Release Behavior of Thin-Walled Microcapsules Composed of

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Langmuir 2001, 17, 2036-2042

Release Behavior of Thin-Walled Microcapsules Composed of Polyelectrolyte Multilayers Xiangyang Shi and Frank Caruso* Max Planck Institute of Colloids and Interfaces, D-14424 Potsdam, Germany Received November 7, 2000. In Final Form: January 5, 2001 The release properties of the fluorescent probe pyrene, PYR, encapsulated in polyelectrolyte multilayer capsules were examined by fluorescence spectroscopy. PYR was encapsulated by the layer-by-layer deposition of oppositely charged polyelectrolytes onto PYR microcrystal templates, and its release was accomplished by exposing the polyelectrolyte-coated microcrystals to ethanol solutions. Solubilization of the hydrophobic probe by ethanol resulted in its expulsion from the core through the permeable polyelectrolyte multilayer shells. The PYR release rate, quantified by following the monomer fluorescence of the solubilized PYR as a function of time, decreased with increasing number of polyelectrolyte layers deposited onto the microcrystal cores. In addition, two amphiphiles (sodium dodecyl sulfate and dipalmitoyl-DL-R-phosphatidylcholine) used to disperse the microcrystals in aqueous solution prior to coating with polyelectrolytes significantly influenced the PYR release rate. The maximum release time, defined as the time required to achieve saturation release, could be varied by a factor of 2-5 depending on the nature of the first adsorbed layer and the number of polyelectrolyte layers composing the shell wall. The influence of flow (i.e., centrifugation vs magnetic stirring) as well as solvent on the PYR release rate is also discussed.

Introduction Encapsulation processes are finding increasing use in areas ranging from medicine and pharmaceutics to agriculture and cosmetics.1-8 They are nowadays widely used to encapsulate a host of materials such as enzymes, hormones, drugs, cells, pesticides, and fragrances. The encapsulation process is often exploited to protect watersoluble substances from degradation and, in the vast majority of cases, to provide the controlled release of various compounds under specified conditions.1 Tailored release of encapsulated materials may be achieved by utilizing encapsulation techniques that provide a semipermeable “membrane shell” and allow adjustment of the membrane thickness and pore size. In addition, the membrane should be strong enough to withstand effects arising out of mechanical agitation. The classical systems of liposomes, microparticles, and microemulsions commonly exploited to encapsulate and deliver various substances do not readily permit manipulation of permeability, mechanical and chemical stability, surface charge, and biocompatibility. It is difficult to achieve control over these parameters using a single-step encapsulation process. However, semipermeable membrane shells with precisely adjusted permeability, stability, chemical functionality, and biocompatibility may be achieved by using a multistep strategy. An attractive and flexible stepwise construction method that lends itself to this task is the layer-by-layer (LbL) technique, which is based on the sequential deposition of oppositely charged individual polymer layers.9

Recently, by utilizing the LbL approach we have encapsulated a variety of materials within semipermeable polyelectrolyte multilayers.10-14 Polymer microparticles11 as well as metal nanoparticles12 have been enveloped with multilayered polyelectrolyte shells. This technique is also amenable for delicate biological materials; protein crystals of catalase have been coated by the sequential adsorption of polyanions and polycations.13 The polymer-encapsulated enzyme was stable against protease degradation and retained 100% of its activity after incubation for 100 min with protease.13 The LbL strategy has also been extended to uncharged microparticles to achieve the encapsulation of hydrophobic, poorly water-soluble low molecular weight compounds, such as pyrene (PYR) and fluorescein diacetate (FDA).14 The PYR and FDA microcrystals were precharged by the self-assembly of amphiphilic compounds onto their surface, thereby rendering them water-dispersible, followed by LbL deposition of oppositely charged polyelectrolytes. In a subsequent step, removal of the encapsulated core materials via their solubilization resulted in the formation of multilayered, thin-walled, intact polyelectrolyte hollow capsules.14 The fact that the cores could be readily removed implies that the polyelectrolyte multilayer shells are permeable, as no rupture of the capsule walls was observed. Their semipermeable nature was further verified by other studies, which demonstrate that under certain conditions the capsules are permeable for low molecular weight dyes and ions11-13 but not for enzymes (e.g., catalase)13 or polymers with molecular

* To whom correspondence should be addressed. Fax: +49 331 567 9202. E-mail: [email protected].

(9) (a) Decher, G.; Hong, J.-D. Ber. Bunsen-Ges. Phys. Chem. 1991, 95, 1430. (b) Decher, G. Science 1997, 277, 123. (10) For a review, see: Caruso, F. Chem.sEur. J. 2000, 6, 413. (11) (a) Caruso, F.; Donath, E.; Mo¨hwald, H. J. Phys. Chem. B 1998, 102, 2011. (b) Donath, E.; Sukhorukov, G. B.; Caruso, F.; Davis, S. A.; Mo¨hwald, H. Angew. Chem., Int. Ed. 1998, 37, 2201. (c) Caruso, F.; Lichtenfeld, H.; Donath, E.; Mo¨hwald, H. Macromolecules 1999, 32, 2317. (d) Caruso, F.; Schu¨ler, C.; Kurth, D. G. Chem. Mater. 1999, 11, 3394. (12) Gittins, D. I.; Caruso, F. Adv. Mater. 2000, 12, 1947. (13) Caruso, F.; Trau, D.; Mo¨hwald, H.; Renneberg, R. Langmuir 2000, 16, 1485. (14) Caruso, F.; Yang, W.; Trau, D.; Renneberg, R. Langmuir 2000, 16, 8932.

(1) Lim, F.; Sun, A. M. Science 1980, 210, 908. (2) Arshady, R. J. Controlled Release 1991, 17, 1. (3) Kreuter, J. Colloidal Drug Delivery Systems; Marcel Dekker: New York, 1994. (4) Pommersheim, R.; Schrezenmeir, Vogt, W. Macromol. Chem. Phys. 1994, 195, 1557. (5) Hari, P. R.; Chandy, T.; Sharma, C. P. J. Microencapsulation 1996, 13, 319. (6) Park, K. Controlled Drug Delivery: Challenges and Strategies; American Chemical Society: Washington, DC, 1997. (7) Langer, R. Nature 1998, 392, 5. (8) Bartkowiak, A.; Hunkeler, D. Chem. Mater. 2000, 12, 206.

10.1021/la001550d CCC: $20.00 © 2001 American Chemical Society Published on Web 02/24/2001

Release Behavior of Thin-Walled Microcapsules

weights about 4000 or greater.15 For hollow polyelectrolyte multilayer capsules produced from microcrystal templates pre-charged with a first layer of amphiphile, significant differences in their morphology were observed by electron microscopy and atomic force microscopy, suggesting different permeability characteristics as a result of the first adsorbed layer.14 One attractive feature of the LbL encapsulation strategy is that the thickness of the polyelectrolyte multilayer shell can be controlled at the nanometer level, with a single polyelectrolyte layer typically being 1-2 nm thick.10,11 This provides a straightforward and simple means to vary the release behavior of encapsulated substances through control of the multilayer thickness. The inclusion of nanoparticles,16 biomacromolecules,17 and/or lipids18,19 in the capsule walls may be an alternative means to control the permeability. Permeability control is desirable when, for example, the capsules are used for immobilization or cell transplantation and where the capsule membrane serves as an immunoprotective barrier. In addition, control over the capsule permeability is crucial for the sustained release of encapsulated active substances. A number of related studies have shown that the use of sequentially deposited polycation/polyanion multilayer coatings on preformed alginate-based microcapsules is effective in controlling their permeability.4,8,20,21 Vogt et al. demonstrated that enzymes could be retained within alginate microcapsules upon coating with polycation/ polyanion multilayers, for example, polyethyleneimine, poly(acrylic acid), poly(N-vinylamine), carboxymethylcellulose, and chitosan.4,20 It was reported that coating of cytochrome C-loaded alginate beads with a multilayer membrane consisting of alternating layers of poly(Nvinylamine) and poly(acrylic acid) or water-soluble anionic cellulose derivatives minimized loss of the protein during storage when at least two polycation/polyanion double layers were used.4,20 However, single-layer membranes were not able to retain cytochrome C.4,20 In another study, it was shown that one double layer of alginate and chitosan deposited on preformed alginate-chitosan capsules had only a minor effect on the permeability of immunoglobulin G (IgG), whereas four double layers yielded a large effect in limiting the diffusion of IgG.21 The decrease in permeability was attributed to the complexed structure of the polyelectrolyte pairs. Furthermore, it was found that the final permeability was dependent on the initial porosity of the uncoated alginate capsules.21 Recently, Bartkowiak and Hunkeler demonstrated the simultaneous regulation of mechanical properties and permeability for microcapsules based on oligochitosan and alginate through control of the reaction conditions.8 The molar mass of the polyanion was shown to influence the mechanical resistance of the capsule while a reduction in membrane (15) Sukhorukov, G. B.; Brumen, M.; Donath, E.; Mo¨hwald, H. J. Phys. Chem. B 1999, 103, 6434. (16) (a) Caruso, F.; Caruso, R. A.; Mo¨hwald, H. Science 1998, 282, 1111. (b) Caruso, F.; Lichtenfeld, H.; Mo¨hwald, H.; Giersig, M. J. Am. Chem. Soc. 1998, 120, 8523. (c) Caruso, F.; Mo¨hwald, H. Langmuir 1999, 15, 8276. (d) Caruso, F.; Caruso, R. A.; Mo¨hwald, H. Chem. Mater. 1999, 11, 3309. (17) (a) Caruso, F.; Mo¨hwald, H. J. Am. Chem. Soc. 1999, 121, 6039. (b) Caruso, F.; Fiedler, H.; Haage, K. Colloids Surf., A 2000, 169, 287. (c) Schu¨ler, C.; Caruso, F. Macromol. Rapid Commun. 2000, 21, 750. (d) Caruso, F.; Schu¨ler, C. Langmuir 2000, 16, 9595. (18) Moya, S.; Sukhorukov, G. B.; Auch, M.; Donath, E.; Mohwald, H. J. Colloid Interface Sci. 1999, 216, 297. (19) Moya, S.; Donath, E.; Sukhorukov, G. B.; Auch, M.; Ba¨umler, H.; Lichtenfeld, H.; Mo¨hwald, H. Macromolecules 2000, 33, 4538. (20) Rilling, P.; Walter, T.; Pommersheim, R.; Vogt, W. J. Membr. Sci. 1997, 129, 283. (21) Gaserod, O.; Sannes, A.; Skjak-Brk, G. Biomaterials 1999, 20, 773.

Langmuir, Vol. 17, No. 6, 2001 2037 Scheme 1. Schematic of Experimental Procedurea

a PYR microcrystals were first precoated (by self-assembly) with either PSS, SDS, or DPPC, followed by further coating with oppositely charged polyelectrolytes by using the layerby-layer deposition method. Subsequently, the coated microcrystals were exposed to solvent (ethanol or ethanol/water mixtures) to solubilize and release PYR through the semipermeable polyelectrolyte multilayer capsules.

permeability was achieved by increasing the polyanion concentration. Subsequent alginate coatings (i.e., postencapsulation) were also found to reduce the membrane permeability.8 These earlier investigations demonstrate the usefulness of polycation/polyanion multilayers in controlling the release of encapsulated materials. In the present study, we systematically investigate the release behavior of PYR from microcapsules composed of polyelectrolyte multilayers (membrane shell) of various thicknesses. The release of solubilized PYR from precharged PYR microcrystals coated with polyelectrolyte multilayers is examined by fluorescence spectroscopy. PYR release was induced by exposure of the precoated, poorly water-soluble PYR microcrystals to ethanol/water mixtures (Scheme 1). It is demonstrated that the release properties of low molecular weight materials (such as PYR) through polyelectrolyte multilayer capsules can be tailored by varying the number of polyelectrolyte layers as well as the amphiphile type used as the first adsorbed layer. Additionally, the effects of applied flow (centrifugation versus magnetic stirring) on PYR release were compared, and the influence of solvent quality, which was found to alter the release properties of PYR, was also examined. The main motivations for this work were to examine the role of polyelectrolyte multilayers and the influence of preadsorbed amphiphilic layers on the release properties of encapsulated (crystalline) low molecular weight compounds. An understanding of such release characteristics is not only of scientific interest but is also of great relevance for related systems in immunoassay applications where they can be employed as labels with enhanced sensitivity.22 Hence, the systems investigated can be considered as useful models for practical and novel release applications. Experimental Section Materials. Pyrene (PYR), the polycation, poly(allylamine hydrochloride) (PAH), Mw 15 000, and the polyanion, poly(sodium 4-styrenesulfonate) (PSS), Mw 70 000, were obtained from Aldrich. Sodium dodecyl sulfate (SDS) was also from Aldrich, and (22) (a) Yang, W.; Trau, D.; Renneberg, R.; Yu, N. T.; Caruso, F. J. Colloid Interface Sci. 2001, 234, 356. (b) Trau, D.; Renneberg, R.; Caruso, F. European Patent Application, 2000.

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Shi and Caruso

Chart 1. Molecular Structures of the Compounds Used in This Study

Figure 1. Fluorescence spectra of PYR in solution as a result of ethanol dissolution of bare PYR microcrystal cores. Spectra are shown for (a) 2 min and (b) 8 min after the addition of the uncoated microcrystals to ethanol solution. The excitation wavelength was 350 nm. The inset shows the PYR dissolutiontime profile; the fluorescence intensity was monitored at 373 nm (monomer emission). Time zero corresponds to the time at which the microcrystals were added to the ethanol solution.

dipalmitoyl-DL-R-phosphatidylcholine (DPPC) was purchased from Sigma. Ethanol (absolute) was obtained from Merck. All materials were used as received, except for PSS, which was dialyzed against Milli-Q water (Mw cutoff 14 000) and lyophilized before use. The water used in all experiments was passed through a Millipore Milli-Q Plus 185 purification system and had a resistivity exceeding 18.2 MΩ cm. Assembly of Polyelectrolyte Multilayers onto PYR Microcrystals. The LbL assembly of the oppositely charged polyelectrolytes onto PYR microcrystals (most in the size range of 1-10 µm) was carried out as described earlier.14 Specifically, 100 mg of finely milled “core” particles was first thoroughly mixed with 100 mL of three different dispersing agents (Chart 1): 6 mg mL-1 PSS, 0.25 wt % DPPC, or 0.3 wt % SDS. The crystals were suspended by immediate sonication for 5 min, and the suspension was allowed to stand for 30 min, during which time the larger crystals sedimented. In some cases, gentle centrifugation was performed to settle the larger microcrystals. The resulting product was purified by washing twice with water and finally resuspending in water. The precharged microcrystal particles were then LbL-coated with PAH and PSS with slight modifications of the procedure described elsewhere.14 (In all cases, the polyelectrolyte concentration was sufficiently higher than that required for saturation coverage of the microcrystal surface.) When DPPC was used as the first adsorbed layer, 10 mL of a PSS solution (5 mg mL-1, containing 0.5 M NaCl) was added to a 15 mL centrifuge tube containing 2 mL of the DPPC-coated, dispersed microcrystals. PAH solution (10 mL of 5 mg mL-1, containing 0.5 M NaCl) was added when PSS or SDS was adsorbed onto the microcrystals. After an adsorption time of 15 min for PAH or PSS adsorption, the suspension was centrifuged at 10000g for 10 min. The supernatant was then removed, and the coated microcrystals were washed by three alternate cycles of centrifuging and resuspending the particles in pure water. Additional polyelectrolyte layers bearing an opposite charge to those already adsorbed on the microcrystals were deposited in identical fashion until the desired number of multilayers was achieved.

PYR Release Experiments. A portion (10 µL) of the coated microcrystal suspension was quickly added into a 4 mL cuvette, which contained 3 mL of ethanol or 80% ethanol (mixed with water), and stirred gently by using a stirrer bar of 5 mm length. The stirring speed was kept constant throughout all experiments. After a defined time interval (2, 5, 10 min, etc.), the fluorescence of the (air-equilibrated) solution was determined in situ by using a LS-50B fluorescence spectrophotometer. The fluorescence emission intensity of PYR was measured directly by using an excitation wavelength (λex) of 350 nm and monitoring the emission (λem) from 360 to 600 nm. Both the excitation and emission slit openings were set at 5 nm. Maximum PYR release was taken as that at which the PYR fluorescence (at 373 nm, peak fluorescence) remained constant with time. The volume of the capsules is less than 0.3% of that of the solution; thus, the contribution of PYR emission from within the interior of the capsules to the measured fluorescence intensity (assuming an equilibrium between inside and outside the capsules) is negligibly small. As control experiments, the dissolution characteristics of bare PYR microcrystals were also studied as outlined above.

Results and Discussion Fluorescence spectroscopy is an appropriate technique to follow the dissolution and release behavior of PYR from the polyelectrolyte microcapsules. In pure water solution, the fluorescence from PYR in the microcrystals is predominantly excimer emission,23 which has a broad peak centered at 480 nm (data not shown). As PYR is released into bulk solution, as a result of solubilization by ethanol, there is a sudden increase in the monomer fluorescence intensity (λ ∼ 373 nm).23 Figure 1 shows typical fluorescence spectra of PYR in solution resulting from dissolution of uncoated PYR microcrystal cores in ethanol solution (control experiment). The inset displays the time profile for PYR dissolution, which reaches a maximum within 8 min. Because the PYR excimer emission is small (typically ca. 5% of that observed for the monomer), the rate of PYR (23) See, for example: (a) Gra¨tzel, M.; Thomas, J. K. Modern Fluorescence Spectroscopy; Wehry, E. L., Ed.; Plenum: New York, 1976; Vol. 2. (b) Zachariase, K. A.; Kozankiewicz, B.; Ku¨hnle, W. Surfactants in Solution; Mittal, K. L., Ed.; Plenum: New York, 1983. (c) Winnik, M. A. Polymer Surfaces and Interfaces; West, W. J., Munro, H. S., Eds.; Wiley: New York, 1987; pp 1-31. (d) Rangarajan, B.; Coons, L. S.; Scranton, A. B. Biomaterials 1996, 17, 649.

Release Behavior of Thin-Walled Microcapsules

Langmuir, Vol. 17, No. 6, 2001 2039 Table 1. Maximum PYR Release Times for the Polyelectrolyte (PE) Multilayer Capsules Formed on Amphiphile-Coated Microcrystals, As Determined by Fluorescence Spectroscopy release time (min)a system

+prelayer

+4 PE layers

+12 PE layers

+20 PE layers

PYR-PSS PYR-SDS PYR-DPPC

150 60 120

160 90 180

210 200 270

280 290 340

a

Figure 2. PYR release-time profiles for microcrystals coated with a different number of polyelectrolyte multilayers. The solvent used was 100% ethanol.

release from polyelectrolyte-coated microcrystals can be followed in situ by monitoring the increase in monomer emission as a function of time. Influence of Polyelectrolyte Layer Number on PYR Release. It has been established that the thickness of the polyelectrolyte multilayer shell increases regularly with the consecutive adsorption of polycations and polyanions onto colloidal templates.10-12 Hence, the thickness of the polyelectrolyte shell can be tuned with nanometer precision simply by varying the number of polyelectrolyte layers deposited. By measurement of the heights of airdried hollow polyelectrolyte capsules produced by coating PYR and fluorescein diacetate microcrystals and subsequently removing the core by ethanol dissolution, it was shown that the average thickness per polyelectrolyte layer was approximately 1-1.5 nm.14 On the basis of these data, the thickness of the polyelectrolyte multilayers in the current work can be estimated to range from ca. 1 to 30 nm, that is, thin-walled microcapsules composed of 1-20 polyelectrolyte layers. Accordingly, the release rate of encapsulated substances would be expected to decrease with increasing shell thickness. Figure 2 shows the release-time profiles for PYR microcapsules coated with a different number (1, 5, 13, or 21) of polyelectrolyte layers. PSS0L refers to one layer of PSS adsorbed on the PYR microcrystal surface. Similarly, PSS4L denotes that PSS was the first (precharging) layer coated onto the PYR microcrystal, followed by four additional polyelectrolyte layers (i.e., (PAH/PSS)2). As can be seen, the rate of release progressively decreases as the number of polyelectrolyte layers forming the membrane shell increases. There appear to be several stages in the dissolution of the PYR microcrystals and the release behavior of solubilized PYR. (The coated microcrystals have a large size distribution (ca. 1-10 µm) and a range of shapes (close to spherical, rodlike, square, rectangular, etc.), making accurate quantification of the data in terms of diffusion and permeability difficult.) Qualitatively, the dissolution and release process can be described as follows. Core dissolution occurs, and there may be a change in the properties of the polyelectrolyte multilayer shell because of an osmotic pressure increase as a result of solubilization of PYR.13 (In a previous study, solubilization of catalase crystals encapsulated within polyelectrolyte multilayers (and not able to diffuse out) resulted in a dramatic morphology change of the polyelectrolyte multilayer capsule shape because of osmotic pressure effects.13)

The error in the values is estimated as (5%.

Further PYR solubilization may create the situation where a time-independent concentration gradient between the microcapsule interior and the bulk solution exists. Upon complete dissolution of the core, an equilibrium is set up between the PYR concentration within the microcapsule and that in the bulk, which results in a constant concentration of PYR in the bulk and is evidenced as a plateau in the release profiles (taken as maximum release). Confirmation of PYR release was provided in our earlier study where hollow polyelectrolyte capsules were obtained after exposure of polyelectrolyte-coated PYR microcrystals to ethanol.14 The time to achieve maximum PYR release increases with the number of polyelectrolyte multilayers deposited onto the microcrystals (Figure 2). The time for maximum release is a factor of ∼2 longer for PYR microcrystals coated with 21 polyelectrolyte layers compared with those coated with a single PSS layer. The release data is summarized in Table 1. In all cases, the times for maximum release are significantly longer than those previously employed (