1230
Langmuir 2005, 21, 1230-1237
Controlling the Internal Structure of Giant Unilamellar Vesicles by Means of Reversible Temperature Dependent Sol-Gel Transition of Internalized Poly(N-isopropyl acrylamide) Aldo Jesorka,* Martin Markstro¨m, and Owe Orwar Department of Chemistry and Bioscience, Chalmers University of Technology, SE-41296 Go¨ teborg, Sweden Received August 31, 2004. In Final Form: November 8, 2004 In this work, we present preparation and basic applications of lipid-bilayer-enclosed picoliter volumes (microcontainers) of solutions of poly(N-isopropylacrylamide) (PNIPAAm). Giant unilamellar vesicles (GUVs) were prepared from phospholipids using a standard swelling procedure and subsequently surface immobilized. Clear, slightly viscous solutions of PNIPAAm of varying concentration in aqueous buffer were directly pressure-microinjected into the GUVs, using a submicrometer-sized, pointed capillary. The GUV was subjected to changing temperature over a 21-40 °C range. The typical phase transition of the polymeric material upon heating and cooling across the lower critical solution temperature was followed using optical microscopy and shown to be reversible over multiple sequential heating/cooling cycles without compromising the integrity of the GUV membrane. Fluorescent, carboxylic acid modified 200 nm latex beads, co-injected with the PNIPAAm solution, were temperature-reversibly immobilized during the phase transition, practically freezing the Brownian motion of the entrapped particles in the volume. Furthermore, a co-injected water soluble fluorescent polysaccharide-dye conjugate was shown not to migrate from the aqueous phase into the hydrophobic polymer part upon heating, whereas the fluorescent beads were completely but reversibly immobilized in the hydrophobic domains of dense polymer agglomerates. The system reported here provides a feasible method for the reversible stabilization and solidification of GUV interior volumes, e.g., as a micrometer-sized model system for controlled drug release.
Introduction Giant unilamellar vesicles are micrometer-sized compartments made from surfactant materials. They are typically composed of lipid membranes and can harbor volumes smaller than 10-12 L in a biomimetic environment.1 The fluid character of the membrane gives the vesicles dynamic properties, which means that introduction of liquid through the membrane can be easily achieved, e.g., by electroinjection.2 The nature of the liquid content of each vesicle can therefore be freely chosen, provided that the filling solution does not disrupt, destabilize, or dissolve the membrane. Soft matter nanofluidic devices based on phospholipid membrane vesicles and interconnecting lipid nanotubes have been developed quite recently,3 and a number of related studies has been conducted, e.g., the controlled generation of the micrometer scale networks, their structure and topology,4,5 as well as modification with biomacromolecules within the compartments and nanotubes.6 As was shown previously, these vesicles and vesiclenanotube networks are useful systems to study a variety * Corresponding author. E-mail:
[email protected]. Fax: +46-31-7722785. Telephone: +46-31-7723069. (1) Menger, F. M.; Angelova, M. I. Acc. Chem. Res. 1998, 31, 789797. (2) Karlsson, M.; Nolkrantz, K.; Davidson, M. J.; Stro¨mberg, A.; Ryttse´n, F.; A° kerman, B.; Orwar, O. Anal. Chem. 2000, 72 (23), 58575862. (3) Karlsson, A.; Karlsson, R.; Karlsson, M.; Cans, A.-S.; Stro¨mberg, A.; Ryttse´n, F.; Orwar, O. Nature 2001, 409, 150-152. (4) Karlsson, M.; Sott, K.; Cans, A.-S.; Karlsson, A.; Karlsson, R.; Orwar, O. Langmuir 2001, 17, 6754-6758. (5) Sott, K.; Karlsson, M.; Pihl, J.; Hurtig, J.; Lobovkina, T.; Orwar, O. Langmuir 2003, 19, 3904-3910. (6) Davidson, M.; Karlsson, M.; Sinclair, J.; Sott, K.; Orwar, O. J. Am. Chem. Soc. 2003, 125, 374-378.
of functional aspects, e.g., applications as models for cellular functions7 or the interaction with functional membrane proteins.8 Reversible control of concentration and migration of compounds dissolved or suspended in the internal solution of such vesicles has not been achieved to present. The goals of the study presented here were the evaluation of a polymer-based reversible sol/hydrogel system enclosed in single giant unilamellar vesicles (GUVs) and the control of the sol/gel process within the vesicle by temperature change. One important rationale for the study is the stabilization of GUVs, aiming at a biocompatible, nonliquid but simple internal structure, practically the most basic feature of a biological cell. Fundamental differences in kinetics in cellular environments are attributed to macromolecular crowding, resulting from hindered diffusion, size exclusion, and limited molecular motion in such systems.9 A cell-sized membrane compartment featuring reversible macromolecular crowding represents a model for quantitative studies of diffusioncontrolled kinetics in comparison to macroscopic chemical reactors.10,11 A second point of interest is the temporary, reversible immobilization and/or release of chemical and biological material within the vesicle,12-14 often in the context of artificial cells.15,16 (7) Cans, A.-S.; Wittenberg, N.; Karlsson, R.; Sombers, L.; Karlsson, M.; Orwar, O.; Ewing, A. Proc. Natl. Acad. Sci. U.S.A. 2003, 1009, 400-404. (8) Roux, A.; Capello, G.; Cartaud, J.; Prost, J.; Goud, G.; Bassereau, P. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5394-5399. (9) Schnell, S.; Turner, T. E. Prog. Biophys. Mol. Biol. 2004, 85, 235260. (10) Dagdug, L.; Berezhkovskii, A. M.; Shvartsman, S. Y.; Weiss, G. H. J. Chem. Phys. 2003, 119 (23), 12473. (11) Dagdug, L.; Berezhkovskii, A.; Bezrukov, S. M.; Weiss, G. H. J. Chem. Phys. 2003, 118 (5), 2367.
10.1021/la047822k CCC: $30.25 © 2005 American Chemical Society Published on Web 01/19/2005
Controlling the Internal Structure of GUVs
Langmuir, Vol. 21, No. 4, 2005 1231
Figure 1. Experimental setup, combining an optical microscope, femtoliter-pressure injection, and micromanipulation with a thin-film resistive heat stage and a micrometer-sized thermoelement-based temperature measurement setup.
To address some of those issues, especially the unsupported, unstructured internal volume, a hydrogel filling of a vesicle presents an interesting possibility. So far, publications on gel-filled nanometer-sized and giant vesicle suspensions have appeared to address questions such as particle formation or shape and kinetics of the membrane during osmotic shrinkage.17,18 An attractive material for reversible sol-gel transition studies is the water soluble poly(N-isopropylacrylamide) (PNIPAAm). This polymer with its reversible, temperature responsive sol/gel phase transition upon crossing the lower critical solution temperature (LCST) of 32 °C in pure water has been used for several applications, such as size separation and extraction, enzyme and cell immobilization, and drug delivery systems.19-24 It has been shown in bulk experiments that the diffusion properties of (12) Kiser, P. F.; Wilson, G.; Needham, D. J. Controlled Release 2000, 68 (1), 9-22. (13) Giant Vesicles, Perspectives in Supramolecular Chemistry; Luisi, P. L., Walde, P., Eds.; John Wiley & Sons Ltd.: Chichester, 2000. (14) Apel, C.; Mautner, M.; Deamer, D. W. Biochem. Biophys. Acta, Biomembr. 2002, 1559, 1-9. (15) Bachmann, P. A.; Luisi, P. L.; Lang, J. Nature 1992, 357, 57-59. (16) Fischer, A.; Oberholzer, T.; Luisi, P. L. Biochim. Biophys. Acta 2000, 1467, 177-188. (17) Monshipouri, M.; Rudolph, A. S. J. Microencapsulation 1995, 12 (2), 117-127. (18) Viallat, A.; Dalous, J.; Abkarian, M. Biophys. J. 2004, 86, 21792187. (19) Bae, Y. H.; Okano, T.; Hsu, R.; Kim, S. W. Makromol. Chem. Rapid Commun. 1987, 8, 481-485. (20) Hoffman, A. S.; Afrassiabi, A.; Dong, L. C. J. Controlled Release 1986, 4, 213-222.
materials embedded in temperature sensitive hydrogels can be altered significantly by changing the temperature.25 Ultimately, though not entirely within the scope of this early study, reversible separation of specific organic material from the aqueous phase or concentration of hydrophilic material in the aqueous phase or even electromigration and electroseparation of charged biological material in such a GUV/sol-gel system is desired. Materials and Methods Experimental Setup. The experimental setup is schematically displayed in Figure 1. All important elements of the setup are described in detail in the following paragraphs. Giant unilamellar vesicles (10-50 µm in diameter) were immobilized on a microscope coverslip of 0.2 mm thickness, coated with an electrically conducting metal film on the underside. For the experiments, the coverslip was placed under an inverted microscope equipped with a 40× high numerical aperture objective. To introduce polymer solution, a borosilicate capillary (
