Force Required to Disassemble Block Copolymer Micelles in Water

May 13, 2010 - The force needed to disassemble block copolymer micelles of poly(acrylic acid)-polyfluorene-poly(acrylic acid) in water is found to be ...
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Force Required to Disassemble Block Copolymer Micelles in Water Ying Yu, Guanglu Wu, Kai Liu, and Xi Zhang* Key Lab of Organic Optoelectronics and Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084, PR China Received March 29, 2010. Revised Manuscript Received May 6, 2010 The force required to disassemble block copolymer micelles in water has been directly investigated via atomic force microscopy-based single-molecule force spectroscopy. The force needed to disassemble block copolymer micelles of poly(acrylic acid)-polyfluorene-poly(acrylic acid) in water is found to be 23 pN. The force increases as the stretching velocity increases, indicating that micelle disassembly is a dynamic process. In addition, the disassembly force is sensitive to the properties of the solvents. This study represents the first attempt to employ single-molecule force spectroscopy to directly measure the force needed to disassemble block copolymer micelles in water.

Introduction Polymer micelles are defined as nanoscale polymer particles with a core-shell structure that are formed from block or graft copolymers. The chemical structure of the shell can be modified to improve the interaction of the micelles with the environment, allowing issues such as solubility, biocompatibility, and environmental selectivity to be addressed.1-4 Although any components can be chosen for the core, the selection of appropriate materials can produce polymer micelles with diverse functions such as drug embedding, controlled loading and release, and interesting optoelectronic properties. The wide range of potential applications encourages researchers to study polymer micelles.5,6 Although significant progress has been made, understanding the driving force of the micelles is thought to be important for controlling the properties and the stability of polymer micelles. The force required to disassemble block copolymer micelles is too small to be detected easily. Wu et al. employed ultrafiltration to study polymer micelles composed of polystyrene and polyisoprene block copolymers in n-hexane and indirectly revealed its very weak flow-rate-dependent hydrodynamic force.7 However, the force needed to disassemble micelles formed in aqueous solution has not been directly measured. Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) has been proven to be a powerful method for the measurement of microcosmic forces.8-13 Various types of interactions, such as covalent bonding, host-guest recognition, π-π interactions, intercalating forces, and hydrogen bonding *To whom correspondence should be addressed. E-mail: xi@mail. tsinghua.edu.cn. Tel: þ86-10-62796283. Fax: þ86-10-62771149.

(1) Zhang, L.; Eisenberg, A. Science 1995, 268, 1728. (2) Nishiyama, N.; Yokoyama, M.; Aoyagi, T.; Okano, T.; Sakurai, Y.; Kataoka, K. Langmuir 1998, 15, 377. (3) Zhang, G.; Niu, A.; Peng, S.; Jiang, M.; Tu, Y.; Li, M.; Wu, C. Acc. Chem. Res. 2001, 34, 249. (4) Zhang, L.; Shen, H.; Eisenberg, A. Macromolecules 1997, 30, 1001. (5) Kwon, G. S.; Kataoka, K. Adv. Drug Delivery Rev. 1995, 16, 295. (6) Lu, S.; Fan, Q.-L.; Liu, S.-Y.; Chua, S.-J.; Huang, W. Macromolecules 2002, 35, 9875. (7) Hong, L.; Jin, F.; Li, J.; Lu, Y.; Wu, C. Macromolecules 2008, 41, 8220. (8) Smith, S. B.; Finzi, L.; Bustamante, C. Science 1992, 258, 1122. (9) Zhang, W.; Zhang, X. Prog. Polym. Sci. 2003, 28, 1271. (10) Giannotti, M. I.; Vancso, G. J. ChemPhysChem 2007, 8, 2290. (11) Zhang, X.; Liu, C.; Wang, Z. Polymer 2008, 49, 3353. (12) Hugel, T.; Seitz, M. Macromol. Rapid Commun. 2001, 22, 989. (13) Janshoff, A.; Neitzert, M.; Oberdorfer, Y.; Fuchs, H. Angew. Chem., Int. Ed. 2000, 39, 3213.

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have been directly measured using SMFS with specially designed systems.14-19 SMFS not only allows for interactions to be directly measured but also provides new physical insight into the interactions by combining static and dynamic force spectroscopy.17,20 SMFS has also been used to study the suprastructures of macromolecules, allowing the relationship between force patterns and suprastructures to be established.21-27 This letter represents the first attempt to directly investigate the force required to disassemble block copolymer micelles in water using AFM-based SMFS, with the aim of understanding both the nature of the driving force and the disassembly process of the block copolymer micelles.

Experimental Section We have chosen a poly(acrylic acid)-polyfluorene-poly(acrylic acid) (PAA-PF-PAA) triblock copolymer, as shown in Figure 1a. The large conjugated structure of the hydrophobic region of this block copolymer allows it to form stable micelles in water.6 We first dissolved the copolymer in DMSO, which is a good solvent for both the PAA and PF segments, and then added the solution to water dropwise. Water is a poor solvent for the hydrophobic PF segment but a good solvent for the PAA segment, causing a micelle solution of low concentration to form. The composition of DMSO/water was fixed at 1/100 v/v. (14) Grandbois, M.; Beyer, M.; Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. Science 1999, 283, 1727. (15) Sch€onherr, H.; Beulen, M. W. J.; Bugler, J.; Huskens, J.; van Veggel, F. C. J. M.; Reinhoudt, D. N.; Vancso, G. J. J. Am. Chem. Soc. 2000, 122, 4963. (16) Zhang, Y.; Liu, C.; Shi, W.; Wang, Z.; Dai, L.; Zhang, X. Langmuir 2007, 23, 7911. (17) Liu, C.; Jiang, Z.; Zhang, Y.; Wang, Z.; Zhang, X.; Feng, F.; Wang, S. Langmuir 2007, 23, 9140. (18) Zou, S.; Sch€onherr, H.; Vancso, G. J. J. Am. Chem. Soc. 2005, 127, 11230. (19) Eckel, R.; Wilking, S. D.; Becker, A.; Sewald, N.; Ros, R.; Anselmetti, D. Angew. Chem., Int. Ed. 2005, 44, 3921. (20) Zhang, Y.; Yu, Y.; Jiang, Z.; Xu, H.; Wang, Z.; Zhang, X.; Oda, M.; Ishizuka, T.; Jiang, D.; Chi, L.; Fuchs, H. Langmuir 2009, 25, 6627. (21) Li, H.; Rief, M.; Oesterhelt, F.; Gaub, H. E. Adv. Mater. 1998, 10, 316. (22) Guan, Z.; Roland, J. T.; Bai, J. Z.; Ma, S. X.; McIntire, T. M.; Nguyen, M. J. Am. Chem. Soc. 2004, 126, 2058. (23) Gunari, N.; Walker, G. C. Langmuir 2008, 24, 5197. (24) Zhang, W.; Machon, C.; Orta, A.; Phillips, N.; Roberts, C. J.; Allen, S.; Soultanas, P. J. Mol. Biol. 2008, 377, 706. (25) Li, H.; Wang, H.-C.; Cao, Yi; Sharma, D.; Wang, M. J. Mol. Biol. 2008, 379, 871. (26) Walther, K. A.; Gr€ater, F.; Dougan, L.; Badilla, C. L.; Berne, B. J.; Fernandez, J. M. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 7916. (27) Cao, Y.; Yoo, T.; Li, H. B. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 11152.

Published on Web 05/13/2010

DOI: 10.1021/la101235e

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Figure 1. (a) Chemical structure of the PAA-PF-PAA block copolymer. (b) Illustration of a micelle bridging the AFM tip and the substrate.

Figure 2. AFM image of adsorbed micelles on a silicon slide. To prepare the sample, a silicon slide was dipped for 10 min into an aqueous solution containing a micelle concentration of 110-1 mg mL-1 and then dried using nitrogen gas. The average radius of the particles measured using AFM is about 12 nm, which was determined by measuring the fwhm of the peak. The average height of the particles is about 3 nm. Dynamic light scattering (DLS) data indicated that particles that formed with an average radius of 6.3 nm are dominant. AFM observation, as shown in Figure 2, showed that the particles are well separated at the surface and have an average radius of 12 nm. The size measured by AFM is larger than that determined by DLS. This can be attributed to the adsorption of micelles onto a substrate, enlarging their size along the substrate while decreasing their height. Moreover, the tip-broadening effect can also contribute to the larger lateral size determined using AFM. To implement the disassembly of a polymer micelle, PAA-PFPAA was immobilized on an AFM tip made of Si3N4. The AFM tip was first silanized using a mixed solution of 3-aminopropyldimethylethoxysilane/octadecyldimethylmethoxysilane (1/100 v/v) and then immersed in a DMSO solution containing PAA-PF-PAA (1  10-3 mg mL-1) in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. This allowed one or more carboxyl groups from PAA-PF-PAA to be covalently attached to the surface of the silanized tip. Because the SMFS experiments were carried out in aqueous solution, the block copolymer attached to the AFM tip could form a micelle, with the PF segment as the core and the PAA segment as the shell. When the tip with an attached micelle was brought into contact with an NH2-covered quartz substrate, a molecular bridge formed between the AFM tip and the substrate, as shown in Figure 1b. Retracting the piezotube caused the AFM tip and the substrate to separate. This caused the micelle to disassemble, allowing the disassembly force to be directly measured using SMFS.

Results and Discussion In a PAA-PF-PAA block copolymer micelle, the PAA segments should wrap around the hydrophobic PF segment, minimizing the 9184 DOI: 10.1021/la101235e

Yu et al.

interfacial energy between the PF segment and H2O by keeping them separate. In the disassembly process of a micelle, the enveloping PAA segment is continuously pulled away from the PF segment by the applied force, which should require a constant force until the micelle is totally disassembled. Such a possible disassembly process is reflected by a typical single-molecule force-extension curve (in brief, a force curve) such as that shown in Figure 3a. More typical force curves are shown in Figure S1. Force curves are characterized by a shoulderlike plateau (part I in Figure 3a) before a peak (part II in Figure 3a). The peak corresponds to the stretching process of a fully extended copolymer chain. Part II of all of the force curves can be well fit using a modified freely jointed chain model with the parameters of a narrow distribution, which provides evidence that the force signals are from the elongation of single polymer chains, such as that shown in Figure S2. Part I in Figure 3a corresponds to the disassembly process of a micelle, and the heights of the plateaus represent the forces needed to disassemble the micelles. A statistical analysis of the heights of the plateaus in all of the curves showed that the most probable height of these plateaus at a stretching velocity of 1 μm s-1 is 23 pN from the Gaussian fitting (Figure 3b). The fact that the distribution of the heights of the plateaus can be fitted using a Gaussian function provides further evidence that the force signals are from single polymer chains. To demonstrate how the shoulderlike plateaus are related to the disassembly of copolymer micelles, the length of the plateau was also statistically analyzed. The most probable length of these plateaus is around 15 nm according to Gaussian fitting, as shown in Figure 3c. The length of the plateau corresponds to the increase in length after the micelle is disassembled. According to the estimation in the Supporting Information, the average length of 15 nm seems a rational length required for a flexible PAA chain to encompass a PF segment in water. The dependence of the disassembly of a copolymer micelle on the stretching velocity was studied further to determine whether the process is dynamic. Several tens of shoulderlike plateau events were obtained at different stretching velocities (Figure S3), and the most probable force at each stretching velocity was determined using Gaussian fitting. The most probable force was plotted against stretching velocity on a logarithmic scale as shown in Figure 3b. The graph clearly shows that the force increases as the stretching velocity increases. The fact that the disassembly force depends on the stretching velocity indicates that the disassembly of a micelle is a dynamic process.28,29 It has been widely reported that the plateaus attributed to the desorption process of polymer chains from substrates do not depend on the stretching velocity.30,31 Therefore, the dependence of the disassembly force on the stretching velocity supports the viewpoint that shoulderlike plateaus result from the disassembly of block copolymer micelles rather than the desorption of polymer from the substrate. To provide further evidence that the shoulderlike plateaus in the force curves result from the disassembly of micelles composed of PAA-PF-PAA block copolymer, two control experiments were carried out. First, homopolymerized PAA with a length that is similar to that of the block copolymer was stretched using the same conditions as for PAA-PF-PAA. Simple force curves were obtained; none contained shoulderlike plateaus (Figure 4). The force rises monotonically with the increasing extension and then drops to zero rapidly at the rupture point. Second, PAA-PF-PAA (28) Bell, G. I. Science 1978, 200, 618. (29) Evans, E.; Ritchie, K. Biophys. J. 1997, 72, 1541. (30) Cui, S.; Liu, C.; Wang, Z.; Zhang, X.; Strandman, S.; Tenhu, H. Macromolecules 2004, 37, 946. (31) Yu, Y.; Yao, Y.; Wang, L.; Li, Z. Langmuir 2009, 26, 3275.

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Figure 3. (a) Typical force curve and illustrations of the corresponding disassembly process. (b) Histogram of the force of the shoulderlike plateaus at a stretching velocity of 1 μm s-1, with the most probable force determined to be 23 pN from the Gaussian fitting. (c) Histogram of the length of the shoulderlike plateaus at different stretching velocities; the most probable length is estimated to be 15 nm from the Gaussian fitting. (d) Plot of the most probable forces of the plateaus as a function of the corresponding stretching velocity on a logarithmic scale.

Figure 4. Comparison of the normalized force curves for stretching a copolymer in water (blue), a homopolymerized PAA in water (red), and a copolymer in ethanol (black). The shoulderlike plateau was found to be unique for the experiment investigating copolymers in water.

stretching experiments were performed in ethanol. Interestingly, shoulderlike plateaus were not present in the force curves. The PF segment is expected to be more soluble in ethanol than in water. The micelles of PAA-PF-PAA that formed in ethanol are much more loosely packed than those that formed in water. This means that the disassembly force required for micelles in organic solvents such as ethanol should be much smaller than that in water (23 pN). According to Wu et al.,7 the force might be as small as 1 pN, which is comparable to the noise level of an AFM-based SMFS instrument. This explains why the disassembly force is not directly detected using AFM-based SMFS. The PAA-PF-PAA micelles disassemble more easily in organic solvent, showing that the force required to disassemble the micelles is sensitive to the properties of the solvent. In the above experiments, the block copolymer was covalently linked to the AFM tip through an amide bond to increase the probability of stretching. However, other characterizations of the micelles are carried out in solution or on a substrate. To investigate whether covalent bonding of the micelle to the AFM Langmuir 2010, 26(12), 9183–9186

Figure 5. Histogram of the forces of the plateaus obtained on different substrates with adsorbed homopolymerized PAA or polymer micelles. All of the curves were obtained at a stretching velocity of 1 μm s-1. (a) Substrates covered with amino groups with adsorbed block copolymer micelles. (b) Substrates covered with amino groups with adsorbed homopolymerized PAA chains. (c) Substrates covered with hydroxyl groups with adsorbed copolymer micelles. (d) Substrates covered with hydroxyl groups with adsorbed homopolymerized PAA chains.

tip had any influence on the micelles, the experiments were also performed using an alternative method. The micelles were first adsorbed onto a substrate. A bare AFM tip was placed in contact with the substrate and then retracted. From these experiments, force curves with plateaus were obtained but the rupture points were not as high as that found using the previous method (Figure S4). Statistical analysis was performed on the heights of the plateaus, giving two different types of plateaus with different heights, as shown in Figure 5a. One type of plateau had a most probable force of 22 pN, similar to that calculated previously for the shoulderlike plateau, whereas the other type of plateau had a DOI: 10.1021/la101235e

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most probable force of 76 pN. Homopolymerized PAA was also studied using the same method and conditions. Only one type of plateau was obtained, which had a most probable force of 77 pN, as shown in Figure 5b. These results reveal that the plateaus with a higher force obtained in the experiment using PAA-PF-PAA can be ascribed to the desorption of PAA from the substrate covered with amino groups. As another control experiment, the micelles were adsorbed onto a substrate covered with hydroxyl groups. Because there are no electrostatic interactions between PAA and the hydroxyl groups on the substrate, the desorption force of the PAA segment from the substrate obviously decreased, as shown in Figure 5c. Figure 5d shows that plateaus resulting from homopolymerized PAA desorbing from the hydroxyl-modified substrate exhibited a similar decrease. However, plateaus with a lower force resulting from the disassembly of the micelles showed less change, indicating that these plateaus are not influenced by the different substrates. This result confirms that these plateaus arise from the interactions within the micelle because they are not dependent on the properties of the substrate.

Conclusions The force required to disassemble block copolymer micelles in water was investigated directly using AFM-based SMFS. The force needed to disassemble PAA-PF-PAA block copolymer micelles in water is 23 pN, and the disassembly is found to be a

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dynamic process. The disassembly events disappear in ethanol, indicating that the force needed to disassemble a micelle is sensitive to the properties of the solvent. As far as we know, this work is the first report of the force required to disassemble a block copolymer micelle in water using SMFS. Systemic studies of the force required to disassemble block copolymer micelles consisting of different monomers in different solvents will improve our understanding of the disassembly mechanism of polymer micelles and increase our ability to fabricate controlled polymer micellebased functional devices. Acknowledgment. This work was financially supported by the National Natural Foundation of China (20834003), an NSFCDFG joint grant (TRR-61), and the National Basic Research Program (2007CB808000). We thank Prof. Daoyong Chen, Zhenghua Jiang, and Yingheng Zhang for helpful discussions. Supporting Information Available: Force-extension curves for disassembling PAA-PF-PAA block copolymer micelles. The modified freely jointed chain (M-FJC) model. Force curves at different stretching velocities. Typical force curves obtained on substrates covered with amino groups with adsorbed micelles. Estimation of the length of the shoulderlike plateaus. This material is available free of charge via the Internet at http://pubs.acs.org.

Langmuir 2010, 26(12), 9183–9186