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Ultrasonically Induced Opening of Polyelectrolyte Microcontainers Dmitry G. Shchukin,* Dmitry A. Gorin, and Helmuth Mo¨hwald Max-Planck Institute of Colloids and Interfaces, D14424 Potsdam, Germany ReceiVed April 18, 2006. In Final Form: May 31, 2006 The effect of ultrasonic treatments of different intensity and duration on the integrity and permeability of polyelectrolyte capsules was investigated both in poly(allylamine)/poly(styrene sulfonate) and Fe3O4/poly(allylamine)/poly(styrene sulfonate) polyelectrolyte capsules. Ultrasonic treatment of polyelectrolyte capsules induces the destruction of the polyelectrolyte shell and the release of the encapsulated material even at short (5 s) sonification times. The presence of magnetite nanoparticles significantly improves the efficiency of the ultrasonically stimulated release of the encapsulated compounds and enables magnetically controlled delivery to the desired site before ultrasonic treatment. Release of the encapsulated compound induced at ultrasonic power comparable to those of ultrasonic generators applied in medicine, demonstrating practical application of the ultrasonically triggered capsule opening in medicine.
Introduction Targeted delivery and controlled release of drugs have a high priority for further development in modern medicine, material science, and biochemistry.1,2 In particular, this research area is important for curing dangerous diseases such as cancer or pulmonary tuberculosis. In this case, highly toxic but effective drugs are usually employed. To decrease their toxicity, it is desirable to treat only the damaged part of the organism, escaping the negative influence of the toxic drugs on the rest of the body. The most promising approach to meet this challenge is to entrap toxic drugs in the inner volume of the microcontainers. Their shell will prevent any contact with healthy tissue during the targeted delivery of drugs before they reach damaged cells. To date, polymer3,4 and polyelectrolyte5,6 capsules, fluoroalcane microemulsions,7 micelles,8 hydrogels,9 and porous hydroxyapatites10 have been developed to encapsulate and transfer various materials dispersed on the micro- and nanolevel. Each microcontainer type has specific advantages and drawbacks; however, most of them can be functionalized by magnetic nanoparticles11,12 or molecular materials possessing specific recognition properties13,14 to perform targeted delivery of the compounds inside. * To whom correspondence should be addressed. E-mail:
[email protected]; phone: +49 (0)331-567-9257; fax: +49 (0)331-567-9202. (1) Lu, D. R.; Oie, S. Cellular Drug DeliVery: Principles and Practice; Humana Press: Totowa, NJ, 2004. (2) Junginger, H. E. Drug Targeting and DeliVery; Taylor & Francis: Oxford, UK, 1992. (3) Lee, D.; Rubner, M. F.; Cohen, R. E. Chem. Mater. 2005, 17, 1099-1105. (4) Croll, L. M.; Stover, H. D. H. Pure Appl. Chem. 2004, 76, 1365-1374. (5) Ai, H.; Jones, S. A.; de Villiers, M. M.; Lvov, Y. M. J. Controlled Release 2003, 86, 59-68. (6) Sukhorukov, G. B.; Donath, E.; Davis, S.; Lichtenfeld, H.; Caruso, F.; Popov, V. I.; Mo¨hwald, H. Polym. AdV. Technol. 1998, 9, 759-763. (7) Cui, Z. G.; Fountain, W.; Clark, M.; Jay, M.; Mumper, R. J. Pharm. Res. 2003, 20, 16-23. (8) Ideta, R.; Yanagi, Y.; Tamaki, Y.; Tasaka, F.; Harada, A.; Kataoka, K. FEBS Lett. 2004, 557, 21-25. (9) Uchiyama, T.; Kiritoshi, Y.; Watanabe, J.; Ishihara, K. Biomaterials 2003, 24, 5183-5190. (10) Mizushima, Y.; Ikoma, T.; Tanaka, J.; Hoshi, K.; Ishihara, T.; Ogawa, Y.; Ueno, A. J. Controlled Release 2006, 110, 260-265. (11) Shchukin, D. G.; Radtchenko, I. L.; Sukhorukov, G. B. J. Phys. Chem. 2003, 107, 86-90. (12) Menager, C.; Sandre, O.; Mangili, J.; Cabuil, V. Polymer 2004, 45, 24752481. (13) Heuberger, R.; Sukhorukov, G.; Voros, J.; Textor, M.; Mo¨hwald, H. AdV. Funct. Mater. 2005, 15, 357-366. (14) Jule, E.; Nagasaki, Y.; Kataoka, K. Bioconjugate Chem. 2003, 14, 177186.
Besides targeted delivery, a tool for controlled release of the encapsulated material at a desired place has to be elaborated for successful operation of the whole delivery system. This can be performed by introducing biodegradable polymers into the microcontainer shell,15,16 adsorbing IR-sensitive or magnetic nanoparticles, followed by treatment by IR light at around 1 µm wavelength or by alternating magnetic fields.17,18 However, the methods developed so far are not universal and have considerable shortcomings: biodegradable microcontainers can be destroyed on their way to the damaged cells; application of IR irradiation demands focusing on the microcontainers to attain the intensity required to split their shell, which is difficult to perform in vivo deep under the skin; and alternating magnetic fields of high intensity (1200 Oe) are required to accomplish a magnetically induced release of the encapsulated drug. Another approach for the remote-activated release of the encapsulated materials from the microcontainers is described.19 This approach is based on the effect of acoustic cavitation in liquids under ultrasonic vibrations with frequency more than 20 kHz. Ultrasonic treatment is already employed in many fields (e.g., fabrication of nanoparticles20-22 and surface cleaning23), including medicine for diagnosing and curing several diseases.24,25 Ultrasonic waves have enough power and penetrating ability to damage the microcontainer shell and, as a consequence, to release an encapsulated drug in vivo, deep inside the body.26 Moreover, the existence of already developed and FDA approved medical ultrasonic processors and instruments opens an avenue for (15) Chun, K. W.; Cho, K. C.; Kim, S. H.; Jeong, J. H.; Park, T. J. J. Biomater. Sci., Polym. Ed. 2004, 15, 1341-1353. (16) Liu, H.; Finn, N.; Yates, M. Z. Langmuir 2005, 41, 379-385. (17) Lu, Z.; Prouty, M. D.; Guo, Z.; Golub, V. O.; Kumar, C. S. S. R.; Lvov, Y. M. Langmuir 2005, 21, 2042-2050. (18) Skirtach, A. G.; Dejugnat, C.; Braun, D.; Susha, A. S.; Rogach, A. L.; Parak, W. J.; Mo¨hwald, H.; Sukhorukov, G. B. Nano Lett. 2005, 5, 1371-1377. (19) Antipov, A. A.; Mamedov, A.; Sukhorukov, G.; Mo¨hwald, H.; Kotov, N.; Skirtach, A. Remote control release of encapsulated materials. German Patent W02005107701, Nov 17, 2005. (20) Okitsu, K.; Teo, B. M.; Ashokkumar, M.; Grieser, F. Aust. J. Chem. 2005, 58, 667-670. (21) Prozorov, T.; Prozorov, R.; Suslick, K. S. J. Am. Chem. Soc. 2004, 126, 13890-13891. (22) Dhas, N. A.; Suslick, K. S. J. Am. Chem. Soc. 2005, 127, 2368-2369. (23) Muthukumaran, S.; Yang, K.; Seuren, A.; Kentish, S.; Ashokkumar, M.; Stevens, G. W.; Grieser, F. Sep. Purif. Technol. 2004, 39, 99-107. (24) Turgay, E.; Salcudean, S.; Rohling, R. Ultrasound Med. Biol. 2006, 32, 221-235. (25) Levy, D. M.; Silverman, E. D.; Massicotte, M. P.; McCrindle, B. W.; Yeung, R. S. M. J. Rheumatol. 2005, 32, 928-934. (26) Liu, H. L.; McDannold, N.; Hynynen, K. Med. Phys. 2005, 32, 12701280.
10.1021/la061047m CCC: $33.50 © 2006 American Chemical Society Published on Web 07/12/2006
Opening Polyelectrolyte Capsules Via Ultrasound
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Figure 1. Schematic representation of the procedure employed for the ultrasonically induced opening of dextran-loaded Fe3O4/polyelectrolyte capsules. Fe3O4/poly(allylamine)/poly(styrene sulfonate) capsules were suspended in water, then concentrated by an external magnet directly under the sonotrode and sonicated.
Figure 2. SEM (bottom panels) and fluorescence confocal (top panels) images of the initial Fe3O4/polyelectrolyte capsules and Fe3O4/ polyelectrolyte capsules treated by ultrasound for 1 min at different powers.
practical utilization of ultrasound-assisted release of the encapsulated drugs. The combination of modern ultrasound-assisted diagnostic protocols with methods for targeted delivery of the entrapped drugs can provide high selectivity and accuracy for the targeted medical treatment with minimal toxic effect. In the present paper, we investigate the possibility of using ultrasonic treatment as a remote trigger for the release of materials encapsulated inside layer-by-layer assembled polyelectrolyte capsules. Layered polyelectrolyte capsules were chosen as prospective delivery microcontainers because of the high versatility of their properties achieved by changing the components (e.g., polyelectrolytes, lipids, nanoparticles, and biopolymers) of the polyelectrolyte shell and their order in the shell, as well as the ability to easily control encapsulation and release by changing the pH, ionic strength, and temperature of the surrounding medium.27-29 Experimental Methods Materials. Sodium poly(styrene sulfonate) (PSS, MW ∼ 70 000), poly(allylamine hydrochloride) (PAH, MW ∼ 50 000), tetramethylrhodamine isothiocyanate-labeled PAH (TRITC-PAH), fluorescein isothiocyanate-labeled PAH (FITC-PAH), FITC-labeled dextran (27) Ko¨hler, K.; Shchukin, D. G.; Mo¨hwald, H.; Sukhorukov, G. B. J. Phys. Chem. B 2005, 109, 18250-18259.
(FITC-dextran, MW ∼ 20 000) were purchased from Aldrich and used without additional purification. Monodisperse polystyrene microparticles of 10.25 µm diameter and 8 nm citrate-stabilized Fe3O4 sol were respectively purchased from Microparticles GmbH, Berlin, Germany. The water was prepared in a three-stage Millipore Milli-Q Plus 185 purification system and had a resistivity higher than 18 MΩ‚cm. Capsule Preparation and FITC-Dextran Encapsulation. Hollow PAH/PSS capsules were prepared by alternating the deposition of PAH and PSS monolayers on the polystyrene template particles from 2 mg/mL PAH and 2 mg/mL PSS aqueous solutions in the presence of 0.5 M NaCl and at 15 min of incubation time for each monolayer. Polystyrene/polyelectrolyte particles were washed after each adsorption step three times with deionized water. The total number of monolayers was 16 for each sample. Magnetite nanoparticles (saturation polarization is 22 mT) were adsorbed from an 8 mg/mL suspension, replacing negatively charged PSS layers in the third, fourth, fifth, and sixth bilayers. Then, the polystyrene template was dissolved in tetrahydrofuran (THF) for 6 h, centrifuged, and washed again with THF four times. The overall structure of the Fe3O4-containing capsule shell was (PAH/PSS)2(PAH/Fe3O4)4(PAH/ (28) Ibarz, G.; Dahne, L.; Donath, E.; Mo¨hwald, H. AdV. Mater. 2001, 13, 1324-1327. (29) Antipov, A. A.; Sukhorukov, G. B.; Mo¨hwald, H. Langmuir 2003, 19, 2444-2448.
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Results and Discussion
Figure 3. Fluorescence confocal images of the polyelectrolyte (A) and Fe3O4/polyelectrolyte (B) capsules treated for 1 min by ultrasound (500 W). PSS)2; that of pure polyelectrolyte capsules was (PAH/PSS)8. More detailed information on the capsule fabrication can be found elsewhere.6 FITC-dextran was loaded inside the resulting capsules by the thermal encapsulation method:30 empty capsules were mixed with FITC-dextran (2 mg/mL) for 1 h and incubated at 70 °C for 90 min. The elevated temperature causes shell densification, which prevents the spontaneous release of entrapped dextran into water.27,30 Ultrasonic treatment was performed by a 500 W Sonics & Materials VCX500 ultrasonic processor operating at 20 kHz and equipped with 1.3 cm diameter Ti horn. A cylindrical glass vessel (2.8 cm inner diameter) with a total volume of 20 mL was used for ultrasonic irradiation. The vessel was closed during sonification. Characterization. Confocal microscopy images of polyelectrolyte capsules in solution were obtained on a Leica TCS SP scanning system equipped with a 100× oil immersion objective operating in fluorescence mode. Fluorescence spectra of the released FITCdextran were recorded using a Spex Fluorolog 212 spectrofluorimeter (488 nm excitation line). For scanning electron microscopy (SEM) analysis, a drop of each sample solution was applied to a glass wafer with sequential drying. Then the samples were sputtered with gold, and measurements were conducted using a Gemini Leo 1550 instrument. (30) Ko¨hler, K.; Shchukin, D. G.; Mo¨hwald, H.; Sukhorukov, G. B. Macromolecules 2004, 37, 9546-9550.
The effects of ultrasound are primarily derived from acoustic cavitation,31 where microbubble collapse in liquids results in an enormous concentration of energy, even at low input power, converting the surface energy and kinetic energy of the liquid motion into the heat of the microbubble contents and chemical energy of the compounds formed. Ultrasonic treatment can induce a wide range of physicochemical consequences applicable for the synthesis and modification of both organic and inorganic materials (e.g., dispergation,19,20 surface modification and microannealing,32 and the formation of bimetallic nanoparticles33,34). The effect of ultrasonic treatment can be enhanced by passing the acoustic waves through the media of different densities.35 These properties of ultrasonic waves exhibit the necessary conditions for applying ultrasound as a tool for the remotely controlled opening of the loaded microcontainers. To demonstrate the possibility of an ultrasonically induced opening, we employed layer-by-layer-assembled polyelectrolyte capsules. The polyelectrolyte shells of the capsules are modified by several monolayers of nanosized Fe3O4 to increase the density gradient across the water/shell interface and to impart magnetic properties to the polyelectrolyte capsules, providing their targeted delivery regulated by constant magnetic field. The experimental procedure is schematically depicted in Figure 1. First, the capsule suspension was concentrated by a magnet at a desired site of the reactor vessel directly under the sonotrode. Then, the suspension was treated by ultrasound (20 kHz) at different intensities and sonification times and analyzed. Figure 2 represents the results of ultrasonic irradiation of the Fe3O4-containing polyelectrolyte capsules at fixed time (1 min) and different powers. Ultrasound has a drastic influence on the integrity of the Fe3O4/polyelectrolyte capsules. One minute of the ultrasonic treatment leads to the complete destruction of the capsule shell. The size of the shell residues depends on the intensity of the acoustic waves. At relatively low (100 W) power, one can observe small shreds of polyelectrolyte shell with an average size of around 2 µm. An increase in the power input results in an increase in the size of the shell residues. At high (500 W) power, the polyelectrolyte capsule is decomposed into two to three pieces that are ∼ 16 µm in size. The counterintuitive effect of smaller fragments could be explained by the differences in the formation mechanism of the cavitation microbubbles in heterogeneous mixtures such as suspensions and colloid solutions.36,37 The formation of microbubbles prevails at low power on heterogeneous solid/liquid (polyelectrolyte/water) interfaces, mainly on Fe3O4 nanoparticles. The following collapse of these microbubbles initiates the distortion of the polyelectrolyte shell integrity simultaneously at several places, resulting in the shreds of polyelectrolyte complex of 1-3 µm size, as seen in Figure 2a. Increasing the ultrasonic power, the ratio of the cavitation microbubbles formed homogeneously in the water, both surrounding the capsule shell and inside the capsule volume, is increased in size but decreased in number.38 These cavitation (31) Rae, J.; Ashokkumar, M.; Eulaerts, O.; von Sonntag, C.; Reisse, J.; Grieser, F. Ultrason. Sonochem. 2005, 12, 325-329. (32) Kim, J.-K.; Kim, N.-K.; Park, B.-O. J. Mater. Sci. 2000, 35, 4995-4999. (33) Vinodgopal, K.; He, Y.; Ashokkumar, M.; Grieser, F. J. Phys. Chem. B 2006, 110, 3849-3852. (34) Mizukoshi, Y.; Fujimoto, T.; Nagata, Y.; Oshima, R.; Maeda, Y. J. Phys. Chem. B 2000, 104, 6028-6032. (35) Suslick, K. S., Ed. Ultrasound: Its Chemical, Physical, and Biological Effects; VCH Publishers: New York, 1988. (36) Hiraga, K.; Nakano, K.; Suzuki, T. S.; Sakka, Y. J. Am. Ceram. Soc. 2002, 85, 2763-2770. (37) Miller, D. L.; Williams, A. R.; Morris, J. E.; Chrisler, W. B. Ultrasonics 1998, 36, 947-952. (38) Endo, H. J. Am. Acoustic Soc. 1994, 95, 2409-2415.
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Figure 4. SEM (bottom panels) and fluorescence confocal (top panels) images of the Fe3O4/polyelectrolyte capsules, which were incubated at 70 °C for 90 min, treated by ultrasound (500 W) for different time durations.
microbubbles generated either inside the polyelectrolyte capsule or in the surrounding water media rarely affect the polyelectrolyte shell, resulting in shell breakage into two or three pieces. The other possible explanation of the fragment size could be the partial removal of magnetite nanoparticles out of the polyelectrolyte shell at high acoustic energies. The presence of inorganic nanoparticles in the shell of the polyelectrolyte capsules drastically increases their sensitivity to the ultrasonic treatment. Sonification of the pure polyelectrolyte capsules and Fe3O4/polyelectrolyte capsules under the same conditions (500 W, 1 min) revealed different results (Figure 3). Polyelectrolyte capsules without inorganic nanoparticles inside are only deformed after sonification, while Fe3O4/polyelectrolyte capsules, as also shown before, are ruptured into several pieces of polyelectrolyte complex particles. Two explanations of the observed phenomenon can be taken into consideration: the low elasticity and, as a consequence, high brittleness of the Fe3O4/ polyelectrolyte shell, and a higher density gradient, which leads to the reflection and superposition of acoustic waves, increasing the cavitation yield at the water/Fe3O4/polyelectrolyte interface compared to the water/polyelectrolyte interface. As demonstrated by Fery et al.,39 the shell of the polyelectrolyte capsules reinforced by inorganic nanoparticles revealed a pronounced stiffness increase up to a factor of 8. Moreover, pure polyelectrolyte capsules show the onset of buckling instability already at deformations on the order of 10%, while reinforced capsules did not a buckle even for the highest deformations applied, which were around 20%. Hence, the presence of inorganic nanoparticles or other materials increasing the density contrast and decreasing the elasticity of the microcontainer shell is of key importance to achieve high efficiency of the ultrasonically stimulated remote release of encapsulated compounds. The shell of the as-prepared polyelectrolyte capsule possesses controlled permeability properties for only large molecular weight compounds.28,29 However, an entirely controlled capsule permeability cannot be achieved for species with low molecular weight and size, which freely penetrate the polyelectrolyte shell. A new (39) Dubreuil, F.; Shchukin, D. G.; Sukhorukov, G. B.; Fery, A. Macromol. Rapid Commun. 2004, 25, 1078-1081.
Figure 5. SEM images of dextran-containing Fe3O4/polyelectrolyte capsules, which were incubated at 70 °C for 90 min, before (A) and after (B) 1 min of ultrasonic treatment at 100 W. (C) Fluorescence spectra of FITC-dextran in the supernatant solution of the capsule suspension before and after 1 min of ultrasonic treatment at 100 W. The excitation wavelength is 488 nm. The fluorescence signal appeared from previously encapsulated FITC-dextran.
encapsulation method was recently elaborated to entrap and transport low molecular weight species inside standard polyelectrolyte capsules.27,30 The principal of the method is the thermal treatment of the polyelectrolyte capsule suspension in the presence
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of the dissolved low molecular weight compound. Continuous heating of the polyelectrolyte capsules in water results in the reorganization of polyelectrolyte multilayers, an increase in the capsule shell thickness and density, and thus a lowering of the permeability of the polyelectrolyte shell. The influence of the sonification on the permeability and release properties of the temperature-treated Fe3O4/polyelectrolyte capsules was investigated because a considerable percentage of the drugs used for selective medical treatment have low molecular weights. Figure 4 demonstrates the effect of the ultrasound on heated (70 °C, 90 min) Fe3O4/polyelectrolyte capsules. Heated capsules are more stable under ultrasonic treatment than initial ones. However, their deformation and shell rupture are also observed, even after 5 s of ultrasonic treatment, which confirms the high efficiency of the ultrasound for remote triggering of the release from the inner volume of the polyelectrolyte capsules. Increasing the sonification time decreases the size of the shell residues. The remote, ultrasonically initiated release of the encapsulated compounds was performed for Fe3O4/poly(allylamine)/PSS capsules loaded with FITC-dextran by the thermal encapsulation method. Dextran-loaded polyelectrolyte capsules were placed under the sonotrode by an external magnet and then treated by ultrasound at 20 kHz and 100 W intensity, which is similar to the frequency and power parameters employed in medicine. Sonification of the loaded polyelectrolyte capsules led to complete splitting of the polyelectrolyte shell (Figure 5a,b) and release of the encapsulated dextran. As one can see in Figure 5c, no fluorescence signal was observed from the aqueous supernatant solution before ultrasonic treatment, which shows the absence of the spontaneous release of FITC-dextran from intact polyelectrolyte capsules. A fluorescent signal of high intensity at
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500-550 nm, corresponding to FITC-dextran fluorescence in aqueous media, appeared after 1 min of ultrasonic treatment. Additional incubation of the sample in aqueous solution after sonification did not increase the fluorescence intensity, which confirms the complete release of the encapsulated dextran and instantaneous shell destruction during ultrasonic treatment. In conclusion, we demonstrated an approach for the remote opening of polyelectrolyte capsules, which can be used as a carrier system in drug delivery, by applying ultrasound at low frequencies to the capsule suspension. Poly(allylamine)/ poly(styrene sulfonate) capsules containing Fe3O4 nanoparticles in the shell were used as model delivery and depot carriers for 20 kDa FITC-dextran. Besides the possibility of magnetically controlled delivery, the presence of magnetite nanoparticles significantly improves the efficiency of the ultrasonically stimulated release of the encapsulated compounds. The demonstrated release of FITC-dextran from polyelectrolyte capsules at intensities comparable to those of ultrasonic generators already applied in medicine exhibits a valid practical application of the proposed approach in medicine for addressable delivery and the remotely controlled release of toxic drugs, decreasing their negative effect on the undamaged parts of the organism. The remote opening of polyelectrolyte microcontainers can be also facilitated by coupling ultrasonic treatment with electromagnetic field. Acknowledgment. The work was supported by the EU project NANOCAPSULE (contract # MIF1-2004-002642). The authors thank R. Pitschke and J. Hartmann for electron microscopy analysis. LA061047M
