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Toxicity Evaluation of Poly(Anhydride) Nanoparticles As Carriers for Oral Administration of Drugs

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Toxicity Evaluation of Poly(Anhydride) Nanoparticles As Carriers for Oral Administration of Drugs Toxicity evaluation of poly(anhydride) nanoparticles as carriers for oral administration of drugs Patricia Ojer Ojer TESIS DOCTORAL Pamplona, octubre 2013 TESIS DOCTORAL Facultad de Farmacia Departamento de Farmacia y Tecnología Farmacéutica Departamento de Farmacología y Toxicología Toxicity evaluation of poly(anhydride) nanoparticles as carriers for oral administration of drugs Trabajo presentado por Dña.Patricia Ojer Ojer para obtener el Grado de Doctor Fdo. Patricia Ojer Ojer Pamplona, octubre 2013 El presente trabajo, titulado “Toxicity evaluation of poly(anhydride) nanoparticles as carriers for oral administration of drugs”, presentado por DÑA. PATRICIA OJER OJER para optar al grado de Doctor por la Universidad de Navarra en el área de tecnología Farmacéutica y Toxicología, ha sido realizado bajo nuestra dirección en los Departamentos de Farmacia y Tecnología Farmacéutica y de Farmacología y Toxicología de la Universidad de Navarra. Estimamos que puede ser presentado al tribunal que lo ha de juzgar. Y para que así conste, firman la presente: Fdo.: Dr. Juan M. Irache Garreta Fdo.: Dra. Adela López de Cerain Salsamendi Pamplona, octubre 2013 Esta tesis doctoral se ha llevado a cabo gracias a las ayudas predoctorales de Formación (convocatoria 2007) y de movilidad internacional (convocatoria 2010) dentro del plan de Formación y de I+D del departamento de educación del Gobierno de Navarra y a la ayuda para la formación del personal investigador de la Asociación de Amigos de la Universidad de Navarra. Las investigaciones realizadas en el presente trabajo se han desarrollado dentro del proyecto CAN “Nanotecnología y medicamentos” (REF 10828). A mis padres, Inma y Vicente, y a mi hermano, Daniel. “Lo esencial es invisible para los ojos” Antoine de Saint-Exupéry AGRADECIMIENTOS Deseo expresar mi agradecimiento a todas las personas e instituciones que durante estos últimos años han contribuido a que la presente tesis doctoral sea una realidad. En primer lugar, quisiera agradecer especialmente a mis directores, el Dr. Juan Manuel Irache y la Dra. Adela López de Cerain, la oportunidad de realizar esta tesis doctoral. Gracias por su inestimable labor de dirección, su estímulo para seguir creciendo intelectualmente y sobre todo, por la confianza depositada en mí y en mi trabajo. Gracias a la Universidad de Navarra y en especial a los investigadores, profesores y personal de los Departamentos de Farmacia y Tecnología Farmacéutica y de Farmacología y Toxicología, gracias por la ayuda prestada cuando la he necesitado. A la asociación de Amigos y el Departamento de Educación del Gobierno de Navarra, por la aportación económica recibida a través de sus programas de ayudas para la realización de tesis doctorales y estancias en el extranjero. I am grateful to the Department of Pharmaceutical Technology and Biopharmaceutics of the University of Vienna, in particular to Prof. Franz Gabor and Prof. Michael Wirth for giving me the opportunity to join their group. Thanks to those people who I met in Vienna, and especially to Lukas Neutsch for your friendliness and your “incubation times”. A mis compañeros y amigos de los laboratorios de Galénica y Toxicología, ha sido un placer poder compartir con todos vosotros estos años, no sólo el trabajo dentro del laboratorio sino también los grandes momentos vividos fuera. A Hugo. Muchísimas gracias por toda tu ayuda en estos años, porque no sólo te has limitado a realizar tu trabajo sino que además lo has hecho mucho más divertido, aunque la radio ayudaba bastante. Gracias por quedarte a trabajar horas que no te correspondían y por venir los fines de semana. A Irene Esparza. Gracias por tu apoyo incondicional, tu sabiduría, tu infinita paciencia pero sobre todo gracias por tu amistad, siempre dispuesta a ayudar y a escuchar aunque para ello tuvieses que sacar al día 25 horas. A todas las personas que durante estos años han formado parte de mi vida, besonders Camillo, well er an mich mehr geglaubt hat als ich selbst. Todo esto no hubiera sido posible sin el cariño de mi familia, y en especial de mis padres. Este es también vuestro premio. TABLE OF CONTENTS ABBREVIATIONS.........................................................................................................................................17 CHAPTER 1......................................................................................................................................................21 GENERAL INTRODUCTION CHAPTER 2 .....................................................................................................................................................79 AIM AND OBJECTIVES CHAPTER 3 .....................................................................................................................................................83 EXPERIMENTAL DESIGN CHAPTER 4 .....................................................................................................................................................87 Spray-drying of poly(anhydride) nanoparticles for drug/antigen delivery CHAPTER 5 ...................................................................................................................................................113 Cytotoxicity and cell interaction studies of bioadhesive poly(anhydride) nanoparticles for oral antigen/drug delivery CHAPTER 6 ...................................................................................................................................................143 Toxicity studies of poly(anhydride) nanoparticles as carriers for oral drug delivery CHAPTER 7 ...................................................................................................................................................169 Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration CHAPTER 8 ...................................................................................................................................................195 GENERAL DISCUSSION CHAPTER 9 ...................................................................................................................................................207 CONCLUSIONS/CONCLUSIONES 15 ABREVIATIONS ABREVIATIONS %ID/g: Percentage of injected dose per gram 99mTc: 99mTechnetium ABI-007: Abraxane® ADA: Adenosine Deaminase AFM: Atomic Force Microscopy ALT: Alanine Transaminase APCs: Antigen-Presenting Cells AST: Aspartate Transaminase ATO: Atovaquone ATP: Adenosine Thriphosphate BCS: Biopharmaceutics Classification System BSA: Bovine Serum Albumin BSA-FITC: Bovine Serum Albumin-Fluorescein Isothiocyanate Conjugate Bw: Body weight CdSe: Cadmium Selenide CdTe: Cadmium Telluride CLSM: Confocal Laser Scanning Microscopy CMC: Critical Micelle Concentration CNTs: Carbon Nanotubes CT: Computed Tomography CTB: Cholera Toxin B subunit CYP: Cytochrome P450 metabolizing enzyme system DCFA: 2′7′-dichlorofluorescein diacetate DISS: Digital Image System DMF: Dimethylformamide DOX: Doxorubicin DPBS: Dulbecco´s Phosphate Buffered Saline DTXL: Docetaxel DTNB: 5,5-dithio-bis-2-nitrobenzoic acid EASAC: European Academies Science Advisory Council ELSD: Evaporative Light Scattering Detection EMA: European Medicines Agency EPR: Enhanced Permeability and Retention ESR: Erythrocyte Sedimentation Rate FDA: Food and Drug Administration FP7: Seventh Framework Program GCSF: Granulocyte Colony-Stimulating Factor GIT: Gastrointestinal Tract HGB: Hemoglobin HCT: Hematocrit 17 ABREVIATIONS HER2: Human Epidermal growth factor Receptor 2 HGF: Hepatocytes Growth Factor HIV: Human Immunodeficiency Virus HPCD: 2-hydroxipropyl-β-cyclodextrin HPLC: High Performance Liquid Chromatography HPMA: N-(2-Hydroxypropyl) methacrylamide HS: Hot Saline antigenic extract HSA: Human Serum Albumin i.m.: intramuscular i.r.: intravitreous i.t.: intrathecal i.v.: intravenous ILSI RF/RSI: International Life Sciences Institute Research Foundation/Risk Science Institute InAs: Indium Arsenide InP: Indium Phosphide IRIV: Immunopotentiating Reconstituted Influenza Virosome ISO: International Organization for Standardization ITLC: Instant Thin Layer Chromatography JRC: Joint Research Centre of the European Commission LD50: Lethal Dose 50% LDH: Lactate dehydrogenase LPS: Lipopolysaccharide LUVs: Large Unilamellar Vesicles MCH: Mean corpuscular hemoglobin MCHC: Mean Corpuscular Hemoglobin Concentration MCV: Mean Corpuscular Volume MLVs: Mutilamellar Vesicles MPS: Mononuclear Phagocyte System MTS: 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt MTT: 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide MWCNT: Multi-Walled Carbon Nanotubes NCS: Nanotoxicological Classification System NCTR: National Center of Toxicological Research NOAEL: No Observed Adverse Effect Level NNI: National Nanotechnology Initiative NP: Conventional poly(anhydride) nanoparticles NP-HPCD: Nanoparticles containing 2-hydroxypropyl-β-cyclodextrin OECD: Organisation for Economic Co-operation and Development Omp: Outer membrane proteins OVA: Ovalbumin PACA: Poly(akyl cyanocrylates) 18 ABREVIATIONS PAMAM: Poly(amidoamine) PBS: Phosphate buffered saline PCL: Poly(Ɛ-caprolactone) PCS: Photon Correlation Spectroscopy PDI: Polydispersity Index PEG: Polyethylene glycol PEG-NP: Pegylated poly(anhydride) nanoparticles P-gp: P-glycoprotein PLA: Poly(lactic acid) PLDsis: Phospholipidosis PLGA: Poly(lactide-co-glycolide) PLT: Platelet count QDs: Quantum Dots RBC: Red Blood Corpuscles count RES: Reticular
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