Charge-Controlled Permeability of Polyelectrolyte Microcapsules

Scanning force microscopy (SFM), UV-vis, Raman spectroscopy, and ζ-potential confirm the existence of the PSS molecules in the CaCO3 particles and th...
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J. Phys. Chem. B 2005, 109, 13159-13165

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Charge-Controlled Permeability of Polyelectrolyte Microcapsules Weijun Tong,†,‡ Wenfei Dong,‡ Changyou Gao,*,† and Helmuth Mo1 hwald‡ Department of Polymer Science and Engineering, Zhejiang UniVersity, Hangzhou 310027, China, and Max-Planck-Institute of Colloids and Interfaces, 14424 Potsdam, Germany ReceiVed: March 3, 2005; In Final Form: April 25, 2005

Multilayer microcapsules showing unique charge-controlled permeability have been successfully fabricated by employing poly(styrene sulfonate) (PSS)-doped CaCO3 particles as templates. Encapsulation of the PSS molecules is thus achieved after core removal. Scanning force microscopy (SFM), UV-vis, Raman spectroscopy, and ζ-potential confirm the existence of the PSS molecules in the CaCO3 particles and the resultant microcapsules, which are initially incorporated during the core fabrication process. A part of these additionally introduced PSS molecules interacts with PAH molecules residing on the inner surface of the multilayer wall to form a stable complex, while the other part is intertwined in the capsule wall or in a free state. Capsules with this structure possess many special features, such as highly sensitive permeability tuned by probe charge and environmentally controlled gating. They can completely reject negatively charged probes, but attract positively charged species to form a higher concentration in the capsule interior, as evidenced by confocal microscopy. For example, the capsules completely exclude dextran labeled with fluorescein isothiocyanate (FITC-dextran), but are permeable for dextran labeled with tetramethylrhodamine isothiocyanate (TRITC-dextran) having similar molecular mass (from 4 to 70 kDa), although there are only few charged dyes in a dextran chain. By reversing the charge of the probes through pH change, or by suppressing charge repulsion through salt addition, the permeation can be readily switched for proteins such as albumin or small dyes such as fluorescein sodium salt.

Introduction Hollow capsules are of great interest due to their potential applications and fundamental importance as new colloidal structures in areas such as medicine, drug delivery, artificial cells or viruses, and catalysis. Recently a novel type of hollow microcapsule has been fabricated by means of alternating deposition of oppositely charged polyelectrolytes1 on colloidal templates. After formation of polyelectrolyte multilayers, the templates are removed to yield hollow capsules.2 Using this fabrication technique, capsules with well-controlled size and shape, finely tuned wall thickness, and variable wall composition have been produced. For example, microcapsules with customized physicochemical properties can be obtained by incorporation of one or more functional components such as biomacromolecules, lipids, photoactive dyes, nanoparticles, as well as multivalent ions onto the capsule wall or into the capsule interior.3 For various applications, a defined and switchable permeability of the capsules is required so that encapsulation and subsequent release can be realized. Previous studies have shown that the permeability of the polyelectrolyte microcapsules can be readily tuned by factors such as layer number,4 pH value,5 ionic strength6 and polarity of the solution,7 as well as annealing8 and resealing9 after core removal. More recently, charge repulsion and attraction between the capsules and the substances in bulk solution have been found to exhibit also a pronounced effect on the permeability of the polyelectrolyte microcapsules. For example, when charged oligomers are polymerized in a preformed capsule, both the capsule interior and the capsule † ‡

Zhejiang University. Max-Planck-Institute of Colloids and Interfaces.

wall are modified with the as-synthesized polyelectrolytes. As a result, the selective permeability of the capsule is dramatically enhanced: even small molecules with the same charge as the polyelectrolytes are fully rejected.10 In another case, capsules composed of chitosan/chitosan sulfate have also shown surface charge controlled permeability.11 Our previous studies have revealed that various water soluble substances can be spontaneously deposited into the preformed capsules templated on melamine formaldehyde particles.12 Its supposed mechanism is closely associated with the remaining charged complexes confined in intact capsules. On the other hand, much attention has been paid to polyelectrolyte multilayer microcapsules filled with neutral or charged polymers as well, which can be prepared by a variety of methods.10,13 Such “filled” capsules represent a novel type of nanoengineered composite microstructures and show unique physicochemical properties. For instance, a polarity gradient across the capsule wall can be established through which poorly water soluble materials such as most drugs can be precipitated in the capsule interior.14 A pH difference, on the other hand, can catalyze a spatial synthesis of nanoparticles inside the capsules.15 We report here an observation of very strong chargecontrolled permeation for capsules templated on CaCO3 microparticles, which are prepared in the presence of poly (styrene sulfonate) (PSS), a negatively charged polyelectrolyte. The PSS molecules are initially incorporated during fabrication of the CaCO3 crystal matrix and then are entrapped inside the microcapsules after core removal. Scanning electron microscopy (SEM), scanning force microscopy (SFM), confocal Raman, and UV-vis spectroscopy are employed to investigate the structure of the microcapsules. Confocal laser scanning microscopy

10.1021/jp0511092 CCC: $30.25 © 2005 American Chemical Society Published on Web 06/16/2005

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(CLSM) is used to investigate the permeability of the capsules for small dye molecules as well as macromolecules with different charges. Tuned by the ionic strength of the bulk solution, the switchable permeability for negatively charged molecules is further illustrated. As shall be illustrated, this method is simpler and can yield more stable capsules with very sharp gating property compared with previous strategies. Experimental Section Materials. Sodium poly(styrene sulfonate) (PSS, MW ∼ 70K), poly(allylamine hydrochloride) (PAH, MW ∼ 65K, calcium nitrate tetrahydrate (Ca(NO3)2‚4H2O), sodium carbonate (Na2CO3), disodium ethylenediaminetetraacetate dihydrate (EDTA), rhodamine 6G (Rd6G), fluorescein sodium (FL), fluorescein isothiocyanate labeled bovine albumin (FITCalbumin), FITC-dextran (MW ∼ 4K and ∼70K), tetramethylrhodamine isothiocyanate labeled dextran (TRITC-dextran, MW ∼ 4.4K and ∼65-76K) were all obtained from SigmaAldrich. All compounds 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. Methods. Fabrication of CaCO3 Particles. To prepare the CaCO3 particles, PSS was completely dissolved in 200 mL of 0.025 M calcium nitrate solution in a beaker under magnetic agitation (∼600 rpm), into which an equal volume of 0.025 M sodium carbonate solution in another beaker was rapidly poured at room temperature. The final PSS concentration was 2 mg/ mL. At the end of the reaction, the precipitated CaCO3 particles were collected and washed using a membrane filtration apparatus equipped with a cellulose filter having pore size of 0.45 µm. Layer-by-Layer Coating and Capsule Fabrication. Alternative adsorption of polyelectrolytes (2 mg/mL) onto the CaCO3 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 300 g for 5 min. After assembly of five polyelectrolyte bilayers, the coated particles were incubated in 0.02M EDTA solution for 30 min under shaking. The resultant capsules were centrifuged at 1500 g for 5 min with three washings in fresh EDTA solution. Finally, the capsules were washed with water three times. As the CaCO3 particles possess a negatively charged surface, the first layer was PAH, while the outermost layer in this study was always PSS. Scanning Electron Microscopy (SEM). For SEM analysis, samples were prepared by applying a drop of the particle or capsule suspension (concentration 106-107 particles or capsules per mL) on to a glass slide and drying overnight. After sputtering with gold, the samples were measured using 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 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 in tapping mode. 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. Equal amounts of capsule suspension and Rd6G (0.02 mg/mL), FL (2 mg/mL), TRITC-dextran (2 mg/mL), FITC-dextran (2 mg/mL), or FITC-albumin (2 mg/mL) were mixed. Observations were performed immediately or 2 h later.

Figure 1. SEM images of (a) bare CaCO3 particles and (b) (PAH/ PSS)5 microcapsules templated on these particles.

Raman Spectroscopy. Raman spectroscopy and Raman imaging of the CaCO3 particles and the polyelectrolyte capsules were performed in water under ambient conditions using a confocal Raman microscope (CRM200, Witec) equipped with a piezo scanner (P-500, Physik Instrument) and high NA microscope objectives (60×, NA ) 0.80 or 100× oil, NA ) 1.25, Nikon). In a typical experiment, a circularly polarized laser (CrystaLaser, λ ) 532 nm) was focused on the samples with a diffraction limited spot size (∼λ/2). An avalanche photodiode detector (APD) was used to record high-resolution Raman images. UV-Vis Spectroscopy. The CaCO3 microparticles and the resultant microcapsules were dried at 90 °C overnight to a constant weight (W0) and then were dissolved in 0.1 M HCl and 0.1 M NaOH solutions, respectively. The UV-vis absorption spectra were recorded using a Cary 50 UV-visible spectrophotometer. The absorbance at 225 nm corresponding to the phenyl groups was used to calculate the amount (WPSS) of PSS by referring to a calibration curve obtained from pure PSS at the same solvent conditions. The percentage of PSS was thus calculated as 100% × WPSS/W0. ζ-Potential Measurement. The ζ-potentials of the CaCO3 microparticles and the microcapsules were measured in water using a Zetasizer Nanoinstrument Nano Z. Each value was averaged from five parallel measurements. Small CaCO3 particles (∼2 µm) were used in order to avoid quick sedimentation. Results and Discussion Biomimetic synthesis of biominerals such as CaCO3 crystals in the presence of organic templates and/or additives has been extensively investigated in recent years, as reviewed by Co¨lfen.16 It has been shown that special functional low molecular weight and polymeric additives can strongly influence the crystallization of CaCO3,17 including complex liquidlike morphologies18 or stabilized amorphous CaCO3.19 Usually organic additives with complex functional groups are used to control nucleation, growth, and alignment of the crystals. It is found that double hydrophilic block copolymers are much more effective to control crystal growth.20 More recently, simple polyelectrolyte has been used to control the crystallization of carbonates.21 Figure 1a shows that spherical CaCO3 microparticles with average diameter of 8.2 ( 0.8µm were obtained through mineralization of the solutions of Ca(NO3)2 and Na2CO3 in the presence of PSS. The homogeneity of both the particle size and the surface morphology is very promising for these particles being used as template for multilayer deposition. Since the CaCO3 microparticles are synthesized in PSS solution, a negatively charged surface can thus be expected, as evidenced by ζ-potential measurement (-13 ( 0.3mV). Hence the assembly is started from the positively charged PAH. After

Charge-Controlled Permeability of Microcapsules

Figure 2. CLSM images of the microcapsules of Figure 1 incubated in solutions of (a) fluorescein for 2 h and (b) Rd6G for a few seconds.

Figure 3. Schematic illustration to depict the formation process of CaCO3 microparticles.

five bilayers were assembled (PSS was the outmost layer), the CaCO3 templates were removed by exposure to 0.02 M EDTA (pH 7) to yield hollow capsules. The hollow nature and integrity are clearly demonstrated by SEM observation (Figure 1b). The folds and creases are rather typical for collapsed hollow capsules due to evaporation of the solvent and low mechanical strength of the capsule wall. These capsules can also be well-dispersed in water and resemble almost the macroscopic shape and size of their CaCO3 template (φ 8.2 ( 0.8 µm). Their diameter, 8.4 ( 0.7 µm, is almost unchanged. When a suspension of the microcapsules was mixed with fluorescein (FL), a negatively charged small molecule dye, surprisingly, the capsule interior remained dark even after incubation for 2h (Figure 2a). This would mean that FL cannot permeate into the interior of the hollow capsules or the capsules can exclude the small dyes completely. Only after one month was a small number of the FL molecules found inside the capsules (data not shown). When the negatively charged FL was substituted by a positively charged small dye, such as Rd6G, in sharp contrast, a more intense fluorescence was observed from the capsule interior than from the bulk (Figure 2b) even after incubation for just a couple of seconds. These results reveal the surprising fact that the permeability of the as-prepared capsules is highly selective to the charge of

J. Phys. Chem. B, Vol. 109, No. 27, 2005 13161 the molecules being encapsulated: exclusion of negatively charged molecules but attraction of positively charged ones. This is very different from the capsules with the same composition but templated on melamine formaldehyde (MF) particles, which are permeable to small dye molecules and even polymers, regardless of their charges.22 A reasonable explanation for this phenomenon should be exclusively attributed to the microstructure of the resultant capsules that are templated on the PSS modified CaCO3 particles. According to the mechanism of particle formation, it is quite reasonable that the CaCO3 microparticles are assembled from a number of tiny crystallites or nanoparticles that have previously been stabilized by the PSS molecules. These PSS molecules are then incorporated into the crystal matrix and on the particle surface (Figure 3). Core removal will then release these negatively charged PSS molecules, which are confined in the microcapsules and thus repel molecules with the same charge. The magnified SEM image (Figure 4a) indeed shows that the typical surface texture of the particle is very rough and not smooth. It is built from smaller, nearly spherical building blocks with a typical diameter of 50-100 nm. The formation of this structure may arise from a mesoscale self-assembly process in the present case. The polyelectrolyte-stabilized amorphous nanoparticles (Figure 3b) act as precursor for the sequential mesoscale self-assembly and finally form micron-sized particles (Figure 3c). The driving force of formation of the spherical aggregate is likely the minimization of the surface energy.21b Confocal Raman spectroscopy was employed to visualize PSS in the CaCO3 microparticles. A typical Raman spectrum corresponding to a signal from the center of the CaCO3 particle is shown in Figure 4b. Apart from the most pronounced peak at 1084 cm-1, which is assigned to the carbonate group, vibrations of the sulfonate group at 1128 cm-1 and the aromatic ring quadrant stretching at 1600 cm-1 are clearly visible. Hence, one can conclude that there undoubtedly exists some amount of PSS molecules in the CaCO3 particle. By UV-vis spectroscopy the amount of PSS in the CaCO3 particles was found to be ∼6 wt %. Using these PSS-doped particles as template, one can thus investigate the filled microcapsule structure, since the permeability of the capsule walls is limited to macromolecules with larger molecular weight.22b The confocal Raman spectra of five bilayer PAH/PSS microcapsules show strong bands at 1128 and 1600 cm-1 at positions on both the capsule wall (Figure 5a) and the interior (Figure 5b), demonstrating the existence of PSS molecules inside the hollow capsules. Note that signals from the capsule wall may also partially be contributed by PSS originating from the core, because the method itself does not

Figure 4. (a) SEM image to show the typical surface texture of the CaCO3 particle. (b) Confocal Raman spectrum recorded from the center of a CaCO3 microparticle.

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Tong et al. the rough surface.25 Adsorption of the PSS molecules onto the capsule wall, most likely on the inner surface, should be responsible for this thick wall, too, since the inner surface is dominated with PAH.10 Let us roughly estimate the released and retained PSS amount in one capsule taking into account the SFM and UV-vis data. Since the average radius (r) is 4.1 × 10-4 cm, the volume (V) and weight (W) per CaCO3 particle can be calculated by eqs 1 and 2, respectively.

V ) (4/3)πr3 ) 2.9 × 10-10 cm3

(1)

W ) FV ) 2.5 g‚cm-3 × 2.9 × 10-10 cm3 ) 7.3 × 10-10 g (2) Figure 5. Raman spectra recorded from (a) capsule wall and (b) capsule interior. Since the settings of the apparatus have not been changed between recording of parts a and b, the relative intensities can be compared.

have enough spatial resolution to distinguish these two contributions. After dissolution of the capsules and destruction of the complex in 0.1 M NaOH,23 the PSS fraction of the polyelectrolyte in or on the capsules was quantified as 78 wt % by UVvis spectra taking the absorbance at 225 nm. To reveal the distribution of the PSS molecules, the microcapsules were further subjected to characterization by SFM. In the typical SFM image of intact capsules (Figure 6a), the most obvious feature is that more than 80% of the capsules (for homogeneity, see Figure 1b also) have shown the huge folds with a height of 1500-3500 nm. From the profile an average height of 116 ( 15 nm for the double wall thickness is derived. To measure this wall thickness without free PSS, the capsule was ruptured by means of ultrasonication, followed by washings to remove the released PSS. The broken capsules have a double wall thickness of 113 ( 9 nm, which roughly matches the height of the intact capsules. The biggest change, however, is that the huge fold has completely disappeared (Figure 6b). We presume that due to the rather small pore size and compact structure deduced from Figure 4a, the polyelectrolyte used for multilayer fabrication cannot diffuse inside. Assuming now that the huge fold is attributed to the incorporation of PSS, which may lead to incomplete collapse, this change is quite reasonable, since the PSS can be released. Compared to the thickness (15 nm) of (PSS/PAH)5 multilayers on a smooth substrate,24 the wall thickness of 55 nm is much larger, which may partly result from

The density (F) is the average density of the PSS-doped CaCO3, which is estimated by taking into account the density of pure CaCO3 (2.7 g‚cm-3) and PSS (1.2 g‚cm-3). Hence, the PSS weight in a CaCO3 particle is 6% × 7.3 × 10-10 g ) 4.4 × 10-11 g. If a thickness (δ) of 5-10 nm for the adsorbed PSS is subtracted from the value measured by SFM, the wall thickness can be estimated as 50 nm. The weight of a capsule wall (Wcapw) can be calculated by eq 3, assuming a multilayer density of 1.2 g‚cm-3.

Wcapw ) 4πr2d × 1.2 g‚cm-3 ) 1.2 × 10-11 g

(3)

Elemental analysis has characterized 27% PAH in PAH/PSS complex formed at neutral pH.26c Yet quartz crystal microbalance (QCM)26a and ellipsometry26b have measured a much higher value of 40% for five bilayers of PAH/PSS assembled in 0.5 M NaCl solution. Since the capsules have a multilayer structure, the PAH percentage of 40% should be more reliable in the present case. Therefore, the PAH amount in the capsule wall is 40% × 1.2 × 10-11 g ) 4.8 × 10-12 g. The PSS amount in the fabricated capsules was quantified as 78 wt % by UVvis spectra. Suppose the remaining 22% of the mass is all attributed to PAH in the wall, the real weight of a capsule (Wcap) should be 4.8 × 10-12 g/(1 - 0.78) ) 2.2 × 10-11 g. Hence, the entrapped PSS in a capsule is 2.2 × 10-11 g - 1.2 × 10-11 g ) 1 × 10-11 g, which is only about 23% of the amount of PSS (4.4 × 10-11 g) originating from the CaCO3. This would mean that about 77% of PSS originating from the CaCO3 particle

Figure 6. SFM images and their corresponding height profiles for dried capsules on mica. (a) Intact capsule with free PSS trapped inside and (b) broken capsule without free PSS inside.

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Figure 7. Schematic illustration to show the formation process and the topology of the as-prepared microcapsules. For details, see the text.

has been released to the outside (released amount: 4.4 × 10-11 g - 1 × 10-11 g ) 3.4 × 10-11 g). If these remaining free PSS molecules accumulated into a triangular prism after drying (Figure 6a), whose area is estimated as one-third to one-fourth of the equatorial plane of the capsule, the height (h) of the prism can be estimated as 1300-2000 nm by eq 4.

1 × 10-11g ) (1/3-1/4) × (1/3)πr2 × h × 1.2 g‚cm-3

(4)

This value matches roughly with the value measured from SFM (1500-3500 nm). As pointed out above, the incomplete collapse of the capsules may induce a somewhat thicker apparent wall by SFM. Furthermore, the remaining PSS corresponds to a concentration of about 3.4 wt % (1× 10-11 g/2.9 × 10-10 cm3). This value is not high enough to induce apparent swelling of a capsule with a wall thickness of 50 nm.13b,27 According to this analysis and based on the results of SEM, CLSM, SFM, Raman, and UV-vis, the topology of the capsule as well as its formation process can then be explained. Due to the rough surface of the CaCO3 particle (Figure 4a), a larger amount of polyelectrolytes would be adsorbed and even entrapped into the nanopores on the surface for the first few layers (Figure 7a).25b It is reasonable to assume that the rough surface would adsorb a relatively larger amount of polyelectrolytes for the first layer than the later layers, resulting in excess PAH located mainly in the inner surface of the multilayers. These excess PAH molecules will then combine with a part of the PSS molecules released from the CaCO3 particle during core removal (Figure 7b), forming a complex layer that is tightly bound onto the inner surface of the multilayers (Figure 7c). During the core dissolution and subsequent washing, a fraction of the unbound PSS is released from the capsule interior through the pores or defects on the capsule wall, while a small part of PSS is still encapsulated (Figure 7c). This encapsulation is largely dependent on the resealing of the big pores or defects by the formation of the complex in the inner surface of the wall and by the entrapment of the PSS molecules in the multilayer wall. We thus further postulate that some PSS segments on the complex should extend into solution, forming a brushlike interface to reduce the charge repulsion between PSS molecules. The electrostatic interaction in the complex neutralizes most charges so that no apparent capsule swelling is observed. Together with physical entanglement, the complex is stably connected with the multilayer wall. Consequently, the same wall thickness is measured for both broken and intact capsules. Furthermore, it is highly probable that substances permeate through water-filled pores in the polyelectrolyte multilayers.4a The entrapment of the PSS molecules in the multilayer wall would likely decrease both the number and the size of the waterfilled pores. Hence, the “resealing” effect created by the negatively charged PSS will build a very negatively charged

Figure 8. CLSM images of the same microcapsules incubated in solutions of FITC-dextran with molecular mass of (a) 4 kDa and (c) 70 kDa for 2 h and of TRITC-dextran with molecular mass of (b) 4.4 kDa and (d) 65-76 kDa for a few seconds. Scale bar ) 10 µm.

layer in the capsule wall, by which the permeation of negatively charged substances is blocked. As release of most PSS molecules has been confirmed by the above calculation using the UV-vis data, the intertwining of PSS molecules in the multilayer is confirmed by ζ-potential characterization. The ζ-potential of a colloidal particle is mainly determined by its surface charge. A negative ζ-potential of -42.1 ( 0.8 mV was measured for capsules with PSS as the outmost layer, a value quite consistent with that of multilayers templated on MF particles.28 However, a nearly neutral value of -0.8 ( 0.7 mV was detected for capsules with PAH as the outmost layer, which is far below the normal value (+50 mV)28 of capsules templated on MF particles. This reduction of positive surface charge can be only explained by the fact that PSS molecules have been intertwined in the multilayer. Capsules with this structure possess many special features, such as highly sensitive permeability tuned by probe charge and environmentally controlled gating. For example, dextran, a natural polysaccharide, is generally regarded as neutral, regardless of labeling with fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC). However, when solutions of FITC-dextran and TRITC-dextran with molecular mass of ∼4 or ∼70 kDa were mixed with the capsule suspension, substantially different permeation was observed (Figure 8). The capsule interior remained dark after incubation for 2 h in FITC-dextran solutions, regardless of the molecular weight (Figure 8a,c), indicating that the FITC-dextran did not permeate. In the time scale we investigated, this blocking effect lasted at least for 1 month (data not shown). When incubated in TRITC-dextran solution for a few seconds, in sharp contrast, stronger fluorescence was recorded from the capsule interior than from the bulk (Figure 8b,d), demonstrating a higher probe concentration in the capsule interior. (Note that the level of fluorescence coming from the wall is 1.5-2 times higher than that from the interior, which means that more FITC-dextran molecules were adsorbed in the wall.) This effect has previously been recognized as spontaneous deposition driven by charged polymers or complexes in the capsules.12 The opposite is

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Figure 9. CLSM images to show the selective permeability of the capsules to albumin. (a) At pH 7, albumin is negatively charged and hence cannot permeate; (b) at pH 2.5, the charge is reversed; hence, albumin can permeate.

apparently caused by the slight difference of molecular charges originating from the pendent fluorescent groups. Since dextran is neutral, labeling with FITC and TRITC lends the polymer negative or positive charges,11 respectively. Although the charge density of both molecules is very low, this slight difference may already lead to an effect of complete repulsion or attraction. It is worth mentioning that there are only 4-5 dye charges in a dextran chain with MW ∼ 70K. The very few dyes can be regarded as “signal” molecules, by which the permeation of macromolecules is strictly controlled. In other words, the capsules have very high sensitivity and selectivity to the properties of molecules, illustrated here with charge behavior. In some sense this kind of capsule can be used to mimic cell functions, such as selective permeation of ions through ion channels. We have to mention that for the charge-dominated permeability control the cutoff molecular mass detected so far is approximately 70 kDa. Beyond this value, the permeation depends more likely on molecular size instead of charge. For example, when TRITC-dextran with a molecular mass of 155 kDa was used, no fluorescence was recorded from the capsule interior after incubation for 2 h. This is understandable, since the pore size in the capsule wall is finite. Since the permeability of the capsules is largely dependent on the charge of the permeant, charge reversion of the probes may then affect permeability switching for the same molecules. It is known that a pH change can cause charge reversion of some molecules, in particular biomacromolecules such as proteins and enzymes. For example, at pH 7.0 negatively charged albumin (-15.4 ( 3.6 mV)12 could not permeate through the capsule walls (Figure 9a). After the pH value was decreased below the isoelectric point of albumin (4.2), for example 2.5, FITC-albumin was positively charged (+44.6 ( 1.1 mV).12 Consequently, these protein molecules could freely permeate into the capsule interior (Figure 9b). However, before drawing further conclusions, we should be aware that this drastic pH change may also change the wall permeability. More systematic experiments will be needed to clarify this. Apart from modulation of the probe charge, another approach to switch the permeability of the capsules is addition of salt, because the electrostatic forces can be effectively screened by electrolytes. Figure 10 shows that fluorescein sodium salt (FL) could not permeate into the capsules in pure water. However, when the water was substituted with 0.5 M NaCl, FL permeated immediately. The switch on and off can be repeated at least for five times, as illustrated in Figure 10. Besides screening the repulsion between the PSS and FL molecules, salt may also induce lateral shrinkage of the multilayers to enlarge the pores.6 Both are beneficial to enhance the permeability. The reversible

Tong et al.

Figure 10. CLSM images to show the switch on and off cycles tuned by addition and removal of salt, respectively. Fluorescein is used as a probe. Scale bar ) 10 µm.

switching offers the microcapsules a great opportunity for practical applications, such as smart sensors and intelligent drug delivery vehicles. Conclusions We demonstrate that multilayer microcapsules containing free polyelectrolytes, exemplified here with PSS, have been successfully fabricated through a simple entrapment method. While these novel capsules exhibit unique charge-controlled permeability, no prominent capsule swelling is observed. Encapsulation of the PSS molecules is achieved by layer-by-layer coating of CaCO3 microparticles with rough surface, which are fabricated using PSS as a morphology control reagent. By UV-vis characterization the PSS percentage in the CaCO3 particles is determined as 6 wt %. After CaCO3 removal with EDTA, most PSS molecules are released into bulk solution and rinsed off. A small part of the PSS molecules are intertwined into the multilayers, adsorbed on or combined with PAH molecules residing on the inner surface, while the other part is in a free state. Capsules with this topology can completely resist permeation of negatively charged probes but can attract positively charged species to accumulate in the capsule interior. The selectivity of the permeability is very pronounced. For dextran labeled with a few charged dyes, the permeability is sharply dependent on the sign of the charge. By reversing the charge of the probes through a pH change, or by suppressing charge repulsion through salt addition, permeation can be readily switched for proteins or small dyes with negative charge such as FL. Acknowledgment. We thank Prof. J. C. Shen for his continuous support and stimulating discussions. A. Heilig and R. Pitschke 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). References and Notes (1) (a) Decher, G. Science 1997, 277, 1232. (b) Decher, G.; Hong, J. D. Makromol. Chem., Macromol. Symp. 1991, 46, 321. (c) Keller, S. W.; Johnson, S. A.; Brigham, E. S.; Yonemoto, E. H.; Mallouck, T. E. J. Am. Chem. Soc. 1995, 117, 12879. (d) Ruths, J.; Essler, F.; Decher, G.; Riegler, H. Langmuir 2000, 16, 8871. (2) Donath, E.; Sukhorukov, G. B.; Caruso, F.; Davis, S. A.; Mo¨hwald, H. Angew. Chem., Int. Ed. 1998, 37, 2202. (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,

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