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Langmuir 2007, 23, 2224-2230
Biocompatible Polymer Vesicles from Biamphiphilic Triblock Copolymers and Their Interaction with Bovine Serum Albumin Alexander Wittemann,*,†,‡ Tony Azzam,† and Adi Eisenberg† Department of Chemistry, McGill UniVersity, 801 Sherbrooke Street West, Montreal, Quebec, Canada H3A 2K6, and Physikalische Chemie I, UniVersita¨t Bayreuth, UniVersita¨tsstrasse 30, 95440 Bayreuth, Germany ReceiVed September 25, 2006. In Final Form: NoVember 14, 2006 The self-assembly of the biamphiphilic triblock copolymer poly(ethylene oxide)-b-poly(caprolactone)-b-poly(acrylic acid) into polymer vesicles is studied. The vesicles provide both biocompatibility and biodegradability. Moreover, the biamphiphilic nature of the triblock copolymer provides different surface properties in the interior and in the outer interface of the vesicles. Preparation of the aggregates by direct dissolution of the copolymer in a solution of albumin does not alter the morphology of the aggregates, and thus, they have the potential to immobilize protein molecules. Since a part of the protein is encapsulated in the interior of the vesicles, they can be used as nanocontainers. A further fraction of the protein is bound to the outer interface, which is primarily composed of the poly(acrylic acid) tails. Immobilization of protein on the outer interface can stabilize the colloidal particles and also provide them with a biofunctional component.
Introduction
* To whom correspondence should be addressed. E-mail:
[email protected]. Phone: +49 921 55 2776. Fax: +49 921 55 2780. † McGill University. ‡ Universita ¨ t Bayreuth.
In recent years, much work has been devoted to robust polymer vesicles made of synthetic amphiphilic block copolymers.16-25 Such vesicles might be particularly suitable for a long-time storage of encapsulants. Ideally, submicrometer-sized capsules for entrapped enzymes should be robust, but also permeable for substrate molecules and their reaction products. Polymer vesicles were also prepared from copolymers which can be degraded under physiological conditions. Such polymer vesicles are more permeable and allow the release of their encapsulants in a manner akin to that found in liposomes.26-28 Often block copolymer aggregates are prepared by first dissolving the copolymer in an organic solvent common for all blocks.11,16,17 The self-assembly is induced by adding water, which is a poor solvent for the hydrophobic block, to the copolymer solution.18,19,22,23 This procedure is not applicable to the encapsulation of enzymes, since organic solvents can denature the proteins. However, polymer vesicles can also be prepared by direct dissolution of block copolymers in aqueous solution.28,29 Encapsulation of proteins can be achieved if the vesicles are formed in solutions of the protein. Self-assembly in the absence of organic solvent can also be achieved by film rehydration or electroformation.30-32 Both techniques were successfully applied to encapsulate protein molecules.30-32 Meier and co-workers
(1) Vriezema, D. M.; Aragone`s, M. C.; Elemans, J. A. A. W.; Cornelissen, J. J. L. M.; Rowan, A. E.; Nolte, R. J. M. Chem. ReV. 2005, 105, 1445. (2) Sukhorukov, G. B.; Brumen, M.; Donath, E.; Mo¨hwald, H. J. Phys. Chem. B 1999, 103, 6434. (3) Antipov, A. A.; Sukhorukov, G. B.; Donath, E.; Mo¨hwald, H. J. Phys. Chem. B 2001, 105, 2281. (4) Gao, C.; Leporatti, S.; Moya, S.; Donath, E.; Mo¨hwald, H. Langmuir 2001, 17, 3491. (5) Balabushevich, N. G.; Tiourina, O. P.; Volodkin, D. V.; Larionova, N. I.; Sukhorukov, G. B. Biomacromolecules 2003, 4, 1191. (6) Lvov, Y.; Antipov, A. A.; Mamedov, A.; Mo¨hwald, H.; Sukhorukov, G. B. Nano Lett. 2001, 1, 125. (7) Lian, T.; Ho, R. J. Y. J. Pharm. Sci. 2001, 90, 667. (8) Hausschild, S.; Lipprandt, U.; Rumplecker, A.; Borchert, U.; Rank, A.; Schubert, R.; Fo¨rster, S. Small 2005, 1, 1177. (9) Chakrabarti, A. C.; Breaker, R. R.; Joyce, G. F.; Deamer, D. W. J. Mol. EVol. 1994, 39, 555. (10) O ¨ zden, M. Y.; Hasirci, V. N. Biochim. Biophys. Acta 1991, 1075, 102. (11) Discher, B. M.; Hammer, D. A.; Bates, F. S.; Discher, D. E. Curr. Opin. Colloid Interface Sci. 2000, 5, 125. (12) Gravano, S. M.; Borden, M.; von Werne, T.; Doerffler, E. M.; Salazar, G.; Chen, A.; Kisak, E.; Zasadzinski, J. A.; Patten, T. E.; Longo, M. L. Langmuir 2002, 18, 1938. (13) Mu, M.; Ning, F.; Jiang, M.; Chen, D. Langmuir 2003, 19, 9994.
(14) Lawson, G. E.; Lee, Y.; Singh, A. Langmuir 2003, 19, 6401. (15) Sakai, H.; Takeoka, S.; Park, S. I.; Kose, T.; Nishide, H.; Izumi, Y.; Yoshizu, A.; Kobayashi, K.; Tsuchida, E. Bioconjugate Chem. 1997, 8, 23. (16) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967. (17) Lim Soo, P.; Eisenberg, A. J. Polym. Sci., Part B: Polym. Phys. 2004, 42, 923. (18) Zhang, L.; Eisenberg, A. Science 1995, 268, 1728. (19) Zhang, L.; Eisenberg, A. J. Am. Chem. Soc. 1996, 118, 3168. (20) Antonietti, M.; Fo¨rster, S. AdV. Mater. 2003, 15, 1323. (21) Discher, B. M.; Won, Y.-Y.; Ege, D. S.; Lee, J. C.-M., Bates, F. S.; Discher, D. E.; Hammer, D. A. Science 1999, 284, 1143. (22) Luo, L.; Eisenberg, A. J. Am. Chem. Soc. 2001, 123, 1012. (23) Luo, L.; Eisenberg, A. Langmuir 2001, 17, 6804. (24) Ding, J.; Liu, G. Macromolecules 1997, 30, 655. (25) Kukula, H.; Schlaad, H.; Antonietti, M.; Fo¨rster, S. J. Am. Chem. Soc. 2002, 124, 1658. (26) Meng, F.; Hiemstra, Ch.; Engbers, G. H. M.; Feijen, J. Macromolecules 2003, 36, 3004. (27) Meng, F.; Engbers, G. H. M.; Feijen, J. J. Controlled Release 2004, 101, 187. (28) Ahmed, F.; Discher, D. E. J. Controlled Release 2004, 96, 37. (29) Bryskhe, K.; Jansson, J.; Topgaard, D.; Schille´n, K.; Olsson, U. J. Phys. Chem. B 2004, 108, 9710.
In nature, biochemical reactions take place in confined micrometer- or submicrometer-sized environments. Compartmentalization of guest molecules such as hydrophilic drugs or enzymes in space and time is also of considerable interest for chemists in the design of highly efficient conversions or even cascade reactions.1 Hollow capsules in the submicrometer range to be used as carriers of immobilized enzymes or drugs can be prepared by alternate deposition of oppositely charged polyelectrolytes (LBL) on colloidal templates.2-6 An alternative approach is the self-assembly of amphiphiles into vesicular structures, which is provided by nature itself. Lipids self-assemble into semipermeable vesicles (liposomes) in aqueous solution; these liposomes are known to encapsulate hydrophilic drugs as well as proteins.1,7-10 However, their wall is rather thin and thus lacks robustness.11-13 Attempts were made to improve the robustness by cross-linking the lipid molecules14 or by employing poly(ethylene oxide)-conjugated phospholipids.15
10.1021/la062805b CCC: $37.00 © 2007 American Chemical Society Published on Web 01/05/2007
Polymer Vesicles from Triblock Copolymers
studied copolymer vesicles containing channel proteins in the wall which mediate the diffusion of substrate molecules to enzymes in the interior of the capsules.33-35 The authors showed that the enzymatic activity is retained in such a microcompartment. Thus, these systems are appropriate to create submicrometersized plants. Napoli et al. prepared oxidation-sensitive polymer vesicles.36 Self-destruction of the vesicular structure is achieved through production of hydrogen peroxide, which is produced by encapsulated glucose oxidase. Such vesicles are of potential use as drug delivery systems. Polymer vesicles obtained from biamphiphilic ABC block copolymers may provide different properties on the outer surface and in the interior. For instance, enzyme molecules might be selectively immobilized on either the inner or the outer surface, which might allow selective immobilization. Up to now, only a few studies have been devoted to this class of polymer vesicles.37-39 Recently, we presented the synthesis of biamphiphilic triblock copolymers of poly(ethylene oxide)-b-poly(caprolactone)-b-poly(acrylic acid) (PEO-b-PCL-b-PAA) prepared by a combination of ROP (ring-opening polymerization) and ATRP (atom transfer radical polymerization).40 PEO is a hydrophilic, nontoxic polymer which has been approved by the United States Food and Drug Administration (FDA) for medical uses.41 It is known for its low protein adsorption and low cell adhesion. PCL has also been approved by the FDA. The hydrophobic polymer is reabsorbable, which means that it can be digested by the body and disposed of without a trace.42,43 PAA is a bioadhesive, FDA-approved polymer which has good and instantaneous mucoadhesive properties.44,46 It provides numerous advantages as a coating material for stabilization and surface modification. Preliminary experiments showed that polymer vesicles and large compound micelles (LCMs) can be prepared by direct dissolution of the triblock copolymer PEO-b-PCL-b-PAA in aqueous solutions. These aggregates are biocompatible and biodegradable, as each block of the copolymer is approved for food and drug consumption and the PCL block, which forms the wall, can be degraded by hydrolysis.47,48 Moreover, the biamphiphilic nature of the triblock copolymer can yield polymer vesicles of different surfaces inside and outside.49,22 (30) Lee, J. C.-M.; Bermudez, H.; Discher, B. M.; Sheehan, M. A.; Won, Y.-Y.; Bates, F. S.; Discher, D. E. Biotechnol. Bioeng. 2001, 73, 135. (31) Brannan, A. K.; Bates, F. S. Macromolecules 2004, 37, 8816. (32) Arifin, D. R.; Palmer, A. F. Biomacromolecules 2005, 6, 2172. (33) Nardin, C.; Meier, W. ReV. Mol. Biotechnol. 2002, 90, 17. (34) Benito, S. M.; Sauer, M.; Meier, W. Encycl. Nanosci. Nanotechnol. 2004, 6, 301. (35) Sauer, M.; Meier, W. In Polymer nanocontainers for drug deliVery; Svenson, S., Ed.; ACS Symposium Series 879 (Carrier-based drug delivery); American Chemical Society: Washington, DC, 2004; p 224. (36) Napoli, A.; Boerakker, M. J.; Tirelli, N.; Nolte, R. J. M.; Sommerdijk, N. A. J. M., Hubbell, J. Langmuir 2004, 20, 3487. (37) Yu, G.-E.; Eisenberg, A. Macromolecules 1998, 31, 5546. (38) Stoenescu, R.; Meier, W. Chem. Commun. 2002, 3016. (39) Liu, F.; Eisenberg, A. J. Am. Chem. Soc. 2003, 125, 15059. (40) Azzam, T.; Wittemann, A.; Eisenberg, A. In preparation. (41) Sinha, V. R.; Aggarwal, A.; Trehan, A. Am. J. Drug DeliVery 2004, 2, 157. (42) Rohner, D.; Hutmacher, D. W.; Cheng, T. K.; Oberholzer, M.; Hammer, B. J. Biomed. Mater. Res., B 2003, 66B, 574. (43) Thomas, V.; Dean, D. R.; Vohra, Y. K. Curr. Nanosci. 2006, 2, 155. (44) Bernkop-Schnu¨rch, A.; Leitner, V.; Moser, V. Drug DeV. Ind. Pharm. 2004, 30, 1. (45) Hornof, M.; Weyenburg, W.; Annick, L.; Bernkop-Schnu¨rch, A. J. Controlled Release 2003, 89, 419. (46) Luessen, H. L.; Verhoef, J. C.; Borchard, G.; Lehr, C. M.; de Boer, A. G.; Junginger, H. E. Pharm. Res. 1995, 12, 1293. (47) Tjong, S. C.; Xu, Y.; Meng, Y. Z. Polymer 1999, 40, 3703. (48) Zhao, Y.; Hu, T.; Lv, Z.; Wang, S.; Wu, C. J. Polym. Sci., Part B: Polym. Phys. 1999, 37, 3288. (49) Luo, L.; Eisenberg, A. Angew. Chem., Int. Ed. 2002, 41, 1001.
Langmuir, Vol. 23, No. 4, 2007 2225 Table 1. Characteristics of the Biamphiphilic Triblock Copolymers copolymera
Mn(NMR)b
Mn(SEC)c
PDIc
PEO45-b-PCL85-b-PAA110 PEO45-b-PCL85-b-PAA200 PEO45-b-PCL133-b-PAA165 PEO45-b-PCL150-b-PAA80
19500 26100 29000 24800
21200 39000 43100 39500
1.29 1.42 1.45 1.39
a The composition of the copolymers was derived from 1H NMR spectra of the precursor PEO-b-PCL-b-PtBA. b Number-average molecular weights Mn of PEO-b-PCL-b-PAA as calculated from the composition given in the first column. c Mn and polydispersity indices (PDI ) Mw/Mn, where Mw is the weight-average molecular weight) of the precursor PEO-b-PCL-b-PtBA as determined by size exclusion chromatography.
In the present study we expose the self-assembled aggregates to a biological compound. Fluorescence-labeled bovine serum albumin (FITC-BSA) is applied as a hydrophilic model protein. The direct self-assembly of the triblock copolymer in the protein solution is investigated. Immobilization of the hydrophilic protein molecules is achieved either by encapsulation or by adsorption of the protein on the corona of the aggregates. The different natures of the inner and outer interface of the vesicles permit preferential adsorption of the protein to one of the interfaces. Experimental Section Materials. Poly(ethylene oxide) monomethyl ether (Mn ≈ 2000), -caprolactone, stannous(II) octoate, 2-bromoisobutyryl bromide (98%), tert-butyl acrylate (tBA), triethylamine, copper bromide (CuBr), N,N,N′,N′,N′′-pentamethyldiethylenetriamine (PMDETA) and trimethylsilyl iodide ((TMS)I) were purchased from Aldrich. PEO, -caprolactone, tBA, triethylamine, tetrahydrofuran, and toluene were purified as decribed in ref 40. Bovine serum albumin, fluorescein isothiocyanate conjugate (12 mol of FITC/mol of BSA, Sigma), and Nile red (Molecular Probes) were used without further purification. Deionized water obtained from a reverse osmosis water purification system (Millipore Academic) was used throughout the entire study. All other chemicals and solvents were of analytical grade and were used as received. Synthesis of the Triblock Copolymers PEO-b-PCL-b-PAA. The synthesis of the biamphiphilic triblock copolymers listed in Table 1 is described in detail in ref 40. Briefly, the diblock copolymer PEO-b-PCL with a hydroxyl end group at the PCL block was synthesized by ring-opening polymerization according to the method of Bogdanov et al.50 Poly(ethylene oxide) monomethyl ether was used as the initiator and stannous octoate as the catalyst. The terminal hydroxyl end groups of the PCL block were acetylated with 2-bromoisobutyryl bromide to obtain PEO-b-PCL-Br, which serves as the macroinitiator for the synthesis of the third block by ATRP using tBA as the monomer and CuBr/PMDETA as the catalyst.40 PEO-b-PCL-b-PtBA was converted into PEO-b-PCL-b-PAA by hydrolysis with (TMS)I according to a protocol established by Zhang et al.51 (TMS)I is an effective reagent for the hydrolysis of PtBA, but it is mild enough to avoid degradation of the PCL block. Analysis of the Triblock Copolymers PEO-b-PCL-b-PAA. The average molecular weight and polydispersity of the copolymers were determined by size exclusion chromatography (SEC) in THF at room temperature (Table 1). The instrument was equipped with a Waters 510 liquid chromatography pump, two (HR1 and HR4) Styragel columns connected in series, and a refractive index detector (Varian RI-4). The calibration was performed using polystyrene standards of narrow molecular weight distribution (Scientific Polymer Products Inc., Ontario, NY). NMR spectra were recorded on a Varian XL-300 spectrometer to determine the composition of the triblock copolymer before (50) Bogdanov, B.; Vidts, A.; Van Den Bulcke, A.; Verbeeck, R.; Schacht, E. Polymer 1998, 39, 1631. (51) Zhang, Q.; Remsen, E. E.; Wooley, K. L. J. Am. Chem. Soc. 2000, 122, 3642.
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hydrolysis. CDCl3 and d6-DMSO were used as solvents and TMS as the internal reference. Since the degree of polymerization (DP) of the narrowly disperse PEO block is known (45), the total molecular weight of the triblock copolymer can be calculated (Table 1). 1H NMR (CDCl3): δ 1.05-1.98 (m), 2.15-2.4 (m), 3.38 (s), 3.5-3.8 (s, br), and 3.95-4.15 (t). Preparation of the Aggregates. Self-assembly of the aggregates of the triblock copolymers PEO-b-PCL-b-PAA was accomplished by direct dissolution of the copolymer in 10 mM MOPS buffer (pH 7.1) to yield a 0.1 wt % solution. The buffer solution ensured a constant pH and ionic strength during aggregation. The oxygen-rich backbone of PCL results in a weak but partial hydrophilicity of the hydrophobic block, which does allow the self-assembly of the aggregates to proceed slowly.40 Visual inspection revealed that solid polymer was still present to a certain extent in the turbid samples during the first hours of stirring at room temperature. To ensure complete conversion of the solid copolymer into self-assembled aggregates, each sample was vigorously stirred for at least 4 days. PCL is known for its stability against hydrolysis under mild conditions.52 Thus, chain scission of the PCL block is not expected during the extended equilibration time. Immobilization of Bovine Serum Albumin. The aggregation was performed in solutions of fluorescein-labeled BSA (FITCBSA) in 10 mM MOPS buffer (pH 7.1) of various concentrations. After 4 days of equilibration, unbound protein was removed by exhaustive dialysis. Control experiments proved that the applied dialysis membrane (Spectra/Por Biotech PVDF membrane, MWCO 500000) provides for efficient removal of free protein. The progress of the dialysis was monitored via the fluorescence and absorption of the protein outside the dialysis bag and continued until no further protein could be removed. A UV/vis spectrometer (Varian Cary 300 Bio) was used to determine the amount of protein which was bound onto the aggregates. The spectra were recorded over a wavelength range of 200-800 nm. Two individual scans acquired with 0.2 nm resolution at a scan rate of 120 nm/min were averaged. Spectra of the immobilized protein were obtained after subtraction of the spectra of the bare aggregates. A calibration curve of the absorption of FITC-BSA at 465 and 491 nm was used to determine the amount of immobilized protein. Transmission Electron Microscopy (TEM). TEM studies of the self-assembled aggregates were performed on a JEOL 2000FX instrument operating at an accelerating voltage of 80 kV. One drop of the aqueous suspensions was deposited onto a copper grid coated with carbon (Electron Microscopy Science, Hatfield, PA). Excess solvent was swept away by touching the edge of the grids with filter paper (Whatman-1). The grids were allowed to dry at ambient temperature for 24 h. TEM images were recorded digitally by a CCD camera (Gatan 792 Bioscan) and processed with a digital imaging system (Gatan Digital Micrograph 3.9). Dynamic Light Scattering (DLS). DLS measurements were performed on a Brookhaven Instruments goniometer system equipped with a Compass 315M-150 laser (Coherent Technologies) which provides a wavelength of 532 nm and a BI9000 AT digital correlator. Autocorrelation functions were recorded at 60°, 75°, 90°, 105°, and 120°. Hydrodynamic radii were obtained by a cumulant analysis53 from the correlation functions. ζ Potential Measurements. The pH dependence on the electrophoretic mobilities of the aggregates was measured on a Malvern Zetasizer Nano ZS in conjunction with an MPT2 autotitrator (Malvern). The electrophoretic mobilities (u) were converted into ζ potentials via the Smoluchowski equation ζ ) uη/0, where η denotes the viscosity and 0 the permittivity of the suspension). Laser Scanning Confocal Microscopy (LSCM). The hydrophobic dye Nile red was used to selectively stain the hydrophobic walls of the self-assembled aggregates. A 6 µL sample of a solution of Nile red in DMF was added to 2 mL of a 0.025 wt % suspension of the aggregates. After 1 day of equilibration one drop of the
suspension was placed on an adhesive microscope slide (Canadawide Scientific Polysine microscope slides). The aggregates were viewed with an inverted confocal laser scanning microscope (Zeiss LSM 510, 63× oil objective) equipped with an argon laser and a helium/ neon mixed gas laser with excitation wavelengths of 488 and 543 nm, respectively. Clear differentiation between the fluorescence of the FITC labels and Nile red was achieved by an optical band-pass filter for use in the 505-530 nm wavelength range and a low-pass filter designed for transmittance of a wavelength of 560 nm. Scans at a resolution of 1024 × 1024 pixels were taken in the line-averaging mode. Micrographs were analyzed by LSM software (Zeiss LSM 510, version 2.8 SP1).
(52) Baras, B.; Benoit, M.-A.; Dupre´, L.; Poulain-Godefroy, O.; Schacht, A.M.; Capron, A.; Gillard, J.; Riveau, G. Infect. Immun. 1999, 67, 2643. (53) Frisken, B. J. Appl. Opt. 2001, 40, 4087.
(54) Nardin, C.; Hirt, T.; Leukel, J.; Meier, W. Langmuir 2000, 16, 1035. (55) Graff, A.; Sauer, M.; Van Gelder, P.; Meier, W. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5064.
Results and Discussion Analysis of PEO-b-PCL-b-PAA Aggregates. Polydispersities are reported as the weight-average molecular weight Mw divided by the number-average molecular weight Mn. Typical ratios for the triblock copolymers are 1.29-1.45 (Table 1). One might speculate that such a polydispersity might prevent the formation of well-defined polymer vesicles. However, Meier and co-workers showed that polydispersity does not present a severe limitation to vesicle formation.54-55 The authors prepared vesicles from an ABA triblock copolymer consisting of a hydrophobic midblock of poly(dimethylsiloxane) and two hydrophilic blocks of poly(2-methyloxazoline). A polydispersity of 1.7 was reported for the triblock copolymer.54,55 The fact that polydisperity does not play a decisive role in the present case is corroborated by TEM. Typical aggregates obtained from the biamphiphilic triblock copolymers are shown in Figure 1. Vesicles are the dominant type of aggregates which were obtained from the copolymers PEO45-b-PCL85-b-PAA110 and PEO45-b-PCL85-b-PAA200. The lengths of the PCL blocks are the same for both polymers, as they were made of the same precursor diblock copolymer PEO45-b-PCL77-Br. The deviation in the length of the PCL block of the triblock from the diblock copolymers can be attributed to experimental error, which arises from the determination of the block length from NMR data. However, an increase in the length of the PCL block of the triblock was found for all triblock copolymers. We attribute this to a loss of a small fraction of triblock copolymer during the purification by precipitation from methanol/H2O.40 This fraction would contain molecules of lower mass which are more soluble in the precipitating solvent mixture. Since PCL forms the wall of the vesicular aggregates and as the length of the PCL block is the same for both PEO45-b-PCL85-b-PAA110 and PEO45-bPCL85-b-PAA200, TEM images revealed a constant wall thickness of 12 nm for aggregates of both copolymers. In addition to vesicles, a few LCMs were also found (Figure 1) which are similar in size to the vesicles. As different morphologies often coexist, it is hard sometimes to obtain one exclusive morphology by self-assembly. However, the absolute number of LCMs observed by TEM was negligible compared to the number of vesicles obtained from these copolymers. Thus, light scattering can be applied to reveal characteristic properties of the vesicles. DLS measurements were performed to elucidate the selfassembly of PEO45-b-PCL85-b-PAA110. The analysis was carried out using the methods of cumulants. The moment-based time autocorrelation function g2(τ) is given by53
(
g2(τ) ) B + β exp(-2Γτ) 1 +
µ2 2 µ3 3 τ - τ - ... 2! 3!
)
2
(1)
Polymer Vesicles from Triblock Copolymers
Langmuir, Vol. 23, No. 4, 2007 2227
Figure 2. DLS analysis of PEO45-b-PCL85-PAA110 vesicles in the presence of 10 mM MOPS buffer (pH 7.2). Dependence of the decay rate (Dq2) on the square root of the scattering vector (q2). The linear dependence indicates that only translational diffusion occurs. Triangles: aggregates prepared in the absence (open symbols) or presence (closed symbols) of 1 mg/mL FITC-BSA. Squares: Aggregates were ultrasonically treated during their preparation. Circles: Aggregates were prepared at 60 °C. See the text for further explanation.
Figure 1. Schematic illustration and TEM images of the aggregates obtained from the triblock copolymer PEO45-b-PCL85-b-PAA200. (A) Polymer vesicles: The hydrophobic PCL blocks form the vesicle wall of 12 nm thickness, while the hydrophilic PAA block is preferentially segregated to the outside and the shorter hydrophilic PEO block primarily toward the interior. (B) Large compound micelles (LCMs): LCMs are assemblies of reverse micelles in which the hydrophilic blocks form the core of the heterogeneities and the hydrophobic blocks surround the heterogeneities and thus form the continuous phase of the LCM.18,19 The hydrophilic chains on the outer surface give the LCMs considerable stability, which prevents agglomeration of the particles.18
B is the long-time value of g2(τ). Γ ) Dq2 denotes the decay rate, where D is the diffusion coefficient of the aggregates and q is the magnitude of the scattering vector (q ) (4πn0/λ) sin(θ/2), with θ the scattering angle, n0 the solution refractive index, and λ the wavelength of the scattered light). Figure 2 presents a plot of Dq2 versus q2 for the self-assembled aggregates. The linear dependence indicates that only translational diffusion occurs. Thus, the slope gives the apparent diffusion coefficient of the aggregates in the highly diluted suspensions. A consistent hydrodynamic radius rH of the aggregates can be calculated from the Stokes-Einstein equation, which reads
rH ) kT/6πηD
(2)
kT denotes the thermal energy, and η is the solvent viscosity. A hydrodynamic diameter of 242 nm was found for aggregates of PEO45-b-PCL85-b-PAA110 which were prepared by direct dissolution of the copolymer in 10 mM MOPS buffer at pH 7.2. It should be noted that DLS radii are dominated by the largest species in the suspension. In addition, factors which facilitate the self-assembly were studied by DLS. Self-assembly proceeds
much faster if the samples are treated ultrasonically. Moreover, a significantly smaller hydrodynamic diameter of 220 nm was found. Even smaller aggregates of 182 nm could be obtained if the samples were prepared at a temperature of 60 °C. This temperature is near the melting temperatures of PCL, which forms the wall of the vesicles. Thus, external stimuli such as temperature or ultrasonication can be applied to facilitate the formation of the aggregates. Previously, we studied aggregates prepared from PEO45-bPCL133-b-PAA165.40 Unlike the experiments described above, both vesicles of 20 nm wall thickness but also LCMs are the two dominant types of aggregates. The formation of LCMs, and thus the lower content of vesicles, might be related to the considerable length of the hydrophobic block of 930 bonds between pairs of atoms (i.e., 133 × 7, where 133 is the DP and 7 is the number of bonds between atoms in each repeat unit). Moreover, the hydrophilic blocks have to be of a certain length to obtain stable aggregates. For example, aggregates of PEO45-b-PCL150-b-PAA80 did not form stable suspensions. Because of the large size of some of the aggregates and the lower extent of stabilization by the shorter PAA block, these particles are subject to settling due to gravity. Corona chains can be directed preferentially to either the inside or the outside of the vesicles. Recently, Luo and Eisenberg prepared vesicles from a mixture of polystyrene-b-poly(acrylic acid) (PS-b-PAA) and polystyrene-b-poly(vinylpyridine) (PSb-P(4-VP)).49 Although the hydrophilic blocks of the copolymers are either acidic or basic, vesicles with preferentially segregated acidic and basic corona chains could be obtained.49 Since the triblock copolymer is biamphiphilic in the present study, in principle either PAA or PEO or, because of the compatibility of both blocks, eventually a mixture of both may form the corona of the vesicles. However, one would expect that, primarily, PAA is segregated to the outer interface since the PAA block is longer than the PEO block. Fluorescence quenching experiments have shown that longer blocks are preferentially segregated to the outer interface.23 Moreover, preferential segregation of charged groups to the outer interface minimizes the corona chain repulsion.39 This is further corroborated by the ζ potentials of the aggregates shown in Figure 3. Addition of base strongly affects the ζ potentials of aggregates prepared from the triblock copolymers in aqueous solution. Moreover, ζ potentials of aggregates prepared from the precursor diblock PEO-b-PCL-Br are approximately zero, independent of the pH (Figure 3). Thus,
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Figure 3. ζ potentials as a function of pH. Circles: aggregates prepared from the triblock copolymer PEO45-b-PCL85-PAA110 in water. Squares: aggregates prepared from the precursor diblock copolymer PEO45-b-PCL77-Br.
the carboxylic groups of the triblock copolymers are accessible to base, and the pattern of the ζ potentials in Figure 3 is related to the degree of dissociation of the carboxylic groups (pK ) 4.2). Full dissociation is observed at a pH above 5.5. Thus, the PAA blocks are preferentially segregated to the outer interface and thus form primarily the corona of the vesicles (Figure 1). Above pH 4, the absolute values of the ζ potential are beyond 40 mV. Therefore, the suspension of the self-assembled aggregates is very stable. Immobilization of Fluorescence-Labeled BSA. Albumin is the most abundant plasma protein and contributes 80% of the osmotic blood pressure.56,57 The protein is believed to have several physiological functions, but the ability to bind low-molecular weight ligands including drugs might be considered as the most important one.56 Despite its physiological role, BSA is used to stabilize colloidal particles, preventing especially unspecific agglomerates in immunoassays. Thus, BSA was chosen as a model protein to provide the biocompatible vesicular aggregates with a biofunctional component, which might be a first step toward an artificial cell. Biofunctional vesicles can be prepared by either binding the protein to the corona or encapsulation in the interior of the aggregates or a combination of both. In the present study, immobilization was accomplished by direct dissolution of the biamphiphilic triblock copolymer in a solution of FITC-BSA (Figure 4). Proteins are polyampholytes as they carry both positive and negative charges on their surface. Depending on the pH, proteins have either a net positive or negative charge, which is balanced at the isoelectric point. Thus, proteins interact strongly with linear polyelectrolytes of opposite charge. However, proteins form soluble complexes with polyelectrolytes even under a pH where the polyelectrolytes and the proteins are like-charged. Dubin and co-workers explained this unexpected finding by the presence of “patches” of opposite charge on the surface of the proteins.58,59 Comprehensive reviews on complexes between proteins and polyelectrolytes were given by de Kruif and co-workers60 and by Dubin and co-workers.58 In the present study, soluble complexes of the PAA blocks of the copolymer and the protein might be formed right after dissolution of the copolymer in the protein solution. Such complexes might possibly affect the size and the morphology of the aggregates or even prevent their formation. However, DLS (56) Lindup, W. E. In Plasma protein binding of drugsssome basic and clinical aspects; Bridges, J. W., Chasseaud, L. F., Gibson, G. G., Eds.; Progress in Drug Metabolism, Vol. 10; Taylor & Francis Ltd.: Levittown, PA, 1987. (57) Carter, D. C.; Ho, J. X. AdV. Protein Chem. 1994, 45, 153. (58) Cooper, C. L.; Dubin, P. L.; Kayitmazer, A. B.; Turksen, S. Curr. Opin. Colloid Interface Sci. 2005, 10, 52. (59) Seyrek, E.; Dubin, P. L.; Tribet, Ch.; Gamble, E. A. Biomacromolecules 2003, 4, 273. (60) de Kruif, C. G.; Weinbreck, F.; de Vries, R. Curr. Opin. Colloid Interface Sci. 2004, 9, 340 and references therein.
Figure 4. Immobilization of FITC-BSA. The aggregates are prepared by direct dissolution of the triblock copolymer in solutions of the protein. Thus, protein molecules can be encapsulated in the interior of the vesicles but also adsorbed on their outer interface, which is primarily composed of PAA. TEM images show spots of low transmission in the interior of the vesicular aggregates which might be due to encapsulated protein. See the text for further explanation.
Figure 5. Absorption spectrum of PEO45-b-PCL85-b-PAA110 vesicles with immobilized FITC-BSA (short dashed line). Subtraction of the spectrum of unloaded vesicles (long dashed line) gives the spectrum of the immobilized FITC-BSA (solid line).
did not detect any changes in the hydrodynamic radii of the aggregates whether they were formed in pure buffer solution or in protein solution (Figure 2). This is further corroborated by the TEM images (Figure 4), which demonstrate that the wall thickness of the vesicles is not affected either. Thus, complexes of proteins and the PAA blocks do not influence the structure of the selfassembled aggregates. The assembly into vesicular aggregates by direct dissolution of the triblock copolymer in a protein solution is thus appropriate to encapsulate protein molecules in their interior. In addition, adsorption onto the walls of the vesicles might occur. The total amount of adsorbed protein was determined by UV/vis spectroscopy after removal of unbound protein by exhaustive dialysis. The DLS studies showed that the structure of the aggregates is not altered by the protein. Hence, subtraction of the spectrum of vesicles with immobilized protein from the spectrum of the unloaded aggregates results in the spectrum of the immobilized protein (Figure 5). Table 2 shows the efficiency of the uptake of protein molecules. Efficiency on the order of 80% appears rather high. However, careful reference experiments of samples of a known protein amount proved that the method indeed allows a quantitative determination of the protein concentration in the suspension of the aggregates. Further control experiments with free FITC-BSA in solution showed that the dialysis membrane provides an efficient removal of free protein. An explanation for
Polymer Vesicles from Triblock Copolymers
Langmuir, Vol. 23, No. 4, 2007 2229 Table 2. Immobilization Efficiencya
copolymer
morphology
dH (nm)
c0,FITC-BSA (g/L)
cb,FITC-BSA (g/L)
eff (%)
PEO45-b-PCL85-b-PAA 110 PEO45-b-PCL85-b-PAA 110 PEO45-b-PCL133-b-PAA165 PEO45-b-PCL85-b-PAA 200
vesicles, few LCMs vesicles, few LCMs vesicles, LCMs vesicles, few LCMs
242 242 398 330
1.00 2.00 2.00 2.00
0.89 1.66 1.87 1.69
89 83 93 84
a Immobilization was accomplished by direct dissolution of 0.1 wt % copolymer in a solution of FITC-BSA in 10 mM MOPS buffer (pH 7.2). dH is the hydrodynamic diameter as determined by DLS, c0,FITC-BSA denotes the initial protein concentration, cb,FITC-BSA refers to the protein concentration in the suspension after exhaustive dialysis, and eff is the binding efficiency.
the highly efficient protein binding has to be found in the nature of immobilization, which will be discussed in more detail. Vesicles prepared in a protein solution encapsulate protein molecules in their interior.30-32 TEM images indicate that a certain fraction of the protein is encapsulated in the vesicles. One sees small spots of low transmission in the interior of dried vesicles (Figure 4). These dark spots were found on all images of vesicles which were prepared in the presence of proteins, whereas none could be observed for vesicles prepared in the pure buffer solution. Thus, these spots have to stem from entrapped protein molecules. ζ potential measurements revealed that the biamphiphilic nature of the triblock copolymer leads to the formation of vesicles where the PAA blocks are preferentially segregated to the outer interface while the PEO blocks primarily form the inner interface.40 Thus, the inner interface of the vesicular aggregates resembles a dense PEO brush. PEO brushes are well-known to suppress protein adsorption over long time scales.61,62 Hence, the entrapped protein molecules might remain in solution. Even if a minor part of the protein is adsorbed onto the inner wall of the vesicles, one has to expect free protein inside the vesicles of concentration similar to the protein concentration during the formation of the aggregates. Drying of the vesicles leads to solid agglomerates of the free protein in the interior, which can be seen as dark spots in the TEM images (Figure 4). In addition to this, protein molecules might adsorb to the outer interface, which is preferentially composed of PAA. Recently, it was shown that BSA and other protein molecules bind strongly to a densely packed layer of poly(acrylic acid) chains even under conditions where both components are like-charged.63-68 The reason for this surprising observation is ascribed to the release of numerous counterions of the PAA layer and the protein, which leads to a gain in entropy and thus presents a strong driving force for the adsorption.64 A review on the so-called polyelectrolytemediated protein adsorption (PMPA) was given recently.69 Hence, one should expect the PMPA also to take place on the outer interface of the aggregates. LSCM was used to image the aggregates. Either the fluorescence of the dye incorporated in the wall of the structures or the fluorescence of the dye binding to the protein can be imaged (Figure 6). As LSCM has a high resolution, even single aggregates can be monitored. However, vesicular structures cannot be seen in the LSCM images as the aggregates are in the submicrometer (61) Zolk, M.; Eisert, F.; Pipper, J.; Herrwerth, S.; Eck, W.; Buck, M.; Grunze, M. Langmuir 2000, 16, 5849. (62) Ostuni, E.; Grzybowski, B. A.; Mrksich, M.; Roberts, C. S.; Whitesides, G. M. Langmuir 2002, 19, 1861. (63) Wittemann, A.; Haupt, B.; Ballauff, M. Phys. Chem. Chem. Phys 2003, 5, 1671. (64) Wittemann, A.; Haupt, B.; Ballauff, M. Prog. Colloid Polym. Sci. 2006, 133, 58. (65) Wittemann, A.; Ballauff, M. Anal. Chem. 2004, 76, 2813. (66) Anikin, K.; Ro¨cker, C.; Wittemann, A.; Wiedenmann, J.; Ballauff, M.; Nienhaus, U. J. Phys. Chem. B. 2005, 109, 5418. (67) Czeslik, C.; Jansen, R.; Ballauff, M.; Wittemann, A.; Royer, C. A.; Gratton, E.; Hazlett, T. Phys. ReV. E 2004, 69, 021401. (68) Hollmann, O.; Czeslik, C. Langmuir 2006, 22, 3300. (69) Wittemann, A.; Ballauff, M. Phys. Chem. Chem. Phys. 2006, 8, 5269.
Figure 6. LSCM images of PEO45-b-PCL85-b-PAA110 aggregates with immobilized FITC-BSA. The hydrophobic dye, Nile red, was used as the fluorescent probe for the hydrophobic wall of the aggregates. The micrographs and their individual RG channels referring to the fluorescence of FITC-BSA and Nile red, respectively, were taken at two different magnifications as indicated by the bars.
range. Extremely bright spots which come from the fluorescence of FITC-BSA are visible at the location of the aggregates (Figure 6). The sizes of these spots are governed by the convolution of the aggregate size with the optical resolution of the microscope. As mentioned above, segregation of the triblock copolymers leads to a preferential orientation of the PAA blocks to the outer interface, whereas the inner interface of the vesicles is mainly composed of PEO and should thus repel protein molecules. Even if single PAA chains are oriented to the inner interface, they cannot efficiently bind protein molecules. Single polyelectrolyte chains cannot ensure strongly correlated counterions. Thus, the overall electrostatic repulsion between the protein and the likecharged polyelectrolyte cannot be overcome by a gain in entropy due to the release of counterions. Hence, the protein should hardly
2230 Langmuir, Vol. 23, No. 4, 2007
bind to the inner wall (see the discussion in ref 69). Moreover, the concentration of encapsulated protein is not supposed to exceed the bulk concentration. The LSCM studies were performed before and after the removal of unbound protein. In both cases, the LSCM images show a bright fluorescence at the location of the aggregates. Hence, there has to be a strong enrichment of the protein onto the wall of the vesicles, which furthermore explains the high binding efficiencies (Table 2). As we would not expect an enrichment of the protein at the inner interface, the bright green spots in Figure 6 indicate that a large fraction of the protein has to be adsorbed onto the outer wall of the aggregates. This strong adsorption of FITC-BSA onto the outer wall, which is preferentially composed of the PAA blocks, is fully explained by the PMPA.69 The outer interface of the aggregates presents a polyelectrolyte brush. The counterions of the PAA blocks are strongly confined within the brush layer. Positively charged patches on the surface of the FITC-BSA molecules become multivalent counterions of the brush layer. Subsequently, monovalent counterions are released. This leads first to a partial relief of the osmotic pressure within the layer, which is caused by the confined counterions, and second to a gain in entropy.69 Hence, the high binding efficiencies, which cannot be understood on the basis of encapsulation alone, have to be ascribed mainly to the PMPA. In summary, the direct self-assembly of the aggregates in the FITC-BSA solution leads to protein molecules which are encapsulated in the interior but also to protein molecules which are primarily adsorbed on the outer interface (Figure 4). Fluorescence quenching studies should provide further insight into the localization of the protein.23
Wittemann et al.
Conclusions The biamphiphilic triblock copolymer PEO-b-PCL-b-PAA, which is based on fully biocompatible blocks, forms well-defined aggregates by direct self-assembly in aqueous solution. Among those aggregates, well-defined polymer vesicles can be found. Loading of the vesicles is accomplished by adding biofunctional molecules to the aqueous solution during the preparation of the aggregates. The biamphiphilic nature of the block copolymer allows adsorption of protein molecules to the outer interface, which can be used to stabilize the particles from agglomeration but also to functionalize the surface with biomolecules. Moreover, protein molecules such as BSA, which by themselves present a carrier for low-molecular-weight drugs, can be entrapped in the interior of the vesicles. The biodegradable aggregates combine an essential robustness needed for a long-term encapsulation of guest molecules with the ability to release them after a certain lifetime in circulation. Such polymer vesicles may meet the requirements for technical or therapeutic applications requiring both biocompatibility and biodegradability. Acknowledgment. Financial support by the Natural Science and Engineering Research Council of Canada (NSERC) is gratefully acknowledged. A.W. is grateful to the Deutsche Forschungsgemeinschaft (DFG) for support through a Forschungsstipendium. LA062805B
