pubs.acs.org/Langmuir © 2010 American Chemical Society
Covalent Attachment of Polymersomes to Surfaces Stephanie Domes, Volkan Filiz, Jasmin Nitsche, Andreas Fr€omsdorf, and Stephan F€orster* Institute of Physical Chemistry, University of Hamburg, Grindelallee 117, D-20146 Hamburg, Germany Received November 3, 2009. Revised Manuscript Received January 29, 2010 We show that vesicles made of block copolymers with aldehyde end groups can be covalently attached to aminated and non-aminated, untreated glass surfaces. The attached vesicles were sufficiently stable to allow a detailed investigation of vesicle shapes by confocal laser scanning microscopy (CLSM) and AFM in aqueous solutions allowing reconstruction of 3D images of the vesicle structure. Covalently attached PCL-PEO, PLA-PEO, and PI-PEO block copolymer vesicles have different footprint areas and different shapes due to their differences in bilayer stiffness.
Introduction Amphiphilic molecules such as surfactants, lipids, and block copolymers can self-assemble to form vesicles. Vesicles are selfsupported closed bilayer assemblies of amphiphiles that enclose an aqueous interior volume.1 Lipid vesicles or “liposomes” have received considerable attention as model systems for fluid interfaces and biomembranes2 as well as in applications in the area of cosmetics and pharmaceutics.3-5 In recent years block copolymer vesicles or “polymersomes” have attracted increasing interest because of their excellent stability and the potential to control physical, chemical, and biological properties by tailoring of block lengths, block chemistry, and functionalization.6-10 For investigations of vesicle structures in aqueous media it is often desirable to localize or immobilize vesicles on surfaces. This is of particular importance when employing modern microscopy techniques such as atomic force microscopy (AFM), confocal laser scanning microscopy (CLSM), or environmental scanning electron microscopy (ESEM) that allow in situ studies in aqueous media. Usually Brownian motion or convection prevent detailed structural or time-resolved studies of vesicle properties under ambient conditions relevant for fundamental biophysical studies as well as for many applications particularly in diagnostics and therapeutics. The immobilization of vesicles is usually achieved by embedding vesicles into gels11 or by noncovalent interactions with surfaces, most notably by the weak adhesion to glass surfaces in case of lipid vesicles. Adhesion alters the geometry of the adhering vesicles which can be used to calculate the adhesion energy.12 The *To whom correspondence should be addressed. E-mail: forster@chemie. uni-hamburg.de. (1) F€orster, S.; Borchert, U. Polymer vesicles. In Encyclopedia of Polymer Science and Technology, 3rd ed.; Mark, H. E., Ed.; John Wiley: New York, 2005. (2) Lipowsky, R.; Sackmann, E. Structure and Dynamics of Membranes - From Cells to Vesicles; Elsevier Science: Amsterdam, 1995. (3) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem. 1988, 100, 117. (4) Lasic, D. D.; Papahadjopoulos, D. Science 1995, 267, 1275. (5) Drummond, D. C.; Meyer, O.; Hong, K.; Kirpotin, D. B.; Papahadjopoulos, D. Pharmacol. Rev. 1999, 51, 691. (6) Zhang, L. F.; Eisenberg, A. Science 1995, 268, 1728. (7) Zhang, L. F.; Yu, K.; Eisenberg, A. Science 1996, 272, 1777. (8) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967. (9) Lee, J. C. M.; Bermudez, H.; Discher, B. M.; Sheehan, M. A.; Won, Y. Y.; Bates, F. S.; Discher, D. E. Biotechnol. Bioeng. 2000, 73, 135. (10) Antonietti, M.; F€orster, S. Adv. Mater. 2003, 15, 1323. (11) Borchert, U.; Lipprandt, U.; Bilang, M.; Kimpfler, A.; Rank, A.; PeschkaS€uss, R.; Schubert, R.; Lindner, P.; F€orster, S. Langmuir 2006, 22, 5843–5847. (12) Gruhn, T.; Franke, T.; Dimova, R.; Lipowsky, R. Langmuir 2007, 23, 5423– 5429.
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adhesion of intact lipid vesicles has been reported by Sch€onherr et al.13 for sufficiently low concentrations of vesicles. At higher concentrations, lipid vesicles fuse at the surface to form larger vesicles and eventually rupture resulting in bilayer disks, finally merging and forming a flat bilayer film (supported bilayer) on the surface. Stronger adhesion occurs for Coulombic interactions between positively charged lipid vesicles and negatively charged surfaces, where spreading of the vesicles at the bilayer is observed.14 Li et al. reported the immobilization of polymersomes via Coulombic attraction to glass or mica surfaces mediated by Mg2þ ions.15 Since physical adhesion of vesicles will strongly depend on the aqueous environment, a stable covalent chemical attachment of single vesicles to surface without disintegration of vesicles would be highly desirable. For lipid vesicles a covalent attachment was reported for the case of vesicles formed by specially designed diacetylene lipids with amine-functionalized head groups. The vesicles were stabilized prior to surface attachment by UV irradiation that cross-links the bilayer by the formation of polydiacetylenes. The amine-functionalized vesicles were then bound to aldehyde-functionalized glass surfaces.16 The covalent binding of amines and aldehydes via formation of Schiff bases has been previously used by Emoto et al.17 for the covalent attachment of block copolymer micelles to amino-functionalized surfaces. Also in this case it was observed that the micelles were disrupted upon attachment. Only micelles with cross-linked cores maintained their structure on the surface. Here we report that a covalent binding via aldehydes can conveniently be used for the attachment of block copolymer vesicles without disruption of the vesicular structure. Furthermore, we found that a covalent attachment is also possible for unfunctionalized glass or silicon surfaces via stable acetal formation. The attachment is demonstrated for a number of chemically different block copolymer vesicles. We demonstrate the potential of this method for long-term, stable immobilization by performing 3Dtomography using confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM) in aqueous solutions. (13) Sch€onherr, H.; Johnson, J. M.; Lenz, P.; Frank, C. W.; Boxer, S. G. Langmuir 2004, 20, 11600–11606. (14) Bernard, A.-L.; Guedeau-Boudeville, M.-A.; Sandre, O.; Palacin, S.; di Meglio, J.-M.; Jullien, L. Langmuir 2000, 16, 6801–6808. (15) Li, F.; Ketelaar, T.; Cohen-Stuart, M. A.; Sudh€olter, E. J. R.; Leermakers, F. A. M.; Marcelis, A. T. M. Langmuir 2008, 24, 76–82. (16) Kim, J.-M.; Ji, E.-K.; Woo, S. M.; Lee, H.; Ahn, D. J. Adv. Mater. 2003, 15, 1118–1121. (17) Emoto, K.; Nagasaki, Y.; Kataoka, K. Langmuir 1999, 15, 5212–5218.
Published on Web 03/31/2010
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Figure 1. Immobilization of aldehyde-modified block copolymers on an aminated glass surface.
Experimental Section Block Copolymer Synthesis. We investigated vesicles formed by poly(lactide-b-ethylene oxide) (PLA-PEO), poly(caprolactone-b-ethylene oxide) (PCL-PEO), and poly(isoprene-b-ethylene oxide) (PI-PEO). PLA-PEO and PCL-PEO were synthesized by sequential anionic ring-opening polymerization of ethylene oxide and 3,6-dimethyl-1,4-dioxane-2,5-dione18 and ε-caprolactone,19 respectively, using 3,3-diethoxy-1-propanol as an initiator. The conversion of the acetal into an aldehyde end group was conducted after vesicle formation. The vesicle solution was adjusted to pH 2 and stirred for 2 h at room temperature. Afterward, the reaction mixture was neutralized, and the vesicle solution was dialyzed against water to remove the salt and then freeze-dried.20 PI-PEO was synthesized by sequential anionic polymerization of isoprene and ethylene oxide. The terminal OH group was selectively oxidized into the aldehyde by Dess-Martin oxidation.21,22 Although reaction times of 20 min up to 2 h are described in the literature, in the case of polymers a reaction time of 24 h is required. The degrees of polymerization of the block copolymers used in this study were PLA(130)-PEO(84), PCL(191)-PEO(54), and PI(41)-PEO(35). The polymers were characterized by GPC, 1H NMR, and MALDI-TOF-MS to determine the molecular weight, polydispersity, and end-group functionalization. The polydispersity of the PLA-PEO and PCL-PEO block copolymers both are 1.25, and the polydispersity of the PI-PEO block copolymer is 1.06. Vesicle Preparation. For the preparation of the PEO-PLA and PEO-PCL vesicles, these block copolymers were dissolved in chloroform and transferred into a vial. The chloroform phase was covered with a layer of water. Upon stirring and slow evaporation of chloroform, the block copolymers transfer into the aqueous phase to form a vesicle solution of 10 mg/mL polymer concentration in water. The phase transfer was done at 80 °C for 48 h until all chloroform had evaporated. With this procedure, a large amount of giant vesicles with a low amount of insoluble poly(18) Scholz, C.; Iijima, M.; Nagasaki, Y.; Kataoka, K. Macromolecules 1995, 28, 7295. (19) Bogdanov, B.; Vidts, A.; Bulcke, A. V. D.; Verbeeck, R.; Schacht, E. Polymer 1998, 39(8-9), 1631–1336. (20) Xiong, X.-B.; Mahmud, A.; Uludag, H.; Lavasanifar, A. Biomacromolecules 2007, 8, 874–884. (21) Surendra, K.; Srilakshmi Krishnaveni, N.; Arjun Reddy, M.; Nageswar, Y. V. D.; Rama Rao, K. J. Org. Chem. 2003, 68, 2058–2059. (22) Speicher, A.; Bomm, V.; Eicher, T. J. Prakt. Chem. 1996, 338, 588–590.
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mer fractions can be obtained. For the preparation of PI-PEO vesicles, the film-rehydration method was used. After dissolving the block copolymer in chloroform, the chloroform was slowly evaporated and a thin film was obtained. Upon adding water, the film started to swell at room temperature and large unilamellar vesicles were formed. The concentration was 10 mg/mL. Amination of Glass Plates. For the amination of the glass plates (cover slides), the plates were cleaned with ethanol and immersed in a solution of 3-aminopropyltriethoxysilane (APTS) in dry THF (1 mmol/mL) for 2 h. Afterward, excess APTS is removed by rinsing in ethanol.17 Immobilization of Vesicles. The aminated substrate was covered with the vesicle solution in a special sample holder that can be closed to prevent evaporation of water during prolonged investigations and can be opened to immerse or rinse the glass plate with different solutions. After incubation for 12 h the surface was washed with water and investigated using confocal laser scanning microscopy. The vesicles were fluorescently labeled by solubilization of Nile Red into the bilayer or by covalent attachment of fluorescein isothiocyanate-tagged albumin (FITC-albumin). For attachment to unfunctionalized glass plates, the plates were covered with the vesicle solution for periods between 10 min and 12 h reaction time depending on the polymer. The glass plates were washed with water and investigated with a CLSM. Confocal Laser Scanning Microscopy (CLSM). The measurements were performed with a Fluoview FV 1000 FV10-ASW from Olympus equipped with a 60 oil immersion objective. The software Fluoview Ver. 1.6a was used to create the 3D images. Atomic Force Microscopy (AFM). AFM measurements were carried out on a NanoWizard from JPK Instruments in contact mode in water by using gold-coated silicon nitride cantilevers (DNPS, Digital Instruments) with a spring constant of 0.13 ( 0.02 N m-1 and a nominal tip radius of 10 nm. The set point was set in a various range of 100-500 mV.
Results and Discussion By using aldehyde-functionalized block copolymers, the vesicles have a high surface density of aldehyde groups at their inner and outer surface. The aldehyde groups at the outer surface can react with the amino groups of aminated glass surfaces to form imines (Figure 1), leading to a stable covalent attachment of the vesicles to the surface. Rinsing the glass surface with solvent does not remove the attached vesicles. In a control experiment, vesicles Langmuir 2010, 26(10), 6927–6931
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formed by block copolymers with OH-terminal groups do not attach to the glass surface and are completely washed off by rinsing the surface. The covalently attached vesicles do not disintegrate, and their location is stable against Brownian motion and convection. This enables for the first time detailed tomographic in situ studies of their shape using different microscopy techniques. Figure 2 shows a confocal laser scanning microscopy (CLSM) 3D-tomographic reconstruction of PCL-PEO vesicles fluorescently labeled with Nile Red and attached to an aminated glass surface. Because of the covalent attachment, all vesicles are located on the glass surface with a high surface concentration and with all vesicles located in the same focal plane of the microscope, which is of advantage particularly in conventional nonconfocal microscopy. Figure 3 shows a typical 3D-tomographic image of a PCLPEO vesicle. We observe a slightly prolate vesicle shape with a flat footpring area, i.e., a barrel-type shape, where it is covalently attached to the glass surface. Figure 3a shows a cut through the vesicle parallel to and slightly above the glass surface to show the circular cross section. Figure 3b shows a cut perpendicular to the glass surface to show the barrel-type shape. A detailed inspection of the footprint area shows that the bilayer is not smooth but
Figure 2. CLSM image of PEO-PCL vesicles covalently attached to an aminated glass surface. The position of the glass surface is shown by the dashed line.
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slightly irregular and deformed. The irregular shape is due to the rigid mechanical properties of the PCL bilayer, which is in a glassy state at room temperature (Tg = 60 °C). This shows that detailed microscopic studies of vesicle shapes giving new insights are possible by covalent immobilization of vesicles to surfaces. Examples of regular and unregular shapes were also observed for PLA-PEO vesicles. For the purpose of testing the aldehyde terminal groups for biofunctionalization, the vesicles shown in Figure 4 were fluorescently labeled by covalent attachment of FITC-albumin. The aldehyde-functionalized bilayer covalently binds to the amino groups of the protein via Schiff-base formation. This could simply be achieved by incubating the attached vesicles with FITC-albumin and subsequently removing unbound FITC-albumin by washing with solvent. In this way only the outer surface of the vesicles was fluorescently labeled. In Figure 4a we observe the typical, rather smooth shape observed for PLA-PEO vesicles with a smaller footprint area compared to PCL-PEO vesicles. PLA has a similarly high Tg as PCL-PEO and is partially crystalline. By 3D-tomographic reconstruction it possible to image also topologically more complicated shapes such as in Figure 4b. What seems to be a vesicle-in-vesicle topology is a vesicle with an invaginated bilayer, which is why the bilayer patch visible inside the vesicle is fluorescently labeled. Invaginated vescicle shapes occur for vesicles with large bilayer asymmetry and very small interior volume/surface ratios.1 Surprisingly, a covalent attachment of aldehyde-functionalized vesicles is also possible for unfunctionalized glass surfaces, most likely via stable acetal formation with the Si-OH groups. We suppose that the locally high concentration of aldehyde end groups close to the glass surface with its high local concentration of OH groups drives the reaction toward acetal formation. All aldehyde-functionalized PCL-PEO, PLA-PEO, and PI-PEO vesicles investigated in this study could be attached to unfunctionalized glass surfaces. In the following we show the attachment of PI-PEO vesicles as an example. In contrast to the rather inflexible PCL-PEO and PLA-PEO vesicles, vesicles made of PI-PEO are highly deformable and show the highest degree of surface coverage (Figure 5a). Figure 5b shows a cross section of a PI-PEO vesicle covalently attached to an unfunctionalized glass surface. The vesicle structure is not ruptured or spread over the substrate to form a flat, empty bilayer structure but is still intact, albeit with a highly oblate shape and a low volume/surface ratio. Since PI-PEO vesicles solubilize smaller amounts of Nile Red
Figure 3. Cross sections of a CLSM image of a PCL-PEO vesicle covalently attached to an aminated glass surface parallel (a) and perpendicular to the surface (b). As a consequence of the inflexible vesicle membrane, the footprint area is not planar but rather irregular. The position of the glass surface is shown by the dashed line. Langmuir 2010, 26(10), 6927–6931
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Figure 4. Cross sections of CLSM images of PLA-PEO vesicles covalently attached to aminated glass surfaces. The biofunctionalization with FITC-albumin was performed after surface attachment of the vesicles. (a) shows a commonly observed spherical vesicle, and (b) shows a vesicle with a invaginated topology. The position of the glass surface is shown by the dashed line.
Figure 5. CLSM image of a PI-PEO vesicle covalently attached to an unfunctionalized glass surface (a) and cross section of the same image (b). The vesicle shape is flat due to the more flexible vesicle membrane.
Figure 6. AFM images of PI-PEO vesicles covalently attached to unfunctionalized glass surfaces: (a) area with small vesicles; (b) detailed image of a large vesicle that was scanned with higher contact forces at regular intervals. When higher contact forces are applied, the vesicle is deformed in scanning direction, which is to the right-hand side.
compared to PCL-PEO and PLA-PEO vesicles, the images were taken at higher illumination so that also the weak fluorescence of the glass surface due to small amounts of adsorbed dye are observed. So far we have shown the possibility to image covalently attached vesicles by confocal laser scanning microscopy. Our aim was 6930 DOI: 10.1021/la904175u
also to demonstrate the potential to use atomic force microscopy (AFM) to image covalently attached vesicles. This is demonstrated for PI-PEO vesicles which due to their membrane flexibility show an interesting response to the forces exerted by the AFM tip. First, Figure 6a shows the image of a large number of PI-PEO vesicles covalently attached to a glass substrate (cover Langmuir 2010, 26(10), 6927–6931
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Figure 7. Trace (red) and retrace (black) curve of the vesicle image in Figure 6b made with increased contact forces. The flexible vesicle deforms in the scanning direction while the contact area stays at its original position. Because of the covalent attachment, no vesicle movement is observed.
slide). The peculiar arrangement into parallel lines is due to the surface structure (grooves, steps) on the substrate. It is not an artifact of the measurement as can be seen from a repeated scan of the same region shown in the Supporting Information. We also observe that some vesicles appear toroidal, whereas others appear spherical. We have no clear answer to this at the moment but suspect that this is due to different osmotic pressures inside the vesicles. If the osmotic pressure is high, the upper spherical cap of the vesicles withstands the contact force of the AFM tip; if it is low, it will deform, and it is the bilayer rim that elastically responds to the AFM tip. As can be in the AFM image that was subsequently recorded with the same contact force (Supporting Information), more vesicles appear to have toroidal shape, possibly due to a decrease of the osmotic pressure either by their attachment to the surface or by the forces exerted by the AMF tip. Thus, in situ imaging of vesicles that are covalently attached to a surface and immersed in solution is possible and reveals detailed insight into vesicle shapes. A more detailed investigation by AFM shows that the vesicles exhibit some local flexibility and can be deformed while their footprint area of the membrane is firmly attached to the glass surface. When recording an AFM image, the AFM tip scans the surface on adjacent lines. If this is done with low contact forces, the vesicle shape is not affected and AFM images such as in Figure 6a are obtained. Higher contact forces, however, lead to a deformation of the vesicles in the scanning direction of the AFM tip. When recording the image in Figure 6b, higher contact forces were applied at regular intervals. Then the scanned image of the vesicle appears to be shifted in scanning direction, which is to the right-hand side in Figure 6b. What appears to be an artifact of
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the measurement is a consequence of the elastic response of the vesicle membrane to the AFM tip. This shows the possibility to in principle measure the elastic properties of vesicles directly by using AFM, which requires to quantify the shape of the vesicle upon deformation. The deformed shapes of the vesicles upon tracing and retracing with the AFM tip is shown in the recorded height profile in Figure 7. We observe that the vesicle is significantly deformed and elongated in the scanning direction which is to the right upon tracing and to the left upon subsequent retracing. A shift of the vesicle position on the surface is not observed.
Conclusion We have shown that vesicles made of block copolymers with aldehyde end groups can be covalently attached to aminated and non-aminated, untreated glass surfaces. The attached vesicles were sufficiently stable to allow a detailed investigated of the vesicle shapes by CLSM and AFM in aqueous solutions, allowing reconstruction of 3D images of the vesicle structure. Block copolymer vesicles have different footprint areas due to differences in membrane stiffness. PEO-PCL with a glassy bilayer membrane shows a small, deformed footprint area while PI-PEO with high a bilayer flexibility shows the highest footprint area that is possible while maintaining a hollow vesicular shape. We observe that higher forces of the cantilever let to vesicle deformation while the footprint area is fixed to the surface. Supporting Information Available: Image of the second scan of the region shown in Figure 6a. This material is available free of charge via the Internet at http://pubs.acs.org.
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