NANO LETTERS
Electrostatically Assembled Polyelectrolyte/Dendrimer Multilayer Films as Ultrathin Nanoreservoirs
2002 Vol. 2, No. 4 415-418
Ajay J. Khopade and Frank Caruso* Max Planck Institute of Colloids and Interfaces, D-14424 Potsdam, Germany Received December 10, 2001; Revised Manuscript Received January 16, 2002
ABSTRACT We report the preparation of loadable ultrathin, multilayered polyelectrolyte/dendrimer films by the sequential electrostatic deposition of negatively charged poly(styrenesulfonate) (PSS) and a positively charged fourth generation poly(amidoamine) dendrimer (4G PAMAM). Multilayers were first constructed on planar supports to examine their layer-by-layer growth. Quartz crystal microbalance (QCM) measurements showed regular growth for each layer deposited, while UV−vis spectrophotometry revealed an adsorption−desorption trend to film formation, with partial PSS removal upon deposition of 4G PAMAM. PSS/4G PAMAM films were subsequently constructed on spherical latex colloids. Fluorescence spectroscopy showed that the films, when exposed to dye-containing solutions, acted as nanoreservoirs, sequestering the charged molecules from solution due to the presence of the oppositely charged dendrimer. Release of the entrapped dye molecules was subsequently achieved by concentration-dependent diffusion in isotonic saline solutions, illustrating the potential of the dendrimer-based films as systems for the uptake and release of various compounds.
Dendrimers, perfect monodisperse macromolecules with a regular and highly branched three-dimensional structure, have attracted wide attention from scientists in various fields.1,2 The combination of a discrete number of functionalities and high local densities of active groups in dendrimers makes them unique molecules with a diverse range of applications. They can be used as sensors, catalysts, gene delivery agents and drug carriers for controlled release and site-specific delivery. The dendritic core also offers a unique microenvironment, making them interesting candidates for hostguest chemistry.1 The assembly of dendrimers to form thin films on surfaces has been performed by various groups,2 with several studies focusing on the preparation of multilayer films.3-9 Tsukruk et al. prepared self-assembled multilayer films from oppositely charged dendrimers.3 Multilayered dendrimer-based assemblies, including dendrimer-avidin/biotin, dendrimer/ enzyme, and dendrimer/polyelectrolyte films, have also been constructed.4-9 Most studies, however, have focused on the preparation aspects of film formation on planar supports only; there have been no reports on the creation of multilayered dendrimer films on colloids. Furthermore, these previous investigations3-9 have not dealt with the subsequent loading of the dendrimer films, i.e., their use as nanoreservoirs, which is an essential requirement if such films are to be utilized as specific reactor systems or in drug delivery applications.1 * Corresponding author. Fax: +49 331 567 9202. E-mail: frank.caruso@ mpikg-golm.mpg.de. 10.1021/nl015696o CCC: $22.00 Published on Web 02/08/2002
© 2002 American Chemical Society
Herein, we report a versatile layer-by-layer approach to prepare poly(styrenesulfonate) (PSS)/fourth generation poly(amidoamine) dendrimer (4G PAMAM) multilayer films of defined thickness and composition under physiological salt concentration by exploiting electrostatic interactions. We first prepare the films on planar supports to elucidate the factors that govern the formation of stable films, and then construct PSS/4G PAMAM films on colloid substrates. We are primarily interested in exploiting these ultrathin films as highcapacity loadable nanoreservoirs, a property that is potentially afforded by using dendrimer-coated particles, due to the inherently high surface areas associated with colloids. Dendrimer-coated particles prepared by using colloid templates are especially attractive for drug targeting, sustained release applications,10 as well as for separation processes and as nanoreactor thin films (e.g., for nanoparticle formation), as dendrimers allow the entrapment of various species within them.1 In this communication, we demonstrate loading of the films with a model fluorescent dye and its subsequent release under physiological conditions. Multilayer films were prepared on quartz crystal microbalance (QCM) electrodes and poly(ethyleneimine)-modified quartz slides by the sequential deposition of PSS (Aldrich) and 4G PAMAM (Aldrich) from 1 mg mL-1 solutions containing 0.154 M NaCl (isotonic to physiological fluid). The films were rinsed with water several times and then dried with nitrogen after each layer was deposited. The layers were formed from 4G PAMAM solutions of pH 10.3 and from PSS solutions of pH 6.8. Multilayer growth was driven
Figure 1. Multilayer film growth of PSS and 4G PAMAM on (a) gold-coated QCM electrodes and (b) quartz substrates, as studied by UV-vis spectrophotometry. The first layer deposited was PEI, followed by alternating layers of PSS and 4G PAMAM. The QCM data show regular layer growth. In (b) the PSS absorbance at 227 nm is plotted versus layer (or bilayer) number. The absorption spectra after PSS (solid line) and 4G PAMAM dendrimer (dotted line) adsorption are shown in the inset.
primarily by the electrostatic attraction between the amino and sulfonate groups of 4G PAMAM and PSS, respectively. Formation of the PSS/4G PAMAM multilayer films was followed by using a QCM, which showed that regular growth of the films occurs (Figure 1a). The average frequency changes are -37 ( 9 Hz and -140 ( 20 Hz for each 4G PAMAM and PSS layer, respectively.11 The frequency change corresponding to the dendrimer layer is ca. 32 ( 8 ng (1 Hz ) 0.87 ng), which is lower than that expected for monolayer coverage (ca. 47 ng), assuming close-packing of the dendrimer. Overall, the QCM data provide unequivocal evidence for the successful formation of polyelectrolyte/ dendrimer multilayers. UV-vis experiments were also performed to monitor the growth of the PSS/4G PAMAM films. Although regular film growth was observed for deposition of each bilayer (Figure 1b), the UV-vis data revealed an adsorption-desorption trend to film formation: the absorption peak at 227 nm (due to the phenyl group in PSS) decreased after the dendrimer adsorption step, indicat416
ing that PSS was removed from the film. (The possibility of dendrimer and/or PSS removal cannot be directly discerned solely from the QCM data due to the simultaneous processes of desorption of one species and adsorption of the other.) The PSS stripped off may be that which is loosely adsorbed onto the dendrimer outermost layer of the film, subsequently forming polyelectrolyte/dendrimer complexes in solution.12 Notwithstanding, a sufficient quantity of PSS (average of 65% over 10-layer assembly for 3 films from UV-vis measurements) remains in the film to promote further multilayer growth. We have previously observed a similar adsorption-desorption behavior for polyelectrolyte/dye multilayer films.13 Ellipsometry measurements on a 10-layer PSS/ 4G PAMAM film yielded an average thickness of around 3.5 nm per bilayer. Since the dendrimer has a diameter of 4.5 nm,3 and the PSS layer is likely to be around 1-2 nm,14-17 this value suggests that less than a monolayer of dendrimer remained in the film for each dendrimer/PSS adsorption cycle. Deformation of the dendrimer could have also occurred upon adsorption and polyelectrolyte complexation.12b The morphology of the polyelectrolyte/dendrimer films was examined by using an atomic force microscope (AFM) (Nanoscope IIIa, Santa Barbara, CA) in tapping mode. The films were uniform, with a root-mean-squared (RMS) roughness of ca. 0.6 ( 0.1 nm (over 1 µm2 area) and grain size of ca. 60 ( 10 nm for films with 10 layers. This RMS value obtained is similar to those reported (0.81.0 nm) for multilayer films composed of linear polyelectrolytes.14 To prepare ultrathin polyelectrolyte/dendrimer coatings on colloids, PSS/4G PAMAM multilayers were adsorbed onto particles using the conditions employed for the planar supports and by applying the layer-by-layer assembling protocol to colloids.15-17 (No drying step was employed after deposition of each layer.11) Stepwise growth was confirmed by the successful recharging of the particle (polystyrene, PS, 925 nm, Polysciences) surface with each deposition cycle:16 the ζ-potential of the particles alternated between -30 mV (PSS) and +45 mV (4G PAMAM) with each coating step, suggesting multilayer growth occurred on the particles. The coated particles were characterized by transmission electron microscopy (TEM, Philips CM12 microscope). TEM images of the coated particles showed a slight increase in surface roughness (Figure 2) (compared with the uncoated particles), confirming the adsorption of dendrimer and polyelectrolyte. Complexes of 4G PAMAM, a hierarchical polymer, and a long chain polyelectrolyte (e.g., PSS of ca. Mw 70 000) are significantly different from those made of linear, oppositely charged polyelectrolytes, in that the dendritic core offers the possibility of selectively entrapping guest molecules.1 4,5 Carboxyfluorescein (CF), a model acidic substance, was entrapped in the PSS/4G PAMAM multilayers by exposure of the films on planar supports and colloids to an aqueous CF solution (20 µg mL-1) for 3 h (excess CF was removed by washing with water). Steady-state fluorescence measurements of the “air-dried” multilayer films on quartz slides showed that the fluorescence intensity of the film (due to CF) increased and that the spectral peak position Nano Lett., Vol. 2, No. 4, 2002
Figure 2. TEM micrograph of 925 nm PS particles coated with PEI/(PSS/4 G PAMAM)4 (9 layers total). The inset is a higher magnification of one of the spheres, showing the surface roughness due to the multilayers deposited. Uncoated particles display entirely smooth and featureless surfaces at these magnifications.
shifted to higher wavelength (from ca. 526 nm to ca. 530 nm) with increasing layer number, reflecting increasing amounts of CF entrapped in the multilayers (Figure 3a). From the QCM data, 18 ( 5% w/w of CF was entrapped in the films (a range of 4-10 layers was studied), also showing the feasibility of loading charged dyes in PSS/4G PAMAM multilayer films. Since CF does not bind to PSS,16 the loading observed is due to the interaction of CF with the dendrimer. CF most likely binds to the 4G PAMAM amino (tertiary and primary) groups present in the core and/or at the surface that are not utilized in the electrostatic binding with PSS. It is important to note that, in contrast to our previous work utilizing multilayer films of linear polyelectrolytes,16 CF bound to the PSS/4G PAMAM films even when PSS was the outermost layer. This lends further support that the interaction of CF occurred with the dendrimer in the films. The PSS/4G PAMAM-coated PS particles (9 layers, PSS outer layer) were also subsequently loaded with CF, and confocal laser scanning microscopy (CLSM) images (Leica, Germany) were taken (Figure 3b). The fluorescence, due to entrapped CF in the dendrimer-based shell, seen as a ring in the image, confirms their dye loading potential. No binding of CF to the negatively charged PS particles (control) was observed, in agreement with our earlier study.16 To demonstrate release of the entrapped dye, the CFloaded films on the quartz and polystyrene sphere supports were exposed to a stirred solution of 20 mL of 0.154 M NaCl, pH 6.5 at room temperature. At periodic intervals, the filmcoated substrates were removed or separated (centrifugation was used for the particles) from the CF solution, rinsed with water, air-dried, and the fluorescence spectra recorded. (A fluorescence microscope was used to measure the fluorescence of the particles after drying on a glass slide.) The Nano Lett., Vol. 2, No. 4, 2002
Figure 3. CF loading experiments for PSS/4G PAMAM multilayer films formed on (a) quartz substrates and (b) 925 nm PS particles. (a) Fluorescence spectra of CF entrapped in the multilayer films formed on quartz slides. The total number of layers in the film are shown. Excitation wavelength ) 450 nm. (b) CLSM image of 925 nm PS particles coated with PSS/4G PAMAM multilayers (9 layers, outermost PSS layer) and loaded with CF.
percentage released was calculated relative to the initial CF amount in the films. The release profile of CF from a quartzsupported 10-layer film in 20 mL of 0.154 M NaCl (pH 6.5) was Fickian.18 After an initial burst, about 80-90% of the dye was released in 4 h (Figure 4). The CF release characteristics (Fickian-type) were similar for the thin films coated on particles. The overall CF release profile and released amount (after 4 h) were similar for films with 4G PAMAM or PSS as the outermost layer. This demonstrates the potential utility of such films for sustained release applications. From UV-vis measurements on the multilayers on quartz, no loss of PSS from the assembled films was observed after CF release, indicating that the films remained intact. As an extension of the coated colloids, hollow polyelectrolyte/dendrimer capsules (or capsular colloids) were obtained after removal of the core (1 µm melamine formaldehyde,19 MF (Microparticles GmbH), in place of PS particles) with 0.1 M HCl from the PSS/dendrimer multilayer-coated particles. The thin and highly porous capsules were fragile during the core removal procedure; therefore, they were 417
dendrimer multilayer films constructed on planar substrates as well as on colloids. The films prepared present novel loaded nanoreservoir-based systems that are potentially useful for a range of controlled release applications, particularly when coupled to biopolymers,19a to obtain biocompatible and biodegradable systems. Experiments to increase the stability of the multilayers by cross-linking and to make them biomimetic, by lipid and polymer brush grafting, are currently underway. In addition, the films reported represent potentially interesting nanoreactor systems in which nanoparticle synthesis (i.e., within the dendrimers) may be conducted, allowing the synthesis of nanoparticles on planar or colloid supports.
Figure 4. Typical release profile of CF-loaded PSS/4G PAMAM multilayers (10 layers) on a quartz slide in 0.154 M NaCl at room temperature. The blank (quartz/PEI) film shows negligible loading and insignificant release.
Acknowledgment. A.J.K. acknowledges the Alexander von Humboldt Foundation for a research fellowship. This work was also funded by the BMBF under the Biofuture initiative. P. Schuetz, U. Bloeck (Hahn-Meitner Institute, Berlin) and C. Pilz are thanked for assistance with the TEM and CLSM measurements, and H. Mo¨hwald for supporting the work. References
Figure 5. TEM micrograph of hollow PSS/4G PAMAM capsules formed after removal of the MF core from coated particles (similar to those shown in Figure 2b). The hollow polyelectrolyte/dendrimer capsules (9 layers) were stabilized with an additional outer coat of PAH/PSS.
prepared containing an outer coat of PSS/poly(allylamine hydrochloride) (PAH)/PSS. Figure 5 shows the capsules obtained were stable and consisted of folds due to drying effects, displaying a similar morphology to other hollow capsules made of linear polyelectrolytes.19 These capsular colloids represent interesting free-standing systems for loading and controlled release, as they can exhibit elastic properties, similar to biological cells. We have demonstrated the successful layer-by-layer formation and subsequent dye loading of polyelectrolyte/
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(1) Bosman, A. W.; Janssen, H. M.; Meijer, E. W. Chem. ReV. 1999, 99, 1665. (2) Tully, D. C.; Frechet, J. M. J. Chem. Commun. 2001, 1229. (3) Tsukruk, V. V. AdV. Mater. 1998, 10, 253. (4) Anzai, J.; Kobayashi, Y.; Nakamura, N.; Nishimura, M.; Hoshi, T. Langmuir 1997, 15, 2221. (5) Cheng, L.; Cox, J. A. Electrochem. Commun. 2001, 3, 285. (6) He, J. A.; Valluzzi, R.; Yang, K.; Dolukhanyan, T.; Sung, C. M.; Tripathy, S. K.; Samuelson, L.; Balogh, L.; Tomalia, D. A. Chem. Mater. 1999, 11, 3268. (7) Watanabe, S.; Regen, S. L. J. Am. Chem. Soc. 1994, 116, 8855. (8) Yoon, H. C.; Kim, H. S. Anal. Chem. 2000, 72, 922. (9) Wang, J. F.; Chen, J. Y.; Jia, X. R.; Cao, W. X.; Li, M. Q. Chem. Commun. 2000, 6, 511. (10) Chouly, C.; Pouliquen, D.; Lucet, I.; Jeune, J. J.; Jallet, P. J. Microencapsulation 1996, 13, 245. (11) Multilayer films could also be prepared without a drying step between each layer. These experiments showed a total QCM frequency change 20% higher for a 10-layer PSS/4G PAMAM film (when dried after the 10 layers were deposited), compared with the same film that was prepared with intermediate nitrogen drying steps. 9-MHz gold-coated QCM electrodes (Kyushu Dentsu, Japan) were used. (12) (a) Miura, N.; Dubin, P. L.; Moorefield, C. N.; Newkome, G. R. Langmuir 1999, 15, 4245. (b) Welch, P.; Muthukumar, M. Macromolecules 2000, 33, 6159. (13) Tedeschi, C.; Caruso, F.; Mo¨hwald, H.; Kirstein, S. J. Am. Chem. Soc. 2000, 122, 5841. (14) (a) Caruso, F.; Furlong, D. N.; Ariga, K.; Ichinose, I.; Kunitake, T. Langmuir 1998, 14, 4559. (b) Sukhorukov, G. B.; Schmitt, J.; Decher, G. Ber. Bunsen-Ges. Phys. Chem. 1996, 100, 948. (15) Caruso, F.; Caruso, R. A.; Mo¨hwald, H. Science 1998, 282, 1111. (16) Caruso, F.; Lichtenfeld, H.; Donath, E.; Mo¨hwald, H. Macromolecules 1999, 32, 2317. (17) Caruso, F. AdV. Mater. 2001, 13, 11. (18) Huang, X.; Brazel, C. S. J. Controlled Release 2001, 73, 121. (19) (a) Schu¨ler, C.; Caruso, F. Biomacromolecules 2001, 2, 921. (b) Pastoriza-Santos, I.; Scho¨ler, B.; Caruso, F. AdV. Functional Mater. 2001, 11, 122. (c) Donath, E.; Sukhorukov, G. B.; Caruso, F.; Davis, S. A.; Mo¨hwald, H. Angew. Chem., Int. Ed. Engl. 1998, 37, 2201.
NL015696O
Nano Lett., Vol. 2, No. 4, 2002
