Freezing of Binary Colloidal Systems for the Formation of Hierarchy

Synopsis. Cryo-etch scanning electron microscopy (cryo-etch SEM) of binary colloidal gels composed of colloidal silica nanoparticles in the 1−40 nm ...
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Chem. Mater. 2006, 18, 554-559

Freezing of Binary Colloidal Systems for the Formation of Hierarchy Assemblies Maria L. Ferrer,† Rocio Esquembre,‡ Ilida Ortega,† C. Reyes Mateo,‡ and Francisco del Monte*,† Instituto de Ciencia de Materiales de Madrid-ICMM, Consejo Superior de InVestigaciones Cientificas-CSIC, Campus de Cantoblanco, 28049 Madrid, Spain, and Instituto de Biologı´a Molecular y Celular, UniVersidad Miguel Herna´ ndez, Elche, 03202 Alicante, Spain ReceiVed September 16, 2005. ReVised Manuscript ReceiVed NoVember 7, 2005

Cryo-etch scanning electron microscopy (cryo-etch SEM) of aqueous gels composed of colloidal silica nanoparticles in the 1-40 nm range and liposomes of ∼200 nm gave unique morphologies. The aqueous gels are frozen at subcooled liquid nitrogen and fractured to obtain a fresh surface. High-vacuum sublimation of ice from the freshly exposed surface (etching) results in the formation of a hierarchy assembly, characterized by granular fences composed of colloidal silica and liposomes surrounded by empty areas in which amorphous ice originally resided. The biocompatible character of this ice segregation induced self-assembly (ISISA) process that allows for the preservation of the structural integrity of liposomes within the assembly is demonstrated by fluorescence anisotropy performed at the binary colloidal aqueous gels and differential scanning calorimetry and electron microscopy at the hierarchy assembly. The resulting assembly shows an interesting dual character, with one colloidal entity supporting the structure (e.g., silica) and the other providing functionality (e.g., liposomes).

Introduction Liposomes are spherical closed vesicles of phospholipid bilayers with an entrapped aqueous phase, and may consist of one (LUV) or more (MLV) bilayers ranging in size from 20 to 500 nm, and occasionally as large as 5 µm.1 Liposomes as models of living cells present an enormous opportunity for incorporating cellular properties into these artificial vesicles and hence the idea of developing liposomes for biosensors and bioelectronic devices.2 Liposomes packed three-dimensionally (3D) like neuron networks in the brain may also be applicable to future biochips.3 For this purpose, it is necessary to develop suitable methods for assembling liposomes into organized arrays or patterns for designing architectures that are capable of performing such biochip functions. Attempts to immobilize patterning liposomes have successfully been done on different surfaces, such as polymers and silicon wafers.4 Several interesting approaches * To whom correspondence should be addressed. Fax: 34 91 3720623. E-mail: [email protected]. † Instituto de Ciencia de Materiales de Madrid-ICMM. ‡ Universidad Miguel Herna ´ ndez.

(1) Lasic, D. D. Liposomes: From Physics to Applications; Elsevier: Amsterdam, 1993. (2) (a) Rangin, M.; Basu, A. J. Am. Chem. Soc. 2004, 126 (16), 50385039. (b) Yoshina-Ishii, C.; Miller, G. P.; Kraft, M. L.; Kool, E. T.; Boxer, S. G. J. Am. Chem. Soc. 2005, 127 (5), 1356-1357. (c) Santos, M.; Roy, B. C.; Goicoechea, H.; Campiglia, A. D.; Mallik, S. J. Am. Chem. Soc. 2004, 126 (34), 10738-10745. (3) (a) Kuhnert, L.; Agladze, K. I.; Krinsky, V. I. Nature 1989, 337, 244247. (b) Patolsky, F.; Lichtenstein, A.; Willner, I. J. Am. Chem. Soc. 2001, 123 (22), 5194-5205. (4) (a) Kim, J. M.; Jung, H. S.; Park, J. W.; Yukimasa, T.; Oka, H.; Lee, H. Y.; Kawai, T. J. Am. Chem. Soc. 2005, 127 (7), 2358-2362. (b) Vermette, P.; Griesser, H. J.; Kambouris, P.; Meagher, L. Biomacromolecules 2004, 5 (4), 1496-1502. (c) Benes, M.; Billy, D.; Benda, A.; Speijer, H.; Hof, M.; Hermens, W. T. Langmuir 2004, 20 (23), 10129-10137.

have also recently been described for the formation of 3D patterned liposomes. Among others, Vermette and coworkers induced liposome aggregation by NeutrAvidinbiotin adsorption to the surfaces of the vesicles,5 Raghavan and co-workers added an associating biopolymer (e.g., chitosan) to surfactant vesicles,6 and Chen and co-workers induced aggregation of zwitterionic oligolamellar liposomes by DNA.7 Meanwhile, Menger and co-workers reported the ability of gemini surfactants to interdigitate and form vesicles, which ultimately rearrange into three-dimensional assemblies at subfreezing temperatures.8 Unfortunately, the absence of further associating molecules limits the stability of the assembly to that of the vesicles, that is, the whole assembly collapses in an irreversible process above the gel transition temperature (Tm), at which vesicles are in the fluid state. Herein, we plan to prepare an aqueous sol composed of colloidal silica and liposomes and subject it to subfreezing temperatures right after gelation. High-vacuum sublimation (etching) of ice from a freshly exposed surface of the freezing gel results in the formation of silica fences surrounding empty areas at which amorphous ice originally resided. With this approach, we attempt to form a hierarchical hybrid assembly through an ice segregation induced self-assembly (ISISA) process, which provides a second level of space organization (e.g., silica fences) on top of that of the vesicles. Cryo-etch SEM has been demonstrated to result in assembly formation (5) Vermette, P.; Taylor, S.; Dunstan, D.; Meagher, L. Langmuir 2002, 18 (2), 505-511. (6) Lee, J.-H.; Gustin, J. P.; Chen, T.; Payne, G. F.; Raghavan, S. R. Langmuir 2005, 21, 26-33. (7) Wu, C.-M.; Chen, H.-L.; Liou, W.; Lin, T.-L.; Jeng, U.-S. Biomacromolecules 2004, 5 (6), 2324-2328. (8) Menger, F. M.; Peresypkin, A. V. J. Am. Chem. Soc. 2003, 125, 53405345.

10.1021/cm052087z CCC: $33.50 © 2006 American Chemical Society Published on Web 12/14/2005

Freezing of Binary Colloidal Systems To Form Hierarchy Assemblies

from saline and surfactant solutions.9 Meanwhile, aqueous silica gels have been reported to form microhoneycombs in a similar freezing process (e.g., slowly immersed in liquid nitrogen).10 More intriguing, however, is the application of the ISISA process to a binary colloidal system. Thus, we first studied a model system such as submicrometer silica particles (∼290 nm) dispersed within the aqueous silica gel composed of colloidal particles of 1-40 nm,11 which is binary just in terms of colloidal size, not in composition. The submicrometer silica particles were prepared through a modified procedure of the Sto¨ber method.12 The size of the submicrometer silica particles is thus selected because of its similarity with that of liposomes (290 vs 200 nm). Finally, we studied the assembly formation from binary colloidal aqueous gels composed of stable liposomes and colloidal silica (of ∼200 and 1-40 nm size, respectively). It is worthwhile to note that the full preservation of the liposome structure (e.g., vesicles) below and above Tm would allow for the reversibility of the liposome phase transition, which is required in most liposome applications. Thus, the current work also attempts to study the biocompatible character of the whole ISISA process, from sol to gel to freeze-dried assembly. Sol-gel materials have shown their ability for hosting different organic molecular assemblies (e.g., micelles and proteins, among others) with a full preservation of their molecular organization.13 Actually, surfactant-induced organized organic structures (e.g., micelles) are used as templates for the preparation of nanostructured mesoporous materials.13a However, in regard to the encapsulation of biological entities (e.g., proteins, enzymes, and even whole cells), special care must be taken with the presence of denaturing agents (e.g., alcohol released during hydrolysis and condensation of alkoxide precursors).14 Liposomes are also case sensitive, and the use of alcoholfree sol-gel routes is required in order to preserve the integrity of the phospholipid bilayer during encapsulation.15 In this paper, we use an alcohol-free sol-gel route recently described by our group.14c The liposomes under study are composed of a negatively charged phospholipid such as 1,2dimyristoyl-sn-glycero-3-phosphatidic acid (DMPA), and their stability within the aqueous silica gel is studied by steady-state fluorescence anisotropy. Differential scanning (9) (a) Menger, F. M.; Zhang, H.; Caran, K. L.; Seredyuk, V. A.; Apkarian, R. P. J. Am. Chem. Soc. 2002, 124, 1140-1141. (b) Menger, F. M.; Galloway, A. L.; Chlebowski, M. E.; Apkarian, R. P. J. Am. Chem. Soc. 2004, 126, 5987-5989. (c) Liang, J.; Ma, Y.; Zheng, Y.; Davis, H. T.; Chang, H.-T.; Binder, D.; Abbas, S.; Hsu, F.-L. Langmuir 2001, 17, 6447-6454. (10) Mukai, S. R.; Nishihara, H.; Tamon, H. Chem. Commun. 2004, 874875. (11) Brinker, C. J.; Scherer, G. W. Sol Gel Science; Academic Press: San Diego, 1990. (12) Sto¨ber, W.; Fink, A.; Bohn, E. J. J. Colloid Interface Sci. 1968, 26, 62. (13) (a) Kresge, C. T.; Leonowicz, M. E.; Roth, W. J.; Vartuli, J. C.; Beck, J. S. Nature 1992, 359, 710. (b) Braun, S.; Rappoport, S.; Zusman, R.; Avnir, D.; Ottolenghi, M. Mater. Lett. 1990, 10, 1. (c) Ellerby, L. M.; Nishida, C. R.; Nishida, F.; Yamanaka, S. A.; Dunn, B.; Valentine, J. S.; Zink, J. I. Science 1992, 255, 1113. (14) (a) Gill, I.; Ballesteros, A. J. Am. Chem. Soc. 1998, 120, 8587. (b) Bhathia, R. B.; Brinker, C. F.; Gupta, A. K.; Singh, A. K. Chem. Mater. 2000, 12, 2434. (c) Ferrer, M. L.; del Monte, F.; Levy, D. Chem. Mater. 2002, 14, 3619. (15) Besanger, T.; Zhang, Y.; Brennan, J. D. J. Phys. Chem. B 2002, 106 (41), 10535-10542.

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calorimetry (DSC) and transmission electron microscopy (TEM) are used for study of the structural integrity of liposomes at the hierarchy assembly, after freeze-drying of the binary colloidal aqueous gel. Experimental Section Materials. Tetramethyl orthosilicate (TMOS) and tris-(hydroxymethyl)-aminomethane (Tris) were purchased from Aldrich Chemical Co. The phospholipid 1,2-dimyristoyl-sn-glycero-3phosphatidic acid (DMPA) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). The fluorescence probe 1,6-diphenyl-1,3,5hexatriene (DPH) was purchased from Molecular Probles (Eugene, OR). Water was distilled and deionized. Sample Preparation. Liposome formation. DMPA phospholipid was dissolved in a small amount of chloroform and methanol. The solvent was removed first by evaporation under a dry nitrogen gas stream, and was subsequently kept under a vacuum for 3 h. Multilamellar vesicles (MLVs) were obtained by rehydration of the dried lipid films in the appropriate buffered solution heated above the liposome phase-transition temperature (∼60 °C). Large unilamellar vesicles (LUVs) of 100 and 200 nm mean diameter were obtained by a 20-fold extrusion process of the MLV suspension through a carbonate membrane filter in an Avanti Miniextruder suited at 60 °C.16 Both the concentration of the resulting DMPA solutions and the size of the DMPA liposomes were individually adjusted for fluorescence measurements and assembly formation, e.g., 20 mM and 100 nm, and 120 mM and 200 nm, respectively. DPH was the fluorescence probe used for fluorescence anisotropy measurements. In this case, aliquots of DPH in N,N′-dimethylformamide (1 × 10-3 M) were directly added to the lipid dispersion (20 mM) prior to the extrusion process. The final probe:lipid molar ratio was 1:200. Solutions were incubated for 30 min at well above the phospholipid phase-transition temperature to facilitate the probe incorporation in the liposome, and were then subjected to the extrusion process. The membrane size was 100 nm to minimize light scattering, which distorts anisotropy measurements. Preparation of Submicrometer Silica Particles. Uniform silica particles of different mean diameters (e.g., 290 nm) were prepared by a procedure published by Matijevic and co-workers.17 In short, tetraethyl orthosilicate is hydrolyzed in an alcohol medium in the presence of water and ammonia at 40 °C. Details of the reactant concentrations are given in the Supporting Information. Preparation of Gels for Fluorescence Measurements. Liposomes were encapsulated in pure silica matrixes through the alcohol-free sol-gel route recently described by our group.14c In short, 4.41 mL of TMOS, 2.16 mL of H2O, and 0.06 mL of HCl (0.62 M) were mixed under vigorous stirring at 4 °C in a closed vessel. After 50 min, 1 mL of the resulting sol was mixed with 1 mL of deionized water, and the solution was subjected to rotoevaporation for a weight loss of ∼0.6 g (i.e., 0.6 g is approximately the alcohol mass resulting from alkoxyde hydrolysis). The aqueous sol was mixed with 1 mL of a diluted buffered suspension of DPH doped liposomes (0.2 mM DMPA, 50 mM Tris buffer, 100 mM NaCl, pH 7.5) in a disposable cuvette of poly(methyl methacrylate). Gelation occurs readily after mixing. After gelation, monoliths were wet aged in a Tris buffer (50 mM, pH 7.5) and NaCl (100 mM) solution at 4 °C for up to 15 days. (16) Subbarao, N. K.; MacDonald, R. I.; Takesita, K.; MacDonald, R. C. Biochim. Biophys. Acta 1991, 1063, 147. (17) Hsu, W. P.; Yu, R.; Matijevic, E. J. Colloid Interface Sci. 1993, 156, 56-65.

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Preparation of Gels for Cryo-Etch SEM. Aqueous silica gels were prepared from the aqueous silica sol described above for fluorescence measurements but diluted (e.g., 5 volumes of 200 mM Tris buffer, pH 7.5, was added to 1 volume of the aqueous sol). Binary colloidal aqueous gels composed of submicrometric and nanometric silica particles were prepared by adding 5 volumes of a suspension of submicrometric colloidal silica (∼5 wt %) in Tris buffer (200 mM, pH 7.5) to 1 volume of the aqueous silica sol. Binary colloidal aqueous gels composed of liposomes and silica nanoparticles were prepared by adding 5 volumes of a suspension of liposomes (120 mM, ∼4 wt %) in Tris buffer (50 mM, pH 7.5) to 1 volume of the aqueous silica sol. Sample Characterization. Fluorescence Measurements. Fluorescence anisotropy measurements were carried out in a Cary Eclipse spectrofluorometer (Varian) interfaced with a Peltier cell and fitted with thin film polarizers. The steady-state anisotropy 〈r〉, defined by eq 1 〈r〉 )

IVV - GIVH IVV + 2GIVH

(1)

was obtained as a function of temperature by measuring the vertical and horizontal components of the fluorescence emission with excitation vertical and horizontal to the emission axis. The G factor (G ) IHV/IHH) corrects for the transmissivity bias introduced by the detection system. Samples were excited at 360 nm (slit width 5 nm), and the polarized emission was detected at 430 nm (slit width 5 nm). Cryo-Etch SEM Experiments. Small fragments of the aqueous gels (both silica and binary colloidal gels) are mechanically fixed onto the specimen holder of a cryotransfer system (Oxford CT1500), plunged into subcooled liquid nitrogen, and then transferred to a preparation unit via an air lock transfer device. The frozen specimens are cryofractured and transferred directly via a second air lock to the microscope cold stage, where they are etched for 2 min at -90 °C. After ice sublimation and in the preparation unit, the etched surfaces are sputter coated with gold for 10 min at a sputter current of 10 mV. The thickness of the resulting gold film is within the 5-10 nm range, which allows for the undistorted observation of the liposomes and silica spheres with diameters of ca. 200 nm. Samples are subsequently transferred onto the cold stage of the scanning electron microscope chamber. Fractured surfaces are observed with a DSM 960 Zeiss scanning electron microscope at -135 °C under the following conditions: acceleration potential, 15 kV; working distance, 10 mm; and probe current, 5-10 nA. Differential Scanning Temperature Measurements. Thermotropic phase transition parameters of DMPA LUVs were measured by a Seiko 220CU calorimeter. Freeze-dried and rehydrated (50 mM Tris buffer) DMPA-silica colloidal binary assemblies were loaded on hermetically sealed aluminum pans. DSC scans consisted of three heating/cooling cycles in a temperature range between 10 and 70 °C at a scan rate of 5 °C/min. The data from the first scan were always disregarded in order to avoid mixing artifacts.18 Experiments were carried out in triplicate.

Results and Discussion As mentioned in the Introduction, the use of alcohol-free sol-gel routes is required for preserving the integrity of the phospholipid bilayer during encapsulation in aqueous silica gels.14 However, long-term storage of encapsulated DPPC (18) Castillo, J. A.; Pinazo, A.; Carilla, J.; Infante, M. R.; Alsina, M. A.; Haro, I.; Clape´s, P. Langmuir 2004, 20, 3379-3387.

liposomes also results in a reduction of the natural dynamic motions of the bilayers. Such a poor viability was attributed to the physical constraint exerted on the bilayer by the characteristic shrinkage of the sol-gel silica matrixes during the aging process.15,19 Moreover, and as reported for living cells,20 lack of viability could also be due to liposome interaction with the strongly acidic silanol groups located at the porous surface of the host matrix. Herein, we plan to use a negatively charged phospholipid (e.g., DMPA) to minimize the chemical interactions established between the outer layer of the liposomes and the silanol groups. Furthermore, the sol-gel process involved in liposome encapsulation has always been performed at temperatures well above the phase-transition temperature of DMPA (∼50 °C),21 to promote the matrix growing around the liposomes in their fluid state, at which the liposome volume is at a maximum.22 Under these circumstances, one could expect to minimize constraining effects exerted by the host matrix on the liposome structure. After gelation, the encapsulated liposomes were cooled to the gel state (e.g., below Tm) and studied by steady-state fluorescence anisotropy measurements. For this purpose, a fluorescence probe such as diphenylhexatriene (DPH) was incorporated into the bilayer membrane of the liposomes. This fluorescence technique resembles the natural dynamic motions of the bilayer and is a common tool used for the study of Tm.23 Phospholipids undergo a characteristic gelliquid crystalline phase transition at a temperature range that, in principle, is a function of the molecular structure, e.g., Tm largely differs between liposomes with acyl chains, branched chains, or those carrying bulky side groups. Nonetheless, Tm also depends on the environment surrounding the liposomes. For example, the Tm of phosphatidic acidbased liposomes (e.g., DMPA) shifts as soon as the pH differs from neutral as a consequence of electrostatic repulsion that pushes the headgroups apart.24 Thus, Tm is considered to be a liposome fingerprint, and its determination through fluorescence anisotropy provides accurate information regarding the structural integrity of the lipidic bilayer. Figure 1 shows the changes in DPH anisotropy for free and encapsulated DMPA LUVs 1 day after encapsulation. The reversibility of the phase transitions confirms that the changes in anisotropy are related to phase transitions and not to a rupturing of the encapsulated liposomes. The shape of the phase transition is a perfect sinusoidal curve, which is indicative of the preservation of the integrity of the liposome structure. Moreover, it is worthwhile to note the cooperative nature of the process, as it denotes the sharpness (19) Sen, P.; Mukherjee, S.; Patra, A.; Bhattacharyya, K. J. Phys. Chem. B 2005, 109, 3319-3323. (20) (a) Nassif, N.; Bouvet, O.; Rager, M. N.; Roux, C.; Coradin, T.; Livage, J. Nat. Mater. 2002, 1, 42. (b) Ferrer, M. L.; Yuste, L.; Rojo, F.; del Monte, F. Chem. Mater. 2003, 15, 3614-3618. (21) See http://www.avantilipids.com/ (22) Wilkinson, D. A.; Nagle, J. F. Anal. Biochem. 1978, 84, 263-271. (23) (a) Duportail, G.; Brochon, J. C.; Lianos, P. Biophys. Chem. 1993, 45 (3), 227-234. (b) Hutterer, R.; Schneider, F. W.; Sprinz, H.; Hof, M. Biophys. Chem. 1996, 61 (2-3), 151-160. (c) Castanho, M.; Prieto, M. Biophys. J. 1995, 69 (1), 155-168. (24) Betageri, G. V.; Kulkarni, S. B. Microspheres, Microcapsules and Liposomes. Volume 1: Preparation and Chemical Applications; Arshady, R., Ed.; Citus Books: London, 1999; Chapter 18.

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of the transition. The SEM micrograph shown in Figure 2 reveals well-rounded liposomes homogeneously dispersed within the aqueous silica gel. Note that the liposome size is ∼100 nm, ensuring the absence of light scattering in fluorescence measurements. Meanwhile, the Tm found for encapsulated DMPA LUVs is slightly shifted to a higher temperature than that found in solution (54 versus 48 °C, respectively). High Tm values are indicative of an increase in ordering in micelle and vesicle structures; for ionic surfactants in solution, the increase occurs in the presence of electrolytes as a result of the reduction of the electrical surface potential of headgroups, which allows for a further packaging of the whole structure.25 Sol-gel silica matrixes have shown their ability to act as electrolytes for ionic micelles.26 Thus, in the current case, Tm shifts must be ascribed to the nature of the chemical interactions established between the outer layer of the bilayer structure of DMPA liposomes and the silanol groups placed at the porous surface

of the host matrix. Both species are negatively charged, and as mentioned above,26 electrostatic repulsion ultimately determines the further packaging of the whole lipid bilayer. It is worthwhile to note that encapsulation causes no detrimental effect to the bilayer structure of DMPA liposomes, which is preserved for more than 15 days after encapsulation (see the Supporting Information). However, as shown in Figure 2, this aqueous silica gel fails in the formation of colloidal assemblies when subjected to subfreezing temperatures (also see the Supporting Information). The formation of a dense rather than an open structure in the cryo-etch SEM results from the close packing of the colloidal silica particles (∼40 nm in diameter)11 that form the gels. However, as mentioned in the Introduction, silica microhoneycombs have been successfully prepared when aqueous silica gels formed from sodium silicate solutions are subjected to a temperature of 77 K.10 There are several experimental differences in the preparation of the silicate solution that is frozen (e.g., the silica precursor and pH, among others). The freezing process is even different; whereas the silica microhoneycombs are obtained through a unidirectional immersion of fresh aqueous silica gels in liquid nitrogen at a constant speed (e.g., 6 and 20 cm/h), our sample was plunge-frozen in subcooled liquid nitrogen, in a procedure similar to those reported by Menger et al.8,9 The former process promotes the pseudosteady formation of ice crystals of a polygonal shape (normal ice is hexagonal),27 which ultimately, besides having the ability for water to free itself of solutes during freezing, acts as template for the silica assembly. Meanwhile, the quenchfreezing process pursues the formation of amorphous rather than crystalline ice, given that the freezing temperature is well below the glass-transition temperature of water, e.g., Tg ≈ -137 °C. Nonetheless, it is important to note that Tg is not an equilibrium transition temperature and that, being only quasithermodynamic, Tg may vary with experimental conditions.28 One of the most interesting aspects of amorphous ice is its density, which is higher than that of normal crystalline ice (0.934 g/L for amorphous or cubic ice vs 0.917 g/L for hexagonal ice).9a A limited volume increase upon water freezing is ultimately the key that allows for the preservation of liposome structural integrity in the resulting assembly. Otherwise, freezing results in deformed and unstable vesicles.29 Taking into account that the preservation of liposome stability is our main interest, the absence of hexagonal ice is a capital aspect that guided us in the use of this plunge-freezing process. However, we believe that the freezing process does not play such an important role for the formation of the assembly. Actually, the appearance of assemblies obtained for aqueous gels of different chemical compositions (e.g., gels obtained from saline, surfactant, or silicate solutions)8-10 is similar no matter the freezing process. Thus, we thought that the lack of success in the formation of assemblies from our aqueous gel was related to the silica (SiO2) concentration at the starting sol (e.g., 2.25 M, ∼13.5 wt %), which is above

(25) Tandford, C. J. Phys. Chem. 1974, 78, 2469. (26) Ferrer, M. L.; del Monte, F.; Levy, D. J. Phys. Chem. B 2001, 105 (45), 11076-11080.

(27) Lekner, J. Phys. B 1998, 252, 149-159. (28) Williams, E.; Angell, C. A. J. Phys. Chem. 1977, 81, 232-237. (29) Caffrey, M. Biochim. Biophys. Acta 1987, 896, 123-127.

Figure 1. Changes in steady-state anisotropy vs temperature for DPH within DMPA liposomes encapsulated in silica gels, wet aged in a buffered solution (50 mM Tris buffer, 100 mM NaCl, pH 7.5) (open triangle), and freely suspended in an identical buffered solution (open circle).

Figure 2. Cryo-etch SEM of DMPA liposomes (∼100 nm in size) encapsulated in the aqueous silica gel and subjected to subcooled liquid nitrogen (bar is 2 µm). The inset at the upper-right corner of the figure represents a sphere of diameter 100 nm. Arrows indicate some representative liposomes.

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Figure 3. Cryo-etch SEM of a diluted aqueous silica gel subjected to subcooled liquid nitrogen (bar is 20 µm). Inset: detail of silica fence (bar is 2.5 µm).

that typically used in the formation of the above-mentioned assemblies (within the 1-10 wt % range). Thus, assembly formation occurs (Figure 3) when dilution of the aqueous silica sol (∼5-fold) is accomplished, for a final SiO2 concentration of 0.75 M (∼4.5 wt %). Note that water dilution can never be a problem for liposome stability, and the resulting gel must keep being biocompatible. Actually, sol dilution has been described as being of great help for the preservation of the integrity of the cell membrane during sol-gel encapsulation of bacteria.30 At this stage, we are prepared to study the freezing behavior of a binary colloidal system. In our first approach, we chose as a model system the one based on submicrometer silica particles dispersed within the aqueous silica gel. As mentioned above, in this case, the term binary just applies to the size of colloids involved in the assembly formation, e.g., the colloidal silica nanoparticles of 1-40 nm forming the aqueous gel and the submicrometer silica particles of ∼290 nm. Figure 4 shows the assembly that resulted from the quench freezing of such a binary system. The submicrometer particles are clearly observed at the fences, which are also composed of nanometer colloidal particles. Note that the submicrometer particle concentration is below that required to fully fill the fences (i.e., uncompleted sintering is observed between submicrometer particles). This later observation indicates that the assembly is supported mainly by the colloidal silica nanoparticles rather than by the submicrometer silica particles. Following a similar approach, we can now prepare a binary colloidal system composed of DMPA liposomes of ∼200 nm and colloidal silica nanoparticles. Submission at subfreezing temperatures of such a binary system results in the formation of granular fences as a result of liposomes packing within colloidal silica nanoparticles (Figure 5). Note that the (30) Premkumar, J. R.; Lev, O.; Rosen, R.; Belkin, S. AdV. Mater. 2001, 13, 1773.

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Figure 4. Cryo-etch SEM of a binary colloidal system composed of submicrometer particles of 290 nm dispersed within the aqueous silica gel and subjected to subcooled liquid nitrogen (bar is 5 µm). The inset at the upper right corner of the figure represents a sphere of diameter 290 nm.

Figure 5. Cryo-etch SEM of a binary colloidal system composed of DMPA liposomes (∼200 nm in size) dispersed within the aqueous silica gel and subjected to subcooled liquid nitrogen (bar is 5 µm). The inset at the upperright corner of the figure represents a sphere of diameter 200 nm. Arrows indicate some representative liposomes.

granule size is in good concordance with that of the liposomes dispersed within the sol (∼200 nm). The integrity of the liposome bilayer should be preserved in the assembly given the experimental conditions used for its preparation (e.g., the absence of alcohol during the gel formation process as well as of hexagonal ice crystals during the freezing process). Unfortunately, no fluorescence anisotropy data can be collected from these samples because of light scattering. To clarify this issue and as an alternative technique to fluorescence anisotropy, we performed DSC on hierarchy assemblies obtained after application of ISISA to the binary colloidal aqueous gel, i.e., plunge-frozen in liquid

Freezing of Binary Colloidal Systems To Form Hierarchy Assemblies

Figure 6. DSC scan of a vacuum-dried and rehydrated hierarchy assembly composed of colloidal silica and DMPA liposomes.

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resembles the presence of additives in those gels used for fluorescence measurements that are not in the gels used for assembly formation (e.g., NaCl and DPH), i.e., additives, even at very low concentrations, are well-known to alter Tm.32 Actually, the Tm obtained by fluorescence anisotropy in gels without NaCl is also shifted (∼38 °C, see the Supporting Information). Furthermore, the DSC scan of liposomes incorporated at the hierarchy assembly reveals hysteresis effects (the main endothermic peak in the heating run appears at higher temperatures than that in the cooling run (∼45 vs ∼42 °C, respectively), in concordance with previous data reported for liposomes encapsulated within aqueous silica gels.15 Conclusions

Figure 7. TEM micrograph of a vacuum-dried hierarchy assembly composed of colloidal silica and DMPA liposomes (bar is 200 nm).

helium, aged for 4 h in liquid nitrogen, and dried under vacuum to allow for water sublimation (e.g., freeze-drying). The hierarchy assembly is mostly preserved after such a drying process (see the Supporting Information). Prior to taking DSC measurements, we rehydrated dried assemblies to allow liposomes to be in regular conditions for the phase transition.31 The DSC scans of liposomes integrated within the assembly clearly reveal the reversibility in the liposome phase transition, i.e., the endothermic peak at ∼45 °C that appears in the heating run and the exothermic one at ∼42 °C that appears in the cooling run (Figure 6) are maintained for different cycles. These results confirm that the bilayer structure is preserved and maintains its functionality regardless of the liposome integration within the silica colloids at the assembly. Figure 7 shows well-rounded shaped liposomes surrounded by colloidal silica, which indeed corroborates the preservation of the bilayer structural integrity. Under these circumstances and given that the assembly is mainly supported by the silica colloids, liposomes have freedom in membrane fluidity without having detrimental effects on the assembly structural stability. The DSC scans also reveal that Tm is shifted as compared to that found by fluorescence anisotropy. This behavior (31) Miyajima, K. AdV. Drug DeliVery ReV. 1997, 24, 151-159.

Cryo-etch SEM has shown how the application of the ISISA process to binary colloidal aqueous gels results in the formation of hierarchy assemblies at certain colloid weight percentages (e.g., 4.5 wt % colloidal silica nanoparticles and 4.5 wt % submicrometer silica particles or liposomes). We have also demonstrated the biocompatible character of the ISISA process, which allows liposomes to keep their characteristic phase transition reversible and hence their functionality, regardless of their integration within the assembly. It is worthwhile to note that the reversibility of the phase transition causes no detrimental effect on the structural integrity of the hierarchy assembly, which fully relies on the silica fences formed by the colloidal silica nanoparticles. The dual character of the assembly allows for sharing of tasks (one entity supporting the structure, the other providing functionality), which may open the path to promising applications. Eventually, ternary assemblies formed from colloidal silica and liposomes of two different compositions could be prepared. In that case, the individual response of each of the liposomes integrated within the assembly could be individually triggered to two independent stimuli (e.g., two different temperatures),33 which would be of tremendous interest in drug-delivery applications. Acknowledgment. The authors thank CSIC and Instituto de Salud Carlos III for Grants 200460F027 and PI020606, respectively. Fundacio´n Domingo Martı´nez and TPA Inc. are also acknowledged for valuable support. M.L.F. also thanks MEC for a Ramon y Cajal research contract. Prof. Carmen Ascaso and Fernando Pinto are acknowledged for fruitful discussions and assistance with cryo-etch SEM experiments. Prof. Rosa Rojas is acknowledged for helpful discussions with DSC experiments. Supporting Information Available: Experimental details, cryoetch SEM and SEM micrographs, and fluorescence anisotropy curves (pdf). This material is available free of charge via the Internet at http://pubs.acs.org. CM052087Z (32) (a) Black, S. G.; Dixon, G. S. Biochemistry 1981, 20, 6740-6744. (b) Crommelin, D. J. A.; Bommel, E. M. G. Pharm. Res. 1984, 1, 159-163. (33) Chen, W.-H.; Regen, S. L. J. Am. Chem. Soc. 2005, 127 (18), 65386539.