Multicompartment Films Made of Alternate Polyelectrolyte Multilayers

Maria Muniz , Heather Gibson , Wei-Zen Wei , Haipeng Liu , and Guangzhao Mao ... ACS Macro Letters 2012 1 (7), 797-801 .... The Journal of Physica...
0 downloads 0 Views 344KB Size
7298

Langmuir 2004, 20, 7298-7302

Multicompartment Films Made of Alternate Polyelectrolyte Multilayers of Exponential and Linear Growth Juan Me´ndez Garza,† Pierre Schaaf,‡ Sylvaine Muller,§ Vincent Ball,‡ Jean-Franc¸ ois Stoltz,§ Jean-Claude Voegel,*,† and Philippe Lavalle† INSERM Unite´ 595, Faculte´ de Chirurgie Dentaire, Universite´ Louis Pasteur, 11 rue Humann, F-67085 Strasbourg Cedex, France, Institut Charles Sadron (CNRS UPR 22), 6 rue Boussingault, F-67083 Strasbourg Cedex, France, and Me´ canique et Inge´ nierie Cellulaire et Tissulaire, UMR CNRS 7563 - LEMTA, Faculte´ de Me´ decine, 54500 Vandœuvre-le` s-Nancy, France Received April 8, 2004. In Final Form: May 14, 2004 The layer by layer deposition process of polyelectrolytes is used to construct films equipped with several compartments containing “free polyelectrolytes”. Each compartment corresponds to a stratum of an exponentially growing polyelectrolyte multilayer film, and two consecutive compartments are separated by a stratum composed of a linearly growing multilayer that acts as a barrier preventing polyelectrolyte diffusion from one compartment to another. We use hyaluronic acid/poly(L-lysine) as the system to build the compartments and the poly(styrene sulfonate)/poly(allylamine) system for the barrier. Using confocal microscopy, it is shown that poly(L-lysine) diffuses only within the compartment in which it was initially introduced during the film construction and is thus unable to cross the barriers. Using fluorescein isothiocyanate as a pH indicator, it is also shown that although poly(styrene sulfonate)/poly(allylamine) multilayers act as a barrier for polyelectrolytes, they do not prevent proton diffusion through the film. Such films open the route for multiple functionalization of biomaterial coatings.

Introduction The modification of solid substrates by surface treatments has become a major concern over the past decades. To this end, numerous strategies were developed and recently an emerging method was proposed to prepare, in a simple manner, easily tunable interfacial coatings. The approach is based on the layer by layer buildup of films called “polyelectrolyte multilayers” obtained by the alternate deposition of polycations and polyanions on solid surfaces. Described in 1992 by Decher et al.,1,2 these films have received, since their discovery, considerable attention and present numerous potential applications in fields ranging from electro-optical devices3 to separation processes4-6 and biomaterial surface coatings.7-15 * To whom correspondence should be addressed. Phone: 33 3 90 24 33 87. Fax: 33 3 90 24 33 79. E-mail: jean-claude.voegel@ medecine.u-strasbg.fr. † INSERM Unite ´ 595, Faculte´ de Chirurgie Dentaire, Universite´ Louis Pasteur. ‡ Institut Charles Sadron (CNRS UPR 22). § Me ´ canique et Inge´nierie Cellulaire et Tissulaire, UMR CNRS 7563 - LEMTA, Faculte´ de Me´decine. (1) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1992, 210, 831. (2) Decher, G. Science 1997, 277, 1232. (3) Onitsuka, O.; Fou, A. C.; Ferreira, M.; Hsieh, B. R.; Rubner, M. F. J. Appl. Phys. 1996, 80, 4067. (4) Liu, X. Y.; Bruening, M. L. Chem. Mater. 2004, 16, 351. (5) Harris, J. J.; Bruening, M. L. Langmuir 2000, 16, 2006. (6) Kotov, N. A.; Magonov, S.; Tropsha, E. Chem. Mater. 1998, 10, 886. (7) Hammond, P. T. Curr. Opin. Colloid Interface Sci. 1999, 4, 430. (8) Bertrand, P.; Jonas, A.; Laschewsky, A.; Legras, R. Macromol. Rapid Commun. 2000, 21, 319. (9) Eckle, M.; Decher, G. Nano Lett. 2001, 1, 45. (10) Chluba, J.; Voegel, J. C.; Decher, G.; Erbacher, P.; Schaaf, P.; Ogier, J. Biomacromolecules 2001, 2, 800. (11) Jessel, N.; Atalar, F.; Lavalle, P.; Mutterer, J.; Decher, G.; Schaaf, P.; Voegel, J.-C.; Ogier, J. Adv. Mater. 2003, 15, 692. (12) Thierry, B.; Winnik, F. M.; Merhi, Y.; Tabrizian, M. J. Am. Chem. Soc. 2003, 125, 7494.

When a polyelectrolyte multilayer ending with a polyanion layer is brought in contact with a polycation solution, the polycations interact with the polyanions at the film/ solution interface and form a new layer of complexes which will ultimately present an excess of positive charges at the outer surface. A similar process takes place when a polyanion solution is brought in contact with a film ending with a polycation layer. Even if all these films are constituted of polyanion/polycation complexes, they do not all share the same buildup mechanism. The first investigated polyelectrolyte multilayers described in the literature presented a linear growth of both the mass and thickness with the number of deposition steps. Poly(styrene sulfonate)/poly(allylamine) (PSS/PAH)16-19 or poly(acrylic acid)/poly(allylamine)20 constitute a typical example of such systems. Their structure is stratified, each polyelectrolyte layer interpenetrating only a few of the neighboring ones, and their growth mechanism involves mainly electrostatic interactions between the incoming polyelectrolyte molecules from the solution and the polyelectrolytes of opposite charge forming the outer layer of the film.21,22 The motor of the growth mechanism (13) Thierry, B.; Winnik, F. M.; Merhi, Y.; Silver, J.; Tabrizian, M. Biomacromolecules 2003, 4, 1564. (14) Grant, G. G. S.; Koktysh, D. S.; Yun, B.; Matts, R. L.; Kotov, N. A. Biomed. Microdevices 2001, 3, 301. (15) Ai, H.; Jones, S. A.; Lvov, Y. M. Cell Biochem. Biophys. 2003, 39, 23. (16) Ramsden, J. J.; Lvov, Y. M.; Decher, G. Thin Solid Films 1995, 254, 246. (17) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3422. (18) Ladam, G.; Schaad, P.; Voegel, J. C.; Schaaf, P.; Decher, G.; Cuisinier, F. Langmuir 2000, 16, 1249. (19) Picart, C.; Ladam, G.; Senger, B.; Voegel, J.-C.; Schaaf, P.; Cuisinier, F. J. G.; Gergely, C. J. Chem. Phys. 2001, 115, 1086. (20) Shiratori, S. S.; Rubner, M. F. Macromolecules 2000, 33, 4213. (21) Yoo, D.; Shiratori, S. S.; Rubner, M. F. Macromolecules 1998, 31, 4309. (22) Lo¨sche, M.; Schmitt, J.; Decher, G.; Bouwman, W. G.; Kjaer, K. Macromolecules 1998, 31, 8893.

10.1021/la049106o CCC: $27.50 © 2004 American Chemical Society Published on Web 07/21/2004

Multicompartment Films of Polyelectrolyte Layers

is provided by the charge overcompensation that appears after each deposition step. The existence of such a charge overcompensation is demonstrated by the alternating sign of the zeta potential during the film buildup.18,23 More recently, a new type of polyelectrolyte multilayer characterized by an exponential increase of both the mass and the thickness of the film with the number of deposition steps was discovered. The exponential growth mechanism of polyelectrolyte multilayers built with a copolymer of acrylamide and [3-(2-methylpropionamido)propyl]trimethylammonium chloride]/poly(acrylic acid)24 or poly(hexyl viologen)/poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate)25 or poly(diallyldimethylammonium chloride)/poly(styrenesulfonate)26 systems was attributed to an increase of the film surface roughness with the number of deposited layers. An alternative explanation based on the diffusion, “in” and “out” of the whole film, of at least one of the polyelectrolytes constituting the multilayer was proposed later.27 The validity of this explanation was demonstrated for hyaluronic acid/poly(L-lysine),28 hyaluronic acid/chitosan,29 poly(L-glutamic acid)/poly(allylamine)30 and poly(L-glutamic acid)/poly(Llysine)31,32 films. In particular, observations of the hyaluronic acid/poly(L-lysine) (HA/PLL) system showed that PLL diffuses in and out of the whole film during the buildup of each bilayer whereas HA does not diffuse through the multilayer. For this system, it was shown that after the deposition of 10 bilayers the films become very flat so that an exponential increase mechanism due to an increase of the film roughness can be totally ruled out.27 The alternating sign of the zeta potential during the film buildup can be also observed for exponentially growing multilayers. Due to the diffusion of at least one of the polyelectrolytes within the multilayer, one could take advantage of such systems to design new drug delivery devices or smart material coatings. Recent studies suggested also that the deposition of two poly(styrene sulfonate)/poly(allylamine) bilayers on top of a poly(Lglutamic acid)/poly(L-lysine) multilayer should be sufficient to prevent diffusion of further adsorbed poly(Lglutamic acid) and poly(L-lysine) into the underlying poly(L-glutamic acid)/poly(L-lysine) film.33 Two bilayers of (PSS/PAH) seemed thus to be sufficient to act as a barrier for the further polyelectrolyte diffusion into the film. The concept of barriers separating compartments in polyelectrolyte multilayers was recently introduced by Jonas and co-workers.34 These authors used multilayers that grow (23) Caruso, F.; Donath, E.; Mo¨hwald, H. J. Phys. Chem. B 1998, 102, 2011. (24) Schoeler, B.; Poptoshev, E.; Caruso, F. Macromolecules 2003, 36, 5258. (25) DeLongchamp, D. M.; Kastantin, M.; Hammond, P. T. Chem. Mater. 2003, 15, 1575. (26) McAloney, R. A.; Sinyor, M.; Dudnik, V.; Goh, M. C. Langmuir 2001, 17, 6655. (27) Picart, C.; Lavalle, P.; Hubert, P.; Cuisinier, F. J. G.; Decher, G.; Schaaf, P.; Voegel, J. C. Langmuir 2001, 17, 7414. (28) Picart, C.; Mutterer, J.; Richert, L.; Luo, Y.; Prestwich, G. D.; Schaaf, P.; Voegel, J. C.; Lavalle, P. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 12531. (29) Richert, L.; Lavalle, P.; Payan, E.; Zheng, X. S.; Prestwich, G. D.; Stoltz, J. F.; Schaaf, P.; Voegel, J.-C.; Picart, C. Langmuir 2004, 20, 448. (30) Boulmedais, F.; Ball, V.; Schwinte, P.; Frisch, B.; Schaaf, P.; Voegel, J. C. Langmuir 2003, 19, 440. (31) Lavalle, P.; Gergely, C.; Cuisinier, F. J. G.; Decher, G.; Schaaf, P.; Voegel, J. C.; Picart, C. Macromolecules 2002, 35, 4458. (32) Lavalle, P.; Vivet, V.; Jessel, N.; Decher, G.; Mesini, P. J.; Voegel, J.-C.; Schaaf, P. Macromolecules 2004, 37, 1159. (33) Boulmedais, F.; Bozonnet, M.; Schwinte´, P.; Voegel, J.-C.; Schaaf, P. Langmuir 2003, 19, 9873. (34) Jonas, A. T.; Pe´ralta, S.; Habib-Jiwan, J.-L.; Nicol, E. Oral communication, 2004; Presented at the 227th ACS Meeting, Anaheim, CA; Talk No. 78.

Langmuir, Vol. 20, No. 17, 2004 7299

Figure 1. Inverted QCM-D frequency shift (-∆f/ν) after deposition of each polycation and polyanion layer on a quartz crystal coated with SiO2. Data were obtained at 15 MHz in liquid conditions (NaCl 0.15 M, pH ) 5.9). The film is finally made of three parts: a first part (part I) made of 5 (PLL/HA) bilayers is directly deposited on the crystal (4), followed by a second part (part II) constituted of 5 (PAH/PSS) bilayers (O) and a third one (part III) made of 5 (PLL/HA) bilayers (3). Black lines correspond to three independent fits: from PLL1 to HA5, the data points are fitted with an exponential law (-∆f/ν ) beci, with b ) 35.51 and c ) 0.27), from PSS5 to PSS10 the data points are fitted with a linear law (-∆f/ν ) a + bi, with a ) 422.6 and b ) 23.7), and from PLL11 to HA14 the data points are again adjusted with the exponential law (-∆f/ν ) a + beci, with a ) 799.2, b ) 0.41, and c ) 0.26). i corresponds to the number of deposited layers (from i ) 1 for PLL1 to i ) 30 for HA15).

linearly with the number of deposition steps both for the compartments and the barriers. Multicompartment films using the barrier concept are also currently investigated by the group of Hammond at MIT.35 In this article, we will make use of the barrier effect to build films equipped with several compartments composed of exponentially growing multilayers (HA/PLL in our example) separated by linearly growing (PSS/PAH)n barriers. We will show, in particular, that PLL can diffuse within a given compartment but remains confined in it. We will also show that although (PSS/PAH)n acts as a barrier toward polyelectrolyte diffusion, it allows the diffusion of protons so that the pH can be changed within the compartments. This work should open the route toward the buildup of complex, multicompartment films of high loading capacity and containing different active molecules. By using only degradable polyelectrolytes, one should be able to construct devices with controlled sequential and tunable activity releases. Materials and Methods Poly(L-lysine) (PLL, MW ) 5.7 × 104 Da), fluorescein isothiocyanate labeled poly(L-lysine) (PLLFITC, MW ) 5.0 × 104 Da), poly(sodium 4-styrenesulfonate) (PSS, MW ) 7.0 × 104 Da), and poly(allylamine hydrochloride) (PAH, MW ) 7.0 × 104 Da) were purchased from Sigma (St. Quentin Fallavier, France), and hyaluronic acid (HA, MW ) 4.0 × 105 Da) from BioIberica (Barcelona, Spain). Polyelectrolyte solutions were prepared by dissolution of adequate amounts of polyelectrolyte powders in 0.15 M NaCl solution (pH ) 5.9). The final polyelectrolyte concentrations were 1 mg mL-1. All solutions were prepared using ultrapure water (Milli Q-plus system, Millipore) with a resistivity of 18.2 MΩ cm. (35) Hammond, P. T. Oral communication, 2004; Presented at the 227th ACS Meeting, Anaheim, CA; Talk No. 74.

7300

Langmuir, Vol. 20, No. 17, 2004

Garza et al.

Figure 2. Vertical section image of a (PLL/HA)30/PLLFITC/HA/(PAH/PSS)30/(PLL/HA)30/(PAH/PSS)30/(PLL/HA)30/PLLFITC multilayered film architecture in solution (NaCl 0.15 M, pH ) 5.9) observed by CLSM. The horizontal dashed line indicates the position of the glass slide on which the polyelectrolyte multilayers have been deposited. Indications on the right side of the image give the structural interpretations of the section observed.

Figure 3. Vertical section image of a (PLL/HA)30/PLLFITC/HA/(PAH/PSS)30/(PLL/HA)30/(PAH/PSS)30/(PLL/HA)30/PLLFITC multilayered film architecture in solution (NaCl 0.15 M, pH ) 5.9) observed by CLSM. The horizontal short-dashed line indicates the position of the glass slide on which the polyelectrolyte multilayer has been deposited. The vertical long-dashed lines specify the position of the bleached zone. Image A is acquired immediately after the end of the bleach, and image B is taken 35 min later. Indications on the right side of the images give the structural interpretations of the section observed. For the buildup of the multilayers, we used an automatized dipping robot (Riegler & Kirstein GmbH, Berlin, Germany). Glass slides (VWR, Fontenay sous Bois, France) used as the initial substrate were first dipped in a polycation solution (PLL) for 10 min. Then, a rinsing step was performed by dipping the substrates for 10 min in 0.15 M NaCl solution. The polyanion (HA) was then deposited in the same manner. The buildup process is pursued by the alternated deposition of PLL and HA. After the deposition of n bilayers, the film will be denoted (PLL/HA)n. The same method was applied for PAH (polycation) and PSS (polyanion) deposition leading to (PAH/PSS)n multilayers. The dye-conjugated polyelectrolytes were adsorbed in the same way at a certain stage of the buildup process. The buildup process of the multilayered films was monitored in situ by quartz crystal microbalance-dissipation using the axial flow chamber QAFC 302 (QCM-D, D300, Q-Sense, Go¨tenborg, Sweden). The QCM-D technique consists of measuring the resonance frequency and dissipation changes (∆f and ∆D, respectively) of a quartz crystal induced by polyelectrolyte adsorption on the crystal, in comparison with the crystal in contact with NaCl solution. The quartz crystal is excited at its fundamental frequency (5 MHz), and the measurements are performed

at the third overtone (denoted as ν) corresponding to 15 MHz. Changes in the resonance frequency, ∆f, during each adsorption step are measured. A shift in ∆f/ν can be associated, in first approximation, with a variation of the mass adsorbed to the crystal. The crystal used here is coated with a ≈50 nm thick SiO2 film deposited by active sputter-coating. The measurement methodology has been addressed in detail elsewhere and is applied in the present work.27 Confocal laser scanning microscopy (CLSM) investigations of the films are performed in liquid conditions. CLSM observations were carried out with a Zeiss LSM 510 microscope using a ×40/ 1.4 oil immersion objective and with 0.4 µm z-section intervals. FITC fluorescence was detected after excitation at 488 nm with cutoff dichroic mirror 488 nm and emission band-pass filter 505530 nm (green). Virtual vertical sections can be visualized, hence allowing the determination of the thickness of the film. All the experiments are performed in liquid conditions (NaCl 0.15 M, pH ) 5.9), and the multilayer films were never dried.

Results and Discussion The buildup of a film corresponding to (PLL/HA)5/ (PAH/PSS)5/(PLL/HA)5 was first followed with QCM-D

Multicompartment Films of Polyelectrolyte Layers

Langmuir, Vol. 20, No. 17, 2004 7301

Figure 4. Vertical section image of a (PLL/HA)30/PLLFITC/HA/(PAH/PSS)30/(PLL/HA)30/(PAH/PSS)30/(PLL/HA)30/PLLFITC multilayered film architecture observed by CLSM. (A) Image acquired in the presence of NaCl 0.15 M, pH ) 5.9 solution; (B) after 15 s in NaCl 0.15 M, pH ) 4.0 solution (fluorescence in the upper compartment has disappeared). After 30 s, no more fluorescence is observed over the entire film section (experiment not shown). Fluorescence gains and laser intensity are kept constant for all images. Indications on the right side of the images give the structural interpretations of the section observed.

(Figure 1). The inverted frequency shift (-∆f/ν) increases exponentially with the number of deposited layers n in the first part (part I) of the film corresponding to the buildup of (PLL/HA)5. This exponential growth results from the ability of the PLL chains to diffuse in and out of the whole film during each deposition step as described above. The deposition of the following (PAH/PSS)5 multilayers corresponding to the second part (part II) of the film leads to a linear growth regime where no diffusion of PAH or PSS through the architecture should occur. The third and last part of the film (part III) is again made of 5 (PLL/HA) bilayers which also present an exponential growth. The exponential growth regimes observed for the (PLL/HA) parts in the first and third compartments are both exponential in nature. When fitted with an exponential law of the type ∆f/ν ) a + beci, where i corresponds to the number of deposited layers (from i ) 1 for PLL1 to i ) 30 for HA15), they lead to the same parameter c (0.27 and 0.26 for the buildup regimes of the first and third domains, respectively) but to different parameters b (23.64 and 0.41, respectively). This is expected for two identical exponential growth processes starting with different initial conditions. Indeed, the parameter c directly describes the film growth whereas b reflects the state of the first deposited layers. This result strongly suggests that the exponential growth process of the (PLL/HA) films is not influenced by the substrate on which the deposition takes place. It also suggests that the (PSS/PAH)5 multilayer acts as a real barrier against PLL diffusion from compartment 1 to compartment 3. This is in accordance with previous studies performed with optical waveguide lightmode spectroscopy and Fourier transform infrared spectroscopy in attenuated total reflection configuration which suggested that two (PSS/PAH) bilayers were sufficient to

act as a barrier for the diffusion of PGA or PLL chains.33 We will now directly show this barrier effect by means of confocal laser scanning microscopy. To directly demonstrate the barrier effect for PLL diffusion of a compartment made of (PAH/PSS) multilayers, we built a film containing three compartments. Figure 2 corresponds to a (PLL/HA)30/PLLFITC/HA/(PAH/ PSS)30/(PLL/HA)30/(PAH/PSS)30/(PLL/HA)30/PLLFITC architecture observed with CLSM. Only the first (located on the bottom of the film) and the third compartments (located on the top of the film) contained PLLFITC molecules; the second compartment located in the middle of the film was built using unlabeled polypeptides. The image section shows from the bottom to the top of the film a first intense green band, a second large black band, and a third green band. Each band has a thickness ranging between 4.3 and 4.6 µm and corresponds to a (PLL/HA)30 multilayer film. The first and the third compartments appear green due to the use of FITC-labeled PLL molecules for the buildup of the ending layer of both compartments. This proves again the diffusion of these labeled molecules through the entire corresponding compartment. The black band observed in the middle part of the film is easily explained by the absence of PLLFITC inside the second compartment and directly proves that PLLFITC can diffuse neither from compartment 3 into compartment 2 nor from compartment 1 to compartment 2: the (PAH/PSS)30 multilayer thus acts as a perfect barrier toward the diffusion of PLL. The weaker green intensity of the third compartment could be the result of some PLLFITC diffusion out of the upper part of the film during a 1 day storage before CLSM imaging.36 Similar images, presenting exactly the same structure and the same green band distributions, were obtained with films stored over 1 month

7302

Langmuir, Vol. 20, No. 17, 2004

in a 0.15 M NaCl solution at pH ) 5.9 at ambient temperature. This seems to indicate that the PLLFITC diffusion out of compartment 3 takes place only during the first hours or days after the film construction. Next, we tested the ability of PLL molecules to diffuse inside the compartment constituted of PLL/HA complexes. For this purpose, we performed experiments32 where we locally bleached the multilayer film. Figure 3 corresponds to a similar film as previously described in Figure 2: again, three (PLL/HA) compartments constituted this film and only the first and the third compartments contained PLLFITC that was introduced in the last buildup steps of each compartment. If one illuminates a zone of the film by applying for several minutes the laser light with maximal power and using green filters, a bleach in the green fluorescence domain appears immediately in the section of compartments 1 and 3 which are labeled. This is seen in Figure 3A. Then, after 35 min, the green fluorescence is almost entirely recovered in these two compartments, suggesting a diffusion of PLLFITC chains. The fact that the second compartment remains black indicates again that PLL diffuses laterally inside a given compartment but does not cross the (PSS/PAH)30 barrier. After having constructed a three-compartment film, we now turn to the question of whether there is still communication possible between the lower compartment and the solution in contact with the film by the diffusion of small ions through the (PSS/PAH)n barrier. We thus tested the ability of H3O+ ions to cross (PSS/PAH)30 barriers. We used a film similar to the ones described in Figures 3 and 4. The FITC dye bound to PLL was used as a pH indicator and hence as a H3O+ sensor. The fluorescence emission of FITC is dramatically reduced at acidic pH, and finally at pH ) 4, the intensity is close to zero.37 Figure 4A shows a CLSM section of the (PLL/HA)30/ PLLFITC/HA/(PAH/PSS)30/(PLL/HA)30/(PAH/PSS)30/(PLL/ HA)30/PLLFITC film in a pH ) 5.9 solution. The film appears with two green bands separated by a black one, as in Figure 2. Changing the pH of the solution from 5.9 to 4 leads after 15 s to the extinction of the fluorescence located in the third (upper) compartment and to a decrease of the green intensity of the first (lower) compartment (Figure 4B). After 30 s, no more fluorescence was visible over the entire film. This indicates that the (PSS/PAH)30 multilayer does not constitute a barrier against H3O+ diffusion. The diffusion process through the barrier is however not instantaneous since approximately 30 s is needed for these ions to reach the lowest compartment. Diffusion of H3O+ is probably slower in the (PSS/PAH) multilayer films than in the (PLL/HA) film due to the larger degree of hydration of these later architectures.38 Finally, by bringing the film again in contact with a solution at pH ) 5.9, one recovers (36) Lavalle, P.; Picart, C.; Mutterer, J.; Gergely, C.; Reiss, H.; Voegel, J.-C.; Senger, B.; Schaaf, P. J. Phys. Chem. B 2004, 108, 636. (37) pH Indicators. In Handbook of fluorescent probes and research products, 9th ed.; Molecular Probes: Eugene, OR, 2004; Chapter 21.

Garza et al.

the initial image A with the two green compartments separated by the black one, showing that the pH changes did not alter the film (image not shown). Conclusion In this paper, we show the possibility to build multilayered films with different compartments by alternating the construction of exponentially and linearly growing films. The linear growing multilayer acts as a barrier toward the diffusion of the polyelectrolytes but does not hinder the diffusion of small ions such as protons. When compared to linearly growing films in which active biomolecules are embedded at different depths and that can also act as multicompartment systems, this new type of film can be used to store much larger amounts of material due to the exponential growth of the compartments acting as reservoirs. The buildup of films equipped with several compartments opens the route for multiple functionalization of biomaterial coatings. In particular, this approach allows an easy embedding of biologically active peptides bound to the diffusing polyelectrolytes and included in the compartments that act as reservoirs. The inclusion of different peptides in different compartments could also allow the induction of a cascade of cellular reactions after deposition of cells on the top of the film.11 Such a strategy would however require barriers that are degradable by the cells, and we are currently working on this aspect. Another aspect of main importance results from the ability of small ions to diffuse through the whole film structure. This property could allow the storage of pH- or ionactivated molecules in a given compartment and their activation when needed simply by changing the pH or by adding small ions reacting with the embedded molecules. For example, different enzymes present in the different available compartments could be activated selectively by simply tuning the pH of the solution in contact with the multilayer. Acknowledgment. This work was supported by the program ACI “Nanosciences” (NR204) from the ministe`re Franc¸ ais De´le´gue´ a` la Recherche. J.M.G. is indebted to CONACyT (Consejo Nacional de Ciencia y Tecnologia, Me´xico) for financial support. We are grateful to Je´roˆme Mutterer of the Institut de Biologie Mole´culaire des Plantes, CNRS/ULP (Strasbourg, France), for his assistance with the CLSM. The CLSM platform used in this study was cofinanced by the Re´gion Alsace, the Universite´ Louis Pasteur, and the Association pour la Recherche sur le Cancer. LA049106O (38) Richert, L.; Boulmedais, F.; Lavalle, P.; Mutterer, J.; Ferreux, E.; Decher, G.; Schaaf, P.; Voegel, J.-C.; Picart, C. Biomacromolecules 2004, 5, 284.