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Chem. Mater. 2005, 17, 4610-4616
Manipulating the Properties of Polyelectrolyte Microcapsules by Glutaraldehyde Cross-Linking Weijun Tong,†,‡ Changyou Gao,*,† and Helmuth Mo¨hwald‡ Department of Polymer Science and Engineering, Zhejiang UniVersity, Hangzhou 310027, China, and Max-Planck-Institute of Colloids and Interfaces, 14424 Potsdam, Germany ReceiVed April 7, 2005. ReVised Manuscript ReceiVed July 5, 2005
The stability of hollow microcapsules against environmental alterations such as pH, osmotic pressure, and temperature is a critical issue for practical applications. It is demonstrated here that multilayer capsules assembled from poly(allylamine hydrochloride) (PAH) and sodium poly(styrene sulfonate) (PSS) can be considerably stabilized by cross-linking of only the PAH component with glutaraldehyde (GA). Formation of a Schiff base between the aldehyde and the amine groups was evidenced by UV-vis spectroscopy. After cross-linking by 2% GA for 2 h, an apparently thicker capsule wall was obtained with higher folds, and no alteration of the macroscopic topology of the capsules was observed after incubation in 0.1 M NaOH for 24 h. The cross-linking significantly improved the mechanical strength of the capsules to resist osmotic pressure induced invagination. Consequently, both the critical pressure and the elasticity modulus (680 MPa) of the capsule wall were doubled compared with that of the control. The crosslinking also greatly lowered the permeability of the capsule wall, as evidenced by confocal laser scanning microscopy and fluorescence recovery after photobleaching. Quantitative analysis revealed that the permeation coefficient for dextran (Mw ∼ 250 kD) was reduced by a factor of 3 after cross-linking.
Introduction The layer-by-layer (LbL) assembly technique has received great interest in the past decade as a versatile method for construction of multilayer thin films with tailored architecture and properties.1 Recently, this strategy was successfully extended to three-dimensional curved surfaces by assembly of polyelectrolytes onto colloidal particles. Following a core removal procedure, nano- and microcapsules have been further fabricated with well-controlled size and shape, finely tuned capsule wall thickness, and variable wall composition.2,3 These novel hollow capsules have shown potential applications as drug delivery vehicle, biosensor, and microreactor as well as microcontainer.4 One of the critical questions for various applications is shell permeability control so that loading and subsequent release can be realized in a desired manner. Numerous attempts have already been engaged in * Author to whom correspondence should be addressed. E-mail:
[email protected]. Fax: +86-571-87951948. † Zhejiang University. ‡ Max-Planck-Institute of Colloids and Interfaces.
(1) (a) Decher, G.; Schlenoff, J. B. Multilayer Thin Films: Sequential Assembly of Nanocomposite Materials; Wiley-VCH: Weinheim, Germany, 2002. (b) Decher, G. Science 1997, 277, 1232. (2) (a) Donath, E.; Sukhorukov, G. B.; Caruso, F.; Davis, S. A.; Mo¨hwald, H. Angew. Chem., Int. Ed. 1998, 37, 2202. (b) Sukhorukov, G. B.; Donath, E.; Davis, S.; Lichtenfeld, H.; Caruso, F.; Popov, V. I.; Mo¨hwald, H. Polym. AdV. Technol. 1998, 9, 1. (c) Caruso, F.; Caruso, R. A.; Mo¨hwald, H. Science 1998, 282, 1111. (3) (a) Schuler, C.; Caruso, F. Biomacromolecules 2001, 2, 921. (b) Moya, S.; Donath, E.; Sukhorukov, G. B.; Auch, M.; Baumler, H.; Lichtenfeld, H.; Mo¨hwald, H. Macromolecules 2000, 33, 4538. (c) Sukhorukov, G. B.; Da¨hne, L.; Hartmann, J.; Donath, E.; Mo¨hwald, H. AdV. Mater. 2000, 12, 112. (d) Dai, Z. F.; Da¨hne, L.; Mo¨hwald, H.; Tiersch, B. Angew. Chem., Int. Ed. 2002, 41, 4019. (e) Radtchenko, I. L.; Sukhorukov, G. B.; Leporatti, S.; Khomutov, G. B.; Donath, E.; Mo¨hwald, H. J. Colloid Interface Sci. 2000, 230, 272.
controlling the permeability of the capsules, typically by varying the properties of the capsule walls.5 Alteration of the layer number is a straightforward pathway.6 Yet, it is rather time-consuming since a significant change of permeability requires many layers. The other pathway relies on modulation of the ambient conditions such as pH value, ionic strength, and polarity of the solution.7 Another critical question is the mechanical stability of the microcapsules. From practical consideration, these capsules should be stable enough to withstand a wide range of pH change, solvent etching, and shear stress. One of the promising pathways is covalent cross-linking of the capsule walls, which has been initially established on planar substrates and then has been extended to curved capsule walls. For example, by incorporation of the photosensitive diazoresin (DAR) in an LbL manner, the charge interaction in the multilayers can be converted into covalent bonding via UV irradiation.8 The cross-linked multilayers exhibit increased stability against solvent etching. Appling this (4) (a) Moya, S.; Da¨hne, L.; Voigt, A.; Leporatti, S.; Donath, E.; Mo¨hwald, H. Colloids Surf., A 2001, 27, 183. (b) Caruso, F.; Trau, D.; Mo¨hwald, H.; Renneberg, R. Langmuir 2000, 16, 1485. (c) McShane, M. J.; Brown, J. Q.; Guice, K. B.; Lvov, Y. M. J. Nanosci. Nanotechnol. 2002, 2, 411. (d) Shchukin, D. G.; Sukhorukov, G. B. AdV. Mater. 2004, 16, 671. (5) For a review, see: Antipov, A. A.; Sukhorukov, G. B. AdV. Colloid Interface 2004, 111, 49. (6) Antipov, A. A.; Sukhorukov, G. B.; Donath, E.; Mo¨hwald, H. J. Phys. Chem. B 2001, 105, 2281. (7) (a) Antipov, A. A.; Sukhorukov, G. B.; Leporatti, S.; Radtchenko, I. L.; Donath, E.; Mo¨hwald, H. Colloids Surf., A 2002, 198, 535. (b) Sukhorukov, G. B.; Antipov, A. A.; Voigt, A.; Donath, E.; Mo¨hwald, H. Macromol. Rapid Commun. 2001, 22, 44. (c) Ibarz, G.; Da¨hne, L.; Donath, E.; Mo¨hwald, H. AdV. Mater. 2001, 13, 1324. (d) Antipov, A. A.; Sukhorukov, G. B.; Mo¨hwald, H. Langmuir 2003, 19, 2444. (e) Lvov, Y.; Antipov, A. A.; Mamedov, A.; Mo¨hwald, H.; Sukhorukov, G. B. Nano Lett. 2001, 1, 125.
10.1021/cm0507516 CCC: $30.25 © 2005 American Chemical Society Published on Web 08/11/2005
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Scheme 1. Schematic Illustration to Show the Reaction between (a) Monomeric and (b) Polymeric GA with PAH
technique on polyelectrolyte capsules has improved their stability against solvent etching and osmotic pressure.9 More recently, macromolecules have been entrapped in DAR-based capsules10 through cross-linking the walls via UV irradiation which leads to the decrease of the cutoff molecular weight. For those preformed multilayers and polyelectrolyte capsules, other techniques have also been adopted. For example, weak poly(allylamine hydrochloride)/poly(acrylic acid) (PAH/PAA) multilayers become stable over a wide pH range and highly impermeable after formation of a nylonlike structure at elevated temperature.11 Hydrogen-bonded multilayers composed of polyacrylamide (PAAm) and poly(acrylic acid) (PAA) can also be stabilized to neutral pH by thermo- or photoinduced cross-linking reactions12 or watersoluble carbodiimide chemistry.13 With this chemistry, weak polyelectrolyte capsules14 are similarly cross-linked, resulting in stable capsules against solvent etching and pH alteration. The aforementioned techniques are largely based on a reaction between the functional groups of the two components in the multilayers. Neutron reflectivity shows that there is a large overlap between segments of the adjacent layers,15 resulting in a finite monomer density along the layer normal for both types of polyelectrolytes.1b Therefore, it is possible to improve the multilayer stability by cross-linking just one component. For example, stability of the hydrogen-bonded multilayer microcapsules is improved by carbodiimide chemistry using ethylenediamine as a foreign cross-linking reagent for the poly(methacrylic acid) component.16 The amino groups of fourth-generation poly(amidoamine) den(8) (a) Zhong, H.; Wang, J.; Jia, X.; Li, Y.; Qin, Y.; Chen, J.; Zhao, X.; Cao, W.; Li, M.; Wei, Y. Macromol. Rapid Commun. 2001, 22, 583. (b) Chen, J.; Luo, H.; Cao, W. Polym. Int. 2000, 49, 382. (c) Cao, T.; Yang, S.; Yang, Y.; Huang, C.; Cao, W. Langmuir 2001, 17, 6034. (9) Pastoriza-Santos, I.; Scholer, B.; Caruso, F. AdV. Funct. Mater. 2001, 11, 122. (10) Zhu, H. G.; McShane, M. J. Langmuir 2005, 21, 424. (11) Harris, J. J.; DeRose, P. M.; Bruening, M. L. J. Am. Chem. Soc. 1999, 121, 1978. (12) Yang, S. Y.; Rubner, M. F. J. Am. Chem. Soc. 2002, 124, 2100. (13) Yang, S. Y.; Lee, D.; Cohen, R. E.; Rubner, M. F. Langmuir 2004, 20, 5978-5981. (14) (a) Schuetz, P.; Caruso, F. AdV. Funct. Mater. 2003, 13, 929. (b) Mauser, T.; Dejugnat, C.; Sukhorukov, G. B. Macromol. Rapid Commun. 2004, 25, 1781. (15) (a) Schmitt, J.; Gru¨newald, T.; Decher, G.; Pershan, P. S.; Kjaer, K.; Lo¨sche, M. Macromolecules 1993, 26, 7058. (b) Baur, J. W.; Rubner, M. F.; Reynolds, J. R.; Kim, S. Langmuir 1999, 15, 6460. (c) Laurent, D.; Schlenoff, J. B. Langmuir 1997, 13, 1552. (16) Kozlovskaya, V.; Ok, S.; Sousa, A.; Libera, M.; Sukhishvili, S. A. Macromolecules 2003, 36, 8590.
drimer (4G PAMAM) in sodium poly(styrene sulfonate) (PSS)/4G PAMAM multilayers have also been cross-linked and activated by glutaraldehyde (GA) treatment for biological applications.17 Herein, hollow multilayer PAH/PSS microcapsules are treated with GA to manipulate their properties, in particular, stability and mechanical strength. The amino groups of PAH molecules in the capsule shells readily react with aldehyde groups of GA (Scheme 1) at very mild conditions.18 Various characterizations are then performed to reveal the chemical, morphological, and mechanical alterations. GA has been used more frequently as a cross-linking reagent, since it is less expensive, nontoxic, and highly soluble in aqueous solution.19 The solution of GA consists of mixtures of free mono- and polymeric GA.19b Both of them exhibit the ability to react with primary amine groups. Evidence suggests that the conjugated aldehyde moieties in the polymers yield more stable reaction products, whereas mono-GA yields hydrolyzable labile entities.20 Polymerization of GA takes place quickly under alkaline pH. Yet, even at pH 5 the aqueous solution of GA contains polymeric moieties. Consequently, aqueous GA reacts with the amino groups as an unsaturated polymer to yield stabilized bonds (Schiff base) by resonance (Scheme 1b).21 Experimental Section Materials. Sodium poly(styrene sulfonate) (PSS, Mw ∼ 70 kD), poly(allylamine hydrochloride) (PAH, Mw ∼ 65 kD), FITC-dextran (Mw ∼ 464 kD and ∼250 kD), FITC-albumin, manganese sulfate hydrate (MnSO4‚H2O), disodium ethylenediaminetetraacetate dihydrate (EDTA), ammoniumhydrogencarbonate (NH4HCO3), and glutaraldehyde (GA, 30 wt % solution in water) were all obtained from Sigma-Aldrich. All chemicals were used as received. The water used in all experiments was prepared in a three-stage Millipore Milli-Q Plus 185 purification system and had a resistivity higher than 18.2 MΩ. Spherical MnCO3 microparticles with a (17) Khopade, A. J.; Caruso, F. Langmuir 2003, 19, 6219. (18) (a) Meyers, W. E.; Royer, G. P. J. Am. Chem. Soc. 1977, 99, 6141. (b) Chanda, M.; Rempel, G. L. React. Polym. 1995, 25, 25. (c) Tuncel, D.; Matthews, J. R.; Anderson, H. L. AdV. Funct. Mater. 2004, 14, 85. (19) (a) Walt, D. R.; Agayn, V. I. TrAC-Trend. Anal. Chem. 1994, 13, 425. (b) Jayakrishnan, A.; Jameela, S. R. Biomaterials 1996, 17, 471. (c) DeSantis, G.; Jones, J. B. Curr. Opin. Biotechnol. 1999, 10, 324. (20) Rembaum, A.; Levy, J.; Gupta, A.; Margel, S. Polym. Prepr. (Am. Chem. Soc., DiV. Polym. Chem.) 1978, 19, 648. (21) Monsan, P.; Puzo, G.; Mazarguil, H. Biochemie 1975, 57, 1281.
4612 Chem. Mater., Vol. 17, No. 18, 2005 diameter of 7∼8 µm were synthesized by mixing MnSO4 and NH4HCO3 solutions.22 Melamine formaldehyde (MF) microparticles with an average diameter of 3.97 µm were purchased from Microparticles GmbH, Berlin, Germany. FITC-PAH was prepared following the procedure described in the literature.23 Methods. Layer-by-Layer Assembly, GA Cross-Linking and Hollow Capsule Fabrication. Adsorption of the polyelectrolytes (2 mg/mL) onto the MnCO3 microparticles (∼1% w/w in suspension) was conducted in 0.5 M NaCl solution for 10 min followed by three washings in water. The excess polyelectrolytes were removed by centrifugation at 300g for 5 min. After assembly of five bilayers of PAH/PSS (PSS being the last layer), the coated particles were incubated in 2% GA solution for a desired time at room temperature. They were then incubated in 0.1 M HCl solution for 20 min under continuous shaking. The resultant capsules were centrifuged at 1500g for 5 min and were washed three times with 0.01 M EDTA solution to rinse off the Mn2+. Finally, the capsules were washed with water three times. For elasticity modulus measurement by an osmotic pressure method (see below),24 positively charged melamine formaldehyde (MF) microparticles with an average diameter of 3.97 µm were used as templates because of their narrower size distribution. After assembly of five bilayers of PSS/PAH (PAH being the last layer), the particles were subjected to 0.1 M HCl to decompose the cores. After dissolution of the cores, cross-linking of the hollow microcapsules was conducted by mixing with GA at a final concentration of 2.5% at room temperature for 1 h. Multilayer Buildup on Planar Substrates and Cross-Linked with GA. Silicon wafers and quartz slides were first treated with “piranha” solution for 1 h and were thoroughly rinsed with water (caution: piranha solution, containing 3:7 H2O2:H2SO4, reacts violently with many organic materials and should be used with extreme care, and it should not be stored in sealed containers). The pretreated substrates were sequentially immersed into PAH and PSS solutions (both 2 mg/mL, in 0.5 M NaCl) for 10 min, with three rinses in water at each interval. After 10 layers were assembled (PSS being the outmost layer), the multilayer was dried with nitrogen flow and was further treated at room temperature overnight. For GA cross-linking, the assembled multilayers without drying were incubated in 2% GA solution for a desirable time at room temperature, followed by a thorough rinse with water and then dried as described above. Stability of the Capsules and the Multilayers. To test the chemical stability, the capsules or multilayers on quartz slides were incubated in 0.1 M NaOH for a given time and then were washed with water three times. UV-vis spectroscopy was employed to characterize the change of absorbance at 225 nm. To observe the thermal response, the capsule suspension in a test tube was incubated at 90 °C in water for 2 h in a water bath. Scanning Electron Microscopy (SEM). Samples were prepared by applying a drop of the capsule suspension onto a glass slide. After drying overnight, the samples were sputtered with gold and were measured by a Gemini Leo 1550 instrument at an operation voltage of 3 keV. Scanning Force Microscopy (SFM). A drop of sample suspension was applied to freshly cleaved mica and was dried in air. The images were obtained by means of a Digital Instruments nanoscope IIIa Multimode SFM (Digital Instruments Inc., Santa Barbara, CA) in air at room temperature using a tapping mode. (22) Antipov, A. A.; Shchukin, D.; Fedutik, Y.; Petrov, A. I.; Sukhorukov, G. B.; Mo¨hwald, H. Colloids Surf., A 2003, 224, 175. (23) von Klitzing, R.; Mo¨hwald, H. Langmuir 1995, 11, 3554. (24) (a) Gao, C.; Donath, E.; Moya, S.; Dudnik, V.; Mo¨hwald, H. Eur. Phys. J. E 2001, 5, 21. (b) Gao, C. Y.; Leporatti, S.; Moya, S.; Donath, E.; Mo¨hwald, H. Langmuir 2001, 17, 3491.
Tong et al. Confocal Laser Scanning Microscopy (CLSM). Confocal images were taken with a Leica confocal scanning system mounted to a Leica Aristoplan and equipped with a 100× oil immersion objective with a numerical aperture (NA) of 1.4. To investigate the cutoff molecular weight, an equal volume of capsule suspension and FITCdextran (2 mg/mL) was mixed and observed after 20 min. For visualization, FITC-PAH was used to stain the capsules. Elasticity Modulus Measurement by Osmotic Pressure Method.24 For clear visualization in polyelectrolyte solution, the capsules were coated first with a layer of FITC-albumin. Since the modulus of albumin should be far smaller than that of the cross-linked polyelectrolyte complex, the influence of the adsorbed albumin on the final modulus is negligible. Moreover, the albumin layer may seal off the existing pores on the capsule shells, thus enhancing the sensitivity of osmotic-induced invagination. Equal amounts of capsule solution and FITC-albumin solution (1 mg/mL) were mixed together and were stored at 4 °C at least for 2 h. The suspension was centrifuged at 4000 rpm (1145g) and was washed three times in H2O to remove the excess FITC-albumin. The solution of FITCalbumin labeled capsules was rapidly mixed with an equal amount of PSS solution of different concentration. CLSM was employed to determine the number of deformed and undeformed capsules. The total number of capsules counted for each PSS concentration was at least 200. The deformation ratio was defined as the number of deformed capsules divided by the total number of capsules. The critical PSS concentration was defined as the concentration necessary to induce the invagination of 50% of the intact capsules. The critical pressure was found by referring to a calibration curve.24a Quantification of Permeability. Permeability of the capsule to FITC-dextran (Mw ∼ 250 kD) was quantified by means of fluorescence recovery after photobleaching (FRAP). To follow diffusion of the fluorescent probe, the capsule interior was photochemically bleached using a laser line of 488 nm. The laser beam was focused onto a spot inside the capsule at 100% intensity. The time of bleaching was set long enough to bleach all the fluorescent probes in the capsules. The time interval between images was varied depending on duration of the recovery. Quantitative data were obtained by measuring the fluorescence intensity in a defined area (ROI, analysis protocol provided by the CLSM software). UV-Vis Spectroscopy. The UV-vis absorption spectra of the hollow capsules were recorded in water by a Cary 50 UV-visible spectrophotometer. The absorbance of PSS at 225 nm was used as the inner standard to normalize the curves. The absorbance of multilayers deposited on the quartz slides was recorded as well. Ellipsometry. Ellipsometry was performed using an optical null ellipsometer (Multiskop Ellipsometer, Optrel GmbH) with a HeNe laser at 632.8 nm at an incidence angle of 70°. Multilayers were deposited on silicon wafers, and their thicknesses were calculated by assuming a refractive index of 1.5.
Results and Discussion Fabrication of Cross-Linked Microcapsules. As depicted in Scheme 1, reaction between the aldehyde and the amine groups will produce -CdN- bonds (Schiff base) on the capsule walls, which should exhibit absorbance in the UV region. Therefore, the cross-linking was first followed and verified by UV-vis spectroscopy (Figure 1). The broad absorbance in the region of 250-350 nm increased along with the treatment time. The absorbance at 270 nm as a function of time is shown in the inset, from which one can see that the reaction proceeds very fast at the initial stage and then decreases gradually. Actually, cross-linking for 5 min was already long enough to effectively improve the
Properties of Polyelectrolyte Microcapsules
Figure 1. UV-vis spectra of hollow (PAH/PSS)5 capsules cross-linked with GA for different times. From bottom to top: 0, 5, 20, 60, and 120 min, respectively. The inset shows the absorbance at 270 nm as a function of time.
stability of the capsules against alkaline treatment (see below). One can thus conclude that the cross-linking degree can be tuned conveniently by the reaction time. The capsules cross-linked for 2 h were further subjected to SEM and SFM characterizations to reveal their morphological change. Figure 2 shows that both the control (Figure 2a, c) and the cross-linked capsules (Figure 2b, d) collapsed as a result of evaporation of the water content during the drying procedure. Similar to other techniques, cross-linking by GA could not produce capsules with hard enough wall structure to resist collapse. However, compared with the control capsules, the cross-linked capsules have higher folds (Figure 2b), as particularly quantified by the SFM detection (Figure 2d, over 1000 nm). Surprisingly, the double wall thickness of the cross-linked capsules is approximately 64 ( 2 nm, which is about 50% higher than that of the control capsules (44 ( 2 nm). By contrast, on planar silicon wafer only a slight increase of layer thickness was measured by ellipsometry, namely, 15.6 ( 0.3 nm and 17.6 ( 0.4 nm for (PAH/PSS)5 multilayers before and after GA cross-linking, respectively. This gives a hint that the cross-linked capsules might not be completely collapsed because the wall may have become stiffer, so that the polyelectrolyte wall cannot closely approach as that of the control. No apparent size variation and capsule coagulation have been observed after crosslinking (Figure 3). Improved Chemical, Mechanical, and Thermal Stability by Cross-Linking. As cross-linking can produce covalent bonds besides charge interaction in the capsule walls, the stability and mechanical strength of the capsules should thus be improved. For example, exposure of PAH/PSS capsules templated on MnCO3 particles to 0.1 M NaOH solution causes complete dissolution in a couple of seconds.25 After treatment with GA for only 5 min, the chemical stability of the same capsules was greatly improved as shown in Figure 3b. Most of the capsules survived at least for 2 h in 0.1 M NaOH solution, although part of them was dissolved to form floccules (Figure 3e). Around the floccules, the aggregation of the capsules was observed as well (Figure 3e). After treatment with GA for 2 h, however, the capsules exhibited
Chem. Mater., Vol. 17, No. 18, 2005 4613
very high stability against the NaOH solution of the same concentration even for 24 h (Figure 3c, f). The capsules could be still well dispersed in water and could keep their intact structure as those initially existing in pure water (Figure 3a, d). These results would also mean that in principle the chemical stability of the capsules can be tuned at least by the cross-linking time. Dissolution of the PAH/PSS polyelectrolyte capsules at high pH is the result of deprotonation of PAH, leading to disassembly of the polyelectrolyte multilayer films. Apparently, GA cross-linking provides additional covalent bonding, which can maintain the macroscopic topology of the capsules after breakage of the electrostatic interaction at high pH. The PSS chains should be kinetically trapped within the crosslinked network through entanglements, which prevents quick leaching of PSS from the cross-linked capsules. Nonetheless, slow release of the PSS molecules is inevitable. For instance, after 2 h incubation of the 2 h cross-linked multilayers in 0.1 M NaOH, the absorbance of PSS at 225 nm decreased by about 10%. To explore alteration of the mechanical strength, capsule deformation induced by osmotic pressure difference in concentrated PSS solution was adopted. In the theoretical model, based on continuum mechanics (1), which describes the relationship between the critical pressure differences Pc at which an indentation of the capsules occurs, the elasticity modulus µ, the capsule wall thickness δ, and the capsule radius R were recently employed.24 Pc ) 4µ
(Rδ)
2
(1)
Figure 4 shows that both un-cross-linked and cross-linked PAH/PSS capsules underwent a shape transition from spherical (Figure 4a, d) to a cup shape at certain critical PSS concentrations (Figure 4b, e). At still higher PSS concentration, the capsules shrunk further and lost most of their internal volume (Figure 4c, f). The capsules with a spherical shape in Figure 4c, f represent broken ones. They are easily distinguished by the absence of shrinking. In such capsules, PSS can equilibrate through sufficiently large holes.24 The invaginated capsules in Figure 4d are formed in the repeated centrifugation procedure during cross-linking and washings. A typical sigmoid shape is obtained when the ratio of deformed capsules is plotted versus PSS concentration curve (Figure 5). At lower polyelectrolyte concentrations, the osmotic pressure is not large enough to overcome the elastic restoring force of the capsule walls. Thus, capsules do not invaginate at low PSS concentrations. At large enough PSS (25) Dejugnat, C.; Sukhorukov, G. B. Langmuir 2004, 20, 7265. (26) (a) Lo¨sche, M.; Schmitt, J.; Decher, G.; Bouwman, W. G.; Kjaer, K. Macromolecules 1998, 31, 8893. (b) Farhat, T.; Yassin, G.; Dubas, S. T.; Schlenoff, J. B. Langmuir 1999, 15, 6621. (c) Ruths, J.; Essler, F.; Decher, G.; Riegler, H. Langmuir 2000, 16, 8871. (d) Harris, J. J.; Bruening, M. L. Langmuir 2000, 16, 2006. (27) Leporatti, S.; Gao, C.; Voigt, A.; Donath, E.; Mo¨hwald, H. Eur. Phys. J. E 2001, 5, 13. (28) Ko¨hler, K.; Shchukin, D. G.; Sukhorukov, G. B.; Mo¨hwald, H. Macromolecules 2004, 37, 9546. (29) Ibarz, G.; Da¨hne, L.; Donath, E.; Mo¨hwald, H. Macromol. Rapid Commun. 2002, 23, 474. (30) Gao, C. Y.; Moya, S.; Lichtenfeld, H.; Casoli, A.; Fiedler, H.; Donath, E.; Mo¨hwald, H. Macromol. Mater. Eng. 2001, 286, 355.
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Figure 2. SEM (a, b) and SFM (c, d) images of (PAH/PSS)5 hollow capsules before (a, c) and after 2 h cross-linking with 2% GA (b, d).
Figure 3. CLSM fluorescence images of (a) un-cross-linked control capsules, (b) capsules cross-linked with 2% GA for 5 min and subsequently incubated in 0.1 M NaOH for 2 h, and (c) capsules cross-linked with 2% GA for 2 h and subsequently incubated in 0.1 M NaOH for 24 h. (d), (e), and (f) are their corresponding transmission images.
concentrations, all capsules, except broken ones, were invaginated. A three-tangent evaluation technique as described before24 is used to estimate the critical PSS concentrations, at which the shape transition occurs. As can be seen
from Figure 5, the concentrations amount to 3.6 wt % and 7.25 wt % PSS which correspond to 2.6 × 105 and 5.8 × 105 N/m2 for the un-cross-linked and the cross-linked capsules, respectively. Their radii are measured as 2.06 (
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Figure 4. CLSM images to show the capsule deformation process in a concentration series of PSS solution. (a), (b), and (c) are control capsules suspended in 0%, 6%, and 8% (wt) PSS solutions, respectively. (d), (e), and (f) are capsules cross-linked with 2.5% GA for 1 h and suspended in 0%, 8%, and 12% (wt) PSS solutions, respectively. Five bilayers of PSS/PAH are assembled on MF particles with a diameter of 3.97 µm, followed by removal of the cores by 0.1 M HCl.
Figure 6. CLSM images to show (PAH/PSS)5 capsules after annealing at 90 °C for 2 h in water. (a) Control capsules, (b) capsules cross-linked with 2% GA for 2 h. a and b have the exact same scale bar. Figure 5. Percentage of deformed capsules as a function of PSS concentration. The capsule information and cross-linking conditions are the same as that in Figure 4.
0.06 µm and 2.12 ( 0.05 µm by CLSM in wet state, and their wall thicknesses are measured as 22.1 ( 2.4 nm and 39 ( 2.2 nm by SFM in dry state, respectively. However, the wall thickness of the cross-linked capsules could not be used directly because of the incomplete collapse. Judging from the fact that both original and cross-linked multilayers have similar thickness on silicon wafers, it is reasonable to assume that in water they should have the same wall thickness, too. With a wall thickness of 30.9 nm (calculated from AFM image of the un-cross-linked capsules in dry state and adding 40% of water content26 taking into account multilayer swelling), the elastic modulus was calculated as 290 and 680 MPa for the un-cross-linked and the cross-linked capsules, respectively. This would mean that cross-linking has indeed increased the hardness of the multilayers.
As a result of covalent cross-linking, the stability of the capsules against thermally induced shrinkage is also improved dramatically. For example, previous studies reveal that incubation of the PSS/PAH microcapsules at 70 °C for 2 h has caused capsule shrinkage and increase of wall thickness.27 This effect is largely enhanced when the incubation temperature is increased to 120 °C. Incubation for 20 min readily leads to pronounced capsule shrinking accompanied by a wall thickness increase by g10 times.28 In the present case, the un-cross-linked capsules reduced their diameter from 7.4 ( 0.3 µm to 5.3 ( 0.4 µm after incubation at 90 °C for 2 h (Figure 6a). However, at the same incubation conditions, no size variation was observed for the capsules cross-linked with GA for 2 h (Figure 6b). The diameter changed from 7.3 ( 0.5 µm to 7.2 ( 0.5 µm. Capsule shrinkage is understood as a result of chain rearrangement toward a direction of an energetically favored coiled conformation.27,28 This evolution is accelerated by thermal energy since the probability of temporary breakage of ion
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Figure 7. CLSM images of capsules incubated in FD-460kD solution for 20 min. (a) Control capsules, (b) capsules cross-linked with 2% GA for 2 h.
pairs is higher at elevated temperature. After covalent crosslinking, however, the mobility of the polymer chains and segments is restricted, and thus the macroscopic topology of the capsules can be retained even though the breakage of ion pairs can still occur. Permeability Change after Cross-Linking. CLSM investigations revealed that the un-cross-linked capsules rejected completely FITC-dextran with molecular weight of 2000 kD (FD-2000 kD) but were permeable to FD-460kD (Figure 7a). After cross-linking, FD-460kD was completely rejected from the capsules too (Figure 7b). Although the precise difference of the cutoff molecular weight between these two types of capsules is not yet clear, conclusions can still be drawn that the cross-linking has lowered the cutoff molecular weight. Larger pores should also exist in the capsule wall since polymers with a radius of gyration of 10∼30 nm such as dextran are permeable.29 These pores are created during the core removal procedure as a result of osmotic pressure induced capsule swelling.30 Cross-linking should thus endow the capsules with a stronger ability to reduce the extent of swelling. The existence of pores is not contradictory with the osmotic-induced invagination. Albumin can seal off the pores to some extent. More importantly, the normal pressure is dependent on the equilibration time between the capsule interior and the bulk. With sufficiently high concentration and fast enough mixing rate, even NaCl and other low molecular weight oligomers can create enough transient pressure to induce capsule invagination (data not shown). Quantitative comparison was made by fluorescence recovery after photobleaching (FRAP) using FD-250kD as a probe. A slower recovery rate was recorded for the crosslinked capsules (Figure 8). Following eq 2 established in the literature,29,31 the recovery curve of the fluorescence intensity I(t) as a function of time t can be described: I(t) ) I0 + (Is - I0)(1 - e-3Pt/R)
(2)
Is and I0 denote the fluorescence at tf∞ and t ) 0, respectively. P is the permeability, and R is the radius of the capsules assumed as a sphere. Taking the half time of recovery, the permeability can thus be calculated. After crosslinking, the permeability coefficient decreased by a factor of 3, that is, from 3.7 × 10-8 m/s (for the control) to 1.2 × 10-8 m/s. Investigations are underway to explore the detailed (31) Ibarz, G.; Da¨hne, L.; Donath, E.; Mo¨hwald, H. Chem. Mater. 2002, 14, 4059.
Figure 8. Typical fluorescence recovery curve recorded from the capsule interiors after photobleaching. (a) Control capsules, (b) capsules cross-linked with 2% GA for 2 h. The dashed line presents the initial intensity in the capsule interior before photobleaching.
relationship between the cross-linking conditions and the cutoff molecular weight and permeability. Conclusions The properties of the polyelectrolyte microcapsules, exemplified here with PAH/PSS microcapsules templated on MnCO3 and MF particles, can be well tailored by glutaraldehyde (GA) cross-linking. UV-vis characterization reveals that the reaction between the aldehyde and the amine groups proceeds very fast at the initial stage and then decreases gradually. Upon formation of the covalent bonds, the capsules exhibit higher stability against dissolution in concentrated alkaline solution (0.1 M NaOH). The critical pressure, at which invagination of capsules takes place, is doubled by cross-linking via 2.5% GA for 1 h. Estimated from the critical pressure, the elasticity modulus of the crosslinked capsule wall has been increased from 290 MPa (for the control) to 680 MPa. Another effect caused by the crosslinking is that the thermal stability against shrinking at elevated temperature is dramatically improved, for example, no apparent size reduction is measured after treatment at 90 °C for 2 h. Cross-linking has also decreased the permeability of the capsules. All these results demonstrate that significant improvement of capsule stability can be realized by crosslinking only one component of the multilayer capsules, for example, PAH in the present study. Noting that other multilayer systems assembled from components containing amino groups such as polyamines, proteins, enzymes, and polysaccharides may be similarly cross-linked as well, the method described here possesses wide applicability. Acknowledgment. We thank Prof. J.C. Shen for his continuous support and stimulating discussions. A. Heilig and Dr. D.Y. Wang are greatly acknowledged for their assistance in SFM and SEM measurements. W.J. Tong and C.Y. Gao thank the Max-Planck Society for a visiting scientist grant. This study is financially supported by the Natural Science Foundation of China (No. 20434030 and 90206006) and the National Science Fund for Distinguished Young Scholars of China (No. 50425311). CM0507516
