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Aggregation Behavior of Poly(ethylene glycol-bl-propylene sulfide) Di- and Triblock Copolymers in Aqueous Solution Simona Cerritelli,†, Conlin P. O’Neil,†, Diana Velluto,†, Antonella Fontana,‡ Marc Adrian,§ Jacques Dubochet,§ and Jeffrey A. Hubbell*,† Institute for Bioengineering and Institute for Chemical Sciences and Engineering, Ecole Polytechnique F ed erale de Lausanne (EPFL), Station 15, CH-1015 Lausanne, Switzerland, ‡Department of Pharmaceutical Sciences, University G. d’Annunzio, 66013 Chieti Scalo, Italy, and §Laboratory for Ultrastructural Analysis, University of Lausanne, CH-1016 Lausanne, Switzerland. These authors contributed equally. )
†
Received February 23, 2009. Revised Manuscript Received July 16, 2009 Block copolymers of poly(ethylene glycol)-bl-poly(propylene sulfide) (PEG-PPS) have recently emerged as a new macromolecular amphiphile capable of forming a wide range of morphologies when dispersed in water. To understand better the relationship between stability and morphology in terms of the relative and absolute block compositions, we have synthesized a collection of PEG-PPS block copolymers and quantified their critical aggregation concentration and observed their morphology using cryogenic transmission electron microscopy after thin film hydration with extrusion and after solvent dispersion from tetrahydrofuran, a solvent for both blocks. By understanding the relationship between aggregate character and block copolymer architecture, we have observed that whereas the relative block lengths control morphology, the stability of the aggregates upon dilution is determined by the absolute block length of the hydrophobic PPS block. We have compared results obtained with PEG-PPS to those obtained with poly(ethylene glycol)-blpoly(propylene oxide)-bl-poly(ethylene glycol) block copolymers (Pluronics). The results reveal that the PEG-PPS aggregates are substantially more stable than Pluronic aggregates, by more than an order of magnitude. PEG-PPS can form a wide variety of stable or metastable morphologies in dilute solution within normal time and temperature ranges, whereas Pluronics can generally form only spherical micelles under the same conditions. On the basis of these results, block copolymers of PEG with poly(propylene sulfide) may present distinct advantages over those with poly(propylene glycol) for a number of applications.
Introduction One of the main challenges in drug delivery using self-assembling systems for the encapsulation and release of small molecules, polypeptides, or nucleic acid-based therapeutics is the stability of the formed mesophases. One of the limitations in exploiting these materials is the relatively high critical aggregation concentration (cac) of conventional amphiphiles and macroamphiphiles, which can limit circulation times in vivo. Moreover, it is desirable that the polymer systems display a wide variety of morphologies in solution to be useful in delivering both hydrophobic and hydrophilic molecules. Dilution in the circulatory system (sink conditions) of aggregates from lipid or block copolymer amphiphiles displaying a very high cac results in their dissociation1-4 and a consequent burst release or loss of the encapsulated compound from the carrier. In terms of morphology, lamellar aggregates such as polymersomes (block copolymer vesicles) are useful for deliverying hydrophilic drugs, including polypeptides, or nucleic acid therapeutics5 whereas micelles are well suited for the delivery of amphipathic or hydrophobic compounds.6,7 It would be advantageous to develop a block copolymer system that displays both a low cac and the ability to form a variety of stable *Corresponding author. E-mail:
[email protected]. (1) Senior, J. H. Crit. Rev. Ther. Drug 1987, 3, 123–93. (2) Photos, P. J.; Bacakova, L.; Discher, B.; Bates, F. S.; Discher, D. E. J. Controlled Release 2003, 90, 323–334. (3) Lasic, D. D.; Papahadjopoulos, D. Medical Applications of Liposomes; Elsevier Science: New York, 1998. (4) Lasic, D. D. Trends Biotechnol. 1998, 16, 307–321. (5) Discher, D. E.; Ortiz, V.; Srinivas, G.; Klein, M. L.; Kim, Y.; Christian, D.; Cai, S.; Photos, P.; Ahmed, F. Prog. Polym. Sci. 2007, 32, 838–857. (6) Torchilin, V. P. J. Controlled Release 2001, 73, 137–172. (7) Torchilin, V. P. Cell. Mol. Life Sci. 2004, 61, 2549–2559.
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morphologies in solution by either controlling the absolute and relative block compositions or by controlling the preparation method. The ability of block copolymers to self-assemble above a certain cac when dispersed in solvents that selectively solubilize only one of their domains has been studied for the last several decades.8-10 The driving force for the aggregation is the tendency of the hydrophobic domains to minimize their contact with water and for the hydrophilic chains to orient themselves toward the aqueous phase and become solvated, resulting in an interfacial tension on the hydrophobic core surface. Poly(ethylene glycol)-blpoly(propylene oxide)-bl-poly(ethylene glycol) block copolymers (Pluronics) have been used to deliver various drugs because of their relative low cac compared to low molecular weight surfactants and the consequent greater stability under sink conditions as experienced under in vivo conditions. In dilute systems, block copolymers generally form spherical micelles, worm-like micelles, and vesicles,11 mimicking the well-established states of aggregation created when low-molecular-weight amphiphiles (LMWSs) are dissolved in water. However, block copolymers can also create a wide range of other morphologies that are not possible with conventional surfactants. The formation of this extraordinary variety of aggregates in the case of block copolymers is mainly due to the low mobility10,12 of the hydrophobic block, which can be considered to be almost frozen when the dispersing liquid is an (8) Zhang, L.; Eisenberg, A. Science 1995, 268, 1728–1731. (9) Discher, B. M.; W., Y.-Y.; Ege, D. S.; Lee, J. C.-M.; Bates, F. S.; Discher, D. E.; Hammer, D. A. Science 1999, 284, 1143–1146. (10) Jain, S. B.; Frank, S. Science 2003, 300, 460–464. (11) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967–973. (12) Zhang, L.; Eisenberg, A. Macromolecules 1999, 32, 2239–2249.
Published on Web 08/27/2009
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incompatible solvent such as water. This is in contrast to classical surfactant micelles, where the exchange of monomers between micelles and the bulk solution occurs within microseconds.13 Therefore, although the morphology of LMWSs are mainly controlled by thermodynamics, kinetic effects can determine the formation of a wide variety of metastable aggregates in the case of block copolymers.14 The reasons for the increasing interest of researchers in block copolymers is mainly based on the extended physical and chemical properties of block copolymer aggregates with respect to those formed by LMWS with respect to their potential stealthy qualities when using nonionic blocks such as polyethylene glycol (PEG) and the ease with which morphological variations can be induced. Simple alterations of block copolymers to their relative and absolute block composition and concentration,8,12 temperature,15 the addition of ions,16,17 the addition of organic solvents,12 and modification of the protocols for preparation14 allow for many different control variables to modify the mesophase morphology by altering the volume fraction of the hydrophilic and hydrophobic blocks or by changing the Flory-Huggins interaction parameter between the formed aggregate and the solution in which it is dispersed. In this sense, block copolymers can be designed and synthesized to form specific morphologies (polymersomes, worm-like micelles, or sperhical micelles), which can take that form under appropriate processing conditions for a therapeutic compound to be encapsulated. Here, we compare the self-assembling properties of poly(ethylene glycol)-bl-poly(propylene sulfide) (PEG-PPS), diblock, and triblock copolymers to those of Pluronics by quantifying their cac’s and by exploring morphology diagrams based both on block composition and on the preparation protocol. A great deal of research has been performed to document the aggregation behavior of commercially available Pluronics.16-19 Such block copolymers are homologous to PEG-PPS, bearing an oxygen atom instead of sulfur in the hydrophobic domain. One of the main differences between these two groups of block copolymers arises from the relative hydrophobicity of the sulfur atom compared to the oxygen atom in the hydrophobic block. Both blocks display similar Tg values20,21 such that at room temperature they have some character of fluidity in the hydrophobic domain. This confers at least some level of intrinsic mobility to the hydrophobic core even at room temperature. However, the less hydrophobic Pluronic block copolymers cannot adopt stable higher-ordered structures such as lamellae in dilute solution because these are not thermodynamically favorable. This is because the partitioning coefficient c log P for Pluronic block copolymers is relatively close to that of water (-1.38 for water, 3.00 for EG8PO15 Pluronic).22 In contrast, PEG-PPS is able to form polymersomes that are stable over long periods of time under dilution and within normal temperature ranges23 as a result
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of the very large mismatch in c log P (13.07 for EG8PS15) for water and the subsequent larger interfacial tension (σ). Another difference is the ability of PEG-PPS to form aggregates, the stability of which can be tuned14 by the method chosen for their preparation, whereas aggregates obtained from Pluronics (except for micelles) have been demonstrated to be relatively unstable. For example, polymersomes formed from Pluronic L121 (EG5PO68EG5) change their morphology over a period of hours.24 We used the fluorescence probe technique to measure the cac of PEG-PPS diblock and triblock copolymers and commercially available Pluronic block copolymers. We obtained results from pyrene, characterizing the polarity of the solubilizing microenvironment by following its spectral behavior in the presence of block copolymer aggregates. We also examined the role of the preparation protocol on mesophase morphology and stability, using both solvent dispersion from THF into water and thin film hydration followed by extrusion. In the first protocol, hypothesized to yield structures close to their thermodynamic equilibrium, the copolymer was dissolved in THF, a good solvent for both blocks, and then dispersed in water, a selective solvent for the hydrophilic domain. The dispersion of THF into the bulk water drives the aggregation of the hydrophobic blocks. In the hydrophobic core of the aggregates, perhaps still swollen by the organic solvent,12 the polymer chains reorganize to achieve a minimum interfacial free energy. In spite of reorganization within the aggregate, the presence of water surrounding the hydrophobic core can dramatically slow the exchange of the polymer chains between the aggregate and the bulk solution. In the second protocol, hypothesized to yield morphologies farther from equilibrium, a thin solvent-free copolymer film was hydrated and subsequently extruded through a nanoporous membrane. In this case, both intra- and interaggregate exchange of the polymer chains are minimal because reorganization within the aggregate is very limited. Thus, these aggregates are more kinetically frozen in their initial morphology, giving rise to the formation of complex, nonergotic structures. The morphological behavior of the PEGPPS preparations was analyzed using cryogenic transmission electron microscopy (cryo-TEM), whereas the microviscosity and cac of the preparations were measured fluorimetrically using the pyrene method.The goal of this research is to gain better insight into the behavior of the relatively new PEG-PPS block copolymer system. By discovering where the boundaries in the relative and absolute block compositions are in relation to stability and morphology, we can design novel block copolymers suited for specific applications. In this sense, we can prepare block copolymers that can form polymersomes to encapsulate hydrophilic materials and micelles for amphipathic and hydrophobic compounds while conferring the maximum stability for those drug carriers under sink conditions.
Materials and Methods (13) Clint, J. H. Surfactant Aggregation; Blackie & Son Ltd: London, 1992. (14) Cerritelli, S.; Fontana, A.; Velluto, D.; Adrian, M.; Dubochet, J.; De Maria, P.; Hubbell, J. A. Macromolecules 2005, 38, 7845–7851. (15) Nivaggioli, T.; Alexandridis, P.; Hatton, T. A.; Yekta, A.; Winnik, M. A. Langmuir 1995, 11, 730–737. (16) Alexandridis, P. Macromolecules 1994, 27, 2414–2425. (17) Nivaggioli, T.; Tsao, B.; Alexandridis, P.; Hatton, T. A. Langmuir 1995, 11, 119–126. (18) Colin Booth, D. A. Macromol. Rapid Commun. 2000, 21, 501–527. (19) Kabanov, A. V.; Batrakova, E. V.; Alakhov, V. Y. J. Controlled Release 2002, 82, 189–212. (20) Nicol, E.; Nicolai, T.; Durand, D. Macromolecules 1999, 32, 7530–7536. (21) Jannasch, P. Polymer 2000, 41, 6701–6707. (22) Tetko, I. V.; Gasteiger, J.; Todeschini, R.; Mauri, A.; Livingstone, D.; Ertl, P.; Palyulin, V. A.; Radchenko, E. V.; Zefirov, N. S.; Makarenko, A. S.; Tanchuk, V. Y.; Prokopenko, V. V. J. Comput.-Aided Mol. Des. 2005, 19, 453–63. (23) Napoli, A. Doctoral Thesis ETH Zurich 2003, no. 26750.
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Synthesis of PEG-PPS Block Copolymers. Block copolymers of PEG-PPS were synthesized as described elsewhere.25,26 Preparation of fluorescent PEG-PPS is described in the Supporting Information. Purified polymers were analyzed via gel permeation chromatography (GPC) using Waters Styragel THF columns (HR 2, 3, and 4) and via both refractive index and UV/vis detectors using THF as the mobile phase at 1 mL/min and at 40 °C. We also characterized the polymers using 1H NMR in (24) Schillen, K.; Bryskhe, K.; Mel’nikova, Y. S. Macromolecules 1999, 32, 6885–6888. (25) Velluto, D.; Demurtas, D.; Hubbell, J. A. Mol. Pharm. 2008, 5, 632–642. (26) Napoli, A.; Tirelli, N.; Kilcher, G.; Hubbell, J. A. Macromolecules 2001, 34, 8913–8917.
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Cerritelli et al. Table 1. Characterization of PEG-PPS Using GPC, 1H NMR, cac, and Cryo-TEM
name
Mw
fPEG
nPPS
cac TF (M)a
morphology from TFa
cac SD (M)b
E17S64E17 6246 0.24 64 1.5 10-5 V 1.3 10-5 8152 0.49 56 1.3 10-5 S 9.1 10-6 E44S56E44 9635 0.42 76 3.7 10-6 VþC 2.9 10-6 E44S76E44 -6 VþC 4.5 10-6 E44S107E44 11 934 0.34 107 8.8 10 SþC 4.1 10-6 E44S112E44 12 304 0.33 112 3.0 10-6 network 2.0 10-6 E44S290E44 25 500 0.16 290 1.5 10-6 4150 0.48 29 2.7 10-5 S 2.2 10-5 E45S29 -6 5559 0.36 48 8.8 10 S 7.3 10-6 E45S48 4744 0.42 37 1.4 10-5 S 1.1 10-5 E45S37 5262 0.38 44 1.3 10-5 C 9.4 10-6 E45S44 3568 0.21 38 9.7 10-6 V 1.0 10-5 E17S38 -6 6746 0.3 64 7.6 10 VþC 7.0 10-6 E45S64 a Thin film hydration with extrusion: V, vesicles; C, cylinders; and S spherical micelles. cylinders; S, spherical micelles; and O, unimolecular sheets.
CDCl3, D1 10 s, with 32 scans per sample and with TMS as an internal standard. The results are summarized in Table 1. Size measurements were made using cryo-TEM. Sample Preparation. The two sample preparation protocols have been reported previously in the literature.14 For the solvent dispersion method, a stock solution of the polymer was dissolved in THF, and a few microliters of this were diluted in 10 mmol PBS at pH 7.4. The second protocol is the common thin film hydration followed by extrusion (200 nm pores) of the polymeric suspension to obtain uniformly sized dispersions. All sample preparations and handling were performed at room temperature. Fluorescence Measurements. Pyrene is a fluorescent probe used to evaluate the cac of block copolymers and is also used to characterize the physical characteristics of the hydrophobic domain of block copolymer aggregates. Because of its relative hydrophobicity, pyrene partitions into hydrophobic environments such as the core of micelles. The polarity dependence of the emission of pyrene is expressed in terms of the ratio I1/I3, which represents the intensities of λem bands I and III. The I1/I3 ratio decreases as the block copolymer concentration increases as a result of the tendency of the block copolymer to self-organize into preaggregates bearing a hydrophobic region in which the pyrene solubilizes. As the concentration of pyrene is increased, the I1/I3 ratio reaches a stable minimum, indicating that true aggregates with an optimum aggregation number form. The onset of this minimum corresponds to the cac of the polymer. Pyrene is also able to form excimers, and the ratio of the fluorescence intensity of the excimer to the monomer (IE/IM) provides information on the distribution of the probe molecules in the aggregate and on the microviscosity of the system. A high ratio corresponds to a low microviscosity. Steady-state fluorescence spectra were recorded at 25 °C using a Jasco FP662 spectrofluorometer with a band pass of 5 nm. A given volume of pyrene (98%, Aldrich) stock solution in ethanol (2 10-3 M) was added to the aggregate suspension under stirring and was left to equilibrate for at least 3 min before performing the measurement. The excitation wavelength chosen for the measurements was λex = 335 nm, and no significant difference in the excitation spectra was found between the λex of pyrene in water and that in the aggregate. The monomer emission was read at λem = 377 (I1) and λ em = 386 (I3) whereas the excimer emission was read at λ em = 480 nm. Two concentrations of pyrene were chosen: 2 10-6 M to study the monomer emission (and to determine the corresponding I1/I3) and 5 10-5 M to study the emission of the excimer (and to determine the corresponding IE/IM). Further details of the sample preparation are described in the literature.14 To confirm the behavior of these aggregates upon dilution, we exploited the self-quenching of fluorescein isothiocyanate (FITC)-derivatized block copolymers in the micellar phase. In particular, we studied two block copolymers, E112S57-FITC and (27) O’Neil, C. P.; van der Vlies, A. J.; Velluto, D.; Wandrey, C.; Demurtas, D.; Dubochet, J.; Hubbell, J. A. J. Controlled Release 2009, 137, 146–151.
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morphology from SDb V S S S SþC SþCþVþO S S S S V S b Solvent dispersion from
TF size (nm)a
SD size (nm)b
148 141 8 8 17/22 17 45/30 20 21/30 28 20 17/58 7 8/16 24 128 60 16 22/30 8 309/102 141 50/8 8 THF into water: V, vesicles; C,
E45S17-FITC.27 By using these derivatized copolymers, we could monitor the increase in fluorescence as an indicator of the dissociation of copolymeric micelles. As the control, results were compared to the same samples dissociated with low-molecularweight surfactant Triton X-100. To confirm that the emission was above the background fluorescence, we compared the results against control samples of the buffers used for the dilution. The above-mentioned F€ orster resonance energy transfer (FRET) technique can also be used as an alternative method to measure the cac of PEG-PPS block copolymers. Cryo-Transmission Electron Microscopy. Cryo-TEM was used to investigate the morphology of PEG-PPS prepared using both protocols described above. For measurement from solvent dispersion, a few microliters of a stock solution in THF at 10% block copolymer were diluted in 10 mmol PBS at pH 7.4 to give a final concentration from 0.5 to 1%. Samples prepared from thin film hydration and extrusion were made at a concentration of 1 wt % copolymer in 10 mmol PBS. The specimens for analysis were prepared by application of a 5 μL drop of the aqueous aggregate suspension on a 400 mesh copper grid coated with a porous carbon film. Excess suspension was blotted away, resulting in a 40-200 nm film on the carbon support. The sample was rapidly vitrified by immersion in liquid ethane and transferred to the cryo-electron microscope. The samples were observed using a Philips CM12 (FEI, The Netherlands) operating at 80 keV in transmission mode and at a temperature never exceeding -160 °C. Images were recorded on CCD camera using the low dose technique at a nominal defocalizaiton of 2 to 10 μm with magnification of up to 35 000.28,29 Samples were observed within 24 h of preparation.
Results and Discussion Aggregation Study: Critical Aggregation Concentration (cac). As suggested by Astafieva30 more than a decade ago, the cac for amphiphiles can be defined as the concentration below which only single chains are present and above which aggregates and single chains can be found in solution. Measurement of the cac can give information on the thermodynamic stability of the formed aggregates in solution. At the cac, free polymer chains are in equilibrium with the aggregates, and the free energy of micellization is given by ΔG° = RT ln cac. As discussed by Tanford,31 it is difficult to define a precise cac for each amphiphile, and it would be more proper to refer to a (28) Adrian, M.; Dubochet, J.; Lepault, J.; McDowall, A. W. Nature 1984, 308, 32–36. (29) Dubochet, J.; Adrian, M.; Chang, J. J.; Homo, J. C.; Lepault, J.; McDowall, A. W.; Schultz, P. Q. Rev. Biophys. 1988, 21, 129–228. (30) Astafieva, I.; Zhong, X. F.; Eisenberg, A. Macromolecules 1993, 26, 7339– 7352. (31) Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes; John Wiley & Sons: New York, 1973.
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critical transition interval of concentrations in which aggregation occurs. This is even more relevant when considering block copolymers rather than classical LMWS because the complexity of the system is increased by the substantially higher Mw and the resultant higher stability.32 Both of these factors influence chain mobility and allow for kinetic factors to play a crucial role in aggregation phenomena. Indeed, in contrast to LMWS association, the aggregation curve of a block copolymer generally displays a higher curvature when the aggregation process is taking place over a large concentration range. The determination of the cac can then be rather ambiguous compared to that of simple surfactant molecules. An example is the contradictory amount of data that can be found in the literature on the cac of Pluronic block copolymers, which can vary by 1 or 2 orders of magnitude from one publication to another. Many authors explain such differences as the effect of inhomogeneous samples as a result of the lack of block composition consistency from one lot to another and the unusually high polydispersity values for Pluronic block copolymers. Alexandridis33 addressed this topic directly by claiming that a misinterpretation of surface tension data occurred when considering the first rather than the second inflection of the aggregation curve. Kabanov34 considered that the real cac could be obtained by simply ignoring the eventual step due to the presence of a second inflection “and determined the cmc (critical micellar concentration) by the crossing point of the straight lines that continue the surface tension vs log concentration curves before and after the step”. A similar misinterpretation can arise from data obtained by using the fluorescence probe method. Some authors consider the cac to be the concentration at the onset of the decrease in the I1/I3 ratio35 whereas other authors36,37 believe that the concentration at the approach of a stable, low I1/I3 ratio is better. In some papers, the cac corresponds to the inflection point.38 The disagreements arise again from the fact that the critical transition interval from monomers to proper micelles is quite large overall for block copolymers. At this time, we believe that it is best to use one accepted measurement and analytical method to characterize the cac in order to compare PEG-PPS to Pluronic block copolymers. To answer the criticism relating to the variability of the composition of commercially available Pluronic block copolymers, we have fully characterized the Pluronics used in this study using GPC and 1H NMR in both CDCl3 and d6-DMSO to obtain absolute block compositions and polydispersity data. In this way, we avoid problems comparing our acquired data to the literature and provide a set of data for others to compare as long as they also characterize the Pluronics used in subsequent studies. We have studied the aggregation behavior of PEG-PPS diblock and triblock copolymers over the range of Mw from about 6000 to 26 000 Da. A list of the investigated copolymers is reported in Figure 1, where the copolymers are indicated as ExSy, or ExSyEx, with E representing the PEG domain, S representing the PPS domain, and x and y representing the repeating units in the respective polymer chains.
(32) Discher, D. E.; Ahmed, F. Annu. Rev. Biomed. Eng. 2006, 8, 323–341. (33) Alexandridis, P.; Athanassiou, V.; Fukuda, S.; Hatton, T. A. Langmuir 1994, 10, 2604–2612. (34) Kabanov, A. V.; Nazarova, I. R.; Astafieva, I. V.; Batrakova, E. V.; Alakhov, V. Y.; Yaroslavov, A. A.; Kabanov, V. A. Macromolecules 1995, 28, 2303–2314. (35) Vasilescu, M.; Caragheorgheopol, A.; Caldararu, H. Adv. Colloid Interface Sci. 2001, 89-90, 169–194. (36) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99, 2039– 2044. (37) Chen, W. Y. Macromolecules 1995, 28, 8604. (38) Wolszczak, M.; Miller, J. J. Photochem. Photobiol., A 2002, 147, 45–54.
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Figure 1. General molecular structure of the PEG-PPS block copolymers and schematics of the various architectures under study. EnSmEn represents the block copolymer, with E being the PEG block and S being the PPS block and m and n corresponding to the degrees of polymerization of the blocks in each copolymer.
Figure 2. Sigmoid curve of the I1/I3 ratio (open squares, left axis) versus concentration (bottom axis) and the I1 intensity (filled circles, right axis) of E45S56E45 triblock copolymer using the pyrene method. The cac can be calculated either from the ratio of the pyrene monomer emissions I1/I3 or by plotting I1. The results using both methods lead to similar values.
Figure 2 is an example of the sigmoid curve of aggregation obtained by plotting the emission intensity ratio (I1/I3) of the pyrene probe against the concentration of the block copolymers. The decrease in the I1/I3 ratio over a large interval of polymer concentration indicates a slow increase in the hydrophobicity of the environment experienced by the probe as the concentration of the copolymer increases. It has been suggested that the aggregation process of Pluronic block copolymers in water is particularly complex and consists of different steps: first, the formation of monomolecular micelles; second, the reorganization of the polymer coils upon increasing the polymer concentration; and finally, DOI: 10.1021/la900649m
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Figure 3. cac of PEG-PPS block copolymers from thin film hydration (A) and solvent dispersion from THF (B).
Figure 4. cac of PEG-PPS block copolymers as a function of the degree of polymerization of the PS block (n PS) shown for samples prepared from solvent dispersion (9) and thin film hydration (2). Both triblocks (A) and diblocks (B) display decreasing cac’s with increasing n PS.
the formation of mature micelles with the final shape and aggregation number.39 Because of the previously observed presence of premicellar aggregates in low-concentration solutions of E45S76E4514 and the similarity of PEG-PPS to Pluronics, we expect similar behavior here. Therefore, we take as the cac the concentration at which I1/I3 reaches a maximum plateau, which should indicate the prevailing presence in solution of micelles with an optimum aggregation number. The cac has also been measured by plotting the pyrene intensities of the first emission band, I1, versus the copolymer concentration. It is known that the intensity of I1 increases significantly in the presence of nonpolar surroundings. This reflects the increase in the lifetime of the excited state of pyrene.30,40 In the case of micelle formation, the pyrene will be solubilized in the hydrophobic core with a consequent increase of the pyrene emission intensity. An example is reported in Figure 2, where the pyrene emission intensity I1 and the I1/I3 ratio are plotted against block copolymer concentration. The increase in the I1 pyrene emission intensity registered after increasing the copolymer concentration correlates quite well with the plateau of the I1/I3 sigmoid plot. Similar results were obtained for all of the block copolymers investigated, as shown in Figure 3 for aggregation from thin film hydration (A) and solvent dispersion (B). The cac’s of the Pluronics have been extensively studied.16,33,34,41,42 It has been shown that the cac linearly decreases with the increasing length of the hydrophobic PPO block33,42 as (39) Turro, N. J.; Chung, C. Macromolecules 1984, 17, 2123–2126. (40) Wilhelm, M. Macromolecules 1991, 24, 1033. (41) Alexandridis, P. Colloids Surf., A 1995, 96, 1–46. (42) Kozlov, M. Y.; Melik-Nubarov, N. S.; Batrakova, E. V.; Kabanov, A. V. Macromolecules 2000, 33, 3305–3313.
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expected, considering that the aggregation process is driven by the hydrophobic effect. Moreover, from the same studies, the effect of PEG on the aggregation behavior of Pluronics seems much less pronounced, and an increase in PEG length causes only a slight increase in the cac of Pluronic block copolymers. The cac’s of all PEG-PPS copolymers are reported in Figures 4 and 5. In Figure 4, the effect of the hydrophobic block on the aggregation of the copolymers can be observed. As with the Pluronics, an increase in the length of the hydrophobic block causes a decrease in the cac. It is interesting that for the corresponding diblocks, after an initial drastic decrease in the cac on increasing the n PPS, a plateau is reached where, independently of the number of PPS units, the cac does not decrease further. Similar nonlinear behavior for the aggregation of block copolymers has been observed for several different polyelectrolyte amphiphilic copolymers, including poly(styrene-bl-sodium acrylate) copolymer, PS-bl-PANa,30 and poly(hydrogenated isoprene-bl-sodium styrene sulfonate) copolymer, Pip.h2-blPSSNa.43 This has been attributed to the interfacial and electromechanical limit γ, which prevents further decreases in the cac.32 The effect of the PEG chains on the cac is shown in Figure 5. A higher PEG/PPS Mw ratio, or fPEG (Mw PEG/(Mw PEG þ Mw PPS), causes an increase in the corresponding cac, starting from block copolymers with at least 30-40% PEG. This is more obvious for the diblock copolymers than for the triblock copolymers because of the greater number of polymers and diversity of investigated block compositions. However, the increase in the cac (43) Kaewsaiha, P.; Matsumoto, K.; Matsuoka, H. Langmuir 2005, 21, 9938– 9945.
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Figure 5. cac of PEG-PPS block copolymers as a function of the mass fraction of PEG in the copolymer for triblocks (A) and diblocks (B) for samples prepared from solvent dispersion (9) and thin film hydration (2). Although there is a trend with the triblocks, there is a good correlation between diblock fPEG values and the cac. Because the cac is determined primarily by the absolute hydrophobic block length rather than the ratio or relative block lengths, PEG-PPS displaying the same n PS but very different fPEG values will have similar cac’s. For example, in A, fPEG = 0.24 and fPEG = 0.49 display very similar cac values despite the difference in their n EG’s.
Figure 6. Increasing micelle core microviscosity is indicated by the decreasing excimer-to-monomer ratio of pyrene (IE/IM) as the degree of polymerization of PPS (n PS) increases. Triblock (A) and diblock (B) copolymers were examined after aggregate formation by solvent dispersion (9) and thin film hydration (2).
with increasing relative hydrophilic composition (fPEG) is mainly due to the decrease in the n PS, which is the primary factor governing both stability and self-assembly. This is clear from the differences between Figures 4 and 5. In Figure 4, clear trends can be seen with both diblock and triblock copolymers. However, in Figure 5, outliers can be observed. Upon closer examination, the outlier in Figure 5A at fPEG = 0.24 is composed of E17S64E17, which displays a cac similar to that for E45S56E45 with fPEG = 0.49, despite the difference in the relative block compositions. Therefore, as with other block copolymer systems, aggregation and stability are directly related to the size and chemical composition of the hydrophobic block. It is difficult to evaluate whether the aggregation behavior of the triblock copolymers is similar to that of the diblock copolymers because of the lower number of triblock (TB) copolymers analyzed and the slightly more scattered data obtained for the triblock copolymers. However, a different behavior for diblock and triblock copolymers had already been proposed to explain the differences in chain mobility observed in the study of the oxidative degradation of monolayers of PEG-PPS DB and TB copolymers.44 It is theorized that kinetic factors could play a central role in the aggregation process because triblock copolymers have to accommodate two PEG chains for each hydrophobic block (44) Napoli, A.; Bermudez, H.; Hubbell, J. A. Langmuir 2005, 21, 9149–9153.
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whereas diblocks can adopt simple linear conformations above the cac. Therefore, the chain conformation in a triblock copolymer micelle would display the two PEG chains on the surface, and the PPS chain in the core is forced into a coiled configuration. Triblock and diblock copolymers have also been analyzed in terms of the microviscosity of the PPS core by measuring the pyrene excimer emission with respect to the monomer emission (IE/IM). The term microviscosity is used to distinguish the viscosity of the probe environment in the interior of the aggregate from that in the aqueous medium. Figure 6 shows the linear correlation between the microviscosity and the number of PPS units for both triblock and diblock copolymers. The length of the hydrophobic block causes an increase in the aggregate core viscosity, and this effect is particularly evident in the investigated diblock copolymers. Triblock copolymers generally display a more viscous core with respect to those of diblocks; however, the correlation is less clear for reasons mentioned above. To confirm the behavior of these block copolymers upon dilution, we used PEG-PPS micelles with fluorescein covalently bound to the terminus PPS block, as previously reported.27 The formed micelles force the fluorescein moieties into close proximity at the micelle surface, causing a mutual quenching effect known as FRET. When the micelles dissociate, either at low concentration or with a surfactant, the fluorescence emission will thus increase DOI: 10.1021/la900649m
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Figure 7. FRET ratio of FITC-derivatized copolymers: (A) E45S17-FITC and (B) E45S17-FITC. The results are reported in terms of the Triton X-100/sample ratio in PBS alone. Table 2. Analysis of Pluronic Block Copolymers by GPC, 1H NMR, and cac 1
GPC composition
Mp
Mw
Mn
PDI
EO12-PO30-EO12 3507 3516 3208 1.10 EO122-PO44-EO122 9427 8498 7732 1.10 EO128-PO74-EO128 12 961 10 994 9574 1.15 (EO69-PO22)4 12 873 11 349 10071 1.13 EO69-PO45-EO69 8067 7905 7115 1.11 a Prepared by bulk or thin film hydration. b Analysis in d6-DMSO and CDCl3.
dramatically as the distance between fluorescein groups increases. In the case of the two polymers tested, E112S57-FITC and E45S17-FITC, the difference in fluorescence was greater than 500%. We already know that Triton X-100 is a good surfactant to use for the dissociation of PEG-PPS aggregates.45 By measuring the emission upon dilution in parallel with the same samples dissociated with Triton X-100, we could confirm the total dissociation upon dilution. The analysis by FRET demonstrated cac values that were considerably lower than determined by the pyrene method. These two methods produce somewhat complementary information, with the pyrene method giving the cac for micelles closer to their ideal equilibrium aggregation numbers and the FRET method giving values corresponding to nearly complete dissociation of the block copolymers in solution. As shown in Table 2, measured cac values of Pluronics are substantially higher than those of analogous PEG-PPS copolymers. If considering Pluronic F-127 with E45S29 from Table 1, it can be seen that the cac of PEG-PPS is more than 100 lower. Furthermore, compared with the same PEG-PPS above to the most stable Pluronic tested, we can observe that PEG-PPS with the smallest n PS is still ∼10 more stable. Because the cac is related to aggregate stability in vivo, one can predict that PEGPPS will offer more favorable drug delivery characteristics than will Pluronics. Cryo-TEM Analysis and Morphology Diagram. All of the investigated block copolymers were characterized by cryo-TEM analysis, which provided images of the polymeric aggregates prepared by both protocols. The images in the Supporting Information (supplemental Figures 1 and 2) reveal the existence of several isotropic dispersions of discrete morphologies as well as samples containing a variety of morphologies. Such images have (45) Cerritelli, S.; Velluto, D.; Hubbell, J. A.; Fontana, A. J. Polym. Sci., Part A 2008, 46, 2477–2487.
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H NMR
Mnb
fPEG
cac TF (mg/mL)a
cac TF (M)a
2797 13 320 15 598 17 335 8744
0.37 0.81 0.72 0.70 0.70
1.2 19.0 15.0 27.0 4.2
3.3 10-4 2.0 10-3 1.2 10-3 2.1 10-3 5.1 10-4
Figure 8. Selected cryo-TEM images of PEG-PPS aggregates in solution. Here we see PEG-PPS polymersomes (E17S38, from thin film hydration with extrusion, A), wormlike micelles (E45S112E45, from solvent dispersion, B), and spherical micelles (E45S44, from solvent dispersion, C). Scale bars represent 400 nm (A, C) and 200 nm (B).
allowed us to generate a morphology diagram based on fPEG for the investigated triblock and diblock copolymers (Figure 8). A clear dependence of the morphology of the aggregates on fPEG can be observed. Morphologies obtained by thin film hydration formed a wide variety of structures: spherical micelles or vesicles at high or at low fPEG, respectively. At intermediate hydrophilic fractions, a variety of morphologies can coexist, including wormlike micelles, rodlike micelles, Y junctions, and micelles. The morphology diagram is in actuality a continuum, with isotropic dispersions of vesicles or micelles occurring on the far left and right of the diagram, respectively (except E45S290E45, which forms unimolecular sheets and other morphologies.) However, aggregates obtained by solvent dispersion using THF generally form only isotropic dispersions of either vesicles or micelles. The differences in morphology obtained by the two methods are primarily due to the fact that the solvent dispersion method (tetrahydrofuran into water) leads to structures that are at or close to their thermodynamic equilibrium in solution whereas with the thin film method the block copolymer is rehydrated and dispersed in a kinetically trapped phase that will slowly reach equilibrium.14 Langmuir 2009, 25(19), 11328–11335
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Figure 9. Morphology diagrams of the PEG-PPS block copolymers obtained by block copolymers prepared either by thin film hydration (A) or solvent dispersion (B). Four main types of architectures are marked on the graphs: unimolecular sheets (lamellae), vesicles (V), worm-like micelles or cylinders (C), and spherical micelles (S). The compositions of the diblock (•) and triblock (2) copolymers are shown and are plotted according to fPEG.
The relatively high hydrophobicity of the PPS blocks compared to that of the PPO block of Pluronics allows these PPS-based block copolymers to adopt a wider array of metastable kinetically trapped morphologies that are not present in most commercially available Pluronic block copolymers. Wormlike micelles have been shown to be particularly useful in some biomedical applications, owing to their increased loading capacity and longer circulation times in blood compared to spherical micellar aggregates.46,47 Because of the slow dissociation times of PEG-PPS wormlike micelles, these morphologies could be of interest for hydrophobic drug delivery to the circulatory system over prolonged time periods. PEG-PPS can form many useful morphologies in solution that can be exploited as carriers for hydrophilic (proteins, peptides, or small molecules) or hydrophobic therapeutics (small molecule drugs) in either polymersomes or micelles, respectively, whereas Pluronic block copolymers typically form only relatively unstable spherical micelles in solution. The morphology diagram shown in Figure 9 should allow a further understanding of the relationship of relative block composition and morphology so as to allow the design of block copolymers with particular applications in mind, and the cac data will provide information on how to prepare these morphologies with the optimal stability.
absolute block composition in terms of n PS, the morphology is controlled by the relative block composition, fPEG. At high values of fPEG, the polymers generally form micelles, and vesicle morphologies form at low fPEG values. Aggregates obtained by thin film hydration can also form nonergotic structures at intermediate fPEG values that are not possible with low-molecular-weight surfactants such as wormlike micelles and Y junctions. By using FRET with micelles bearing fluorescein on the terminus of the hydrophobic core-forming polymer block, we were able to observe the dissociation of the formed aggregates via fluorimetry. The results confirm what we have observed with pyrene, that the block copolymer dissociation is complex and that complete dissociation occurs at very low dilution compared to low-molecular-weight amphiphiles and PPG-based macroamphiphiles. We have demonstrated that PEG-PPS block copolymers display superior stability and morphology control compared to Pluronic block copolymers. These features make PEG-PPS block copolymers a promising new material for both theoretical and applied research. By applying knowledge of the relationship between mesophase morphology and block copolymer architecture, we can now design PEG-PPS block copolymers to form micelles, wormlike micelles, or vesicles, depending on fPEG and the method of preparation, for specific biomedical applications.
Conclusions Block copolymers composed of PEG-PPS represent an emerging new material that displays both a desirably low cac and tunable morphology. Although stability is determined by the
Acknowledgment. We thank Letizia Gullifa for measuring the cac of the Pluronic block copolymers.
(46) Smart, T.; Lomas, H.; Massignani, M.; Flores-Merino, M. V.; Perez, L. R.; Battaglia, G. Nano Today 2008, 3, 38–46. (47) Geng, Y.; Dalhaimer, P.; Cai, S.; Tsai, R.; Tewari, M.; Minko, T.; Discher, D. E. Nat. Nano 2007, 2, 249–255.
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Supporting Information Available: Cryo-TEM images for all PEG-PPS polymers tested using the two preparation methods, synthesis and preparation of PEG-PPS-FITC, and FRET cac analysis of the raw data. This material is available free of charge via the Internet at http://pubs.acs. org.
DOI: 10.1021/la900649m
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