ARTICLE pubs.acs.org/Langmuir
Aggregation and Thermoresponsive Properties of New Star Block Copolymers with a Cholic Acid Core Cancan Li,†,‡ Christine Lavigueur,† and X. X. Zhu*,† † ‡
Department of Chemistry, Universite de Montreal, C.P. 6128, Succursale Centre-ville, Montreal, QC H3C 3J7, Canada Department of Applied Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
bS Supporting Information ABSTRACT: Poly(allyl glycidyl ether) (PAGE) and poly(ethylene glycol) (PEG) blocks were sequentially grown via anionic polymerization to form four block copolymer arms on a cholic acid (CA) core, yielding star block copolymers (CA(AGE8-b-EGn)4) with low polydispersities (ca. 1.05). The introduction of PAGE segments into CA(PEG)4 significantly reduced their crystallinity. The polymers can aggregate in water at room temperature above their critical aggregation concentration. The copolymers are thermoresponsive; their behavior in aqueous solutions was studied by the use of UV visible spectroscopy, dynamic light scattering, and transmission electron microscopy. Their cloud points vary from 13 to 55 °C with increasing length of the PEG segments. Double thermoresponsive behavior was observed with short PEG segments because of a two-step transition process: small micelles are formed upon heating and then further aggregate into micellar clusters through the association of PEG chains.
’ INTRODUCTION Star polymers are branched structures in which several arms are attached to a central core. Compared to linear polymers of equal molecular weight, they present a very compact structure with a high density of functional groups. They also typically exhibit better solubility and lower viscosity as well as different thermal properties.1,2 Therefore, they are promising candidates for various applications in fields such as encapsulation, drug delivery, adhesives, and compatibilizers.3 6 Cholic acid (CA), a naturally occurring bile acid, is a good starting point to prepare a variety of star polymers because of its rigid steroid skeleton and four functional groups. In the body, this facially amphiphilic molecule aids in the digestion of fats through the formation of mixed micelles;7 bile salts can also self-assemble on their own in aqueous solutions.8 Several polymers derived from bile acids have shown interesting aggregation properties and good biocompatibility.9 15 We have previously developed an efficient procedure for the preparation of well-defined star polymers based on bile acids via anionic polymerization.12,16 Both poly(ethylene glycol) (PEG) star polymers, CA(EGn)4, and poly(allyl glycidyl ether) (PAGE) star polymers, CA(AGEn)4, were synthesized. CA(PEG)4 star polymers were found to form micelles in water, driven by the facial amphiphilicity of cholic acid.12 Because of the strong hydrophobicity of the PAGE chains, CA(AGEn)4 star polymers are not soluble in water.16 However, the introduction of amines onto the allyl groups of CA(AGEn)4 and the subsequent acetylation impart their derivatives with interesting thermoresponsive properties in water over a wide range of temperature (5 75 °C).16 The biological origin and thermoresponsive properties of these polymers make them appealing candidates r 2011 American Chemical Society
for various biomedical applications; for example, the self-assembled aggregates formed by bile acid-based star polymers could be used for drug delivery. Other applications being considered for bile acid derivatives include sensing and the formation of biodegradable hydrogels.7 Allyl glycidyl ether (AGE) has previously been copolymerized with ethylene oxide (EO) to form random or block copolymers.17 22 Amphiphilic PAGE-b-PEG block copolymers and their derivatives could form small micelles with narrow size distributions in water, with PAGE (or PAGE modified with pendant groups) forming the hydrophobic core and PEG forming the hydrophilic corona.17,18 Furthermore, the use of PAGE provides a platform for the introduction of various functional groups.17,18,20 22 Because of the biocompatibility of PEG,23 26 PEG-b-PAGE block copolymers may be potential candidates for biomedical applications, and they have been proposed for the preparation of micelles for drug delivery.17,18 So far, reports on random or block copolymers of ethylene oxide and AGE have focused only on the functionalization of the vinyl groups and have been limited to linear copolymers. We have synthesized several well-defined CA(AGE8-bEGn)4 copolymers by varying the number of units in the PEG blocks from 8 to 40 while keeping the length of the PAGE blocks constant. The aggregation and the thermoresponsive properties of the block copolymers in aqueous solutions have been studied.
Received: June 10, 2011 Revised: July 27, 2011 Published: July 29, 2011 11174
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Table 1. Characterization of CA(AGE8)4 and the CA(AGE8-b-EGn)4 Copolymers Mn (g/mol) polymer
theoretical
NMR c
MALDI-TOF MS
SEC
PDIa
cac (millimolal, mm)b
CP (°C)
CA(AGE8)4
4100
3800
3200
4000
1.03
CA(AGE8-b-EG8)4
5600
5100d
4600
6300
1.04
CA(AGE8-b-EG12)4
6200
5700d
5600
8300
1.04
9.0
30, 35
CA(AGE8-b-EG16)4
7000
6800d
6300
8700
1.05
8.5
40
CA(AGE8-b-EG40)4
11 200
8800d
7600
10 500
1.04
10.1
55
13
Obtained from SEC measurements. b Obtained from viscosity measurements; error approximately (0.5 mm. c Calculated from the ratio of the NMR peak integrations of CHd (5.8 ppm) to 18,19,21-CH3 signals. d Calculated from the ratio of NMR peak integrations of CH2 (3.6 ppm) to 18,19,21-CH3 signals. NMR spectra are shown in Figure S1. a
Scheme 1. Structure of the Star Block Copolymers Studied
’ EXPERIMENTAL SECTION The anionic polymerization of CA(AGE8-b-EGn)4 was carried out as follows. The cholic acid derivative with four hydroxyl groups used to initiate the polymerization was prepared from cholic acid in two steps according to our previously published procedures (yield 93%).12,27 The first block, PAGE, was introduced following a previous report.16 The polymerization was allowed to proceed overnight (ca. 15 h), after which 5 mL of the CA(AGE8)4 solution was withdrawn from the mixture with the aid of a double-ended needle using positive pressure. After purification, 1H NMR spectroscopy was used to verify that complete conversion of the AGE monomers was achieved; MALDI-TOF mass spectrometry (MS) and size exclusion chromatography (SEC) analysis also confirmed that the expected molecular weight was obtained. The desired amount of dry ethylene oxide for the polymerization of the second block was introduced into the reaction mixture, and the polymerization was allowed to proceed overnight according to a literature procedure.12 CA(AGE8)4 and CA(AGE8-b-EGn)4 were both purified in the same manner. The reaction mixtures were extracted with dichloromethane (3 100 mL) to remove naphthalene, and DMSO was evaporated under reduced pressure. The product was dissolved in THF and filtered to remove salts, and THF was evaporated under reduced pressure. The samples were dialyzed against water and then freeze dried. Yield >85%. The samples prepared are listed in Table 1.
’ RESULTS AND DISCUSSION Preparation of CA(AGE8-b-EGn)4. The star block copolymers depicted in Scheme 1 were prepared by anionic polymerization from a cholic acid derivative bearing four hydroxyl groups. Good control of the polymerization of the first block is essential to the synthesis of block copolymers. SEC analysis of CA(AGE8)4 showed that the polymerization of AGE onto the CA core
yielded a polymer with a narrow molecular weight distribution (PDI = 1.04). Molecular weight measurements by 1H NMR spectroscopy, MALDI-TOF MS, and SEC confirmed that the expected molecular weight was obtained, indicating the complete conversion of the monomers. After the addition of the second monomer, the molecular weight of the copolymers clearly shifted toward higher values and the integration of the 1H NMR peaks of the methylene groups (ca. 3.80 ppm) increased, indicating the successful addition of the second block onto the polymer. The PDIs remained quite narrow (ca. 1.05), which indicates that polymers with well-controlled chain lengths were obtained. To verify that four arms were attached to the cholic acid core, 1 H NMR spectroscopy experiments were conducted as described in a previous publication.12 Trifluoroacetic anhydride was added to polymer samples in deuterated chloroform, causing a change in the chemical shift of the methylene groups adjacent to the alcohol end group of the PEG chains from 3.80 to 4.55 ppm. The ratio of the integration of these methylene groups to that of the three methyl groups on the cholic acid core was observed to be 8:9, indicating the presence of four chains on each cholic acid molecule. The theoretical molecular weights of the polymers were calculated from the reaction stoichiometry and are presented in Table 1 along with the molecular weights obtained by 1H NMR, MALDI-TOF MS, and SEC. The numbers of repeat units listed are those obtained from the theoretical molecular weight. By MALDI-TOF MS, a series of distinct peaks corresponding to different numbers of repeat units were observed for CA(AGE8)4 but only a broad peak was observed for the block copolymers. The NMR and MALDI-TOF MS results generally correspond well to the theoretical values. The data for CA(AGE8-b-EG40)4 has a greater discrepancy with the theoretical molecular weight; there is a much greater uncertainty in measuring the molecular weight of heavier polymers, but it is still probable that this polymer is shorter than expected on the basis of the reaction stoichiometry. The molecular weights measured by SEC are expected to deviate from the theoretical values because of the lack of appropriate standards for these star polymers. Thermal Properties. Because the melting points of crystalline polymers may depend on the thermal history of the samples,28 all DSC thermograms were recorded during the second heating at 20 °C/min. In our previous work, CA(EGn)4 (n g 6) star polymers showed both crystallization and melting points.12 The introduction of a PAGE segment significantly changes the thermal properties of the star polymers: the block copolymers with shorter PEG chains, CA(AGE8-b-EGn)4 with n = 8, 12, and 16, have an obvious glass-transition temperature (Tg) 11175
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Figure 1. Transmittance at 500 nm of CA(AGE8-b-EGn)4 aqueous solutions (1.0 mg/mL) as a function of temperature, with a heating rate of 0.1 °C/min: (a) n = 8, (b) n = 12, (c) n = 16, and (d) n = 40.
at 68.5 °C, without any crystallization or melting point. The same behavior was observed for the parent polymer CA(AGE8)4. In contrast, CA(AGE8-b-EG40)4 shows an obvious glass transition, a sharp crystallization peak, and a broad melting point. It is known that PEG block copolymers comprising an amorphous block generally have a lower degree of crystallinity as compared to that of PEG28 30 and that they can present coexisting crystalline and amorphous domains.28,29 The ordered phase of PEG may completely disappear after the addition of an amorphous second block if the glass-transition temperature of this block is lower than the crystallization temperature of PEG.29 In the case of CA(AGE8-b-EGn)4, the hydrophobic PAGE blocks and the star architecture may disturb the organization of the PEG blocks, and crystallinity can be restored only if the PEG chains are long enough. Thus, the star block copolymers are amorphous for n = 8, 12 and 16 and semicrystalline for n = 40. Aggregation Properties. Both linear PAGE-b-PEG copolymers and star-shaped CA(PEG)4 are known to self-assemble in aqueous media because of their amphiphilic nature; the CA(AGE8-b-EGn)4 copolymers are expected to do the same. The critical aggregation concentrations (cac’s) of the polymers obtained by viscosity measurements are listed in Table 1. Block copolymer CA(AGE8-b-EG8)4 does not have a measurable cac at room temperature because of its high hydrophobicity and consequent poor solubility in water; aggregates were always present, even at the lowest concentrations tested (0.2 mm). Block copolymers with longer PEG blocks all have a measurable cac, indicating that they maintain a reasonable balance of hydrophilicity, allowing full solubilization at low concentration, and hydrophobicity, causing the formation of stable aggregates at higher concentrations. In general, cac’s are expected to increase with increasing hydrophilicity because of the improved solubilization of the polymer.31 The results obtained are consistent with this trend: the cac is below 0.2 mm when the PEG block has only 8 repeat units, and increases to approximately 9 mm when the length of the PEG block is increased to 12, 16, or 40 units. Only a slight difference in cac is observed among these last three polymers, which may not be statistically significant. The aggregation of the star block copolymers may make them useful in the solubilization of hydrophobic compounds such as drugs. UV Visible Spectroscopy Studies. All of the block copolymers are soluble in cold water, forming clear, stable solutions (1 mg/mL). Upon heating, the solutions become turbid at a characteristic temperature, which is known as the cloud point
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Figure 2. Temperature dependence of the hydrodynamic diameter (Dh) of CA(AGE8-b-EGn)4 copolymers in aqueous solutions (1.0 mg/ mL): (a) n = 8; (b) n = 12, also shown in the inset to emphasize the first step (circled zone); (c) n = 16; and (d) n = 40.
(CP). Figure 1 shows the temperature dependence of the transmittance of copolymer solutions, and the CPs are listed in Table 1. At 25 °C, the solution of CA(AGE8-b-EG8)4 is turbid, indicating that it is above its cac. The solutions of the other polymers are still clear at this temperature, which is expected because they are at concentrations below their measured cac’s (1 mg/mL is between 0.1 and 0.2 mm for these polymers). The cloud point of CA(AGE8-b-EGn)4 copolymers can be tuned over a broad temperature range by varying the length of the PEG segments. As expected, the CP increases with the length of the PEG blocks, going from 13 °C for CA(AGE8-b-EG8)4 to 55 °C for CA(AGE8-b-EG40)4. The transition is broader for the copolymers with a shorter PEG block and becomes sharper when the PEG block is longer. Moreover, a two-step transition can be observed for those with a shorter PEG sequence; this is especially clear for CA(AGE8-b-EG12)4, with CPs at 30 and 35 °C. We and others have previously observed double thermoresponsive behavior in block copolymers containing two different thermoresponsive segments.32 34 Each block causes a distinct transition, with a CP related to the one observed for its corresponding homopolymer. The double thermoresponsive behavior of CA-(AGE8-b-EG12)4 is rather unexpected because the PAGE block is not known to be thermoresponsive. The two steps therefore cannot be ascribed to individual transitions pertaining to each of the blocks but must rather be caused by a two-step self-assembly process. All of the phase transitions are reversible, with only a small hysteresis that can be ascribed to additional interchain hydrogen bonds formed in the collapsed state at higher temperatures. This phenomena has been extensively studied in other thermoresponsive polymers.35 Dynamic Light Scattering Studies. DLS was used to study the phase transitions of the block copolymer solutions in water. Figure 2 shows the change in the hydrodynamic diameter (Dh) of the polymer aggregates during the heating process. CA(AGE8-b-EG12)4, which showed a clear two-step transition by UV visible spectroscopy, also displayed a two-step change in Dh. At 20 °C, a Dh of 15 nm was measured, which likely corresponds to single polymer chains in solution (or very small aggregates such as dimers or trimers). Upon heating to 32 °C, the Dh increased to approximately 40 nm, which is consistent with the size expected for micelles. In the temperature range of 30 34 °C, these small aggregates coexisted with larger ones 11176
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Figure 3. Size distribution histograms and representative TEM micrographs of aggregates formed upon heating aqueous solutions (1.0 mg/mL) of the CA(AGE8-b-EGn)4 polymers: (a) n = 12, 45 °C; (b) n = 16, 60 °C; and (c) n = 40, 64 °C.
(ca. 300 nm) but with the great majority of the polymers present as small micelles, as detailed in the Supporting Information. The appearance of these micelles and a few aggregates leads to the first decrease in transmittance observed in the UV visible experiment. When the solution was heated to 36 °C, a Dh of ca. 300 nm was measured. This size is too large to correspond to simple micelles and is likely caused by their aggregation to form micellar clusters. The aggregation of the micelles corresponds to the second decrease in transmittance, which occurs in the range of 34 36 °C. Further increasing the temperature to above 36 °C leads to a decrease in Dh to approximately 190 nm, which may be explained by the dehydration of the PEG blocks. For the polymer with the shortest PEG blocks, CA(AGE8-bEG8)4, a Dh of approximately 70 nm was measured at 1 °C: some aggregates, probably micelles, are present even at the lowest temperature, and single polymer chains were never observed by DLS. However, the solutions are completely clear at this temperature, with a transmittance of 100%. The Dh of the aggregates then increased to a maximum value of approximately 140 nm at 10 °C and decreased to approximately 130 nm upon heating to 25 °C. The change in the Dh of the aggregates was gradual, which may account for the broad transition observed in the UV visible experiment. In the case of the two polymers with the longest PEG blocks, CA(AGE8-b-EG16)4 and CA(AGE8-b-EG40)4, the Dh first remained constant at around 15 nm at low temperatures, indicating that the polymers are present as single chains. Upon further heating, the Dh dramatically increased to a maximum value (ca. 1000 nm) before finally decreasing to a constant value (ca. 700 800 nm). Both large aggregates and small micelles were observed at the same time in the transition-temperature range for these polymers, but micelles were never observed on their own. It
appears that the micelles start to aggregate as soon as they are formed. Furthermore, the micellar clusters obtained at high temperatures have a much higher Dh than those obtained with the two polymers having shorter PEG blocks. The coexistence of large aggregates and micelles has been observed previously in aqueous micellar solutions of PEG-based block copolymers.36 39 Larger aggregates were regarded as the result of the clustering of the initial spherical micelles, caused by the interpenetration of the PEG chains between micelles.37 TEM Studies. TEM was used to study the size and morphology of the particles formed by the star block copolymers upon heating. Figure 3 shows representative TEM micrographs and size distribution histograms obtained for the CA(AGE8-b-EGn)4 polymers with n = 12, 16, and 40. TEM micrographs of CA(AGE8-b-EG12)4 samples prepared at 45 °C showed the presence of roughly spherical aggregates, their most common size being approximately 170 nm. This is in close agreement with the hydrodynamic diameter measured by DLS (ca. 190 nm), which is always expected to be slightly larger because it is a measure of the size of the fully hydrated aggregates. For CA(AGE8-b-EG16)4 and CA(AGE8-b-EG40)4, the modes of the TEM size distribution are approximately 170 and 190 nm, respectively, but with larger particles observed with sizes of up to 800 nm. In contrast, DLS had revealed only the presence of the larger aggregates, with sizes of approximately 800 and 700 nm. In DLS, it is not uncommon for smaller particles to be eclipsed by the presence of larger ones, which scatter much more light, because the scattering intensity scales roughly as the sixth power of the diameter. Thermoresponsive Self-Assembly. Each of the four star block copolymers studied exhibited different behavior upon heating, ultimately resulting in the formation of aggregates 11177
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Langmuir Scheme 2. Schematic Representation of the Self-Assembly of CA(AGE8-b-EGn)4 Polymers in Aqueous Solution upon Heatinga
a
The exact number of micelles in a cluster is not known. For n = 8, free polymers are not observed; for n = 12, all steps are distinctly observed; for n = 16 and 40, micelles start aggregating as soon as they are formed and free micelles are not observed.
having very different hydrodynamic diameters. On the basis of the DLS and TEM results, we propose a model explaining the phase transitions undergone by all four polymers, as schematically illustrated in Scheme 2. At low temperatures, the polymers with sufficiently long PEG blocks (n = 12, 16, 40) are fully soluble in water. A hydrodynamic diameter of approximately 15 nm is measured in the size intensity distribution, which could correspond to single star polymers in solution, likely in a spherical unimolecular micelle conformation with the PEG blocks folded to envelop the hydrophobic cholic acid core and PAGE blocks. The star architecture and the individual micelle conformation could explain that the measured size is larger than that expected for linear polymers with comparable molecular weights; however, it is also possible that this size corresponds to small aggregates of a few star polymers rather than to single molecules. The three polymers with longer PEG blocks are fully soluble at 25 °C, as expected because the thermoresponsive self-assembly studies were performed at concentrations below their cac’s. The only exception is CA(AGE8-b-EG8)4, which is the most hydrophobic of the polymers studied. A Dh of 60 nm was measured at 1 °C, indicating the presence of aggregates even at very low temperature Upon heating, CA(AGE8-b-EG12)4 first forms small aggregates (Dh = 20 60 nm) and then larger ones (Dh = 200 nm) in a distinct second step. The first aggregates formed are likely micelles in which the star polymers act as surfactants, implying that all four arms of the star polymers remain on the same side of the CA core. The micelles then aggregate together to form micellar clusters as the PEG blocks start to dehydrate at higher temperatures. The more hydrophobic CA(AGE8-b-EG8)4 also goes from micelles to micellar clusters upon heating but does not present a transition corresponding to the initial formation of the micelles because they are present even at very low temperatures. The two polymers with the longest PEG blocks (n = 16 and 40) displayed a single transition by UV vis spectroscopy and DLS: from single polymer chains to very large aggregates. It is likely that small micelles were formed, as for the other two polymers, but quickly started aggregating. DLS shows the formation of much larger aggregates when the PEG blocks are longer, but TEM experiments showed that a majority of the aggregates formed are in fact of a very similar size for polymers with n = 12, 16, or 40 and only a few larger aggregates are present for the last
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two. Upon further heating, all polymers show a decrease in Dh, which can be attributed to the dehydration of the PEG blocks.
’ CONCLUSIONS Star-shaped block copolymers CA(AGE8-b-EGn)4 with welldefined molar masses and low polydispersity indices (
