Morán Badenas, María del Carmen

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PREPARACIÓ DE PARTÍCULES GEL D´ADN COM A SISTEMES D´ALLIBERAMENT CONTROLAT Seminaris de Recerca de la Facultat de Farmàcia 16 d´octubre 2012 M. Carmen Morán Grup d´Interacció de Tensioactius amb Membranes Cel.lulars Departament de Fisiologia-Facultat de Farmàcia Gene delivery  • Degradation on the bloodstream by the endogenous nucleases • Is too large to cross the cellular membranes  •  TO CONCENTRATE ENCAPSULATED MATERIAL •  TO PROTECT ENCAPSULATED MATERIAL •  TO RELEASE ENCAPSULATED MATERIAL IN A CONTROLLED WAY Encapsulation OPPOSITELY CHARGED POLYELECTROLYTES (OR SURFACTANTS AND POLYELECTROLYTES) ASSOCIATIVE PHASE SEPARATION  VISCOUS LIQUID GEL, LIQUID CRYSTAL  OR PRECIPITATE Polyelectrolyte Complexation     Gelation formed at  WATER/WATER                       emulsion type interfaces      Encapsulation      Associative phase separation Aims  High content DNA reservoir   Without adding any kind of cross-linker or organic solvent    MAIN ADVANTAGES    Gelation formed at  WATER/WATER                       emulsion type interfaces     Encapsulation     Associative phase separation INTERNAL POLYELECTROLYTE (DNA) OPPOSITELY CHARGED POLYELECTROLYTES COMPLEX   Aims How To Prepare DNA Gel Particles  CATIONIC AGENT dsDNA ssDNA Reference SINGLE-TAIL SURFACTANTS CTAB x x Langmuir, 23, 6478–6481( 2007)  Biomacromolecules, 8, 3886–3892 (2007) DTAX x x Soft Matter,  8, 3200–3211 (2012) ALA, LAM x x Langmuir, 26, 10606–10613 (2010) DOUBLE-TAIL SURFACTANTS DDAB x x Soft Matter, 7, 2001–2010 (2011) POLYSACCHARIDES CH (MW) x J. Dispersion Sci. Technol., 30, 1494–1499 (2009) PROTEINS LS, PS x Langmuir, 23, 6478–6481( 2007) Langmuir, 25, 10263–10270 (2009)                                                                  REVIEW                        Soft Matter,  6, 3143-3156 (2010) Systems Phase map: ds-DNA (Salmon testes)-CTAB System   Charge Equilibration 1 Φ 2 Φ Surfactant-DNA Gel Particles LYSOZYME from chicken egg white   INTERNAL POLYELECTROLYTE (DNA) PROTEIN-POLYELECTROLYTE COMPLEX   Protein-DNA Gel Particles CHITOSAN   R=[ds-DNA]/[CH+]  INTERNAL POLYELECTROLYTE (DNA) POLYELECTROLYTE-POLYELECTROLYTE COMPLEX   Polysaccharide-DNA Gel Particles PHYSICOCHEMICAL CHARACTERIZATION DEGREE OF DNA ENTRAPMENT PARTICLE MORPHOLOGY SECONDARY STRUCTURE OF DNA SWELLING / DISSOLUTION BEHAVIOUR KINETICS OF DNA RELEASE DEGREE OF COMPLEXATION In vitro BIOCOMPATIBILITY HAEMOCOMPATIBILITY CYTOTOXICITY LOADING EFFICIENCY(%) = [(total amount of DNA)-(non-bound DNA)]     x 100               total amount of DNA Degree of DNA entrapment Spectrophotometric determination OD260   Degree of DNA Entrapment LOADING CAPACITY(%) = [(total amount of DNA)-(non-bound DNA)]     x 100                weight of particles Degree of DNA entrapment Spectrophotometric determination OD260   Degree of DNA Entrapment + 1 16 + 1 12 12 + 1 12 + 2 + 1 12 12 LOADING CAPACITY(%) = [(total amount of DNA)-(non-bound DNA)]     x 100                weight of particles Degree of DNA entrapment Spectrophotometric determination OD260   Degree of DNA Entrapment PS 5.1 KDa +21 LS 14.1 KDa +9 LOADING CAPACITY(%) = [(total amount of DNA)-(non-bound DNA)]     x 100                weight of particles Degree of DNA entrapment Spectrophotometric determination OD260   Degree of DNA Entrapment Degree of DNA entrapment Spectrophotometric determination OD260   Magnetically  stirred DNA distribution  + 1 16 + 1 12 12 + 1 12 + 2 + 1 12 Degree of DNA entrapment Spectrophotometric determination OD260   Magnetically  stirred DNA distribution  Degree of DNA entrapment Spectrophotometric determination OD260   Magnetically  stirred DNA distribution  SOLID PARTICLES             CORE-SHELL PARTICLES Degree of DNA entrapment Spectrophotometric determination OD260   Magnetically  stirred DNA distribution  CTAB ALA AO-ssDNA: 640 nm AO-dsDNA: 530 nm ALA-dsDNA 12(+2)  LAM-dsDNA 12 (+1) CTAB-dsDNA 16 (+1) Secondary structure of DNA Fluorescence Microscopy •SURFACTANT SYSTEMS DTAB-dsDNA 12 (+1) DDAB-dsDNA 12 12 (+1) ALA-ssDNA 12 (+2) LAM-ssDNA 12 (+1) CTAB-ssDNA 16 (+1)  DTAB-ssDNA 12 (+1)  DDAB-ssDNA 12 12 (+1) Secundary Structure of DNA  DNA complexed (%) 79 ± 10 94 ± 1.2 99 ± 0.2 99 ± 0.3 97 ± 1.5 Particle morphology  Scanning Electron Microscopy •SURFACTANT SYSTEMS Outer  Inner  Particle Morphology CTAB-dsDNA 16 (+1) CTAB-ssDNA 16 (+1) DTAB-dsDNA 12 (+1) DTAB-ssDNA 12 (+1) DNA complexed (%) 52 ± 10 70 ± 12 99 ± 0.3 94 ± 1.2 Particle morphology  Scanning Electron Microscopy •SURFACTANT SYSTEMS Outer  DDAB-dsDNA 12 12 (+1)  DDAB-ssDNA 12 12 (+1) NR-hydrophobic lipid: 657 nm Particle Morphology Particle morphology  Scanning Electron Microscopy •SURFACTANT SYSTEMS Outer  DDAB-dsDNA 12 12 (+1) DDAB-ssDNA 12 12 (+1) AO-ssDNA: 640 nm AO-dsDNA: 530 nm Particle Morphology Secondary structure of DNA Fluorescence Microscopy •PROTEIN SYSTEMS LS-dsDNA PS-dsDNA LSPS70-dsDNA LSPS85-dsDNA LSPS15-dsDNA LSPS30-dsDNA LSPS50-dsDNA Increasing PS content Secundary Structure of DNA  AO-ssDNA: 640 nm AO-dsDNA: 530 nm Secondary structure of DNA Fluorescence Microscopy •POLYSACCHARIDE SYSTEMS R: 3 R:12 R: 25 R: 20 R=[ds-DNA]/[CHIT] with [ds-DNA] equal at 60 mM   Increasing CHIT content Secundary Structure of DNA  AO-ssDNA: 640 nm AO-dsDNA: 530 nm SUPERNATANTS PARTICLES Rw > 1  SWELLING Rw= 1 Rw> 1 DISSOLUTION In vitro Kinetics  In vitro Kinetics + 1 16 Swelling and dissolution behaviour Relative weight (RW=Wt/Wi)  + 1 12 12 + 1 12 + 2 Swelling and Dissolution Behaviour + 1 12 12 + 1 16 DNA release from particles Spectrophotometric determination OD260   DNA Release From Particles + 1 12 12 + 1 12 + 2 + 1 12 12 Swelling and dissolution behaviour Relative weight (RW=Wt/Wi)          Swelling and Dissolution Behaviour DNA release from particles Spectrophotometric determination OD260   DNA Release From Particles Swelling and dissolution behaviour Relative weight  (RW=Wt/Wi )  Swelling and Dissolution Behaviour DNA release from particles Spectrophotometric determination OD260   DNA Release From Particles I. Haemocompatibility: Surfactant-DNA gel particles Morán et al., Soft Matter,  8, 3200–3211 (2012) Br-N+Cl-F3C-S-O-OO• HYDROPHOBIC CHAIN LENGTH  • NUMBER OF CHARGES  • COUNTERION INFLUENCE    ?  . . CENTRIFUGATION  10, 000 rpm 5 min RED BLOOD CELLS SURFACTANT SOLUTIONS DNA GEL PARTICLES 1: Solutions or DNA gel particles  2: PBS buffer 3: RBC dispersion 12 3 INCUBATION room temperature  10- 360 min  Haemolysis assay CENTRIFUGATION  10, 000 rpm 5 min RED BLOOD CELLS SURFACTANT SOLUTIONS DNA GEL PARTICLES 0% HAEMOLYSIS isotonic PBS buffer  100% HAEMOLYSIS  bidistelled water   OD: 540 nm 0% 20% 100% 50% Haemolysis assay Haemolysis in solution Spectrophotometric determination OD540   Haemolysis in solution System DTAB DTAC DTATf HC50 (µg/mL) 510 443 468 Haemolysis induced by surfactants in DTA-DNA gel particles   STRONGLY DEPENDENT OF THE SURFACTANT-DNA INTERACTION   FAVOURED IN THE CASE OF ss-DNA DUE TO ITS AMPHIPHILIC CHARACTER  DTATf < DTAC< DTAB Haemolysis vs. DNA release ds-DNA released= 100 µg/mL DTAB: 80% DTAC: 60%  DTATf: 30% ss-DNA released= 6 µg/mL DTAB: 70% DTAC: 50%  DTATf: 20% Haemolysis vs. DNA Release Surfactant DTAB DTAC DTATf α   values 0.261 0.372 0.13-0.153 Electro neutral complexes 1. S. B. Rao   et al.,  J. Biomed. Mater. Res. 1997, 34, 21-28.  2. B. L. Bales et al.,  J. Phys. Chem. B.  2002, 106, 1926-1939. 3. F. S. Lima et al., Langmuir  2011, 27, 4319-4323. 1 - α  Degree of counterion dissociation (α)  content (µg/mL) 1030 830 490 Degree of counterion dissociation (α ) Haemolysis   DTAB > DTAC> DTATf II. Cytotoxicity: Mixed-Protein DNA gel particles LS-dsDNA LSPS70-dsDNA LSPS85-dsDNA LSPS15-dsDNA LSPS30-dsDNA LSPS50-dsDNA Increasing PS content PS-dsDNA Novel non-viral vector Biocompatible Protective Biodegradable Controlled release Long-acting Reproducible . . . . . ? Morán et al., Macromolecular Bioscience  (2012) 75cm2 culture flasks  HeLa  HUMAN EPITHELIAL CARCINOMA 3T3 FIBROBLASTS Day 1 Culture preparation Formation of the cell monolayer    37 ºC 5% CO2 24 h A B C D E G F H 2 1 4 3 6 7 5 8 10 11 12 9 80% confluence        Cell counting: EB/AO Trypsinization of adherent cells  trypsin/EDTA         1 x 105 cell/mL (3T3)       Celular suspension          5 x 104 cell/mL (HeLa)     (100 µl/well)   Cytotoxicity in vitro Day 2  Assay Incubation of the cells in the presence of  DNA gel particles A B C D E G F H 2 1 4 3 6 7 5 8 10 11 12 9 37 ºC 5% CO2 24 h A B C D E G F H 2 1 4 3 6 7 5 8 10 11 12 9 C: CONTROL (100 µl/well)  S: SOLUTIONS (100 µl/well) P: PARTICLES (100 µl/well + 1p)   Cytotoxicity in vitro Day 3 End-point measurements Cell death/viability measurements OD: 550 nm %  CELL VIABILITY 37 ºC 5% CO2 3 h A B C D E G F H 2 1 4 3 6 7 5 8 10 11 12 9 DMSO agitation  10 min A B C D E G F H 2 1 4 3 6 7 5 8 10 11 12 9 Cytotoxicity in vitro HeLa human epithelial carcinoma  3T3  fibroblasts IC50 (LS) > 2000 µg/mL IC50 (PS) = 140-250 µg/mL Cytotoxicity  in vitro  Cytotoxicity in vitro: Proteins in solution tincubation=24 h R=[ds-DNA]/[P+]=1 with [ds-DNA] and [P+] equal at 60 mM   PROTEIN SOLUTION  1 PARTICLE  FOR WELL PLATE * Signiﬁcant differences (p<0.05) from the corresponding protein solution Cytotoxicity in vitro induced by protein- DNA gel particles at millimeter scale R=[ds-DNA]/[P+]=1 with [ds-DNA] and [P+] equal at 2.5 mM                         Morán  et al., Soft  Matter 5, 2538-2542 (2009) System Mean size (nm) P. I.  CTAB-dsDNA CTAB-ssDNA 139 ± 8 126 ± 1 0.456 ± 0.01 0.403 ± 0.01 PS-dsDNA PS-ssDNA 387 ± 8 343 ± 10 0.782 ± 0.30 0.140 ± 0.03 LS-dsDNA LS-ssDNA 9208 ± 2800 9450 ± 2000 0.313 ± 0.40 0.781 ± 0.50 Gene delivery: Endocytosis R=[ds-DNA]/[P+]=1 with [ds-DNA] and [P+] equal at 2.5 mM   tincubation=24 h PROTEIN SOLUTION  PARTICLE DISPERSION * Signiﬁcant differences (p<0.05) from the corresponding protein solution Cytotoxicity in vitro induced by protein- DNA gel particles at submicron scale R=[ds-DNA]/[P+]=1 with [ds-DNA] and [P+] equal at 2.5 mM   method of Bradford Determination of the protein content and degree of complexation System 3T3 /IC50 (mM) Hela/ IC50 (mM) LS-DNA < 0.16 < 0.16 LSPS7.5-DNA* > 1.25 0.88 LSPS15-DNA > 1.25 0.92 LSPS30-DNA* > 1.25 0.32 LSPS50-DNA > 1.25 0.92 LSPS70-DNA 0.80 0.60 LSPS85-DNA* 0.75 0.68 PS-DNA > 1.25 > 1.25 * Signiﬁcant differences (p<0.05) between 3T3 and HeLa for the same conditions Relative viabilities of tumour and non-tumour cell lines HeLa PS-(AO/DNA)  particles dispersion  ½ dilution (250 µL)  tincubation=2 h  40x magnification FM CM CM FM Internalization of protein-DNA gel particles at submicron scale  Highlights  The magnitude of DNA entrapment can be controlled and controlled release systems achieved through the formation of an oppositely charged polyelectrolytes complex network giving rise to these DNA gel particles. The decrease in haemolysis and cytotoxicity as well as the formation of a releasable high DNA content reservoir renders these DNA gel particles promising DNA vehicles for use as a nonviral gene delivery system.  Acknowledgments Dr. M.Pilar Vinardell Grup d´Interacció de Tensioactius amb Membranes Cel.lulars (ITMC)  Dr. María Rosa Infante Dr. Ramon Pons Dr. Pere Clapés  Dr. Maria Graça Miguel Dr. Björn Lindman Andreia Jorge Dr. Emanuela Corsini   Valentina Galbiati MICINN       Ramon y Cajal contract RyC 2009-04683  CTQ2009-14151-C02-02    MAT2012-38047-C02-01 Generalitat de Catalunya (2009SGR1331) Dr. Montse Mitjans  Dr. M.Carmen Morán Dr. Verónica Martínez  Daniele Nogueira Albert Botta Tania Alonso Joana Fortuño Maragda Pérez  Laia Vidal 

Preparació de partícules gel d'ADN com a sistemes d'alliberament controlat (Seminaris de Recerca 2012)

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