pH and Temperature Responsive Polymeric Micelles and

May 21, 2010 - The aqueous solution behavior of novel polypeptide-based double hydrophilic block copolymers (DHBCs), namely, ...
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pH and Temperature Responsive Polymeric Micelles and Polymersomes by Self-Assembly of Poly[2-(dimethylamino)ethyl methacrylate]b-Poly(glutamic acid) Double Hydrophilic Block Copolymers Willy Agut,†,‡ Annie Br^ulet,§ Christophe Schatz,†,‡ Daniel Taton,*,†,‡ and Sebastien Lecommandoux*,†,‡ †

Universit e de Bordeaux, ENSCBP, 16 avenue Pey Berland, 33607 Pessac Cedex, France, ‡CNRS, Laboratoire eon Brillouin, UMR12 de Chimie des Polym eres Organiques, UMR5629, Pessac, France, and §Laboratoire L (CEA-CNRS), C.E. Saclay, 91191 Gif sur Yvette, France Received February 7, 2010. Revised Manuscript Received April 28, 2010

The aqueous solution behavior of novel polypeptide-based double hydrophilic block copolymers (DHBCs), namely, poly[2-(dimethylamino)ethyl methacrylate]-b-poly(glutamic acid) (PDMAEMA-b-PGA), exhibiting pH- and temperature-responsiveness is presented using a combination of scattering techniques (light and neutron) and transmission electron microscopy. Close to the isoelectric point (IEP), direct or inverse electrostatic polymersomes are generated by electrostatic interactions developing between the two charged blocks and driving the formation of the hydrophobic membrane of the polymersomes, with the latter being stabilized in water by uncompensated charges. Under basic conditions, that is, when PDMAEMA is uncharged, the thermosensitivity of the DHBCs relates to the lower critical solution temperature (LCST) behavior of PDMAEMA around 40 °C. As a consequence, at pH = 11 and below this LCST, free chains of DHBC unimers are evidenced, while above the LCST the hydrophobicity of PDMAEMA drives the self-assembly of the DHBCs in a reversible manner. In this case, spherical polymeric micelles or polymersomes are obtained, depending on the PGA block length. These possibilities of variation in size and shape of morphologies that can be achieved as a function of temperature and/or pH variations open new routes in the development of multiresponsive nanocarriers for biomedical applications.

Introduction Block copolymer self-assembly is an efficient way to generate nanoscale micellar structures in dilute solutions, which may serve in several applications as nanoreactors or nanovehicles in biology-related fields.1-3 The self-assembly process can be driven by different noncovalent interactions, including hydrophobic ones, hydrogen bondings, and metal coordination or electrostatic interactions. The hydrophobic effect has been widely used to selfassemble amphiphilic block copolymers in aqueous solutions.4-8 Double hydrophilic block copolymers (DHBCs) are a particular class of block copolymers that generally associate a polyelectrolyte block with a stabilizing block in water, but they can also be composed of two polyelectrolytes.9 DHBCs can self-associate to form micellelike structures in water, which can be induced by a change of pH, by temperature, or by ionic strength variations. Upon applying one of these stimuli, one block turns insoluble in *To whom correspondence should be addressed. E-mail: [email protected] (D.T.); [email protected] (S.L.). (1) Hadjichristidis, N.; Pispas, S.; Floudas, G. A. Block Copolymers: Synthetic Strategies, Physical Properties, and Applications ; Wiley Interscience, John Wiley & Sons, Inc.: Hoboken, NJ, 2003. (2) Block Copolymers in Nanoscience; Lazzari, M., Liu, G., Lecommandoux, S., Eds.; Wiley-VCH Verlag GmbH & Co, KGaA: Weinheim, 2006. (3) (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. (4) Antonietti, M.; F€orster, S. Adv. Mater. 2003, 15, 1323. (5) Gohy, J.-F. Adv. Polym. Sci. 2005, 190, 65. (6) Th€unemann, A. F.; M€uller, M.; Dautzenberg, H.; Joanny, J.-F.; L€owen, H. Adv. Polym. Sci. 2004, 166, 113. (7) Riess, G. Prog. Polym. Sci. 2003, 28, 1107. (8) Rodriguez-Hernandez, J.; Checot, F.; Gnanou, Y.; Lecommandoux, S. Prog. Polym. Sci. 2005, 30, 691. (9) For a review on DHBCs, see: (a) Colfen, H. Macromol. Rapid Commun. 2001, 22, 219–252. (b) Nakashima, K.; Bahadur, P. Adv. Colloid Interface Sci. 2006, 123-126, 75.

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water while the other stabilizes the colloidal aggregates. Currently, nanoassemblies made from DHBCs are considered as promising nanovectors in drug controlled release applications.10-13 Welldefined nanostructures can also be achieved by self-assembly of block copolymers via the formation of hydrogen bondings.14-17 Benefit can be taken from such interactions to tune both the size and the morphology of self-assemblies by minimizing hydrogen bondings between polymer blocks and solvent molecules.18,19 A third strategy based on metal-ligand coordinations can be used to direct the self-assembly of block copolymers.20 On this principle, and in order to develop drug delivery applications, Kataoka et al. reported the self-assembly of poly(ethylene glycol)-b-poly(glutamic acid) (PEG-b-PGA) by forming a polymer-metal complex between dichloro(1,2-diamino-cyclohexane)platinum(II) (DACHPt), an antitumor drug, and carboxylic groups of the PGA blocks.21 (10) (a) Nishiyama, N.; Bae, Y.; Miyata, K.; Fukushima, S.; Kataoka, K. Drug Discovery Today: Technol. 2005, 2, 21–26. (b) Nishiyama, N.; Kataoka, K. Pharmacol. Ther. 2006, 112, 630–648. (11) Torchilin, V. P. Adv. Drug Delivery Rev. 2006, 58, 1532–1555. (12) Francis, F. F.; Cristea, M.; Winnik, F. M. Pure Appl. Chem. 2004, 76, 1321. (13) Wong, S. Y.; Pelet, J. M.; Putnam, D. Prog. Polym. Sci. 2007, 32, 799–837. (14) Ilhan, F.; Galow, T. H.; Gray, M.; Clavier, G.; Rotello, V. M. J. Am. Chem. Soc. 2000, 122, 5895. (15) Deans, R.; Ilhan, F.; Rotello, V. M. Macromolecules 1999, 32, 4956. (16) Thibault, R. J.; Hotchkiss, P. J.; Gray, M.; Rotello, V. M. J. Am. Chem. Soc. 2003, 125, 11249. (17) Uzun, O.; Sanyal, A.; Nakade, H.; Thibault, R. J.; Rotello, V. M. J. Am. Chem. Soc. 2004, 126, 14773. (18) For a review on polymeric assemblies via metal-ligand interactions, see: (a) Fustin, C.-A.; Guillet, P.; Schubert, U. S.; Gohy, J.-F. Adv. Mater. 2007, 19, 1665. (b) Gohy, J.-F. Coord. Chem. Rev. 2009, 253, 2214. (19) Lefevre, N.; Fustin, C.-A.; Varshney, S. K.; Gohy, J.-F. Polymer 2007, 48, 2306–2311. (20) Lefevre, N.; Fustin, C.-A.; Gohy, J.-F. Langmuir 2007, 23, 4618–4622. (21) Cabral, H.; Nishiyama, N.; Kataoka, K. J. Controlled Release 2007, 121, 146–155.

Published on Web 05/21/2010

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Finally, the self-assembly of block copolymers can be triggered by a cooperative formation of ionic bonds between two oppositely charged polyelectrolytes. As their formation is electrostatically driven, the resulting aggregates can respond to a change of ionic strength, or to a pH variation in the case of weak polyelectrolytes. Such aggregates can be obtained by selfassembling two diblock copolymers AB and AC, where A is a neutral hydrophilic block, and B and C are blocks of opposite charge.22-25 The group of Kataoka has first described the formation of polymeric vesicles, using PEG-b-poly(aspartic acid) and PEG-b-poly([2-aminoethyl]-R,β-aspartamide) block copolymers with similar molecular weight and composition.22c These peculiar electrostatic vesicles have been named polyion complexes or PICsomes.22 More generally speaking, polymer vesicles have a great potential in the biomedical field due to their structural resemblance with primitive biological cells, giving polymersomes unique features such as chemical stability, ability of encapsulating actives in their membranes and/or in their shell, and tunable surface functionalization.26,27 Alternatively, micellar aggregates can be formed by mixing a charged A block and a BC diblock copolymer, with B being charged and C neutral.28-31 Kabanov et al. have thus reported the formation of block ionomer complexes (BICs) using poly(N-ethyl-4-vinylpyridinium) cation (PEVPþ) complexed with poly(ethylene oxide)-b-poly(sodium methacrylate) (PEO-PMANa).28 Colloidal electrostatic complexes were stabilized, either by the PEO block when the diblock copolymer interacted with a charged homopolymer or by the excess of charges when complexes resulted from the interaction between two oppositely charged polyelectrolytes. As for Gohy et al., they have reported the formation of stable electrostatic polymersomes from a DHBC, namely, poly(2-(dimethylamino)ethyl methacrylate)-b-poly(methacrylic acid) (PMAA49-b-PDMAEMA11).32 In this case, electrostatic polymersomes were observed due to the development of electrostatic interactions between charged blocks forming the hydrophobic membrane which was stabilized by the excess of negative charges brought by PMAA units. In this contribution, we describe the aqueous solution behavior of novel DHBCs, namely, poly[2-(dimethylamino)ethyl methacrylate]-b-poly(glutamic acid) (PDMAEMA-b-PGA) (see Scheme 1), exhibiting pH- and temperature-responsiveness. These DHBCs (22) (a) Park, J. S.; Akiyama, Y.; Yamasaki, Y.; Kataoka, K. Langmuir 2007, 23, 138–146. (b) Kishimura, A.; Koide, A.; Osada, K.; Yamasaki, Y.; Kataoka, K. Angew. Chem., Int. Ed. 2007, 46, 6085–6088. (c) Koide, A.; Kishimura, A.; Osada, K.; Jang, W. D.; Yamasaki, Y.; Kataoka, K. J. Am. Chem. Soc. 2006, 128, 5988–5989. (d) Kakizawa, Y.; Harada, A.; Kataoka, K. J. Am. Chem. Soc. 1999, 121, 11247– 11248. (e) Harada, A.; Kataoka, K. Science 1999, 283, 65–67. (f) Harada, A.; Kataoka, K. Macromolecules 1995, 28, 5294–5299. (23) Gohy, J. F.; Varshney, S. K.; Jerome, R. Macromolecules 2001, 34, 3361– 3366. (24) Adams, D. J.; Rogers, S. H.; Schuetz, P. J. Colloid Interface Sci. 2008, 322, 448–456. (25) Voets, I. K.; Moll, P. M.; Aqil, A.; Jerome, C.; Detrembleur, C.; Waard, P. d.; Keizer, A. D.; Stuart, M. A. C. J. Phys. Chem. B 2008, 112, 10833–10840. (26) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967–973. (27) For a recent review on polymersomes and their potential applications, see : Meng, F.; Zhong, Z.; Feijen, J. Biomacromolecules 2009, 10, 197–209. (28) Kabanov, A. V.; Bronich, T. K.; Kabanov, V. A.; Yu, K.; Eisenberg, A. Macromolecules 1996, 29, 6797–6802. (29) (a) Cohen Stuart, M. A.; Besseling, N. A. M.; Fokkink, R. G. Langmuir 1998, 14, 6846–6849. (b) Voets, I. K.; van der Burgh, S.; Farago, B.; Fokkink, R.; Kovacevic, D.; Hellweg, T.; de Keizer, A.; Cohen Stuart, M. A. Macromolecules 2007, 40, 8476–8482. (30) (a) Gohy, J. F.; Varshney, S. K.; Jerome, R. Macromolecules 2001, 34, 2745–2747. (b) Gohy, J.-F.; Varshney, S. K.; Antoun, S.; Jerome, R. Macromolecules 2000, 33, 9298–9305. (31) Weaver, J. V. M.; Armes, S. P.; Liu, S. Macromolecules 2003, 36, 9994– 9998. (32) Gohy, J.-F.; Creutz, S.; Garcia, M.; Mahltig, B.; Stamm, M.; Jerome, R. Macromolecules 2000, 33, 6378–6387.

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Article Scheme 1. Chemical Structure of the Poly[2-(dimethylamino)ethyl methacrylate]-b-Poly(glutamic acid) PDMAEMA-b-PGA DHBCs under Study; x and y Represent the Number Average Degree of Polymerization of PDMAEMA and PGA Blocks, Respectively

are also potentially zwitterionic (amphoteric) polymer compounds,33 as a function of pH. PDMAEMA, a polybase widely studied as DNA condensing agent,34 is a multiresponsive polymer in itself as it responds both to pH and temperature under defined conditions. PDMAEMA is indeed freely soluble in water at room temperature whatever the pH and becomes thermoresponsive at neutral and basic pH with a lower critical solution temperature (LCST) in water depending on both its molar mass (32-46 °C at pH = 8)35 and pH (40-80 °C between pH = 11 and pH = 7).36 PGA is a biocompatible and pH-sensitive polypeptide, neutral at low pH and negatively charged at pH above pKa. As is the case of many other synthetic polypeptides, PGA can also undergo a reversible transition from a rodlike R-helix conformation to a coil conformation by a pH variation.37-39 Herein, we demonstrate that self-assembly in water of PDMAEMA-b-PGA DHBCs is driven either by hydrophobic effect or by electrostatic interactions according to pH and temperature conditions. Manipulation of these new DHBCs based on PDMAEMA and PGA allowed us to generate miscellaneous submicrometer size range aggregates in water.

Experimental Section 1. Materials and Methods. 1.1. Synthesis of PDMAEMA-b-PGA Double Hydrophilic Block Copolymers. These compounds were obtained from poly(γ-benzyl-L-glutamate)-bpoly[2-(dimethylamino)ethyl methacrylate] (PBLG-b-PDMAEMA) block copolymers whose synthesis has already been described by us in a previous report.40 The deprotection of the benzyl groups of the PBLG blocks was carried following an already reported procedure.37 Into a tetrahydrofuran (THF) solution (25 mg/mL) of the PDMAEMA-b-PBLG 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 solubilized in distilled water, dialyzed during 3 days, and lyophilized. The deprotection of the PBLG blocks was monitored by 1H NMR, following the disappearance of the benzyl protons at δ = 7.5 and 5 ppm (see Figure 1). 1.2. Preparation of Block Copolymer Solutions. PDMAEMA-b-PGA copolymers varying in block composition were dissolved in aqueous solution at 1 mg/mL. Sodium chloride was added to vary the ionic strength of the solutions, pH was adjusted (33) Lowe, A. B.; McCormick, C. L. Chem. Rev. 2002, 102, 4177. (34) Rungsardthong, U.; Ehtezazi, T.; Bailey, L.; Armes, S. P.; Garnett, M. C.; Stolnik, S. Biomacromolecules 2003, 4, 683–690. (35) Butun, V.; Armes, S. P.; Billingham, N. C. Polymer 2001, 42, 5993–6008. (36) Plamper, F. A.; Ruppel, M.; Schmalz, A.; Borisov, O.; Ballauff, M.; Muller, A. H. E. Macromolecules 2007, 40, 8361–8366. (37) Deming, T. J. Nature 1997, 390, 386. (38) Klok, H.-A.; Lecommandoux, S. Adv. Polym. Sci. 2006, 202, 75. (39) Kricheldorf, H. R. Angew. Chem., Int. Ed. 2006, 45, 5752. (40) (a) Agut, W.; Taton, D.; Lecommandoux, S. Macromolecules 2007, 40, 5653–5661. (b) Agut, W.; Agnaou, R.; Lecommandoux, S.; Taton, D. Macromol. Rapid Commun. 2008, 29, 1147–1155.

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Figure 1. 1H NMR (400 MHz) of PDMAEMA-b-PBLG (in black, DMSO-d6) and PDMAEMA-b-PBLG (in red, D2O).

Figure 2. pH (0) and conductivity (O) titration curves of PDMAEMA85-b-PGA37 in aqueous solution. (Polymer concentration 0.05 wt %, 25 °C, no added salt.)

Table 1 PDMAEMAxb-PGAy compositiona

Mn PGA block (g/mol)

85-37 85-77 85-186

4800 9900 24 000

Mn PGA content copolymer (g/mol) PDIb (% wt) IEPc IEPd IEPe 18 100 23 200 37 300

1.23 1.20 1.25

26 43 64

8.5 7.6 4.5

8.5 6.8 3.9

7.1 6.1 4

a x and y correspond to the degrees of polymerization of the PDMAEMA and PGA blocks, respectively. b Determined by SEC in DMF with LiBr (1 g/L) at 60 °C before PBLG deprotection. c IEP determined by electrophoretic mobility measurements. d IEP determined from the mid-adsorption value (λ = 550 nm) when varying the pH of copolymer solutions from pH 11 to 2 (addition of 100 μL of 0.1 M HCl solution). e IEP calculated according to ref 41.

with HCl or NaOH solutions, and temperature was adjusted using a thermostat bath. Size or zeta potential measurements were performed 10 min after.

2. Characterization. 2.1. Nuclear Magnetic Resonance (NMR) Spectroscopy. 1H NMR spectra were recorded on a Bruker AC 400 spectrometer.

2.2. Electophoretic Mobility Measurements. The isoelectric point (IEP) of the amphoteric PDMAEMA-b-PGA DHBCs was obtained by determining the zero value of electrophoretic mobility by varying the pH of 0.1 wt % copolymer solutions, using a Malvern Zetasizer DTS 3000. 2.3. Dynamic Light Scattering (DLS). DLS experiments were performed using an ALV Laser goniometer, with a 22 mW linear polarized laser (632.8 nm HeNe) and an ALV-5000/EPP Multiple Tau Digital correlator with a 125 ns initial sampling time. The samples were studied at different temperatures. The accessible scattering angular range varied from 40° up to 120°. The solutions were introduced into 10 mm diameter glass cells. Data were acquired with the ALV-Correlator Control software, and the analysis time was set for each sample at 120 s. Relaxation time distributions of aggregates were derived from a CONTIN analysis. The hydrodynamic radius (RH) could be calculated from the diffusion coefficient using the StokesEinstein relation. 2.4. Transmission Electron Microscopy (TEM). TEM observations were conducted on a Hitachi H7650 microscope operating at 80 kV. Images were taken with a GATAN Orius camera (11 megapixels). Samples for TEM observations were prepared by spraying a 0.1% wt block copolymer solution onto copper grids under an overpressure of nitrogen. 10548 DOI: 10.1021/la1005693

Figure 3. Electrophoretic mobility (μE) versus pH curve for a 0.5% wt aqueous solution of the PDMAEMA-b-PGA diblock copolymers at 25 °C. (Polymer concentration 0.05 wt %, 25 °C, no added salt.)

2.5. Small Angle Neutron Scattering (SANS). SANS experiments were performed at the Leon Brillouin Laboratory (Orphee 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 A˚-1. Solutions of PDMAEMA-b-PGA at 0.1 wt % in D2O were introduced in a 5 mm thickness rectangular quartz cell and studied at 60 °C. The blank sample was pure D2O. Data treatment was done with the 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.41 The signal of the pure D2O blank sample was first subtracted. (41) 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 4. Hydrodynamic radius RH and normalized absorbance (λ = 550 nm) as a function of pH measured for 0.1 wt % aqueous solutions of PDMAEMA85-b-PGA37, PDMAEMA85-b-PGA77, and PDMAEMA85-b-PGA186 diblock copolymers. (Polymer concentration 0.1 wt %, 25 °C, no added salt.)

Results and Discussion 1. Synthesis and Characterization of PDMAEMA-bPGA DHBCs. The DHBCs based on PDMAEMA and PGA were prepared from well-defined poly(γ-benzyl-L-glutamate)-bpoly[2-(dimethylamino)ethyl methacrylate] (PBLG-b-PDMAEMA) diblock copolymers whose synthesis is reported elsewhere.40 The deprotection of the PBLG blocks into poly(glutamic acid) (PGA) ones was carried out under basic conditions affording the targeted PDMAEMA-b-PGA DHBCs selectively and quantitatively (Scheme 1). This was verified by 1H NMR spectroscopy showing the complete disappearance of the benzyl protons at δ = 7.5 and 5 ppm of PBLG (Figure 1). In this way, three PDMAEMA-b-PGA DHBCs with different PGA block lengths were successfully synthesized (Table 1). 2. pH-Induced Self-Assembly in Water at 25 °C. 2.1. Titration: pH and Conductivity Measurements. Figure 2 shows both the pH and conductivity titration curves of a 0.05% wt aqueous solution of the PDMAEMA85-b-PGA37 DHBC. One can distinguish two transition points corresponding to the beginning of the ionization of the amine moiety of the PDMAEMA block and the end of the protonation of the carboxylic group of the PGA segment. All the ionizable groups were titrated between these two inflection points, with the difference in pKa being too Langmuir 2010, 26(13), 10546–10554

weak to separate PGA (pKa,PGA= 4.1)42 and PDMAEMA protonation (pKa,PDMAEMA = 7.7).43 A good concordance between both techniques can be noted, even if the number of moles corresponding to the titrated functions (between the two inflection points) is slightly different from the calculated one. It is worth pointing out that a particular domain of insolubility was observed between pH 7 and 9, owing to electrostatic interactions between the oppositely charged blocks leading to the formation of large aggregates; this characterizes the behavior of polyampholytes.33 As a matter of fact, it is difficult to accurately titrate functional groups near the isoelectric point.44-46 2.2. IEP Determination. For the same reasons mentioned above, the determination of IEP values of the polyampholytes was not highly accurate. A first method used theoretical equations reported by Patrickios et al.:43 (42) Gil, E. S.; Hudson, S. M. Prog. Polym. Sci. 2004, 29, 1173–1222. (43) Patrickios, C. S.; Hertler, W. R.; Abbott, N. L.; Hatton, T. A. Macromolecules 1994, 27, 930–937. (44) Bekturov, E. A.; Kudaibergenov, S. E.; Rafikov, S. R. Rev. Macromol. Chem. Phys. 1990, C30, 233. (45) Merle, Y. J. Phys. Chem. 1987, 91, 3092–3098. (46) Mao, B. W.; Gan, L. H.; Gan, Y. Y.; Tam, K. C.; Tan, O. K. Polymer 2005, 46, 10045–10055.

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Figure 5. Autocorrelation function C(q,t) and distribution of the relaxation time at 90° by CONTIN analysis for a 1 mg/mL solution of PDMAEMA85-b-PGA186 at pH = 6.2 and zero added salt. The inset shows the relaxation frequency as a function of q2.

For acid-rich polyampholytes: pHIEP ¼ pKa, PGA

3 2 8 !1=2 9  2 < 1-R = R 1 R 4 7 6 - log4 þ 10ðpKa, PGA - pKa, PDMAEMA Þ þ 5 ; 2: R R R

For base-rich polyampholytes: pHIEP ¼ pKa, PDMAEMA

2 8 3 !1=2 9  2 < = 1-R 4 61 1 - R 7 þ þ log4 þ 10ðpKa, PGA - pKa, PDMAEMA Þ 5 ; 2: R R R

In these equations, R is the molar ratio between acidic and basic functions. Another method is to determine the zero value of the electrophoretic mobility (μE) of a block copolymer aqueous solution as a function of pH, as shown in Figure 3. Lastly, IEP can be determined by absorbance at λ = 550 nm as the value at the midpoint of the pH range of precipitation, as illustrated in Figure 4. As summarized in Table 1, IEP values determined from these three methods are very close. For each block copolymer composition, a region where electrostatic interactions between both blocks drive their self-aggregation is observed. Consequently, a large increase of the absorbance is noted. Depending on the PGA length, the IEP varies from 4 to 8.5 and, as expected, IEP values decrease by increasing the PGA block length. 2.3. Light Scattering and Turbidity Studies. Hydrodynamic sizes of block copolymers were measured in aqueous solutions by dynamic light scattering (DLS), as a function of pH. Absorbance measurements by UV were also performed at λ = 550 nm. All DHBCs adopt the same general behavior in water as a function of pH, with three domains (A, B, and C) being distinguished (Figure 4). Above the IEP (region C), both the sizes and the absorbance are very low. In this range of pH, block copolymers behave as free chains (unimers) with both blocks being fully soluble; PGA blocks are negatively charged, while PDMAEMA segments are neutral but remain soluble at 25 °C below their 10550 DOI: 10.1021/la1005693

Figure 6. (a) TEM picture of electrostatic vesicles obtained from PDMAEMA85-b-PGA186 copolymer at pH 6.2 (0.1 wt %). (b) High magnification of the previous TEM image. (c) Schematic representation of the electrostatic vesicles.

LCST (39 °C). The same trend is observed below the IEP (region A) where the block copolymer chains are also fully soluble. It has been reported that PGA-based block copolymers can self-assemble in acidic media due to the PGA change of conformation from a coil to a R-helix.47,48 In our case, the hydrophilicity of the PDMAEMA block is apparently high enough to solubilize PDMAEMA-b-PGA block copolymers as unimers. Finally, in (47) (a) Rodriguez-Hernandez, J.; Lecommandoux, S. J. Am. Chem. Soc. 2005, 127, 2026–2027. (b) Checot, F.; Rodriguez-Hernandez, J.; Gnanou, Y.; Lecommandoux, S. Biomol. Eng. 2007, 24, 81–85. (c) Babin, J.; Leroy, C.; Lecommandoux, S.; Borsali, R.; Gnanou, Y.; Taton, D. Chem. Commun. 2005, 15, 1993–1995. (48) Rao, J.; Luo, Z.; Ge, Z.; Liu, H.; Liu, S. Biomacromolecules 2007, 8, 3871– 3878.

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Figure 7. Autocorrelation functions C(q,t) and relaxation time distribution at 90° as revealed by CONTIN analysis for 1 mg/mL solutions of PDMAEMA85-b-PGA77 at pH = 4-6 and zero added salt.

Figure 9. Light scattering intensity as a function of temperature for 0.1 wt % aqueous solutions of PDMAEMA homopolymer and PDMAEMA85-b-PGA37, PDMAEMA85-b-PGA77, and PDMAEMA85-b-PGA186 diblock copolymers at pH 11.

Figure 10. Temperature-dependent 1H NMR sprectra of PDMAEMA85-b-PGA77 in D2O at pH 11.

Figure 8. TEM pictures of electrostatic vesicles from PDMAEMA85-b-PGA77 copolymer at pH 4 (a), 5 (b), and 6 (c) for a concentration 0.1 wt %. (d) Schematic representation of the electrostatic polymersomes. Scale bar represents 100 nm (a, b) and 200 nm (c).

the intermediate B region, the strong increase of the absorbance and the hydrodynamic radius is obviously due to the aggregation mediated by electrostatic interactions between oppositely charged blocks. 2.4. pH-Responsive Self-Assembly of PDMAEMA-b-PGA. As depicted in Figure 4, peculiar pH domains where PMAEMA85-b-PGA77 and PDMAEMA85-b-PGA186 block copolymers form nanoparticles around 100 nm in size can be isolated. For a better understanding of this behavior, DLS experiments have been performed in water for PDMAEMA85-b-PGA186 at pH = 6.2 and for PDMAEMA85-b-PGA77 at pH = 4-6. Figure 5 Langmuir 2010, 26(13), 10546–10554

shows the relaxation time distribution and the angular dependence of the PDMAEMA85-b-PGA186 DHBC at pH 6.2 in water at 25 °C. A monodisperse relaxation time distribution with a hydrodynamic radius RH = 60 nm is observed. The linear evolution of the frequency over q2 suggests a diffusive behavior. TEM analysis of PDMAEMA85-b-PGA186 at pH = 6.2 clearly reveals the formation of polymersomes with a diameter in good agreement with light scattering measurement (Figure 6). A simple calculation from the Henderson-Hasselbalch law, pH = pKa þ log (R/(1 - R)), where R is the dissociation degree, allowed us to approximate the fraction of charged groups within each block. At pH = 6.2 and considering the respective pKa of each group, more than 90% of amino functions are protonated whereas the PGA block is almost fully deprotonated. Formation of the vesicular membrane is thus explained by the development of electrostatic interactions between oppositely charged groups with DOI: 10.1021/la1005693

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Figure 11. Small angle neutron scattering for 0.1 wt % aqueous solutions of PDMAEMA85-b-PGA37 and PDMAEMA85-b-PGA77 in D2O at 60 °C and pH 11. The dashed lines are fitting curves by a hollow sphere model (dark) and Gaussian chains (clear) with Rg = 10 nm (upper curve) or Rg = 5 nm (lower curve).

an electrostatic stabilization provided by the large excess of carboxylate functions, as schematically described in Figure 6c. As mentioned above, electrostatic polymersomes have already been observed in the case of self-assembled asymmetrical PMAA49-b-PDMAEMA11 DHBCs.32 The occurrence of such “electrostatic polymersomes” was confirmed by studying the influence of added salts on light intensity scattered by vesicles. For 1 M of NaCl added, the initial turbid solution became transparent and the scattered intensity decreased dramatically, as a result of the vesicle disassembly by a charge screening effect. The same studies were performed on the PDMAEMA85-b-PGA77 DHBC at pH = 4-6. DLS analyses revealed sizes varying from 100 to 300 nm and a relatively narrow relaxation time distribution whatever the pH (Figure 7). TEM imaging confirmed the size range and also showed the formation of vesicular aggregates, similar to those obtained from PDMAEMA85-b-PGA186 at pH 6.2 (Figure 8). Here also, we propose that the vesicle membrane is formed by electrostatic interactions between protonated amines of PDMAEMA blocks and carboxylate groups of PGA blocks; in this case, the polymersomes are stabilized by the excess of amino groups. This is in agreement with the IEP value of this block copolymer (IEP ∼ 7, Table 1) and the positive values of electrophoretic mobility at pH = 4-6 (Figure 3). The observed decrease in size of vesicles with pH may be related to two parameters: the charge ratio and the change of conformation of PGA blocks, from a coil at pH = 6 to a R-helix at pH = 4.47,48 The PDAMEMA block is almost fully charged between pH = 4 and pH = 6, whereas the degree of ionization of PGA blocks decreases from pH = 6 to pH = 4. The ion pairing is thus far more efficient at pH = 6 than at pH = 4, and it may be expected that both the aggregation number and the size of the vesicles vary in the same manner. Figure 4 also clearly evidences the progressive dissolution of the polymersomes as pH decreases. The known coil to helix transition of PGA blocks when pH decreases should favor the formation of a more hydrophobic and dense membrane leading to persistent polymersomes, but such an effect is not observed in the present case. 3. Temperature-Induced Self-Assembly of PDMAEMAb-PGA DHBCs at pH = 11. In addition to their pH-responsiveness described above, DHBCs based on PDMAEMA and PGA are expected to selectively respond to temperature variations since PDMAEMA exhibits a LCST at approximately 39 °C. 10552 DOI: 10.1021/la1005693

Figure 12. Small angle neutron scattering for a 0.1 wt % aqueous

solutions of PDMAEMA85-b-PGA186 in D2O at 60 °C and pH = 11. The dashed lines are fitting curves by a simple sphere model (dark) and Gaussian chain (clear) with Rg = 5 nm.

The effect of temperature on the solution behavior of PDMAEMA85-b-PGA37, PDMAEMA85-b-PGA7, and PDMAEMA85-bPGA186 DHBCs as well as on that of PDMAEMA85 homopolymer was studied in water at pH = 11, that is, the pH threshold for PDMAEMA to exhibit a LCST behavior.36 In this range of pH, no electrostatic interactions between PDMAEMA and PGA blocks are expected. LCSTs of homo and block copolymers were determined in a simple manner by light scattering (Figure 9). The strong increase in scattered light defines the LCST where the PDMAEMA segment becomes insoluble. The three block copolymers show similar thermoresponsive behavior like the PDMAEMA homopolymer. However, the presence of the hydrophilic PGA part logically increases the LCST, as it has been observed on other systems based on this polymer.49-51 In our study, the LCST is shifted from 38 °C for the (49) Zhang, X.; Li, J.; Li, W.; Zhang, A. Biomacromolecules 2007, 8, 3557–3567. (50) Yusa, S.; Yamago, S.; Sugahara, M.; Morikawa, S.; Yamamoto, T.; Morishima, Y. Macromolecules 2007, 40, 5907–5915. (51) Deng, L.; Shi, K.; Zhang, Y.; Wang, H.; Zeng, J.; Guo, X.; Du, Z.; Zhang, B. J. Colloid Interface Sci. 2008, 323, 169–175.

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Figure 13. Schematic representation of the different morphologies obtained from polypeptide-based multiresponsive block copolymers.

homopolymer to 48 °C for the PDMAEMA85-b-PGA186 diblock copolymer. Hence, the longer the hydrophilic block is, the higher the LCST value is. For a better understanding of the formation of these nano-objects, the thermally induced aggregation of PDMAEMA85-b-PGA77 in D2O was monitored at pH = 11 by 1 H NMR spectroscopy (Figure 10). At 25 °C, PDMAEMA85-b-PGA77 is fully solvated, and hence, characteristic peaks of each block are detected. Near the LCST (T = 45 °C), the intensity of the peaks due to PDMAEMA progressively decreases, indicating a less hydrated state of this block though the suspension remains macroscopically stable. This loss of solubility can be ascribed to the formation of PDMAEMA-based cores of micellar aggregates. Further increasing the temperature from 45 to 75 °C induced an almost complete disappearance of signals due to PDMAEMA, witnessing its higher hydrophobicity. Since the PGA blocks form the hydrophilic stabilizing corona, characteristic protons of this block remain unchanged in the NMR spectrum. These results are consistent with previous experiments performed by us on another type of polypeptide-based DHBC, namely, a Jeffamine-b-PGA diblock copolymer, where Jeffamine is a statistical copolymer made of propylene oxide and ethylene oxide units also exhibiting a LCST behavior.52 Interestingly, even if the scattered intensity measured by DLS increases well above the LCST, well-defined aggregates are only obtained at high temperatures. These thermally induced self-assembled nanoparticles based on PDMAEMA-b-PGA DHBCs have been further investigated by small angle neutron scattering (SANS) in D2O at 60 °C. Figure 11 shows the scattered intensity for PDMAEMA85-b-PGA37 (Figure 11a) and PDMAEMA85-b-PGA77 (Figure 11b). The vesicular morphology of both samples can be demonstrated using scaling arguments. The q-2 decrease of the SANS intensity attests to the presence of a lamellar interface which corresponds to the hydrophobic membrane of the vesicles formed by neutral and insoluble PDMAEMA blocks. At a higher q range (2  10-3 < q < 4  10-3), the q-4 variation of the experimental curve depicts the interface between the vesicle surface and water molecules. Vesicles are stabilized by PGA chains, as attested by the q-5/3 dependence of the scattered intensity in a high q range, which is the hallmark of free polymer chains in good solvent. (52) Agut, W.; Br^ulet, A.; Taton, D.; Lecommandoux, S. Langmuir 2007, 23, 11526–11533.

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Experimental curves have been fitted by the form factor of a hollow sphere: PðqÞ ¼

  16π2 sinðqRe Þ - qRe cosðqRe Þ - sinðqRi Þ þ qRi cosðqRi Þ 2 q6 VðRe Þ - VðRi Þ

ð1Þ where Re and Ri correspond, respectively, to the external and internal radius of the shell and V(Ri) (V(Re)) is the volume of the sphere of radius Ri (Re). Values of the average radius (R) and the thickness of the vesicle membrane (δ) were obtained by fitting experimental curves to this form factor using a homemade program,53,54 taking into account the resolution function of the spectrometer.55 These values were the same for both PDMAEMA85-b-PGA37 and PDMAEMA85-b-PGA77: R = 110 nm and δ = 15 nm.56 However, the fit was not good at high q (q > 2.10-2) because it did not include the contribution of the PGA hydrophilic corona. Hence, the form factor of a Gaussian chain, the Debye function,57 can be added: PDebye ðqÞ ¼

2ðe - x - 1 þ xÞ with x ¼ q2 Rg 2 x2

ð2Þ

The two curves calculated with Rg = 10 nm and Rg = 5 nm are shown in Figure 11a. Unfortunately, the q range in which we roughly observe a q-5/3 dependence corresponds to the asymptotic range of the Debye function, and consequently, the scattering curve is no more sensitive to the radius of gyration of chains inside the corona. So, it is not possible from this simple data analysis to get accurate information concerning the extension of PGA chains inside the corona. In addition, the SANS curve of the PDMAEMA85-b-PGA186 diblock copolymer evidenced the absence of q-2 dependence of the SANS intensity (Figure 12), meaning that these nano-objects cannot be vesicles but are more likely polymeric micelles. (53) Checot, F.; Br^ulet, A.; Oberdisse, J.; Gnanou, Y.; Mondain-Monval, O.; Lecommandoux, S. Langmuir 2005, 21, 4308–4315. (54) Oberdisse, J.; Couve, C.; Appell, J.; Berret, J. F.; Ligoure, C.; Porte, G. Langmuir 1996, 12, 1212–1218. (55) (a) Pedersen, J. S. J. Phys. IV 1993, 3, 491. (b) Lairez, D. J. Phys. IV 1999, 9, 67. (56) The same vesicle membrane thickness (δ = 15 nm) was directly determined from the slope (δ2/12) of the asymptotic representation ln(q2 I(q)) versus q2. (57) Debye, P. J. Phys. Colloid Chem. 1947, 51, 18–32.

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To fit these data, we used the form factor of a homogeneous sphere of radius Rs expressed as " Psphere ðqÞ ¼

3ðsinðqRs Þ - qRs cosðqRs ÞÞ ðqRs Þ3

#2 ð3Þ

Micelles with a radius Rs = 110 nm (corresponding to the value actually measured by DLS), stabilized by a PGA corona with Rg of about 5 nm, have been obtained. Here again, the PGA chain extension is not determined with a high accuracy. Finally, depending on the weight ratio of the hydrophilic PGA block, we can conclude that the various morphologies found for PDMAEMA85-b-PGA37 and PDMAEMA85-b-PGA77 (polymersomes) and PDMAEMA85-b-PGA186 (polymeric micelles) in water at 60 °C are in good agreement with those observed for other amphiphilic block copolymers, as described by Discher and Eisenberg26 (Figure 13).

Conclusion Poly[2-(dimethylamino)ethyl methacrylate]-b-poly(glutamic acid) (PDMAEMA-b-PGA) double hydrophilic block copolymers (DHBCs) can be readily synthesized from poly(γ-benzylL-glutamate)-b-poly[2-(dimethylamino)ethyl methacrylate] amphiphilic block copolymer precursors. Self-assembly in water of these PDMAEMA-b-PGA DHBCs can be selectively triggered by a variation of either the pH or the temperature. In the former case,

10554 DOI: 10.1021/la1005693

the self-assembly is driven by electrostatic interactions, whereas it is induced by the hydrophobic effect in the latter case. Investigations into the self-assembled nanostructures generated from such doubly responsive DHBCs by neutron and/or dynamic light scattering and transmission electron microscopy reveal the formation of either electrostatic polymersomes or spherical polymeric micelles, according to the pH and temperature conditions (Figure 13). The resulting morphologies also depend on the overall composition of the DHBCs. For pH values below and above the isoelectric point (IEP), the DHBCs behave as unimers owing to the high solubility of the PDMAEMA block. Close to the IEP, direct or inverse electrostatic polymersomes are generated by electrostatic interactions developing between the two charged blocks driving the formation of the hydrophobic membrane of the polymersomes, which is stabilized in water by uncompensated charges. The thermosensitivity of the DHBCs relates to the LCST behavior of PDMAEMA around 40 °C. Thus, at pH = 11 and below the LCST of PDMAEMA, free chains of DHBC unimers are evidenced, while above the LCST the hydrophobicity of PDMAEMA drives the self-assembly of the DHBCs in a reversible manner. In this case, spherical polymeric micelles or polymersomes are obtained, depending on the PGA block length. These possibilities of variation in size and shape of morphologies that can be achieved, because the temperature and the pH can be independently varied, open new routes in the development of stimuli-responsive nanocarriers for biomedical applications.

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