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The layer-by-layer self-assembly. The powerful technique for drug delivery applications.

The layer-by-layer self-assembly. The powerful technique for drug delivery applications. D. Volodkin 1 , A. Skirtach 1 , G. Sukhorukov 3 , J-C. Voegel 2 , V. Ball 2 , H. Möhwald 1 1 - Max-Planck Institute for Colloids and Interfaces, D-14424 Potsdam, Germany;

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The layer-by-layer self-assembly. The powerful technique for drug delivery applications.

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  1. The layer-by-layer self-assembly. The powerful technique for drug delivery applications. D. Volodkin1, A. Skirtach1, G. Sukhorukov3, J-C. Voegel2, V. Ball2, H. Möhwald1 1 - Max-Planck Institute for Colloids and Interfaces, D-14424 Potsdam, Germany; 2 - INSERM U-595, ULP, Medical Dep., 11 rue Humann, 67085 Strasbourg, France; 3 - Queen Mary University, London, England. NanoMed 2009, 4-6 March 2009

  2. OUTLINE • 1. The principle of the polyelectrolyte Layer-by-Layer (LbL) assembly. • 2. LbL films for bio-applications. • 3. LbL planar films for drug delivery. • a). Liposome-containing films. Design and stability. • b). Externally stimulated (remote) release by IR-light. • 4. LbL free-standing films (microcapsules) as drug delivery vehicles. • a). CaCO3-templated gel-like microcapsules. • b). Intracellular drug remote release by IR-light . • - Conclusions Core dissolution

  3. - - Strong polyelectrolytes Weak polyelectrolytes Chitosan Poly(styrene sulfonate) Polyacrylic acid Polylysine Polydiallyl Dimethyl Ammonium Chloride Hyaluronic acid Polyallylamine - - - - - - - + + + + + + + + + + 1. The principleofthepolyelectrolyte Layer-by-Layer (LbL) assembly. G. Decher, Science, 1997, 277, 1232. Exponential regime Linear regime 1 layers 100-200 nm in average 1 layers 1-2 nm Low polymer mobility High polymer mobility

  4. 1. The principleofthepolyelectrolyte Layer-by-Layer (LbL) assembly. Advantages of the LbL films • Tune thickness to nanometre scale (number of layers) • Formation on any surface (complex geometries, miniaturized) • Mild conditions to prepare the films • Automized (dipping robots, spraying, spin coating) G. Decher, Science, 1997, 277, 1232. K. Ariga et al. Phys. Chem. Chem. Phys., 2007, 9, 2319. Z. Tang et al. Adv. Mater., 2006, 18, 3203.

  5. 2. LbLfilmsfor bio-applications. Biocompatiblepolymers - hualyronicacid, chitosan, chondroitinsulfate, heparin, etc Bioactivefilms - DNA, proteins, peptides, growthfactors, etc BIOAPPLICATIONS Cellinteractionwiththefilms - Celladhesiveandresistantfilms - Cell-induceddegradation Drug releasecarriers - releaseinducedbychange in pH, ionicstrength, T, self-degradation Ai, H. et al Cell Biochem. Biophys.2003,39, 23. Tang, Z et al Adv Mater2006,18, 3203. Ariga, K. et al, Macromol. Biosci. 2008, 8, 981.

  6. Biomedical device (implant) 3. LbL planar films for drug delivery. 1. Tune mechanical properties of surface. 2. Controll interaction with blood, cells, etc. 3. Films as drug reservoirs. Delivery to a target (tissue) LbL film loaded with drugs (pharmaceuticals, antibiotics, growth factors, etc) Highly efficient site-specific drug delivery

  7. NH2 Biomedical device (implant) 3. LbL planar films for drug delivery. Liposome-containing films. Design and stability. Liposomes – reservoirs for drug molecuels 129 nm, controlled permeability, biocompatibility HA - hyaluronic acid PLL - polylysine (PLL/HA)24 ~ 7 µm - Homogeneous - Stable (physiological conditions) - 70-80% water (hydrogel) Liposomes: DPPC/DPPG/Cholesterol, 80-60/10-30/10 % 10 mM TRIS-buffer, pH 7.4, 15 mM NaCL PLL – 28 kDa, HA – 400 kDa Volodkin D.V. et al (2008) Soft Matter, 4, 122.

  8. 3. LbL planar films for drug delivery. Liposome-containing films. Design and stability. Supported lipid bilayer I. Reviakine, Langmuir, 2000, 16, 1806. R. P. Richter, R Langmuir, 2006, 22, 3497. PLL coating (stabilization) Volodkin D.V. et al (2007) Biochim. Biophys. Acta., 1768, 280. Volodkin D.V. et al (2007) J. Control. Release, 117, 111. Volodkin, D.V. et al (2007)Colloids Surf. A., 303, 89.

  9. Lip10(30) – 10(30)% of DPPG Lipid-polymer and interpolymer interactions. A crucial role for vesicle embedding. poly-(sodium 4-styrenesulfonate) PSS + Polyanion hyaluronic acid HA poly-L-glutamic acid PGA C A B Lip10 + PSS Lip30 + PSS Lip10 + PGA Lip30 + PGA + Polyanion Lip10 + HA Lip30 + HA Interaction enthalpies (microcalorimetry) Liposome  surface PLL  polyanion in the film 12 1 stronger that 2 embedding OK (B, C) 2 stronger that 1 embedding failed (A) Volodkin D.V. et al (2009) Soft Matter, DOI 10.1039/b815048f.

  10. Lip30 Normalized fluorescence, a.u. Lip10 Atomic Force Microscopy (PLL/HA)12/Lip-PLL/HA/PLL/HA Number of liposome «interlayers» Lip in the film 45oC Lip in solution CF released, % 311±50 nm Lip in the film 21±6 nm 25oC Time, min 3. LbL planar films for drug delivery. Liposome-containing films. Design and stability. Liposome amount in the film is adjusted by Lip „interlayer“ number and Lip charge Liposomes are immersed in the film Volodkin D.V. et al (2008) Soft Matter, 4, 122. Volodkin D.V. et al (2008) Adv. Planar Lipid Bilayers and Liposomes, 8, pp 1-25.

  11. Number of bacteria, cfu/ml AgNO3 release at 37oC, % AgNO3, M Time, min AgNO3 solution Film Liposome-containing films as antibacterial coatings Up to 1 µg of AgNO3 per cm2 1M AgNO3 Ttrans. = 35oC E. coli Population changes versus AgNO3 concentration for AgNO3 in solution and AgNO3 in the film. T=37oC. Initial bacteria concentration ~106 cfu/ml. Malcher M. et al, Langmuir (2008), 24, 10209.

  12. 3. LbL planar films for drug delivery. Externally stimulated (remote) release by IR-light. Liposomes are sensitive The film is sensitive Gold nanoparticles (absorb light) Light irradiation = Energy absorption: heat + photoluminescence

  13. Abs. 520 nm , nm Normalized scattered intensity Abs. Normalized scattered intensity 650 nm Diameter / nm Diameter / nm , nm Liposome-GoldNanoparticle assemblies. Light response. near IR-light „Biological window“ 700-900 nm Volodkin D.V. et al (2009) Angew. Chem. Int. Ed., 48, 1807.

  14. Liposome-Gold Nanoparticle complexes. Light response. Light Cargo release Light =800 nm Green - caroxyfluorescein Volodkin D.V. et al (2009) Angew. Chem. Int. Ed., 48, 1807.

  15. 3. LbL planar films for drug delivery. Externally stimulated (remote) release by IR-light. Liposomes are sensitive The film is sensitive Gold nanoparticles (absorb light) Light irradiation = Energy absorption: heat + photoluminescence

  16. Surface-mediated remote DNA transfection by IR-light stimulation Collaboration with Dr. C. Duschl, Dr. A. Lankenau; IBMT, Golm Wissenschaftspark. The idea IR-light transfection (remote, to single cell) Cell Cell DNA „layer“ (up to 10 µm) Gold nanoparticles (PLL/HA)x

  17. Surface-mediated remote DNA transfection by IR-light stimulation Collaboration with Dr. C. Duschl, Dr. A. Lankenau; IBMT, Golm Wissenschaftspark. film (green) red (DNA) 3T3 cells spread on the film surface Gold nanoparticle aggregates IR-light

  18. 4. LbL free-standing films (microcapsules) as drug delivery vehicles. Core dissolution LbL Sacrificial template (core) LbL coated template Microcapsule Sukhorukov, G. B. atal.Colloid Surf A-Physicochem Eng Asp. 1998, 137, 253

  19. CaCO3-templated gel-like microcapsules. LbL Gel-likecapsule (bead) EDTA (PAH/PSS)3 Molar ratio PAH:PSS= 1.34±0.08 CaCO3 microparticles PAH – poly(allylaminehydrochloride) PSS – polystyrenesulfonate Volodkin D. V., et al, Langmuir, 2004, 20 (8), 3398. Sukhorukov G.B. et al, J. Mater. Chem., 2004, 14, 2073. Confocal fluorescent microscopy Raman confocal microscopy

  20. - Particlediameter:2- 20mkm - Pore diameter: 20 - 80 nm - Surfacearea:13  7 m2/g (Brunauer–Emmett–Teller method) Na2CO3 + CaCl2 20 m 2 m CaCO3-templated gel-like microcapsules. • Spherical and homogeneously sized (vaterite form, no calcite) • Biocompatible, inexpensive • Non-aggregated, easily decomposable (EDTA, pH 7.0 or pH < 4-5) Volodkin D. V., Petrov A. I., Prevot M., Sukhorukov G. B., Langmuir, 2004, 20 (8), 3398.

  21. 5 m Passive and active ways to encapsulate proteins in the capsules. passive High protein capacity (300 mg/ml) due to large internal surface Protein Protein EDTA LbL Na2CO3 + CaCl2 + Protein Co-synthesis active (PAH/PSS)3 with Lactalbumine-rhodamine Volodkin D.V. et al. Biomacromolecules, 2004, 5, 1962 Petrov A.I., Volodkin D.V. and Sukhorukov G.B. Biotech. Prog., 2005, 21(3), 918.

  22. Antigen deliery system for vaccination Biocompatible capsules (Alg/PLL)3 with Asp f2 antigen (green, from Aspergillus fumigatus) Control Asf f2 D (492 nm) IgG 1 IgG 2A Phagosytosis of microcapsules with Asp f2 Uptake by human peripheral blood monocytes Asp f2 specific antibody production induced by encapsulated antigens Markvicheva E., et al.XIIth Int. Workshop on Bioencapsulation, Vitoria-Gasteiz, September 24-26, p. 89-92.

  23. Microcapsules for remote intracellular drug release Capsules modified with gold nanoparticles Skirtach, A. et al Angew. Chem. Int. Ed. (2006), 45, 4612.

  24. Conclusions • LbL technique is a powerful approach to make surface-supported films for biomedical applications. • Gel-like micrometer-sized HA/PLL films can serve as matrix for liposomes embedding to provide T-triggered release. • Both HA/PLL films and liposomes could be modified with gold nanoparticles to control drug release by a near-IR light stimulation. • Antibacterial activity if HA/PLL films with liposomes filled with AgNO3 is demonstrated. • Gel-like microcapsules are made by templating on sacrificial CaCO3 porous particles by LbL deposition. Highly developed surface allows protein loading up to 30% w/w. The capsules could be used for drug delivery (e.g. vaccination) by various administration routes dictated by capsule size.

  25. ACKNOLEDGEMENT Prof. Helmuth Möhwald(MPI KGF, Germany) Prof. Sukhorukov G.B. (Queen Mary University, London, England) Prof. Jean-Claude Voegel(INSERM, France) Prof. Vincent Ball (INSERM, France) Prof. Pierre Schaaf(ICS, CNRS, France) Dr. Rumen Krastev(MPI KGF, Germany) Dr. Andre Skirtach (MPI KGF, Germany) Dr. YouriArntz(INSERM, France) Dr. FouziaBoulmedais(ICS, CNRS, France) Heidi Zastrow(MPI KGF, Germany) Marta Malcher(IGL WUT, Poland) • Support • EU6 project BIOCOATING (Marie-Curie fellowship), • - DAAD grant, number A/03/01495, • French Ministryof Education grant, • INSERM grantforyoungscientists, • SofjaKovalevskaja program of the Alexander von Humboldt Foundation. • Thankyouforattention

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