pubs.acs.org/Langmuir © 2009 American Chemical Society
Surface-Supported Multilayers Decorated with Bio-active Material Aimed at Light-Triggered Drug Delivery† D. V. Volodkin,* N. Madaboosi, J. Blacklock, A. G. Skirtach, and H. M€ohwald Max-Planck Institute of Colloids and Interfaces, Research Campus Golm, Potsdam, D-14424 Germany Received April 30, 2009. Revised Manuscript Received July 16, 2009 In this work, we report on the functionalization of layer-by-layer films with gold nanoparticles, microcapsules, and DNA molecules by spontaneous incorporation into the film. Exponentially growing films from biopolymers, namely, hyaluronic acid (HA) and poly-L-lysine (PLL), and linearly growing films from the synthetic polymers, namely, poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH), were examined for the embedding. The studied (PLL/HA)24/PLL and (PAH/PSS)24/PAH films are later named HA/PLL and PSS/PAH films, respectively. The HA/PLL film has been found to be more efficient for both particle and DNA embedding than PSS/PAH because of spontaneous PLL transport from the interior of the whole HA/PLL film to the surface in order to make additional contact with embedded particles or DNA. DNA and nanoparticles can be immobilized in HA/PLL films, reaching loading capacities of 1.5 and 100 μg/cm2, respectively. The capacities of PSS/PAH films are 5 and 12 times lower than that for films made from biopolymers. Polyelectrolyte microcapsules adsorb irreversibly on the HA/PLL film surface as single particles whereas very poor interaction was observed for PSS/PAH. This intrinsic property of the HA/PLL film is due to the high mobility of PLL within the film whereas the structure of the PSS/PAH film is “frozen in”. Gold nanoparticles and DNA form micrometer-sized aggregates or patches on the HA/PLL film surface. The diffusion of nanoparticles and DNA into the HA/PLL film is restricted at room temperature, but DNA diffusion is triggered by heating to 70 °C, leading to homogeneous filling of the film with DNA. The film has not only a high loading capacity but also can be activated by “biofriendly” near-infrared (IR) laser light, thanks to the gold nanoparticle aggregates on the film surface. Composite HA/PLL films with embedded gold nanoparticles and DNA can be activated by light, resulting in DNA release. We assume that the mechanism of the release is dependent on the disturbance in bonding between “doping” PLL and DNA, which is induced by local thermal decomposition of the HA/PLL network in the film when the film is exposed to IR light. Remote IR-light activation of dextran-filled microcapsules modified by gold nanoparticles and integrated into the HA/PLL film is also demonstrated, revealing an alternative release pathway using immobilized light-sensitive carriers (microcapsules).
Nowadays, research in smart biomaterials is one of the most dynamically developing areas in modern nanotechnology, aimed at biomedical applications. The polymer layer-by-layer (LbL) technique1,2 is a relatively new but very powerful approach to engineering films with defined architecture.3-6 It offers excellent characteristics such as fine film tuning in terms of thickness, mechanics, chemistry, stability, and so forth. Besides, the LbL films can be formed on surfaces with complex or miniaturized geometries and also spherical sacrificial templates, allowing the formulation of free-standing structures such as microcapsules to be used as drug-loaded microcarriers.7-11 The LbL method is based on the alternating adsorption of † Part of the “Langmuir 25th Year: Self-assembled polyelectrolyte multilayers: structure and function” special issue. *Corresponding author. E-mail:
[email protected]. Tel: þ49-(0)-331-567-9440. Fax: þ49-(0)-331-567-9202.
(1) Decher, G.; Hong, J.-D. Makromol. Chem. 1991, 46, 321. (2) Decher, G. Science 1997, 277, 1232–1237. (3) Decher, G.; Schlenoff, J. B. In Multilayer Thin Films: Sequential Assembly of Nanocomposite Materials; Decher, G., Schlenoff, J. B., Eds; Wiley-VCH: Weinheim, Germany, 2003; p 524. (4) Ariga, K.; Hill, J. P.; Li, Q. Phys. Chem. Chem. Phys. 2007, 9, 2319–2340. (5) Wang, Y.; Angelatos, A. S.; Caruso, F. Chem. Mater. 2008, 20, 848–858. (6) Ariga, K.; Hill, J. P.; Lee, M. V.; Vinu, A.; Charvet, R.; Acharya, S. Sci. Technol. Adv. Mater. 2008, 9, 014109. (7) Peyratout, C. S.; Dahne, L. Angew. Chem., Int. Ed. 2004, 43, 3762–3783. (8) Sukhorukov, G. B.; Mohwald, H. Trends Biotechnol. 2007, 25, 93–98. (9) Volodkin, D. V.; Larionova, N. I.; Sukhorukov, G. B. Biomacromolecules 2004, 5, 1962–1972. (10) Volodkin, D. V.; Petrov, A. I.; Prevot, M.; Sukhorukov, G. B. Langmuir 2004, 20, 3398–3406. (11) De Geest, B. G.; Sanders, N. N.; Sukhorukov, G. B.; Demeester, J.; De Smedt, S. C. Chem. Soc. Rev. 2007, 36, 636–649.
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polymers having the ability to interact with each other. Electrostatic interactions,1 hydrogen bonding,12 and hydrophobic13 and host-guest interactions14-16 can alternatively or simultaneously be the main driving forces to assemble the films. A variety of biomacromolecules have been successfully employed for LbL film buildup: lipids,17,18 proteins and enzymes,19-22 polypeptides,23 nucleic acids,24-28 polysaccharides,29 and even (12) Inoue, H.; Sato, K.; Anzai, J. Biomacromolecules 2005, 6, 27–29. (13) Stockton, W. B.; Rubner, M. F. Macromolecules 1997, 30, 2717–2725. (14) Cho, J.; Caruso, F. Macromolecules 2003, 36, 2845–2851. (15) Sukhishvili, S. A.; Granick, S. J. Am. Chem. Soc. 2000, 122, 9550–9551. (16) Sukhishvili, S. A.; Granick, S. Macromolecules 2002, 35, 301–310. (17) Cassier, T.; Sinner, A.; Offenhauser, A.; Mohwald, H. Colloids Surf., B 1999, 15, 215–225. (18) Moya, S.; Donath, E.; Sukhorukov, G. B.; Auch, M.; Baumler, H.; Lichtenfeld, H.; Mohwald, H. Macromolecules 2000, 33, 4538–4544. (19) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117–6123. (20) Anzai, J. I.; Hoshi, T.; Nakamura, N. Langmuir 2000, 16, 6306–6311. (21) Jessel, N.; Atalar, F.; Lavalle, P.; Mutterer, J.; Decher, G.; Schaaf, P.; Voegel, J.-C.; Ogier, J. Adv. Mater. 2003, 15, 692–695. (22) Ladam, G.; Schaaf, P.; Cuisinier, F. J.; Decher, G.; Voegel, J.-C. Langmuir 2001, 17, 878–882. (23) Boulmedais, F.; Ball, V.; Schwinte, P.; Frisch, B.; Schaaf, P.; Voegel, J.-C. Langmuir 2003, 19, 440–445. (24) Lvov, Y.; Decher, G.; Sukhorukov, G. B. Macromolecules 1993, 26, 5396– 5399. (25) Sukhorukov, G. B.; Montrel, M. M.; Petrov, A. I.; Shabarchina, L. I.; Sukhorukov, B. I. Biosens. Bioelectron. 1996, 11, 913–922. (26) Pei, R.; Cui, X.; Yang, X.; Wang, E. Biomacromolecules 2001, 2, 463–468. (27) Zhang, J.; Chua, L. S.; Lynn, D. M. Langmuir 2004, 20, 8015–8021. (28) Jewell, C. M.; Zhang, J.; Fredin, N. J.; Lynn, D. M. J. Controlled Release 2005, 106, 214–223. (29) Richert, L.; Lavalle, P.; Payan, E.; Shu, X. Z.; Prestwich, G. D.; Stoltz, J.-F.; Schaaf, P.; Voegel, J.-C.; Picart, C. Langmuir 2004, 20, 448–458.
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large objects such as liposomes30-33 and viruses.34 High activity in the research of LbL films has catalyzed the introduction of this method for biomedical applications that aim at engineering nano(micro)scale drug delivery/release systems.35-40 The properties of biomacromolecule-containing films were reported by Ai.36 The film-incorporated enzymes can retain their catalytic activity and, moreover, have a high tolerance to harsh conditions.36,41-43 DNA has been embedded in LbL films44 by assembly with natural and synthetic polycations24-28 and can be transcriptionally viable.27 Free-standing LbL films have been shown to be promising for intracellular drug delivery.45-47 The LbL films used for drug delivery applications have to provide a living organism with a sufficient number of active molecules, for instance, to provide it with growth factors or antibiotics, reduce bacterial colonization, or guide the subsequent cellular response.48,49 It is also essential to confer the LbL film/ biocoating with active molecules that could be released from the film in order to have an “on demand” response at a definite time. Remote release or release on demand is desired in medicine in order to minimize drug toxicity by effective local drug delivery. IR-light-stimulated remote release is of special interest because of the external control of light intensity and modulation and its noninvasive character, which are undoubtedly advantageous characteristics. Gold nanoparticles have been employed for the functionalization of drug-containing vehicles such as phospholi(30) Michel, M.; Vautier, D.; Voegel, J.-C.; Schaaf, P.; Ball, V. Langmuir 2004, 20, 4835–4839. (31) Volodkin, D.; Schaaf, P.; Mohwald, H.; Voegel, J.-C.; Ball, V. Soft Matter 2009, 5, 1394–1405. (32) Volodkin, D. V.; Arntz, Y.; Schaaf, P.; M€ohwald, H.; Voegel, J.-C.; Ball, V. Soft Matter 2008, 4, 122–130. (33) Volodkin, D. V.; Michel, M.; Schaaf, P.; Voegel, J.-C.; Mohwald, H.; Ball, V. Liposome Embedding into Polyelectrolyte Multilayers: A New Way to Create Drug Reservoirs at Solid-Liquid Interfaces. In Advances in Planar Lipid Bilayers and Liposomes; Liu, A. L., Ed.; Elsevier: Amsterdam, 2008; Vol. 8. (34) Dimitrova, M.; Arntz, Y.; Lavalle, P.; Meyer, F.; Wolf, M.; Schuster, C.; Haikel, Y.; Voegel, J.-C.; Ogier, J. Adv. Funct. Mater. 2007, 17, 233–245. (35) Tang, Z.; Wang, Y.; Podsiadlo, P.; Kotov, N. A. Adv. Mater. 2006, 18, 3203–3224. (36) Ai, H.; Jones, S. A.; Lvov, Y. M. Cell Biochem. Biophys. 2003, 39, 23–43. (37) Ariga, K.; Hill, J. P.; Ji, G. Macromol. Biosci. 2008, 8, 981–990. (38) Volodkin, D. V.; Mohwald, H. Polyelectrolyte Multilayers for Drug Delivery. In Encyclopedia of Surface and Colloid Science; Somasundaran, P., Ed.; Taylor & Francis: Boca Raton, FL, 2009; in press. (39) Skirtach, A. G.; Kreft, O. In Nanotechnology in Drug Delivery; de Villiers, M. M., Aramwit, P.; Kwon, G.S., Eds.; Springer: Berlin, 2009; DOI: 10.1007/978-0387-77667-5. (40) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3427–3433. (41) Onda, M.; Ariga, K.; Kunitake, T. J. Biosci. Bioeng. 1999, 87, 69–75. (42) Disawal, S.; Qiu, J.; Elmore, B. B.; Lvov, Y. M. Colloids Surf., B 2003, 32, 145–156. (43) Schwinte, P.; Voegel, J.-C.; Picart, C.; Haikel, Y.; Schaaf, P.; Szalontai, B. J. Phys. Chem. B 2001, 15, 11906–11916. (44) Jewell, C. A.; Lynn, D. M. Adv. Drug Delivery Rev. 2008, 60, 979–999. (45) De Koker, S.; De Geest, B. G.; Cuvelier, C.; Ferdinande, L.; Deckers, W.; Hennink, W. E.; De Smedt, S.; Mertens, N. Adv. Funct. Mater. 2007, 17, 3754– 3763. (46) Skirtach, A. G.; Javier, A. M.; Kreft, O.; Kohler, K.; Alberola, A. P.; Mohwald, H.; Parak, W. J.; Sukhorukov, G. B. Angew. Chem., Int. Ed. 2006, 45, 4612–4617. (47) Javier, A. M.; del Pino, P.; Bedard, M. F.; Ho, D.; Skirtach, A. G.; Sukhorukov, G. B.; Plank, C.; Parak, W. J. Langmuir 2008, 24, 12517–12520. (48) Hirano, Y.; Mooney, D. J. Adv. Mater. 2004, 16, 17–25. (49) Crouzier, T.; Ren, K.; Nicolas, C.; Roy, C.; Picart, C. Small 2009, 5, 598– 608. (50) Volodkin, D. V.; Skirtach, A. G.; Mohwald, H. Angew. Chem., Int. Ed. 2009, 48, 1807–1809. (51) Skirtach, A. G.; Karageorgiev, P.; Bedard, M. F.; Sukhorukov, G. B.; Mohwald, H. J. Am. Chem. Soc. 2008, 130, 11572–11573. (52) Wu, G. H.; Milkhailovsky, A.; Khant, H. A.; Fu, C.; Chiu, W.; Zasadzinski, J. A. J. Am. Chem. Soc. 2008, 130, 8175–8177. (53) Skirtach, A. G.; Dejugnat, C.; Braun, D.; Susha, A. S.; Rogach, A. L.; Parak, W. J.; Mohwald, H.; Sukhorukov, G. B. Nano Lett. 2005, 5, 1371–1377. (54) Bedard, M. F.; Braun, D.; Sukhorukov, G. B.; Skirtach, A. G. ACS Nano 2008, 2, 1807–1816.
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pid vesicles and polymer microcapsules50-54 by providing lightresponsivity and control of the vehicle membrane permeability. Thus, both the amounts of loaded substances and their controlled release from LbL films are of fundamental importance. Engineering LbL films with high, controlled loading capacity that are able to release a sufficient number of functional molecules by invasive stimulus on demand is a challenge in modern biomaterial science. In this study, we report on the functionalization of exponentially and linearly growing LbL films (i.e., HA/PLL and PSS/PAH films) with gold nanoparticles, polyelectrolyte microcapsules, and DNA. The interaction of these films with the particles and DNA has been investigated while taking into account the different mobilities of polymers in the studied films. Lightstimulated release of film-embedded DNA as well as microencapsulated dextran by external stimulation with IR light is shown.
Experimental Section Materials. PLL with a viscosimetric molecular mass of 1530 kDa (ref P7890), PLL labeled with FITC (PLL-FITC, ref P3543, 15-30 kDa), PAH (ref 28 322-3, 70 kDa), PSS (ref 24 305-1, 70 kDa), hydrophilic citrate-stabilized colloidal gold nanoparticles (diameter 20 nm), ethidium bromide solution 0.07% (EtBr, ref E1510), rhodamine-dextran (ref R9379, 70 kDa), and poly(diallyldimethylammonium chloride) (PDADMAC, 200350 kDa) were purchased from Sigma-Aldrich (Germany). The nanoparticle concentration was determined to be 1012 particles/mL. HA with a viscosimetric molecular mass of 360 kDa was purchased from Lifecore Biomedical (ref 002570). gWiz high-expression luciferase plasmid (6732 bp) (DNA) was purchased from Aldevron and was used without purification. Throughout this study, 10 mM Tris containing 15 mM NaCl at pH 7.4 was used and will be mentioned in the text as Tris buffer. The water used in all experiments was prepared in a three-stage Millipore Milli-Q Plus 185 purification system and had a resistivity higher than 18.2 MΩ cm. Preparation of the Films. The polyelectrolyte multilayer films were prepared by the LbL technique using a dipping robot (Riegler & Kirstein GmbH, Berlin, Germany). The films were deposited either onto a microscopy cover glass (12 mm in diameter, Marienfeld GmbH, Germany) or on dialysis membranes from regenerate cellulose with a cutoff of 10 kDa (ref 132 118, Spectropor). Before deposition, the glass slides were cleaned by consecutive incubation in hot solutions (60 °C) of 2% (v/v) Hellmanex (Hellma GmbH, Germany) and 0.1 M HCl for 15 min for each solution, followed by multiple rinsing steps with pure water. The membrane was used without further processing. Film buildup was pursued at 25 °C by alternating dipping of the glass slides into polymer solutions (0.5 mg mL-1) in Tris buffer with an intermediate washing step with buffer. Before deposition, polyelectrolyte solutions were filtered through a 0.45 μm filter. Each dipping step lasted for 10 min. To prepare the film labeled with FITC, PLL-FITC was added to the PLL solution used for film deposition at 30:1 w/w unlabeled PLL/labeled,. To quantify the amount of PLL in a film or DNA embedded in a film, the film prepared with PLL-FITC or containing DNA labeled with EtBr was removed from the glass substrate by the addition of 200 μL of 0.05 M NaOH followed by 5 min of incubation in this solution. This treatment allows for the complete removal of the multilayer film by the deprotonation of PLL molecules. Then, 1.35 mL of 0.1 M Tris buffer (pH 8.0, containing 0.1 M NaCl) was added. The fluorescence intensity was then measured with a spectrofluorometer (Fluoromax-4, Horiba Jobin Yvon). The excitation wavelength was set at 490(510) nm and the emission was measured at 520(595) nm for FITC-labeled PLL and EtBr-labeled DNA, respectively. The amount of PLL or DNA in the film has been measured using a calibration curve for the PLL solution with PLL-FITC or DNA-EtBr solution. Adsorption of Microcapsules, Gold Nanoparticles, and DNA. Polyelectrolyte microcapsules (PDADMAC/nanoparticles/PSS)4 Langmuir 2009, 25(24), 14037–14043
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containing gold nanoparticles were prepared according to the method described elsewhere with some modifications.54 The capsules were made using the layer-by-layer deposition technique and 4.78 μm SiO2 particles (Microparticles GmbH, Germany) as sacrificial cores. Polyelectrolytes were dissolved in 0.5 M NaCl at a concentration of 0.5 mg/mL. SiO2 templates with PDADMAC as the outermost layer were resuspended in a gold nanoparticle suspension (5 1011 nanoparticles/mL) that was allowed to incubate for half a minute in 0.1 M NaCl. After polyelectrolyte and NP deposition, coated SiO2 particles were further dissolved in HF (0.3 M) solution, and the sample was then washed with water until the pH of the solution reached 5. The encapsulation of rhodamine-dextran was done by heating a mixture of capsules in 0.1 mg/mL rhodamine-dextran water solution for 20 min at 54 °C, leading to capsule shrinkage to a diameter of about 2 μm. The samples were allowed to cool for 5 min and washed twice with water to remove nonencapsulated rhodamine-dextran. Furthermore, the microcapsules were adsorbed on the (HA/PLL)24/PLL film by the addition of 50 μL of the capsule suspension (2 106 capsules/mL in TRIS buffer) on top of the glass coverslip coated with the freshly prepared film. After 1 h of incubation, the film was intensively washed three times with TRIS buffer. To deposit gold nanoparticles or DNA on the films, colloidal gold nanoparticles and DNA-EtBr were used in the experiments. One milliliter of the nanoparticle water solution or 50 μL of DNA (0.8 mg/mL) solution in Tris buffer was added to a well containing a freshly prepared (PLL/HA)24/PLL or (PAH/PSS)24/PAH film at the bottom and incubated for 2 days or 2 h at room temperature for the adsorption of nanoparticles or DNA, respectively. UVvis absorption spectra of the films with adsorbed nanoparticles and supernatants with the nanoparticle suspension and DNA were recorded. To prepare DNA labeled with EtBr, 0.25 mL of EtBr was added to 0.5 mL of the water DNA solution. NaCl solution was then added to obtain a final concentration of 15 mM NaCl. The mixture was vortex mixed for 30 min and dialyzed against Tris buffer for 48 h. The final DNA concentration was 0.8 mg mL-1. A composite HA/PLL film with embedded DNA and gold nanoparticles was prepared by nanoparticle deposition, followed by DNA deposition as described above. Confocal Laser Scanning Microscopy (CLSM). CLSM micrographs were taken with a Leica confocal scanning system mounted onto a Leica Aristoplan and equipped with a 100 oilimmersion objective with a numerical aperture of 1.4. The excitation wavelength was 488 nm for PLL-FITC-containing films and 552 nm for films with DNA-EtBr. Remote Activation. Experiments were conducted according to the previously described procedures employed for the remote release of encapsulated materials.55 Briefly, the films were marked with a black marker, and the images were recorded on a confocal scanning microscope. Then, the holder with the film was positioned in the setup, and the same site was identified by the position of the marker. Subsequently, the film was exposed to a near-IR laser operating at 830 nm. The laser power used to remotely activate the film or the immobilized capsules is in the range of 20-30 mW with 1 s of laser light exposure time. The light beam was focused onto the sample in an area of around 1 μm through a microscope objective with 100 magnification (numerical aperture 1.25, Edmund Scientific). After this step, the holder with the film was placed back into the confocal microscope, and another CLSM image was taken at the same site that was identified by the marked position.
Results and Discussion Two LbL films, namely, (PAH/PSS)24/PAH and (PLL/HA)24/ PLL, were studied for their interaction with stiff particles having diameters in the nano- and microscale ranges (gold nanoparticles (55) Skirtach, A. G.; Antipov, A. A.; Shchukin, D. G.; Sukhorukov, G. B. Langmuir 2004, 20, 6988–6992.
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and polyelectrolyte microcapsules with diameters of 20 nm and 1 μm, respectively) as well as with relatively flexible DNA. The films will be mentioned in the text as HA/PLL and PSS/PAH, respectively. The last deposited polyelectrolyte was either PAH or PLL for the films composed of synthetic or natural polymers, respectively. This is needed to provide interaction with the particles or DNA molecules that are oppositely (negatively) charged. The films have been chosen because of different internal structure in terms of polymer mobility whereas the chemistry is similar because both polymers, PAH and PLL, have primary amino group in their structure. In our recent study, we have shown that HA/PLL films grow in the exponential regime using 400 kDa HA and 28 kDa PLL and that a very thin, noncontinuous film was formed for larger PLL (280 kDa) as a result of steric restrictions on larger polymers penetrating the film.32 The HA/ PLL film used in this study belongs to the exponentially growing category and is present in dynamic equilibrium or a mixed configuration56 and has a thickness in the micrometer range, thanks to the high PLL mobility.56-59 The HA/PLL film with 30 bilayers has a thickness of around 5 μm.60 The mechanism of HA/PLL film formation has been described by so-called polymer diffusion “in” and “out”. Jourdainne56 and Crouzier61 have recently demonstrated that the diffusion coefficient of PLL within the film can reach values on the order of 10-1 μm2 s-1 or even higher, which is close to the diffusion in water, whereas HA molecules are immobilized. In this regard, the PSS/PAH film is much thinner; the thickness of the (PAH/PSS)24/PAH film made at 15 mM NaCl can be estimated to be around 50 nm.62 The film is characterized by the low mobility of the polymers with diffusion coefficients of the polymers within the range of 10-8 to 10-6 μm2 s-1;63 therefore, the film is in the “frozen in” state. As soon as the interaction of charged particles or DNA with the LbL film is driven by the interaction with polymers as film constituents, one expects different behavior of the examined films in contact with charged particles and DNA. The principal scheme of the interaction of the LbL film with the particles and DNA is presented in Figure 1. The scheme summarizes some experimental findings of this study that helps the reader comfortably understand the main message of this work. The PSS/PAH film is characterized by the low mobility of polymers within the film. The interaction of particles or DNA with this film leads to their adsorption on the film surface as a result of complexation with the chains of PAH that are present on the film surface as a terminating layer (Figure 1B-A,B-D). The situation is different in the case of the HA/PLL film. PLL, as a polymer responsible for interaction with the examined particles and DNA, is pulled from the whole film to the film surface in order to make more contacts with the adsorbing molecules and particles. The accumulation of PLL on the film surface induces a large number of adsorbed particles and DNA on the film (Figure 1B-C,B-F) with no diffusion into the film. We believe that diffusion is restricted by their strong interaction with flexible (56) Jourdainne, L.; Lecuyer, S.; Arntz, Y.; Picart, C.; Schaaf, P.; Senger, B.; Voegel, J. C.; Lavalle, P.; Charitat, T. Langmuir 2008, 24, 7842–7847. (57) Lavalle, P. P.; Picart, C.; Cuisinier, F. J. G.; Decher, G.; Schaaf, P.; Voegel, J. C. Biophys. J. 2002, 82, 53a–53a. (58) Picart, C.; Lavalle, P.; Hubert, P.; Cuisinier, F. J. G.; Decher, G.; Schaaf, P.; Voegel, J. C. Langmuir 2001, 17, 7414–7424. (59) Boulmedais, F.; Ball, V.; Schwinte, P.; Frisch, B.; Schaaf, P.; Voegel, J. C. Langmuir 2003, 19, 440–445. (60) Jourdainne, L.; Arntz, Y.; Senger, B.; Debry, C.; Voegel, J.-C.; Schaaf, P.; Lavalle, P. Macromolecules 2007, 40, 316–321. (61) Crouzier, T.; Picart, C. Biomacromolecules 2009, 10, 433–442. (62) Ladam, G.; Schaad, P.; Voegel, J. C.; Schaaf, P.; Decher, G.; Cuisinier, F. Langmuir 2000, 16, 1249–1255. (63) Klitzing, R. v.; Mohwald, H. Macromolecules 1996, 29, 6901–6906.
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Figure 1. Principal scheme of interaction of the LbL films (B), namely, PSS/PAH and HA/PLL, with the gold nanoparticles (gray spheres) and DNA (in blue). A vertical cross-section of the film is present. The particles and DNA interact only with the surface PAH groups of the PSS/PAH film (B-A and B-D); however, they can be accumulated in large quantities as a result of the interaction with PLL “doping” from the whole interior of the HA/PLL film (B-C and B-F). Diffusion of DNA into the HA/PLL film can be triggered by heating to 70 °C (F-E).
PLL molecules. The high mobility of PLL in the film allows the examined particles or DNA to adopt the thermodynamically most stable conformation, being complexed with PLL. This strong complexation prevents the diffusion of material from the bulk into the film. Furthermore, we describe the experimental findings to support the scenario present in Figure 1. First, we have proven that embedding of the gold nanoparticles or DNA is mediated by complexation with PLL but not by salt ions, which can be present in the film in substantial amounts and are able to induce particle aggregation50 or salt DNA out. The film with adsorbed particles has been doped from bulk solution with additional amount of either PLL (0.5 mg/mL in Tris buffer) or NaCl (15 mM in Tris buffer), and a fresh nanoparticle suspension was added. In the case of PLL external doping of the film, nanoparticle adsorption proceeds further, but this is not the case for external salt doping. Micrometer-sized polyelectrolyte capsules adsorb on the HA/ PLL film (Figure 2C,F). The capsules adsorb preferentially as single particles, which was proven by optical microscopy enlarged images on the top of the film (data not shown). The capsules that adhere to the PSS/PAH film or sediment on it can be easily removed from the film surface by washing, whereas being once adsorbed on the HA/PLL film, the capsules stay immobilized. The surface charge of the microcapsules used in this experiment is almost compensated for overall,64 but their strong attachment to the film can be attributed to low uncompensated for negative charge and cooperative interaction with flexible PLL as described above. Such a strong interaction is not typical for the adsorption of large polymer microcapsules onto the LbL films, which can be done by strong bonding such as covalent coupling.65 The adsorption of smaller objects (i.e., 20 nm gold nanoparticles and DNA molecules) results in the formation of inhomogeneous structures (aggregates) on the HA/PLL film as can be seen from optical transmission and fluorescence images (Figure 2A,B,D,E). Similar structures were reported by Crouzier when growth factor rhBMP2 had been adsorbed onto the HA/ PLL film.49 Surface-located aggregates of gold nanoparticles and DNA are composed of the particles or DNA molecules in complex with PLL. The aggregates have dimensions in the (64) Kohler, K.; Mohwald, H.; Sukhorukov, G. B. J. Phys. Chem. B 2006, 110, 24002–24010. (65) Wang, B.; Zhao, Q. H.; Wang, F.; Gao, C. Y. Angew. Chem., Int. Ed. 2006, 45, 1560–1563.
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Figure 2. Transmission images of gold nanoparticles (A,D), DNA-EtBr (B), and microcapsules (C) adsorbed onto the (PLL/ HA)24/PLL film. Fluorescent microscopy images of DNA (E) and microcapsules (F) on the film surface. Microcapsules are filled with dextran-rhodamine.
micrometer ranges of 1-5 and 5-15 μm for the nanoparticles and DNA, respectively. Aggregation occurs by a nucleation and growth mechanism. First, adsorbed particles or DNA molecules compensate their charge by complexation with surface PLL. PLL molecules pulled from the film to the surface make additional Langmuir 2009, 25(24), 14037–14043
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Figure 3. Absorption spectra of (PLL/HA)24/PLL (A) and (PAH/ PSS)24/PAH (B) films after incubation with gold nanoparticles.
contact with the particle or DNA, which induces the transport of a larger number of particles or DNA from the bulk and further growth of the nanoparticle-PLL or DNA-PLL complex. Thus, the newly formed aggregate is continuously growing to a definite size until charge compensation occurs in the aggregate. The (PLL/ HA)24/PLL film adsorb 1.5 μg of DNA per cm2 after incubation of the film with DNA solution for 2 h. This was found by analyzing the amount of embedded DNA in the film. In the case of gold nanoparticles, the mass of embedded nanoparticles has been found to be about 100 μg/cm2 (all of the nanoparticles were adsorbed on the film from solution), which is comparable to the PLL mass in the film on the basis of the PLL quantification experiment. The PLL content in the film was found to be 94 μg/ cm2. Such a high loading capacity can undoubtedly be attributed to the doping PLL. The optical transmission image of DNA “patches” allows us to visualize them clearly (the bare film is transparent), revealing very condensed DNA packing. In transmission or fluorescence imaging, no defined structures have been registered for the PSS/PAH film in contact with either gold nanoparticles or DNA under the same conditions as for HA/PLL films (data not shown). This means that both nanoparticles and DNA exhibit much weaker interactions with the PSS/PAH film, resulting in a considerably smaller amount of adsorbed material. After 2 h of incubation of the films with DNA and 2 days with gold nanoparticles, the amount of adsorbed material was found to be 5 and 12 times less for the PSS/PAH films than for the HA/PLL films, respectively. The absorption spectra of HA/PLL and PSS/PAH films modified with gold nanoparticles are presented in Figure 3A,B, respectively. A much lower peak intensity for the PSS/PAH film indicates a lower adsorption of nanoparticles that were present as single particles (Figure 3B). This peak position (maximum at 520 nm) is the same as for a water suspension of gold nanoparticles. The HA/PLL film adsorbs many more gold nanoparticles and the spectrum shows a peak with a maximum at around 530 nm and a large shoulder at the higher wavelengths, indicating particle aggregation. Figure 4 shows photographs of the HA/PLL and PSS/PAH films (cuvettes 2 and 3, respectively) after contact with a solution of nanoparticles (the solution is present in cuvette 1). All of the nanoparticles were taken up from solution and adsorbed onto the HA/PLL film (cuvette 2) whereas very little adsorption of the nanoparticles has been observed for the PSS/ PAH film (cuvette 3). Note that the PSS/PAH-layered glass in cuvette 3 is not very visible because of the very poor adsorption of gold nanoparticles. Experimental proof of PLL doping onto the film surface in order to embed DNA is given in Figure 4B. Increasing fluorescence on the surface of the film is related to Langmuir 2009, 25(24), 14037–14043
Figure 4. (A) Plastic cuvettes with gold nanoparticle suspension before (1) and after incubation with (PLL/HA)24/PLL (2) and (PAH/PSS)24/PAH (3) films. The glass in cuvette 3 is not very visible because of the very poor adsorption of gold nanoparticles. Fluorescence microscopy (B) and transmission (C) images of a (PLL/HA)24/PLL film with embedded DNA, where the DNA incubation time is 23 min. The scale bar is 10 μm.
DNA complexation with PLL-FITC, which is doped from the film interior. The DNA-PLL complex has micrometer dimensions and can be seen in an optical microscope (Figure 4C). Furthermore, we consider the HA/PLL film to be a matrix not only permitting the embedding of a large amount of material showing reservoir properties but also providing a chance to manipulate a diffusion of the film material in and out. All examined large microcapsules, gold nanoparticles, and DNA are embedded on the HA/PLL film surface. Restricted diffusion of microcapsules is expected because of the large capsule size (around 1 μm). However, even small nanoparticles with a diameter of 20 nm and DNA molecules do not diffuse into the film (at least 2 days after adsorption), as proven in our current research by electron microscopy for nanoparticles66 and as can be concluded from the DNA stack profile in Figure 5, plot 1. Srivastava67 reported the strong accumulation of 4 nm quantum dots in the exponentially growing PDADMAC/poly(acrylic acid) films, which can spontaneously diffuse within the film but also partially diffuse out of the film with time, indicating no significant diffusion problems. We assume that because of the strong interaction of the gold nanoparticles with doping PLL or because of their large size, 20 nm gold nanoparticles do not diffuse into the HA/PLL film. The diffusion of DNA-EtBr can be triggered by a temperature increase (Figure 1FE). The stack profiles of the PLL-FITC and DNA-EtBr distribution within the HA/PLL film are shown in Figure 5 before (1) and after (2) heating to 70 °C for 30 min. The profiles are obtained by CLSM z slicing perpendicular to the film surface. After heating, the intensity profile of PLL-FITC (green) is decreased, which is attributed to the photobleaching of PLLFITC as shown by a control experiment using the same system but without the heating step. Previously, we have shown that the HA/ PLL film is stable up to 50 °C.31 From this experiment, one can suggest that a higher temperature (70 °C) does not result in (66) Volodkin, D. V.; Delcea, M.; M€ohwald, H.; Skirtach, A. G. ACS Appl. Mater. Interfaces , 200910.1021/am900269c (67) Srivastava, S.; Ball, V.; Podsiadlo, P.; Lee, J.; Ho, P.; Kotov, N. A. J. Am. Chem. Soc. 2008, 130, 3748–3749.
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Figure 5. Stack profiles obtained by CLSM for (PLL/HA)24/PLL with embedded DNA labeled with EtBr. The film was prepared with PLL-FITC. Before (1) and after (2) heating to 70 °C for 30 min. The green curve corresponds to the PLL-FITC stack profile, and the red one corresponds to the DNA-EtBr stack profile. The distance on the y axis is from the top of the film to the bottom.
significant film decomposition. DNA is located on the film surface before heating, but the peak corresponding to DNA is shifted after heating to the position corresponding to the polyelectrolyte film (Figure 5, plots 1 and 2). This undoubtedly indicates the diffusion of DNA into the film, which can be substantiated on quantitative terms. Integral fluorescence from DNA-EtBr before and after heating is the same, which means that there is no release of DNA molecules during heating. The shift of the DNA profile indicates that the embedded DNA is replaced from the film surface into the film interior as a result of temperature-triggered diffusion. z slicing does not give the correct thickness of the film or DNA “layer” on its top because of its focal resolution limits on the z axis. However, it allows the identification of a shift in the profile and making quantative conclusions considering integral fluorescence. DNA molecules that diffuse into the film need free PLL amino groups with which to form electrostatic interactions. Around half of all PLL amino groups in the HA/PLL film are in a complex with carboxylic groups of HA, and the other half are free.61 We believe that high content of free amino groups and the high mobility of PLL make possible the embedding of DNA. The restriction of DNA diffusion at room temperature can be caused by the formation of a strong DNAPLL complex on the film surface that prevents further DNA diffusion into the film, which, however, could be achieved at higher temperatures when the DNA mobility is increased. 14042 DOI: 10.1021/la9015433
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Light-Triggered DNA Release. DNA strongly interacts with the film as a result of the formation of a stable complex with PLL, which is doped from the film. However, if the PLLDNA interaction is disturbed, then DNA molecules may be released from the film. Furthermore, we suggest that the lightinduced decomposition of the HA/PLL network in the film can lead to the release of embedded DNA. The HA/PLL film functionalized with gold nanoparticles becomes active in response to the “biologically friendly” IR laser at a power above 20 mW. This activation is characterized by localized heating of the film as a result of the conversion of absorbed light energy into heat. Gold nanoparticles serve as absorption centers for energy supplied by a laser beam.68,69 We use this property for remote activation of the films by a laser operating at 830 nm. Figure 6 shows a CSLM image of a HA/PLL film (PLL is labeled with FITC) with embedded gold nanoparticles before and after light illumination (images A and B, respectively). Decreased film fluorescence is evidence of the local heating on the films when absorbed light energy is converted to heat. The polymers supposedly get pushed out of the film; however, the film is not recovered and does not proceed to decomposition after light irradiation, which could be due to thermal cross-linking between amino groups of PLL and carboxylic groups of HA induced at high local heating. The crosslinking is reported to take place at temperatures above 130 °C.70,71 The threshold when the locally affected area is visible on the film is around 20 mW at an 830 nm laser light wavelength. Figure 6C,D shows a confocal image of the DNA “layer” on the film top visualized before (C) and after (D) stimulation with laser light. Scanning has been performed in the position corresponding to the peak maximum in the stack profiles identified for the film (Figure 5, plot 1, green curve) and DNA on the film (Figure 5, plot 1, red curve). It is also to be noted that the position of the DNA layer that was imaged remained the same as that of the film underneath, with no lateral movement. Gold nanoparticles are localized under the DNA layer. The significant decrease in the intensity of the DNA image after light stimulation shows the release of DNA molecules from the film We suggest this mechanism of DNA release, believing that the distortion of doping PLL-DNA interaction is responsible for the DNA release but not high-temperature destruction of the DNA molecules due to the high temperature of the light-stimulated nanoparticle aggregates. The temperature profile for the aggregates is relatively sharp, and temperature close to room temperature can be reached in the vicinity of the aggregates within a distance of a few micrometers from the aggregates.72 DNA is located at relatively long distances from the nanoparticle aggregates, that is, in the micrometer range (around 6 μm) as can be concluded from Figure 5 (plot 1). The distance between the maximum in the DNA and film (labeled by PLL-FITC) profile is 8 μm, and that of half of the film is 2 μm. DNA release was observed not only in the areas where the film was destroyed (black areas on Figure 6B) but also between the areas, and the distribution of the rest of the DNA on the film after irradiation is more homogeneous than before irradiation (Figure 6D). This suggests that large DNA aggregates (Figure 6C) are removed from the film top because (68) Lee, J.; Govorov, A. O.; Kotov, N. A. Angew. Chem., Int. Ed. 2005, 44, 7439–7442. (69) Govorov, A. O.; Richardson, H. H. Nano Today 2007, 2, 30–38. (70) Harris, J. J.; DeRose, P. M.; Bruening, M. L. J. Am. Chem. Soc. 1999, 121, 1978. (71) Lee, S. W.; Kim, B.-S.; Chen, S.; Shao-Horn, Y.; Hammond, P. T. J. Am. Chem. Soc. 2009, 131, 671–679. (72) Skirtach, A. G.; Dejugnat, C.; Braun, D.; Susha, A. S.; Rogach, A. L.; Sukhorukov, G. B. J. Phys. Chem. C 2007, 111, 555–564.
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Article
Figure 7. CLSM images of gold-nanoparticle-modified microcapsule (PDADMAC/PSS)4/gold nanoparticles embedded in the (PLL/HA)24/PLL film before (A) and after (B) exposure to nearIR light. The scale bar is 1 μm. The light power is 20 mW, the exposure time is 1s, and the wavelength is 830 nm.
no disruption. The LbL film with embedded microcapules represents an alternative drug delivery carrier where drug release is triggered by permeability changes in the capsule wall. Here, we show the proof of the concept that film-immobilized capsules can be used for remote release of its content. If the film allows capsule embedding, such as the PLL/HA film shown in this work, then remote release from surface-supported lightsensitive capsules is possible. The surface-supported films based on biopolymers can be used as coatings for medical devices implanted under the skin or in soft tissue, which is easily accessible by IR light that can penetrate relatively deeply.73
Conclusions
Figure 6. (PLL/HA)24/PLL film with embedded DNA before (A, C) and after (B, D) irradiation with IR laser light. PLL in the film is labeled with FITC, and DNA is labeled with EtBr. The scale bar is 5 μm. (E) Schematic presentation of the suggested mechanism of DNA release induced by the distortion of the DNA-doping PLL interaction as a result of partial thermal film decomposition around nanoparticle aggregates.
DNA interaction with the film is weakened. Our future study will be devoted to the analysis of the activity of the released DNA. The HA/PLL film with deposited nanoparticles possesses light-active properties. As shown above, the film has a strong ability to embed materials of different sizes (from small nanoparticles to large capsules). Thus, the film can host lightsensitive carriers such as microcapsules modified with gold nanoparticles. The microcapsules were premodified with gold nanoparticles and were successfully embedded in the film (Figure 2C-F and Figure 7A). The microcapsule was subjected to the laser beam and released its contents as a result of localized permeability changes in their walls (Figure 7A,B). The insets of the images show transmission microscopy images of the microcapsules before and after exposure to light. The capsules maintain their spherical shape after light irradiation, indicating
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The HA/PLL film studied here possesses high loading capacity as a result of polymer doping onto the film surface that results in the accumulation of a large amount of adsorbing material, which is many times less for the PSS/PAH film that has low polymer mobility. Microcapsules, gold nanoparticles, and DNA can be embedded in the HA/PLL film and located on the film surface. The diffusion of embedded DNA into the film can be triggered by heating. The HA/PLL film with adsorbed gold nanoparticles and DNA possesses remote release features by stimulation with “biofriendly” IR light. DNA release from the film modified with gold nanoparticles is supposed to be caused by local destruction of the polymer network in the film followed by the blocking of PLL-DNA bonding and, as a result, the release of DNA molecules from the film. Laser activation of film-supported capsules shows the remote release of encapsulated dextran. This study can serve future biomedical applications in tissue engineering and biocoatings where high loading capacity together with remote release functionalities is demanded. Light-triggered DNA transfection to a single cell can also be achieved by this approach. Acknowledgment. The work is supported by a Marie-Curie fellowship (EU6 project BIOCOATING) as well as the PICT2006-01365 (Max-Planck Society-Argentine SeCyt). (73) Vriezema, D. M.; Aragones, M. C.; Elemans, J. A. A. W.; Cornelissen, J. J. L. M.; Rowan, A. E.; Nolte, R. J. M. Chem. Rev. 2005, 105, 1445–1489.
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