Embedded Silver Ions-Containing Liposomes in Polyelectrolyte

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Langmuir 2008, 24, 10209-10215

10209

Embedded Silver Ions-Containing Liposomes in Polyelectrolyte Multilayers: Cargos Films for Antibacterial Agents Marta Malcher,†,‡,§ Dmitry Volodkin,*,†,|,⊥,# Be´atrice Heurtault,‡ Philippe Andre´,∇ Pierre Schaaf,O Helmuth Mo¨hwald,# Jean-Claude Voegel,|,⊥ Adam Sokolowski,§ Vincent Ball,|,⊥ Fouzia Boulmedais,O and Benoit Frisch‡ De´partement de Chimie Bioorganique, Institut Gilbert Laustriat, UMR 7175 CNRS/UniVersite´ Louis Pasteur, Illkirch, France, Wroclaw UniVersity of Technology, Faculty of Chemistry, Wroclaw, Poland, Institut National de la Sante´ et de la Recherche Me´dicale, Unite´ 595, Strasbourg, France, UniVersite´ Louis Pasteur, Faculte´ de Chirurgie Dentaire, Strasbourg, France, Max-Planck Institute for Colloids and Interfaces, Potsdam, Germany, De´partement de Pharmacologie, Institut Gilbert Laustriat, UMR 7175 CNRS/UniVersite´ Louis Pasteur, Illkirch, France, and Centre National de la Recherche Scientifique, Institut Charles Sadron (ICS), UPR 22 Strasbourg, France ReceiVed May 13, 2008. ReVised Manuscript ReceiVed July 7, 2008 A new antibacterial coating made of poly(L-lysine)/hyaluronic acid (PLL/HA) multilayer films and liposome aggregates loaded with silver ions was designed. Liposomes filled with an AgNO3 solution were first aggregated by the addition of PLL in solution. The obtained micrometer-sized aggregates were then deposited on a PLL/HA multilayer film, playing the role of a spacer with the support. Finally, HA/PLL/HA capping layers were deposited on top of the architecture to form a composite AgNO3 coating. Release of encapsulated AgNO3 from this composite coating was followed and triggered upon temperature increase over the transition temperature of vesicles, found to be equal to 34 °C. After determination of the minimal inhibitory concentration (MIC) of AgNO3 in solution, the antibacterial activity of the AgNO3 coating was investigated against Escherichia coli. A 4-log reduction in the number of viable E. coli cells was observed after contact for 120 min with a 120 ng/cm2 AgNO3 coating. In comparison, no bactericidal activity was found for PLL/HA films previously dipped in an AgNO3 solution and for PLL/HA films with liposome aggregates containing no AgNO3 solution. The strong bactericidal effect could be linked to the diffusion of silver ions out of the AgNO3 coating, leading to an important bactericidal concentration close to the membrane of the bacteria. A simple method to prepare antibacterial coatings loaded with a high and controlled amount of AgNO3 is therefore proposed. This procedure is far superior to that soaking AgNO3 or Ag nanoparticles into a coating. In principle, other small bactericidal chemicals like antibiotics could be encapsulated by this method. This study opens a new route to modify surfaces with small solutes that are not permeating phospholipid membranes below the phase transition temperature.

Introduction Bacterial infections at the site of implanted medical devices present a serious source of problems, leading, if untreated, to chronic microbial infection, inflammation, tissue necrosis to septicaemia and eventually to death.1,2 Such infectious diseases can, for example, be due to Staphylococcus epidermidis and Escherichia coli, in the case of intravascular catheter associated infections,1,3 or Staphylococcus aureus, in the case of metallic implants.4 In the long term, certain medical implants in the body suffers from a progressive accumulation of proteins, bacteria, and cells on their surface, developing a biofilm.5 Commonly in * Corresponding author. Phone: +49-331-5679440. Fax: +49-3315679202. E-mail: [email protected]. † Both authors contributed equally. ‡ De´partement de Chimie Bioorganique, Institut Gilbert Laustriat. § Wroclaw University of Technology. | Institut National de la Sante´ et de la Recherche Me´dicale. ⊥ Universite´ Louis Pasteur. # Max-Planck Institute for Colloids and Interfaces. ∇ De´partement de Pharmacologie, Institut Gilbert Laustriat. O Centre National de la Recherche Scientifique.

(1) Rupp, M. E.; Archer, G. L. Clin. Infect Dis. 1994, 19, 231–43. (2) Stickler, D.; McLean, R. Cells Mater. 1995, 5, 167–182. (3) Dankert, J.; Hogt, A. H.; Feijen, J. CRC Crit. ReV. Biocompat. 1986, 2, 219–301. (4) Barth, E.; Myrvik, Q. M.; Wagner, W.; Gristina, A. G. Biomaterials 1989, 10, 325–8. (5) Vacheethasanee, K.; Marchant, R. E. In Handbook of Bacterial Adhesion: Principles, Methods, and Applications; An, Y. H., Friedman, R. J., Eds.; Humana Press: Totowa, NJ, 2000; Chapter 5, pp 73-90.

postsurgery therapy, antibiotic treatments (delivered orally or by injection) are used to avoid bacterial growth. However, new antibacterial strategies are needed in the situation where more and more bacteria are antibiotic-resistant.6,7 Surface treatments have been developed to reduce bacterial adhesion by modifying the physicochemical properties of the substrate in order to reduce the interactions between bacteria and the substrate. These “passive” coatings are based on polyethylene glycol (PEG)8-11 or hydrophilic polyurethane12,13 surface modification and render the surface hydrophilic, allowing a limited bacterial adhesion and proliferation. A recent alternative approach was proposed based on “active” coatings by integrating antibacterial agents at the implant site. The advantage of a direct delivery at the site of implantation is the high local concentration that can be delivered during the critical short-term postimplantation period (several hours) without reaching the systemic toxicity level of the agent. (6) Ofek, I.; Doyle, R. J. In Bacterial Adhesion to Cells and Tissues; Chapman Hall: New York, 1994. (7) Hetrick, E. M.; Schoenfisch, M. H. Chem. Soc. ReV. 2006, 35, 780–789. (8) Boulmedais, F.; Frisch, B.; Etienne, O.; Lavalle, P.; Picart, C.; Ogier, J.; Voegel, J.-C.; Schaaf, P.; Egles, C. Biomaterials 2004, 25, 2003–2011. (9) Kingshott, P.; Wei, J.; Bagge-Ravn, D.; Gadegaard, N.; Gram, L. Langmuir 2003, 19, 6912–6921. (10) Park, K. D.; Kim, Y. S.; Han, D. K.; Kim, Y. H.; Lee, E. H.; Suh, H.; Choi, K. S. Biomaterials 1998, 19, 851–9. (11) Razatos, A.; Ong, Y. L.; Boulay, F.; Elbert, D. L.; Hubbell, J. A.; Sharma, M. M.; Georgiou, G. Langmuir 2000, 16, 9155–9158. (12) Nagel, J. A.; Dickinson, R. B.; Cooper, S. L. J. Biomater. Sci. Polym. Ed. 1996, 7, 769–80. (13) Pulat, M.; Akdogan, A.; Ozkan, S. J. Biomater. Appl. 2002, 16, 293–303.

10.1021/la8014755 CCC: $40.75  2008 American Chemical Society Published on Web 08/13/2008

10210 Langmuir, Vol. 24, No. 18, 2008

Antibiotic-coated surfaces (penicillin or ciprofloxacin) are thus proposed,7,14 but their effectiveness is limited. The inhibitory and bactericidal properties of silver have been known for centuries.15 Nontoxic to human cells, silver and silver ions can act against a broad spectrum of bacterial strains. Silver is commercially used in numerous products, including water sterilization and therapeutic applications.16 Polyelectrolyte multilayer films, obtained by the alternate deposition of cationic and anionic polymers, emerged as a simple approach to functionalize surfaces in a controlled way.17,18 These films grow either linearly,19,20 or exponentially.21-23 Such films find a wide application in surface treatments,24,25 membranes,26,27 chemical or biological detections,28,29 biotechnology,30-32 and biomaterials.33-35 In particular, this technique also allows preparation of nanoarchitectures exhibiting specific properties in the control of cell activation.36,37 Moreover, these multilayer films can act as reservoirs for active molecules38 and are therefore potentially suited for the development of local drug delivery systems.39 An antiadhesive film was developed leading to a reduction in adhesion by about 92% of E. coli cells compared to bare substrate.8 LbL films with embedded antimicrobial peptides,40 embedded hydrophobic bactericide,41 or films built with chitosan, a polyelectrolyte used as an antibacterial agent,42,43 were investigated. LbL films with inserted silver nanoparticles (14) Schierholz, J. M.; Steinhauser, H.; Rump, A. F. E.; Berkels, R.; Pulverer, G. Biomaterials 1997, 18, 839–844. (15) Sykes, G. In Disinfection and Sterilization; Van Nostrand: Princeton, NJ, 1958. (16) Clement, J. L.; Jarrett, P. S. Metal-Based Drugs 1994, 1, 467–482. (17) Decher, G. Science 1997, 277, 1232–1237. (18) Decher, G.; Hong, J.-D. Makromol. Chem. 1991, 46, 321. (19) Ladam, G.; Schaad, P.; Voegel, J.-C.; Schaaf, P.; Decher, G.; Cuisinier, F. J. G. Langmuir 2000, 16, 1249–1255. (20) Shiratori, S. S.; Rubner, M. F. Macromolecules 2000, 33, 4213–4219. (21) Hubbell, J. A. Curr. Opin. Biotechnol. 1999, 10, 123–129. (22) Boulmedais, F.; Ball, V.; Schwinte´, P.; Frisch, B.; Schaaf, P.; Voegel, J.-C. Langmuir 2003, 19, 440–445. (23) Lavalle, P.; Picart, C.; Cuisinier, F. J. G.; Decher, G.; Schaaf, P.; Voegel, J.-C. Biophys. J. 2002, 82, 53a–53a. (24) Bravo, J.; Zhai, L.; Wu, Z. Z.; Cohen, R. E.; Rubner, M. F. Langmuir 2007, 23, 7293–7298. (25) Wu, Z. Z.; Walish, J.; Nolte, A.; Zhai, L.; Cohen, R. E.; Rubner, M. F. AdV. Mater. 2006, 18, 2699–2702. (26) Dubas, S. T.; Farhat, T. R.; Schlenoff, J. B. J. Am. Chem. Soc. 2001, 123, 5368–5369. (27) Kotov, N. A.; Magonov, S.; Tropsha, E. Chem. Mater. 1998, 10, 886– 895. (28) Brusatori, M. A.; Van Tassel, P. R. Biosensors Bioelectron. 2003, 18, 1269–1277. (29) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3427–3433. (30) Hillberg, A. L.; Tabrizian, M. Biomacromolecules 2006, 7, 2742–2750. (31) Jewell, C. M.; Fuchs, S. M.; Flessner, R. M.; Raines, R. T.; Lynn, D. M. Biomacromolecules 2007, 8, 857–863. (32) Zelikin, A. N.; Quinn, J. F.; Caruso, F. Biomacromolecules 2006, 7, 27–30. (33) Thierry, B.; Winnik, F. M.; Merhi, Y.; Silver, J.; Tabrizian, M. Biomacromolecules 2003, 4, 1564–1571. (34) Vodouhe, C.; Schmittbuhl, M.; Boulmedais, F.; Bagnard, D.; Vautier, D.; Schaaf, P.; Egles, C.; Voegel, J.-C.; Ogier, J. Biomaterials 2005, 26, 545–554. (35) Vautier, D.; Karsten, V.; Egles, C.; Chluba, J.; Schaaf, P.; Voegel, J.-C.; Ogier, J. J. Biomater. Sci.sPolym. Ed. 2002, 13, 713–732. (36) Jessel, N.; Oulad-Abdeighani, M.; Meyer, F.; Lavalle, P.; Haikel, Y.; Schaaf, P.; Voegel, J.-C. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 8618–8621. (37) Chluba, J.; Voegel, J.-C.; Decher, G.; Erbacher, P.; Schaaf, P.; Ogier, J. Biomacromolecules 2001, 2, 800–805. (38) Me´ndez Garza, J.; Schaaf, P.; Muller, S.; Ball, V.; Stoltz, J.-F.; Voegel, J.-C.; Lavalle, P Langmuir 2004, 20, 7298–7302. (39) Boulmedais, F.; Tang, C. S.; Keller, B.; Vo¨ro¨s, J. AdV. Funct. Mater. 2006, 16, 63–70. (40) Etienne, O.; Picart, C.; Taddei, C.; Haikel, Y.; Dimarcq, J. L.; Schaaf, P.; Voegel, J. C.; Ogier, J. A.; Egles, C. Antimicrob. Agents Chemother. 2004, 48, 3662–9. (41) Nguyen, P. M.; Zacharia, N. S.; Verploegen, E.; Hammond, P. T. Chem. Mater. 2007, 19, 5524–5530. (42) Fu, J. H.; Ji, J.; Yuan, W. Y.; Shen, J. C. Biomaterials 2005, 26, 6684– 6692. (43) Bratskaya, S.; Marinin, D.; Simon, F.; Synytska, A.; Zschoche, S.; Busscher, H. J.; Jager, D.; van der Mei, H. C. Biomacromolecules 2007, 8, 2960–2968.

Malcher et al.

were also designed to induce an antibacterial effect.44-49 However, the main point is not the presence of metallic silver on the catheter, but the ability of the silver to be dissociated into silver ions.50,51 Silver ions, having antibacterial properties, are not necessarily present at a surface coated with metallic silver. Lok et al.52 showed that the antibacterial activities of silver nanoparticles are dependent on chemisorbed Ag+ ions, formed owing to extreme sensitivity to oxygen. Possible modes for the release of chemisorbed Ag+ species may involve direct association between the nanoparticles and the bacteria. In vivo studies of silver coatings failed to demonstrate a decrease of bacterial adhesion.53,54 Silver ions, having antibacterial properties, are not actively released by these coatings and are not necessarily present at a surface coated with metallic silver. Liposomes are well-known and widely used in medical applications55,56 due to their small size and the high integrity of the lipid bilayer. They allow small amphiphilic molecules and water soluble species to be kept and released.57 Thus, surfaces with immobilized vesicles are perfect candidates for drug delivery applications.58,59 Our recent studies were devoted to the buildup of liposome-containing polyelectrolyte multilayer films,60-64 with liposomes playing the role of reservoirs with controlled release. However, the amount of immobilized active compound is low due to the limited thickness of one layer of liposomes corresponding to approximately 100 nm in thickness.65 Our recent findings on complexation of vesicles with polyelectrolytes suggest a way to overcome the low loading capacity of such films.66-71 With a fast injection of the vesicles into a PLL solution, obtained (44) Fu, J. H.; Ji, J.; Fan, D. Z.; Shen, J. C. J. Biomed. Mater. Res. A 2006, 79A, 665–674. (45) Shi, Z.; Neoh, K. G.; Zhong, S. P.; Yung, L. Y.; Kang, E. T.; Wang, W. J Biomed. Mater. Res. A 2006, 76, 826–34. (46) Dai, J. H.; Bruening, M. L. Nano Lett. 2002, 2, 497–501. (47) Lee, D.; Cohen, R. E.; Rubner, M. F. Langmuir 2005, 21, 9651–9659. (48) Li, Z.; Lee, D.; Sheng, X. X.; Cohen, R. E.; Rubner, M. F. Langmuir 2006, 22, 9820–9823. (49) Lee, D.; Rubner, M. F.; Cohen, R. E. Chem. Mater. 2005, 17, 1099–1105. (50) Kumar, R.; Howdle, S.; Munstedt, H. J. Biomed. Mater. Res. B 2005, 75, 311–9. (51) Kumar, R.; Munstedt, H. Biomaterials 2005, 26, 2081–8. (52) Lok, C. N.; Ho, C. M.; Chen, R.; He, Q. Y.; Yu, W. Y.; Sun, H.; Tam, P. K. H.; Chiu, J. F.; Che, C. M. J. Biol. Inorg. Chem. 2007, 12, 527–534. (53) Masse, A.; Bruno, A.; Bosetti, M.; Biasibetti, A.; Cannas, M.; Gallinaro, P. J. Biomed. Mater. Res. 2000, 53, 600–604. (54) Sheehan, E.; McKenna, J.; Mulhall, K. J.; Marks, P.; McCormack, D. J. Orthop. Res. 2004, 22, 39–43. (55) Lasic, D. D. In Liposomes: From Physics to Applications; Elsevier: Amsterdam, 1993. (56) Lasic, D. D.; Papahadjopoulos, D. In Medical Applications of Liposomes; Elsevier: Amsterdam, 1998. (57) Graff, A.; Winterhalter, M.; Meier, W. Langmuir 2001, 17, 919–923. (58) Brochu, H.; Vermette, P. Langmuir 2007, 23, 7679–7686. (59) Christensen, S. M.; Stamou, D. Soft Matter 2007, 3, 828–836. (60) Michel, M.; Izquierdo, A.; Decher, G.; Voegel, J. C.; Schaaf, P.; Ball, V. Langmuir 2005, 21, 7854–7859. (61) Michel, M.; Vautier, D.; Voegel, J. C.; Schaaf, P.; Ball, V. Langmuir 2004, 20, 4835–4839. (62) Volodkin, D.; Ball, V.; Schaaf, P.; Voegel, J. C.; Mo¨hwald, H. Biochim. Biophys. Acta 2007, 1768, 280–290. (63) Volodkin, D.; Mo¨hwald, H.; Voegel, J. C.; Ball, V. J. Controlled Release 2007, 117, 111–120. (64) Volodkin, D. V.; Ball, V.; Voegel, J. C.; Mo¨hwald, H.; Dimova, R.; Marchi-Artzner, V. Colloids Surf. A 2007, 303, 89–96. (65) Volodkin, D. V.; Arntz, Y.; Schaaf, P.; Mo¨hwald, H.; Voegel, J.-C.; Ball, V. Soft Matter 2008, 4, 122–130. (66) Ge, L. Q.; Mo¨hwald, H.; Li, J. B. Colloids Surf. A 2003, 221, 49–53. (67) Kabanov, A. V.; Nazarova, I. R.; Astafieva, I. V.; Batrakova, E. V.; Alakhov, V. Y.; Yaroslavov, A. A.; Kabanov, V. A. Macromolecules 1996, 29, 8999–8999. (68) Kabanov, V. A.; Yaroslavov, A. A. J. Controlled Release 2002, 78, 267– 271. (69) Kawakami, K.; Nishihara, Y.; Hirano, K. J. Phys. Chem. B 2001, 105, 2374–2385. (70) Yaroslavov, A. A.; Efimova, A. A.; Lobyshev, V. I.; Kabanov, V. A. Biochim. Biophys. Acta 2002, 1560, 14–24. (71) Yaroslavov, A. A.; Kiseliova, E. A.; Udalykh, O. Y.; Kabanov, V. A. Langmuir 1998, 14, 5160–5163.

Embedded SilVer Ions-Containing Liposomes

complexes can be changed from isolated polymer-coated vesicles to vesicle aggregates, depending on the polymer/lipid ratio. PLLstabilized liposomes can be used as containers filled with active compounds able to release them upon heating over the lipid transition temperature. The same is true not only for single vesicles but also for vesicle aggregates. The main goal of this work was to obtain biocompatible surfaces with an antibacterial activity by using the innovative approach of surface functionalization. The controlled release of silver ions, which can act against a wide range of bacteria, has been pursued as an alternative strategy for reducing bacterial proliferation. To this aim, surfaces were functionalized by AgNO3-containing liposome aggregates (AgNO3-liposome aggregates) embedded in PLL/HA multilayer films. The obtained architecture was named AgNO3 coating. Liposome aggregates were used as silver ion reservoirs for coating of solid surfaces. Release of silver ions from the coating was investigated at room temperature and at 37 °C. The bactericidal effect of AgNO3 coatings was characterized.

Experimental Section Materials. The used lipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DPPG), were purchased from Avanti Polar Lipids. Cholesterol (CL, ref C-8667), 4-(2-hydroxyethyl)piperazine-1ethanesulfonic acid (HEPES, ref H3375), poly-L-lysine hydrobromide with MW ) 28 kDa (PLL, ref P7890), and Triton X-100 (ref 23,4729) were purchased from Sigma-Aldrich. Hyaluronic acid with MW ) 420 kDa (HA, ref 002570) and 5(6)-carboxyfluorescein (CF, ref 21877) were respectively purchased from Lifecore Biomedical and Fluka. Silver nitrate (AgNO3), sodium iodide (NaI), and sodium nitrate (NaNO3) were purchased from VWR Prolabo. Sephadex G-75 (G75120) was purchased from Sigma-Aldrich. All products except PLL were used without further purification. Microscopy cover glasses with a diameter of 12 or 30 mm were purchased from Marlenfeld GmBH. Throughout this study, 10 mM HEPES buffer prepared at pH 7.4 containing 150 mM NaNO3 was used and will be mentioned in the text as HEPES. The water used in all experiments was prepared in a three-stage Millipore Milli-Q Plus 185 purification system and had a resistivity of 18.2 MΩ cm. Purification of PLL. PLL used in this study contains bromide ions that have to be removed in order to maintain the integrity of AgNO3-liposomes, which could be destroyed by the formation of insoluble AgBr in contact with PLL solution. Bromide counterions were eliminated by a 10 M NaOH solution dropped in to 1 mL of PLL solution (at a concentration of 13.5 mg/mL) to reach a concentration of 1 M NaOH. At this concentration, all the amino groups of PLL become neutral and then PLL precipitates. The formed gel was centrifuged during 5 min at 10 000g and the supernatant removed. After the addition of an identical volume of 1 M NaOH solution, the gel was again centrifuged. This step was repeated four times to remove completely bromide ions. Two milliliters of HEPES buffer was finally added to the precipitate at the desired concentration. The quasiquantitative removal of bromide counterions from the PLL solution was checked by the absence of turbidity at the addition of 1 M AgNO3. Preparation of CF- and AgNO3-Liposomes. Unilamellar phospholipid vesicles filled with CF or AgNO3 solutions were prepared by the ultrasonication method. Briefly, 2 mg of CL was added to the lipid solution in chloroform containing 12 mg of DPPC, 4 mg of DMPC, and 2 mg of DPPG, with DPPG previously dissolved in chloroform:methanol (1:1 v/v) solution. The ratio of the components DPPC/DMPC/DPPG/CL was 60/20/10/10 w/w. The final mixture (4-5 mL) was placed in 100 mL round-bottom flask. The solvent was removed by rotary evaporation at 35 °C and intensive rotation of 200 rpm under a pressure of 300 mbar during 20 min and then overnight at 17 mbar at room temperature. The thin lipid film was hydrated at 55 °C with 4 mL of 1 M AgNO3 solution at a desired

Langmuir, Vol. 24, No. 18, 2008 10211 concentration in HEPES or a similar volume of 0.2 mg/mL CF in HEPES solution. Then, the lipid suspension was transferred in a glass tube and sonicated during 90 min (40 W, 50% impulse time) by Vibra-Cell VC 600 (Sonics & Materials). The glass tube was put in a water bath to maintain the temperature in the range of 50-65 °C. The temperature of the lipid suspension was measured from time to time during the procedure. The titanium particles originating from the sonicator tip were removed by centrifugation. One milliliter of slightly turbid suspension of vesicles was subjected to a 1 × 20 cm2 glass column filled with Sephadex G-75 (gel volume 12 mL), previously equilibrated with HEPES. The fraction of the collected liposomes was stored at 4 °C. Dynamic Light Scattering and Electrophoretic Mobility. The average hydrodynamic diameter of the vesicles and their electrophoretic mobility were respectively determined by dynamic light scattering (DLS) and laser Doppler electrophoresis. These experiments were carried out at 25 °C using a Malvern Zetasizer 4 apparatus (Malvern Instruments). The ζ-potential was calculated from the measured electrophoretic mobility using the Smolukovski equation. CF- and AgNO3-containing liposomes are respectively denoted CF-liposomes and AgNO3-liposomes. PLL/HA Multilayer Films. The buildup of PLL/HA multilayer films by the layer-by-layer technique consisted of sequential dipping during 10 min in the polycation (PLL) and in the polyanion (HA) solutions with intermediate washing steps with HEPES by means of a dipping robot (Riegler & Kirstein GmbH). PLL samples were previously purified from bromide ions as described above. Before use, the polyelectrolyte solutions prepared at 0.5 mg/mL were filtrated through a 0.22 µm filter. PLL/HA films were built on 12 mm glass slides. Glass slides were cleaned by successive incubations at 60 °C in 2% (w/v) Hellmanex (Hellma GmbH), 0.01 M sodium dodecyl sulfate, and 0.1 M HCl during 15 min for each solution followed by multiple rinsing with pure Milli-Q water. The obtained structure is denoted (PLL/HA)20 for a film with 20 PLL/HA pairs of layers and has a thickness of about 5 µm.72 Preparation of AgNO3 or CF Coatings. First AgNO3-liposome aggregates were prepared by injecting 2 mL of AgNO3-liposome solution into 2 mL of 0.1 mg/mL PLL solution. The formed suspension was centrifuged during 2-3 min at 1000g, and the supernatant was washed twice with HEPES to remove the excess of PLL and finally resuspended in 300 µL of the same buffer to obtain AgNO3-liposome aggregates. The concentration of liposomes was chosen to have a final molar ratio of PLL amino groups/DPPG equal to 2. THe AgNO3 coating was prepared as follows: 70 µL of AgNO3-liposome aggregates was deposited on a (PLL/HA)20 film, built on a glass slide, and dried 2 h under the laminar flow of a hood. To tune the AgNO3 content of the coating, AgNO3-liposome aggregate solution was diluted at a desired concentration and adsorbed on the film. Additional HA/PLL/HA layers were deposited on the architecture by alternating immersion of the glass slide in HA and PLL solutions separated by rinsing steps. The total amount of AgNO3 in the formed coating was determined by gravimetric analysis of silver ion release after film dissolution (see “Release of Silver Ions from AgNO3 Coatings” in the Experimental Section). The final architecture (PLL/HA)20-AgNO3-liposome aggregates-HA/PLL/ HA, called AgNO3 coating, was stored at 4 °C in HEPES solution. In order to prepare CF coatings, the same protocol was used with CF-liposomes. Loading CF in CF Coatings. The loading of CF coatings was achieved by using the liposomes filled with CF. To determine the loading, the fluorescence intensity of the supernatant was measured after the complete dissolution of the CF coating. For this procedure, a first treatment was applied to obtain a complete removal of the polyelectrolyte multilayer film after deprotonation of PLL: 200 µL of a 0.05 M NaOH solution was deposited during 5 min on the CF coating. Then, a treatment was applied to reach the decomposition of the lipid bilayer: 1 mL of Tris buffer solution (0.5 M Tris, 0.25 M NaCl, pH 8.3) with 1% of Triton X100 was added to the supernatant and the obtained mixture was heated to 75 °C for 20 min and then (72) Francius, G.; Hemmerle, J.; Ball, V.; Lavalle, P.; Picart, C.; Voegel, J. C.; Schaaf, P.; Senger, B. J. Phys. Chem. B 2007, 111, 8299–8306.

10212 Langmuir, Vol. 24, No. 18, 2008 cooled to room temperature. The fluorescence intensity of CF was then measured with a VersaFluor fluorometer (BioRad) with an excitation wavelength at 490 nm and the emission was measured at 520 nm. A calibration curve of CF in the medium used for the destruction of the coating was then established. We assumed that all vesicles prepared were aggregated with PLL before deposition on the film, yielding the lipid content. The results are presented as the average value of at least three independent experiments with their standard deviation. Release of Silver Ions from AgNO3 Coatings. The release of silver ions from the AgNO3 coating was studied at room temperature and at 37 °C by the determination of the silver ions present in the supernatant of the coating. Gravimetrical analysis was performed on AgNO3 coatings prepared on 30 mm spherical cover glasses with 1 mL of supernatant. To this aim, 0.8 mL of the liposome aggregates (see paragraph above) were placed on 30 mm cover glasses previously covered with (PLL/HA)20 film and dried under a hood. The dry samples were hydrated with 1 mL of HEPES and stored at room temperature or 37 °C. At defined time intervals, 200 µL aliquots were withdrawn and mixed with the same volume of 10-3 M NaI. After washing with water and centrifugation, the AgI precipitates were placed on weighed 12 mm spherical microscopy cover glasses and calcined at 300 °C until a constant weight. Then the amount of AgNO3 released was calculated at each time interval. One hundred percent of released AgNO3 corresponds to the total amount of silver ions deposited on the glass slide followed by the drying and rehydration procedure. To determine this value, the film was dissolved by addition of 1 mL of Tris buffer solution (0.5 M Tris, 0.25 M NaCl, pH 8.3) with 1% of Triton X100 followed by heating to 75 °C for 20 min. Then gravimetric analysis was done as described above. Three samples were used to determine the standard deviation. Minimal Inhibitory Concentration (MIC) Determination of AgNO3 in Solution. Gram-negative bacteria, E. coli strain CIP 53 126, were used as a model system and were cultivated on tryptic soy agar (TSA) medium and incubated at 37 °C. The cell suspension was diluted with HEPES buffer/Muller Hinton broth (Biokar Diagnostics, BK108HA) (10:2 v/v) to obtain a final bacteria concentration in the range of (1-3) × 106 colony-forming units (cfu)/mL. The minimal inhibitory concentration (MIC) is defined as the lowest concentration of an antimicrobial drug that inhibits the visible growth of a microorganism after overnight incubation. One milliliter of the prepared bacterial suspension was mixed with the same volume of AgNO3 solution at different concentrations prepared by consecutive dilutions by a factor 2 starting from a concentration of 10-2 M. All tubes were incubated at 37 °C for 20 h. The last tube remaining transparent in the determined range is defined as the MIC. Antibacterial Activity Test of AgNO3 in Solution and AgNO3 Coatings. The antibacterial activity of AgNO3 in solution and in AgNO3 coatings were determined by following the evolution of the number of viable E. coli cells with time. E. coli cell suspension was prepared as described above with a final concentration of bacteria in the range (1-3) × 106 cfu/mL. One milliliter of this bacterial suspension was placed in 24-well culture plates containing either AgNO3 solution at a given concentration or a glass covered by the AgNO3 coating with the desired content in silver ions. These plates were incubated at 37 °C and rotated at 100 rpm (Aerotron INFORS AGCH-4103). At defined time intervals, the aliquots (50 µL) of the cells were withdrawn and consecutively diluted (10×) with Muller Hinton broth. Viable bacteria were determined by plating 100 µL of each dilution on TSA medium and counting the developed colonies after incubation at 37 °C during 20 h. All experiments were done in triplicate and are presented with standard deviation.

Results and Discussion Bactericidal Property of AgNO3 in Solution. As a first step, the MIC of AgNO3 in solution was determined for E. coli. In our case, MIC is equal to 22 µg/mL (1.3 × 10-4 mol/L). The MIC of AgNO3 was reported as 2.3573 or 3.9 µg/mL74 for E. coli (73) Kong, H.; Jang, J. Langmuir 2008, 24, 2051–2056.

Malcher et al.

Figure 1. Evolution of the number of viable E. coli cells versus time in the presence of AgNO3 solutions. Bacteria were put into contact with AgNO3 solutions at a concentration of (O) 6 MIC, (b) 150 MIC, (∆) 300 MIC, and (2) 450 MIC. The initial concentration of bacteria was around 106 cfu/mL. The MIC of AgNO3 in solution was equal to 22 µg/mL.

cells cultivated in Luria-Bertani broth. The MIC of silver ions is typically found in the range of 5-40 µg/mL,75 depending on the local environment, i.e. the growth media. The use of Muller Hinton broth could explain the 10-fold increase of the MIC of AgNO3 compared to the value found for Luria-Bertani broth. In a second step, the kinetics of bacteria growth in AgNO3 solutions at various concentrations was studied (Figure 1). For incubation times up to 2 h, a strong decrease of the viable E. coli cells is always observed for the whole range of explored silver ion concentration from 6 to 450 MIC. At 300 and 450 MIC of AgNO3 in solution, a 5-log reduction of the bacteria number was found respectively for an incubation time of 75 and 15 min. For the more concentrated AgNO3 solution, i.e. 450 MIC, the antibacterial effect of silver ions becomes very efficient in terms of time of contact between bacteria and silver ions. Liposomes Characterization. The first step to build a liposome-based biocoating is to determine the right lipid composition of the vesicles. Liposomes were prepared with a phase transition temperature close to 37 °C since this temperature corresponds to the optimal temperature for growth of many bacteria. This is also physiological temperature. DPPC/DPPG/ CL vesicles (80/10/10 w/w) characterized in a previous study63 were compared to DPPC/DMPC/DPPG/CL vesicles (60/20/10/ 10 w/w). DPPG provides a negative charge for the vesicles, enabling their complexation with polycations (PLL) by means of electrostatic interactions, and CL provides rigidity to the vesicles. DPPC/DPPG/CL vesicles do not release their cargo for a long time (up to 1 week or more) if the temperature is kept below the transition temperature, determined to be 41 °C. Addition of a lipid having a lower transition temperature than this former composition should decrease the transition temperature of the final mixture. DMPC is a perfect candidate, as it is a saturated lipid like DPPC with a shorter carbon chain and it has a transition temperature of 23 °C. Taking into account that the addition of a new lipid to the lipid mixture can decrease the transition temperature proportionally to its content,76 the final DPPC/DMPC/DPPG/CL ratio of 60/20/10/10 w/w should lead to a transition temperature of about 37 °C. The respective transition (74) Keleher, J.; Bashant, J.; Heldt, N.; Johnson, L.; Li, Y. Z. World J. Microbiol. Biotechnol. 2002, 18, 133–139. (75) Burrell, R. E. Ostomy Wound Manage. 2003, 49, 19–24. (76) Taylor, K.; Craig, D. In Liposomes: A Practical Approach, 2nd ed.; Torchilin, V., Weissig, V., Eds.; Oxford University Press: Boston, MA, 2003; pp 79-103..

Embedded SilVer Ions-Containing Liposomes

Figure 2. Mean count rate, measured by DLS, as a function of the temperature for (b) DPPC/DMPC/DPPG/CL (60/20/10/10 w/w) and (O) DPPC/DPPG/CL (80/10/10 w/w) liposomes. The phase transition temperature, corresponding to the inflection point of the curves, is indicated with vertical dashed lines.

temperatures of the prepared DPPC/DPPG/CL and DPPC/DMPC/ DPPG/CL were determined by DLS measurements (Figure 2). The method is based on the modification of the optical properties of aqueous surfactant solutions upon phase transition in the phospholipid bilayer membrane.62,77 The mean count rate (the average number of detected photons per second), i.e. light scattering of the vesicles, was found to change dramatically when the transition temperature is reached. For DPPC/DPPG/CL vesicles, the curve of mean count rate versus temperature decreases when the temperature was set above 40 °C. This result agrees with previous data where the transition temperature was found to be close to 41 °C for such vesicles.78 DPPC/DMPC/ DPPG/CL vesicles differ from DPPC/DPPG/CL ones just by substituting 20% of DPPC by DMPC. Due to this fact, we can suppose that the DPPC/DMPC/DPPG/CL vesicles should behave similarly, but with a reduced transition temperature that can be detected correctly by DLS. The shape of the curve allows effective determination of the phase transition temperature for these vesicles. It lies around 34 °C and is close to the expected one (37 °C). Liposome Aggregation with PLL. In the present study, we investigate the complexation of DPPC/DMPC/DPPG/CL vesicles by the cationic polypeptide PLL. The complexation profile has a cupola-like shape and shows clearly a strong liposome aggregation over a wide range of PLL amino group/DPPG molar ratio of 0.2-10 (Supporting Information, Figure S-1). When the charge of the PLL coating is almost neutralized by the liposome surface charge (i.e., the isoelectric point), the PLL-coated particles have a strong tendency to form aggregates. The reason for liposome aggregation may be a nonhomogeneous overcompensation of the surface charge of the liposomes by the PLL molecules. The DPPC/DMPC/DPPG/CL vesicle complexation profile is wider than that of DPPC/DPPG/CL vesicles for the same content of charged DPPG. Indeed, their aggregation has been observed at a ratio of 0.4-1.5.62-64 The stronger aggregation of DPPC/DMPC/DPPG/CL vesicles could be due to the smaller size of the uncoated vesicles compared to uncoated DPPC/DPPG/ CL vesicles, respectively 83 ( 5 and 129 ( 2 nm. Bridge aggregation has a significant effect on the aggregation of small particles when the size of the bridging agent (in this case PLL) is comparable to the size of the vesicles. DPPC/DMPC/DPPG/ CL vesicle aggregates at a molar PLL amino group/DPPG ratio (77) Michel, N.; Fabiano, A. S.; Polidori, A.; Jack, R.; Pucci, B. Chem. Phys. Lipids 2006, 139, 11–19. (78) Lipid Data Bank, www.lipidat.chemistry.ohio-state.edu.

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equal to 2 were selected for the preparation of AgNO3 coatings [i.e., their deposition on (PLL/HA)20 polyelectrolyte films] because they can contain large amounts of encapsulated silver ions and are positively charged. Indeed, such aggregates have a diameter of about 1 µm (Supporting Information, Figure S-1) and a ζ-potential of 32 ( 4 mV. The positive surface charges could provide electrostatic bonding of the aggregates to the negatively charged (PLL/HA)20 multilayers. The stability of aggregates of vesicles is very important for this study, since the vesicles should not release the cargo till the release is needed. We have demonstrated in a previous study that PLL-covered liposomes are able to keep not only the encapsulated dyes, like carboxyfluorescein,63 but also small ions, like ferrocyanide ions.60 In this last case, we demonstrated (by means of cyclic voltametry) that the spontaneous release from the internal lumen of the vesicles is pretty slow as it extends over more than 12 h. However, ferrocyanide ions should be considered as less favorable ions for long-term encapsulation in vesicles. We have also encapsulated calcium ions in vesicles and showed that upon PLL coating no significant calcium release was found after long-term storage. Indeed, at least part of these calcium ions were used to induce the precipitation of calcium phosphates inside of the vesicles which also contained an enzyme, alkaline phosphatase. This triggered the production of phosphate anions upon permeation of an amphiphilic phosphate-containing substrate through the polyelectrolyte multilayer film as well as through the lipid bilayer.79 The group of Kabanov has also demonstrated, using conductometry, that sodium chloride encapsulated in the vesicles is not released from the internal lumen when poly(4-vinylpyridine) is adsorbed on the surface of the vesicles.71 However, the adsorption of quaternized poly(4-vinylpyridine) induced some ion leakage from the vesicles, showing that the incorporation of aliphatic chains from the polycation induces some disorder in the lipid bilayer. PLL is devoid of hydrophobic side chains, and we previously demonstrated that it does not change the main phase transition temperature of DPPC/DPPG-containing vesicles.63 In this study, we have shown that PLL adsorption does not induce release of encapsulated silver ions from the internal lumen of the vesicles upon aggregation. This was checked by mixing the supernatant of aggregated vesicles with 0.15 M NaCl solution, which did not induce any visible precipitation of AgCl, which should be observed in the supernatant once aggregated vesicles would release encapsulated AgNO3. Fabrication of AgNO3 and CF Coatings. AgNO3-liposomes were obtained by hydration of the thin lipid film with 4 mL of AgNO3 solution of 1 M (170 mg/mL). After the formation of AgNO3-liposome aggregates by addition of PLL, AgNO3 coatings were designed on a first step by deposition of AgNO3-liposome aggregates diluted at a given concentration on (PLL/HA)20 multilayers. After water evaporation at room temperature, the deposited vesicles formed a thin micron-sized coating and were maintained at the surface by adding HA/PLL/ HA capping layers (Scheme 1). Additional HA/PLL/HA layers were deposited on the architecture by alternated immersion of the glass slide in HA and PLL solutions separated by rinsing steps. The amount of silver ions in the final coating was found by film destruction followed by gravimetric analysis of silver ions (see Experimental Section). To evaluate the effect of (PLL/HA)20 matrix on the stability of the liposome aggregates deposited on it, CF coatings were realized using the same procedure as with Ag-liposome (79) Michel, M.; Arntz, Y.; Fleith, G.; Toquant, J.; Haikel, Y.; Voegel, J.-C.; Schaaf, P.; Ball, V. Langmuir 2006, 22, 2358–2364.

10214 Langmuir, Vol. 24, No. 18, 2008

Malcher et al. Scheme 1. Design of AgNO3 Coatingsa

a (a f b) Liposomes complexation with PLL resulted in the formation of AgNO3-liposome aggregates. (b f c) Deposition of AgNO3-liposome aggregates on (PLL/HA)20 film followed by additional coating with HA/PLL/HA layers.

aggregates. After the dissolution of the coating, the fluorescence intensity of the supernatant was measured to determine the CF quantity embedded into CF coatings. It was found that 56 ( 12% of the initially deposited CF remains in the film. This suggests that part of the vesicles lost their integrity upon drying, probably those located on the surface because of water evaporation. However, a large fraction of the vesicles that are more deeply embedded in the aggregates are stabilized by PLL and the underlying hydrated PLL/HA film. This stabilization is confirmed by the fact that when the CF-liposome aggregates are deposited on a bare glass substrate, only 15 ( 4% of the initially deposited CF-liposomes remains on the surface. The hydrogel-like PLL/ HA film plays the role of a spacer, which not only provides a negatively charged surface for the attachment of positively charged vesicles but also contains a quite high amount of water, rendering the surface soft enough to bind the aggregates to the surface. Moreover, in the scope of the design of a transient bactericidal coating especially for implants, PLL/HA film constitutes a very good candidate. It is known to be nonadhesive for numerous cell types.80-82 This property could be very useful to limit or inhibit cellular adhesion in addition to the bactericidal effect of the coating. The film loading capacity was also investigated. The loading of the film was tuned by using CF-liposome aggregate suspensions diluted at different concentrations, deposited on (PLL/ HA)20 multilayers, and capped by HA/PLL/HA layers. To evaluate the film loading, CF coatings were dissolved, and the fluorescence of CF in the supernatant was measured. Figure 3 represents the fluorescence intensity of the supernatant after the dissolution of the CF coating. CF encapsulated in the film increases linearly with the concentration of deposited suspension of the CF-liposome aggregates. Triggered Release of AgNO3. DPPC/DMPC/DPPG/CL liposomes were chosen to build AgNO3-liposome aggregates because of their phase transition temperature close to 37 °C, optimal for the growth of numerous bacteria. Upon crossing the transition temperature due to the increased lipid mobility, the permeabilization of either single liposome or liposome aggregates (80) Richert, L.; Schneider, A.; Vautier, D.; Vodouhe, C.; Jessel, N.; Payan, E.; Schaaf, P.; Voegel, J.-C.; Picart, C. Cell Biochem. Biophys. 2006, 44, 273– 285. (81) Richert, L.; Boulmedais, F.; Lavalle, P.; Mutterer, J.; Ferreux, E.; Decher, G.; Schaaf, P.; Voegel, J.-C.; Picart, C. Biomacromolecules 2004, 5, 284–294. (82) Mhamdi, L.; Picart, C.; Lagneau, C.; Othmane, A.; Grosgogeat, B.; Jaffrezic-Renault, N.; Ponsonnet, L. Mater. Sci. Eng. C 2006, 26, 273–281.

Figure 3. CF encapsulated in CF coatings versus the lipid content deposited on the film. CF fluorescence was measured in the supernatant after the decomposition of CF coating, and the lipid content is assumed to be equal to the amount adsorbed. The dotted line is a linear fit going through zero. The error bars correspond to the standard deviation of three different samples.

Figure 4. Release of silver ions from AgNO3 coating versus time at (b) room temperature and (O) 37 °C. Silver ions content in the supernatant was determined by gravimetrical analysis.

is dramatically increased. The PLL coating does not induce any changes in the behavior of “solid” liposomes; however, it increases the permeability of “fluid” liposomes.63 The release of silver ions entrapped in AgNO3 coatings was studied by gravimetry analysis of the supernatant (Figure 4). At defined time intervals, aliquots were withdrawn from the supernatant and mixed with a NaI solution to form AgI precipitates. The precipitates are then

Embedded SilVer Ions-Containing Liposomes

Figure 5. Number of viable E. coli cells after an incubation of 20 h at 37 °C versus AgNO3 concentration encapsulated into AgNO3 coating. The initial concentration of bacteria put into contact with AgNO3 coatings was around 106 cfu/mL. The AgNO3 content is expressed as the amount of AgNO3 encapsulated per cm2.

weighted and calcinated till a constant weight to determine the Ag+ amount released by the AgNO3 coating. No silver ions were detected in the supernatant up to 3 h at room temperature. When the AgNO3 coating is stored at 37 °C, the situation becomes strongly different. After 30 min of incubation, 11 ( 6% of the silver ions encapsulated in the coating is released. More than 54 ( 17% of the entrapped AgNO3 is released within 90 min, indicating a continuous release of ions. At 37 °C, the AgNO3 coating loses about 100% of its content within 15 h. This quite fast release of Ag+ ions could have a very efficient antibacterial property. However, this could also be a weak point of the coating concerning its lifetime. A slower release of Ag+ ions could give a longer lifetime but a weaker efficiency against bacteria. In the present study, we were interested in validating the use of this coating as an antimicrobial treatment of surfaces. Future studies will deal with tuning the release of silver ions. The storage of AgNO3 coating was also followed. The coating is quite stable upon storage up to 1 day; however, long-term storage leads to a partial leaking of the active compound. Indeed, after 5 days at 4 °C, the coating keeps still 32% of the initially entrapped AgNO3. This loss of material could be due to a spontaneous release of AgNO3 or erosion of AgNO3-liposome aggregates. The vesicle aggregates used in this study (about 1 µm in diameter) have an excess of charge and a strain in the structure that can destabilize the lipid membrane domains, increasing the membrane permeability. Antibacterial Activity of AgNO3 Coating. The antibacterial activity of AgNO3 coatings was tested against E. coli with different content in AgNO3. As a control, (PLL/HA)20 films previously dipped in 0.1 M AgNO3 were put into contact with an E. coli bacterial suspension. Such films had no antibacterial activity. Moreover, PLL/ HA films prepared with liposome aggregates without AgNO3 have also no antibacterial activity. Figure 5 presents the concentration of viable E. coli cells in contact for 20 h with various AgNO3 coatings. A very small effect is found up to 11 ng/cm2. Then, the viability decreases strongly with complete bacteria death for concentrations above 42 ng/cm2. For AgNO3 coatings with 21 ng/cm2 AgNO3, a 5-log reduction of the bacteria number is observed compared to a coating with no AgNO3 encapsulated. Liposomes represent perfect containers for small solutes due to (i) their low lipid membrane permeability, (ii) a possible temperature control of their permeability, and (iii) the possibility to embed a large amount of active compounds in a controlled manner. In a further step, the decrease of viable bacteria with time was followed in contact with a highly charged coating of 120 ng/cm2 during the time.

Langmuir, Vol. 24, No. 18, 2008 10215

Figure 6. Evolution of the number of viable E. coli cells as a function of the incubation time in contact with AgNO3 coating (120 ng/cm2 in AgNO3).

Kinetics of Bacteria Growth in the Presence of AgNO3 Coating: Typical Decay Kinetics. The decay kinetics with a 120 ng/cm2 AgNO3 coating was monitored over about 2.5 h (Figure 6). After 2 h of incubation, a 4-log reduction of the number of viable bacteria was observed. A nearly complete bacteria death is observed after 2.5 h of contact with the AgNO3 coating. The progressive release of AgNO3 through the liposome walls induces a high local concentration on the surface of the coating in the neighborhood of bacteria. Ag+ ions react with the cell membranes of bacteria and disrupt crucial metabolic proteins and enzymes, leading to cell death. The present strategy is also to be compared with multilayers prepared with embedded silver nanoparticles in the poly(acrylic acid)/quaternized poly(ethylene imine) system.45 The antimicrobial polyelectrolyte multilayers, obtained by in situ wet phase reduction of Ag+ ions into silver nanoparticle, can release silver ions into the surroundings as was shown by the Kirby-Bauer test. These coatings show an about 2-log reduction of the number of viable E. coli cells after 2 h. Thus the AgNO3-liposome aggregate based system has a fast and strong performance against bacteria growth compared to other multilayer coatings.

Conclusions Liposome aggregates filled with AgNO3 solution leading to AgNO3 coating were prepared with a temperature control of silver ions in the supernatant. The AgNO3 coatings can be embedded in PLL/HA films. The architecture showed a strong and rapid antibacterial effect against E. coli. A contact of 120 min with an AgNO3 coating (with 120 ng/cm2 AgNO3 concentration) induces a 4-log reduction of the bacterial population. PLL/HA films could not be directly loaded with noticeable amounts of silver ions to induce bacterial death. The AgNO3 coating represents a solvent-based antibacterial system through release of bactericidal agent from the film under external stimuli. In the next coming steps, we foresee performing experiments aimed to prepare silver ions containing coatings with increased stability and with control of release over a longer period of time. Acknowledgment. M.M. was supported by a doctoral fellowship of the Ministe`re de l’Enseignement Supe´rieur et de la Recherche and the Polish Ministry of Education grant no. 3T08D03330. D.V. was supported by the Ministe`re de l’Enseignement Supe´rieur et de la Recherche and by the EU6 project BIOCOATING. Supporting Information Available: Figure S-1 showing the average diameter of DPPC/DMPC/DPPG/CL liposomes/PLL complexes formed as a function of the molar PLL amino group/DPPG ratio. This information is available free of charge via the Internet at http://pubs.acs.org. LA8014755