Microencapsulated Lipid Nanoparticles for Pulmonary Delivery of Biopharmaceutical Agents
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UNIVERSIDADE DE LISBOA FACULDADE DE FARMÁCIA Microencapsulated lipid nanoparticles for pulmonary delivery of biopharmaceutical agents Diana Patrícia Rodrigues Gaspar Orientador: Professor Doutor António José Leitão das Neves Almeida Co-orientadores: Professora Doutora María del Carmen Remuñán López Professor Doutor Pablo Antelo Taboada Tese especialmente elaborada para obtenção do grau de Doutor em Farmácia na especialidade de Tecnologia Farmacêutica 2017 UNIVERSIDADE DE LISBOA FACULDADE DE FARMÁCIA Microencapsulated lipid nanoparticles for pulmonary delivery of biopharmaceutical agents Diana Patrícia Rodrigues Gaspar Orientador: Professor Doutor António José Leitão das Neves Almeida Co-orientadores: Professora Doutora María del Carmen Remuñán López Professor Doutor Pablo Antelo Taboada Tese especialmente elaborada para obtenção do grau de Doutor em Farmácia na especialidade de Tecnologia Farmacêutica Júri Presidente: Doutora Matilde da Luz dos Santos Duque da Fonseca e Castro, Professora Catedrática e Directora da Faculdade de Farmácia da Universidade de Lisboa Vogais: Doutora María del Carmen Remuñán-López, Titular de Universidad da Facultad de Farmacia da Universidad de Santiago de Compostela, Espanha, orientadora Doutor Domingos de Carvalho Ferreira, Professor Catedrático da Faculdade de Farmácia da Universidade do Porto Doutora Ana Margarida Moutinho Grenha, Professora Auxiliar da Faculdade de Ciências e Tecnologia da Universidade do Algarve Doutora Helena Maria Cabral Marques, Professora Associada com Agregação da Faculdade de Farmácia da Universidade de Lisboa Doutor Jorge Manuel Barreto Vítor, Professor Auxiliar da Faculdade de Farmácia da Universidade de Lisboa Doutora Maria Manuela de Jesus Guilherme Gaspar, Investigadora Auxiliar da Faculdade de Farmácia da Universidade de Lisboa 2017 ↠ Nothing is impossible, the word itself says I'm possible! ↞ Audrey Hepburn Table of Contents Table of Contents Acknowledgements................................................................................................ xi Abstract.................................................................................................................. xv Resumen................................................................................................................. xvii Resumo.................................................................................................................. xix List of Abbreviations and Acronyms..................................................................... xxiii List of Figures........................................................................................................ xxv List of Tables......................................................................................................... xxxi Aims and Organization of the Thesis.................................................................... xxxiii Chapter 1. General introduction – Particle engineering by nanoparticle microencapsulation for pulmonary delivery………………………………..... 1 Abstract.................................................................................................................. 3 Graphical abstract.................................................................................................. 4 1.1. Drug delivery systems based on nanoparticles……………………………... 5 1.2. Types of nanoparticles……………………………………………………… 8 1.2.1. Liposomes……………………………………………………………. 9 1.2.2. Nanoparticles based on solid lipids………………………………….. 10 1.3. Inhaled drug delivery through nanoparticles……………………………….. 19 1.4. Deposition of inhaled particles in the respiratory tract……………………... 21 1.5. Fate of inhaled particles in the lung………………………………………… 24 1.6. Techniques for nanoparticles microencapsulation by particle engineering… 26 1.6.1. Spray-drying…………………………………………………………. 27 1.6.2. Spray-freeze-drying………………………………………………….. 36 1.6.3. Other microencapsulation techniques………………………………... 40 1.6.3.1. Milling……………………………………………………….. 40 1.6.3.2. Supercritical fluids technology………………………………. 41 1.7. Nanoparticles recovery after microencapsulation…………………………... 43 1.8. Conclusions…………………………………………………………………. 44 1.9. References…………………………………………………………………... 45 Chapter 2. Rifabutin-loaded solid lipid nanoparticles for inhaled antitubercular therapy: physicochemical and in vitro studies......................... 65 Abstract.................................................................................................................. 67 Graphical abstract.................................................................................................. 67 2.1. Introduction..................................................................................................... 69 2.2. Materials......................................................................................................... 70 2.3. Methods.......................................................................................................... 71 2.3.1. RFB solubility studies........................................................................... 71 2.3.2. Formulation of SLN.............................................................................. 71 2.3.3. Characterization of SLN....................................................................... 71 2.3.3.1. Particle size, surface charge and physical stability.................. 71 2.3.3.2. Encapsulation efficiency and drug loading.............................. 72 2.3.3.3. Stability studies........................................................................ 73 2.3.3.4. Transmission electron microscopy analysis (TEM)................. 73 2.3.3.5. Thermal analysis using dynamic light scattering (DLS).......... 74 2.3.3.6. Differential scanning calorimetry (DSC) studies..................... 74 2.3.4. In vitro cell viability studies................................................................. 74 vii Table of Contents 2.3.5. Quantitative SLN uptake assessment.................................................... 75 2.3.6. Fluorescence Microscopy..................................................................... 76 2.3.7. In vitro RFB release studies.................................................................. 76 2.3.8. Statistical analysis................................................................................. 77 2.4. Results and Discussion................................................................................... 78 2.4.1. SLN characterization............................................................................ 78 2.4.2. Stability studies..................................................................................... 79 2.4.3. DLS thermal analysis.......................................................................... 83 2.4.4. DSC analysis......................................................................................... 84 2.4.5. In vitro cell viability studies................................................................. 86 2.4.6. Intracellular SLN uptake studies.......................................................... 89 2.4.7. In vitro RFB release studies from SLN................................................ 90 2.5. Conclusions...……………………………………………………………….. 92 2.6. References...………………………………………………………………… 93 Chapter 3. Microencapsulated solid lipid nanoparticles as a hybrid platform for pulmonary antibiotic delivery………………………………….. 99 Abstract.................................................................................................................. 101 Graphical abstract.................................................................................................. 101 3.1. Introduction…………………………………………………………………. 103 3.2. Materials……………………………………………………………………. 104 3.2.1. Chemicals……………………………………………………………. 104 3.2.2. Animals………………………………………………………………. 105 3.2.3. Mycobacterial strains............................................................................ 105 3.3. Methods…………………………………………………………………….. 105 3.3.1. Preparation of RFB-loaded SLN…………………………………….. 105 3.3.2. Characterization of RFB-loaded SLN……………………………….. 105 3.3.3. Determination of association efficiency and drug loading of RFB...... 106 3.3.4. Preparation of dry powders containing SLN........................................ 107 3.3.5. Characterization of microspheres size, morphology and moisture content…………………………………………………………………………… 108 3.3.6. Structural characterization of SLN-loaded microspheres using CLSM..................................................................................................................... 108 3.3.7. Determination of the dry powders flow properties…………………... 109 3.3.8. Isothermal titration calorimetry of SLN and spray-drying excipients.. 109 3.3.9. In vitro deposition of dry powders using a twin-stage liquid impinger 111 3.3.10. SLN recovery from microspheres....................................................... 111 3.3.11. In vitro RFB release from microencapsulated RFB-loaded SLN....... 112 3.3.12. In vitro activity of RFB formulations against M. avium strain DSMZ 44157 by MTS assay................................................................................. 112 3.3.13. In vivo fate of RFB formulations........................................................ 112 3.3.13.1. Biodistribution studies......................................................... 112 3.3.13.2. RFB extraction from blood and tissues................................ 113 3.3.13.3. Determination of RFB by HPLC......................................... 113 3.3.13.4. Preparation of standard solutions for