Inherent Charge-Shifting Polyelectrolyte Multilayer Blends: A Facile

Jun 30, 2011 - E-mail: [email protected] (P.T.H.); [email protected] (K.C.). ...... Vazquez, Eduardo; Dewitt, David M.; Hammond, Paula T.; Lynn, Da...
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ARTICLE pubs.acs.org/Biomac

Inherent Charge-Shifting Polyelectrolyte Multilayer Blends: A Facile Route for Tunable Protein Release from Surfaces Jinkee Hong,†,‡ Byeong-Su Kim,† Kookheon Char,*,‡ and Paula T. Hammond*,† †

Department of Chemical Engineering, Institute for Soldier Nanotechnologies, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States ‡ School of Chemical and Biological Engineering, The National Creative Research Initiative Center for Intelligent Hybrids, The WCU Program of Chemical Convergence for Energy & Environment, College of Engineering, Seoul National University, Seoul 151-744, Republic of Korea

bS Supporting Information ABSTRACT: Recent research has highlighted degradable multilayer films that enable the programmed release of different therapeutics. Multilayers constructed by the layer-by-layer (LbL) deposition that can undergo disassembly have been demonstrated to be of considerable interest, particularly for biomedical surface coatings due to their versatility and mild aqueous processing conditions, enabling the inclusion of biologic drugs with high activity. In this study, we examine the controlled release of a protein using a different mechanism for film disassembly, the gradual dissociation of film interactions under release conditions. Poly(β-amino ester)s and poly(Llysine) (PLL) were used as the positively charged multilayer components coassembled with a model negatively charged antigen protein, ovalbumin (Ova). The release of the protein from these multilayer films is dominated by the slow shift in the charge of components under physiological pH conditions rather than by hydrolytic degradative release. The time scale of release can be varied over almost 2 orders of magnitude by varying the ratio of the two polyamines in the deposition solution. The highly versatile and tunable properties of these films form a basis for designing controlled and sequential delivery of drug coatings using a variety of polyions.

’ INTRODUCTION One of the key areas of growth in the pharmaceutical industry is the development of biologic drugs, drugs that have proteins or peptides as a major component; these therapeutics present challenges in their encapsulation and release using traditional polymeric systems that require heat and organic solvents for processing and also yield relatively low drug loadings. New polymeric biomaterials that would enable controlled protein release for improved therapeutic outcomes, particularly in localized release, can lead to a much more widespread potential for biomedical and biological applications.1 6 Thin films that provide the sustained release of active biomolecules such as proteins, peptides, or enzymes from surfaces for local delivery have the potential to broaden the development of new delivery coatings for biomedical devices, regenerative tissue scaffolds, and artificial organs.7 15 Polyelectrolyte multilayers constructed by the layer-by-layer (LbL) deposition have attracted considerable interest, particularly for biomedical surface coatings because of their versatility, mild aqueous processing conditions, and ability to coat virtually any substrate conformally.16 21 These systems offer great promise for the controlled release from surfaces; a significant level of this control relies on introducing mechanisms of film release, r 2011 American Chemical Society

usually via the systematic breakdown of the film. Diverse multilayer films have been investigated for the controlled release of active therapeutic agents by tuning many parameters such as ionic strength,22 temperature,23 electroactivation,24 enzymatic,25 and pH treatment26 of polymer films. We and other groups have introduced and explored the use of hydrolytically degradable polyions as a means of regulating the release; in these systems, we introduce a hydrolytically degradable component, a cationic polyester, which undergoes degradation and erosion, thus releasing the film components.27 29 However, the release kinetics of the constituents of LbL assembled multilayers can rely on a number of factors, including the degradability of one of the components, the out-diffusion of film components, and the disassembly of multilayer structures via the loss or change in secondary interactions that were employed to assemble the film. Such interactions can be intentionally manipulated via the use of charge-shifting polyelectrolytes that gradually shift from positive to negative charges upon hydrolysis30,31 or the use of electrochemical redox to “erase” charge from one of the Received: April 25, 2011 Revised: June 29, 2011 Published: June 30, 2011 2975

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Biomacromolecules components.32,33 In this work, we examine the use of known weak polycations that undergo a shift in charge density under physiological release conditions that trigger the disassembly of films and the manipulation of two different polycations to tune the release period of a common protein from minutes to days. Our group recently reported the surface delivery of a model antigen protein, ovalbumin (Ova), from a degradable polyelectrolyte multilayer film platform for transcutaneous vaccine applications.34 The film incorporated a hydrolytically degradable polycation, poly(β-amino ester) (PBAE), to control the release kinetics of the protein antigen upon exposure to physiological condition. To our surprise, however, the release kinetics were not altered by the variation of hydrophobicity of the poly(β-amino ester). Instead, all films released the incorporated Ova within a short period of time (∼30 min) that was much faster than the rate of hydrolytic degradation of the PBAE. We postulated that the release was based on the lowering of positive charge density of the PBAE and the loss of strong interactions with negatively charged Ova under the release conditions, thus leading to destabilization and release of Ova over very short time periods. As a result, we were prompted to search for a new system to control the loading and release kinetics more precisely with simple preparation processes and commercially available biomaterials. In this context, we report herein multilayer thin films capable of releasing model proteins in a controlled manner by simply blending two different pH-sensitive, like-charged polyions during deposition based on the distinct pKa and charge density differences between the polymers. Caruso and coworkers have reported that the construction of multilayer films from blended polyelectrolyte solutions can be used to exert control over many film properties.35 37 Along with PBAE, containing tertiary amines along its backbone repeats, we chose a polypeptide, poly(L-lysine) (PLL), which contains more basic primary amines as side groups and a higher overall charge density along its backbone. Both weak polyelectrolytes are highly dependent on solution pH, influencing the surface adsorption behavior as well as the internal film structure. One interesting benefit of using weak polyelectrolytes in controlling the drug release lies in the fact that the dissociation of interactions within the multilayer films can be easily tuned by a simple change in pH. For example, Ova containing multilayer films prepared at pH 6.0 would release the negatively charged Ova upon exposure to physiological pH condition (pH 7.4). We have completed the detailed analysis of loading and release kinetics of (Poly1/Ova)n and (PLL/Ova)n multilayer systems independently and also compared their individual release kinetics with those of polycation blend systems with different ratios of the two components. We found that the variation of the ratio between two polycations can significantly alter the release kinetics as well as the loading of Ova with a notably broad range of release time from 0.3 to 79 h. We believe that the approach presented in this study for the controlled release of charged molecules provides insights into how multilayer films can be designed for passive but highly controlled disassembly simply by manipulating systems that are bound by weak interactions at or near their point of dissociation. The use of these principles can yield drug release systems with a great range of release kinetics using simple polymers as components. Of particular interest is the ability to use commercially available FDA-approved components with this kind of approach. Also, the fine-tuning of the release kinetics of constituents is anticipated to provide a means of satisfying specific requirements

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of active therapeutic release over a wide range of time spans for multiple biomedical applications.

’ EXPERIMENTAL SECTION Materials. Poly(β-amino ester)s were synthesized as previously described.27 PLL (Mw = 80 kDa) was purchased from Sigma (St. Louis, MO) and ovalbumin (Ova), Fluorescein, and Texas RED-conjugated Ova, were purchased from Invitrogen (Eugene, OR). Preparation of Polyelectrolyte Solutions. Poly1 and PLL were dissolved in pH 6 in 100 mM sodium acetate buffer (NaOAc) at a concentration of 2.0 mg/mL. Solution pH values were adjusted to 6.0 with the addition of 0.10 M HCl or 0.10 M NaOH. Film Construction. LbL multilayer films were assembled with a modified programmable Carl Zeiss HMS DS50 slide stainer. Typically, films were constructed on a silicon wafer and glass slide with approximate size of 2  1.4 in2, which were cleaned initially with a Piranha solution (sulfuric acid/hydrogen peroxide 70/30 v/v%) and negatively charged by heating at 70 °C for 20 min in a 5:1:1 vol % mixture of water, hydrogen peroxide, and a 29% ammonia solution (RCA solution). The substrate was then dipped in positively charged polyelectrolyte solutions (either Poly1 or PLL solution in 100 mM NaOAc buffer, pH 6.0) for 10 min, followed by three sequential rinsing steps with pH-adjusted water for 1 min each. Then, the substrate is dipped in negatively charged Ova (0.10 mg/mL in 100 mM NaOAc buffer, pH 6.0) for 10 min and exposed to the same rinsing steps as described above to achieve the desired numbers of bilayer film. The prepared films were dried and stored in air. In addition, blend films were prepared via the same procedure above, except the positively charged Poly 1 and PLL were mixed in a different volume ratio prior to the deposition. Film and Release Characterization. Film growth was determined by profilometry (Tencor P-10) at five different predetermined locations on the film surface. Release experiments were conducted by immersing a prepared multilayer film into a 20 mL vial containing 3.0 mL of phosphate-buffered saline (PBS: water-based salt solution containing sodium chloride, sodium phosphate, potassium chloride, and potassium phosphate, the osmolarity and ion concentration of the solution usually similar with human body) at room temperature. At a series of different time points, films were transferred to another vial, and fresh PBS solution of same volume was introduced. Ova release from the multilayer film was followed by measuring the fluorescence spectra (Quantamaster Fluorometer, PTI) of released Fluorescein-labeled and Texas-Red conjugated Ova in a PBS solution (absorption 494 and 596 nm/fluorescence emission 520 and 615 nm maxima, respectively). Because the absorption spectra of Texas-Red overlaps with the emission spectra of Fluorescein, we fabricated two sets of multilayers films. In one set, unlabeled Ova was first layered with PLL (PLL/Ova)20, followed by incorporation of Ova-FL with Poly1 (Poly1/Ova-FL)20. In the other set, (PLL/Ova-TR)20 was layered first, followed by (Poly1/Ova), where the Ova was unlabeled. Release fractions were collected at different predetermined time intervals for each set of multilayer films and plotted on the same scale for comparison. The changes in the film thickness were also monitored by the thickness of film after each time point in PBS. All release and film degradation studies were performed in triplicate.

’ RESULTS AND DISCUSSION To prepare drug delivery coatings with sequential and tunable drug release, we utilized two different types of positively charged biodegradable materials: a linear poly(β-amino ester) (Poly1), which contains tertiary amines along its polymer backbone with a pKa at approximately 5.5 to 6.0, and PLL, a known linear polyisopeptide with a pendant primary amine and a pK a of ∼9. 38 Poly1 is known to degrade hydrolytically under 2976

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Figure 1. (a) Chemical structures of polycations used in the present study. (b) Schematic illustrations of (Poly1/Ova)20 and (PLL/Ova)20 multilayer films. (c) Growth curve of electrostatically assembled (Poly1/Ova)n (9) and (PLL/Ova)n (2) multilayer films as a function of bilayer number. The film thickness was measured with profilometry in dried state.

physiological conditions, with degradation taking place over multiple days at pH 7.4. This polyion has been used in our previous work to generate hydrolytically degradable multilayers; for example, we found that when it is included in LbL films with tetralayer architectures with a range of polyanions and a lysozyme protein, degradation at pH 7.4 at room temperature can take up to 30 days, and release at physiological pH and temperature can yield films that degrade over a period of several days.29 In contrast, for this work, we do not utilize the hydrolytic nature of Poly1 to induce the film degradation over these longer time scales; rather, both Poly 1 and PLL act as weak polyelectrolytes that enable film erosion through the gradual dissociation of electrostatic interactions upon changes in pH (Figure 1a). As a model protein, we examined the assembly of multilayers containing the relatively weakly charged Ova, a 45 kDa globular protein with a net charge of 12 at pH 7.0, which is routinely used as a model antigen in immunology.39 The net charge density of Ova is much lower than that of many other proteins we examined for hydrolytic release. First, we prepared multilayer films using the LbL assembly method through the alternating adsorption of positively charged Poly1 and negatively charged Ova from aqueous solutions onto a silicon wafer based on the electrostatic interactions (Figure 1b). According to previous results, the deposition pH value (pH 6.0) was selected for both Poly1 and Ova (pI ∼4.6) to achieve the maximal incorporation of Ova.34 To maintain the same basis, (PLL/Ova) multilayers were also constructed at pH 6. The formation of (Poly1/Ova)n (n = number of bilayer) and (PLL/Ova)n multilayer films was examined by profilometry. Figure 1c shows the thickness of dried multilayer films placed on silicon wafers as a function of the number of deposition bilayers. The thickness growth curves were approximately linear for both (Poly1/Ova)n and (PLL/Ova)n multilayer films, although the slope is quite different between two film types. The average bilayer thicknesses of (Poly1/Ova)n and (PLL/ Ova)n multilayer films were 8.3 ( 2.9 and 27.8 ( 3.2 nm, respectively. The root-mean-square (rms) roughnesses are 26 ( 8 nm for (Poly1/Ova)20 films and 43.4 ( 6 nm for (PLL/Ova)20 films. The (PLL/Ova)20 multilayer films were observed to be

three times thicker than that of (Poly1/Ova)20 pairs, suggesting that the loading of Ova protein in the (PLL/Ova)20 film should be larger than the Ova loading within the (Poly1/Ova)20 multilayer film due to enhanced electrostatic and hydrogen bonding interactions with the more basic PLL amines. Measurement of the total amount of Ova released from completely dissolved films showed that for films assembled at pH 6, 20 bilayer (Poly1/ Ova)20 and (PLL/Ova)20 multilayer films carried 3.2 and 7.8 μg/ cm2 Ova protein total. We studied the release of Ova from both (Poly1/Ova)20 and (PLL/Ova)20 multilayer films by exposing them under physiological conditions (PBS buffer solution at pH 7.4). Figure 2 shows the disassembly of multilayers and subsequent release of Ova from (Poly1/Ova)20 and (PLL/Ova)20 multilayers determined by independently tracking the decrease in film thickness with profilometry and the release of fluorescein-labeled Ova into buffer solution. Upon incubation in PBS solution, the (Poly1/Ova)20 multilayer film exhibits a rapid release of Ova along with a corresponding decrease in film thickness (Figure 2). As previously reported, >80% disassembly and consequent release of Ova from (Poly1/ Ova)n multilayer films occurred within 6 h.34 We propose that the Poly1 polycation loses some of its charge density at pH 7.4, and the relatively weakly charged and globular Ova is no longer effectively trapped within the LbL matrix, resulting in the diffusion of Ova out of the (Poly1/Ova)n multilayer films upon contact with aqueous solution. The diffusion of Ova from the film further leads to the charge destabilization of the electrostatic assembled multilayer film and subsequent rapid dissolution of the film. The (PLL/Ova)20 films exhibited a relatively slow erosion release profile. Without a burst initial linear release, Ova was continuously released out to 120 h. In comparison with the (Poly1/Ova)20 multilayer, the time taken for the release of half of the Ova (t1/2) was significantly extended from 0.3 h in the first case to 79 h in the case of (PLL/Ova)20 multilayer film. We believe that this difference stems from the differences in the relative basicity of the two polyamines and their respective pKa values as well as in the higher charge density of PLL versus Poly 1. 2977

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Figure 2. Film dissociation of single-component films in PBS buffer (pH 7.4 at 25 °C). (a) Normalized release profiles of Ova and (b) the change of film thickness of (Poly1/Ova)20 (9) and (PLL/Ova)20 (Δ) films as a function of treatment time. All films were prepared in pH 6 deposition condition and released in PBS solution of pH 7.4.

Figure 3. Effect of sequence order in dual film architectures of (PLL/Ova)20 and (Poly1/Ova) 20 on the release profile: Normalized cumulative release from: (a) substrate/(PLL/Ova)20(Poly1/Ova)20 and (b) substrate/(Poly1/Ova)20(PLL/Ova)20 multilayer films. The release experiments were conducted in PBS buffer (pH 7.4 at 25 °C). 9 (green line) and 2 (pink line) indicate Ova released from (Poly1/Ova)20 and (PLL/Ova)20, respectively.

In both cases, Ova is a protein with fairly low surface charge density, thus making multilayers formed with Ova unstable when assembled with either of the polyamines upon exposure to a pH higher than the assembly pH value of 6.0. This is also supported by the fact that the films disassemble slowly at pH values lower than the assembly conditions, as compared with the physiological release condition of pH 7.4 (Supporting Information, Figure S1). A greater affinity of Ova for PLL versus the PBAE is inferred from the differences in the average thickness per bilayer pair and Ova loading. The larger amount of Ova loading is likely due to enhanced electrostatics but may also be attributable to other interactions with the chemical structure of PLL. We could tune the drug release profile by changing the electrostatic interactions between PLL and Ova. Negatively charged Ova starts to diffuse out from the (PLL/Ova)n multilayer films constructed at pH 6 when the film is subjected to the physiological buffer condition of pH 7.4. The amine groups in PLL partially lose their positive charge, and the increased ionic strength of the PBS solution also introduces more charge shielding, further inducing the structural disassembly of PLL chains complexed with Ova. In other words, the control of electrostatic dissociation between PLL and Ova through pH and ionic strength variations led to the relatively slow release of Ova. On the basis of the two distinct release properties of the singlecomponent films with these two different polycations, we have

further investigated the combination of the two films with different sequences of layering. More specifically, the hybrid films of (Poly1/Ova)20 and (PLL/Ova)20 were prepared in a dual film format, such that a film contained an underlying layer of (PLL/Ova)20, followed by surface layers of (Poly1/Ova)20 film (substrate/(PLL/Ova)20/(Poly1/Ova)20) and, conversely, lower layers of (Poly1/Ova)20 film and a top layer of (PLL/Ova)20 (substrate/(Poly1/Ova)20/(PLL/Ova)20) (Figure 3). To analyze quantitatively the release behavior, we examined the release of two different fluorescent-dye-conjugated Ova molecules (Fluorescein and Texas Red). Interestingly, the release profile of each hybrid film shows quite similar behavior regardless of the location of each single-multilayer film component, whether at the top surface or at the bottom of the film; this behavior has been observed in other PBAE release systems and is characteristic of interdiffusion between the films.40 For example, the time required for the half release of Ova (t1/2) was observed to be 0.5 h for the Poly1-based multilayer pair and 77 h for the PLL-based multilayer pair from substrate/(PLL/ Ova)20(Poly1/Ova)20. In the case of substrate/(Poly1/Ova)20 (PLL/Ova)20 film, t1/2 of Poly1- and PLL-based multilayer pairs was measured to be 2.6 and 65 h, respectively. These results suggest that some degree of interlayer diffusion occurs between (PLL/Ova)n and (Poly1/Ova)n multilayer films under aqueous condition during the dipping process. In addition, both films 2978

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Biomacromolecules exhibit similar disassembly profiles. (See Figure S2 of the Supporting Information.) However, the thickness of the substrate/(Poly1/Ova)20(PLL/Ova)20 sample was observed to decrease slightly faster compared with that of substrate/(PLL/ Ova)20(Poly1/Ova)20. In particular, the duration from 25 to 75 h of release time shows a pronounced thickness difference. At 75 h,

Figure 4. Normalized cumulative Ova release curves from different polycation blend compositions in the multilayer films. (a) Comparison of Ova release profiles from a single-multilayer film: (Poly1/Ova)20 (9, green line), (Poly190-PLL10/Ova)20 (f, black line), (Poly170-PLL30/ Ova)20 (b, blue line), (Poly150-PLL50/Ova)20 ([, orange line), and (PLL/Ova)n (2, pink line). (b) (left) The amount of Ova loaded in the multilayer films measured by a fluorimeter and (right) the release half time of Ova from the multilayer films. Films were prepared with various volume ratios of Poly1 and PLL to produce positively charged polyeletrolytes interacting with Ova.

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>90% of the Ova was already released from both Poly1- and PLLbased pairs from substrate/(Poly1/Ova)20(PLL/Ova)20 multilayer films. However, in the case of substrate/(PLL/Ova)20(Poly1/Ova)20, the PLL-based pairs showed ∼60% of the Ova released. Our studies strongly suggest that the release profiles depend not only on the breakdown mechanism of the polymer films but also on the sequence of components during the LbL assembly. These findings further suggest that the interdiffusion during the assembly as well as out-diffusion of the protein throughout the film during release may take place; however, the two different protein/polyamine complexes that form in the LbL film are likely not intermixed or cross-exchanged, as indicated by the separation of release of two labeled Ova proteins in each case. To manipulate both timing and rate of Ova release more precisely, we have introduced blend structures based on Poly1 and PLL within multilayer films. Specifically, we have used mixtures of Poly1 and PLL in aqueous solution for the cationic adsorption step (Figure 4). In Figure 4a, it is apparent that the release time of a blend multilayer (Poly190 + PLL10/Ova)20 (mixed by volume ratio of stock solutions) lies between (Poly1/ Ova)20 and (PLL/Ova)20 multilayer films with t1/2 of 44 h. The mixing ratio of stock solution was varied as a key parameter to control the Ova release timing. The cumulative release profile of multilayers consisting of (Poly1 + PLL/Ova)20 with varying volume ratios of stock solutions 50:50, 70:30, and 90:10 (Poly1/ PLL) exhibits a similar amount of Ova loading and release that scales with the increase in PLL content, implying the important role of PLL due to relatively tighter and stronger electrostatic interactions with Ova. This is also consistent with previous data of the pure (PLL/Ova)20 film, demonstrating the enhanced loading of Ova and the significantly extended release of Ova. This result also allows the demonstration of broad flexibility in tailoring the release profiles based on the blend structure of degradable polyelectrolytes. To take full advantage of the utility of these blend systems, we demonstrated a film with the dual-multilayer format of (Poly190 + PLL10/Ova)20 on top of a base layer of (PLL/Ova)20, as shown in Figure 5. In this case, we seek to observe the effect of increasing the PLL content on the interlayer diffusion of PLL in regulating the release properties of degradable films. As already demonstrated, t1/2 for (Poly190 + PLL10/Ova)20 and (PLL/Ova)20 were determined to be 44 and 79 h, respectively. However in the

Figure 5. Cumulative release profiles of Ova from dual-multilayer films: (Poly190+PLL10/ova)20 surface layer (f, black line) and (PLL/Ova)20 base layer (2, pink line). 2979

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Table 1. Overall Thickness and the Release Half Time of Ova from Various Film Architectures Used in the Present Study release half time film architecture

thickness (nm)

(t1/2) (h)

single-multilayer films 170.7 ( 7.8 389.5 ( 30 9

0.3 44

(Polyl70%-PLL30%/Ova)20

405 8 ( 55.4

49

(Polyl50%-PLL50%/Ova)20

411.5 ( 39.7

47

(PLL/Ova)20

576.8 ( 64.3

79

dual-multilayer films 799.6 ( 102.3

(Polyl/ova)20

top Ova: 0.5 bottom Ova: 77

substrate/(Polyl/ova)20

650.3 ( 71.6

top Ova: 2.6

(PLL/ova)20 substrate/(PLL/ova)20

874.2 ( 56.2

bottom Ova: 65 top Ova: 45

(Poly190%-PLL10%/ova)20

bottom Ova: 67

dual-multilayer film, the half times for release were decreased, with t1/2 of 30 h for the (Poly190 + PLL10/Ova)20 surface layer and 67 h for the base (PLL/Ova)20 multilayer pair. Overall, our approaches of controlling the release kinetics of a model protein through different release mechanisms and component blending are summarized in Table 1 by film thickness and half release time. The possibility to control the protein release kinetics through blending structures in single- and double-multilayer films suggests that this simple and cost-effective strategy would assist in developing biomedical thin films with controllable release profiles.

’ CONCLUSIONS We demonstrated that the broad release timing in the Ova release properties can be sequentially controlled using two different film disassembly kinetics based on the rapid or gradual dissociation of electrostatic interactions upon changes in pH. The release of the Ova protein from multilayer films is mainly due to the dissociation of electrostatic interactions upon changes in pH of weak polyelectrolytes (i.e., Poly 1 and PLL), which is accelerated by the Ova diffusion out of the film. In this study, (Poly1/Ova)n, (PLL/Ova)n, and (Poly1 + PLL/Ova)n multilayer films prepared under pH 6/6 deposition conditions show very different release behavior due to the different pKa values of the two polycations and their corresponding pH-shift from assembly conditions to PBS treatments. By creating dual-multilayer films and multilayer films composed of the blends of two different polycations, we can realize a range of dual release behavior from the separate release of two components from the film at very different times to the tuning of release time for proteins through the variation of the relative amount of two polyions coadsorbed from solution. On the basis of these results, our approach could provide a basis for designing the controlled multiple dosage delivery of drug coatings on a variety of surfaces with the attributes of LbL processing method. ’ ASSOCIATED CONTENT

bS

’ AUTHOR INFORMATION Corresponding Author

(Poly/Ova)20 (Polyl90%-PLL10%Ova)20

substrate/(PLL/ova)20

change in film thickness of multilayer films with deposition cycle. This material is available free of charge via the Internet at http:// pubs.acs.org.

Supporting Information. Normalized release profiles of single-component films in pH 3.5 aqueous solution and the

*E-mail: [email protected] (P.T.H.); [email protected] (K.C.).

’ ACKNOWLEDGMENT This research is funded by the Institute for Soldier Nanotechnologies (ISN) and Singapore-MIT Alliance for Research & Technology (SMART) in Massachusetts Institute of Technology (MIT). Additionally, this work was also financially supported by the National Research Foundation of Korea Grant funded by the Korean Government (MEST) (The National Creative Research Initiative Program for “Intelligent Hybrids Research Center” (2010-0018290), (NRF-2009-0093282), and the WCU Program of Chemical Convergence for Energy and Environment (R3110013) in Seoul National University. ’ REFERENCES (1) Simone, E. A.; Dziubla, T. D.; Colon-Gonzalez, F.; Discher, D. E.; Muzykantov, V. R. Biomacromolecules 2007, 8, 3914–3921. (2) Venkatesh, S.; Sizemore, S. P.; Byrne, M. E. Biomaterials 2007, 28, 717–724. (3) Chung, H. J.; Park, T. G. Nano Today 2009, 4, 429–437. (4) Jay, S. M.; Saltzman, W. M. J. Controlled Release 2009, 134, 26–34. (5) Ali, M.; Byrne, M. E. Pharm. Res. 2009, 26, 714–726. (6) Anandhakumar, S.; Nagaraja, V.; Raichur, A. M. Colloids Surf., B 2010, 78, 266–274. (7) Wood, K. C.; Boedicker, J. Q.; Lynn, D. M.; Hammon, P. T. Langmuir 2005, 21, 1603–1609. (8) Zhong, Y.; Whittington, C. F.; Zhang, L.; Haynie, D. T. Nanomedicine (N.Y., NY, U. S.) 2007, 3, 154–160. (9) Ochs, C. J.; Such, G. K.; Stadler, B.; Caruso, F. Biomacromolecules 2008, 9, 3389–3396. (10) Wang, F.; Li, D.; Li, G. P.; Liu, X. Q.; Dong, S. J. Biomacromolecules 2008, 9, 2645–2652. (11) Tomita, S.; Sato, K.; Anzai, J. I. J. Colloid Interface Sci. 2008, 326, 35–40. (12) Wang, L.; Wang, X.; Xu, M. F.; Chen, D. D.; Sun, J. Q. Langmuir 2008, 24, 1902–1909. (13) Kim, B. S.; Smith, R. C.; Poon, Z.; Hammond, P. T. Langmuir 2009, 25, 14086–14092. (14) Crouzier, T.; Ren, K.; Nicolas, C.; Roy, C.; Picart, C. Small 2009, 5, 598–608. (15) Kittitheeranun, P.; Sanchavanakit, N.; Sajomsang, W.; Dubas, S. T. Langmuir 2010, 26, 6869–6873. (16) Ai, H.; Jones, S. A.; Lvov, Y. M. Cell Biochem. Biophys. 2003, 39, 23–43. (17) Hammond, P. T. Adv. Mater. 2004, 16, 1271–1293. (18) Tang, Z. Y.; Wang, Y.; Podsiadlo, P.; Kotov, N. A. Adv. Mater. 2007, 19, 906–906. (19) Schlenoff, J. B. Langmuir 2009, 25, 14007–14010. (20) Smith, R. C.; Riollano, M.; Leung, A.; Hammond, P. T. Angew. Chem., Int. Ed. 2009, 48, 8974–8977. (21) Boudou, T.; Crouzier, T.; Ren, K. F.; Blin, G.; Picart, C. Adv. Mater. 2010, 22, 441–467. (22) Jomaa, H. W.; Schlenoff, J. B. Macromolecules 2005, 38, 8473–8480. 2980

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dx.doi.org/10.1021/bm200566k |Biomacromolecules 2011, 12, 2975–2981