Thermoresponsive Micelles from Jeffamine-b-poly(l-glutamic acid

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Langmuir 2007, 23, 11526-11533

Thermoresponsive Micelles from Jeffamine-b-poly(L-glutamic acid) Double Hydrophilic Block Copolymers Willy Agut,† Annie Bruˆlet,‡ Daniel Taton,*,† and Se´bastien Lecommandoux*,† Laboratoire de Chimie des Polyme` res Organiques, UniVersite´ Bordeaux 1, ENSCPB-CNRS, 16 AVenue Pey Berland, 33607 Pessac Cedex, France, and Laboratoire Le´ on Brillouin, LLB-CNRS-CEA Saclay, 91191 Gif sur YVette, France ReceiVed May 21, 2007. In Final Form: August 18, 2007 Double hydrophilic block copolymers (DHBC) consisting of a Jeffamine block, a statistical copolymer based on ethylene oxide and propylene oxide units possessing a lower critical solution temperature (LCST) of 30 °C in water, and poly(L-glutamic acid) as a pH-responsive block were synthesized by ring-opening polymerization of γ-benzylL-glutamate N-carboxyanhydride using an amino-terminated Jeffamine macroinitiator, followed by hydrolysis. This DHBC proved thermoresponsive as evidenced by dynamic light scattering and small-angle neutron scattering experiments. Spherical micelles with a Jeffamine core and a poly(L-glutamic acid) corona were formed above the LCST of Jeffamine. The size of the core of such micelles decreased with increasing temperature, with complete core dehydration being achieved at 66 °C. Such behavior, commonly observed for thermosensitive homopolymers forming mesoglobules, is thus demonstrated here for a DHBC that self-assembles to generate thermoresponsive micelles of high colloidal stability.

I. Introduction Because of their self-assembly properties in solution or in the bulk, forming micellar structures in the nanometer size range (e.g., spherical micelles, vesicles, rodlike micelles, etc.), amphiphilic block copolymers have great potential in a variety of applications.1-2 For instance, they can be used as surfactants for the stabilization of dispersions, as vehicles for the encapsulation and controlled release of drugs, and as supports for catalysis.2-6 Stimuli-responsive block copolymers can change their sizes or shapes in response to variations in pH, ionic strength, and temperature.7 For obvious environmental concerns, it is highly desirable to derive stimuli-responsive copolymers that would be soluble in aqueous media. Double hydrophilic block copolymers (DHBC)1,8 that can form doubly stimuli-responsive micellar structures in water (i.e., possessing tuneable responses to pH and/or temperature) are scarcer. For instance, doubly temperaturesensitive block copolymers9 or block copolymers consisting of both temperature-sensitive and pH-sensitive blocks10 have been described. Poly(N-isopropylacrylamide) (PNiPAm)11 and poly* Corresponding authors. E-mail: [email protected], lecommandoux@ enscpb.fr. † Universite ´ Bordeaux 1. ‡ LLB-CNRS-CEA Saclay. (1) Hamley, I. W. The Physics of Block Copolymers; Oxford Science Publication: Oxford, England, 1998. (2) (a) Riess, G. Prog. Polym. Sci. 2003, 28, 1107. (b) Riess, G.; Labbe, C. Macromol. Rapid Commun. 2004, 25, 401. (3) Hadjichristidis, N.; Pispas, S.; Floudas, G. A. Block Copolymers: Synthetic Strategies, Physical Properties, and Applications; Wiley-Interscience: John Wiley & Sons: New York, 2003. (4) Block Copolymers in Nanoscience; Lazzari, M., Liu, G., Lecommandoux, S., Eds. Wiley-VCH: Weinheim, Germany, 2006. (5) (a) Kataoka, K.; Harada, A.; Nagasaki, Y. AdV. Drug DeliVery ReV. 2001, 47, 113. (b) Harada, A.; Kataoka, K. Prog. Polym. Sci. 2006, 31, 949-982. (6) Kabanov, A. V.; Batrakova, E. V.; Alakhov, V. Y. J. Controlled Release 2002, 82, 189. (7) Rodriguez-Hernandez, J.; Che´cot, F.; Gnanou, Y.; Lecommandoux, S. Prog. Polym. Sci. 2005, 30, 691-724. (8) For a review on DHBC, see Co¨lfen, H. Macromol. Rapid Commun. 2001, 22, 219. (9) Weaver, J. V. M.; Armes, S. P.; Bu¨tu¨n, V. Chem. Commun. 2002, 18, 2122-2123. (10) Liu, S.; Billingham, N. C.; Armes, S. P. Angew. Chem., Int. Ed. 2001, 40, 2328-2331.

(N-vinyl caprolactam) (PVCL)12 are typical examples of thermoresponsive homopolymers. Both PNiPAm and PVCL in water undergo a coil-to-globule transition upon heating above their lower critical solution temperature (LCST). However, such responsiveness to temperature is concentration-dependent. Also, the formation of dense and narrowly size distributed mesoscopic globules (mesoglobules) based on PNiPAm or PVCL, as a result of the self-assembly of several single chains, is generally stable only under very dilute conditions.13 A recent contribution, however, showed that it was possible to obtain dense stable colloids of nearly pure PNiPAm in a concentration range of 1-6 wt %.14 When associated with a water-soluble stabilizing block, mesoglobular micelles can be formed at higher concentrations by the self-assembly of corresponding block copolymers, as reported for DHBC based on PNiPAm15 or PVCL.16 In addition, such DHBC was found to mimic the behavior of proteins whose self-assembly can be driven to different ordered and stable quaternary structures without macroscopic precipitation under appropriate conditions.17 Here, we describe the controlled synthesis as well as the aqueous solution properties of rod-coil DHBC composed of a Jeffamine block and a poly(glutamic acid) (PGA) block. Commercial Jeffamine is an amino-terminated random copolymer of ethylene oxide and propylene oxide; it is a thermoresponsive block possessing an LCST of around 30 °C in water. As for PGA, it is a pH-sensitive, biocompatible synthetic polypeptide that can undergo a truly reversible transition from a rodlike R-helix conformation to a coil conformation with changing tempera(11) Kirsh, Y. E. Water Soluble Poly-N-Vinylamides: Synthesis and Physicochemical Properties; Wiley: Chichester, England, 1998. (12) (a) Chan, K.; Pelton, R.; Zhang, J. Langmuir 1999, 15, 4018-20. (b) Aseyev, V.; Hietala, S.; Laukkanen, A.; Nuopponen, M.; Confortini, O.; Du Prez, F. E.; Tenhu, H. Polymer 2005, 46, 7118. (13) Aseyev, V. O.; Tenhu, H.; Winnik, F. M. AdV. Polym. Sci. 2006, 196, 1-85. (14) Balu, C.; Delsanti, M.; Guenoun, P.; Monti, F.; Cloıˆtre, M. Langmuir 2007, 23, 2404-2407. (15) Virtanen, J.; Tenhu, H. Macromolecules 2000, 33, 5970. (16) Lozinsky, V. I.; Simenel, I. A.; Kulakova, V. K.; Kurskaya, E. A.; Babushkina, T. A.; Klimova, T. P. Macromolecules 2003, 36, 7308. (17) Stryer, L. Biochemistry, 3rd ed.; W. H. Freeman: New York, 1988.

10.1021/la701482w CCC: $37.00 © 2007 American Chemical Society Published on Web 10/04/2007

ThermoresponsiVe Block Copolymer Micelles

Figure 1. SEC traces of Jeffamine-b-PBLG50 with the amine way (-) and ammonium way (-‚-) in DMF, LiBr at 60 °C.

ture.18-21 PGA blocks were first synthesized by the ring-opening polymerization of γ-benzyl-L-glutamate N-carboxyanhydride using the Jeffamine macroinitiator, followed by hydrolysis of the benzyl groups. We demonstrate that such doubly responsive DHBC spontaneously and reversibly self-assembles in water at relatively high concentrations. With increasing temperature, these DHBCs form stable core-shell micelles consisting of a Jeffamine core electrosterically stabilized by a PGA corona. Investigations by small-angle neuton scattering allowed us to quantify the hydration state of the core of these micelles as a function of temperature. II. Experimental Section 1. Materials and Methods. 1.1. Materials. Dimethylformamide (DMF) (Scharlau, 99.9%) was dried over molecular sieves (3 and 4 Å) and cryodistilled prior to use. Amino-terminated Jeffamine M-2005 (XTJ-507) (Huntsman, +95%), also noted as P(EO6-rPO29)-NH2, was dried under vacuum at 40 °C overnight. γ-BenzylL-glutamate N-carboxyanhydride (Bz-L-GluNCA) (Isochem, +96%) was used as received. 1.2. Synthesis of Jeffamine-b-PGA Copolymers. In a glovebox, Bz-L-GluNCA (4 g, 15.2mmol) was introduced into a flame-dried Schlenk flask and dissolved in anhydrous DMF (0.1 g/mL). In a separate flask, P(EO6-r-PO29)-NH2 (0.5mmol) was dried overnight under vacuum at 40 °C, dissolved in dry DMF (0.1 g/mL), and added to the first flask under N2 through a cannula. The initiation of Bz-L-GluNCA from the Jeffamine hydrochloride macroinitiator, also noted P(EO6-r-PO29)-NH3Cl, was carried out as follows. Jeffamine was dissolved in THF, and the required amount of aqueous HCl (1 M) was added. After 30 min of reaction and evaporation of the solvents, P(EO6-r-PO29)-NH3Cl was dried overnight under vacuum at 40 °C, redissolved in dry DMF (0.1 g/mL), and added to the Schlenk flask under N2 through a cannula. The solution was stirred for 24 h at room temperature when using P(EO6-r-PO29)-NH2 and for 72 h at 40 °C when using P(EO6-r-PO29)-NH3Cl. The final compound was recovered by precipitation in diethyl ether and dried under vacuum. (See Figure 2a for 1H NMR characterization). Deprotection of the benzyl groups of the poly(γ-benzyl Lglutamate) blocks affording Jeffamine-b-poly(L-glutamic acid) copolymers was carried out as follows. Into a THF solution (25 (18) Kricheldorf, H. R. R-Amino acid-N-carboxyanhydrides and Related Heterocycles; Springer: Berlin, 1987. (b) Kricheldorf, H. R. Angew. Chem., Int. Ed. 2006, 45, 5752. (19) Deming, T. J. Nature 1997, 390, 386. (20) Klok, H. A.; Lecommandoux, S. AdV. Polym. Sci. 2006, 202, 75. (21) Che´cot, F.; Bruˆlet, A.; Oberdisse, J.; Gnanou, Y.; Mondain-Monval, O.; Lecommandoux, S. Langmuir 2005, 21, 4308.

Langmuir, Vol. 23, No. 23, 2007 11527 mg/mL) of the Jeffamine-b-poly(γ-benzyl L-glutamate) copolymer precursor was added 1.5 equiv of KOH per benzyl ester function, and the mixture was stirred at room temperature for 15 h. The solvent was removed under vacuum, and the copolymer was precipitated by the addition of diethyl ether. The solution was centrifuged, and the solvent was removed by decantation. The latter procedure was repeated three times. The powder was dried under vacuum for 24 h and characterized by 1H NMR in D2O (Figure 2b). 1.3. Characterization. 1.3.1. Size-Exclusion Chromatography. The molar masses and polydispersity indices of all materials were determined by size exclusion chromatography (SEC) in DMF with LiBr (1 g/L) as the eluent (0.8 mL/min) at 60 °C using a Waters apparatus (Alliance GPCV2000) equipped with a refractometric detector and calibrated with polystyrene standards. 1.3.2. Nuclear Magnetic Resonance Spectroscopy (NMR). The molar masses of PBLG blocks were determined by 1H NMR in DMSO-d6 (Bruker AC 400 spectrometer) by calculating the molar ratio between the benzyl groups of the PBLG blocks and the methyl groups of the Jeffamine macroinitiator. The deprotection of PBLG blocks can be monitored by 1H NMR in D2O, following the disappearance of the benzyl protons at δ ) 7.5 and 5 ppm. 1.3.3. Dynamic Light Scattering (DLS). DLS experiments were performed using an ALV laser goniometer, with a 22 mW linearly polarized laser (632.8 nm HeNe) and an ALV- 5000/EPP multiple tau digital correlator with a 125 ns initial sampling time. The sample was studied at different temperatures (10, 20, 36, 51, and 66 °C), and the temperature was equilibrated for 30 min after every change. The accessible scattering angular range varied from 40° up to 120°. The solutions were introduced into 10-mm-diameter glass cells. The minimum sample volume required for the experiment was 1 mL. Data were acquired with ALV correlator control software, and the analysis time was fixed for each sample at 300 s. The hydrodynamic radii (RH) values of the micelles were obtained by using CONTIN analysis from solutions in basic conditions (pH >11) at a concentration of 25 mg/mL. Millipore water was thoroughly filtered through 0.1 µm filters and directly employed for the preparation of the solutions. The hydrodynamic radius (RH) could be calculated from the diffusion coefficient using the Stokes-Einstein relation. 1.3.4. Small-Angle Neutron Scattering (SANS). SANS experiments were performed at the Le´on Brillouin Laboratory (Orphe´e reactor, Saclay) on the PACE spectrometer. Two spectrometer configurations have been used in order to cover a q range from 5 × 10-3 to 0.15 Å-1. The main parameters used to calculate the corresponding resolution functions22 are listed in Table 1. The sample (a solution of Jeffamine-b-PGA30 in heavy water D2O, concentration 2.5 wt %) was introduced into a 5-mm-thick rectangular quartz cell and studied at different temperatures (10, 20, 36, 51, and 66 °C). The blank sample was pure D2O. Data treatment was done with Pasidur software (LLB). Absolute values of the scattering intensity (I(q) in cm-1) were obtained from the direct determination of the number of neutrons in the incident beam and the detector cell solid angle.23 The signal of a pure D2O blank sample was first subtracted. The contribution due to incoherent scattering of the 2.5 wt % Jeffamine-b-PGA30 solute was determined by plotting q4I(q) versus q4 of the sample signal. At large q values and for high temperatures (51 and 66 °C), this plot is linear, and its slope gives the incoherent contribution of the Jeffamine-b-PGA30 solute. The magnitude of this slope was around 0.008 cm-1 and was subtracted from the data. Two different models have been used to adjust the data. One is the sphere model. The form factor of a homogeneous sphere of radius Rs can be simply expressed as Psphere(q) )

[

]

3(sin(qRS) - qRS cos(qRS)) (qRS)

3

2

(1)

(22) (a) Mildner, D. R. M.; Carpenter, J. M. J. Appl. Crystallogr. 1984, 17, 249. (b) Pedersen, J. S.; Posselt, D.; Mortensen, K. J. Appl. Crystallogr. 1990, 23, 321. (23) Cotton, J. P. In Neutron, X-ray, and Light Scattering; Lindner, P., Zemb, Th., Eds.; Delta Series; North-Holland: Amsterdam, 1991; p 19.

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Figure 2. 1H NMR of Jeffamine-b-PBLG30 (a, DMSO-d6) and Jeffamine-b-PGA30 (b, D2O) (Bruker, 400 MHz). Table 1. Parameters of the SANS Spectrometer Configuration λ nma

∆λ/λ fwhmb

L1 cmc

D1 cmd

D2 cme

L2 cmf

∆R cmg

1.7 0.6

0.1 0.1

500 250

1.2 1.6

0.7 0.7

470 290

1 1

a Neutron wavelength. b Full width at half-maximum of the neutron wavelength distribution. c Collimation distance. d Diameter of the entrance collimation diaphragm. e Diameter of the exit collimation diaphragm. f Sample-to-detector distance. g Pixel size of the detector.

The values of the radii of micelles cores and the polydispersity were obtained by fitting experimental curves to this form factor using a homemade program21 that also takes into account the experimental resolution functions with the parameters listed in Table 1. These values have been confirmed by the analysis of the SANS data in the Porod approximation.24 Although this model fits the data obtained at high-temperature (51 and 66 °C) very well, we have used another model to better adjust the data obtained at lower temperature. This is the spherical copolymer micelle model developed by Pedersen and al.25,26 to describe the conformation of associated copolymers in solution. The form factor of a spherical copolymer micelle is the sum of four different terms: - the self-correlation term of the spherical core, Psphere(q); - the self-correlation term of the polymer chain inside the corona, Pchain(q); - the cross term between the core and the chains, Score-corona(q); - the cross term between the chains inside the corona, Scorona-corona(q). It can be written as25,26 Pspherical micelle(q) ) Z2βcore2 Psphere(q) + Zβcorona2 Pchain(q) + 2Z2βcoreβcoronaScore-corona(q) + Z(Z - 1)βcorona2 Scorona-corona(q) (2) where Z is the aggregation number of the micelle and βcore and βcorona are the total excess scattering lengths of the core and the corona, (24) Porod, G. Kolloid Z. 1951, 124, 82. (25) (a) Pedersen, J. S.; Svanborg, C.; Almdal, K.; Hamley, S. W.; Young, R. N. Macromolecules 2003, 36, 416. (b) Pedersen, J. S.; Laso, M.; Schurtenberger, P. Phys. ReV. E 1996, 54, 5917.

respectively. They can be calculated as βcore ) Vcore(Fcore - Fsolvent) and βcorona ) Vcorona(Fcorona - Fsolvent), where Vcore and Vcorona are the total volumes of a block in the core and in the corona, respectively. Fcore and Fcorona are the corresponding scattering-length densities, and Fsolvent is that of the surrounding solvent. The expressions of the cross terms, core-corona and coronacorona, in eq 2 depend on the geometry of the core. They are given in refs 25 and 26. Concerning the PGA blocks, because they are not very long, very little difference in their conformation depending on the solvent quality is expected. Thus, for the sake of simplification, we have chosen the Debye function27 for the form factor of the chain inside the corona. This spherical copolymer micelle model requires numerous parameters: Z, the aggregation number of the micelle; Rs, the radius of the spherical core with polydispersity σ; and Rg, the radius of gyration of the chains inside the corona. Moreover, the core contrast between Jeffamine and D2O may vary depending on the rate of hydration (x) of the core. As a result, in eq 2 βcore becomes βcore ) Vcore(1 - x)(Fcore - Fsolvent). Fcorona simply refers to the contrastlength density of PGA chains with respect to D2O. We have then calculated the form factor of a spherical copolymer micelle following eq 2, and as for the sphere model, we have taken into account the experimental resolution functions. This model allows us to adjust the curve obtained at 36 °C, which was not well fitted by the previous sphere model.

III. Results and Discussion 1. Synthesis and Characterization of Jeffamine-b-poly(glutamic acid) DHBC. The synthesis of Jeffamine-b-poly(glutamic acid) DHBC first involved the ring-opening polymerization (ROP) of γ-benzyl-L-glutamate N-carboxyanhydride from an amino end-functionalized statistical copolymer possessing an LCST of around 30 °C in water. This precursor is JeffamineNH2, P(EO6-r-PO29)-NH2, where PEO and PPO stand for poly(ethylene oxide) and poly(propylene oxide), respectively, and the subscripts refer to the average degree of polymerization of each monomer unit. Amphiphilic Jeffamine-b-poly(γ-benzyl(26) Pedersen, J. S. J. Appl. Crystallogr. 2000, 33, 637. (27) Debye, P. J. Phys. Colloid Chem. 1947, 51, 18.

ThermoresponsiVe Block Copolymer Micelles

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Scheme 1. General Scheme for the Synthesis of the Jeffamine-b-PGA Block Copolymer

L-glutamate)

block copolymer, Jeffamine-b-PBLG, was thus obtained and subsequently subjected to hydrolysis of the benzylic groups of the PBLG blocks. It is well documented that primary amino-initiated ROP of N-carboxyanhydrides (NCA) proceeds through the so-called nucleophilic “amine mechanism” that may suffer, however, from side reactions.18 The “activated monomer mechanism” that is initiated by the deprotonation of a NCA forming an “NCA anion” (NCA-) may also take place, resulting in a broadening of the molar mass distribution. This problem of such a dual initiation can be overcome by applying the method of ammonium-mediated ROP of NCA, as recently developed by Schlaad and co-workers28 and also used by others.29-30 This method consists of the protonation (e.g., using HCl) of the primary amino-functional initiators, thus forming amino hydrochloride groups. The latter can be viewed as the dormant species in equilibrium with the active growing amines (Scheme 1). The protons released by this equilibrium then rapidly react with NCA-, which suppresses the activated monomer mechanism, providing polypeptides with narrow molar mass distributions. To achieve well-defined block copolymers free of any homopolymer contaminants, we turned to this ammonium-mediated ROP of NCA by protonating the P(EO6-r-PO29)-NH2 precursor for the ROP of γ-benzyl-L-glutamate NCA. The NH2 group of P(EO6r-PO29)-NH2 was thus transformed into its hydrochloride salt by simple treatment with aqueous HCl in THF. After solvent removal, P(EO6-r-PO29)-NH3Cl was obtained. As also pointed out by Schlaad and co-workers,31 no real kinetic investigations have been reported to support the ammonium mechanism discussed above. It is expected, however, that the rate of the ROP can be influenced by experimental parameters, including the extent of protonation and the nature of the solvent, that is, the polarity of the polymerization medium and the temperature. Here, we have briefly studied the influence of the extent of protonation of the amino groups of the P(EO6-r-PO29)-NH2 precursor on the kinetics of the ROP of the NCA. Following this strategy, several Jeffamineb-PBLG copolymers were successfully synthesized in good yield, using the ROP of γ-benzyl-L-glutamate N-carboxyanhydride from the commercial P(EO6-r-PO29)-NH2 (Figure 1, Table 2). Char(28) Dimitrov, I.; Schlaad, H. Chem. Commun. 2003, 23, 2944. (29) Lutz, J. F.; Schu¨tt, D.; Kubowicz, S. Macromol. Rapid Commun. 2005, 26, 23-28. (30) Dimitrov, I.; Berlinova, I. V.; Vladimirov, N. G. Macromolecules 2006, 39, 2423. (31) Dimitrov, I.; Kukula, H.; Co¨lfen, H. S. Macromol. Symp. 2004, 215, 383.

Table 2. Synthesis of Jeffamine-b-PBLG Block Copolymers as a Function of the Extent of Protonation of Amino Groupsa NH3+ T t [NCA]/ conversion DPPBLG Mn % entry % °C h [NH2] exptb g/molb Mw/Mnc 1 2 3 4 5

0 0 10 75 100

25 25 40 40 40

24 24 72 72 72

30 30 30 30 30

97 98 97 50 45

30 32 33 13 12

8570 9000 9200 4850 4630

1.13 1.12 1.20 1.05 1.05

a Theoretical molar mass: DPn theo ) conv[monomer]/[macroinitiator]. The overall composition was determined by 1H NMR in DMSO-d6. c Determined by SEC in DMF at 60 °C. b

acterization by 1H NMR spectroscopy allowed us to determine the overall composition of amphiphilic Jeffamine-b-PBLG copolymers (Figure S1) whereas SEC in DMF in the presence of LiBr gave the molar mass distribution as a function of the extent of protonation (Table 2). The results in Table 2 show that the protonation of P(EO6-r-PO29)-NH2 precursor leads to slightly better-defined block copolymers, as evidenced by SEC measurements (Figure 1). However, one can note that the rate of ROP is significantly lower when resorting to this ammonium-mediated mechanism: after 3 days of ROP, only 50% of the monomer is consumed for 75% of HCl added (entry 4). Even though a complete detailed kinetic investigation has not been undertaken, it turned out that the higher the extent of protonation, the lower the rate of ROP, Rp ) kp[NH2][NCA] (entries 3-5) because of the lower concentration of propagating amino groups. Next, these amphiphilic block copolymers were deprotected quantitatively (Figure 2) under basic conditions using KOH following an already reported procedure;21 this afforded the targeted Jeffamine-bpoly(glutamic acid) DHBC. 2. Temperature-Induced Self-Assembly of Jeffamine-bpoly(glutamic acid) DHBC in Water. The self-assembly properties of one particular DHBC in water, Jeffamine-b-PGA30 obtained after hydrolysis from entry 2 (Table 2), were investigated as a function of temperature. This copolymer was first dissolved in basic solution (pH >11, c ) 25 mg/mL) at low temperature (15 °C) so as to ensure a homogeneous dispersion. Under such conditions, the PGA block is charged whereas the Jeffamine block is below its LCST. The conformation of the DHBC is thus close to that of a single unimer, as attested to by the low scattering intensity measured by DLS (Figure 3a) and the q-5/3 decrease in SANS intensity (Figure 4). Such a variation is typical of polymer

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Figure 5. Frequency versus q2 measured at different angles for Jeffamine-b-PGA30 (c ) 25 mg/mL, pH 11) for different temperatures above the LCST. (Inset) Γ/q2 () Dapp) versus q2, which attests to the formation of spherical micelles.

observed by DLS and by the increase in the characteristic time deduced from the autocorrelation function (Figure 3b). In addition, the linear dependence of the typical frequency Γ as a function of q2 measured by DLS (Figure 5) attests to the purely diffusive behavior of the particles. The hydrodynamic radius RH of these micelles was found to increase slightly from 11.2 to 13.5 nm when increasing the temperature from 36 to 66 °C. Moreover, the SANS curves obtained under the same conditions can be properly fitted with a sphere form factor (Figure 4). Indeed, the scattering from N identical particles of spherical symmetry with a volume Vp placed in a total volume V can be easily described as a function of the modulus of the scattering vector q:

N I(q) ) (∆F)2Vp2P(q) S(q) V Figure 3. Scattering intensity (a) and auto-correlation function C(q, t) (b) of Jeffamine-b-PGA30 in water obtained by DLS at 90° for different temperatures (c ) 25 mg/mL, pH 11).

P(q) is the form factor of the particles, S(q) is the structure factor, and (∆F)2 is the contrast (i.e., the difference in scattering-length density between the particle and the solvent). In the case of very dilute solutions, the structure factor S(q) f 1, and eq 1 becomes

N I(q) ) (∆F)2Vp2 P(q) V

Figure 4. Variation with temperature of the SANS intensity as a function of q for Jeffamine-b-PGA30 (c ) 25 mg/mL, pH 11). The curves at higher temperatures can be perfectly fitted with a sphere form factor. (The radius R and the log-normal σ distribution values are given in the legend.)

chains in a good solvent. Raising the temperature above the LCST of the Jeffamine block then leads to the formation of well-defined aggregates, as indicated by the increase in intensity

(3)

(4)

The appearance of a sphere form factor upon increasing the temperature above the LCST of the Jeffamine block is explained by the collapse of the chains of the micellar core due to an increase in van der Waals attraction forces. Here, an important behavior is that the micellar structures are protected against precipitation and prove to be stable at high concentrations thanks to the PGA-based blocks: the latter form a protective sheath due to electrostatic and steric (electrosteric) colloidal stabilization. Interestingly, variation of the scattering intensity was also observed by SANS (Figure 4) as the temperature was progressively increased. For instance, the form factor changed from that of free chains to that of spheres between 20 and 36 °C. In addition, from 36 to 66 °C, the scattering intensity at low q progressively increased, which indicated that the contrast or/and the molar mass (aggregation number) of the micelles increased. Previous SANS experiments on other block copolymers21,32 have shown that the scattering intensity of core-shell micelles is mainly due to the core: the corona is so “hairy” that its scattering is negligible. (32) Shusharina, P.; Linse, P.; Khokhlov, A. R. Macromolecules 2000, 33, 8488.

ThermoresponsiVe Block Copolymer Micelles

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Vc )

(34)πR

3 c

) (ZMJeffamine/FJeffamineΝA)

(6)

where Rc is the core radius, MJeffamine is the molar mass of Jeffamine, NA is Avogadro’s number, and FJeffamine is the density of Jeffamine (1.004 g cm-3). The aggregation number is given by

Z)

4πRc3FJeffamineNA 3MJeffamine

(7)

From these equations, we obtained an aggregation number of Z ) 45 for the micelles formed at 66 °C, which is a typical value for spherical micelles. One could also determine the aggregation number from the scattering intensity at zero q. Indeed, under such conditions, P(q f 0) ) 1, and eq 4 can be written as Figure 6. Scattering intensity in the Porod representation (q4I(q)) of Jeffamine-b-PGA30 in water at different temperatures: 36 (0), 51 (O), and 66 °C (×).

In addition, the scattering-length density of PGA in deuterated water was very low (the scattering-length density of hydrated PGA was FPGA ) 5.11 × 1010 cm-2 and that of deuterated water was FD2O ) 6.38 × 1010 cm-2), so the variation in the scattered intensity versus temperature affected only the hydrophobic Jeffamine core of the aggregates. On the basis of this assumption, we developed a model that takes into account the intensity of an assembly of spherical Jeffamine domains (with a scatteringlength density of Fjeff ) 4.17 × 109 cm-2) surrounded by heavy water. From the fit of the experimental curve using a simple sphere model, one could determine the radius of the core Rs ) Rc (Figure 4). In addition, the core radius Rc at different temperatures could also be determined from the Porod representation24 of the SANS scattering intensity, as observed in Figure 6: q* ) 2.7/Rc. It decreased from Rc ) 4.5 to 3.4 nm when the solution temperature increased from 36 to 66 °C. This behavior could be attributed to a change in the water content in the core of the micelles, as previously observed by Hatton and coworkers33,34 for (deuterated ethylene oxide)-(propylene oxide)(deuterated ethylene oxide) dPEO-b-PPO-b-dPEO block copolymers on the basis of SANS experiments. These authors observed from contrast-variation experiments that the aggregation number and PPO content in the core both increased with temperature above the LCST of PPO, with the core radius remaining constant. In the present situation, we assumed that the aggregation number remained constant and only the rate of hydration was changing. From our measurements, further calculations could bring more quantitative information. For example, the aggregation number of the micelles Z is defined as

Z)

M h w,micelle M h w,copolymer

(5)

where M h w,micelle is the average molecular weight of the micelles and M h w,copolymer is the average molecular weight of the copolymer. At high temperature, when completely dehydrated, the Jeffamine block representing the core of the micelles was supposed to be spherical, dense, and homogeneous. Then, the corresponding micelle volume could be expressed on the basis of only the characteristics of the Jeffamine core, that is, (33) Goldmints, I.; von Gottberg, F. K.; Smith, K. A.; Hatton, T. A. Langmuir 1997, 13, 3659-3664. (34) Goldmints, I.; Yu, G.; Booth, C.; Smith, K. A.; Hatton, T. A. Langmuir 1999, 15, 1651-1656.

cNA N (∆F)2Vp2 I(q)qf0 ) (∆F)2Vp2 ) V Mmicelle

(8)

where c ) N/(V)(Mmicelle)/NA is the concentration of the block copolymer in solution and Mmicelle is the molecular weight of the self-assembled micelles. Then, combining eqs 5 and 8, we obtained an aggregation number Z ) 48 at 66 °C, confirming the methodology and the full dehydration of the micelle core at this high temperature. Finally, assuming that the core is completely dehydrated at 66 °C, one could estimate the rate of hydration of the core at lower temperature (36 °C) as the ratio between the core volumes as follows:

rate of hydration ) 1 -

3.43 ) 57% 4.53

The rate of hydration thus found is around 60% at 36 °C and progressively decreased with increasing temperature, with the micelles being completely dehydrated at 66 °C. The radius of the micelle cores correspondingly decreased from 4.5 nm at 36 °C to 3.4 nm at 66 °C (Figures 4 and 6). To better describe the data obtained at low temperature (36 °C) together with high-q behavior for which the sphere model is inaccurate, we have used the spherical copolymer micelle model25,26 previously described in the Experimental Section. We have adjusted the data, first at 66 °C where the spherical shape well described the data and then at lower temperature, 36 °C, where this model fails to describe the high-q-range data well. The best-fit parameters with the two models are listed in Table 3. At high temperature, the best-fit value of Rs obtained with the copolymer micelle model is in very good agreement with that found with the fit to the sphere model. The polydispersity of the core, σ, is slightly smaller because part of the broadening of the scattering curve that makes the oscillation of the sphere form factor disappear now arises from the contribution of the corona and the spreading due to the cross term between the core and the chains. However, at this temperature, whatever the size of the chain (an Rg value between 1 and 5 nm), the calculated form factor in the q range observed does not change: we do not succeed in improving the fit quality in the high-q range as can be observed in Figure 7. The huge decrease observed in this range is due to the spherical shape of the micelle. This observation means that the scattering from the PGA chains is negligible. The scattering signal thus mainly comes from the spherical core, with both models properly describing the experimental curve. As revenge, at lower temperature (36 °C), the spherical micelle model effectively fits the experimental curve (Figure 7). The

11532 Langmuir, Vol. 23, No. 23, 2007

Agut et al.

Table 3. Comparison of the Best-Fit Parameters with the Sphere Model and the Spherical Copolymer Micelle Model sphere model 66 °C 51 °C 36 °C a

spherical copolymer micelle model

Rs nm

σ

Rs nm

σ

Z

Rg nm

xa

3.3 ( 0.05 3.4 ( 0.05 4.5 ( 0.05

0.18 ( 0.02 0.20 ( 0.02 0.20 ( 0.02

3.6 ( 0.05 3.8 ( 0.05 4.0 ( 0.05

0.15 ( 0.02 0.18 ( 0.02 0.2 ( 0.02

45 ( 2 45 ( 2 5(1

2(2 1(1 1.3 ( 0.15

0 0.10 ( 0.05 0.80 ( 0.05

Rate of hydration of the Jeffamine core in D2O.

Figure 7. SANS intensity for Jeffamine-b-PGA30 as a function of q and temperature (c ) 25 mg/mL, pH 11). The curves are fitted with a spherical copolymer micelle model from Pedersen25,26 with the introduction of the rate of hydration in the core, x.

core radius values obtained with the two models are comparable. The aggregation number deduced from the best fit is small, Z ) 5, and the rate of hydration of the core is high, 80%. With this low-Z value and high rate of hydration of the core, the signal arising from the core is much smaller than at higher temperature. The chain contribution then becomes visible and may affect the scattering curve obtained at high q values. The best fit of the experimental curve is obtained with an Rg chain value of 1.3 nm. The fit with the spherical copolymer micelle model better describes the whole experimental curve than does the sphere model. With this more detailed model, we find a rate of hydration of the core of 80%, which is higher than that (57%) deduced

from the Rs values of the sphere model and from the extrapolation at zero q value of the scattering intensity. From this detailed analysis using the spherical copolymer micelle model, one can conclude that even if the formation of nanoparticles is detectable at 36 °C above the LCST of Jeffamine, these aggregates have a low aggregation number and a very high degree of hydration. Real core-shell micelles are obtained at higher temperatures, with a constant aggregation number. This typical behavior is represented schematically in Figure 8. Our observations of the progressive dehydration of the micelles with temperature are consistent with the work of Hatton33,34 and that on homopolymers such as PVCL- and PNiPAm-forming mesoglobules.15,16 However, in the work of Hatton33,34 this water expulsion was accompanied by an increase in the micellar aggregation number, resulting in a constant core size. In the present work, this dehydration occurs with a decrease in the micellar core radius, probably reflecting a constant aggregation number. Because of their change with temperature, these structures can be called “mesoglobular micelles”; they result from the selfassemby in water of DHBCs consisting of one LCST-type block and a polyelectrolyte-stabilizing block.

IV. Conclusions Double hydrophilic block copolymers consisting of a thermoresponsive Jeffamine block and an ionogenic hydrophilic block based on poly(glutamic acid) were synthesized by the ammoniumor amine-mediated ring-opening polymerization of γ-benzylL-glutamate N-carboxyanhydride using an amino-terminated Jeffamine macroinitiator, followed by hydrolysis. Preliminary investigations by DLS demonstrated the thermoresponsiveness of the Jeffamine block in aqueous media: below its LCST

Figure 8. Schematic representation of the formation of mesoglobular micelles with temperature. The core of the micelles is swollen with water at 36 °C (blue) and dehydrated at 66 °C (red).

ThermoresponsiVe Block Copolymer Micelles

(30 °C), DHBC appeared as free chains in good solvent, whereas above the LCST, the Jeffamine block became hydrophobic and DHBC self-assembled into spherical micelles. These findings were further confirmed by SANS experiments. The hydration of the core of such micelles could be further manipulated with temperature. Combining a sphere model and a copolymer micelle model, we quantitatively analyzed our experimental data, especially the hydration rate of the obtained micelles. We showed that the size of the core decreased with increasing temperature as a result of the dehydration of Jeffamine, with the spherical micelles being completely dehydrated only at 66 °C. Such behavior may have interesting applications such as the specific absorption and/or release of small molecules (drugs). Such

Langmuir, Vol. 23, No. 23, 2007 11533

behavior commonly observed for thermosensitive homopolymers forming mesoglobules is evidenced here for DHBC generating thermoresponsive micelles of high colloidal stability. Acknowledgment. We thank Huntsman for the gift of Jeffamine and S. P. Armes for fruitful discussions and are grateful for funding from the French Ministry of Research and Education, CNRS, and Re´gion Aquitaine. Supporting Information Available: 1H NMR spectrum of Jeffamine-b-PBLG30 in DMSO-d6. This material is available free of charge via the Internet at http://pubs.acs.org. LA701482W