25 M trioctylphosphine selenide solution containing 150 μL of dimethylcadmium was swiftly injected in 30 g of a degassed trioctylphosphine oxide solution heated at 350 °C. When the CdSe nanoparticles reached the desired size, the nanoparticles were isolated by precipitation with methanol/butanol and resuspended in hexane. ZnS layers were grown as described in ref 11. After their synthesis, the QDs were precipitated with methanol/butanol and suspended in hexane. Synthesis of N-Boc-O-benzyl-L-serine Phospholipid 2. Activation of the Serine Carboxylic Group. To a solution of N-Boc-Obenzyl-L-serine (1 g, 3.58 mmol, 1 equiv) in 10 mL of dry CH2Cl2 was added N-hydroxysuccinimide (620 mg, 1.5 equiv), N,N’-dicyclohexylcarbodiimide (1.47 g, 2 equiv), and 4-dimethylaminopyridine (20 mg, cat.). The reaction mixture was stirred at rt for 12 h before 15 mL of Et2O was added. The precipitate was filtered off, and the solvent was evaporated under reduced pressure to afford the activated carboxylic acid that was used without further purification in the next step. Coupling of Activated Serine to 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine. To a solution of 1,2-dihexadecanoyl-sn-glycero-3phosphoethanolamine (1 g, 1.45 mmol, 1 equiv) and triethylamine (1.46 g, 10 equiv) in 50 mL of CH2Cl2 was added the activated N-Boc-Obenzyl-serine prepared above (797 mg, 1.4 equiv) and dimethylaminopyridine (20 mg, cat.). The solution was stirred at rt for 48 h, and the solvent was evaporated under reduced pressure. Purification by column chromatography on silica (CH2Cl2: MeOH, 92:8) afforded 2 as a white solid (0.70 g, 50%). 1H NMR (CDCl3): δ 0.86 (t, J = 6.8 Hz, 6H), 1.24 (H-Alk), 1.41 (s, 9H, CH3 Boc), 1.56 (m, 4H), 2.26 (m, 4H), 3.15 (m, 4H), 3.48 (s, 2H), 3.48 (m, 3H), 3.64 (m, 2H), 3.72 (m, 1H), 4.52 (m, 2H), 7.30 (m, 5H, Ar).
Synthesis of O-Benzyl-L-serine-Pegylated Phospholipid 3. Removal of the N-Boc Protecting Group. Phospholipid 2 (600 mg, 0.62 mmol, 1 equiv) was reacted with 5 mL of trifluoroacetic acid in 15 mL of CH2Cl2 at rt for 12 h. The solvents were evaporated under reduced pressure to give the Boc-free amino-phospholipid which was used in the next step without further purification. 1H NMR (CDCl3): δ 0.87 (t, J = 6.8 Hz, 6H), 1.24 (H-Alk), 1.55 (m, 4H), 2.29 (m, 4H), 3.63 (m, 4H), 3.73 (m, 1H), 3.92 (m, 4H), 4.30 (m, 2H), 4.47 (m, 2H), 4.54 (m, 1H), 7.30 (m, 5H, Ar). Coupling of the N-Deprotected Phospholipid with Polyethylene Glycol. To a solution of the above N-deprotected phospholipid (0.5 g, 0.6 mmol, 1 equiv) in 30 mL of dry CH2Cl2 was added N-hydroxysuccinimide activated poly(ethylene glycol) monomethylether carboxylic acid (1.26 g, 1 equiv), triethylamine (0.83 mL, 10 equiv), and dimethylaminopyridine (10 mg, cat.). The solution was stirred at rt for 12 h and concentrated. Purification by column chromatography on silica (CH2Cl2/MeOH, 9:1) afforded 3 as a white solid (1.13 g, 65%). 1H NMR (CDCl3): δ 0.87 (t, J = 6.8 Hz, 6H), 1.24 (H-Alk), 1.57 (m, 4H), 2.27 (m, 4H), 3.37 (s, 3H), 3.48 (m, 4H), 3.56 (m, 1H), 3.63 3.73 (HPEG), 3.93 (m, 4H), 4.32 (m, 2H), 4.47 (m, 2H), 4.52 (m, 1H), 7.29 (m, 5H, Ar).
Synthesis of the Polymerizable Pegylated Phospholipid 1. Removal of the O-Benzyl Protecting Group. To a solution of phospholipid 3 (1 g, 0.35 mmol, 1 equiv) in 100 mL of EtOH/CH2Cl2 (9:1) was added 10 wt % Pd/C (30 mg, cat.). The mixture was air evacuated and vigorously stirred under 1 bar of H2 for 24 h. Pd/C was removed by filtration over Celite, and the solution was concentrated under vacuum to give compound 3b in quantitative yield. 1H NMR
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(CDCl3): δ 0.86 (t, J = 6.8 Hz, 6H), 1.24 (H-Alk), 1.56 (m, 4H), 2.28 (m, 4H), 3.36 (s, 3H), 3.53 (m, 4H), 3.54 (m, 1H), 3.68 (H-PEG), 3.80 (m, 4H), 4.00 (m, 2H), 4.17 (m, 2H), 4.23 (m, 1H). Coupling of the Polymerizable Unit to the Phospholipid. To a solution of the O-deprotected PEG-phopspholipid (824 mg, 0.3 mmol, 1 equiv) in 50 mL of dry THF was added triethylamine (125 μL, 3 equiv) and acryloyl chloride (27 μL, 1.1 equiv). The solution was stirred at 80 °C for 4 h. After cooling to rt, Et2O was added. The precipitate was filtered off, and the solution was concentrated under vacuum. Purification by column chromatography on silica (CHCl3:CH3OH:H2O, 71:25:4) afforded 1 as a white solid (252 mg, 30%). 1H NMR (CDCl3): δ 0.86 (t, J = 6.8 Hz, 6H), 1.24 (H-Alk), 1.56 (m, 4H), 2.28 (m, 4H), 3.36 (s, 3H), 3.54 (m, 4H), 3.54 (m, 1H), 3.68 (H-PEG), 3.80 (m,4H), 4.08 (m, 2H), 4.17 (m, 2H), 4.23 (m, 1H), 5.86 (m, 1H), 6.03 (m, 1H), 6.28 (m, 1H). Encapsulation of the QDs with Phospholipids. Quantum dots in hexane were first precipitated with methanol and redissolved in chloroform (final concentration: 8.5 μM). A total of 50 μL of this solution was added to 40 μL of either commercially available 1,2-dipalmitoyl-snglycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] or newly synthesized phospholipid 1 (20 mg/mL in CHCl3). Then 600 μL of H2O was added, and the heterogeneous system was vigorously stirred while heating at 80 °C. This resulted first in the formation of a microemulsion with concomitant evaporation of chloroform and ultimately in the aqueous transfer of the coated nanocrystals. The latter were isolated from QD aggregates by centrifugation at 14 000 rpm and from free micelles by ultracentrifugation at 100 000 rpm in 35% sucrose in phosphatebuffered saline (PBS). Sucrose was eliminated by centrifugation using a Sartorius Vivaspin 500K disposable system. A stock solution of the encapsulated QDs was obtained by dissolving the coated nanocrystals in 600 μL of water. Electron Beam Polymerization. Polymerization was carried out using electron pulses from a Titan Beta, Inc. linear accelerator delivering electrons of 10 MeV energy.7 In our experiments, 10 ns pulses were used at a repetition rate of 10 Hz. A dose of 20 Gray (Gy) per pulse was determined using Fricke dosimeter.8 Doses determined by this method are analogous to those calculated using the SCN dosimeter in pulse radiolysis. Samples were deoxygenated prior to irradiation and kept under nitrogen.
’ RESULTS AND DISCUSSION Polymerizable amphiphile 1 was synthesized starting from 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine, which was coupled to bis-protected L-serine by initial N-hydroxysuccinimide activation of the carboxylic group (Scheme 2). The Boc protecting group of 2 was then removed under acidic conditions and the resulting amine coupled to poly(ethylene glycol) monomethyl ether carboxylic acid (Mn ∼ 2000). The benzyl protecting group of the serine side chain of 3 was hydrogenolyzed over palladium, and the free alcohol coupled to acryloyl chloride. With phospholipid 1 in hand, micellar encapsulation of quantum dots was performed. The starting CdSe/ZnS nanocrystals were prepared using a previously reported method involving trioctylphosphine/trioctylphosphine oxide as stabilizing agents.12 In a typical encapsulation procedure, water was added to a mixture of QD and PEGphospholipid in chloroform. The heterogeneous system is vigorously stirred while heating, resulting initially in the formation of a milky emulsion with concomitant evaporation of chloroform and ultimately in the aqueous transfer of the coated nanocrystals. The polymerization of coated QD micelles was then initiated by ionizing radiation. To determine the influence of the 4359
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Scheme 2
Figure 2. (a) TEM picture (negative staining) of polymerized QDmicelles. Comparative stability of fluorescence versus time of a reference sample without irradiation (blue line) versus irradiated phospholipidsmicelles QDs (red line) in (b) 20 mM PBS buffer/0.15 M NaCl, pH 7.2, (c) saturated NaCl, and (d) 75 μM HCl. Excitation at 465 nm, fluorescence normalized at 100% to the initial value recorded at 535 nm.
Figure 1. (a) Fluorescence intensity in function of the irradiation dose. (b) Fluorescence spectra of (1) classical PEG-phospholipid QD (PEG 2000-PE), (2) phospholipid-1 QD, (3) phospholipid-1 QD/60 kGy, followed by UV irradiation (365 nm, 100 W, 30 min), and (4) phospholipid-1 QD/60 kGy. All samples had identical absorbance at 350 nm.
irradiation on the photophysical properties of the nanocrystals, QD-micelle solutions (coated with polymerizable phospholipid 1) were irradiated at increasing doses (up to 90 kGy).13 Fluorescence was measured 3 h after irradiation and compared to that of a reference (nonirradiated) QD-micelle solution. The fluorescence intensity of the polymerized QD-micelles decreased linearly as the irradiation dose increased, diminishing to 40% of the control in the case of the sample irradiated with 90 kGy
(Figure 1a). This drop in fluorescence emission could be the consequence of the radiolytic species introducing trapping sites in the QDs.14 To evaluate the effective dose to induce polymerization of the acrylate moiety, irradiation experiments were also undertaken with a 1 mg/mL solution of phospholipid 1 only. Progress of the polymerization was monitored by 1H NMR of aliquots which indicated complete disappearance of the acrylate protons upon exposure to 60 kGy. Accordingly, the colloidal nanocrystal samples were polymerized using a mean dose of 60 kGy, and the resulting QDs retained ca. 60% of their initial fluorescence (Figure 1b, curve 3). Noteworthy was the aging of the irradiated samples whose fluorescence dropped dramatically after a few days. Indeed, 6 days after exposure to the electron source, the residual fluorescence of the 60 kGy sample was only 20% of the initial value. However, aging of the samples could be stopped and reversed using intense UV illumination, as up to 80% of the initial 4360
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Langmuir fluorescence was restored after 100 W irradiation at 365 nm, for 30 min (Figure 1b, curve 4). This phenomenon has already been observed and commented on by others.15 QDs coated with polymeric micelles are highly fluorescent and exhibit nearly identical absorption and emission spectra to that of classical (nonpolymerizable) PEG-phospholipid QDs (PEG2000-PE from Avanti polar lipid) (Figure 1b, curves 1 and 2). Transmission electron microscopy pictures (Figure 2a) shows that the nanocrystals are individualized; the samples are also stable as no aggregation was observed in pure H2O over a period of several months. As polymerization around the nanocrystal is expected to induce stabilization of the multicomponent system, assessment the robustness of the polymeric micelle-QDs was undertaken. Comparative stability studies versus classical phospholipid micelle-QDs were thus run under various conditions (e.g., saline and acidic). The polymerized colloidal nanocrystals were suspended in different aqueous solutions, and their fluorescence compared, as a function of time, to that of a nonpolymerized sample. First stability experiments were conducted in saturated NaCl (Figure 2c) where the spontaneous extinction of fluorescence of the nonpolymerized micelle-QDs was observed. In contrast, the polymeric micelle-QDs were much more stable and retained 50% of their fluorescence after exposure for 2 days to hypersaline conditions. The slope of the decrease in fluorescence intensity changed with time, which indicated complex kinetics for the alteration process. As aqueous buffers are known to alter fluorescence properties of nanocrystals,16 experiments were also conducted in pH 7.2 PBS solutions (Figure 2b). While the fluorescence of the polymeric coated QDs is stable, a rapid decrease was observed for the nonpolymerized phospholipid micelle-QDs; after 500 s, a drop of 55% of the initial fluorescence was detected. Similarly for acidic media (75 mM HCl, Figure 2d), a 65% drop in fluorescence was detected after 500 s. Again, the polymeric micelle-QDs exhibited higher stability. Integrity of the nonpolymerized micelle-QDs was assessed in PBS by dynamic light scattering (DLS) which indicated no variation of the hydrodynamic diameter and therefore no aggregation of the QDs. There is thus an overall increase of the fluorescence stability induced by polymerization of the micelle around the QDs, and the polymeric layer is most likely acting as a diffusion barrier against ionic salts, thus preventing alteration of the nanocrystals.16 Polymeric stabilization of micellar amphiphiles around QDs is a key step toward applications in harsh conditions such as electroporation-based cellular delivery, and higher fluorescence stability is expected when these new materials are applied to imaging experiments.
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’ ACKNOWLEDGMENT This work was partly funded by the National Research Agency of France (Visen, Dot-Imager, and IVF-proteomic ANR projects). Dr. Alexander Yuen is gratefully acknowledged for helpful discussions. ’ REFERENCES (1) (a) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538. (b) Smith, A. M.; Gao, X. H.; Nie, S. M. Photochem. Photobiol. 2004, 80, 377. (c) Genin, E.; Carion, O.; Mahler, B.; Dubertret, B.; Arhel, N.; Charneau, P.; Doris, E.; Mioskowski, C. J. Am. Chem. Soc. 2008, 130, 8596. (2) (a) Chan, W. C. W.; Nie, S. Science 1998, 281, 2016. (b) Kim, S.; Bawendi, M. G. J. Am. Chem. Soc. 2003, 125, 14652. (c) Dubois, F.; Mahler, B.; Dubertret, B.; Doris, E.; Mioskowski, C. J. Am. Chem. Soc. 2007, 129, 482. (3) (a) Kloepfer, J. A.; Bradforth, S. E.; Nadeau, J. L. J. Phys. Chem. B 2005, 109, 9996. (b) Aldana, J.; Wang, Y. A.; Peng, X. J. Am. Chem. Soc. 2001, 123, 8844. (4) (a) Depalo, N.; Mallardi, A.; Comparelli, R.; Striccoli, M.; Agostiano, A.; Curri, M. L. J. Colloid Interface Sci. 2008, 325, 558. (b) Carion, O.; Malher, B.; Pons, T.; Dubertret, B. Nat. Protoc. 2007, 2, 2383. (5) Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libchaber, A. Science 2002, 298, 1759. (6) Lewinski, N.; Colvin, V.; Drezek, R. Small 2008, 4, 26. (7) Mialocq, J.-C.; Hickel, B.; Baldacchino, G.; Juillard, M. J. Chim. Phys. 1999, 96, 35. (8) Fricke, H.; Hart, E. J. In Radiation Dosimetry, 2nd ed.; Attix, F. H., Roesch, W. C., Eds.; Academic Press: New York and London, 1966; Vol. 2, pp 167 232. (9) Murray, C. B.; Norris, D. J.; Bawendi, M. G. J. Am. Chem. Soc. 1993, 115, 8706–8715. (10) Hines, M. A.; Guyot-Sionnest, P. J. Phys. Chem. 1996, 100, 468–471. (11) Dabbousi, B. O.; Rodriguez-Viejo, J.; Mikulec, F. V.; Heine, J. R.; Mattoussi, H.; Ober, R.; Jensen, K. F.; Bawendi, M. G. J. Phys. Chem. B 1997, 101, 9463–9475. (12) Travert-Branger, N.; Dubois, F.; Carion, O.; Carrot, G.; Malher, B.; Dubertret, B.; Doris, E.; Mioskowski, C. Langmuir 2008, 24, 3016. (13) For example, the recommended dose in EU and U.S. for sterilization is 25 kGy. (14) Kumar, A.; Janata, E.; Henglein, A. J. Phys. Chem. 1988, 92, 2587. (15) (a) Tsay, J. M.; Doose, S.; Pinaud, F.; Weiss, S. J. Phys. Chem. B 2005, 109, 1669. (b) Carrillo-Carrion, C.; Cardenas, S.; Simonet, B. M.; Valcarcel, M. Chem. Commun. 2009, 5214. (16) Boldt, K.; Bruns, O. T.; Gaponik, N.; Eychm€uller, A. J. Phys. Chem. B 2006, 110, 1959.
’ CONCLUSIONS In summary, we describe here a new polymeric approach toward the aqueous stabilization of micellar phospholipids-QDs. Fluorescent colloidal nanocrystals were obtained by encapsulation with amphiphilic phospholipids followed by pulse-radiolysismediated polymerization. An overall enhancement of the fluorescence stability of the nanocrystals was observed under various conditions. ’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected] (B.D.);
[email protected] (E.D.). 4361
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