pubs.acs.org/Langmuir © 2009 American Chemical Society
Frozen Cyclohexane-in-Water Emulsion as a Sacrificial Template for the Synthesis of Multilayered Polyelectrolyte Microcapsules Sachin Khapli,*,† Jin Ryoun Kim,†, ‡ Jin Kim Montclare,†, § Rastislav Levicky,†, ‡ Maurizio Porfiri,†, # and Stavroula Sofou†, ‡ † Center for Co-operative Bioactive Systems, ‡Department of Chemical and Biological Engineering, §Department of Chemical and Biological Sciences and #Department of Mechanical and Aerospace Engineering, Polytechnic Institute of New York University (NYU-POLY), 6 Metrotech Center, Brooklyn, New York 11201
Received March 23, 2009. Revised Manuscript Received May 20, 2009 This paper reports the application of frozen cyclohexane-in-water emulsions as sacrificial templates for the fabrication of hollow microcapsules through layer-by-layer assembly of polyelectrolytes, poly(styrenesulfonate sodium salt), and poly(allylamine hydrochloride). Extraction of the cyclohexane phase from frozen emulsions stabilized with 11 polyelectrolyte layers by compatibilization with 30% v/v ethanol leads to the formation of water-filled microcapsules while preserving the spherical geometry. The majority of microcapsules (>90%) are prepared with intact polyelectrolyte membranes as measured by their deformation induced by osmotic pressure. This work provides a new route for the synthesis of hollow multilayered microcapsules under mild operating conditions.
Layer-by-layer (LbL) assembly of polyelectrolytes on dissolvable colloidal templates is a versatile tool for the synthesis of microcapsules. The method was first reported by Donath et al.1,2 and later followed by numerous studies that extended its utility from polymeric microcapsules to a variety of building blocks such as nanoparticles,2 dendrimers,3 and biomolecules including DNA,4 polysaccharides,5 proteins,6 and lipids.7 Microcapsules have been prepared with control over size ranging from 0.1 to 10 μm as well as control over wall thickness from 10 to 100 nm.1,2,8,9 With judicious selection of the building blocks, such as stimuli-responsive biomolecules, these microcapsules can be designed to respond to various physicochemical stimuli, including pH,7,10-12 temperature,13 and ionic strength.13,14 The tailored responsiveness makes these microcapsules ideal candidates for a variety of biomedical applications *Corresponding author (e-mail:
[email protected]). (1) Donath, E.; Sukhorukov, G. B.; Caruso, F.; Davis, S. A.; M€ohwald, H. Angew. Chem., Int. Ed. 1998, 37(16), 2202–2205. (2) Caruso, F.; Caruso, R.; M€ohwald, H. Science 1998, 282, 1111–1114. (3) Kim, B.-S.; Lebedeva, O. V.; Koynov, K.; Gong, H.; Caminade, A.-M.; Majoral, J.-P.; Vinogradova, O. I. Macromolecules 2006, 39, 5479–5483. (4) Wang, Z.; Qian, L.; Wang, Z.; Yang, F.; Yang, X. Colloids Surf. A: Physiochem. Eng. Aspects 2008, 326, 29–36. (5) Yu, L.; Gao, Y.; Yue, X.; Liu, S.; Dai, Z. Langmuir 2008, 24, 13723–13729. (6) An, Z.; Tao, C.; Lu, G.; M€ohwald, H.; Zheng, S.; Cui, Y.; Li, J. Chem. Mater. 2005, 17, 2514–2519. (7) An, Z.; M€ohwald, H.; Li, J. Biomacromolecules 2006, 7, 580–585. (8) Shenoy, D. B.; Antipov, A. A.; Sukhorukov, G. B.; M€ohwald, H. Biomacromolecules 2003, 4, 265–272. (9) Schuler, C.; Caruso, F. Biomacromolecules 2001, 2, 921–926. (10) Mauser, T.; Dejugnat, C.; M€ohwald, H.; Sukhorukov, G. B. Langmuir 2006, 22, 5888–5893. (11) Shutava, T.; Prouty, M.; Kommireddy, D.; Lvov, Y. Macromolecules 2005, 38, 2850–2858. (12) Tong, W.; Gao, C.; M€ohwald, H. Macromolecules 2006, 39, 335–340. (13) Gao, C.; Leporatti, S.; Moya, S.; Donath, E.; M€ohwald, H. Chem.;Eur. J. 2003, 9(4), 915–920. (14) Lebedeva, O. V.; Kim, B.-S.; Vasilev, K.; Vinogradova, O. I. J. Colloid Interface Sci. 2005, 284, 455–462. (15) Liu, X.; Gao, C.; Shen, J.; M€ohwald, H. Macromol. Biosci. 2005, 5, 1209– 1219. (16) Borodina, T.; Markvicheva, E.; Kunizhev, S.; M€ohwald, H.; Sukhorukov, G. B.; Kreft, O. Macromol. Rapid Commun. 2007, 28, 1894–1899. (17) Zhao, Q.; Zhang, S.; Tong, W.; Gao, C.; Shen, J. Eur. Polym. J. 2006, 42, 3341–3351. (18) Kreft, O.; Prevot, M.; M€ohwald, H.; Sukhorukov, G. B. Angew. Chem., Int. Ed. 2007, 46, 5605–5608.
9728 DOI: 10.1021/la901020j
including drug carriers for controlled release,15-17 confined bioreactors,18 microencapsulation,19-21 and model structures for artificial cells.22,23 Despite these potential applications, there are few templates available for the incorporation of biomolecules as building blocks while maintaining their intrinsic properties. Most of the colloidal templates have limited utility due to challenges related to incomplete dissolution of the template material24 and harsh chemical treatments for template removal.25 As an alternative, an oil-inwater type of emulsion can be considered as a dissolvable template. Emulsions have been conventionally utilized for the synthesis of microcapsules; however, there are few reports of LbL assembly of polyelectrolytes on these templates. This is mainly due to the lack of effective techniques for the removal of excess polyelectrolytes during LbL assembly with the stability of the emulsion preserved. In particular, among the few available literature reports, McClements et al.27 have shown that the stability of emulsions used in the food industry against freeze and thaw procedures can be improved by the addition of multilayers of proteins and polysaccharides across the oil-water interface. Their experimental protocols are based on the addition of just enough polyelectrolyte during each layer deposition step and can lead to the flocculation and agglomeration of a fraction of emulsion droplets. Similar protocols have been adopted by M€ ohwald et al. for (19) Caruso, F.; Yang, W.; Trau, D.; Renneberg, R. Langmuir 2000, 16, 8932– 8936. (20) Caruso, F.; Trau, D.; M€ohwald, H.; Renneberg, R. Langmuir 2000, 16, 1485–1488. (21) Shchukin, D. G.; Patel, A. A.; Sukhorukov, G. B.; Lvov, Y. M. J. Am. Chem. Soc. 2004, 126, 3374–3375. (22) Tiourina, O. P.; Radtchenko, I.; Sukhorukov, G. B.; M€ohwald, H. J. Membr. Biol. 2002, 190, 9–16. (23) Katagiri, K.; Caruso, F. Adv. Mater. 2005, 17(6), 738–743. (24) Busse, K.; Kressler, J.; Knorr, J.; Bornemann, S.; Arnold, M.; Thomann, R. Macromol. Mater. Eng. 2001, 286(6), 355–361. (25) Kreft, O.; Georgieva, R.; Baumler, H.; Steup, M.; Muller-Rober, B.; Sukhorukov, G. B.; M€ohwald, H. Macromol. Rapid Commun. 2006, 27, 435–440. (26) Sukhorukov, G. B.; Volodkin, D. V.; Gunther, A. M.; Petrov, A. I.; Shenoy, D. B.; M€ohwald, H. J. Mater. Chem. 2004, 14, 2073–2081. (27) Guzey, D.; McClements, D. J. Adv. Colloid Interface Sci. 2006, 128-130, 227–248.
Published on Web 06/09/2009
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encapsulation of toluene droplets with polyglutamate/polyelectrolyte layers.28 Recently, M€ohwald et al. have used the LbL method to coat dodecane droplets with poly(styrenesulfonate sodium salt) (PSS)/poly(diallyldimethylammonium chloride) (PDADMAC) polyelectrolyte layers using creaming-based separation of excess polyelectrolytes.29 They have performed welldefined multilayer assembly on oil droplets; however, removal of the oil cores to create hollow microcapsules has not been attempted. So far, there has been only one report of using an emulsion-based sacrificial template for the fabrication of microcapsules by the LbL method: Tjipto et al. have used liquid crystal emulsion of a nematic liquid crystal, 5CB (40 -pentyl-4-cyanobiphenyl), and removed the liquid crystal core by ethanol extraction to yield hollow microcapsules.30 In this paper, we present a novel template in the form of frozen cyclohexane droplets dispersed in water. LbL assembly of PSS/ PAH (poly(allylamine hydrochloride)) multilayers is performed at the interface of cyclohexane-in-water emulsions below the freezing point of cyclohexane. Cyclohexane is chosen as the oil phase because of its low boiling point (81.0 °C) and unusually high melting point31 (6.5 °C). Thus, oil droplets can be frozen below 6.5 °C, increasing the stability of the emulsion against droplet coalescence. Freezing also reduces the loss of volatile cyclohexane phase by suppressing evaporation. After the oil droplets have been frozen, this template can be handled just like any other solid templates for LbL assembly, enabling utilization of microfiltration and centrifugation for removal of excess polyelectrolyte. Cyclohexane, being a volatile compound, can be easily removed by subsequent evaporation, lyophilization, or ethanolassisted compatibilization. In this approach, the cyclohexane is removed through molecular diffusion across the polyelectrolyte multilayer membrane, enabling nearly complete removal of the template. In addition, this procedure does not require any harsh chemical treatments.
creaming tendency. This concentration was derived by systematically increasing the emulsifier concentration, [PSS], in a series of experiments and visually observing the resulting emulsions for creaming stability. Next, the PAH layer was deposited by the addition of PAH solution immediately after dilution of the emulsion with 0.1 M NaCl solution (40 mL). The concentration of PAH during this step was maintained at 0.5 mg mL-1. This emulsion was allowed to cream overnight, 48 mL of the subnatant containing excess of PAH solution as well as PAH/PSS complexes was removed, and the emulsion was replenished with an equal volume of 0.1 M NaCl solution filtered through 0.22 μm filter. After redispersion, the emulsion was further divided into five aliquots of 10 mL each and allowed to stand for 15 min. The creamy layer that developed on top during this procedure was discarded. Next, the PSS layer was formed by the addition of PSS solution to make the concentration of PSS = 0.75 mg mL-1. Freezing of Emulsions. Freezing of emulsions was carried out in a 4 °C refrigerator. Ten milliliters of emulsion cooled to 10 °C was added into 0.1 M NaCl (40 mL) solution at 1 °C and allowed to stir for 5 min in an ice bath. Stirring was then stopped, and the emulsion was kept in the ice bath for another 15 min. No macroscopic aggregates were observed during this cooling step with the multilayer coated emulsion. Layer by Layer Assembly. Ten milliliters of PSS-coated frozen emulsion was centrifuged at 50g for 4 min at T = 0 °C (Beckman Coulter, Avanti J-E centrifuge). The subnatant (9 mL) was discarded, and the floating turbid layer was redispersed in an equal volume of ice-cooled 0.1 M NaCl solution at T =1 °C. This procedure was performed twice, followed by the addition of PAH solution to make the final PAH concentration, [PAH] = 0.5 mg mL-1. The frozen emulsion was then transferred into an ice bath kept in a 4 °C refrigerator. For each layer deposition step, the adsorption time was 20 min, and the emulsion was stirred after each 5 min time interval to counter gravity-induced concentration gradients. The same polyelectrolyte concentration (0.5 mg mL-1) was used for both PSS and PAH deposition steps. All solutions were stored in an ice bath for the entire duration of the experiment.
Experimental Section
Lyophilization of Frozen Cyclohexane in Water Emulsion. Freeze-drying of emulsions was carried out using a Lab-
Materials. Cyclohexane (>99%), hexadecane (>98%), ethanol (95%), poly(4-styrenesulfonate sodium salt) (PSS, MW 75 kDa, 30 wt % solution), poly(allylamine hydrochloride) (PAH, MW 56 kDa), and fluorescein isothiocynate (FITC) were purchased from Sigma-Aldrich Co. FITC-labeled PAH was synthesized following a reported procedure.19 All chemicals were used as purchased without further purification. Milli-Q deionized water (specific electric conductivity=18.2 MΩ 3 cm) was used to prepare all aqueous solutions. Emulsification. Cyclohexane-in-water emulsions were prepared by sonicating cyclohexane (2 mL) with PSS solution (10 mL, containing 0.1 M NaCl; [PSS] = 0.12 mg mL-1) using a tip sonicator (Branson Sonifier 150, operating at a power level of 10 W for 6 30 s cycles with sufficient cooling between). Emulsions were immediately diluted by the addition of 0.1 M NaCl solution (40 mL) at room temperature. Hexadecane-in-water emulsions were also prepared following the same protocol as above. Multilayer Coating of Emulsions. Emulsions, as prepared above, were coated with extra polyelectrolyte layers to impart additional stability prior to the freezing step. First, a PSS layer was added with the minimum possible concentration of the emulsifier (0.12 mg mL-1) that gave emulsions without significant (28) Teng, X.; Shchukin, D. G.; M€ohwald, H. Adv. Funct. Mater. 2007, 17, 1273–1278. (29) Grigoriev, D. O.; Bukreeva, T.; M€ohwald, H.; Shchukin, D. G. Langmuir 2008, 24, 999–1004. (30) Tjipto, E.; Cadwell, K. D.; Quinn, J. F.; Johnston, A. P. R.; Abbott, N. L.; Caruso, F. Nano Lett. 2006, 6, 2243–2248.
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conco lyophilizer (model 775320). Samples were prepared by pouring small droplets of the emulsion (droplet volume=1 μL) into a glass vial filled with liquid nitrogen and storing the frozen globules at -20 °C for 12 h. The frozen solid was then freeze-dried for 12 h at a base pressure of 0.021 mbar. The condenser temperature was maintained at -54 °C throughout the freezedrying procedure. Lyophilized microcapsules were redispersed into 0.1 M NaCl solution by sonication.
Compatibilization of Frozen Cyclohexane in Water Emulsion. After deposition of 11 polyelectrolyte layers, 10 mL of frozen emulsion was centrifuged at 50g for 4 min and 9 mL of subnatant was replaced with an equal volume of Millipore water. This procedure was repeated twice. After the second centrifugation cycle, frozen droplets were redispersed in 30% v/v ethanol solution in Millipore water and stored in an ice bath for 15 min. Microcapsules were obtained after room temperature compatibilization for 12 h and were further purified by three washings with Millipore water. Fluorescence Microscopy. Images were acquired on an Olympus IX70 inverted fluorescence microscope equipped with 10, 20, 40, and 70 objectives and optical filters for FITC/ RITC. Images were captured with a CCD camera and analyzed with MetaMorph software (version 7.5). Confocal Microscopy. Confocal images were acquired with a Leica TCS SP2 Confocal system using a 100 oil-immersion objective in the fluorescence mode. Leica confocal software (Leica Lite version 2.61) was used for analyzing the images. Zeta Potential Measurements. Zeta potentials of frozen emulsions were measured at 3 °C by a Zetasizer Nano ZS 90 DOI: 10.1021/la901020j
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instrument (Malvern Instruments Ltd., U.K.). Each measurement was repeated three times and for five different samples. Reported values represent the average of these measurements. Corresponding error bars represent the average peak widths of the zeta potential distributions.
Deformation of Microcapsules Induced by Osmotic Pressure. Microcapsules were incubated with PSS solution containing 1.6 wt % PSS for 10 min to induce deformations in the polyelectrolyte shells. Confocal fluorescence microscopy was then performed to visualize the deformations. Osmotic pressure of the PSS solution was measured with an osmometer, based on the technique of freezing point depression (Advanced Instruments Inc., model 3250). Osmolarity reading of the osmometer was converted into osmotic pressure using van’t Hoff equation, π = RTC, where C is the osmolarity of the solution (expressed in mol/m-3) and R is the universal gas constant.
Results and Discussion We performed LbL assembly of PSS/PAH on frozen cyclohexane droplets in water at 4 °C with centrifugation after each layer deposition step to remove nonadsorbed polyelectrolyte. Up to 11 layers of PSS/PAH were deposited, and ethanol-assisted compatibilization was used for removal of the cyclohexane phase to obtain hollow microcapsules. In what follows, we report a detailed discussion of each step. Emulsification. Cyclohexane was dispersed at a volume fraction of 20% in 0.1 M NaCl solution containing PSS as an emulsifier. Emulsions, as prepared above, were polydisperse with droplet sizes ranging from 500 nm to 3 μm (from optical microscopy). The emulsions very fairly stable against creaming, as evidenced by lack of a visible creamy layer even after about 4 h of standing at room temperature. However, when emulsions were allowed to stand overnight, it was observed that creaming led to the formation of a small fraction of droplets with significantly larger diameters (>10 μm). This observation suggested that even with a negatively charged polyelectrolyte layer (zeta potential = -80 mV at 3 °C and pH 6.4 in 0.1 M NaCl solution), droplet agglomeration was possible when the droplets were forced into close proximity within the creamy layer. Droplet coalescence was subsequently avoided by incubating the emulsions at room temperature on a rotisserie mixer overnight, which effectively suppressed the formation of a creamy layer. In contrast, when hexadecane was dispersed in water under identical conditions, the resulting emulsions were found to be stable against creaming for at least 96 h. Because the aqueous solubility of hexadecane32 (
