pubs.acs.org/Langmuir © 2009 American Chemical Society
Capsosomes: Subcompartmentalizing Polyelectrolyte Capsules Using Liposomes :: Brigitte Stadler,† Rona Chandrawati,† Kenneth Goldie, ‡ and Frank Caruso†,* †
Centre for Nanoscience and Nanotechnology, Department of Chemical and Biomolecular Engineering, The University of Melbourne, Parkville, Victoria 3010, Australia, and ‡Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria 3010, Australia Received January 18, 2009. Revised Manuscript Received March 10, 2009 Next-generation therapeutic approaches are expected to rely on the engineering of multifunctional particle carriers that can mimic specific cellular functions. The key features of such particles are the semipermeable nature of the shell for communication with the external environment and multiple nanosized individual subcompartments confined within a micron-sized structurally stable scaffold for conducting specific reactions. Herein, we report the formation of capsosomes, a new class of polyelectrolyte capsules containing structurally intact liposomes as cargo. The multilayer film assembly of polyelectrolytes (poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH)) and liposomes (50 nm 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)) was characterized on planar substrates using quartz crystal microbalance with dissipation monitoring, and these findings were then correlated to the film growth of the polyelectrolytes and structurally intact liposomes on silica particles. Upon removal of the silica template core, stable capsosomes, containing one or two layers of intact liposomes as cargo, were obtained. This novel platform, capsosomes, which combines the advantages of two systems, liposomes and polyelectrolyte capsules, is expected to find diverse applications in biomedicine, in particular for the creation of artificial cells or organelles where the performance of reactions within a confined environment is a prerequisite.
Introduction Bioinspired nanoengineered vehicles, which can perform simple cellular activity, are expected to have an impact on nextgeneration therapeutic concepts.1 They are much simpler in design than biological cells but yet possess the two key features of multiple subcompartments where (enzymatic) reactions can be processed, and defined permeability, which permits controlled interactions with the environment. However, most of the currently considered systems, such as liposomes, micelles, or polymer capsules, are relatively simple, single-compartment vessels equipped with certain functionalities. Liposomes, with their ability to encapsulate smaller objects, biocompatibility, ease of preparation via self-assembly, and small size (50-400 nm), are attractive drug carriers.2,3 Their properties have been continuously improved in order to overcome some of their limitations, which include instability under in vivo conditions and the lack of control over degradability.4 Methods to enhance their performance include stabilizing liposomes by polymer layers5-7 or incorporating polymerizable lipid amphiphiles into the membrane.8,9 Vesosomes, smaller vesicles encapsulated within a bigger vesicle, extend the liposome-based drug carrier approach, providing the opportunity to deliver multiple drugs *To whom correspondence should be addressed. E-mail: fcaruso@ unimelb.edu.au. (1) Zhang, Y.; Ruder, W. C.; Leduc, P. R. Trends Biotechnol. 2008, 26, 14–20. (2) Andresen, T. L.; Jensen, S. S.; Jorgensen, K. Prog. Lipid Res. 2005, 44, 68–97. (3) Drummond, D. C.; Zignani, M.; Leroux, J. C. Prog. Lipid Res. 2000, 39, 409– 460. (4) Cattel, L.; Ceruti, M.; Dosio, F. Tumori 2003, 89, 237–249. (5) Ciobanu, M.; Heurtault, B.; Schultz, P.; Ruhlmann, C.; Muller, C. D.; Frisch, B. Int. J. Pharm. 2007, 344, 154–157. (6) Lee, S. M.; Chen, H.; Dettmer, C. M.; O’Halloran, T. V.; Nguyen, S. T. J. Am. Chem. Soc. 2007, 129, 15096–15097. (7) Moghimi, S. M.; Szebeni, J. Prog. Lipid Res. 2003, 42, 463–478. (8) Mueller, A.; O’Brien, D. F. Chem. Rev. 2002, 102, 727–757. (9) O’Brien, D. F.; Armitage, B.; Benedicto, A.; Bennett, D. E.; Lamparski, H. G.; Lee, Y. S.; Srisiri, W.; Sisson, T. M. Acc. Chem. Res. 1998, 31, 861–868.
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with controlled release profiles.10 Apart from serving as drug delivery vehicles, multicompartmentalized liposomes provide a confined environment to perform small-scale (bio)chemical reactions.11 Release of the cargo from the trapped liposomes and the subsequent reagent mixing, a key point, can be conducted by making use of the inherently high permeability of lipid membranes at their phase transition temperature.12 Inducing pores into the lipid membrane, e.g., by incorporating gramicidin13 or by interaction with certain polymers,14 enables the controlled diffusion of molecules between the liposome interior and exterior. Polymeric vesicles, consisting of synthetic biocompatible amphiphilic copolymer building blocks, offer an alternative route to create nanocontainers by self-assembly with high control over their properties, mainly via the choice of the constituent polymers.15,16 Controlled surface chemistry allows for cell-specific uptake of the vesicles.17 The incorporation of pores into the polymer membranes enables the triggered interaction of the trapped cargo with the environment.18 A first approach to mimicking eukaryotic cells has recently been reported by creating polymeric multivesicle assemblies equipped with pH sensitive channels.19 (10) Kisak, E. T.; Coldren, B.; Evans, C. A.; Boyer, C.; Zasadzinski, J. A. Curr. Med. Chem. 2004, 11, 199–219. (11) Bolinger, P. Y.; Stamou, D.; Vogel, H. Angew. Chem., Int. Ed. 2008, 47, 5544–5549. (12) Bolinger, P. Y.; Stamou, D.; Vogel, H. J. Am. Chem. Soc. 2004, 126, 8594– 8595. (13) Stamou, D.; Duschl, C.; Delamarche, E.; Vogel, H. Angew., Chem., Int. Ed. 2003, 42, 5580–5583. (14) Binder, W. H. Angew. Chem., Int. Ed. 2008, 47, 3092–3095. (15) Discher, B. M.; Hammer, D. A.; Bates, F. S.; Discher, D. E. Curr. Opin. Colloid Interface Sci. 2000, 5, 125–131. (16) Kita-Tokarczyk, K.; Grumelard, J.; Haefele, T.; Meier, W. Polymer 2005, 46, 3540–3563. (17) Ben-Haim, N.; Broz, P.; Marsch, S.; Meier, W.; Hunziker, P. Nano Lett. 2008, 8, 1368–1373. (18) Broz, P.; Driamov, S.; Ziegler, J.; Ben-Haim, N.; Marsch, S.; Meier, W.; Hunziker, P. Nano Lett. 2006, 6, 2349–2353. (19) Chiu, H. C.; Lin, Y. W.; Huang, Y. F.; Chuang, C. K.; Chern, C. S. Angew. Chem., Int. Ed. 2008, 47, 1875–1878.
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Polyelectrolyte capsules,20 prepared by layer-by-layer assembly (LbL),21 are promising drug carriers or microreactors. The LbL assembly of oppositely charged polyelectrolytes,22-24 or functional polymers that can be linked together, e.g., via hydrogen bonding,25 DNA-DNA hybridization,26 or via covalent linking (i.e., click chemistry27,28) onto a colloidal template followed by the subsequent removal of the template leads to hollow polymeric capsules. Capsules have been loaded with a range of materials, from small molecules29,30 to plasmid DNA.31 In addition, the surface of the capsules can be modified to yield low-fouling capsules by the adsorption of a poly (ethylene glycol)-based layer32,33 or targeted capsules by binding antibodies.34 Furthermore, the shell characteristics can be engineered and adapted as required. Properties such as the degradation35-38 or the permeability39-41 of the capsules can be tailored by designing the building blocks used to assemble the shell. For instance, lipid bilayers have been incorporated as a barrier layer into the polymer capsule shell to control the diffusion of small molecules.42-45 A first step toward the creation of a bioreactor by separating the capsule volume in two subcompartments, which can be later recombined using laser light as a trigger, was recently reported.46 Although an interesting approach, these dual-compartment capsules do not provide any additional protection for fragile cargo over single-compartment capsules, and the number of (20) Johnston, A. P. R.; Cortez, C.; Angelatos, A. S.; Caruso, F. Curr. Opin. Colloid Interface Sci. 2006, 11, 203–209. (21) Decher, G. Science 1997, 277, 1232–1237. (22) Balabushevich, N. G.; Tiourina, O. P.; Volodkin, D. V.; Larionova, N. I.; Sukhorukov, G. B. Biomacromolecules 2003, 4, 1191–1197. :: (23) Berth, G.; Voigt, A.; Dautzenberg, H.; Donath, E.; Mohwald, H. Biomacromolecules 2002, 3, 579–590. (24) Yu, A. M.; Wang, Y. J.; Barlow, E.; Caruso, F. Adv. Mater. 2005, 17, 1737– 1741. (25) Zelikin, A. N.; Quinn, J. F.; Caruso, F. Biomacromolecules 2006, 7, 27–30. (26) Johnston, A. P. R.; Read, E. S.; Caruso, F. Nano Lett. 2005, 5, 953–956. (27) Such, G. K.; Tjipto, E.; Postma, A.; Johnston, A. P. R.; Caruso, F. Nano Lett. 2007, 7, 1706–1710. (28) De Geest, B. G.; Van Camp, W.; Du Prez, F. E.; De Smedt, S. C.; Demeester, J.; Hennink, W. E. Chem. Commun. 2008, 2, 190–192. :: (29) Sukhorukov, G. B.; Antipov, A. A.; Voigt, A.; Donath, E.; Mohwald, H. Macromol. Rapid Commun. 2001, 22, 44–46. (30) Han, B. S.; Shen, B. Y.; Wang, Z. H.; Shi, M. M.; Li, H. W.; Peng, C. H.; Zhao, Q. H. Polym. Adv. Technol. 2008, 19, 36–46. (31) Zelikin, A. N.; Becker, A. L.; Johnston, A. P. R.; Wark, K. L.; Turatti, F.; Caruso, F. ACS Nano 2007, 1, 63–69. (32) Kinnane, C. R.; Wark, K.; Such, G. K.; Johnston, A. P. R.; Caruso, F. Small 2009, 5, 444–448. :: :: :: (33) Heuberger, R.; Sukhorukov, G.; Voros, J.; Textor, M.; Mohwald, H. Adv. Funct. Mater. 2005, 15, 357–366. (34) (a) Cortez, C.; Tomaskovic-Crook, E.; Johnston, A. P. R.; Radt, B.; Cody, S. H.; Scott, A. M.; Nice, E. C.; Heath, J. K.; Caruso, F. Adv. Mater. 2006, 18, 1998–2003. (b) Cortez, C.; Tomaskovic-Crook, E.; Johnston, A. P. R.; Scott, A. M.; Nice, E. C.; Heath, J. K.; Caruso, F. ACS Nano 2007, 1, 93–102. (35) De Geest, B. G.; Vandenbroucke, R. E.; Guenther, A. M.; Sukhorukov, G. B.; Hennink, W. E.; Sanders, N. N.; Demeester, J.; De Smedt, S. C. Adv. Mater. 2006, 18, 1005–1009. (36) De Koker, S.; De Geest, B. G.; Cuvelier, C.; Ferdinande, L.; Deckers, W.; Hennink, W. E.; De Smedt, S.; Mertens, N. Adv. Funct. Mater. 2007, 17, 3754– 3763. (37) Zelikin, A. N.; Li, Q.; Caruso, F. Chem. Mater. 2008, 20, 2655–2661. (38) Itoh, Y.; Matsusaki, M.; Kida, T.; Akashi, M. Chem. Lett. 2008, 37, 238– 239. (39) Angelatos, A. S.; Johnston, A. P. R.; Wang, Y. J.; Caruso, F. Langmuir 2007, 23, 4554–4562. :: (40) Dai, Z. F.; Dahne, L.; Mohwald, H.; Tiersch, B. Angew. Chem., Int. Ed. 2002, 41, 4019–4022. (41) Georgieva, R.; Moya, S.; Hin, M.; Mitlohner, R.; Donath, E.; Kiesewetter, :: H.; Mohwald, H.; Baumler, H. Biomacromolecules 2002, 3, 517–524. (42) Katagiri, K.; Caruso, F. Macromolecules 2004, 37, 9947–9953. (43) Katagiri, K.; Caruso, F. Adv. Mater. 2005, 17, 738–743. :: (44) Li, J. B.; Mohwald, H.; An, Z. H.; Lu, G. Soft Matter 2005, 1, 259–264. (45) Moya, S.; Donath, E.; Sukhorukov, G. B.; Auch, M.; Baumler, H.; :: Lichtenfeld, H.; Mohwald, H. Macromolecules 2000, 33, 4538–4544. :: (46) Kreft, O.; Skirtach, A. G.; Sukhorukov, G. B.; Mohwald, H. Adv. Mater. 2007, 19, 3142–3145.
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potential subcompartments is limited. Small solid colloids have been embedded in the shell of polymer capsules;47 however, soft and larger (bio)objects, such as adenoviral vectors48 or intact liposomes,49,50 have only been incorporated into films on planar surfaces. No studies have reported the formation of polyelectrolyte multilayers with embedded (and structurally intact) liposomes on colloidal particles, with either the particle core present or after core removal. With the aim to yield a novel colloidal biomedical platform, herein we report the formation of capsosomes, an approach that combines liposomes and polyelectrolyte capsules. This combination not only preserves the advantages of both systems while eliminating some of the shortcomings but also offers new opportunities to tailor the structural properties or to implement functionality. The sealed aqueous 3D environment offered by liposomes provides a protective barrier for the encapsulation of fragile, low molecular weight biomolecules and therapeutic agents within polyelectrolyte capsules. In particular, biomolecules such as enzymes, which are prone to denaturation and subsequent loss of functionality upon interaction with surfaces, polymers, or harsh environmental conditions could be encapsulated within the liposomes. In doing so, the enzymes can remain protected at physiological conditions during LbL assembly of the film or during removal of the template particle, the latter being important for the generation of capsules. The polyelectrolyte capsules, on the other hand, can provide the structural integrity to embed a large number of liposomes within their semipermeable shell, thus providing thousands of individual subcompartments, with the exact number depending on the size of the liposomes (50-200 nm), that permit confined reactions. Furthermore, the semipermeable nature of polyelectrolyte capsules enables the controlled interaction between the interior and exterior of the capsules; that is, small molecules can freely diffuse across the polymer membrane, while larger molecules remain trapped inside. This is a crucial prerequisite in the development of multifunctional colloidal platforms aimed for use in therapeutic applications. The liposomal subcompartments will also enable the coencapsulation of multiple hydrophobic and hydrophilic cargo within larger structurally stable polymer carriers. In the current study, we chose poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) as the polyelectrolytes to LbL assemble the carrier capsules, and 50 nm zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes as the cargo (Scheme 1). PSS and PAH are known to yield structurally stable, nonaggregated capsules with well-defined properties. In the context of the present study, the nondegradable nature of these capsules is a distinct advantage as this will provide capsules with high structural integrity over an extended period of time for use under conditions where they are expected to act as an artificial cell organelle. We characterized and optimized the adsorption of DOPC liposomes into PSS/PAH films on planar substrates by using quartz crystal microbalance with dissipation monitoring (QCM-D). We also embedded multiple layers of liposomes separated by PSS/PAH layers to create multistrata films. The polyelectrolyte-liposome film assembly on planar substrates was extended to colloidal silica spheres, and following the removal of the silica core, stable and intact capsosomes were (47) Caruso, F. Chem.;Eur. J. 2000, 6, 413–419. (48) Dimitrova, M.; Arntz, Y.; Lavalle, P.; Meyer, F.; Wolf, M.; Schuster, C.; Haikel, Y.; Voegel, J. C.; Ogier, J. Adv. Funct. Mater. 2007, 17, 233–245. (49) Michel, A.; Izquierdo, A.; Decher, G.; Voegel, J. C.; Schaaf, P.; Ball, V. Langmuir 2005, 21, 7854–7859. (50) Volodkin, D.; Arntz, Y.; Schaaf, P.; Moehwald, H.; Voegel, J. C.; Ball, V. Soft Matter 2008, 4, 122–130.
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Article Scheme 1. Schematic Illustration of Capsosome Formationa
a A silica particle (i), which is coated with a number of precursor polyelectrolyte layers (at least one PAH layer) and has a PAH final layer (ii-iv), is exposed to DOPC liposomes (v). Subsequent polyelectrolyte layering can be performed (vi). At least three capping polyelectrolyte layers are added to protect the final layer of liposomes prior to the silica core removal (vii).
obtained. The presence of structurally intact liposomes in the polymer capsules was verified by a combination of confocal laser scanning microscopy and electron microscopy.
were separated from the free dye using a Sephadex column (NAP10, GE Healthcare, USA).
Materials and Methods
were used to determine the adsorption characteristics of PAH and PSS in the presence of liposomes. The silica-coated crystals (QSX 300, Q-sense) were soaked in 2% sodium dodecyl sulfate overnight, washed extensively with water, dried, and placed in a UV cleaner (Bioforce Nanosciences, USA) for 30 min. Immediately after cleaning, the crystals were mounted into the liquidexchange chambers of the instrument, and a baseline in the buffer solution was established. The temperature was kept at 23 ( 0.02 °C throughout all of the experiments. If not otherwise mentioned, a PAH solution (1 mg mL-1, dissolved in HEPES buffer) was used to assemble the initial layer. After the surface was saturated, the polymer solution was replaced with buffer solution. PSS solution (1 mg mL-1, dissolved in HEPES buffer) was then injected into the chambers for incubation. When the surface was saturated, the chambers were washed with buffer solution. Polyelectrolyte layering was continued by the alternate adsorption of PAH and PSS, until the required number of layers was obtained. Then, DOPC liposomes (0.8 mg mL-1, dissolved in HEPES buffer) were added to the chambers and left to incubate until the surface was saturated. The liposome solution was replaced with buffer solution, and the polyelectrolyte layering was continued, as described above. Normalized frequencies using the third overtone are presented. Capsosome Formation. The polyelectrolyte coatings with embedded liposomes on microparticles were assembled via the LbL technique. Silica particles (3 μm, 5 (w/w)%) were washed (1060 g for 30 s) three times in the buffer solution prior to adsorption of the first polyelectrolyte. The particles were suspended in the PAH solution (1 mg mL-1) and left for 15 min. All adsorption steps were performed with constant shaking. The particles were washed three times in the HEPES buffer and then suspended in the PSS solution, left for 15 min, and washed three
Materials. Poly(allylamine hydrochloride) (70 kDa, PAH), poly(styrene sulfonate) (70 kDa, PSS), sodium chloride (NaCl), sodium hydroxide (NaOH), fluorescein, and 4-(2hydroxyethyl)piperazine-1-ethane-sulfonic acid (HEPES) were purchased from Sigma-Aldrich. Silica particles (3 μm) were obtained from Microparticles GmbH, Germany. Zwitterionic lipids, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, phase transition temperature -4 °C), and fluorescent lipids 1-oleoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino] hexanoyl]-sn-glycero-3 phosphocholine (NBD-PC), were purchased from Avanti Polar Lipids, USA. Stock solutions of the lipids (50 mg mL-1) were prepared in chloroform and stored in the freezer. All measurements were carried out in a buffered solution consisting of 10 mM HEPES and 150 mM NaCl (HEPES buffer), with the exception of the electrophoresis experiments, where the NaCl component was omitted. The pH of the HEPES buffer solution was adjusted to pH 7.4 with 3 M NaOH. The buffer solution was made with ultrapure water (Milli-Q gradient A 10 system, resistance 18 MΩ cm, TOC < 4 ppb, Millipore Corporation, USA). Unilaminar liposomes (stored at 4 °C) were prepared by evaporation of the chloroform under nitrogen (1 h), followed by hydration in HEPES buffer (2.5 mg mL-1), and extrusion through 50 nm filters (31 times) to obtain a monodisperse liposome dispersion, as monitored by dynamic light scattering (Malvern Zetasizer). For fluorescent liposomes, 1%(w/w) of NBD-PC was added to the lipid solution. Fluorescein-loaded liposomes were obtained by adding fluorescein to the buffer solution prior to the lipid hydration, and the loaded liposomes Langmuir 2009, 25(12), 6725–6732
Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). QCM-D measurements (Q-sense E4, Sweden)
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times. These two steps were repeated until the required number of polyelectrolyte layers was deposited. The precoated particles were incubated in a liposome solution (0.8 mg mL-1) for 1 h, followed by three washing steps. Then, the previously described polyelectrolyte-based layering was continued, commencing with a layer of PSS after the liposomes, until the required number of layers was achieved, or a second layer of liposomes was adsorbed. After each liposome layer, at least three layers of polyelectrolyte were deposited. The terminating layer was always PSS before the template core was removed. Capsosomes were formed by dissolving the silica core using a 2 M HF/8 M NH4F solution for 3 min, followed by multiple centrifugation (4500g for 5 min)/water washing cycles. Microelectrophoresis. The ζ-potentials of coated particles were measured in a 10 mM HEPES buffer solution (pH 7.4) using a Malvern Zetasizer 4 by taking the average of five successive measurements. Flow Cytometer. A Becton Dickson FACS Calibur flow cytometer using an excitation wavelength of 488 nm was used for all of the flow cytometry experiments. At least 20,000 particles were analyzed in each experiment. Confocal Laser Scanning Microscopy (CLSM). A Leica TCS SP2 AOBS confocal microscope equipped with an argon laser (488 nm), the corresponding filter sets, and a 60 oil immersion objective (Leica, Germany) was used for imaging the fluorescently labeled capsosomes. Transmission Electron Microscopy (TEM). All TEM grids were plasma treated for 10 s prior to use. Five microliters of sample was adsorbed onto a carbon-coated Formvar film mounted on 300 mesh copper grids (ProSciTech, Australia) for 5 min. The grids were blotted and dipped into a 1.5% aqueous uranyl acetate solution for 15 s for negative staining. Caution: Uranyl acetate is toxic and radioactive and should be handled with care. Samples for cryo-transmission electron microscopy (cryoEM) were adsorbed to R3.5/1 holey carbon films mounted on copper grids (Quantifoil, Jena, Germany). After an adsorption time of 1 min, the grids were immediately frozen in liquid ethane using a Vitrobot automated plunging device (FEI Company, Eindhoven, The Netherlands). Specimens for cryo-EM were loaded into a Gatan 626 cryoholder for imaging. Investigations were undertaken using an FEI Company Tecnai TF30 (FEI-Company, Eindhoven, The Netherlands) operated at 200 kV and fitted with a Gatan US1000 2k 2k CCD Camera (Pleasanton, Ca, USA). The cryo-sample temperature was maintained in the microscope at approximately -180 °C. Micrographs were recorded under lowdose conditions at a nominal magnification of 31,000 and at ∼2.5 μm underfocus.
Results and Discussion To develop a reliable process to generate capsosomes, we examined the fundamental steps involved in their assembly. The incorporation of structurally intact 50 nm DOPC liposomes into a polyelectrolyte multilayer film of PAH and PSS was first characterized on planar silica surfaces by QCM-D. The film assembly containing intact liposomes was subsequently performed on colloidal supports (3 μm silica particles), and the findings from the planar and colloidal surfaces were correlated. Finally, structurally stable capsosomes were obtained upon removal of the silica core particles and imaged by CLSM. The presence of structurally intact liposomes within the polymer shell was confirmed using electron microscopy. Layer Assembly on Planar Silica Surfaces. For the polyelectrolyte-liposome film assembly on planar surfaces, we sought to optimize the number of liposomes embedded within the polyelectrolyte multilayer film and to examine the effect of the number of precursor PAH/PSS bilayers on the amount of adsorbed liposomes. This would allow control over the liposome 6728 DOI: 10.1021/la900213a
Figure 1. (a) QCM-D frequency changes upon binding of a first layer of DOPC liposomes as a function of the number of precursor bilayers of PAH and PSS. (b) QCM-D frequency changes for PSS/PAH film growth as a function of surface composition. PSS/ PAH layer build up on PAH-primed (-0-) silica crystals is compared with film growth on SLB-coated (-O-) crystals and the layering on crystals coated with a layer of structurally intact liposomes (-9-).
loading amount within the polyelectrolyte film. The assembly of PAH/PSS in the presence of intact liposomes was monitored using QCM-D. Figure 1a) compares the frequency change of the QCM crystal upon the addition of liposomes as a function of the number of precursor PAH/PSS bilayers. As expected, DOPC liposomes fused into supported lipid bilayers (SLBs) when they were directly adsorbed onto silica-coated crystals.51 The formation of a liposome layer could be observed when a precursor layer of PAH was first adsorbed onto the silica-coated crystal. The changes in frequency (-200 ( 36 Hz) and dissipation (17 ( 6 10-6) were similar to those measured when a layer of 50 nm liposomes is adsorbed onto Au-coated crystals,51 suggesting a high amount of adsorbed structurally intact liposome. The measured frequency change of the crystal upon the addition of liposomes to a PAH-terminated multilayer was not affected by the number of precursor layers of PAH and PSS. Thus, equivalent amounts of liposomes were adsorbed on the polyelectrolyte films, and no measurable penetration of the liposomes into the polyelectrolyte films is likely to have occurred, thus allowing for the precise positioning of the liposomes within the polyelectrolyte film. In good agreement with the literature,52 less than 10% of a liposome layer was adsorbed if the polyelectrolyte film terminated with a layer of PSS. Understanding the effect of the presence of liposomes, large zwitterionic soft objects, on polymer film assembly is crucial in order to gain control over the design of capsosomes. Therefore, the polyelectrolyte film assembly on bare silica was compared to the growth on a substrate precoated with an SLB and a surface precoated with intact liposomes. Figure 1b compares the layer(51) Reimhult, E.; Hook, F.; Kasemo, B. Langmuir 2003, 19, 1681–1691. (52) Kohli, N.; Vaidya, S.; Ofoli, R. Y.; Worden, R. M.; Lee, I. J. Colloid Interface Sci. 2006, 301, 461–469.
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ing characteristic of PSS and PAH as a function of the composition of the preadsorbed layers. The linear film build up on silica crystals with a PAH precursor yielded ΔF = -12 ( 4 Hz and ΔD = 1.1 ( 1.4 10-6 for a PSS layer and ΔF = -6 ( 2 Hz and ΔD = 0.6 ( 0.4 10-6 for a PAH layer (Figure 1b, -0-). A silica QCM crystal coated with an SLB of DOPC did not show any frequency change for the adsorption of the first layer of PSS. However, after immobilizing PAH, linear film growth was observed (PSS, ΔF = -26 ( 3 Hz and ΔD = 1.1 ( 0.5 10-6; PAH, ΔF = -17 ( 3 Hz and ΔD = 2.3 ( 1 10-6; Figure 1b, -O-). Linear film growth was also monitored for crystals precoated with a layer of PAH and a layer of intact liposomes (PSS, ΔF = -65 ( 11 Hz and ΔD = 11.1 ( 4.3 10-6; PAH, ΔF = -11 ( 4 Hz and ΔD = 7.5 ( 2.7 10-6; Figure 1b, -9-). Here, the linear film growth commenced with the first layer of PSS, suggesting that the PSS did not only bind to the liposomes but also to the underlying PAH layer. The next layer of PAH was expected to bind to both the liposomes and the PSS, leading to stable film growth and the incorporation of the liposomes into the layers. The larger film growth on SLB-coated crystals compared to bare surfaces can be attributed to the more pronounced binding of the first PAH layer, leading to an overall larger total change in frequency after three polyelectrolyte bilayers. The presence of intact liposomes did not hinder polyelectrolyte film assembly, but the larger surface area (1.6) of the liposome-coated crystals (assuming a layer of half-spheres) compared to a bare silica surface contributed to the even larger polymer film growth in that case. The large dissipation value further suggests that not only a thicker but also a well hydrated and less dense film was built up on top of structurally intact liposomes. To develop systems where the liposomes are stably entrapped in the polyelectrolyte film and to increase the liposome loading, we examined the formation of multistrata assemblies by embedding a second layer of liposomes into the polyelectrolyte film. This approach allows precise control over the film structure and composition. Figure 2a shows QCM-D data for the growth of a PSS/PAH film with two layers of 50 nm DOPC liposomes. The frequency change (ΔF ) and dissipation change (ΔD) of a silicacoated crystal upon the addition of a precursor layer of PAH were -4 ( 1 Hz and 0.6 ( 0.5 10-6, respectively. A layer of liposomes was then adsorbed, followed by four separation bilayers of PSS and PAH. A second layer of liposomes was immobilized, followed by three capping layers of alternating PSS and PAH. The regular and systematic decrease in QCM frequency observed upon subsequent polyelectrolyte adsorption suggests no significant displacement or rupturing of either the first or second layer of liposomes. In order to design a strata-like film consisting of alternating polyelectrolyte and liposomes layers, the effect of the number of polyelectrolyte separation layers on liposome adsorption was examined. Figure 2b shows the frequency change of a primed crystal upon the adsorption of a second layer of DOPC liposomes. A layer of PAH, DOPC, and a variable number of separation layers of PSS/PAH were used to prime the crystals. The measured changes in frequency (and dissipation) upon binding of the liposomes to PAH increased with an increasing number of polyelectrolyte layers that separated the first layer from the second layer of intact liposomes. Extensive penetration of the liposomes into the multilayer film is unlikely since only little binding of the liposomes to the PSS layer was observed (ΔF = -12 Hz and ΔD = 1 10-6). Furthermore, the liposome binding to the surface did not increase linearly with increasing number of separation layers over the range (1-8 bilayers) Langmuir 2009, 25(12), 6725–6732
Article
Figure 2. (a) Frequency and dissipation changes upon adsorption of a PAH precursor layer, a layer of DOPC liposomes followed by four separation bilayers of PSS and PAH, a second layer of liposomes, and three capping layers of PSS and PAH. (b) QCMD frequency changes upon adsorption of a second layer of liposomes. The silica crystals were primed with a precursor layer of PAH, a layer of liposomes, and a variable number of PSS/PAH separation bilayers.
studied. As a result, adsorption onto the film is more likely than penetration into the film. The measured ∼50% larger ΔF of the QCM crystal upon the binding of liposomes after eight separation bilayers compared to the binding of liposomes after the precursor layers is likely due to a larger available surface area and a more structured and flexible outermost layer, enabling more liposomes to be immobilized. The ability to control the amount of adsorbed liposomes by changing the number of separation layers is a convenient way to tailor the number of embedded liposomes in the multilayer film. Capsosome Formation. The film assembly on colloidal particles and the subsequent formation of capsules containing structurally intact liposomes was investigated using flow cytometry, CLSM, TEM, and cryo-EM. The combination of this multitechnique study on the colloids in addition to the previously discussed results on planar substrates was used to confirm the presence of intact liposomes in the polyelectrolyte capsules. First, the polyelectrolyte assembly on colloidal substrates precoated with a layer of liposomes was characterized. Figure 3a shows the LbL growth of PAH, liposomes, and (PSS/PAH) bilayers on particles, monitored stepwise by microelectrophoresis to determine the ζ-potentials of the colloids. The initial ζ potential of -75 ( 5 mV for the 3 μm silica particles became 9 ( 23 mV after the initial layer of PAH was adsorbed. Although this value is lower than what is usually measured for PAH (20 mV53), the adsorbed amount of polyelectrolyte was sufficient to prevent the liposomes from rupturing. Furthermore, the amount of adsorbed liposomes was found to be independent of the number of precursor layers, as long as the final layer was PAH. The binding of the zwitterionic liposomes led to a ζ-potential of -7 ( 4 mV. The subsequent alternating (53) Kato, N.; Schuetz, P.; Fery, A.; Caruso, F. Macromolecules 2002, 35, 9780–9787.
DOI: 10.1021/la900213a
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Figure 4. (a) CLSM image and (b) negative stained TEM image of (PAH/liposomesNBD/(PSS/PAH)4/PSS) capsosomes. The black arrows identify structurally intact liposomes, while the white arrows indicate areas where the liposomes have been displaced. The inset shows a complete capsosome.
Figure 3. (a) ζ-potential of PAH/PSS-coated silica particles containing one layer of liposomes. The silica core was coated with a precursor layer of PAH, followed by the DOPC liposomes, and the subsequent layering of PSS and PAH. (b) Fluorescence intensity increase after liposomeNBD adsorption onto particles with one precursor layer of PAH (-b-) or nine precursor PAH/PSS layers (-O-), including the fluorescence evolution during the subsequent layering, as measured by flow cytometry. (c) Fluorescence intensity measured after the liposomeNBD adsorption as a function of the number of precursor polyelectrolyte layers.
negative and positive ζ-potentials correspond to PSS and PAH forming the outer layer of the coated particles, respectively. Such alternating reversals in the sign of the ζ-potential are characteristic of the LbL formation of polyelectrolyte multilayers on particles,53 suggesting stepwise layer growth of PSS and PAH, even in the presence of liposomes. The negative and positive ζ-potential difference increased with increasing number of polyelectrolyte layers adsorbed after the liposomes, indicating a diminishing effect of the incorporated liposomes on layering characteristics. The loading capacity of liposomes onto colloids was characterized by the adsorption of NBD-labeled 50 nm DOPC liposomes (liposomesNBD) onto 3 μm silica particles precoated with a different number of precursor layers using flow 6730 DOI: 10.1021/la900213a
cytometry. Figure 3b shows fluorescence data for the layering of polyelectrolytes and liposomesNBD onto particles measured by flow cytometry. A similar increase in intensity was measured, irrespective of whether the liposomesNBD were adsorbed after one precursor layer of PAH (-b-) or after nine precursor layers of PAH/PSS (-O-). In good agreement with the results obtained on planar substrates, a similar amount of liposomes can be added to different thicknesses of the polyelectrolyte film. This flow cytometry data confirmed the presence of the labeled lipids associated with the colloids; however, only the combination with TEM images (see Figures 4 and 5) verifies the presence of structurally intact liposomes. The effect of the number of PAH/PSS precursor layers on the anchoring stability of the liposomes during subsequent polyelectrolyte film assembly was investigated by flow cytometry. A loss of almost 50% in fluorescence intensity was monitored during subsequent polyelectrolyte layering if the liposomesNBD were bound to particles primed with only one precursor layer of PAH (Figure 3b, -b-). This loss was likely caused by displacement and not rupturing of the liposomes since liposome fusion is not expected to affect the measured fluorescent intensity. After the loss of the liposomesNBD during the first polyelectrolyte adsorption step, the changes in the fluorescence intensity measured by flow cytometry remained within experimental error during the subsequent layering and washing, pointing to the incorporation of the remaining intact liposomes into the polyelectrolyte shell. A larger number of precursor polyelectrolyte bilayers reduced the amount of liposomesNBD loss to less than 10% following the deposition of subsequent layers (Figure 3b, -O-), most likely because the underlying polymer film better stabilized the binding between the liposomes and the polyelectrolyte layers. In order to maximize the loading capacity of liposomes onto colloids, the fluorescence intensities of the particles were Langmuir 2009, 25(12), 6725–6732
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Figure 5. (a) CLSM image of capsosomes consisting of (PAH/ PSS)4/PAH/liposomesNBD/PSS/PAH/PSS. (b) A TEM image of a negative stained (PAH/PSS)4/PAH/liposomesNBD/PSS/PAH/PSS capsosome (inset) shows indented structurally intact liposomes and the laminar structure of collapsed liposomes in the polyelectrolyte shell. (c) A cryo-EM image of a (PAH/PSS)4/PAH/liposomesNBD/ PSS/PAH/PSS capsosome embedded in ice (inset) and a close-up of the polyelectrolyte shell, which contains intact liposomes. The liposome membranes are visible as black circles.
measured by flow cytometry after the adsorption of liposomesNBD onto silica particles precoated with a different number of precursor PAH and PSS layers (Figure 3c). The number of precursor polyelectrolyte layers did not affect the intensity measured after the immobilization of liposomesNBD, as long as PAH was the final layer. This indicates that a layer of liposomes was adsorbed at a predefined position in the polyelectrolyte film and that no measurable penetration into the polyelectrolyte film occurred. In contrast, only a low amount of liposomeNBD bound when PSS was the outermost layer. Both of these observations are in good agreement with measurements on planar surfaces using QCM-D (Figure 1a). Langmuir 2009, 25(12), 6725–6732
Article
The structural integrity of the capsosomes and the presence of intact liposomes within the polyelectrolyte shell after template core removal were investigated using CLSM and TEM, respectively. Silica particles coated with PAH/liposomesNBD/(PSS/ PAH)4/PSS or (PAH/PSS)4/PAH/liposomesNBD/PSS/PAH/ PSS were used to obtain capsosomes by dissolving the silica cores, as shown in Figures 4 and 5, respectively. In both cases, the CLSM images in Figures 4a and 5a confirmed the structural integrity of the capsosomes. Although the individual liposomesNBD cannot be resolved with CLSM, the homogeneous green fluorescence intensity suggests a homogeneous layering of the liposomesNBD within the polymer shell. TEM images of negative stained capsosomes confirmed that intact liposomes were present in the polyelectrolyte shell (black arrows in Figures 4b and 5b). In addition, in the case of the PAH/ liposomesNBD/(PSS/PAH)4/PSS capsosome, ∼50 nm holes in the shell (white arrow in Figure 4b) were also observed, indicating that the liposomes were displaced during polyelectrolyte layering, as previously observed by flow cytometry (Figure 3b). Nonetheless, an intact capsosome with an inhomogeneous polyelectrolyte shell was obtained (inset in Figure 4b) when the liposomes were adsorbed onto only one precursor layer of PAH. In contrast, for the (PAH/PSS)4/PAH/liposomesNBD/ PSS/PAH/PSS capsosomes (Figure 5b), TEM images revealed intact capsosomes, indicating that the liposomes remain intact during subsequent polyelectrolyte layering. The absence of holes in the shell is in good agreement with results obtained by flow cytometry (Figure 3b), which suggests negligible liposome displacement during polyelectrolyte layering. A close-up of the polyelectrolyte shell of a negative stained capsosome again shows the lamellar structures and the indented spheres representative of intact liposomes (Figure 5b). A capsosome ((PAH/ PSS)4/PAH/liposomesNBD/PSS/PAH/PSS) embedded in ice and a close-up of the shell imaged via cryo-EM is shown in Figure 5c. The closed black circles in this image confirm the presence of structurally intact liposomes embedded into the polymer shell even after the LbL process and the silica core removal using 2 M HF/8 M NH4F. Although in the current article we do not examine the chemical properties of the lipids after being exposed to 2 M HF/8 M NH4F, our preliminary results using fluoresceinloaded DOPC liposomes suggest that the capsosomes retain the dye molecules (Supporting Information, Figure S1). However, a more detailed study is required to understand the loading efficiency and cargo retention in the liposomes, and to demonstrate that the liposomes are sealed to low molecular weight molecules. Capsosomes represent an opportunity to incorporate intact liposomes in defined regions of a polyelectrolyte shell as a novel approach for multiple drug delivery or as an artificial cell. Incorporating more than just one layer of liposomes into the polyelectrolyte shell would further increase the loading capability of liposomes into the polymer film and subsequently give access to vehicles with a multistrata membrane. Figure 6a represents the normalized fluorescence intensity as measured by flow cytometry upon binding of the second layer of liposomes as a function of the number of separation layers between the two liposome layers. The adsorption of the first layer of liposomes to a PAH precoated silica particle was assumed to be a monolayer. In order to facilitate comparison between the two layers of liposomes, the measured averaged fluorescence intensity (333 ( 41 au) due to the first liposomeNBD immobilization was used to normalize the change in fluorescence signal after the binding of the second layer of liposomesNBD. Increasing the number of PSS/PAH separation bilayers from one to five increased the DOI: 10.1021/la900213a
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normalized fluorescence intensity caused by the liposomesNBD from 50% up to almost 100%, suggesting that a second liposome layer was adsorbed with a density similar to that of the first layer. Three PSS/PAH separation bilayers diminished the effect of the first layer of liposomesNBD. Hence, the fluorescence signal due to the liposomesNBD adsorption became independent of the number of PSS/PAH separation bilayers (leveling off of the curve in Figure 6a). In good agreement with the QCM-D results (Figure 2b), no measurable penetration of the liposomes into the film was observed. The CLSM image in Figure 6b shows the structural integrity of these capsosomes after core removal. The homogeneous green fluorescence indicates that the liposomesNBD in the first layer (on PAH precoated 3 μm silica particles) and the second layer (separated from the first layer by four polyelectrolyte bilayers) were equally distributed in the shell. Negative stained capsosomes imaged by TEM contain many small dark spots in the shell (Figure 6c, inset), which represent intact liposomes. A close-up of the polyelectrolyte shell shows the presence of many intact (indented or collapsed) liposomes (Figure 6c, indicated by white arrows).
Conclusions We have demonstrated the formation of capsosomes through subcompartmentalizing polyelectrolyte capsules by incorporating structurally intact liposomes. Experiments on planar and colloidal substrates showed that the amount of adsorbed, intact 50 nm DOPC liposomes was independent of the number of preadsorbed layers of PAH and PSS when the terminating layer was PAH, indicating the stable and permanent incorporation of liposomes into the polyelectrolyte film. Only negligible liposome binding to PSS was observed. The displacement of the liposomes due to subsequent polyelectrolyte layering was negligible on planar surfaces while on colloidal substrates, the liposome loss decreased with an increasing number of precursor polyelectrolyte layers. Liposome rupturing was only observed upon adsorption onto bare silica surfaces but not when adsorbed onto polyelectrolyte multilayers. A second layer of liposomes could be incorporated, and the amount of adsorbed liposomes increased with increasing number of polyelectrolyte separation layers between the two liposomes layers. Structurally stable capsosomes were observed by CLSM upon the removal of the silica template. In addition, TEM images of the capsosomes verified the presence of structurally intact liposomes. These novel subcompartmentalized microcontainers provide an extension of polyelectrolyte capsules for the creation of multiresponsive microreactors or drug carriers. In particular, the performance of enzymatic cascade reactions within a confined environment, which is the prerequisite for an artificial cell, should be feasible with such systems.
Figure 6. (a) Normalized fluorescence intensity of coated particles after adsorption of the second layer of liposomesNBD as a function of the number of separation layers between the first and the second layer of liposomes. (b) CLSM image of capsosomes containing two layers of liposomes (PAH/liposomesNBD/(PSS/PAH)4/liposomesNBD/PSS/PAH/PSS). (c) A TEM image of a negative stained capsosome (PAH/liposomesNBD/(PSS/PAH)4/liposomesNBD/PSS/ PAH/PSS) (inset) and a close-up of the polymer shell are shown. The arrows indicate examples of structurally intact 50 nm liposomes embedded in the shell.
6732 DOI: 10.1021/la900213a
Acknowledgment. This work was supported by the Swiss National Science Foundation (SNF, PBEZB-118906) as well as the Australian Research Council under the Federation Fellowship and Discovery Project schemes. Supporting Information Available: Fluorescence data on the buildup of multilayers and fluorescein-loaded DOPC liposomes on particles, and differential interference and fluorescence microsopy images of capsosomes cotaining fluorescein-loaded DOPC liposomes. This material is available free of charge via the Internet at http://pubs.acs.org.
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