Multilayered Thin Films that Sustain the Release of Functional DNA

polyelectrolyte assemblies that sustain the release of functional plasmid DNA from the surfaces of model substrates under physiological conditions. Mu...
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Langmuir 2004, 20, 8015-8021

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Multilayered Thin Films that Sustain the Release of Functional DNA under Physiological Conditions Jingtao Zhang, Lynn S. Chua, and David M. Lynn* Department of Chemical and Biological Engineering, University of Wisconsin, Madison, 1415 Engineering Drive, Madison, Wisconsin 53706-1607 Received May 5, 2004. In Final Form: June 21, 2004 The development of thin films and coatings that control the release of DNA from the surfaces of materials could have a significant impact on localized approaches to gene therapy. Here, we report multilayered polyelectrolyte assemblies that sustain the release of functional plasmid DNA from the surfaces of model substrates under physiological conditions. Multilayered assemblies consisting of alternating layers of plasmid DNA encoding for enhanced green fluorescent protein (EGFP) and a synthetic degradable polyamine were deposited on planar silicon and quartz substrates using a layer-by-layer fabrication process. Film growth was monitored by ellipsometry and UV spectrophotometry and correlated linearly with the number of polymer and plasmid layers deposited. In general, the thickness of deposited layers was found to be a function of both the pH and the ionic strength of the polyelectrolyte solutions used. Films up to 100 nm thick were investigated in this study. These assemblies erode gradually upon incubation in phosphatebuffered saline at 37 °C, as determined by ellipsometry and UV spectrophotometry, and sustain the release of incorporated plasmid into the incubation medium for a period of up to 30 h. Characterization of the released plasmid by agarose gel electrophoresis revealed that the DNA was released in a relaxed, open circular, rather than supercoiled, topology; subsequent cell transfection experiments demonstrated that the released plasmid is transcriptionally viable and promotes the expression of EGFP in the COS-7 cell line. These layered materials could represent an approach to the controlled administration of one or more functional DNA constructs from the surfaces of biomedical materials and devices.

Introduction The localized delivery of DNA to cells presents a formidable challenge and an obstacle to the clinical success of gene therapy. Conventional materials do not meet the needs of applications that require the release of DNA from the surfaces of implantable materials or the predictable administration of more than one gene sequence.1-6 Although degradable polymer matrices can be engineered to sustain the release of DNA for prolonged periods,2,3,6 most materials generally lack the functional sophistication required to control both the spatial distribution and temporal release of DNA at the surface of a device or construct.1 Here, we describe a layer-by-layer approach to the assembly of multilayered polyelectrolyte films that makes possible the incorporation and subsequent release of transcriptionally active plasmid DNA from film-coated substrates under physiological conditions. With further development, these materials could represent an effective approach to the controlled administration of multiple gene sequences from the surfaces of biomedical materials and devices.1-5 The layer-by-layer deposition of polyelectrolytes is a well-established method for the fabrication of multilayered polymer films composed of alternating layers of positively and negatively charged materials.7,8 The scope of materials (1) Saltzman, W. M.; Olbricht, W. L. Nat. Rev. Drug Discov. 2002, 1, 177-186. (2) Shea, L. D.; Smiley, E.; Bonadio, J.; Mooney, D. J. Nat. Biotechnol. 1999, 17, 551-554. (3) Saltzman, W. M. Nat. Biotechnol. 1999, 17, 534-535. (4) Klugherz, B. D.; Song, C.; DeFelice, S.; Cui, X.; Lu, Z.; Connolly, J.; Hinson, J. T.; Wilensky, R. L.; Levy, R. J. Hum. Gene Ther. 2002, 13, 443-454. (5) Segura, T.; Shea, L. D. Bioconjugate Chem. 2002, 13, 621-629. (6) Klugherz, B. D.; Jones, P. L.; Cui, X.; Chen, W.; Meneveau, N. F.; DeFelice, S.; Connolly, J.; Wilensky, R. L.; Levy, R. J. Nat. Biotechnol. 2000, 18, 1181-1184. (7) Decher, G. Science 1997, 277, 1232-1237.

that can be incorporated into mutilayered polyelectrolyte assemblies is broad and includes synthetic polymers,8 as well as biological macromolecules, such as proteins9-12 and DNA.13-20 Notable biotechnical applications of this technique include the immobilization of active enzymes11,12,21 and the fabrication of films engineered to either promote or inhibit the attachment of cells22-26 or proteins25,27 to surfaces. In the context of controlled release, (8) Bertrand, P.; Jonas, A.; Laschewsky, A.; Legras, R. Macromol. Rapid Comm. 2000, 21, 319-348. (9) Schwinte, P.; Voegel, J. C.; Picart, C.; Haikel, Y.; Schaaf, P.; Szalontai, B. J. Phys. Chem. B 2001, 105, 11906-11916. (10) Schwinte, P.; Ball, V.; Szalontai, B.; Haikel, Y.; Voegel, J. C.; Schaaf, P. Biomacromolecules 2002, 3, 1135-1143. (11) Jin, W.; Shi, X. Y.; Caruso, F. J. Am. Chem. Soc. 2001, 123, 8121-8122. (12) Tiourina, O. P.; Antipov, A. A.; Sukhorukov, G. B.; Larionova, N. L.; Lvov, Y.; Mohwald, H. Macromol. Biosci. 2001, 1, 209-214. (13) Pei, R. J.; Cui, X. Q.; Yang, X. R.; Wang, E. K. Biomacromolecules 2001, 2, 463-468. (14) Schuler, C.; Caruso, F. Biomacromolecules 2001, 2, 921-926. (15) Shi, X. Y.; Sanedrin, R. J.; Zhou, F. M. J. Phys. Chem. B 2002, 106, 1173-1180. (16) Lvov, Y.; Decher, G.; Sukhorukov, G. Macromolecules 1993, 26, 5396-5399. (17) Caruso, F.; Rodda, E.; Furlong, D. F.; Niikura, K.; Okahata, Y. Anal. Chem. 1997, 69, 2043-2049. (18) Caruso, F.; Furlong, D. N.; Niikura, K.; Okahata, Y. Colloid Surf., B 1998, 10, 199-204. (19) Trubetskoy, V. S.; Loomis, A.; Hagstrom, J. E.; Budker, V. G.; Wolff, J. A. Nucleic Acids Res. 1999, 27, 3090-3095. (20) Serizawa, T.; Yamaguchi, M.; Akashi, M. Angew. Chem., Int. Ed. 2003, 42, 1115-1118. (21) Lvov, Y.; Antipov, A. A.; Mamedov, A.; Mohwald, H.; Sukhorukov, G. B. Nano Lett. 2001, 1, 125-128. (22) Chluba, J.; Voegel, J. C.; Decher, G.; Erbacher, P.; Schaaf, P.; Ogier, J. Biomacromolecules 2001, 2, 800-805. (23) Elbert, D. L.; Herbert, C. B.; Hubbell, J. A. Langmuir 1999, 15, 5355-5362. (24) Tryoen-Toth, P.; Vautier, D.; Haikel, Y.; Voegel, J. C.; Schaaf, P.; Chluba, J.; Ogier, J. J. Biomed. Mater. Res. 2002, 60, 657-667. (25) Richert, L.; Lavalle, P.; Vautier, D.; Senger, B.; Stoltz, J. F.; Schaaf, P.; Voegel, J. C.; Picart, C. Biomacromolecules 2002, 3, 11701178.

10.1021/la048888i CCC: $27.50 © 2004 American Chemical Society Published on Web 07/31/2004

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Scheme 1. Schematic Illustrating the Fabrication (A) and Gradual Erosion (B) of a Multilayered Polyelectrolyte Film Constructed from a Hydrolytically Degradable Cationic Polymer and a Non-Degradable Polyanionic Polymer [e.g., Poly(styrene sulfonate) or DNA]

the layer-by-layer fabrication procedure offers potential advantages over conventional protein and nucleic acid encapsulation strategies, including 1) the ability to control the order and location of multiple polymer layers with nanometer-scale precision and 2) the ability to define the concentrations of incorporated materials simply by varying the number of polymer layers incorporated.7,8 Although numerous reports describe the application of these materials to the sustained release of permeable small molecules,11,12,21,28-32 there are few examples of these assemblies designed to release macromolecular components. Several groups have used changes in ionic strength or environmental pH to trigger the “deconstruction”, or dissolution, of multilayered assemblies fabricated from strong and/or weak polyelectrolytes.14,33-37 In the context of DNA delivery, Schu¨ler et al. used an increase in ionic strength to release herring sperm DNA from hollow assemblies fabricated using a low-molecular-weight polyamine.14 This report represents a significant advance toward the application of these capsules as gene-delivery agents, but this approach is currently limited by the need for relatively high salt concentrations (e.g., from 0.6 to 5.0 M) that are not well-suited for use under physiological conditions. Additionally, this and other past reports12-18,38,39 describing the incorporation of DNA into multilayered assemblies have been restricted to the deposition of nonfunctional DNA or short polynucleotide constructs that are not transcriptionally active when administered to mammalian cells. A recent report describes the layerby-layer deposition of polyelectrolytes onto condensed plasmid DNA nanoparticles,19 and the adsorption of plasmid DNA onto the surface of cationic microparticles has been applied toward the development of new DNA vaccines.40,41 To the best of our knowledge, the sustained release of transcriptionally active plasmid DNA from multilayered polyelectrolyte assemblies has not been reported. The integration of design elements that enable the dissolution, disintegration, or erosion of multilayered polyelectrolyte assemblies under physiological conditions would facilitate the application of these structured materials in therapeutic contexts,42-45 where changes in ionic strength or pH are restricted or undesirable. Toward this goal, we recently reported that the alternating deposition of conventional polyanions with a degradable synthetic cationic polyester yields macromolecular assemblies that erode gradually when incubated under physiological conditions (Scheme 1).46 Here, we report the fabrication of thin films that sustain the release of incorporated plasmid DNA from the surfaces of planar silicon and quartz substrates and describe in full the factors governing plasmid incorporation, film growth, and surface release kinetics for these materials. We further demonstrate that the plasmid released from the surfaces of these materials is released in a transcriptionally active form that promotes significant levels of gene expression

in mammalian cell transfection experiments. The development of structured thin films and coatings that sustain the release of functional plasmid DNA from surfaces could have a significant impact on the development of new localized gene therapies. Materials and Methods General Considerations. Silicon and quartz substrates (0.6 × 2.0 cm) were cleaned with methylene chloride, ethanol, methanol, and deionized water and dried under a stream of nitrogen. Surfaces were then activated by either 1) immersion in a stirred NaOH solution (1.0 M) for 15 min, rinsing with deionized water, dipping briefly into an HCl solution (1.0 M), rinsing again, and drying under a stream of nitrogen or 2) etching in an oxygen plasma etcher for 5 min (Plasma Etch, Carson City, NV). Ellipsometric thicknesses for films deposited on silicon substrates were determined using a Rudolph Research AutoELII ellipsometer (632.8 nm, incident angle ) 70°). Data were processed using the FilmEllipse version 1.1 software package. Relative thicknesses were calculated assuming an average refractive index of 1.54 for the multilayered films. Thicknesses were determined in at least five different standardized locations on each substrate and are presented as an average (with standard deviation) for each substrate. UV-visible absorbance values for films deposited on quartz substrates were recorded on an Ultraspec 2100 Pro spectrophotometer (Amersham Biosciences, Piscataway, NJ). Absorbance values were recorded at a wave(26) Mendelsohn, J. D.; Yang, S. Y.; Hiller, J.; Hochbaum, A. I.; Rubner, M. F. Biomacromolecules 2003, 4, 96-106. (27) Serizawa, T.; Yamaguchi, M.; Akashi, M. Biomacromolecules 2002, 3, 724-731. (28) Qiu, X. P.; Leporatti, S.; Donath, E.; Mohwald, H. Langmuir 2001, 17, 5375-5380. (29) Shi, X. Y.; Caruso, F. Langmuir 2001, 17, 2036-2042. (30) Antipov, A. A.; Sukhorukov, G. B.; Leporatti, S.; Radtchenko, I. L.; Donath, E.; Mohwald, H. Colloid Surf., A 2002, 198, 535-541. (31) Antipov, A. A.; Sukhorukov, G. B.; Donath, E.; Mohwald, H. J. Phys. Chem. B 2001, 105, 2281-2284. (32) Sukhorukov, G. B.; Antipov, A. A.; Voigt, A.; Donath, E.; Mohwald, H. Macromol. Rapid Comm. 2001, 22, 44-46. (33) Sukhishvili, S. A.; Granick, S. J. Am. Chem. Soc. 2000, 122, 9550-9551. (34) Sukhishvili, S. A.; Granick, S. Macromolecules 2002, 35, 301310. (35) Dubas, S. T.; Schlenoff, J. B. Macromolecules 2001, 34, 37363740. (36) Dubas, S. T.; Farhat, T. R.; Schlenoff, J. B. J. Am. Chem. Soc. 2001, 123, 5368-5369. (37) Cho, J.; Caruso, F. Macromolecules 2003, 36, 2845-2851. (38) Lang, J.; Liu, M. J. Phys. Chem. B 1999, 103, 11393-11397. (39) Shchukin, D. G.; Patel, A. A.; Sukhorukov, G. B.; Lvov, Y. M. J. Am. Chem. Soc. 2004, 126, 3374-3375. (40) Singh, M.; Briones, M.; Ott, G.; O’Hagan, D. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 811-816. (41) Trimaille, T.; Pichot, C.; Delair, T. Colloid Surf., A 2003, 221, 39-48. (42) Thierry, B.; Winnik, F. M.; Merhi, Y.; Tabrizian, M. J. Am. Chem. Soc. 2003, 125, 7494-7495. (43) Thierry, B.; Winnik, F. M.; Merhi, Y.; Silver, J.; Tabrizian, M. Biomacromolecules 2003, 4, 1564-1571. (44) Groth, T.; Lendlein, A. Angew. Chem., Int. Ed. 2004, 43, 926928. (45) Serizawa, T.; Yamaguchi, M.; Akashi, M. Macromolecules 2002, 35, 8656-8658. (46) Vazquez, E.; Dewitt, D. M.; Hammond, P. T.; Lynn, D. M. J. Am. Chem. Soc. 2002, 124, 13992-13993.

Thin-Film Sustained Release of Functional DNA length of 260 nm at five different locations on the substrate and were reported as an average (with standard deviation) for each sample. All films were dried under a stream of nitrogen prior to measurement. Materials. Test grade n-type silicon wafers were purchased from Si-Tech, Inc. (Topsfield, MA). Quartz microscope slides were purchased from Chemglass (Vineland, NJ). Linear poly(ethylene imine) (LPEI, MW ) 25 000) was obtained from Polysciences, Inc. (Warrignton, PA). Poly(sodium 4-styrenesulfonate) (SPS, MW ) 70 000), calf thymus DNA, and sodium acetate buffer were purchased from Aldrich Chemical Co. (Milwaukee, WI). Polymer 1 (Mn ) 10 000) was synthesized as previously described.47 Plasmid DNA [pEGFP-N1 (4.7 kb), >95% supercoiled] was obtained from a commercial supplier (Elim Biopharmaceuticals, Inc., San Francisco, CA) or grown in bacterial culture according to established procedures and purified using a commercially available plasmid purification kit (Qiagen, Valencia, CA). All commercial polyelectrolytes were used as received without further purification. Deionized water (18 MΩ) was used for washing steps and to prepare all polymer solutions. All buffers and polymer solutions were filtered through a 0.2 µm membrane syringe filter prior to use unless otherwise noted. Preparation of Polyelectrolyte Solutions. Solutions of polymer 1 used for dipping (5 mM with respect to the molecular weight of the polymer repeat unit) were prepared in sodium acetate buffer (100 mM, pH ) 5.1) and filtered through a 0.2 µm membrane syringe filter prior to use. Solutions of all other synthetic polyelectrolytes (20 mM with respect to the molecular weight of the polymer repeat unit) were prepared in water and were adjusted to pH 5.1 using hydrochloric acid and sodium hydroxide. Solutions of supercoiled plasmid DNA were prepared at a concentration of 1 mg/mL in the desired buffer solution and were not filtered prior to use. Polymer and plasmid solutions used to evaluate the effects of pH or NaCl concentration on film growth were prepared using buffer solutions previously adjusted to desired pH and salt concentrations. Fabrication of Multilayered Films. Films were deposited on planar silicon and quartz substrates precoated with 10 bilayers of LPEI/SPS (terminated with a layer of SPS) to ensure a suitably charged surface for the adsorption of polymer 1. Fabrication of multilayered films was conducted using the alternate dipping method according to the following general protocol: 1) substrates were submerged in a solution of polymer 1 for 5 min, 2) substrates were removed and immersed in an initial water bath for 1 min followed by a second water bath for 1 min, 3) substrates were submerged in a solution of plasmid DNA for 5 min, and 4) substrates were rinsed in the manner described above. This cycle was repeated until the desired number of polymer and DNA layers (typically eight each) had been deposited. Films to be used in degradation experiments were either used immediately or were dried under a stream of dry nitrogen and placed in a vacuum desiccator and stored dry until use to minimize degradation prior to the experiment. Erosion of Multilayered Films and Evaluation of Plasmid Release Kinetics. Experiments designed to investigate the erosion profiles of multilayered polymer 1/plasmid DNA films were performed in the following general manner: film-coated substrates were placed in a plastic UV-transparent cuvette and phosphate-buffered saline (PBS, pH ) 7.4, 137 mM NaCl) was added in an amount sufficient to cover the substrate. The samples were incubated at 37 °C and removed at predetermined intervals (typically every 5 h) for analysis by ellipsometry (for silicon substrates) or UV-visible spectrophotometry (for quartz substrates). For experiments designed to monitor a decrease in film thickness or the loss of plasmid from within a film, the thickness and absorbance (at 260 nm) of each sample was determined in at least five different predetermined locations on the substrate surface, and the sample was returned immediately to the buffer solution. For experiments designed to monitor the concentration of plasmid released into the buffer solution, absorbance readings at 260 nm were made directly on the buffer solution. For plasmid release experiments designed to produce samples for cell transfection experiments (see text), erosion experiments were con(47) Lynn, D. M.; Langer, R. J. Am. Chem. Soc. 2000, 122, 1076110768.

Langmuir, Vol. 20, No. 19, 2004 8017 ducted as above with the following exceptions: at each predetermined time interval (typically 5 h), substrates were removed from the incubation buffer, placed into a new cuvette containing fresh PBS, and the original plasmid-containing solution was stored for analysis. Agarose Gel Electrophoresis Assays. Samples of plasmid DNA collected from film erosion experiments were evaluated by loading 30 µL of plasmid solution into 1% agarose gels (HEPES, 20 mM, pH ) 7.2, 108 V, 45 min). Samples were loaded on the gel with a loading buffer consisting of 50:50 glycerol water (v/v). DNA bands were visualized by ethidium bromide staining. Visualization of DNA in Multilayered Films Using Ethidium Bromide. For experiments designed to visually confirm the presence of plasmid DNA in multilayered films, films deposited on quartz substrates were soaked for 30 min in an aqueous ethidium bromide solution (100 µg/mL). Samples were then rinsed under deionized water for 1 min, dried under a stream of nitrogen, and imaged at 254 nm under the illumination of a handheld UV lamp (UltraViolet Products, Upland, CA). NOTE: Ethidium bromide is a potent mutagen and special care should be taken when handling and disposing of this compound. Cell Transfection Assays. COS-7 cells were grown in 96well plates at an initial seeding density of 12 000 cells/well in 200 µL of growth medium (90% Dulbecco’s modified Eagle’s medium, 10% fetal bovine serum, penicillin 100 units/mL, streptomycin 100 µg/mL). Cells were grown for 24 h, at which time the 50 µL of a Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and plasmid mixture was added directly to the cells according to the general protocol provided by the manufacturer. The Lipofectamine 2000/plasmid transfection milieu was prepared by mixing 25 µL of the plasmid solution collected at each time point during release experiments (arbitrary concentrations but constant volumes) with 25 µL of diluted Lipofectamine 2000 reagent (24 µL stock diluted into 976 µL of water). Fluorescence images were taken after 48 h using an Olympus IX70 microscope and analyzed using the Metavue version 4.6 software package (Universal Imaging Corporation).

Results and Discussion Layer-by-Layer Deposition of Polymer 1/DNA Films onto Planar Silicon and Quartz Substrates. We previously reported that multilayered polyelectrolyte assemblies fabricated from degradable polyamine 1 and conventional polyanions, such as poly(styrene sulfonate) (SPS) and poly(acrylic acid) (PAA), erode gradually over a period of up to 40 h when incubated under physiological conditions.46 On the basis of these initial results, we hypothesized that polymer 1 could also be used to fabricate functional assemblies that would enable both the incorporation and subsequent release of DNA from coated surfaces. As we have independently demonstrated that polymer 1 interacts electrostatically with plasmid DNA to form self-assembled polymer/DNA nanoparticles in

aqueous solution,47 we reasoned that substrates charged with an exposed surface layer of polymer 1 would also be suitable for the direct adsorption of DNA during layerby-layer assembly. Although polymer 1 is hydrolytically degradable, it hydrolyzes relatively slowly under acidic conditions (e.g., t1/2 ) 10 h at pH ) 5.1, 37 °C)47 that allow aqueous fabrication procedures to be carried out under conditions (pH ) 5.1 and ambient temperature) that do not lead to significant polymer or DNA degradation (see below) on the time scale of the fabrication procedure.46 In this study, fabrication times typically ranged from 2 to 4 h depending on the number of polymer layers deposited. We conducted initial experiments demonstrating that films up to 100 nm thick could be fabricated on silicon

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Figure 1. Plot of ellipsometric thickness vs number of polymer 1/DNA bilayer pairs deposited on a silicon substrate using A) a plasmid solution prepared in deionized water and B) a plasmid solution prepared in 100 mM sodium acetate buffer (pH ) 5.1). Black bars represent the thickness of a LPEI/SPS film deposited onto the silicon substrate as an adhesive layer prior to experiment (see text).

substrates using polymer 1 and calf thymus DNA using the alternate dipping fabrication method (data not shown). These results were consistent with the incorporation of calf thymus and other model DNA into films using conventional polycations13-20 and demonstrated that our preliminary work using polymer 1 could be extended to the adsorption and incorporation of nucleic acid material. However, calf thymus DNA is obtained commercially as a high molecular weight, polydisperse material and it is not transcriptionally active when administered to mammalian cells. We therefore selected a commercially available, supercoiled plasmid DNA construct encoding for enhanced green fluorescent protein (pEGFP-N1, 4.7 kb) for use in all subsequent experiments. In addition to serving as a reporter gene in later DNA release and cell transfection experiments, plasmid DNA is both welldefined and monodisperse and may therefore facilitate the extraction of more meaningful relationships between polymer structure, film growth, and material function relative to materials fabricated using polydisperse calf thymus DNA. Subsequent experiments demonstrated that the pEGFP plasmid could be incorporated into multilayered structures using polymer 1. The planar silicon substrates used in these and all later experiments were precoated with multilayered films composed of LPEI and SPS (ca. 15-20 nm thick, with a topmost layer of anionic SPS) to ensure a uniform density of anionic charge on the surface of the substrate suitable for the adsorption of polymer 1. Silicon substrates that were not precoated in this manner did not result in significant polymer adsorption or film growth. The iterative dipping of substrates into dilute aqueous solutions of polymer 1 (5 mM with respect to polymer repeat unit; solutions prepared in 100 mM sodium acetate buffer, pH ) 5.1) and plasmid DNA (1 mg/mL) resulted in the growth of multilayered polymer 1/DNA films ranging from 10 to 100 nm thick. Figure 1a and b shows the increase in ellipsometric thickness for representative films as a function of the number of polymer 1/DNA bilayer pairs deposited using plasmid solutions prepared in either deionized water (Figure 1a) or acetate buffer (pH 5.1, Figure 1b). Attempts to fabricate multilayered polymer 1/DNA films using solutions of plasmid prepared in deionized water did not result in significant deposition for up to five polycation/polyanion dipping cycles (Figure 1a). However, when the pH of the plasmid solution was adjusted to pH 5.1 using acetate buffer (100 mM) the thicknesses of the films deposited increased dramatically. As shown in Figure 1b, the ellipsometric thickness of the films produced in this manner increased linearly in proportion to the number

of polymer 1/DNA bilayer pairs deposited. This deposition profile is consistent with those observed using conventional polyelectrolytes,8 as well as our previous results using polymer 1, and resulted in the deposition of plasmidcontaining films up to 100 nm thick after eight dipping cycles (Figure 1b). We believe that layer growth is enhanced in these experiments by an increase in the cationic character of the topmost adsorbed layer of polymer 1 at lower pH, although the above experiment does not rule out potential contributions from the increased ionic strength of the buffer solution. To investigate the relationship between ionic strength and layer growth in this system, we measured the thickness of films fabricated using dipping solutions adjusted to higher ionic strength using NaCl (e.g., 0.2-1.0 M). In each case, fabrication in the presence of NaCl resulted in the deposition of significantly thinner films (data not shown). Films prepared using plasmid concentrations of 1 mg/mL were generally thicker than films prepared using concentrations of 0.5 or 0.25 mg/mL. As such, all subsequent fabrication experiments using pEGFP were conducted at concentrations of 1 mg/mL in acetate buffer (100 mM, pH 5.1) in the absence of added NaCl to maximize film thickness. UV-visible spectrophotometry has been used as a convenient method to characterize the adsorption of polyelectrolytes onto planar quartz substrates,8 and the absorbance of films at 260 nm (the characteristic maximum absorbance for double-stranded DNA in solution) has been used to monitor layer-by-layer growth for films incorporating DNA.13,15 Although film absorbance values may not be correlated directly with physical thicknesses or with the total amount of DNA incorporated into a film,15 the absorbance of films prepared from either calf thymus DNA13 or a model polydeoxynucleotide15 were found to increase as a linear function of the number of polycation/ poly(nucleic acid) layers adsorbed. To establish the feasibility of this procedure for the characterization of our plasmid-containing materials, we deposited polymer 1/DNA films layer-by-layer onto quartz slides precoated with 10 bilayers of LPEI/SPS, and the absorbance of deposited films was measured periodically (at 260 nm) using a standard UV-visible spectrophotometer. Figure 2 shows a representative plot of absorbance as a function of the number of polymer 1/DNA bilayer pairs adsorbed. The linear relationship between absorbance and bilayer number is consistent with previous observations, as well as the linear increase in ellipsometric film thicknesses observed for deposition of polymer 1/DNA onto silicon substrates (e.g., Figure 1b), and further confirms the stepwise nature of the deposition process for this polycation/plasmid DNA system.

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Figure 2. Plot of absorbance at 260 nm vs the number of polymer 1/pEGFP bilayers for films deposited on a quartz substrate. Substrates were precoated with a film composed of 10 bilayers of LPEI/SPS as an adhesive layer.

Figure 3. Color fluorescence image of two films deposited on quartz substrates and soaked in an aqueous ethidium bromide solution. A) A film fabricated from eight bilayers of polymer 1 and pEGFP plasmid. B) A film fabricated from eight bilayers of polymer 1 and SPS and used as a no-DNA control. The intense fluorescence exhibited by the film A provides direct visual evidence for the incorporation of plasmid using polymer 1 and the layer-by-layer technique. Both films were deposited on quartz substrates precoated with a film composed of 10 bilayers of LPEI/SPS as an adhesive layer (see text).

Direct Visual Evidence of Plasmid Incorporation and Structure. The experiments above provide quantitative data reflecting the layer-by-layer growth of polymer 1/pEGFP films and, by inference, the incorporation of DNA as a component of the material. We performed an additional experiment to more directly confirm the presence and structure of plasmid incorporated into these assemblies by soaking the materials briefly in an aqueous ethidium bromide solution.16,38 Ethidium bromide is a DNA-intercalating dye used widely to visualize doublestranded DNA in applications such as agarose gel electrophoresis.48 The fluorescence intensity of this compound increases dramatically upon intercalation into doublestranded DNA; it does not yield high levels of background fluorescence in the absence of double-stranded DNA, and it does not stain or label single-stranded DNA well. As shown in the color image in Figure 3, a film composed of 8 bilayers of polymer 1/pEGFP exhibited intense fluorescence after soaking in an ethidium bromide solution, while a film composed of 8 bilayers of polymer 1/SPS (used as a non-DNA control) did not, providing direct visual evidence for the incorporation of plasmid into the first film. Although it is not possible to draw more detailed (48) Sambrook, J.; Russel, D. W. Molecular Cloning: A laboratory Manual. 3rd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 2001; Vol. 1, p 1-150.

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conclusions about the specific structure or conformation of the incorporated plasmid from these basic experiments, the intensity of the fluorescence observed in Figure 3 suggests that 1) the plasmid remains substantially doublestranded upon incorporation into the film and 2) the DNA is not condensed or compacted by polymer 1 to an extent that the intercalation of ethidium bromide is inhibited or prevented. Additional experiments will be necessary to more completely determine the structure, topology, and conformation of DNA in these solid films; characterization of the structure and activity of the plasmid upon release are detailed below. Evaluation of Plasmid Release Kinetics and Characterization of Released DNA. The layer-by-layer deposition of polyelectrolytes on surfaces is driven largely by electrostatic attractive forces between charged polymers and oppositely charged surfaces,8 and the overall process is entropically favored through the formation of ‘n’ numbers of small salt molecules as polymers self-assemble into polyvalent ion pairs. The polyvalent nature of these electrostatic interactions yields robust polymer assemblies that are often stable at physiological pH and ionic strength, and the functional irreversibility of these ionically crosslinked networks toward the dissociation of individual layers (i.e., the opposite of the entropically favored deposition process) has been one factor limiting the application of multilayered films to the release of incorporated macromolecular polyelectrolytes. Our initial work demonstrated that multilayered films fabricated from polymer 1 and either SPS or PAA eroded gradually under physiological conditions (e.g., upon incubation PBS at 37 °C).46 Erosion studies conducted with these materials further demonstrated that they eroded more rapidly at pH 7.2 than at pH 5.0, consistent with a mechanism of erosion that is at least partially hydrolytic. These studies suggested that materials incorporating polymer 1 could be used to sustain the release of a functional polyanion, such as DNA, otherwise serving as a structural element in the assembly. To investigate the release of plasmid DNA under physiological conditions, films fabricated from polymer 1 and pEGFP were submerged in PBS (pH ) 7.4, 137 mM NaCl) and incubated at 37 °C in UV-transparent cuvettes. Substrates were removed at predetermined intervals for analysis by ellipsometry (for films deposited on silicon substrates) or UV spectrophotometry (for films deposited on quartz substrates) to monitor either the thickness or the absorbance (at 260 nm) of the films as a function of time. Figure 4a shows a representative plot of ellipsometric thickness vs time for a 55 nm thick film of polymer 1/DNA incubated in PBS, in which average film thickness decreases gradually over a period of 19 h. Figure 4b shows a representative plot of absorbance vs time for a second film deposited on a quartz substrate, in which absorbance is observed to decrease gradually over a 24 h period. These data are consistent with an erosion process in which the incorporated plasmid is released gradually from the surface of the substrate and into the incubation buffer over a period of 20-24 h. Surface release kinetics were determined directly by periodically monitoring the absorbance of the incubation milieu at 260 nm. As shown in Figure 5a for the incubation of a polymer 1/plasmid film ca. 100 nm thick, the absorbance of the buffer solution increased gradually and consistently over a period of 31 h, mirroring a corresponding decrease in the absorbance measured for an identical film over the same time period. DNA concentration was also determined semiquantitatively during this experiment via agarose gel electrophoresis analysis of

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Figure 4. (A) Plot of ellipsometric thickness vs time for a polymer 1/DNA multilayered film incubated in PBS buffer at 37 °C (pH ) 7.4). Black bars represent the thickness of a LPEI/ SPS film deposited onto the silicon substrate as an adhesive layer. (B) Plot of absorbance at 260 nm vs time for a polymer 1/DNA film deposited on a quartz substrate and incubated in PBS buffer at 37 °C.

Figure 5. (A) Plot of absorbance at 260 nm vs time for a 100 nm thick polymer 1/DNA film deposited on quartz substrates and incubated in PBS buffer at 37 °C. Closed circles (b) represent absorbance values measured for the film and correspond to the relative amount of DNA in the film; closed squares (9) represent absorbance values recorded for the incubation buffer and correspond to the concentration of plasmid released into solution. (B) Agarose gel electrophoresis data corresponding to analysis of samples of the incubation buffer solution in part A. Lanes are labeled with the time (in hours) at which samples were removed for analysis and correspond directly to the time axis and sample measurements made in part A. The lane denoted C* corresponds to supercoiled pEGFP used as a control.

samples of the buffer solution removed periodically during the incubation period. As shown in Figure 5b, the concentration of plasmid in the incubation buffer increased continually over a period of 31 h (depicted visually as an increase of the intensity of the white ethidium bromide-

Zhang et al.

stained bands). These data also demonstrate that the plasmid released from these materials is not electrostatically associated or complexed with degraded fragments of cationic polymer 1 within the quantitative limits of the ethidium bromide staining protocol (Figure 5b). The release of plasmid from films deposited on silicon occurred over a similar time period (approximately 30 h) as the release of plasmid coated on quartz substrates. These results most likely reflect the fact that all substrates used in these experiments were precoated with 10 bilayers of LPEI/SPS rather than any particular differences or similarities in the substrates themselves. The gel electrophoresis data in Figure 5b indicate that the majority of the plasmid released from polymer 1/pEGFP films has a structure that is relaxed or opencircular rather than supercoiled (even at time points as early as 1 h) and that a small amount of linearized plasmid is observed. Relaxed plasmid results from the ‘nicking’ or nonspecific cleavage of at least one phosphodiester bond in a closed-circular plasmid structure, leading to ‘relaxation’ of the supercoiled plasmid.49 This form of plasmid DNA is frequently observed during the extended release of plasmid from polymeric matrices2,6,50 and is sometimes associated with fabrication procedures that expose the plasmid to ‘harsh’ chemical or mechanical forces, such as high shear.51 It was somewhat surprising, therefore, to observe the release of relaxed DNA from assemblies fabricated using a room temperature aqueous dipping protocol that generates no notable mechanical forces. We performed several control experiments in an attempt to isolate and identify the elements of our fabrication procedure that may contribute to plasmid nicking. As noted above, the plasmid remains supercoiled over the short time it is exposed to pH 5.1 conditions during dipping procedures (up to 5 h), as well as during the longer times that the released DNA is incubated in or stored in PBS buffer prior to analysis (data not shown). Within the limits of gel electrophoresis analysis, the plasmid is released in a relaxed state to an equal extent regardless of whether the films are deposited onto silicon or quartz and regardless of whether they are dried and stored prior to use or used immediately after fabrication. We did observe the presence of open circular plasmid in separate experiments designed to prepare and degrade self-assembled polymer 1/DNA nanoparticles using a completely different and wellestablished procedure.47 These results suggest that nicking may be related more generally to the self-assembly process itself or the specific structure of polymer 1 than any specific fabrication procedures used in this study. Functional Characterization of Released Plasmidsin Vitro Transfection Assays. Open circular DNA is transcriptionally competent and can yield high levels of gene expression in transfected cells.2,6 To evaluate the transcriptional viability of the plasmid released from the surfaces of these substrates, we conducted gene expression assays using the commercially available lipid-based transfection reagent Lipofectamine 2000 and the COS-7 cell line. Figure 6 shows fluorescence microscopy images of COS-7 cells transfected with plasmid DNA collected over several different 5 h time periods during the incubation of a 100 nm thick polymer 1/DNA film. These experiments were performed in analogy to the experiments (49) Nelson, D. L.; Cox, M. M., Lehninger Principles of Biochemistry. 3rd ed.; Worth Publishers: New York, 2000; p 916. (50) Wang, C.; Ge, Q.; Ting, D.; Nguyen, D.; Shen, H. R.; Chen, J.; Eisen, H. N.; Heller, J.; Langer, R.; Putnam, D. Nat. Mater. 2004, 3, 190-196. (51) Ando, S.; Putnam, D.; Pack, D. W.; Langer, R. J. Pharm. Sci. 1999, 88, 126-130.

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onstrated that released DNA is not complexed with polymer 1. The efficient transfection of most cell lines in vitro generally requires the use of an auxiliary gene transfer agent, such as Lipofectamine or a cationic polymer-based delivery system, that self-assembles plasmid DNA into condensed nanoparticulate structures and/ or enhances cellular uptake.52 However, the use of ‘naked’ or noncomplexed plasmid DNA to transfect cells and tissue in vivo is well precedented,2,6 and we are investigating the application of these thin films to the in vivo delivery of plasmid. The release of naked DNA from microparticles or nanoparticles coated with these assemblies may be useful in the context of the intracellular delivery and release of DNA. We note, however, that the inherent juxtaposition of both polycations and DNA in these multilayered assemblies provides an opportunity to develop subsequent generations of materials that function to release plasmid that is ‘precomplexed’ with or condensed by known and effective polycationic gene delivery agents.

Figure 6. Representative low-magnification fluorescence microscopy images showing relative levels of enhanced green fluorescent protein (EGFP) expression in a confluent monolayer of COS-7 cells. Cells were transfected with samples of plasmid released from a 100 nm thick polymer 1/pEGFP film released over a 31 h period using Lipofectamine 2000 as a transfection agent. As described in the text, plasmid released into PBS buffer was collected in seven discrete batches over seven different time periods. Levels of EGFP observed correspond to amounts of plasmid released and collected over each of the following time periods: a) 0-1, b) 1-6, c) 6-11, d) 11-16, e) 16-21, f) 21-26, and g) 26-31 h.

shown in Figure 5 above, with the exception that the PBS incubation medium was exchanged with fresh buffer at the end of each time period (see Materials and Methods for details). As such, the levels of EGFP expression depicted in Figure 6 do not reflect cumulative levels of released plasmid per se, but rather the relative amounts of plasmid released from the film over seven sequential, discrete time periods. The large number of cells expressing EGFP in these images demonstrates that the plasmid is released from these materials in a form that is transcriptionally viable. This experiment also provides additional evidence that the release of plasmid is sustained over a period of 31 h. The gene expression results in Figure 6 are consistent with gradual decreases in average film thickness observed by ellipsometry and correlate very closely with the release profile determined in Figure 5a, in which the majority of DNA appears to be released from the film during the first 16 h of incubation, followed by an extended period over which the remaining plasmid is released. We used Lipofectamine 2000 to demonstrate the functional nature of the plasmid in the above experiments because the gel electrophoresis data in Figure 5b dem-

Summary Multilayered films up to 100 nm thick containing plasmid DNA and a synthetic degradable cationic polymer were fabricated layer-by-layer onto the surfaces of planar silicon and quartz substrates. The stepwise nature of the deposition process was monitored by ellipsometry and UV spectrophotometry. Both film thickness and absorbance were found to correlate linearly with the number of polymer and DNA layers deposited, in analogy to multilayered materials fabricated from conventional polyelectrolytes, and the thickness of the deposited layers was found to be a function of both the pH and ionic strength of the polyelectrolyte solutions used. Films fabricated from polymer 1 and a plasmid encoding for enhanced green fluorescent protein eroded upon incubation under physiological conditions, releasing the plasmid gradually into the incubation medium over a period of up to 31 h. Characterization of the released DNA revealed that the plasmid is released in an open circular, rather than supercoiled, topology and that it is not electrostatically associated with polymer 1. We further demonstrated that the plasmid released from these materials is transcriptionally active and promoted the expression of high levels of enhanced green fluorescent protein in the COS-7 cell line. The development of approaches that provide for the gradual release of biomacromolecules from multilayered polyelectrolyte films under physiological conditions should facilitate the general evaluation and application of such structured assemblies in multiple therapeutic contexts.44 In the context of gene delivery, the assemblies reported here could represent an approach to the controlled administration of functional DNA constructs from the surfaces of biomedical materials and delivery devices. Acknowledgment. Support for this work was provided by the National Institutes of Health (EB02746), the Arnold and Mabel Beckman Foundation, the National Science Foundation (through the UW Materials Research Science and Engineering Center), and the University of Wisconsin. We thank Prof. Eric Shusta for the use of culture facilities and Prof. Nick Abbott for numerous helpful discussions. LA048888I (52) Luo, D.; Saltzman, W. M. Nat. Biotechnol. 2000, 18, 33-37.