2506
Langmuir 2006, 22, 2506-2510
Two-Compartment Micellar Assemblies Obtained via Aqueous Self-Organization of Synthetic Polymer Building Blocks Andreas F. Thu¨nemann,*,† Stephan Kubowicz,‡ Hans von Berlepsch,§ and Helmuth Mo¨hwald‡ Federal Institute for Materials Research and Testing, Richard Willsta¨tter Strasse 11, 12489 Berlin, Germany, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany, and Free UniVersity of Berlin, Fabeckstrasse 36a, Berlin, Germany ReceiVed December 13, 2005. In Final Form: January 25, 2006 We synthesized a symmetric linear ABCBA pentablock copolymer consisting of poly(ethylene oxide), poly(γbenzyl L-glutamate), and a poly(perfluoro ether) (fluorolink). The different blocks are highly immiscible with each other and form two-compartment micelles of mainly cylindrical shape in aqueous solution with lengths in the range of 100 to 200 nm and diameters of about 24 nm. The poly(perfluoro ether) (C blocks) forms the liquidlike center of the micelles (d ) 6 nm). This is surrounded by a first shell of ca. 2 nm thickness consisting of β-sheets of poly(γ-benzyl L-glutamate) (B blocks) and a second 7 nm shell of poly(ethylene oxide) (A blocks). The A blocks provide water solubility, and the B and C blocks form separated hydrophobic compartments. This work is a contribution to the development of multicompartment micelles devoted to mimic transport proteins such as serum albumins in long-term development.
Introduction The concept of multicompartment micelles that are able to mimic basic properties of natural systems such as serum albumins, which are capable of transporting poorly water-soluble compounds in the blood by their selective uptake and release, is an intriguing example for the use of bottom-up strategies1 in nanotechnology.2 Applications in medicine, pharmacy, biotechnology, and so forth seem to be possible, but the preparation and control of stable multicompartment micellar systems are still at the very beginning. Even less is known about the properties of the existing examples. In the past, various approaches have been presented by Sta¨hler et al.,3,4 Weberskirch et al.,5 and Laschewsky et al.6,7 to realize it experimentally. All approaches are based on the mutual incompatibility of fluorocarbon and hydrocarbon chains within a water-soluble polymer and were reviewed by Laschewsky.8,2 More recently, Lodge et. al.9 presented a cryoTEM investigation of micelles formed by a mixed-arm star block terpolymer in water. They were the first who visualized clearly two separated compartments in a single micelle. Regions that appear to be darker in the TEM pictures were assigned to fluorocarbon-rich compartments, and brighter ones were assigned to hydrocarbon-rich compartments. In this study, we concentrate on a novel linear ABCBA pentablock copolymer (cf. Figure 1). It consists of two hydrophilic poly(ethylene oxide) blocks (PEO, block A), two hydrophobic
Figure 1. Chemical structure of the ABCBA pentablock copolymer (upper). The block lengths are 110 (A block, poly(ethylene oxide)), 8 (B block, poly(γ-benzyl L-glutamate)) and 30 (x . y, C block, poly(perfluoro ether)). The sketch illustrates the self-assembly of the pentablock to spherical and cylindrical micelles with a fluorinated compartment in the center (red and red coils, liquidlike, diameter ) 6 nm) surrounded by a first shell of parallel-stranded β-sheets (blue and blue arrows, shell thickness is 2 nm) and a second shell of water-swollen PEO (brownish coils, thickness is 7 nm).
†
Federal Institute for Materials Research and Testing. Max Planck Institute of Colloids and Interfaces. § Free University of Berlin. ‡
(1) Tu, R. S.; Tirrell, M. AdV. Drug DeliVery ReV. 2004, 56, 1537-1563. (2) Lutz, J. F.; Laschewsky, A. Macromol. Chem. Phys. 2005, 206, 813-817. (3) Stahler, K.; Selb, J.; Candau, F. Langmuir 1999, 15, 7565-7576. (4) Stahler, K.; Selb, J.; Candau, F. Mater. Sci. Eng., C 1999, 10, 171-178. (5) Weberskirch, R.; Preuschen, J.; Spiess, H. W.; Nuyken, O. Macromol. Chem. Phys. 2000, 201, 995-1007. (6) Kotzev, A.; Laschewsky, A.; Adriaensens, P.; Gelan, J. Macromolecules 2002, 35, 1091-1101. (7) Kubowicz, S.; Baussard, J. F.; Lutz, J. F.; Thunemann, A. F.; von Berlepsch, H.; Laschewsky, A. Angew. Chem., Int. Ed. 2005, 44, 5262-5265. (8) Laschewsky, A. Curr. Opin. Colloid Interface Sci. 2003, 8, 274-281. (9) Li, Z. B.; Kesselman, E.; Talmon, Y.; Hillmyer, M. A.; Lodge, T. P. Science 2004, 306, 98-101.
poly(γ-benzyl-L-glutamate) blocks (PBLG, block B), and a hydrophobic perfluoroether block (PFPE, block C) (PEO-bPBLG-b-PFPE-b-PBLG-b-PEO). Materials and Methods Chemicals. N-Carboxy anhydride (NCA) of γ-benzyl-L-glutamate was synthesized in THF in the presence of triphosgene by applying standard procedures.10 R-Methoxy-ω-primary amino-poly(ethylene oxide) (Mn ) 5000 g mol-1; Mw/Mn ) 1.05, DP ) 110) was purchased (10) Penczek, S. Models of Biopolymers by Ring-Opening Polymerization; CRC Press: Boca Raton, FL, 1990; p 388.
10.1021/la0533720 CCC: $33.50 © 2006 American Chemical Society Published on Web 02/16/2006
Two-Compartment Micellar Assemblies from Rapp Polymere GmbH. Prior to polymerization, primary amino functional PEO was dissolved in a mixture of THF and water and treated with aqueous hydrochloric acid to form the corresponding ammonium chloride functional PEO.11,12 Solvents were removed by rotary evaporation, and then the isolated polymer was dissolved in benzene and dried by freeze drying. R-ω-Dicarboxyl perfluoropolyether (Fluorolink C) was purchased from Ausimont GmbH and used as received (Mn ) 3800 g mol-1, DP ) 30, R-R functionalized with carboxylic acid groups). N-hydroxysuccinimide (NHS) and N,N′dicyclohexylcarbodiimide (DCC) were both purchased from Aldrich and used as received. Syntheses. Diblock Copolymer PEO-b-PBLG. The diblock PEOb-PBLG was obtained via a one-pot polymerization of the NCA of γ-benzyl-L-glutamate initiated by an ammonium chloride functional poly(ethylene oxide) macroinitiator.11,12 In a dried flask, we introduced a DMF solution of the PEO macroinitiator and subsequently a DMF solution of the NCA. The mixture was stirred for 5 days under an argon atmosphere. After polymerization, the solvent was concentrated to a minimum amount under high vacuum. The concentrated DMF solution was precipitated in petrol ether and subsequently dried under vacuum. The final sample contained small amounts of low-molecular-weight homopolymer poly(γ-benzyl-Lglutamate), probably initiated by nucleophilic impurities during purification. Homopolymer was separated from the diblock copolymer by selective precipitation of the diblock in cold DMF (-20 °C).13 Final diblock copolymers were analyzed by 1H NMR (DMSOd6) and size-exclusion chromatography (SEC) using N-methylpyrrolidone (NMP) as an eluent. Both measurements indicated that the prepared copolymer PEO-b-PBLG had a very well-defined molecular structure: Mn ) 6000 g‚mol-1, Mw/Mn < 1.05, DPPBLG ) 8. Details of the characterization are described elsewhere.12 Pentablock Copolymer PEO-b-PBLG-b-PFPE-b-PBLG-b-PEO. The coupling of PEO-b-PBLG with R-ω-dicarboxyl perfluoropolyether (PFPE) was assisted by NHS and DCC. The main problem in this coupling reaction was to find a common solvent to dissolve both PEO-b-PBLG and the perfluoropolyether. This was difficult because PFPE is typically soluble only in fluorinated solvents, in which PEO-b-PBLG precipitates. However, because of their polar endgroups R-R-dicarboxyl perfluoropolyethers behave as surfactants in water or polar aprotic solvents.14-16 Thus, in polar solvents, an interfacial coupling reaction between PEO-b-PBLG and PFPE could be carried out at the surface of preformed micelles of PFPE. In dimethylformamide (DMF), PFPE was found to form micelles spontaneously (the studied polymer concentration was around 7 g L-1); these possessed an average hydrodynamic diameter of approximately 15 nm, as evidenced by dynamic light scattering measurements (Figure 2). Therefore, these dilution conditions were used for the coupling reaction. Typically, the R-ω-dicarboxyl perfluoropolyether (1 equiv of COOH functions) was first dissolved in dimethylformamide (DMF) (initial polymer concentration was 7 g L-1). Subsequently, PEO-b-PBLG (1 equiv of NH2 functions), NHS (5 equiv as compared to COOH functions), and DCC (20 equiv as compared to COOH functions) were added to the solution. The mixture was treated with ultrasound for 5 min in an ultrasonic bath and subsequently stirred for 18 h at room temperature. During the reaction, the formation of a white precipitate was observed. The latter corresponds to the urea derivative of DCC, which is not soluble in DMF. After reaction, the precipitate was filtered out, and a clear solution was obtained. Methods. Static Light Scattering (SLS). The static light scattering measurements were performed on a Sofica instrument equipped with a He-Ne laser (λ ) 632.8 nm). The scattered light intensity (11) Dimitrov, I.; Schlaad, H. Chem. Commun. 2003, 23, 2944-2945. (12) Lutz, J. F.; Schutt, D.; Kubowicz, S. Macromol. Rapid Commun. 2005, 26, 23-28. (13) Hruska, Z.; Riess, G.; Goddard, P. Polymer 1993, 34, 1333-1335. (14) Chittofrati, A.; Boselli, V.; Visca, M.; Friberg, S. E. J. Dispersion Sci. Technol. 1994, 15, 711-726. (15) Chittofrati, A.; Lenti, D.; Sanguineti, A.; Visca, M.; Gambi, C.; Senatra, D.; Zhen, Z. Colloids Surf. 1989, 41, 45-59. (16) Kasai, P. H. J. Appl. Polym. Sci. 1995, 57), 797-809.
Langmuir, Vol. 22, No. 6, 2006 2507
Figure 2. Dynamic light scattering measurements of DMF solutions of PFPE (dashed line), a mixture of PFPE and PEO-b-PBLG before the coupling reaction (dotted line), and PFPE/PEO-b-PBLG after the coupling reaction (solid line). was recorded at scattering angles from 30 to 145° at 5° intervals. Toluene was used for calibration to determine the scattering-volumecorrected Rayleigh ratio. All measurements were performed at (25 ( 0.1 °C). A micellar solution (10 mL) was placed into a cylindrical quartz cuvette with a diameter of 2 cm. Prior to measurement, the sample was filtered with a 0.45 µm Millipore syringe filter to free it from dust particles. The cuvettes were extensively cleaned in an ultrasonic bath using a surfactant solution (Hellmanex, Hellma) and subsequently washed several times with distilled water to completely remove any remaining surfactants. Finally, they were washed for 20 min with boiling acetone in a special dust-free chamber and stored in an desiccator. Dynamic Light Scattering (DLS). The dynamic light scattering measurements were performed using a Malvern Instruments particle sizer (HPPS-ET 5002) (Malvern Instruments, U.K.) equipped with a He-Ne laser (λ ) 632.8 nm). The scattering data were recorded at (25 ( 0.1 °C) in back-scattering modus at a scattering angle of 2θ ) 173°. The aqueous sample solution (1.5 mL) was placed into a square 10 mm × 10 mm quartz cuvette. Prior to measurement, the sample was filtered with a 0.45 µm Millipore syringe filter to free it from dust particles. Analytical Ultracentrifugation (AUC). Analytical ultracentrifugation was carried out using a Beckmann-Coulter Optima XL-I ultracentrifuge (Beckmann Coulter, Palo Alto, CA) at (20 ( 0.1) °C and 50 000 min-1 for sedimentation velocity experiments. The sample solutions were placed into a 12 mm double-sector cell of carbon-filled Epon. Detection of the sedimenting boundary was carried out using Rayleigh interference optics equipped with a HeNe laser (λ ) 632.8 nm). Cryo-Transmission Electron Microscopy (cryo-TEM). The samples for cryo-TEM were prepared at room temperature by playing a droplet (10 µL) of the solution onto a hydrophilized perforated carbon film grid (60 s of plasma treated at 8 W using a Baltec Med 020 device). The excess fluid was blotted off to create an ultrathin layer (typical thickness of 100 nm) of solution spanning the holes of the carbon film. The grids were immediately frozen in liquid ethane at its freezing point (-184 °C) using a standard plunging device. Ultrafast cooling is necessary for artifact-free thermal fixation (vitrification) of the aqueous solution avoiding crystallization of the solvent or rearrangement of the assemblies. The vitrified samples were transferred under liquid nitrogen into a Philips CM12 transmission electron microscope using a Gatan cryoholder and cryostage (model 626). Microscopy was carried out at -175 °C (sample temperature) using the microscope’s low-dose protocol at a primary magnification of 58 300. The defocus was chosen to be 2.0 µm. Infra-red Spectroscopy. IR spectra were recorded on a BioRad 6000 FT-IR. All samples were measured in the solid state using a single reflection diamond ART.
Results and Discussion Pentablock Formation by the Coupling Reaction. ABCBA was synthesized in a two-step reaction. First, the diblock copolymer PEO-b-PBLG was synthesized by a ring-opening
2508 Langmuir, Vol. 22, No. 6, 2006
Thu¨nemann et al.
Figure 3. FT-IR spectra of PFPE (a), PEO-b-PBLG (b), and the formed material after the coupling reaction (c). Table 1. Elemental Analysis of PEO-b-PBLG, PFPE, and the Material Obtained after the Coupling Reaction of PEO-b-PBLG and PFPE PEO-b-PBLG/PFPE
PEO-b-PBLG
PFPE
element
exptl
calcd
exptl
calcd
exptl
calcd
%C %H %N %O %F
52 7 2 31 8
49.8 6.6 1.5 28.6 13.5
57 9 2 32
57.3 8.4 1.8 32.5
21 0
20.8 0.1
14 65
15.5 63.7
polymerization of the N-carboxy anhydride of γ-benzyl-Lglutamate using an ammonium chloride-functionalized poly(ethylene oxide) as a macroinitiator containing 110 ethylene oxide units. The PBLG block consists of eight benzyl glutamate units. In a second synthesis step, the PEO-b-PBLG diblock was coupled covalently with PFPE (DP ) 30) using a DCC/NHS-assisted carboxyl-amine coupling reaction in DMF solution. The solution in DMF before and after reaction was analyzed by dynamic light scattering (Figure 2). It can be seen that a new population possessing an average diameter of approximately 140 nm was observed after the reaction, which indicates that the coupling reaction was successful. We attribute this to highly swollen aggregates as expected for the product. To isolate the formed polymer, the reaction mixture was first transferred from DMF to pure water via stepwise dialysis against water/DMF mixtures with successive increases in the water content of the dialysis solution via 30%, 50%, 75%, and finally pure water. Subsequently, polymer was isolated by freeze drying. At last, a white powder was obtained. The latter could not be reliably studied by solvent-dependent analytical techniques such as solution NMR or size-exclusion chromatography because no universal solvent exists for dissolving the three segments composing the resulting copolymer. A single chain cannot be analyzed by NMR or SEC because the formed polymer is a surfactant in most solvents and therefore always self-assembles into nanostructures. Therefore, the final product was characterized using elemental analysis and FT-IR spectroscopy. Table 1 shows the results obtained with elemental analysis. Good agreement between experimental and theoretical numbers was found. Deviations between calculated and observed values are probably due to the relatively high uncertainty in the composition of the PFPE, which was expected from this commercially available compound. In addition, Figure 3 compares the FT-IR spectra measured for PEO-b-PBLG, PFPE, and the formed material. In the spectrum of PFPE, an intense carboxylic acid vibration band is present at 1780 cm-1 whereas it is absent in the spectrum of the formed material. Therefore, we conclude that within experimental error all of the carboxylic acid functions of PFPE form a bond with
Figure 4. Circular dichroism spectra of the AB diblock (dashed line) and the ABCBA pentablock (solid line) in aqueous solution. The maxima are at 198 nm, and the minima are at 226 nm.
the terminal amino functions of the PBLG block. This new formed amide bond is difficult to visualize by FT-IR spectroscopy because it overlaps with the amide bonds of the PBLG block. However, a deformation of the bands in the range of 1700-1650 cm-1 can be observed, which can be attributed to the amide bond of the pentablock. The other vibrational bands of the polymer chain are practically unchanged as expected. In summary, DLS, FT-IR, and elemental analysis prove that the coupling reaction between PEO-b-PBLG and PFPE was successful. However, it must be mentioned that small amounts of triblock copolymer PEO-bPBLG-b-PFPE and unreacted chains of PEO-b-PBLG and PFPE can be present in the final sample. Secondary Structure of the Peptide Blocks. The PBLG block can adopt different conformations of R-helix, β-sheet, and random coil, which probably play a crucial role in the supermolecular ordering of the pentablock. Therefore, we measured the circular dichroism (CD) of the pentablock and the pristine PEO-PBLG diblock to reveal their secondary structures. It can be seen in Figure 4 that the CD spectra of both display a maximum at 198 nm and a minimum at 226 nm. The maximum of the pentablock is slightly broader and the minimum is more pronounced than for the diblock. The shape of both spectra is typical for an R-Lglutamic acid oligomer (DP ) 8) showing a β-sheet conformation in its protonated state.17 Interestingly, we observe bathochromic shifts of ∆λ ) 5 nm for the maximum (from 193 to 198 nm) and ∆λ ) 9 nm for the minimum (from 217 to 226 nm) when comparing the CD spectrum of the R-L-glutamic acid octamer17 with that of our block copolymers. We assume that this is an effect of the benzyl side chains of the peptide block that shifts the π-π* and n-π* transitions slightly to higher wavelengths by intramolecular interactions. From the CD spectrum, we conclude that the PBLG block most likely forms a parallel β-sheet structure in the diblock precursor and in the pentablock. The deeper minimum of the pentablock compared to that of the diblock can be attributed to a higher amount of β-sheet conformation in the pentablock. It is reported that the thermodynamically stable conformation of the poly(γ-benzyl-L-glutamate) homopolymer is an R-helix for the degree of polymerization (DP) > 10 and a β-sheet for DP < 10 and DP > 4.18,19 In the present case, DP ) 8, so our conclusion of β-sheets as the dominant conformation of the B blocks is in agreement with the recent literature. Structure of the Micelles. We expected that micelles are formed because of the immiscibility of the different blocks and (17) Rinaudo, M.; Domard, A. J. Am. Chem. Soc. 1976, 98, 6360-6364. (18) Kricheldorf, H. R. Alpha-Amino Acid-N-Carboxy-Anhydrides and Related Heterocycles. Springer-Verlag: Berlin, 1987. (19) Singh, B. R. Basic Aspects of the Technique and Applications of Infrared Spectroscopy of Peptides and Proteins; American Chemical Society: Washington, DC, 2000; pp 2-37.
Two-Compartment Micellar Assemblies
Figure 5. Guinier plot of the micelles of the pentablock copolymer in water (circle). The straight lines are linear fits resulting tentatively in radii of gyration of Rg ) 95 and 65 nm. The inset shows a Zimm plot of the same data, which results in Rg ) 110 nm (straight line) and is indicative of cylindrical micelles.
Langmuir, Vol. 22, No. 6, 2006 2509
Figure 7. Cryo-TEM picture of spherical and cylindrical micelles in aqueous solution.
Figure 8. TEM image of micelles prepared from aqueous solution, negatively stained with uranyl acetate. Figure 6. Sedimentation coefficient distribution from ultracentrifugation measurements of micelles of the pentablock copolymer in aqueous solution.
the water insolubility of the B and C blocks. Therefore, we measured static and dynamic light scattering of solutions of ABCBA in water. A Guinier plot20 of the static light scattering measurements to determine the radius of gyration, Rg, is shown in Figure 5, but the data systematically deviate from Guninier’s law. Approximations in the lower-q range result in Rg ) 65 and 85 nm in the higher-q range (straight lines in Figure 5). In contrast to the Guinier plot, we found a large linear region in the Zimm plot, indicative of cylindrical micelles, resulting in Rg ) 110 nm (inset of Figure 5). The dynamic light scattering measurements reveal that two different populations of micelles are present with diffusion coefficients of 2.8 × 10-12 and 21.0 × 10-12 m2/s. Tentatively, we attribute this to a large population of cylindrical micelles with Rh ) 90 nm and a small population of spherical micelles with Rh ) 12 nm. We performed a sedimentation velocity measurement with an ultracentrifuge to prove the presence of two different populations. Indeed, the distribution of the sedimentation coefficients clearly shows two separated populations of micelles with a volume ratio of the larger (cylindrical) to smaller (spherical) micelles of 2:1 (cf. Figure 6). Electron microscopy was then performed to reveal the morphology of the micelles more precisely. Cryo-transmission electron microscopy of aqueous micellar solutions of the pentablock was then performed, resulting in the typical image shown in Figure 7. The image reveals a small number of uniformly dispersed spherical micelles with a relatively narrow size distribution of 6 to 7 nm and a large number of cylindrical micelles (20) Guinier, A. Ann. Phys., Paris 1939, 12, 161.
possessing a length of about 40 to 150 nm and a uniform thickness of 5 to 6 nm. We attribute the contrast of the objects to the presence of electron-rich fluorine atoms of the micellar core. The water-soluble PEO corona and the hydrophobic peptide block of the micelles were not directly observable. The overall diameter of the cylindrical micelles can be estimated from their distance to be around 22 to 27 nm. In addition, the TEM image in Figure 8 shows micelles prepared from aqueous solution, negatively stained with uranyl acetate. This image confirms the existence of predominantly cylindrical micelles with lengths of about 100 to 200 nm and a core diameter of about 6 nm. Similar to cryoTEM, a micellar core possessing a uniform thickness of 6 to 7 nm can be observed as a clear area. Moreover, a dark shell is observed around each cylindrical micelle. This contrast is due to a higher local concentration of the staining agent. The latter is probably due to the favored adsorption of the uranyl ions on the corona of the micelles. Therefore, Figure 8 visually confirms the overall dimensions of the micelles. By summarizing the structure information, we assume an onionlike core-shell morphology of coexisting spherical and cylindrical micelles as shown schematically in Figure 1. The aggregation number of the spherical and cylindrical micelles can be estimated from simple geometric considerations. Taking into account the size of the fluorinated core, estimated from cryoTEM (a sphere with a diameter of 7 nm containing C blocks), we determined an aggregation number of about 65 for the spherical micelles. For the cylindrical micelles, the aggregation number was calculated for a micellar length of 100 to 200 nm and a core of 6 nm containing the C blocks. This yields an aggregation number of 1000 to 2000 for the cylindrical micelles. One γ-benzyl-L-glutamate unit in β-sheet conformation has a length of 0.24 nm, and thus the B block has a contour length of about 2 nm. The β-sheet conformation is stretched; therefore, the thickness of the peptide shell can be assumed to be equal to
2510 Langmuir, Vol. 22, No. 6, 2006
the length of the B block. Hence, subtracting from the diameter of 24 nm the core diameter of 7 nm and twice the thickness of the B block, we expect the thickness of the outer PEO corona to be around 7 nm. This value corresponds to about 25% of the fully stretched PEO block, which is reasonable for a coiled conformation of the A block. The structure of the micelle is summarized in Figure 1. Therein, the core-shell-corona micelle is built by a soft liquidlike core formed by the C blocks. In contrast to the soft core, the surrounding shell is comparably stiff because the peptide chains are in the β-sheet conformation. Finally, the outer corona is coiled and swollen by water molecules.
Conclusions We were able to synthesize a pentablock copolymer by combining an R,ω-functionalized perfluoroether with two diblock
Thu¨nemann et al.
copolymers on both ends. The resulting ABCBA pentablock copolymer forms a mixture of a small population of spherical micelles and a large population of cylindrical micelles in water. The morphology of the micelles is most likely a core-shellcorona micelle (i.e., it contains two immiscible compartments (perfluoro ether and oligopeptide) that may be suitable as containers for the selective uptake of active agents). Acknowledgment. This work was supported by the Federal Institute of Materials Research and Testing, the Fraunhofer Society, and the Max Planck Society. We thank J.-F. Lutz for helpful advice and discussions pertaining to syntheses. LA0533720
