Nanoparticles for advanced treatments of arthritic diseases

Universidade do Minho I3Bs - Instituto de Investigação em Biomateriais, Biodegradáveis e Biomiméticos Ana Cláudia Fernandes Lima Nanoparticles for advanced treatments of arthritic diseases Tese de Doutoramento Doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células Estaminais Trabalho efetuado sob a orientação do Professor Doutor Nuno João Meleiro Alves das Neves Outubro de 2019 ii DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Licença concedida aos utilizadores deste trabalho Atribuição CC BY https://creativecommons.org/licenses/by/4.0/ iii ACKNOWLEDGMENTS During this journey, I have been richly blessed by the support, love and kindness of some people that make this thesis possible. To them, I am and will always be grateful. First, I would like to start by expressing my gratitude to my supervisor, Prof. Nuno Neves. No words can express how thankful I am for the patience, availability and inspiration during these last years. Thank you for accepting me as a PhD student and for all the conversations about science and life. You helped me on becoming a more independent researcher and taught me the power of imagination. This thesis would not be possible without all your knowledge and capability to supervise me. To the director of 3B’s Research group, Prof. Rui L. Reis, I am forever grateful for the opportunity to join his group and to develop my PhD at this European Institute of Excellency. Thank you for being an inspiration on pursuing our dreams and ambitions. It’s also difficult to find words to describe how truly grateful I am to have Dr. Helena Ferreira in my life. She had a major contribution in this thesis, with all her help, motivation, support, inspiration, ideas and trust. Thank you, my dearest friend for always be there for me. I would like to acknowledge the coauthors in this thesis and everyone in the 3B’s that helped me. To my friends in 3B’s, we shared the good, the bad, frustrations and achievements, thank you all for the great memories. I have found really special people in this journey, special thanks to Gabriela and Catarina for all the love and friendship. Also mention my gratitude to all my close friends, because “friends are the family you choose”. Finally, I would like to thank all my family for all the unconditional love and support. I am so lucky to have this wonderful family, with all my uncles and cousins being always there for me. Special mention to my adored deceased grandparents and uncle that are always in my heart. This thesis is dedicated to the four most important people in my life: dad, mom, brother and fiancé. To my dad, my king and my hero. To my mom, my strength and my admiration. To my brother, my other half. I fell so blessed for having chosen you as my family. I own you the person that I am today, as you gave the opportunity to follow my dreams and transmitted me important principals. As you say “have courage and be kind”. Last but not the least, to the love of my life, my everything, my fiancé Tiago. It’s difficult to express how much I love and admire you. You are my safe harbor and my happy place. You are always there for me, making me see the brighter side of life. I am so deeply grateful to have found you my love. And I can say that the best is yet to come. I would like to acknowledge the Foundation for Science and Technology (FCT) for my PhD scholarship (PD/BD/11384/2015). iv STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. v ABSTRACT Nanoparticles for advanced treatments of arthritic diseases Arthritic diseases affect more than 350 million people worldwide. Their incidence, prevalence and global burden are increasing, mainly due to the aging of the population. The most common forms of arthritis, osteoarthritis (OA) and rheumatoid arthritis (RA), are chronic inflammatory diseases, which are characterized by synovial inflammation and cartilage and bone destruction. Despite the advances in the pharmaceutical field, there is currently no cure for arthritis. Moreover, current treatments are associated with low efficiency and severe side effects. Recently, nanoparticles (NPs) have emerged as a powerful platform to overcome the present treatment limitations. The goal of this PhD work was to develop novel NPs to treat arthritic conditions. Taking into consideration their main drawbacks, we aimed to design NPs able to enhance the therapeutic index of relevant drugs. Therefore, biological agents were immobilized at the surface of biodegradable polymeric NPs and liposomes to protect, extend and enhance their therapeutic efficacy in OA and RA, respectively. While polymeric NPs were biofunctionalized with anti-IL-6 antibodies to selectively capture and neutralize this key pro-inflammatory cytokine, liposomes were biofunctionalized with anti-IL-23 antibodies in order to inhibit the differentiation of naive CD4+ T cells into Th17 cells, by IL-23 neutralization. Furthermore, enzymaticand redox-responsive polymeric micelles were developed for targeted and controlled drug delivery in arthritis. After accumulation in the inflammatory site, the dexamethasone encapsulated into the polymeric micelles undergo quick release triggered by both redox and glutathione reductase activity. For all NPs formulations, the size distribution, zeta potential and drug immobilization/encapsulation was assessed. Biological tests demonstrated their cytocompatibility in contact with human chondrocytes, macrophages and endothelial cells. Moreover, under inflammatory conditions the NPs were able to demonstrate their enhanced efficacy in comparison with the free drugs. A deeper characterization on the degree of NPs internalization and pathways was performed in a normal and inflammatory scenario by different cells affected by arthritic diseases. The results highlight the importance of considering the targeted cell type when designing functional NPs for specific diseases. Finally, in vivo studies demonstrated the safety and enhanced therapeutic efficacy of the biofunctionalized polymeric NPs immobilizing anti-TNF-α and anti-IL-6 antibodies. In conclusion, as these novel advanced NPs might overcome abovementioned drawbacks, they are promising strategies for radically improving the efficacy of arthritis treatments and severely limiting their side effects. Keywords: Arthritic diseases, Nanoparticles, Antibodies immobilization, Controlled release. vi RESUMO Nanopartículas para o tratamento avançado de doenças artríticas As doenças artríticas afetam mais de 350 milhões de pessoas em todo o mundo. A sua incidência, prevalência e encargos globais estão a aumentar sobretudo devido ao envelhecimento da população. A osteoartrite (OA) e a artrite reumatoide (AR) são as formas mais comuns destas doenças crónicas inflamatórias, que são caracterizadas pela inflamação sinovial e pela destruição da cartilagem e do osso. Apesar dos avanços na área farmacêutica, atualmente não existe cura para estas doenças. Além disso, os tratamentos disponíveis estão associados a uma baixa eficácia e a efeitos adversos graves. Recentemente, as nanopartículas (NPs) emergiram como uma plataforma poderosa capaz de superar as limitações dos tratamentos atuais. Assim, o objetivo desta tese de doutoramento foi o desenvolvimento de novas NPs para o tratamento da artrite. Considerando as atuais limitações, diferentes NPs foram desenhadas de forma a aumentar o índice terapêutico de fármacos. Agentes biológicos foram imobilizados na superfície das NPs e lipossomas de forma a proteger, prolongar e aumentar as suas eficácias terapêuticas no tratamento da OA e AR, respetivamente. Enquanto as NPs foram biofuncionalizadas com anticorpos anti-IL-6 de forma a capturar e neutralizar seletivamente esta citoquina central, os lipossomas foram biofuncionalizados com anticorpos anti-IL-23 de forma a inibir a diferenciação das células T CD4+ em células Th17. Além disso, micelas sensíveis à enzima glutationa redutase e a um ambiente redutor foram desenvolvidas para promover a libertação local e controlada do fármaco. De facto, depois de as NPs se acumularem na articulação inflamada, a dexametasona encapsulada dentro das micelas será facilmente libertada através daqueles mecanismos. Todas as formulações foram avaliadas em relação à homogeneidade do tamanho, ao potencial zeta e à quantidade dos fármacos imobilizada/encapsulada nas NPs. Os testes biológicos demonstraram a sua citocompatibilidade em contacto com condrócitos, macrófagos e células endoteliais humanas. Além disso, sob condições inflamatórias, as NPs revelaram uma maior eficácia em comparação com o fármaco livre. A percentagem e via de internalização das NPs foram avaliadas em condições normais e inflamatórias em diferentes tipos de células. Os resultados reforçam a importância de se considerar o tipo de célula alvo aquando do desenvolvimento das NPs. Finalmente, estudos in vivo demonstraram a segurança e o aumento da eficácia terapêutica das NPs biofuncionalizadas com anticorpos anti-TNF-α e anti-IL-6. Em conclusão, as novas NPs desenvolvidas são estratégias promissoras que podem mudar radicalmente a eficácia dos tratamentos e limitar significativamente os seus efeitos adversos. Palavras-chave: Doenças artríticas, Nanopartículas, Imobilização de anticorpos, Libertação controlada. vii TABLE OF CONTENTS ACKNOWLEDGMENTS _____________________________________________________ III STATEMENT OF INTEGRITY _________________________________________________ IV ABSTRACT ______________________________________________________________ V RESUMO_______________________________________________________________ VI TABLE OF CONTENTS _____________________________________________________ VII LIST OF ABBREVIATIONS ___________________________________________________XVI LIST OF FIGURES _______________________________________________________ XXVI LIST OF TABLES ________________________________________________________ XXXI SHORT CURRICULUM VITAE ______________________________________________ XXXII LIST OF PUBLICATIONS __________________________________________________ XXXIII INTRODUCTION TO THE THESIS FORMAT ____________________________________ XXXVI SECTION 1 ______________________________________________________________ 1 GENERAL INTRODUCTION ___________________________________________________ 1 CHAPTER I - NANOPARTICLES FOR ARTHRITIC DISEASES TREATMENT _________________ 3 Abstract ________________________________________________________________ 3 I-1. Introduction _______________________________________________________ 4 I-2. Arthritic diseases ___________________________________________________ 5 I-2.1. Pathogenesis and molecular targets __________________________________ 5 I-2.2. Treatment ___________________________________________________ 10 I-2.2.1. Analgesics and anti-inflammatory drugs____________________________ 13 I-2.2.2. DMARDs __________________________________________________ 14 I-2.2.3. Biological agents ____________________________________________ 14 I-2.2.4. Gene therapies _____________________________________________ 16 I-3. Nanomedicines ___________________________________________________ 16 I-3.1. Targeting strategies_____________________________________________ 18 I-3.1.1. Cartilage targeting ___________________________________________ 19 I-3.1.2. Inflammation targeting ________________________________________ 20 viii I-3.2. Nanomedicines in Arthritic Diseases _________________________________ 21 I-3.2.1. Liposomes ________________________________________________ 21 I-3.2.2. Polymeric NPs _____________________________________________ 23 I-3.2.3. Micelles __________________________________________________ 25 I-3.2.4. Dendrimers ________________________________________________ 27 I-3.2.5. Inorganic NPs ______________________________________________ 27 I-3.3. NPs internalization _____________________________________________ 30 I-3.3.1. Cellular Uptake Pathways of NPs ________________________________ 31 I-3.3.2. Effect of Physicochemical Properties of NPs on Cellular Uptake __________ 33 I-3.3.3. Effect of Cell Properties over NPs Uptake __________________________ 35 I-4. Conclusions and future perspectives ____________________________________ 36 I-5. Acknowledgment __________________________________________________ 38 I-6. References _______________________________________________________ 38 SECTION 2 _____________________________________________________________ 50 EXPERIMENTAL SECTION __________________________________________________ 50 CHAPTER II - MATERIALS AND METHODS _____________________________________ 52 Overview _______________________________________________________________ 52 II-1. Materials ______________________________________________________ 53 II-1.1. Chitosan ____________________________________________________ 53 II-1.2. Hyaluronic acid _______________________________________________ 54 II-1.3. Lipids ______________________________________________________ 55 II-1.3.1. Fatty acids ________________________________________________ 55 II-1.3.2. Phospholipids______________________________________________ 56 II-1.3.3. Steroids __________________________________________________ 57 II-1.4. Gold nanoparticles _____________________________________________ 58 II-1.5. Glutathione __________________________________________________ 59 II-1.6. Polyethylene glycol _____________________________________________ 60 II-2. Reagents ______________________________________________________ 61 II-3. NPs preparation _________________________________________________ 61 II-3.1. Preparation methods____________________________________________ 61 II-3.1.1. Polyelectrolyte complexation ___________________________________ 61 ix II-3.1.2. Thin-film hydration method ____________________________________ 62 II-3.1.3. Nanoprecipitation ___________________________________________ 62 II-3.2. Chemical coupling reactions ______________________________________ 63 II-3.2.1. EDC/NHS ________________________________________________ 63 II-3.2.2. TBTU ____________________________________________________ 64 II-3.2.3. Thiols ___________________________________________________ 64 II-3.3. Polymeric NPs preparation _______________________________________ 66 II-3.4. LUVs preparation ______________________________________________ 67 II-3.5. Micelles preparation ____________________________________________ 68 II-4. NPs characterization ______________________________________________ 69 II-4.1. Particle size, polydispersity index and zeta potential______________________ 70 II-4.1.1. Size - Dynamic Light Scattering _________________________________ 70 II-4.1.2. Surface electric charge - Zeta potential ____________________________ 71 II-4.2. Stability studies _______________________________________________ 71 II-4.3. Morphology __________________________________________________ 72 II-4.3.1. Scanning electron microscopy __________________________________ 72 II-4.3.2. Transmission Electron Microscopy _______________________________ 72 II-4.3.3. Atomic Force Microscopy _____________________________________ 73 II-4.4. Chemical characterization ________________________________________ 73 II-4.4.1. Fourier Transform Infrared Spectroscopy __________________________ 73 II-5. Bioactive agents _________________________________________________ 74 II-5.1. Antibodies ___________________________________________________ 74 II-5.1.1. Polymeric NPs functionalization _________________________________ 76 II-5.1.2. Liposomes functionalization____________________________________ 76 II-5.2. Dexamethasone _______________________________________________ 77 II-5.2.1. Encapsulation efficiency ______________________________________ 77 II-5.2.2. Release studies ____________________________________________ 78 II-5.2.3. UV-Vis spectrophotometry _____________________________________ 78 II-6. In vitro biological tests _____________________________________________ 79 II-6.1. Cell sources __________________________________________________ 79 II-6.1.1. Human Articular Chondrocytes _________________________________ 80 II-6.1.2. Human monocyte-derived macrophages ___________________________ 80 xvi LIST OF ABBREVIATIONS A α - Alpha 7-AAD - 7-aminoactinomycin D Ab - Antibody AB - Alamar blue AbIA - Anti-type II collagen antibody-induced arthritis ABTS - 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) ADs - Autoimmune diseases ADAMTSs - a disintegrin with thrombospondin motifs AFM - Atomic Force Microscopy AIA - Adjuvant-induced arthritis APRIL - a proliferation-inducing ligand ATR - Attenuated total reflectance Au - Gold AuNPs - Gold nanoparticles B β - Beta BCA - Bicinchoninic acid bDMARDs - Biologic disease-modified anti-rheumatic drugs b-FGF - Basic fibroblast growth factor BMPs - Bone morphogenetic proteins BSA - Bovine serum albumin C C - Carbon CAIA - Collagen antibody-induced arthritis CC - Cholesteryl chloroformate xvii CCL - CC chemokine ligand CD - Cluster of Differentiation CDC - Centers for Disease Control and Prevention cDNA - Complementary deoxyribonucleic acid cfDNA - cell-free deoxyribonucleic acid CFA - Complete Freund’s adjuvant CFSE - Carboxyfluorosuccinimide ester Ch - Chitosan CIA - Collagen‐induced arthritis cm - Centimeter cm-1 - Reciprocal wavelength centimeter CO2 - Carbon dioxide COOH - Carboxyl group COX - Cyclooxygenase enzyme CpG - deoxy-cytidylate-phosphate-deoxy-guanylate Ctr - Control Cu - Cupper Cur - Curcumin D °C - Degree Celsius D - Diffusion coefficient DAMP - Damage-associated molecular patterns DAPI - 4',6'-diamino-2-fenil-indol DD - Degree of deacetylation DDS - Drug delivery system DEAE - Diethylethylamine Dex - Dexamethasone xviii DGAV - Direcção Geral de Alimentação e Veterinária DLS - Dynamic light scattering DMARDs - Disease-modified anti-rheumatic drugs DMEM - Dulbecco’s modified Eagle’s medium DMSO - Dimethyl sulfoxide DNA - Deoxyribonucleic acid DPBS - Dulbecco's Phosphate-Buffered Saline DPPC - Dipalmitoyl phosphatidylcholine dsDNA - Double stranded Deoxyribonucleic acid DS - Dextran sulfate DSPE-PEG-Mal - 1,2-distearoyl-sn-glycero-3-phosphoethanolamineN-[maleimide(polyethyleneglycol)-2000] E EA (EA.hy926) - Human umbilical vein endothelial cell line ECM - Extracellular matrix ECAMs - Endothelial cell adhesion molecules EDC - 1-ethyl-3-(3-(dimethylaminopropyl) carbodiimide EDTA - Ethylenediamine tetraacetic EE - Entrapment efficiency EGCG - (−)-epigallocatechin gallate ELISA - Enzyme-linked Immunosorbent Assay EMA - European Medicines Agency EPC - L-alfa-phosphatidylcholine from egg yolk EPR - Enhanced permeability and retention ER - Endoplasmic reticulum EULAR - European League Against Rheumatism F FA - Folic acid xix FBS - Fetal bovine serum FCT - Foundation for Science and Technology FDA - Food and Drug Administration EGFR - Epidermal growth factor receptor FITC - Fluorescein isothiocyanate FLS - Fibroblast-like synoviocytes FTIR - Fourier transform infrared spectroscopy G GA - Glucosamine GAGs - Glycosaminoglycans GC - Gas chromatography GCs – Glucocorticoids GDF - Growth differentiation factor GM-CSF - Granulocyte-macrophage colony-stimulating factor GR - Glutathione reductase GSH - Glutathione GSSG - Glutathione disulfide H h - Hour H2O2 - Hydrogen peroxide H&E - Hematoxylin and Eosin HA - Hyaluronic acid hACs - Human articular chondrocytes HAP-1 - Synovial fibroblast-homing peptide HEPES - 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPLC - High performance liquid chromatography HRP - Horseradish peroxidase xx Hz - Hertz I 2IT - 2-Iminothiolane IA - Intra-articular ICAM-1 - Intercellular cell-adhesion molecule-1 ICE - Interleukin-1 beta converting enzyme i.e. - “in other words”, form latin id est IFN-γ - Interferon-gamma Ig - Immunoglobulin IGF-1 - Insulin-like growth factor-1 IHC - Immunohistochemistry Ihh - Indian Hedgehog IL - Interleukin IL1ra - Interleukin-1 receptor antagonist IR – Infrared ISO - International Organization for Standardization J JAK - Janus kinase K κ – Kappa KBr - Potassium bromide kDa - Kilodalton kg - Kilogram kV - Kilovolt L L - Liter LPS - Lipopolysaccharide xxi LUVs - Unilamellar liposomes LWT - Limb withdrawal threshold M µ - mu µg - Microgram µL - Microliter µm - Micrometer µM - Micromolar m - Meter M - Molar MEM - Minimum Essential Medium MES - 2-(N-morpholino)ethanesulfonic acid mg - Milligram min - minute miRNA - micro ribonucleic acid mL - Milliliter MLVs - Multilamellar liposomes mm - Millimeter mM – Millimolar mmol – Millimole MMPs - Matrix metalloproteinases mPEG - Methoxypolyethylene glycol amine MRI - Magnetic resonance imaging mRNA - messenger ribonucleic acid mTNF-α - transmembrane tumor necrosis factor alfa MTS - 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfofenyl)-2H-tetrazolium MTV - Multivesicular bodies xxii MTX - Methotrexate mU - Milliunit mV - Millivolt MW - Molecular weight MWCO - Molecular weight cut off N n - Number of independent samples NaCl - Sodium chloride NADH - Nicotinamide adenine dinucleotide NADPH - Nicotinamide adenine dinucleotide phosphate NaHCO3 - Sodium bicarbonate NaOH - Sodium hydroxide NBD Cholesterol - 22-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino)-23,24-Bisnor-5-Cholen-3β-Ol NF-κB - Nuclear factor kappa B NH2 - Amine group NHS - N-hydroxysuccinimide NIR - Near infrared NLS - Nuclear localization signal nm - Nanometers nM - Nanomolar NPC - Nuclear pore complex NPs - Nanoparticles NSAIDs - Non-steroidal anti-inflammatory drugs O O2 - Oxygen OA - Osteoarthritis OARSI - Osteoarthritis Research Society International xxiii OCH3 - Methoxy group OH - Hydroxyl group P p - Statistical level of significance PA - Palmitic acid PAM - Pressure application measurement PAMAM - Poly(amidoamide) PBMCs - Peripheral blood mononuclear cells PBS - Phosphate buffered saline PCL - Polycaprolactone PDI - Polydispersity index pDNA - Plasmid deoxyribonucleic acid PDMA - Poly(2-(diethylamino)ethyl methacrylate) PE - Phosphatidylethanolamine PECs - Polyelectrolyte complexes PEG - Polyethylene glycol pH - Potential hydrogenionic pKa - Negative log of the acid dissociation constant (Ka) PLA - Polylactide PLGA - Poly(lactic-co-glycolic acid) PMA - Phorbol 12-myristate-13-acetate PS - Pristine polystyrene PsA - Psoriatic Arthritis PSA - Polysialic acid R 3Rs - Replacement, Reduction and Refinement RA - Rheumatoid arthritis xxiv RANKL - Receptor activator of the nuclear factor NF-κB ligand RGD - Arginyl-Glycyl-L-Aspartic acid RNA - Ribonucleic acid ROS - Reactive oxygen species Rpm - Rotations per minute RPMI - Roswell Park Memorial Institute RT - Room temperature S s - Seconds SA - Sialic acid SD - Standard deviation SEM - Scanning Electron Microscopy SH - Sulfhydryl group siRNA - small interfering ribonucleic acid SOX - Sry-related HMG box SPIONs - Superparamagnetic iron oxide nanoparticles T 2D - Two-dimensional 3D - Three-dimensional TACE - Tumor necrosis factor-alpha converting enzyme TBTU - 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate TCPS - Tissue culture polystyrene TCZ - Tocilizumab TE - Tris-EDTA TEA - Triethylamine TEM - Transmission Electron Microscopy TGF - Transforming growth factor xxv Th - T helper cells THF - Tetrahydrofuran THP-1 - Human monocyte-like cell line TLC - Thin layer chromatography TLRs - Toll-like receptors TMB - 3,3′,5,5′-Tetramethylbenzidine TNF-α - Tumor necrosis factor-alpha TPP - Triphenylphosphonium cation TRAIL - Tumor necrosis factor-related apoptosis inducing ligand Treg - Regulatory T cells U UV - Ultraviolet UV-Vis - Ultraviolet-visible V VCAM - Vascular cell adhesion molecule VECs - Vascular endothelial cells VEGF - Vascular endothelial growth factor VIP - Vasoactive intestinal peptide (v/v) - Percentage of volume/volume W (w/v) – Percentage of weight/volume (w/w) - Percentage of weight/weight xxxii SHORT CURRICULUM VITAE Ana Cláudia Fernandes Lima was born in 1990 in Guimarães, Portugal. Nowadays, she works as a researcher in 3B's Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, under the supervision of Prof. Nuno M. Neves. She received her MSc degree in 2013 in Pharmaceutical Sciences at the Faculty of Pharmacy of the University of Porto, Portugal with a final grade of 15 (0-20). During the graduation she enrolled in research activities resulting in authorship of a paper entitled “Patulin assessment and fungi identification in organic and conventional fruits and derived products”. In her last year, she had the opportunity to do a threemonth internship at Strathclyde Institute of Pharmacy and Biomedical Sciences in Glasgow, United Kingdom. During that period she was enrolled in the project “Targeting Autophagy during Oncolytic Herpes Simplex Virus type 1 (HSV17+ wild type versus ICP34.5 null mutant HSV1716) cancer treatment”. In 2015, she joined 3B’s Research Group, after being awarded with a grant under the scope of Foundation for Science and Technology (FCT) project Incentivo/SAU/LA0026/2014. In September of the same year, she was awarded with a FCT PhD scholarship (PD/BD/11384/2015), and started the PhD program in Tissue Engineering, Regenerative Medicine and Stem Cells (TERM&SC) in the University of Minho, being the main focus of her research the development of nanoparticles to treat arthritic diseases. During her PhD, she has been involved in the preparation of ICVS/3Bs and FCT project proposals. Additionally, she collaborated with her colleagues in different works, and also with different universities. During 3 months, she was a visiting student at the Faculty of Engineering in Chulalongkorn University, Bangkok, Thailand, studying the biological response of cells encapsulated into silk fibroin DMPG hydrogels. As a result of her research work, she is author or co-author of 7 papers in international journals (2 published, 5 submitted), 2 issued patents, 3 oral presentations and 5 poster presentations. She attended several important international meetings in the field of tissue engineering and regenerative medicine. xxxiii LIST OF PUBLICATIONS The work performed during the PhD period resulted in the publications listed below. Papers in international scientific journals with referees (as first author) 1. Lima A. C., Cunha C., Carvalho A., Ferreira H., Neves N. M. Interleukin-6 Neutralization by Antibodies Immobilized at the Surface of Polymeric Nanoparticles as a Therapeutic Strategy for Arthritic Diseases. Acs Appl Mater Inter . 2018, 10 (16): p. 13839-50. 2. Lima A. C., Ferreira H., Reis R. L., Neves N. M. Biodegradable polymers: an update on drug delivery in bone and cartilage diseases. Expert Opin Drug Deliv . 2019, 16 (8): p. 795-813. 3. Lima A. C., Amorim D., Laranjeira I., Almeida A., Reis R. L., Ferreira H., Pinto-Ribeiro F., Neves N. M. Nanoparticle-mediated neutralization of IL-6 and TNF-α for osteoarthritis treatment. (Submitted) . 2019. 4. Lima A. C., Campos C. F., Cunha C., Carvalho A., Reis R. L., Ferreira H., Neves N. M. Interleukin-23 neutralization by biofunctionalized liposomes encapsulating gold nanoparticles for the treatment of rheumatoid arthritis. (Submitted) . 2019. 5. Lima A. C., Ferreira H., Neves N. M. Enzymaticand redox-responsive polymeric micelles for targeted and controlled drug delivery on arthritic diseases. (Submitted). 2019. 6. Lima A. C., Ferreira H., Neves N. M. Cellular uptake of nanoparticles in an inflammatory arthritis scenario. (Submitted). 2019. Papers in international scientific journals with referees (as co-author) 1. Ferreira H., Amorim D., Lima A. C., Pirraco R., Costa Pinto A. R., Almeida R., Almeida A., Reis R. L., Pinto-Ribeiro F., Neves N. M. In vitro and in vivo compatibility of an injectable hydrogel for neurodegenerative diseases treatment. (Submitted). 2019. Conference abstracts published in international scientific journals (as first author) 1. Lima A. C., Cunha C., Carvalho A., Ferreira H., Neves N. M. Advanced treatment for arthritic diseases based on the capture and inactivation of interleukin-6 by biofunctionalized polymeric nanoparticles. European cells and materials. 2017, Vol. 33 Suppl. 2. xxxiv Conference oral presentations (as first author and speaker) 1. Lima A. C., Cunha C., Carvalho A., Ferreira H., Neves N. M. Advanced treatment for arthritic diseases based on the capture and inactivation of interleukin-6 by biofunctionalized polymeric nanoparticles. TERMIS-EU 2017, Davos, Switzerland. 2. Lima A. C., Cunha C., Carvalho A., Ferreira H., Neves N. M. Capture and neutralization of interleukin-6 by the intra-articular injection of biofunctionalized polymeric nanoparticles as advanced treatment for arthritic diseases. Chem2Nature 2017, Porto, Portugal. 3. Lima A. C., Amorim D., Laranjeira I., Almeida A., Reis R. L., Ferreira H., Pinto-Ribeiro F., Neves N. M. Pro-inflammatory Cytokines neutralization by intra-articular injection of biofunctionalized nanoparticles as advanced treatment for osteoarthritis. TERMIS 2019, Rhodes, Greece. Conference oral presentations (as co-author) 4. Lima A. C., Campos C. F., Cunha C., Carvalho A., Reis R. L., Ferreira H., Neves N. M. IL-23 inactivation by targeted liposomes to mediate the regression of autoimmune diseases. TERMIS 2019, Rhodes, Greece. Conference posters (as first author) 1. Lima A. C., Cunha C., Carvalho A., Ferreira H., Neves N. M. Biofunctionalized polymeric nanoparticles for a prolonged and local capture of pro-inflammatory cytokines in arthritic joints. TERM STEM 2016, Guimarães, Portugal. 2. Lima A. C., Amorim D., Almeida A., Reis R. L., Ferreira H., Pinto-Ribeiro F., Neves N. M. Interleukin-6 and Tumour Necrosis Factor-α neutralization by biofunctionalized nanoparticles after intra-articular injection for osteoarthritis treatment. TERM STEM 2017, Guimarães, Portugal. 3. Lima A. C., Amorim D., Almeida A., Reis R. L., Ferreira H., Pinto-Ribeiro F., Neves N. M. Intra-articular injection of biofunctionalized nanoparticles to neutralize Interleukin-6 and Tumour Necrosis Factor-α to treat osteoarthritis. Chem2Nature 2018, Porto, Portugal. 4. Lima A. C., Amorim D., Laranjeira I., Almeida A., Reis R. L., Ferreira H., Pinto-Ribeiro F., Neves N. M. Neutralization of Pro-inflammatory Cytokines by Intra-articular Injection of Biofunctionalized Nanoparticles as an Advanced Treatment for Osteoarthritis. Society for Biomaterials Annual Meeting and Exposition 2019, Seattle, USA. xxxv 5. Lima A. C., Amorim D., Laranjeira I., Almeida A., Reis R. L., Ferreira H., Pinto-Ribeiro F., Neves N. M. A dual targeting strategy using antibodies immobilized at the surface of biodegradable nanoparticles for local osteoarthritis treatment. Achilles 2019, Porto, Portugal. Patents 1. Lima A. C., Reis R.L., Ferreira H., Neves N. M. Phosphatidylcholine liposomes, methods and uses thereof. ( Filed , 2019). 2. Lima A. C., Reis R.L., Ferreira H., Neves N. M. Polymeric micelle, methods of production and uses thereof. ( Filed , 2019). Awarded grants 1. Foundation for Science and Technology (FCT) PhD scholarship (PD/BD/11384/2015). xxxvi INTRODUCTION TO THE THESIS FORMAT The present thesis is divided into six main sections (1 to 6) containing eight chapters (I to VIII). This structure was adopted to allow for a comprehensive organization of the data presented in the various chapters, preceded by a general introduction (Section 1, Chapter I), as well as an overall materials and methods section (Section 2, Chapter II). Section 3 (Chapters III to V) focuses on the development of the different nanoparticle (NP) formulations to treat arthritic diseases as well as their in vitro evaluation. Section 4 (Chapter VI) provides further evidence regarding the NPs internalization by relevant cells. Section 5 (Chapter VII) describes an in vivo study performed to assess the safety and therapeutic efficacy of the functionalized polymeric NPs. To finalize, Section 6 (Chapter VIII) completes this thesis with the concluding remarks and future perspectives. The main body of the thesis is based on a series of manuscripts either published or submitted for publication in international journals. Each chapter is presented in a manuscript form, i.e., abstract, introduction, experimental section, results, discussion, conclusion and acknowledgements. A list of relevant references is also provided as a subsection within each chapter. The contents of each part and chapter are described below in more detail. Section 1 – General introduction Chapter I – Nanoparticles for arthritic diseases treatment: This chapter provides a general introduction on the pathogenesis and key molecular targets in arthritis, discussing the most successful and novel drugs in arthritis therapy, and also highlights recent trends in nanotechnology-based drug delivery approaches. Section 2 – Experimental section Chapter II – Materials and Methods: A list of the materials and methods used to obtain the results presented in this thesis is provided. The section is intended to provide additional details that are not included in the materials and methods section of published manuscripts. Section 3 – Nanoparticles development and in vitro evaluation In this section, we do report the design and production of novel nanocarriers to enhance the therapeutic efficacy of relevant drugs while reducing their adverse side effects. The experimental work involves the characterization of the developed NPs, the evaluation of the drug loading capacity, the assessment of their in vitro cytocompatibility and their biologic activity. xxxvii Chapter III - Interleukin‑6 neutralization by antibodies immobilized at the surface of polymeric nanoparticles as a therapeutic strategy for arthritic diseases: This chapter describes the production of biodegradable polymeric NPs with anti-IL-6 antibodies immobilized at their surface. The biologic activity of the biofunctionalized NPs was assessed in chondrocytes stimulated with macrophage conditioned medium to model the exposure of the cells to inflammation. Chapter IV - Interleukin-23 neutralization by biofunctionalized liposomes encapsulating gold nanoparticles for the treatment of rheumatoid arthritis: This chapter describes the production of liposomes encapsulating AuNPs and immobilizing anti-IL-23 antibodies. Peripheral blood mononuclear cells of healthy donors and RA patients were activated though Th17 differentiation, being assessed if the biofunctionalized liposomes were able to prevent the production of IL-17A. Chapter V - Enzymaticand redox-responsive polymeric micelles for targeted and controlled drug delivery on arthritic diseases: This chapter describes the development of polymeric micelles responsive to redox and glutathione reductase activity for targeted and controlled delivery of dexamethasone. The biologic efficacy of this strategy was assessed in a co-culture model of inflammatory arthritis. Section 4 – Internalization studies Chapter VIII – Cellular uptake of nanoparticles in an inflammatory arthritis scenario: This chapter aims to study the internalization degree and pathways of the NPs developed in Section 3 in a normal and inflammatory scenario by different cells, namely endothelial cells, chondrocytes and macrophages. Flow cytometry and confocal microscopy analyses were both used to evaluate the cellular NPs uptake. Section 5 – In vivo studies Chapter VII – Nanoparticle-mediated neutralization of IL-6 and TNF-α for osteoarthritis treatment: In this chapter the polymeric NPs developed in Chapter III, but functionalized with anti-TNF-α antibodies beyond anti-IL-6 antibodies, were studied in a co-culture model of inflammatory arthritis and in an experimental carrageenan-induced arthritis rat model. The objective was to confirm the in vitro results and to provide a more substantiated conclusion on their safety and therapeutic efficacy. Section 6 – Concluding remarks Chapter VIII – General conclusions and future perspectives: The final section of the thesis presents the general conclusions and implications, current limitations and potential of the work previously described for the treatment of arthritis. Furthermore, it is highlighted the future directions for the work developed in this thesis in order to move forwards into clinical studies. xxxviii “ Logic will get you from A to B. Imagination will take you everywhere.” Albert Einstein 1 SECTION 1 GENERAL INTRODUCTION 2 Chapter I Nanoparticles for arthritic diseases treatment Chapter I – Nanoparticles for arthritic diseases treatment 3 Chapter I Chapter I - Nanoparticles for arthritic diseases treatment1 ABSTRACT Osteoarthritis (OA) and rheumatoid arthritis (RA), the most common types of arthritis, are chronic inflammatory diseases characterized by synovial joint inflammation and cartilage and bone tissue destruction. Despite the breakthroughs in the field of drug discovery, there is currently no cure for arthritis. Indeed, the unique physiology of the joint capsule protecting the cartilage and bone tissues makes the systemic delivery of free drugs to the joints very challenging. Consequently, effective and targeted delivery systems for arthritic diseases are of utmost importance. Among a wide variety of drug delivery devices, the unique properties of the nanoparticles (NPs) make them highly attractive for the design of carriers that enable a targeted and temporal controlled release of one or more drugs in concentrations within the therapeutic range. Moreover, the increased knowledge about biomaterials science and of the pathophysiology of diseases, biomarkers and targets as well as the development of innovative tools has led to the design of high value-added nanomedicines. However, some challenges persist and are mainly related with an appropriate residence time and a controlled and sustained release over a prolonged period of time of the therapeutic agents. This chapter focuses on the analysis of the pathogenesis and key molecular targets in arthritis, discussing the most successful and novel drugs in arthritis therapy, and also highlight recent trends in the nanotechnology-based drug delivery approaches. Herein, nanomedicines that showed great promise in improving arthritis treatment are reviewed, namely liposomes, polymeric NPs, polymeric micelles, dendrimers and metallic NPs. 1This chapter is based on the following publication: A.C. Lima, H. Ferreira, R. L. Reis, N. M. Neves. Biodegradable polymers: an update on drug delivery in bone and cartilage diseases. Expert Opin Drug Deliv. 2019, 16 (8): p. 795-813. Chapter I – Nanoparticles for arthritic diseases treatment 10 IL-1β Activate leukocytes, endothelial cells, chondrocytes, osteoclast and synovial fibroblasts; Induce MMPs that degrade the cartilage; Inhibits the synthesis of hyaline cartilage. Chondrocytes Osteoblasts Cells forming the synovial membrane Mononuclear cells IL-23 Induces Th17 differentiation. Activated dendritic cells Macrophages Monocytes IL-17A Activation and expression of pro-inflammatory cytokines (IL-6, IL-8, TNF-α), chemokines and MMPs. Stimulated CD4+ T cells Mast cells Figure I-3 – Protein structure of (A) TNF-α, (B) IL-6, (C) IL-1β, (D) IL-23 and (E) IL-17A. I-2.2. Treatment In the last years, the increased knowledge of the physiopathology of arthritic diseases led to a dramatic change in the available treatment modalities. Therefore, the term “drug” is not limited to the conventional therapeutic agents commonly used (e.g. anti-inflammatory drugs), including also recombinant proteins and genes. Recombinant proteins are used as highly effective medical treatments for a wide range of diseases in which a protein is either lacking or present in a deficient amount (e.g. anti- Chapter I – Nanoparticles for arthritic diseases treatment 11 inflammatory cytokines), or is abnormally highly expressed (e.g. antibodies to neutralize the excess of pro-inflammatory cytokines) [39]. Consequently, therapeutic proteins can include antibodies, hormones, growth factors, anticoagulants, blood factors, enzymes, Fc fusion proteins, interferons, interleukins and thrombolytic drugs, which are been produced by recombinant DNA technology. As there is no current cure for OA and RA, the most commonly used therapeutic strategies include analgesics, non-steroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids (GCs) [40]. Moreover, disease-modified anti-rheumatic drugs (DMARDs) and biological agents are the first line of treatment in RA in order to relieve joint damage and control the disease progression. However, despite all recent clinical trials, until date there is no DMARDs and biological agents approved for OA treatment [41]. Taking into consideration the mechanisms of initiation and progression of both diseases, in RA the systemic therapy is generally indicated and appropriated, while in OA the local therapy may offer particular advantages over systemic therapy [42, 43]. Nowadays, IA injections of HA and GCs are standard treatment options for the management of OA-related knee pain. Despite the advances in developing new drugs to the key molecular targets of the disease onset and progression, their modest therapeutic effects are mainly attributed to their poor bioavailability at sites of disease. Indeed, many of these new therapeutic strategies are associated with short half-life, and poor pharmacokinetic distribution to the specific site of disease. Additionally, systemically administered agents with ubiquitous therapeutic targets are associated with severe side effects. In the following section, a brief description of the treatment modalities available for OA and RA will be provided. Table I-2 summarizes the current options in the clinical practice. Chapter I – Nanoparticles for arthritic diseases treatment 12 Table I-2 – Summary of the available treatments for OA and RA. Therapeutic Classification Drugs/Agents Mechanism of action Side Effects Disease Ref Analgesics Acetominophen Prostaglandin inhibitor Hepatic OA [44] NSAIDs Ibuprofen Indometacin Celecoxib Rofecoxib Valdecoxib COXs inhibitors Immunomodulation Gastrointestinal reaction Kidney and cardiac dysfunction OA RA [45] GCs Dexamethasone Hydrocortisone Prednisone Betamethasone Methylprednisolone Immunosuppression Osteoporosis Hyperglycemia Insulin resistance Hypertension OA RA [46] Opioids Hydrocodone Tramadol Analgesia through acting in opioid receptors Gastrointestinal reaction Abuse and addiction OA RA [47] DMARs Methotrexate Hydroxychloroquin Sulfasalazine Clodronate Leflunomide Immunosuppression Disease-modifying activity Myelosuppression Gastrointestinal reaction Liver and kidney dysfunction RA [48] Biological agents Anticytokines Etanercept Infliximab Adalimumab Certolizumab pegol Golimumab TNF-α inhibitor Infection Tuberculosis RA [49] Tocilizumab Sarilumab IL-6 receptor inhibitor Infection Gastrointestinal perforation Anakinra IL-1 receptor inhibitor Infection Denosumab RANKL inhibitor Infection Anti-T cell Abatacept T cell activation inhibitor (binding to CD80 and CD86 receptors) Infection Malignancy Anti-B cell Rituximab B-cell depletion (binding to CD20 receptor) Infection Hypertension Kinase inhibitors Baricitinib Tofacitinib JAK 1 and 2 inhibitors Infection Chapter I – Nanoparticles for arthritic diseases treatment 13 I-2.2.1. Analgesics and anti-inflammatory drugs Analgesics are the most prescribed medicines in OA, mainly due to the lack of more effective treatments approved [50]. Regarding RA, they are no longer considered the first-line treatments, being only used as an adjunctive in symptomatic therapy or during the short phase until a diagnosis is established [51]. Acetaminophen, also known as paracetamol, is a simple analgesic that has both analgesic and antipyretic actions [44]. Due to its relative safety and effectiveness, most guidelines recommend acetaminophen as the first-line oral analgesic for mild-to-moderate OA [52]. For patients with severe symptoms or who do not respond to acetaminophen, more potent drugs should be considered, such as NSAIDs. NSAIDs provide anti-inflammatory and analgesic effects, presenting better pain relief profiles than acetaminophen in moderate-to-severe OA [45]. They inhibit cyclooxygenase (COX) enzyme, which is involved in the conversion of arachidonic acid to prostaglandins. Traditional NSAIDs (e.g. ibuprofen and indometacin) are non-selective COX inhibitors, inhibiting both COX-1 and COX-2. Although the efficacy of NSAIDs for OA treatment has been well documented, the health concerns seriously restrict their extensive application. Indeed, 30% of the patients developed adverse effects, especially gastrointestinal complications [53]. In the last years, selective COX-2 inhibitors (e.g. celecoxib, rofecoxib and valdecoxib) appeared safer than traditional NSAIDs. Nevertheless, they are also associated with potential risk of serious adverse cardiovascular events. Therefore, there is a balance between the efficacy and safety of NSAIDs and, consequently, the benefit/risk ratio should be considered when prescribing these drugs. Osteoarthritis Research Society International (OARSI) guidelines recommend the use of NSAIDs at the minimum effective dose, avoiding their prolonged use. GCs, such as dexamethasone (Dex) and prednisolone, have potent anti-inflammatory and immunesuppressive actions [54]. Despite the good therapeutic outcomes, their use is severely hampered, due to the risk of developing serious side effects, such as osteoporosis, hyperglycemia, insulin resistance and hypertension. Thus, GCs are usually limited in cases of moderate to severe OA, when NSAIDs are not effective [55]. Moreover, in order to minimize their systemic absorbance and avoid possible negative effects, they are frequently administered locally into the OA joint. In RA, the guidelines recommend the administration of low-dose GCs immediately after the diagnosis, followed by longer term modulation of inflammation using DMARDs (in I-2.2.2. section) [46]. Chapter I – Nanoparticles for arthritic diseases treatment 14 When other pharmacological agents are either ineffective or contraindicated, opioids are considered for the treatment of refractory pain in patients with moderate-to-severe OA [52]. Despite the lack of information, it seems that a substantial number of patients with RA are also treated with opioids following the Centers for Disease Control and Prevention (CDC) guideline [56]. Indeed, opioids have outstanding efficacy regarding analgesia, however, they present frequent adverse effects, including nausea, vomiting, dizziness, constipation, sleepiness, tiredness and headache, which may outweigh the benefits in pain relief [57]. Moreover, there are other potential risks on increasing abuse and/or addiction, morbidity and mortality. I-2.2.2. DMARDs A DMARD is defined as a medicine that interferes with signs and symptoms of RA, improves physical function and inhibits progression of joint damage [58]. Traditional DMARDs include methotrexate (MTX), leflunomide, hydroxychloroquine, and sulfasalazine. MTX is currently the first-line therapy for RA, due to its efficacy in reducing the signs and symptoms and preserving cartilage function with relative safety. This drug presents a large dose-titratable range in oral or parenteral formulations and a good ratio benefit/cost [59]. While MTX monotherapy is recommended as an initial pharmacological strategy, it can also be used in combination with a wide variety of other drugs. Indeed, a combination of several DMARDs or a DMARD plus biological agents has been demonstrated to be favorable for the therapy outcome. Nevertheless, their adverse side effects can range from mild (rash, nausea, vomiting, stomatitis) to severe, including hepatotoxicity and bone marrow toxicity [48]. I-2.2.3. Biological agents Biological therapy or immunotherapy is referred to the use of recombinant proteins intended to activate or suppress the activity of the immune system [60]. In inflammatory diseases, such as OA and RA, immunotherapies aim to suppress/reduce the immune system activity (e.g. by neutralizing proinflammatory cytokines and by binding and blocking receptors that trigger immune-cells activation) or to eliminate and regulate immune cells that contribute to tissue damage (e.g. effector lymphocytes). Recently, numerous biological agents have been approved for clinical practice, being even more under the development stage [61]. One of the highest selling class of biologicals since 2009 are the monoclonal antibodies [62]. Indeed, they gained significant attention due to their high specificity and potency [63]. Chapter I – Nanoparticles for arthritic diseases treatment 15 Biological agents for the treatment of arthritic diseases can also be referred as biologic DMARDs (bDMARDs). They present a rapid improvement in clinical symptoms and delayed radiographic progression [64]. However, despite the success of these biological agents, up to 30% of patients with RA still may not respond adequately, and can present an increased risk of infections, along with other side effects, including malignancy, hypertension and administration reactions. The first and most frequently used biological agents in RA are the TNFα inhibitors. Indeed, five different types of TNF-α inhibitors are currently licensed for clinical practice in RA treatment, namely infliximab, entarnecept, adalimumab, certalizumab pegol and golimumab [65]. Based on the currently available literature, TNF inhibitors became the first choice of bDMARDs therapy in RA patients that do not respond or are intolerant to the conventional DMARD treatment. For OA treatment, current evidence from the available studies with TNF-α blockers are still controversial, since while some clinical trials reported promising results, others failed to demonstrate a significant clinical improvement [66-68]. The first humanized anti-IL-6 receptor antibody, tocilizumab, is worldwide approved to treat RA, due to its outstanding clinical efficacy [69]. Recently, sarilumab was also approved, being many other IL-6 inhibitors in development or in clinical trials, including sirukumab and olokizumab. Additionally, IL-6 blocking is also in clinical trials to treat OA [70]. In contrast to the success of TNF-α blockade in arthritis, IL-1β inhibitors, such as anakinra (antagonist of the IL-1 receptor) have less efficiency in the suppression of joint inflammation in patients with RA [71]. Despite being abundantly expressed in RA, the paradox of this modest clinical outcome is not fully understood, but maybe reflects the redundancy of the IL-1 receptor signaling pathways. IL-23 and IL-17A inhibitors are still in clinical trials for RA treatment [32]. For instance, the anti-IL23 inhibitors ustekinumab and guselkumab were approved by the Food and Drug Administration (FDA) for Psoriatic Arthritis (PsA) treatment and moderate to severe plaque psoriasis, respectively [72]. Guselkumab is also in clinical trials for pustular psoriasis (phase III, NCT02343744), PsA (phase III, NCT0315828, NCT03162796), and RA (phase II, NCT01645280). Many other anti-IL-23 antibodies are under clinical evaluation for several immune-mediated conditions. Even though, in a phase II clinical trial, the signs and symptoms of RA were not significantly reduced by guselkumab and ustekinumab after subcutaneous administration [73], more clinical trials should investigate other therapeutic conditions (e.g. route of administration, doses, time of treatment and concomitant administration of other drugs) [74]. The IL-17A inhibitors, ixekizumab and secukinumab, were FDA approved for plaque psoriasis, ankylosing spondylitis and PsA [72]. Ixekizumab and secukinumab improved RA signs and symptoms in RA patients Chapter I – Nanoparticles for arthritic diseases treatment 16 that show an inadequate response to TNF inhibitors, in Phase II and III clinical trials, respectively [75, 76]. I-2.2.4. Gene therapies Gene therapy is based on the intentional modulation of gene expression in specific cells by introducing exogenous nucleic acids to induce the production of proteins (plasmid deoxyribonucleic acid –pDNA, complementary DNA –cDNA, messenger ribonucleic acid –mRNA– and microRNA –miRNA), or to inhibit the transduction of harmful proteins (small interfering RNA –siRNA– or antisense oligonucleotides) [77, 78]. There are two different approaches to deliver nucleic acids to the targeted tissues: direct (using viral or non-viral vectors) or transduced cell-mediated (by in vitro genetic manipulation of cells). Even though in recent years, a vast number of therapeutic gene approaches have demonstrated effectiveness in preclinical models, only a few have moved forward into clinical trials [79]. Indeed, recent studies have demonstrated the efficacy and safety of an ex vivo gene therapy that contains non-transduced and transduced human allogeneic chondrocytes with the TGF-β1 gene [80, 81]. Due to the clinical effectiveness of TissueGene-C (InvossaTM), it was recently approved in Korea for treatment of moderate knee OA, and it is currently in a Phase III clinical trial in the USA. In arthritic diseases, gene therapy essentially focus on increase the expression of secreted proteins, such as growth factors (IGF-1, TGF-β, bone morphogenetic proteins –BMPs, basic fibroblast growth factor –bFGF, growth differentiation factor –GDF-5 and VEGF antagonist) and anti-inflammatory cytokines (IL10, IL-1 receptor antagonist –IL1ra, and anti-inflammatory mediators). More recently it also focus on intracellular and/or signaling components, such as transcription factors (SOX genes and ZNF145) and small, regulatory nucleic acids (miR-23b, miR-140, miR-181b, miR221, miR-145, miR-335) [82]. I-3. NANOMEDICINES Two concepts introduced in the 19th and in the 20th centuries have been revolutionizing the medical field, namely the magic bullet and nanotechnology. The first concept was coined to Paul Ehrlich, in 1900, and is related with a limited effect of the drugs on the cellular target [83]. Therefore, the linking of a targeting moiety to a drug will increase its therapeutic index. Moreover, the assembling of this concept to nanotechnology has provided significant progresses in the diagnostic, treatment and prevention of human Chapter I – Nanoparticles for arthritic diseases treatment 17 diseases. The term nanotechnology has been assigned to Richard Feynman, in 1959 [84], but Norio Taniguchi was the first scientist to use that word at 1974 [85]. Nanotechnology embraces “The design, characterization, production and application of structures, devices, and systems … at the nanometre scale” [86], “with at least one novel/superior characteristic or property” [87]. Although the International Organization for Standardization (ISO/TS 80004-1:2015) defines nanoscale as the “length range approximately from 1 to 100 nm”, there is considerable controversy among the scientific community especially for the upper limit. Indeed, a straight relationship between size and novel effects or functions for different materials does not exist [88]. Therefore, despite the nanoscale definition, in the literature nanostructures frequently include sub-micron particles (1 nm to 1000 nm). The drug delivery field has been advanced and reinforced mainly due to the development of novel and innovative technologies, and the remarkable increase of knowledge about materials science and pathophysiology, biomarkers and targets of the diseases [89]. Thus, the application of nanotechnology in the diagnosis, treatment and prevention of diseases is defined as nanomedicine. It includes NPs, both nanospheres and nanocapsules, liposomes, micelles and dendrimers. In addition, polymer-drug conjugates (including proteins and antibodies-conjugates) are also classified as nanomedicines. With an appropriate nanomedicine it is possible to circumvent important drawbacks of the conventional therapies, namely (i) to decrease the dose of drug administered (by avoiding its metabolism/degradation, clearance and distribution in non-target tissues), (ii) to abolish or drastically reduce the systemic side effects (by targeting delivery, which will enhance the pharmacokinetics and pharmacodynamics of the drug and, consequently, will increase its therapeutic index) and (iii) to reduce the frequency of administration (by the sustained release of therapeutic concentrations of the drug over time). Therefore, an appropriate delivery system can recover withdrawn drugs from the market by overcoming their side effects in nontarget tissues/organs [90-92]. A rational design of a delivery system should consider the nature of the drug to be incorporated (e.g. hydrophobic, hydrophilic or amphipathic), the mechanisms that will control its release (e.g. diffusion, carrier degradation or dissolution, cleavage of chemical bonds, and external, physiological or pathological stimulus) and the disease (e.g. cell/tissue to target or tissue pH and vascularization). Ideally, the drug must be incorporated into the delivery device, being released only in the target cells or tissues in concentrations within the therapeutic range. Moreover, depending on the mechanism of action of the therapeutic agents (e.g. binding to a cell membrane receptor or to an intracellular or nuclear target), the design of delivery carriers should be carefully considered. The selection of the most adequate composition Chapter I – Nanoparticles for arthritic diseases treatment 18 is crucial to obtain a nanocarrier with the desirable drug release properties. Additionally, the preparation method as well as the physicochemical properties of the delivery device (e.g. size and degradation rate in the biological environment) will also influence the release of the drugs [93]. Efforts were also made to achieve a drug release in a pulsatile fashion, triggered by changes in the neighboring milieu (self-regulated delivery systems using different mechanisms, such as pH-sensitive polymers, enzymes, illness markers and pH-dependent drug solubility) or by an external stimulus (externally triggered systems by a magnetic, thermal, ultrasonic, electric or irradiation stimulus) [94-97]. I-3.1. Targeting strategies The targeted delivery of a drug can be either passive or active. Passive targeting is widely investigated mainly in cancer and inflammatory conditions, due to the leaky vasculature or enhanced permeability and retention (EPR) effect [98]. For this and for many other features (e.g. drug release and interaction with cells [99]), the size as well as the surface and shape of the delivery systems are crucial. An active targeting is achieved by attaching to the drug or to the surface of the delivery devices a particular ligand that ideally will bind to a moiety specifically found in a specific organ, tissue or cell of interest (Figure I-4). Chapter I – Nanoparticles for arthritic diseases treatment 19 Figure I-4 – Examples of targeting moieties used to treat arthritic diseases. I-3.1.1. Cartilage targeting The peculiarity of cartilage structures difficult the attainment of drug concentrations required to elicit the desired biological response at the cell and matrix targets. Therefore, there is a huge interest in designing nanocarriers to selectively target cartilage in order to deliver the drug where its therapeutic action is required. Cartilage is avascular what constitutes a major obstacle for drugs as well as for nanomedicines to diffuse and enter in its ECM. Therefore, local administration via intra articular (IA) injection in the joint space has been chosen in detriment of systemic administration to increase the drug bioavailability and to reduce drug dosage, systemic exposure and adverse events. Unfortunately, drugs injected into the joints are normally cleared very quickly (half-life of 0.1 to 6 h), which is even higher in the presence of inflammatory conditions, as in RA and OA. In addition, limited cartilage targeting also limits the therapeutic efficacy of the drugs [100]. To penetrate in the cartilage ECM, the design of a nanocarrier should consider its highly anionic charge and dense nature that leads to a 60 nm mesh size provided by the type II collagen [101] and the  3.2 and 4.4 nm of space between GAGs chains along fetal and mature aggrecan, Chapter I – Nanoparticles for arthritic diseases treatment 26 articular cartilage in CIA mice model. Folate-modified dextran–MTX conjugate micelles (noted as Dex-gMTX/FA) were developed for targeting delivery to macrophages [162]. The micelles shown higher cellular uptake mediated by the folate receptor and higher cytotoxicity toward lipopolysaccharide activated macrophages. Moreover, Dex-g-MTX/FA possessed improved biodistribution at the lesion site and stronger inhibition of pro-inflammatory cytokines, which significant suppressed the synovitis and effectively protected the articular cartilage. In another study, MTX loaded into sialic acid-dextranoctadecanoic acid (SA-Dex-OA/MTX) micelles considerably improved accumulation and transport to arthritic paws presenting a high expression of E-selectin [163]. In a CIA rat model, the micelles significantly inhibited the inflammatory response, diminished the adverse effects of MTX, and increased the bone mineral density. Cationic micelles composed of the diblock copolymer of PLGA and poly(2-(diethylamino)ethyl methacrylate) (PDMA) self-assembled in 40 nm size structures. They scavenge cell-free DNA (cfDNA) derived from RA patients and inhibit the activation of primary synovial fluid monocytes and FLS [164]. As cfDNA exacerbates the pathogenesis of RA, the intravenous injection of the cationic micelles into a CpGinduced mouse or CIA rat mode relieved RA symptoms, including ankle and tissue swelling, and bone and cartilage damage. The positive therapeutic outcomes were also corroborated with the determination of intracellular trafficking, biodistribution, cfDNA levels in systemic circulation and inflamed joints, and cytokine levels in the joints. This innovative work suggests a new direction in treating inflammatory diseases though the effective and safe removal of pathogenic damage-associated molecular patterns (DAMP) molecules. Natural polyphenols, such as curcumin (Cur), were extensively studied as therapeutic agents for various diseases, due to their remarkable anti-inflammatory, antioxidant, antitumor and antimicrobial activities. A novel anti-RA approach composed of HA/Cur micelles was able to overcome the poor bioavailability of Cur, being also able to exert a lubricating action in the joints [165]. When IA injected in a complete Freund’s adjuvant (CFA) and Collagen II RA rat model, the micelles significantly decreased the degree of edema and the expression of pro-inflammatory cytokines (TNF-α and IL-1) and VEGF, which resulted in a marked inhibition of the inflammatory response. Moreover, the friction between the cartilage surfaces of the joints was reduced, protecting the cartilage from further degradation. Chapter I – Nanoparticles for arthritic diseases treatment 27 I-3.2.4. Dendrimers Dendrimers (≈ 2-100 nm in diameter) are highly branched polymeric structures with enhanced functionality, due to the presence of several functional groups at their surface [166, 167]. The functional groups exhibit a high degree of molecular uniformity, which limits the molecular weight distribution and their size. Poly(amidoamide) (PAMAM) dendrimers of generation 5 were conjugated with MTX and FA to target inflammation-activated folate receptor overexpressing macrophages [168]. In an AIA rat model, G5MTX and G5-FA-MTX had similar preventive effects on the development of arthritis as MTX. Importantly, G5-FA-MTX conjugates significantly decreased the spleen toxicity of MTX and, consequently, further studies are needed to determine whether other side effects of MTX are also attenuated. PAMAM dendrimers functionalized with PEG and conjugated with a growth factor, IGF-1, were designed for targeted delivery to chondrocytes and retention within the joint cartilage after IA injection [169]. The cationic nanoformulation was capable of enhancing drug therapeutic lifetime by 10-fold for up to 30 days and cartilage penetration to at least 1 mm within articular joints in 2 days. IGF-1 efficacy was enhanced by the dendrimer-IGF-1 formulation in protecting both cartilage and bone in a rat surgical model of OA, reducing the total area and width of medial tibial cartilage degeneration, as well as total volume of osteophytes in the joint. Dendrimers can be functionalized with other NPs or polymers to combine the advantages and overcome the limitations of both systems. NanoGold-core multifunctional dendrimer were designed for pulsatile chemo-, photothermaland photodynamictherapy of RA [170]. The strategy of comprising gold (Au) NPs, MTX and IR780 into a dendrimer exhibited an important negative role in reactive oxygen species (ROS) generation in vitro and therefore may provide a synergistic opportunity to effectively treat RA. Cationic dendronized polymers (cDenpols) were designed and synthesized using PCL and different generations of cationic PAMAM dendrons to effectively eliminate cfDNA [171]. In a CIA rat model, cDenpols with longer backbones and higher charge densities were preferentially accumulated in the inflamed joint, resulting in the inhibition of joint swelling, synovial hyperplasia and bone destruction. I-3.2.5. Inorganic NPs Inorganic NPs include iron-oxide NPs, silica-Au nanoshells, AuNPs and quantum dots [172, 173]. They are widely applied in nanomedicine, due to their unique size and magnetic properties or enhanced Chapter I – Nanoparticles for arthritic diseases treatment 28 optical absorption. Indeed, theranostic systems (combining both therapeutic treatment and in vivo imaging) can be designed. Hyaluronate/AuNP/Tocilizumab (HA-AuNP/TCZ) complex was developed to synergistically target the VEGF, since AuNPs have angiogenic effects, and the IL-6 receptor (TCZ is a humanized monoclonal antibody against IL-6 receptor) [174]. While in vitro results confirmed the simultaneous antiangiogenic and anti-inflammatory effects of the dual targeting, in vivo results using a CIA mouse model only showed anti-inflammatory therapeutic efficacy. Manganese ferrite and ceria NP-anchored mesoporous silica NPs (MFC-MSNs) were designed to efficiently generate O2 and scavenge ROS for alleviating inflammation through M1 to M2 polarization of macrophages in RA [175]. IA injection of MFC-MSNs to an AIA rat model of RA alleviated hypoxia, inflammation and pathological features in the joint. Additionally, the encapsulation of MTX in the nanocarrier led to its sustained release and to the increment of the therapeutic effect of MFC-MSNs. In imaging, iron oxide NPs, also termed superparamagnetic iron oxide NPs (SPIONs) coated with Dex and glucose were used for the detection and diagnosis of arthritis [176]. In addition, FA was conjugated to these SPIONs for better targeting to the activated macrophages in inflamed sites. The results demonstrated that this imaging system provides an enhanced contrast effect in vivo . Table I-4 – Examples of nanomedicines for arthritic diseases. Type Formulation Drug Targeting rationale Property/function Condition Ref. Liposomes Dex-loaded liposomes Dex Passive targeting through the EPR effect Dex-loaded liposomes demonstrated superior therapeutic efficacy against arthritis and reduced side effects. Arthritis [130] FA-liposomes Betamet hasone Folate receptor of macrophages via FA Due to the selectively accumulation in arthritic rat paws, FA-liposomes encapsulating exhibited superior therapeutic efficacy. RA [137] PEGylated liposomes conjugate with HAP-1 Predniso lone FLS via HAP-1 HAP-1 modified liposomes showed a 10 fold increase localization in affected joints compared to unaffected joints and enhanced therapeutic index in an AIA rat model. Arthritis [139] Chapter I – Nanoparticles for arthritic diseases treatment 29 Polymeric NPs PEG-PLA NPs Betamet hasone - A single injection of the NPs system resulted in complete remission of the inflammatory response after 1 week in AbIA mice, exhibiting higher accumulation in inflamed joints. Arthritis [149] Poly-siRNAthiolated glycol Ch NPs siRNA targeting TNF-α - NPs showed high accumulation at the arthritic joint sites, with significantly inhibition of inflammation and bone erosion in CIA mice. RA [150] Tuftsindecorated alginate NPs encapsulating IL-10 plasmid IL-10 plasmid DNA Activated macrophages of inflamed joints via tuftsin Targeted formulation demonstrated higher transfection efficiency and reduced systemic and joint tissue pro-inflammatory cytokines, which prevented joint damage and delayed the onset of inflammation. Arthritis [155] Micelles PEG-Dex Dex Passive effect PEG-Dex micelles, combined with a pH-responsive hydrazone linker, exhibit higher retention in the inflamed joints and enhanced therapeutic efficacy in an AIA rat model. RA [159] FA-Cholesteryl chloroformate - polysialic acid (FA-CC-PSA) Dex Folate receptor of macrophages via FA In vitro and in vivo results demonstrated the suppression of key pro-inflammatory proteins, improvement of the drug pharmacokinetics and their safety profile. RA [160] Dextran sulfategraftmethotrexate conjugate (DSg-MTX) micelles MTX Scavenger receptor of activated macrophages via DS DS-g-MTX micelles showed higher accumulation in the inflamed joints and stronger antiinflammatory effect, leading to significant alleviation of synovitis and protection of articular cartilage. RA [161] Sialic aciddextranoctadecanoic acid (SA-DexOA) micelles MTX E-selectin receptor of inflammatory vascular endothelial cells via SA SA-Dex-OA/MTX micelles elicited excellent inhibition of inflammatory response and minor adverse effects on liver and kidneys. The synergistic effects between drug and carrier also enhanced bone repair. RA [163] Hyaluronic acid/Curcumin (HA/Cur) nanomicelles Cur IA injection HA/Cur nanomicelles lowered the edema and cartilage degradation in RA rat models, with clear inhibition of the inflammatory response. RA [165] Chapter I – Nanoparticles for arthritic diseases treatment 30 Dendrimers PAMAM-PEGIGF-1 IGF-1 IA injection The nanocarriers enhanced cartilage penetration and joint residence time up to 30 days. A single injection of dendrimer–IGF1 rescued cartilage and bone more effectively than free IGF-1, in a surgical model of rat OA. OA [169] Cationic PCL-gPAMAM dendrons (cDenpols) - - cDenpols eliminates cfDNA and inhibits TLR recognition and nucleic acid-induced inflammation. In a CIA rat model, cDenpols inhibited joint swelling, synovial hyperplasia, and bone destruction. RA [171] Inorganic NPs Hyaluronate/ gold nanoparticle/To cilizumab (HAAuNP/TCZ) complex TCZ - HA-AuNP/TCZ complex showed the dual targeting activity of the binding to VEGF and IL-6R in vitro . The therapeutic effect on a mouse RA model was verified by ELISA, histological, and Western blot analyses. RA [174] Manganese ferrite and ceria NP-anchored mesoporous silica NPs (MFC-MSNs) MTX IA injection MFC-MSNs successfully induced polarization of M1 to M2 macrophages under hypoxic and inflammatory conditions both in vitro and in vivo . The IA injection successfully attenuated inflammation and pathological features in the joint, increasing their therapeutic effect. RA [175] I-3.3. NPs internalization In order to promote a targeted and controlled delivery of the encapsulated drugs from the NPs, it is fundamentally important to understand their uptake by different cells [177]. Indeed, many factors, including physicochemical properties of the NPs, protein-NPs and cell-NPs interactions as well as the cell type and cell’s state affect the mechanism of NPs cellular internalization. The pathway of cellular internalization of the nanomedicine is a key factor in determining their biomedical functions, biodistribution and toxicity [178]. To obtain a high therapeutic efficacy, the nanocarrier should enter into the cells without the induction of cytotoxicity, which deeply relies on the NPs entry pathway and intracellular localization [179]. In addition, since these carriers are usually aimed to Chapter I – Nanoparticles for arthritic diseases treatment 31 deliver the biomolecule to a specific sub-cellular compartment of the cell, intracellular trafficking and fate of NPs is a vital process for its success [180]. Indeed, although most of the nanocarriers can enter into cells via endocytosis, generally they are inevitably entrapped in endosomes, which subsequent merge with lysosomes. The lysosomes are acidic compartments (pH ranging from 4.5 to 5.5) that contain an array of hydrolytic enzymes capable to degrade macromolecules from the secretory, endocytic, autophagic and phagocytic membrane-trafficking pathways [181]. Consequently, the therapeutic molecules that are entrapped inside lysosomes undergo degradation, without providing their therapeutic action. Hence, when designing safe and efficient nanomedicines, it is crucial to understand their cellular uptake and intracellular trafficking. I-3.3.1. Cellular Uptake Pathways of NPs Endocytosis is the major route of cellular uptake of NPs, being an active transport that occurs against the concentration gradient by using energy [182]. It involves the generation of new intracellular membrane-enclosed vesicles from the plasma membrane with a concomitant internalization of lipids, proteins and extracellular fluid (Figure I-5). It is usually classified into two major categories: phagocytosis and pinocytosis. Phagocytosis is an essential mechanism of the immune system defense, being predominantly used by phagocytes, such as macrophages, neutrophils and monocytes [183]. This pathway begins with the recognition of the NPs by the opsonins, such as immunoglobulin (IgG and IgM), complement component and blood serum proteins that attach to the cell surface through Fc receptors and complement receptors [184]. This trigger the polymerization of actin membrane protrusions at the site of ingestion and, consequently, engulfing and digesting larger particles and pathogens. After transporting the opsonized particle into the cell, the formed phagosome will undergo degradation by acidification and enzymolysis in the lysosomes. Conversely to phagocytosis, pinocytosis is present in all types of cells and depending on the proteins involved in the pathways, it is classified to clathrin-mediated endocytosis, caveolae-mediated endocytosis, clathrinand caveolae-independent endocytosis, and micropinocytosis. Clathrin-mediated endocytosis comprises clathrin-coated vesicles formation in the presence of an adaptor protein, Epsin, and other accessory proteins such as dynamin (GTPase) [185]. Briefly, the NPs signaling on the cell surface aligns surface proteins to begin a clathrin-coating process on the inner Chapter I – Nanoparticles for arthritic diseases treatment 32 membrane of the cell [186]. With the assistance of dynamin, a clathrin-coated vesicle with a size of 100150 nm is formed, that internally detaches from the donor membrane. The cell fate seems to be associated with the receptor at the cell surface to which the NPs attach (e.g. NPs could be transferred to lysosomes for degradation or released from the endocytosed vesicles). Caveolae-mediated endocytosis is a pathway dependent on membrane cholesterol, dynamin and cell receptor mediation [187]. The binding of the NPs to the receptors of the plasma membrane, mainly caveolin -1, -2, -3, induce the formation of flask-shaped vesicles. The uncoated invagination initially assumes a flask shape with a body diameter of 60–80 nm and a neck diameter of 10–15 nm. The caveolae vesicles are afterwards cut off from the membrane by dynamin. Caveolae vesicles fuse with caveosomes or multivesicular bodies (MTV), which will move to the endoplasmic reticulum, cytosol or nucleus. Indeed, many pathogens including viruses and bacteria select this way to avoid lysosomal degradation. For the same reason, this pathway is believed to enhance the therapeutic effect of the drugs loaded into the NPs. Clathrinand caveolae-independent endocytosis occurs in cells that are deprived of clathrin and caveolin. The regulatory mechanisms of these pathways are still unknown, but cholesterol-rich microdomains on the plasma membrane, generally referred to as lipid rafts, are involved in this process [188]. Cell fate of the particles entering the cell through this pathway usually includes the delivery to the early endosomes, followed by the transfer to late endosomes and lysosomes. In addition, particles can be also directed to the trans-Golgi network or recycled back to the plasma membrane [189]. Macropinocytosis involves the uptake of large areas of the plasma and, consequently, allows the internalization of big NPs (> 1 µm) [190]. It is a clathrin-, caveolinand dynamin-independent process. In this pathway, tyrosine kinases activate actin polymerization to form protrusions in the cell membrane. After the encapsulation of the particle, the protrusions fuses once again back with the cell membrane. The fate of macropinosomes will depend on the cell type. Chapter I – Nanoparticles for arthritic diseases treatment 33 Figure I-5 – Schematic illustration of endocytic internalization pathways: phagocytosis, macropinocytosis, clathrin-dependent endocytosis, caveolae-dependent endocytosis and clathrin-independent endocytosis. Legend: endoplasmic reticulum (ER), nuclear localization signal (NLS), nuclear pore complex (NPC), triphenylphosphonium cation (TPP) (reprinted with permission [180]). Although endocytosis is widely recognized to be the major processes by which NPs enter into cells, there are also other non-endocytic pathways, including passive diffusion, hole formation, direct microinjection and electroporation [177]. Moreover, recent studies have reported cell-penetrating peptides [191] and membrane fusion between liposomes and cells [192]. Even though those mechanisms are poorly understood, the direct cytosolic delivery of NPs payload via non-endocytic pathways also seems to be an optimal approach to minimize their degradation in the endosomes/lysosomes. I-3.3.2. Effect of Physicochemical Properties of NPs on Cellular Uptake The physicochemical properties of NPs, including size, shape, surface charge, surface hydrophobicity/hydrophilicity and surface functionalization are critical parameters on cellular uptake as it directly affect the uptake level, endocytotic route as well as cytotoxicity of NPs [178]. Chapter I – Nanoparticles for arthritic diseases treatment 34 I-3.3.2.1 Size Size of NPs is a key factor in determining their cellular uptake efficiency, potential toxicity, as well as uptake pathway [193]. Several studies have demonstrated that 50 nm NPs are internalized more efficiently and at a higher uptake rate than smaller particles (around 15–30 nm) or larger particles (around 70–240 nm) [194]. Considering the uptake pathways, the inconsistency of the results on the literature maybe rely on the complexity of controlling other parameters besides size during in vitro and in vivo studies (e.g. agglomeration and aggregation), and also the cell type under evaluation [195-197]. Nevertheless, it seems that small NPs (from a few to several hundred nanometers) enter the cells via pinoor macropinocytosis. NPs in the size range of 120-150 nm are mostly internalized via clathrinor caveolin-mediated endocytosis (the maximum size reported to those pathways was 200 nm [195]), whereas NPs in the size range of 250 nm to 3 μm have been shown to have an optimal in vitro uptake through phagocytosis. I-3.3.2.2 Shape The shape of the NPs also seems to play a pivotal role in their uptake pathway and trafficking inside the cell, however the results are contradictory [178]. While some studies reported a higher uptake of spherical NPs when compared to rod-shaped NPs [194, 198], others describe the opposite [196, 199]. Moreover, there is no conclusion on the pathway selection of NPs considering their shape. I-3.3.2.3 Surface charge Surface charge is an important parameter in the characterization of NPs, as it determines the tendency for aggregation in storage and after administration in the blood and also the interaction with oppositely-charged compounds and the cell membrane. Indeed, highly charged positive or negative NPs present a higher stability, since the Coulombic repulsion forces can overcome the Van der Waals attractive forces between them and, consequently, preventing aggregation [200]. Even though some studies concluded that cationic particles have an increase rate and extent of internalization than negatively charged particles [201], due to the charge attraction between the positive NPs and negative cell membrane surface, the relationship between surface charge and cellular uptake is also contradictory. Indeed, many other studies have shown the successful internalization of negatively charged NPs [202]. Chapter I – Nanoparticles for arthritic diseases treatment 35 Also the cellular pathways present inconsistent results, since a wide plethora of outcomes can be found, for example: (i) positively charged NPs are mainly internalized via macropinocytosis and negatively charged NPs by clathrin-/caveolae-independent endocytosis [203], (ii) both cationic and anionic NPs are internalized mainly by clathrin endocytic pathway [202], and (iii) the use of clathrin-mediated pathway by positively charged NPs and caveolae-mediated pathway by negatively charged NPs [204]. I-3.3.2.4 Surface hydrophobicity/hydrophilicity The hydrophobicity/hydrophilicity of NPs is also important for the interaction with the cellular membrane [193]. The higher affinity for the cell membrane of the hydrophobic NPs improves their cellular uptake in comparison with hydrophilic NPs [205]. I-3.3.2.5 Surface functionalization Surface chemical functionalization of the NPs is a tool that can be used to control distinct properties such as cytotoxicity, stability and cellular internalization [206]. Surface functionalization of NPs predominantly comprises PEG, the negative carboxyl group (-COOH), neutral functional groups like hydroxyl groups (-OH), and the positive amine group (-NH2). Several studies have highlighting the key role of surface chemical functionalization in cellular interactions with NPs. As a representative example, pristine polystyrene (PS) NPs and amino-functionalized polystyrene NPs have demonstrated a higher uptake rate with the amino-functionalized NPs than PS-NPs, and while the former were internalized mainly via clathrin-mediated pathway, the latter was via clathrin-independent endocytosis [207]. In addition, several ligands and peptides were designed to increase the targeted delivery of the NPs into specific cells [208]. 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NanomedicineUk . 2017, 12 (6): p. 581-96. 50 SECTION 2 EXPERIMENTAL SECTION 51 Chapter II Materials and Methods Chapter II – Materials and Methods 58 their structural and dynamic properties, including the stability (improve the resistance to vesicle aggregation), permeability (reduce bilayer permeability to solutes), thickness (increase the packing of the phospholipid molecules), fluidity (reduces the fluidity of the lipid bilayer of the vesicles) and rigidity (the change the fluidity make the vesicles more rigid) [49, 50]. Moreover, since it also influence drug incorporation efficiency (reduce in the case of hydrophobic drugs), the ratio between cholesterol and phospholipids in liposomal formulations needs to be optimized, in order to provide a high stability without reducing the efficacy of drug delivery. Cholesterol (ovine wool, >98%) was acquired from Avanti Polar Lipids (USA) and NBD-Cholesterol (22-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino)-23,24-Bisnor-5-Cholen-3β-Ol) from Thermo Fisher Scientific (USA), which were used for the preparation of LUVs and fluorescent LUVs present in the Chapters IV and VI, respectively. Figure II-5 – Chemical structure of cholesterol. II-1.4. Gold nanoparticles Gold nanoparticles (AuNPs) have been widely employed in the biomedical field, due to their unique properties, such as good biocompatibility, easy synthesis, chemical stability and inertness, facile surface modification and tunable optical properties [51, 52]. Currently there are several techniques to synthetize AuNPs, being the commonly used protocols categorized into: (i) top-down protocols, where physicalchemical processes are used to degrade a bulk material into smaller pieces, achieving the nanometric scale, or (ii) bottom-up protocols, where the syntheses of the NPs starts from smaller precursors, such as metallic salts or molecular seeds that through nucleation form nanostructures [53]. It is possible to synthesize AuNPs with controlled size, shape and surface functionality, for a wide variety of applications. Chapter II – Materials and Methods 59 Computer Assisted Tomography scanning and X-Ray, for example, have been used to monitor the in vivo biodistribution of these contrast agents encapsulated or not into NPs. Moreover, they are promising new drugs for treatment of arthritic diseases attributed to their anti-inflammatory, anti-oxidant and antiangiogenic actions [54]. Stabilized suspension of AuNPs with 20 nm diameter in 0.1 mM phosphate buffered saline (PBS) was acquired from Sigma-Aldrich (USA), and used in the production of LUVs in Chapter IV. II-1.5. Glutathione Glutathione (GSH), also designed as γ-l-glutamyl-l-cysteinyl-glycine, is a tripeptide synthesized by the sequential addition of cysteine to glutamate followed by the addition of glycine (Figure II-6) [55]. It is the most important low MW antioxidant synthesized in cells, since besides removing peroxides, free radicals and many xenobiotic compounds, GSH is also involved in the regulation of the cell cycle. The sulfhydryl group (–SH) of the cysteine is involved in reduction and conjugation reactions where in the present of an antioxidant (or other xenobiotic compound) two GSH molecules become oxidized and join together via a disulfide bond to form glutathione disulfide (GSSG). Furthermore, GSSG can be reduced by glutathione reductase (GR) to regenerate GSH [56]. Thus, GSH is the key regulator of the intracellular redox state, being intracellularly in the range of 1-10 mM, whereas outside cells it is reportedly much lower within the range of 2-20 µM [57]. Recently, drug delivery systems that respond to biochemical differences between the extra and intracellular environments were explored to target and control the delivery of drugs inside cells. Thus, GSH can be used to coat the surface of NPs [58, 59] or to produce NPs, micelles and polymers that are sensible to the redox medium [38, 60-63]. In this thesis, L-glutathione reduced ≥98% was acquired from Sigma-Aldrich (USA), being used in the production of the micelles in Chapters V and VI. Chapter II – Materials and Methods 60 Figure II-6 – Chemical structure of glutathione (GSH). II-1.6. Polyethylene glycol PEG is a linear synthetic polyether that can have a wide range of sizes and terminal functional groups [46]. It is widely used in the pharmaceutical and nanotechnology field, due to its biocompatibility, nonimmunogenicity and good physical properties. Indeed, it can be dissolved in both aqueous and organic solvents, which enhances its applications for end-group derivatization and chemical conjugation to a huge variety of biological molecules, such as polypeptides, polysaccharides, polynucleotides, drugs and other small molecules under mild physiological conditions [64]. Besides being widely applied in the pharmaceutical field as vehicle in oral, topical and intravenous formulations, it has been widely explored for drug delivery and tissue engineering applications [65]. Among the drug delivery field, PEGylation have an important role in avoiding the adsorption of opsonin proteins [66]. Due to the steric repulsive effect, PEG not only increases the blood circulation halflife of the formulation by several orders of magnitude, but also prevents their aggregation during storage [67]. Therefore, PEG reduces the immunogenicity of therapeutic formulations and increases their pharmacokinetic properties. In this thesis, the PEG has a methoxy (OCH3) group in one side and an amine (NH2) group in the other side as terminal functional groups (Figure II-7). Methoxypolyethylene glycol amine 5,000 (extent of labeling: ≥0.17 mmol/g NH2 loading) was acquired from Sigma-Aldrich (USA) and used in the production of the micelles in Chapters V and VI. Chapter II – Materials and Methods 61 Figure II-7 – Chemical structure of methoxypolyethylene glycol amine (mPEG). II-2. REAGENTS Unless addressed otherwise, all the reagents used in this thesis were purchased from Sigma-Aldrich (USA). II-3. NPS PREPARATION Currently, NPs can be prepared by several techniques. In the following sub-sections the preparation methods and chemical coupling reactions performed in this thesis will be described in greater detail. II-3.1. Preparation methods The appropriate method for NPs preparation extremely depends on the characteristics of the polymer used or the biosynthesis molecules and the bioactive agent. Hence, in order to achieve the desirable properties of interest, such as particle size, surface charge, encapsulation efficiency and stability, the preparation method plays a vital role. Different techniques employed in this thesis for the preparation and synthesis of the NPs are discussed below. II-3.1.1. Polyelectrolyte complexation Polyelectrolyte complexes (PECs) are formed due to the electrostatic interactions established between oppositely charged polyions (e.g. polymer-polymer, polymer-drug and polymer-drug-polymer) [68]. PECs are usually obtained by simple, cost-effective and mild methods, presenting the benefit of using organic solvent-free and surfactant-free formulations. The main techniques employed are ionic gelation (cross-linking) or coacervation (phase separation) with the latter not requiring any extra excipient in addition to the polymers and the bioactive molecules. Different parameters are known to influence the Chapter II – Materials and Methods 62 formation of PECs, namely charge density, polyelectrolytes concentration, pH, ionic strength and solvents [69]. In the last years, PECs gained much interest due to their potential applications in the drug delivery field. These systems offer many advantages over conventional delivery systems as they are able to encapsulate different compounds into the polymer matrix at the molecular level, which enhances the efficacy of the biological agents. Nevertheless, drug loading efficacy is normally low and they can also lose the cargo along the time [70]. Polyelectrolyte complexation was used in Chapters III, VI and VII to produce Ch-HA polymeric NPs. II-3.1.2. Thin-film hydration method The thin-film hydration method is one of the simplest ways to prepare LUVs in a research laboratory [71]. This method involves making a thin lipid film in a round-bottom flask by the removal of the organic solvent. Upon the addition and agitation of the dispersion medium, heterogeneous liposomes are formed. After extrusion through polycarbonate membranes, homogeneous liposomes are obtained. It can be used for all different kinds of lipid mixtures, being the drugs encapsulated into the liposomal formulation by introducing them to the aqueous phase in the case of hydrophilic drugs or to the organic phase for hydrophobic drugs. The thin-film hydration method was used in Chapters IV and VI to produce LUVs. II-3.1.3. Nanoprecipitation Nanoprecipitation, also called solvent displacement method or interfacial deposition method, relies on the principle known as Marangoni effect [72]. This method requires the addition of two solvents that are miscible with each other and results in spontaneous formation of NPs by phase separation. The two solvents are selected such that the first solvent (usually organic) dissolves the polymer and the drug. The second system is a non-solvent (usually aqueous solution) in the presence or absence of a surfactant. Hence, this method results in the instantaneous formation of NPs, being an easy and one-step technique that can be easily scaled up [73]. This method is mostly used to encapsulate hydrophobic drugs, but it is also employed in some cases to incorporate hydrophilic drugs. The key parameters in the fabrication procedure are the organic phase injection rate, aqueous phase agitation rate and the organic phase/aqueous phase ratio, which have great influence on the NPs size distribution. Indeed, it can be Chapter II – Materials and Methods 63 synthesized particles with sizes of very narrow distribution, because of the absence of shearing stress [74]. Hence, the nanoprecipitation technique has been widely used in the pharmaceutical and agricultural research as a clean alternative for other drug carrier formulations. Nanoprecipitation method was used in Chapters V and VI to produce polymeric micelles. II-3.2. Chemical coupling reactions Coupling reaction in organic chemistry is a general term for a variety of reactions where two fragments are joined together, normally using coupling reagents. In comparison with the physical methods, chemical coupling reactions result in higher affinities between ligands in the nanocarrier. This increases the biological agent efficacy by improving its bioavailability and targeted delivery, improved safety, extending the molecule half-life in the target tissue and enhancing its stability against chemical and/or enzymatic degradation. The coupling reactions applied in this thesis will be presented in the following sub-sections. II-3.2.1. EDC/NHS Carboiimide reactions have been widely used to couple carboxyl groups to primary amines in a variety of conjugation techniques. 1-ethyl-3-(3-(dimethylaminopropyl) carbodiimide (EDC) reacts with the carboxyl group to form an active ester intermediate, which is stabilized though the presence of Nhydroxysuccinimide (NHS), reacting subsequently with a primary amine to form an amide bond (Figure II-8) [75, 76]. The addition of sulfo-NHS stabilizes the amine-reactive intermediate by converting it into an amine-reactive sulfo-NHS ester, thus increasing the efficiency of EDC-mediated coupling reactions. It is called a ‘zero-length’ cross-linker since the amide linkages are formed without leaving a spacer molecule. This reaction is nontoxic as the remained products and by-products (isourea) can be easily removed in the washing steps [77]. Indeed, several works have demonstrated the in vitro and in vivo cytocompatibility of EDC/NHS chemistry. In Chapter III, VI and VII, EDC/NHS chemistry was used to stabilize the produced Ch-HA NPs and/or immobilize the antibodies at the NPs’ surface. In Chapter V, EDC/NHS chemistry was used to synthesize the copolymers for the micelles preparation. Chapter II – Materials and Methods 64 Figure II-8 – ECD/NHS chemistry (adapted from [76]). II-3.2.2. TBTU 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) is one of the most commonly used coupling reagents for amide bond formation in organic solvents, and it is also called a ‘zero-length’ cross-linker [78]. In the presence of a catalyzer (e.g. triethylamine - TEA), TBTU reacts with the carboxyl group forming an active ester that then reacts with the amine group to form an amide bond (Figure II-9). In Chapter V and VI, TBTU chemistry was used to synthesize the copolymers for the micelles preparation. Figure II-9 – TBTU chemistry (adapted from [78]). II-3.2.3. Thiols Thiols, also called sulfhydryls, are considered analogs of alcohols where OH group is replaced with SH group [79]. They are an integral part of biological systems and have significant roles in protein Chapter II – Materials and Methods 65 structure, detoxification, cell signaling and enzyme activities as well as being cofactors. Despite being used in many chemical reactions and applications [80], in the scope of this thesis thiol-disulfide and thiolmaleimide reactions will be explained in greater detail. The interconversion between thiols and disulfide groups is a redox reaction: the thiol is the reduced state, and the disulfide is the oxidized state. Hence, disulfide bonds are usually formed from the oxidation of sulfhydryl groups (Figure II-10A). Despite being a covalent bond, disulfide bonds are susceptible to reduction while in biological systems. In Chapter V, the oxidation of the thiol group of GSH allows for the encapsulation of a hydrophobic drug into the polymeric micelles. The maleimide group reacts specifically with sulfhydryl groups when the pH of the reaction mixture is between 6.5 and 7.5 (Figure II-10B). The result of this reaction forms a stable thioether linkage that is not reversible (i.e., the bond cannot be cleaved with reducing agents). In order to perform this reaction sometimes is necessary to introduce first the sulfhydryl groups, which can be achieved with 2iminothiolane (2-IT, also known as Traut's reagent) [81]. The cyclic imidothioester reacts with primary amines in a ring-opening reaction regenerating a free sulfhydryl group. In order to prevent disulfide bond formation as a result of the oxidation of the sulfhydryl groups, a chelator (e.g. ethylenediamine tetraacetic acid - EDTA) should be added to the reaction. In Chapter IV, those reactions were performed in order to covalently immobilize the antibodies at the surface of the liposomes. Chapter II – Materials and Methods 66 Figure II-10 – Thiol chemistry: (A) thiol-disulfide reaction, and (B) thiol-maleimide reaction. II-3.3. Polymeric NPs preparation In the Chapters III, VI and VII, Ch-HA NPs were prepared by polyelectrolyte complexation of both natural-based polyelectrolytes (Figure II-11). Figure II-11 – Ch-HA NPs production. For the Ch-HA NPs formation, Ch was dissolved in 1% (v/v) acetic acid, while HA was dissolved in ultrapure water, overnight at room temperature (RT). Both solutions were filtered through a 0.22 μm pore membrane. The NPs formation occurred spontaneously when the HA solution was added dropwise at 1 mL/min rate to the Ch solution under strong magnetic stirring (600 rpm). Different initial polymer A B Chapter II – Materials and Methods 67 concentrations (0.25, 0.5 and 1 mg/mL) and pH values (3, 4, 5, 6 and 7) were used to evaluate the influence of these parameters on the properties of the NPs. During the preparation, the NPs were stabilized through carbodiimide chemistry. EDC/NHS reagents were dissolved in 0.1 M MES buffer (pH 4.7) with 0.9% (w/w) NaCl. Five different EDC/NHS ratios were tested, namely 400/100, 100/400, 200/200, 50/200, and 200/50 mM. To remove unreacted compounds, the NPs were washed twice with ultrapure water by centrifugation (30 min, 4000 rpm at 20 °C) using Vivaspin 300 kDa Filter Units (Fisher Scientific, USA). To avoid NPs aggregation, glucose at 2 mg/mL was added before centrifugation [82]. For the fluorescence biological assays, 200 μL of fluorescein isothiocyanate (FITC, 2 mg/mL in ethanol/water, 1:10) was added to the Ch solution before the NPs formation. In order to assess the optimal production conditions in terms of size distribution and zeta potential, the different parameters evaluated are summarized in Table II-1. The optimal conditions for producing the NPs are highlighted in the table in bold. Table II-1 – Parameters evaluated for CH-HA NPs production. Parameter [CH/HA] (mg/ml) pH (solutions) [EDC/NHS] (mM) Tested conditions 0.25 0.5 1 3 4 5 6 7 400/100 100/400 200/200 50/200 200/50 II-3.4. LUVs preparation In Chapter IV and VI, LUVs were prepared by the thin-film hydration method followed by extrusion (Figure II-13). Chapter II – Materials and Methods 74 technique relies on the absorption of energy from a photon that promotes the transition from a lowerenergy state to a higher-energy (i.e. an excited state), which results in vibrations of molecular bonds (e.g. stretching, bending, twisting, rocking, wagging and out-of-plane deformation) in the infrared (IR) region of the spectrum. Fourier transformation algorithm allied to IR spectroscopy gives a spectrum of IR absorption per frequency/wavelength. Taking into consideration that each compound has a specific IR spectrum and that similar chemical groups absorb in the IR at similar frequencies, FTIR analysis enables to identify the chemical structure of a compound and subsequent chemical modifications. An IR Prestige-21 FTIR spectrometer (Shimadzu, Japan) with the attenuated total reflectance (ATR) technique was used to identify the chemical structure of the produced micelles in Chapter V. A transmittance spectrum was obtained by performing 50 scans in each spectrum over a range of 5004000 cm-1 at a 4 cm-1 resolution by the KBr disk method. II-5. BIOACTIVE AGENTS The bioactive agents used in this thesis as well as their loading/encapsulation efficacy are described in the following sub-sections. II-5.1. Antibodies An antibody (Ab), also known as immunoglobulin (Ig), is a large Y-shaped glycoprotein composed of two different regions, a variable region that is specific of each Ab and a non-variable region that is common for each type of Ab (Figure II-14). The antigens are specifically recognized via the fragment antigen-binding variable region [95]. They are mainly produced by B-cells and used by the immune system to identify and neutralize pathogens, such as bacteria and viruses [96]. In recent years, monoclonal Ab therapy have been approved for over 30 targets and diseases, which had dramatically advanced the therapy of chronic inflammatory diseases and cancer [97]. In this type of immunotherapy, the Ab bind specifically to certain cells or proteins in order to suppress/reduce the immune system activity or to eliminate and regulate immune cells that contribute to tissue damage. Despite their remarkable success, Abs-based therapies are associated with some limitations, including the short half-life of the Abs that decreases their therapeutic efficacy and severe systemic side effects, such as increased risk of infection, malignancy or administration reactions [98]. Chapter II – Materials and Methods 75 Figure II-14 – Antibody structure (adapted from [99]). In this thesis, neutralizing Abs were used due to their ability to block the activity of the antigen after biding to the variable region. Three different Abs were used, namely anti-IL-6 Abs, anti-TNF-α Abs and anti-IL-23 Abs, being their properties summarized in Table II-2. Table II-2 – Summary of antibodies properties. Parameter Anti-IL6 Abs Anti-TNF-α Abs Anti-IL-23 Abs Clonality Monoclonal Monoclonal Polyclonal Monoclonal Host specie Mouse Mouse Rabbit Mouse Species reactivity Human Rat, Human Rat, Human, Cynomolgus monkey, Rhesus monkey Human Application Flow Cytometry, Blocking, Inhibition Assay Immunochemistry, Neutralizing, Sandwich ELISA, Western Blot Flow Cytometry, Inhibition Assay, Neutralizing, Western Blot Neutralizing, Immunochemistry Company Abcam (UK) Abcam (UK) Abcam (UK) Abcam (UK) Chapters III VII VII IV Chapter II – Materials and Methods 76 II-5.1.1. Polymeric NPs functionalization Abs were immobilized at the surface of the optimized Ch-HA NPs, being anti-IL-6 Abs immobilized in Chapter III, and both anti-IL-6 Abs and anti-TNF-α Abs in Chapter VII. To determine the maximum immobilization capacity of the NPs, different concentrations of each Abs were used (from 0 to 20 μg/mL). First, the primary Abs were activated with a solution of 50/200 mM EDC/NHS in 0.1 M MES buffer for 15 min. Then, the NPs were incubated with the activated Abs, overnight at 4 °C. To remove the unbound Abs, the biofunctionalized NPs were washed twice by centrifugation, as previously described. To determine the degree of Abs immobilization, the biofunctionalized NPs were incubated with the secondary Abs for 1 h at RT. NPs without immobilized Abs were used (0 μg/mL) as negative control. After centrifugation, the fluorescence of the unbound secondary Abs (in supernatant) were determined in a microplate reader (Synergie HT, Bio-Tek, USA). The concentration of Abs at the NPs surface corresponds to the difference between the initial and unbound secondary Abs. II-5.1.2. Liposomes functionalization Anti-IL-23 Abs were linked to the maleimide groups of PEG ends after their thiolation with 2IT [47]. For that, a 100-fold molar excess of 2IT was incubated with the Abs (20 µg per 1 mL of LUVs suspension at 30 mM) in the presence of 5 mM EDTA (to avoid the oxidation of the thiol groups) in PBS (pH 8.0) during 1 h at RT [100]. Before linking the thiolated Abs to liposomes, a dialysis (Micro Float-A-Lyzer®, MWCO: 3.5-5 kDa) was performed to remove the excess of 2IT. As thiol groups have a rapid rate of recyclization [101], the buffer replacement was performed each 15-20 min during a period of time lower than 4 h. After LUVs overnight incubation at 4 ºC with the thiolated anti-IL-23 Abs, they were washed twice with HEPES buffer using Vivaspin 300 kDa Filter Units to remove unbound Abs. To quantify the anti-IL-23 Abs immobilized at the LUVs surface, first they were immersed in a solution of 3% (w/v) bovine serum albumin (BSA) for 1 h at RT (to block nonspecific sites), and then the secondary Abs Alexa Fluor® 488 were added. After 1 h at RT, the fluorescence intensity of the supernatant (unbound secondary Abs) was measured using a microplate reader (Synergy HT, BioTek, USA). Chapter II – Materials and Methods 77 II-5.2. Dexamethasone Dexamethasone (Dex), also known as 9α-Fluoro-16α-methylprednisolone, is a potent synthetic member of the glucocorticoids (GCs) class of steroid drugs (Figure II-15), which acts as an antiinflammatory and immunosuppressant [102]. Generally, GCs are among the most commonly prescribed drugs for various inflammatory, autoimmune and allergic disorders. Nevertheless, their use is severely hampered by the risk of developing serious side effects, such as osteoporosis, hyperglycemia, insulin resistance and hypertension. Moreover, the therapeutic efficiency is limited, due to inadequate pharmacokinetics, with low drug bioavailability and off-targeted biodistribution profile [66]. These therapeutic drawbacks can be overcomed by designing nanomedicines, namely drug delivery systems, which have been successfully introduced in the clinic for the treatment of cancer, pain and infectious diseases [103]. In this thesis, Dex ≥ 98% in HPLC was acquired from Sigma-Aldrich (USA), and was encapsulated in the polymeric micelles in the Chapter V. Figure II-15 – Chemical structure of Dexamethasone (Dex). II-5.2.1. Encapsulation efficiency The Dex loading content in the micelles was determined with micelle:Dex feed weight ratios varying from 1:0.2 to 1:0.8 at a micelle concentration of 1 mg/mL. Dex was added to THF solution and then the micelles were produced as previously described. The non-encapsulated Dex was measured in the supernatant of the centrifuged solution using an UV-Vis spectroscopy (Shimadzu, Japan). As Dex is a hydrophobic drug (soluble in organic solvents), samples were diluted in ethanol (0.5:0.5 v/v) in order to allow the Dex dissolution. Chapter II – Materials and Methods 78 Entrapment efficiency (EE) was calculated by measuring the initial concentration and nonencapsulated Dex, according to the following formula: %𝐸𝐸 = (Initial concentration − not encapsulated drug) Initial concentration x 100 II-5.2.2. Release studies The in vitro Dex release profiles of micelles under different external conditions were obtained and measured through a dialysis method. Firstly, 5 mL of micelle suspensions were added to a dialysis system (Micro Float-A-Lyzer®, 3.5 - 5 MWCO) before putting it in a centrifugation tube containing 15 mL PBS (pH 7.4). The tube was shaken at 100 rpm at 37 °C. At the defined time points, an aliquot of solution (0.5 mL) was retrieved from the outside tube with equal volume replenishment. After dilution of the aliquot with 0.5 mL ethanol, Dex concentration was measured using an UV-Vis spectroscopy equipment (Shimadzu, Japan). The enzymaticallyand GSH-responsive properties were evaluated using the same method but with different external media - PBS containing (i) 10 mM of GSH or (ii) 50 mU of GR (with 0.14 mM NADPH and 0.1 mM EDTA). All the characterization experiments were performed in triplicate. II-5.2.3. UV-Vis spectrophotometry UV-Vis spectrophotometry is a very simple, rapid and low cost technique that has been used extensively in analytical chemistry for characterization and quantitative analysis [104]. It uses light in the visible and adjacent regions near ultraviolet (UV) and near infrared (NIR) ranges to acquire the absorption or reflectance spectroscopy of photons of a compound. Hence, it is possible to determine concentrations of an absorbing chemical in solution by using Beer-Lambert law. This is based in the principle that the absorbance of a solution is directly proportional to the concentration of the absorbing species in the solution and the light path length. The concentration can be determined using a calibration curve. In this thesis, UV-Vis spectrophotometry was used to determine the EE and release profile of the Dex loaded into the polymeric micelles in the Chapter V. Chapter II – Materials and Methods 79 II-6. IN VITRO BIOLOGICAL TESTS Several biological assays performed in this thesis are explained in greater detail in the following subsections. After discussing the selection of the cells sources used in this thesis, it is described the cell seeding, the methods used for the study and quantification of metabolic activity and viability and the production of pro-inflammatory cytokines as well as the NPs internalization. II-6.1. Cell sources Inflammatory arthritis compromises many cell types including endothelial cells, chondrocytes, synovial like fibroblasts and inflammatory cells, such as macrophages [105]. Hence, nanomedicines should take in consideration this dynamic environment in order to provide an increased therapeutic action, without affect cell function. In vitro studies performed in this thesis were carried out using both primary cells and cell lines of human origin. Primary cells are isolated directly from a tissue through mechanical, chemical or enzymatic digestion methods [106]. Their use presents some challenges, due to their heterogeneity, sensitivity, lower proliferation rates and limited lifespan in culture. Moreover, they usually require additional nutrients in the culture medium. An important advantage is their accurately representation of the host tissues. In contrast to primary cells, cells lines are immortalized cells that present the ability to proliferate indefinitely either due to a random mutation or due to a programmed modification [107]. They offer several advantages including cost-effectiveness, easy to culture, unlimited supply of cells with high proliferative rates and bypass ethical concerns associated with the use of animaland/or human-derived tissues. Moreover, as they provide a consistent sample, they present reliable and reproducible experimental results. However, they do not always accurately replicate the primary cells and, consequently, results needs to be carefully interpreted. Taking into consideration the dynamic environment of arthritic diseases, two primary cells types, human articular chondrocytes (hACs) and human monocyte-derived macrophages, and two cell lines, human monocyte-like cell line - THP-1 and human umbilical vein endothelial cell line (EA.hy926), were used throughout this thesis. All cells were incubated at 37 °C in a humidified 5% CO2 atmosphere. Chapter II – Materials and Methods 80 II-6.1.1. Human Articular Chondrocytes Chondrocytes, the only cell type of cartilage tissue, have a critical role in the synthesis and turnover of a large volume of ECM components in cartilage including collagen, glycoproteins, proteoglycans and HA [108]. Imbalance in their function leads to degenerative diseases like OA and RA. Thus, hACs from diseased knee arthroplasties, which have a phenotype associated with arthritis disease, are a relevant model to study the biological actions of the developed nanoformulations. In a monolayer culture, hACs acquire a fibrolastic-like morphology after several days in culture [109]. hACs were isolated from knee cartilage samples collected from arthroplasties surgeries biopsies. Samples were obtained through the cooperation agreement between Centro Hospitalar do Alto Ave, Guimaraes, Portugal, and I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, in accordance to the established Protocol (67/CA), and after obtaining the donor informed consent. Cells were isolated by enzymatic digestion, according to a previously described protocol [110]. In detail, human cartilage samples were dissected in small full-depth pieces and washed twice with sterile PBS. Then, samples were digested using 0.25% (w/v) trypsin solution for 30 min at 37 °C under agitation, and after removing the solution, cartilage was washed and incubated with a 2 mg/mL collagenase type II solution overnight at 37 °C under agitation. In the next day, after washing the cells twice with sterile PBS, they were counted and plated at a density of 2×106 cells per 25 cm2 culture flask. hACs cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% Fetal Bovine Serum (FBS, Thermo Fisher Scientific, USA), 10 mM HEPES buffer, 10 mM L-lanyl-L-glutamine, 10 mM MEM nonessential amino acids, 100 units/mL of penicillin, 100 μg/mL of streptomycin and 10 ng/mL of human basic Fibroblast Growth Factor (bFGF, Peprotech, USA). Culture medium was changed every 3 days, until reaching approximately 80 % confluence. Cells were routinely trypsinized with TripLE express (Life Technologies, USA) for 3-5 min at 37 ºC, centrifuged (300 g, 5 min) and re-suspended in T150 cell culture flasks. Cells were used until passage 4. In this thesis, hACs were used in all chapters of Section 3, 4 and 5. II-6.1.2. Human monocyte-derived macrophages Macrophages are an important population of immune cells that play a key role either in maintaining tissue homeostasis or in inflammatory states [111]. The study of human macrophages is frequently Chapter II – Materials and Methods 81 hampered by access to tissue and inability of this cell type to survive in vitro following isolation. Hence, the culture of human monocyte-derived macrophages represent a tool to study macrophages, since monocytes can give rise to tissue macrophages when influenced by certain environmental cues. Despite the advantage of their natural origin, they are not able to replicate in vitro , which limits their applicability. Human monocyte-derived macrophages were generated from peripheral blood mononuclear cells (PBMCs). Buffy coats from healthy donors were acquired after obtaining written informed consent at the Hospital de Braga, Braga, Portugal (SECVS 014/2015). Briefly, PBMCs were enriched from buffy coats by density gradient using Histopaque-1077. The cells in the enriched mononuclear fraction were washed twice in PBS and resuspended in Roswell Park Memorial Institute (RPMI)-1640 media (Thermo Fisher Scientific, USA) with 2 mM glutamine and 2 g/L NaHCO3 supplemented with 10% human serum, 100 units/mL of penicillin, 100 μg/mL of streptomycin and 10 mM HEPES buffer. In the case of frozen PBMCs from RA patients, they were acquired from StemCell Tecnhology (Canada), and used as described in the manufactures’ protocol. In this thesis, healthy human macrophages were used in the Chapter III and IV, while diseased macrophages were used only in Chapter IV. II-6.1.3. Human monocyte-like cell line THP-1 THP-1 designates a spontaneously immortalized monocyte-like cell line, derived from the peripheral blood of a childhood case of acute monocytic leukemia (M5 subtype) [112]. As the human monocytederived macrophages, THP-1 cell line can also give rise to tissue macrophages when stimulated with phorbol 12-myristate-13-acetate (PMA). Hence, taking into consideration the limited availability of PBMCderived monocytes, THP-1 represent a valuable tool for investigating monocyte structure and function in both health and disease [113]. Several publications have compared responses between the THP-1 monocytes and human PBMC-monocytes. In most cases, both types showed relatively similar response patterns, with some variable regarding the degree of expression (e.g. gene expression, surface markers and cytokine secretion). In addition, it is important to emphasize that they are still a cell line and, consequently, present the same abovementioned limitations. THP-1 cell line was kindly provided by Dr. Agostinho Carvalho (Life and Health Science Research Institute, University of Minho, ICVS/3B’s – PT Government Associate Laboratory, Portugal). They were maintained in complete RPMI, containing RPMI-1640 media supplemented with 2 mM of L-glutamine, Chapter II – Materials and Methods 82 100 units/mL of penicillin, 100 μg/mL of streptomycin, 10 mM HEPES buffer and 10% FBS. When started with a frozen cryotube, cells were added to a conical based centrifuge with 5 ml of culture medium. After the centrifugation at low speed (150 g, 5 min), the cell pellet was resuspended at a density of 3 - 5 x 100,000 cells/mL in fresh medium containing 20% FBS. The flask was kept in a vertical position until the cells reach the exponential phase of growth (usually this can take up to 7 days). Once the culture is established, the FBS concentration was reduced to 10%. Cells were maintained at a concentration of 3 - 8 x 100,000 cells/mL, in order to keep the cells in the exponential growth phase. This cell line was used in all chapters of Section 3, 4 and 5. II-6.1.4. Human umbilical vein endothelial cell line EA.hy926 was established by fusing human umbilical vein endothelial cells with the permanent human cell line A549 [114]. Despite culturing them at high passages, EA cells still continue to express a wide range of differentiated endothelial cell properties [115]. As endothelial cells are the main cells of blood vessels, this cell line was used to assess any cytotoxic effects caused by micelles and LUVs as they would be used in systemic injection. Those cells were used in the Chapters IV and V, and in the internalization assays in Chapter VI. Endothelial cells were cultured in DMEM low glucose (D5523) supplemented with 10% FBS, 100 units/mL of penicillin and 100 μg/mL of streptomycin. Culture medium was changed every 3 days, until reaching approximately 80% confluence. Cells were routinely trypsinized with TripLE express for 3-5 min at 37 ºC, centrifuged (300 g, 5 min) and re-suspended in T150 cell culture flasks. II-6.2. Cryopreservation The cryopreservation of the cells used in this thesis was performed using a Statebourne Biosystem 24 cryogenic tank (Statebourne Cryogenics Ltd., UK). Briefly, cell suspensions of 1 × 106 cells/mL were prepared in a cryopreservation solution, consisting of 10 % (v/v) Dimethyl sulfoxide - DMSO (VWR, USA) in FBS, and transferred into 1.5 mL cryovials (VWR, USA). Then, cell suspensions were gently cooled down, first at -20 °C for at least 2 h and then moved to -80 °C freezer for a minimum period of 12 h. The cryovials were subsequently stored at -176 °C in the gas nitrogen phase of the cryogenic tank. Chapter II – Materials and Methods 83 II-6.3. Cell seeding II-6.3.1. Seeding on the bottom of well-plates In Chapter III, IV and V, the cytocompatibility of the nanoformulations in contact with the different cell types was evaluated as described below. Both hACs and EA were seeded at 5×104 cells per well into 24-well plates. To perform the SEM analyses, cells were added to tissue culture polystyrene (TCPS) coverslips in 24-well plates. After cell attachment during 5 h, culture medium was added to complete a final volume of 1 mL. For the induction of THP-1 cell differentiation, cells were seeded at 5×105 cells per well in 24-well plates in cRPMI with 100 nM PMA for 24 h. After incubation, non-adherent cells were removed by aspiration, and the adherent cells were washed three times with cRPMI. To ensure reversion of cells to a resting macrophage phenotype before its stimulation, the cells were incubated for an additional 48 h in cRPMI without PMA. For the stimulation and the retrieval of conditioned media, cells were further incubated for 24 h with 100 ng/mL of lipopolysaccharide (LPS) (and 100 ng/mL of Interferon-gamma, IFN-γ, in Chapter IV) in fresh medium (the supernatants were collected and stored at -80 °C). Regarding the human primary macrophages, isolated monocytes were resuspended in complete RPMI medium and seeded at 5×105 cells per well in 24-well plates for 7 days in the presence of 20 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF). Cells cultured without nanoformulations (only with culture medium) were used as control. After the defined timepoints of culture with different concentrations of the nanoformulations, the different samples in triplicate were washed with sterile PBS and evaluated regarding cell viability, proliferation, total protein synthesis and SEM analyses. In Chapter III, hACs were seeded as previously described to assess the ability of biofunctionalized NPs to capture IL-6. For that, the hACs were stimulated for 24 h with monocyte-derived macrophage conditioned medium containing 500 pg/mL of IL-6. Three different conditions were tested: (i) no treatment, (ii) treatment with biofunctionalized NPs, and (ii) treatment with soluble anti-IL-6 Abs. The Abs were added to the culture medium in a concentration of 1 μg/mL. hACs cultured without macrophage conditioned medium (only culture medium) were used as controls. After 1, 3, 7 and 14 days, samples were collected and evaluated regarding cell viability, proliferation, total protein synthesis and SEM Chapter II – Materials and Methods 90 addition of a stop solution and the absorbance read in a microplate (Synergy HT, BioTek, USA) at 450 nm, with a wavelength correction set at 540 nm. Table II-3 – ELISA procedure summary. IL-6 TNF-α IL-23 IL-17A Capture antibody 100 μL, overnight at RT Blocking 300 μL, 1% BSA in PBS, 1h at RT Sample/Standard 100 μL, 2h at RT Biotinylated detection antibody Rabbit Anti-Human IL-6 100 μL, 2 h at RT Rabbit Anti-Human TNF-α 100 μL, 2 h at RT Goat Anti-Human IL23 100 μL, 2 h at RT Goat Anti-human IL-17A 100 μL, 2 h at RT HRP Avidin-HRP Conjugate 100 μL, 1:2000, 30 min at RT Streptavidin-HRP 100 μL, 20 min at RT Avidin-HRP 100 μL, 30 min at RT Substrate 100 μL of ABTS 100 μL of 1:1 mixture of H2O2 and TMB, 20 min at RT 100 μL of TMB solution, 30 min at RT Stop Solution - - 50 μL stop solution 100 μL stop solution Absorbance 405/560 nm 450/540 nm Sensitivity 2000 pg/mL 3000 pg/mL 8000 pg/mL 250 pg/mL II-6.6. Internalization studies The cellular uptake of the NPs by the cells was performed using flow cytometry and confocal analyses, as described in the following sub-sections. II-6.6.1. Flow cytometry analyses Flow cytometry is a sophisticated instrument with the ability to measure the optical and fluorescence characteristics of a single cell or any other particle in a fluid stream when they pass through a light source [127]. Other parameters can be used to analyze and differentiate the cells including size, granularity and fluorescent features of the cells, resulting from either Abs or dyes. The principle of flow cytometry is related to light scattering and fluorescence emission that occurs as light from the excitation source (commonly a laser beam) strikes the moving cells, giving valuable information about biochemical, Chapter II – Materials and Methods 91 biophysical and molecular aspects of them. Structural and morphological properties of the cells are directly related to the light scattering while the amount of fluorescent probe bound to the cell is proportional to the fluorescence emission derived from a fluorescence probe. Hence, flow cytometry is a powerful tool for detailed analysis of complex populations in a short period of time. In Chapter VI, flow cytometry was performed to quantify and compare the internalization levels along time. Cells were seeded and stimulated as previously described, and afterwards they were incubated with the different fluorescent labelled formulations of NPs for 2, 6 and 24 h at 37 °C in a humidified 5% CO2 atmosphere. Ch-HA NPs and micelles were added at 50 μg/mL and LUVs at 500 µM. After each incubation time, cells were washed twice with PBS in order to remove any cellular debris or noninternalized NPs, and harvested with TripLE express. Afterwards, cells were centrifuged and cell pellets were re-suspended and fixed with 4% formalin in DPBS and kept in the dark at 4 ºC (for less than 1 week) [128]. The analyses of the samples were performed in a BD FACSCalibur™ flow cytometer (Biosciences, Belgium), after and before the addition of 0.11% trypan blue during 1 min, in order to quench the fluorescent signal coming from NPs adsorbed to the cell surface (and also giving an indication of the amount of NPs that were at the cells’ surface) [129]. Data were analyzed using Flowing Software 2.5.1. The results were reported as the mean of the percentage of cellular uptake obtained by measuring 20000 cells and normalized relatively to the cells incubated without NPs. In the same Chapter VI, flow cytometry was also used to study the internalization pathways involved in the cellular uptake of the different NP formulations. After seeding the cells as previously described, they were pre-incubated for 30 min at 37 °C in a humidified 5% CO2 atmosphere with three pharmacological pathway inhibitor solutions: (i) 10 μg/mL of chlorpromazine, (ii) 1 μg/mL of filipin, or (iii) 5 μg/mL of cytochalasin D [20]. Moreover, to study whether the cellular uptake was energy dependent, cells were incubated at 4 ºC for 30 min and then incubated with each NP formulation at 4 ºC. The time of culture was dependent on previous results of the maximum internalization for each cell type and NP formulation. Cells were collected and analyzed by flow cytometric analyses as previously described. II-6.6.2. Confocal microscopy analyses Confocal microscope is broadly used to resolve the detailed structure of specific objects within the cell [130]. Instead of illuminating the whole sample at once, the laser light is focused onto a defined spot Chapter II – Materials and Methods 92 at a specific depth within the sample. Hence, confocal microscopy enables the creation of sharp images of the exact plane of focus, without any disturbing fluorescent light from the background or other regions of the specimen. This tool allows to conveniently visualize structures within thicker objects, and 3D structures can also be analyzed by stacking several images from different optical planes. As such, confocal microscopy has a number of significant advantages over conventional fluorescence microscopy, including increased effective resolution, improved signal-to-noise ratio, depth perception in z-sectioned image, reduced blurring of the image from light scattering and electronic magnification adjustment. Nevertheless, the sample penetration depth is limited. In Chapters III and VI, to assess the NPs internalization and cell morphology microscopy confocal analyses were performed. Cells were seeded as previously described, and at the defined time point (12 h and 6 h for Chapters III and VI, respectively), cells were fixed with 10% formalin in PBS and stored at 4 °C. Then, samples were stained (between each step samples were washed three times with PBS): (i) cell membranes were permeated with 0.2% (v/v) Triton X-100 for 5 min, (ii) nonspecific proteins were blocked with 3% (w/v) BSA for 30 min, (iii) the cytoskeleton was staining with phalloidin (0.25 μg/mL) and cell nuclei labeled with DAPI (1 μg/mL) during 15 min. Images of fluorescent-labeled cells and NPs were obtained by using excitation wavelengths of 405 nm (DAPI), 488 nm (FITC labeled NPs) and 561 nm (phalloidin). Images were acquired using a laser scanning confocal microscopy imaging system (TCS SP8, Leica). II-7. IN VIVO STUDIES The experimental protocol was approved by the Institutional Ethical Commission (SECVS 109/2016) and followed the European Community Council Directive 86/609/EEC and 2010/63/EU concerning the use of animals for scientific purposes. Animal manipulation was performed only by qualified personnel and following the Principle of the 3Rs. Animals were housed in pairs in a limited-access rodent facility, with food and water available ad libitum . The temperature was maintained at 22.0 ± 0.5 °C with a 12/12 h light/dark cycle (starting at 8:00 a.m.). General health parameters were surveyed twice per week by the resident veterinary in order to check for pain and distress, and the weight of each animal was recorded every week throughout the experimental period. On the day of the experiments, animals were left in the experimental room for 1 h Chapter II – Materials and Methods 93 in order to get acquainted with the surroundings. Efforts were always made to minimize the number of animals used per experiment or test and their potential suffering. In vivo studies were performed in an experimental carrageenan-induced arthritis rat model to assess the safety and efficacy of the biofunctionalized Ch-HA NPs in the Chapter VII. II-7.1. Arthritis rat model Several animal models using different species were proposed to study the pathological features of arthritic diseases including pain, synovial inflammation, cartilage degeneration and bone remodeling [118, 131]. They can be categorized into induced or spontaneous models, whereas they are chemically or surgically induced or they occur spontaneously either naturally or due to genetically modification, respectively. Regarding the animal species used in the models of research, they always present advantages and disadvantages. Smaller animal models, such as mice, rats, rabbits and guinea pigs are much easier, cheaper and more readily available than larger animal models, such as horses, pigs and dogs. Nevertheless, the smaller size of those animals makes their anatomical and physiological structure considerably different when compared to humans, which do not happen in larger animal models. Additionally, there are also greater ethical concerns around the use of larger animal models. Based on this, researchers need to make a careful reflection when selecting the animal models. In this thesis, the in vivo assays were performed in a rat model as they have a larger joint in comparison with mouse models, allowing an easier intra-articular (IA) injection of the biofunctionalized NPs. Specifically, arthritis was induced though the IA injection of carrageenan [132]. Structurally, carrageenans are a complex group of polysaccharides composed of repeating galactose-related monomers and are of three main types: lambda, kappa, and iota. The lambda form does not gel strongly at RT and after injection it induces an inflammatory response. Inflammation induced by carrageenan is acute, nonimmune, well-documented and highly reproducible [133, 134]. Fundamental signs of inflammation, including edema, hyperalgesia and erythema, develop immediately by the action of proinflammatory agents. Such agents can be generated in situ at the site of insult or by infiltrating cells as neutrophils that rapidly migrate to sites of inflammation produce pro-inflammatory cytokines, reactive oxygen and other noxious chemicals such as glutamate, prostaglandins, histamine and serotonin [135]. The inflammatory response is usually quantified by increase in paw size (edema), mechanical allodynia Chapter II – Materials and Methods 94 and hyperalgesia, and also by the histological changes in the synovial membrane (development of synovitis) [135]. Therefore, this model has a vital role in testing novel drugs and nanoformulations. II-7.2. Behavioral assessments & clinical parameters Behavioral and clinical parameters were assessed to analyze the edema and nociception of the animals by measuring the knee perimeter, performing the flexion/extension test and the pressure application measurement (PAM), and evaluating the footprint area. II-7.2.1. Evaluation of knee perimeter Measuring the joint volume allows the quantification of the severity and the extension of the edema in the developed animal model [136]. The knee circumference is a widely used technique that measures the articular volume using a measuring tape, from an anatomical point previously established. It is a quick, low cost, accessible and easy to handle technique in comparison with other more recent evaluation methods, such as the Leg-O-Meter (equipment to measure the circumference of the lower limb), the water displacement method, optical electronic methods, computerized tomography, among others, all characterized as expensive devices and more time consuming. Nevertheless, the results may be highly influenced by the researcher training. In this thesis, joint perimeter was assessed as an indirect indicator of the development of an inflammatory state with edema. Knee perimeters of both hind paws were measured using a paper strip with a ruler. II-7.2.2. Flexion/extension test Mechanical allodynia is defined as a painful sensation in response to a non-nociceptive stimulus (e.g. innocuous stimuli like light touch) [137]. Unlike inflammatory hyperalgesia that has a protective role, allodynia has no obvious biological utility. It has been reported a higher mechanical allodynia in arthritic diseases. In this thesis, mechanical allodynia was evaluated by the flexion/extension test, in which animals were submitted to five consecutive flexion/extension movements in both knees. While normal animals do Chapter II – Materials and Methods 95 not vocalize with this stimulus, arthritic rats had an increased number of vocalizations representing high mechanical allodynia. The number of vocalizations during each flexion/extension movement was registered. II-7.2.3. Pressure application measurement Mechanical hyperalgesia is defined as an increased pain sensitivity (i.e. increased pain response produced by a stimulus that normally causes pain) [138]. A classical approach to measure mechanical hyperalgesia is the application of noxious pressure to the primary site of injury [139]. The PAM applies a force range of 0–1500 g and allows an accurate behavioral quantification of the mechanical hypersensitivity in rodents with chronic inflammatory joint pain [140]. In this thesis, the PAM method was used as previously described [141]. Briefly, with the animal securely held, an increasing force was gradually applied across the joint until a behavioral response was observed (paw-withdrawal, freezing of whisker movement, wriggling or vocalization) with a cut-off of 5 s. Limb withdrawal threshold (LWT) was recorded as the peak force (in grams force - gf) applied immediately prior to the behavioral response. LWT was measured twice in both paws at 1 min intervals. The mean LWTs were calculated per animal. II-7.2.4. Footprint area Gait disturbance has also been detected using the knee joint incapacitation test in rats with knee joint arthritis induced by IA injections of carrageenan [142]. Indeed, analyses of the gait gives important information regarding the sensitivity of the animals to the pain. In this thesis, the animals performed the catwalk gait analyses and the footprint area of both feet was measured using the Image J software. II-7.3. Experimental design In vivo studies were divided in two main experiments: (i) assessment of NPs biocompatibility after IA delivery and their therapeutic potential, and (ii) evaluation of the NPs therapeutic efficacy and its Chapter II – Materials and Methods 96 comparison with the injection of both free Abs. Both experiments used a carrageenan-induced inflammatory arthritis model of OA. In the first experiment (Figure II-17A), three days after the arthritis induction through an IA injection of carrageenan into the right knee joint of adult male and female Wistar rats, the development of arthritis was verified as previously described (time point 0) and the animals were treated with NPs or NPs+Abs. The control (SHAM) animals were injected with saline during the induction and then treated with the NPs. At the end of the behavioral session, after 4 days of treatment, animals were sacrificed with a lethal dose of pentobarbital and the knee joints were removed for further histological analyses. In the second in vivo experiment (Figure II-17B), arthritis induction (after 3 days of the carrageenan injection) in male Wistar rats was assessed as previously described (time point 0), and four groups were tested: (i) saline, (ii) NPs, (iii) Abs and (iv) NPs+Abs. At time points 4 and 10, the disease progression was assessed, and in the last time point the animals were sacrificed as described and the joints removed for subsequent histological analyses. Figure II-17 – Experimental design of (A) Experiment 1 - NPs biocompatibility, and (B) Experiment 2 - therapeutic effects of the biofunctionalized NPs. II-7.4. Histological analyses Histological analyses is the gold standard for tissue examination for both qualitative and quantitative measurements in research and medical diagnosis [143]. It is used to assess the inflammation or healing stage and to monitor the presence and distribution of degradation products that dissolved into the surrounding tissue. The process of histological staining takes five key stages: fixation, processing, A B Chapter II – Materials and Methods 97 embedding, sectioning and staining [144]. Different staining is used to identify specific structures, cells and tissues. Joints were fixed with 10% (v/v) of formalin, decalcified in Biodec R (BioOptica, Italy) until all the mineral part of the bone was removed. Then, samples were transferred to histological cassettes, processed and embedded in paraffin. Sagittal sections (5 μm) were cut through the knee joint using a manual rotary microtome (Micron Technology, USA). Afterwards, histological sections were analyzed though hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). II-7.4.1. Hematoxylin & Eosin Staining H&E staining is widely used in histology either in research and in medical diagnosis as it allows the visualization of different cell components [145]. Hematoxylin is a natural basic dye that preferentially stains the acidic components (basophilic cell components), such as nucleic acids in the nucleus. Eosin is a synthetic acidic dye that stains basic components of cells with a pink color, including the cytoplasm or connective tissue. In this thesis, H&E staining was performed following a routine protocol [146]. Briefly, sections were deparaffinized with xylene, rehydrated in ethanol and stained with Gill hematoxylin and alcoholic eosin. Afterwards, the sections were dehydrated and mounted with resinous mounting medium. The histological sections were analyzed under Leica DM750 microscope. II-7.4.2. Immunohistochemistry IHC is a technique to specifically label a cellular antigen (e.g. a protein) in tissue sections using Abs [147]. Hence, these localization methods fundamentally rely on the high specificity, affinity and sensitivity of Ab-antigen interactions. Abs are visualized either directly or indirectly (usually via a secondary Ab), with a stain that is easily detectable under a light or electron microscope. In this thesis, IHC analyses were performed as herein described. After deparaffinization in xylene and rehydration, the tissue sections were subjected to heat-induced antigen-retrieval with sodium citrate buffer (10 mM sodium citrate, 0.05 % (v/v) tween 20 acquired from Bio-Rad (USA), pH 6) for 20 min at 98 ºC. To block nonspecific antigen binding, sections were incubated for 30 min with 3 % (w/v) BSA. Sections were incubated with the mouse anti-IL-6 Abs and rabbit anti-TNF-α Abs at 4 ºC overnight. Then, Chapter II – Materials and Methods 98 sections were incubated with the secondary Abs Alexa Fluor 488 or 594 for 2 h at RT. After removing unbound secondary Abs, the sections were mounted using aqueous mounting medium. For negative controls, the incubation step for primary Abs was replaced with Abs diluent solution alone. The samples were examined using a confocal laser scanning microscope (TCS SP8, Leica). II-8. STATISTICAL ANALYSES All quantitative data are presented as mean ± standard deviation (SD). Statistical analyses were performed using a GraphPad Prism 5.0 software (GraphPad Software, USA). A Shapiro-Wilk normality test was performed to assess data normality. As data do not followed a normal distribution, the Mann–Whitney U test was used when two groups were compared and the Kruskal-Wallis test followed by Dunn’s multiple comparison test when more than two groups were compared. II-9. REFERENCES 1. Islam, S., Bhuiyan, M. A. R. and Islam, M. 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Carbohyd Polym . 2013, 92 (2): p. 1262-79. 106 SECTION 3 NANOPARTICLES DEVELOPMENT AND IN VITRO EVALUATION 107 Chapter III Interleukin-6 neutralization by antibodies immobilized at the surface of polymeric nanoparticles as a therapeutic strategy for arthritic diseases Chapter III – Interleukin-6 neutralization by antibodies immobilized at the surface of polymeric nanoparticles as a therapeutic strategy for arthritic diseases 108 Chapter III Chapter III - Interleukin-6 neutralization by antibodies immobilized at the surface of polymeric nanoparticles as a therapeutic strategy for arthritic diseases1 ABSTRACT Arthritic diseases are disabling conditions affecting millions of patients worldwide. Pro-inflammatory cytokines, particularly interleukin-6 (IL-6), plays a crucial role in inflammation and cartilage destruction. Although the beneficial effects of antibody therapy, its efficacy is limited. Therefore, this work proposes the immobilization of antibodies at the surface of biodegradable polymeric nanoparticles (NPs) to capture and neutralize IL-6. Our system is intended to protect, extend and enhance the therapeutic efficacy after delivery. Chitosan-hyaluronic acid NPs are synthetized as a stable monodisperse population. After determining the maximum immobilization capacity (10 μg/mL), the capture ability was confirmed. Biological assays demonstrate the NPs cytocompatibility with human articular chondrocytes (hACs) and human macrophages. hACs stimulated with macrophage conditioned medium shows the beneficial role of IL-6 capture and neutralization. Biofunctionalized NPs exhibit a prolonged action and stronger efficacy than the free antibodies. In conclusion, this system can be an effective and long lasting treatment for arthritic diseases. 1This chapter is based on the following publication: Lima A. C., Cunha C., Carvalho A., Ferreira H., Neves N. M. Interleukin-6 Neutralization by Antibodies Immobilized at the Surface of Polymeric Nanoparticles as a Therapeutic Strategy for Arthritic Diseases. Acs Appl Mater Inter . 2018, 10 (16): p. 13839-50. Chapter III – Interleukin-6 neutralization by antibodies immobilized at the surface of polymeric nanoparticles as a therapeutic strategy for arthritic diseases 109 III-1. INTRODUCTION Arthritic diseases are inflammatory joint disorders, associated with synovitis and articular cartilage destruction. The most common forms of arthritis are osteoarthritis (OA) and rheumatoid arthritis (RA) [1]. OA, a local degenerative joint disease, is the leading cause of morbidity and disability in the elderly, affecting around 10% of men and 18% of women over 60 years of age [2]. In contrast to OA, RA is a systemic autoimmune disease that usually involves multiple joints, affecting 0.3-1.0% of the general population [2]. Even though joint damage in OA and RA proceeds via different pathways, in both the normal balance of extracellular matrix (ECM) is disrupted and shifts towards degradation [3]. Cartilage disruption is associated with an increase of the levels of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukins (IL, particularly IL-1β and IL-6), mainly produced by mononuclear cells, chondrocytes or synoviocytes [1]. These cytokines increases the production of catabolic factors and downregulates the anabolic mediators [4, 5]. Hence, pro-inflammatory cytokines up-regulates injurious enzymes, especially matrix metalloproteinases. Simultaneously, they inhibit the production of their physiological regulators, stimulate the production of nitric oxide, and hinder the synthesis of ECM components such as collagen type II and aggrecan. IL-6 is a key mediator in the pathophysiology of OA and RA, as it regulates a wide range of fundamental biological activities, including acute-phase responses, inflammation, and immune responses [6]. Indeed, OA and RA patients present high concentrations of this pleiotropic cytokine [7], and many efforts are being made to create biosensors to monitor IL-6 secretion in vivo [8]. In addition, higher levels of IL-6 in OA was found to be a significant predictor of superior risk of cartilage loss and reduced response to treatment [9]. Thus, strategies targeting IL-6 or its receptors are promising treatments for arthritic diseases [10]. The first humanized anti-IL-6 receptor antibody (Ab), tocilizumab, has demonstrated its outstanding clinical efficacy and tolerable safety profile in phase III clinical trials for RA patients, resulting in its worldwide approval to treat moderate-to-severe active RA. This successful approach led to the development of other IL-6 inhibitors, including fully human anti-IL-6 receptor mAb (sarilumab, sirukumab and olokizumab) [11]. This cytokine is being also considered the most interesting new target for OA treatment with clinical trials currently in progress [ClinicalTrials.gov Identifier: NCT02477059]. Although these treatments seem very attractive, systemic injection of biological agents are associated with serious side effects, such as risk of infection, administration reactions, congestive heart failure, demyelinating diseases, hyperlipidemia, among other conditions that affect patient health [12]. In this Chapter III – Interleukin-6 neutralization by antibodies immobilized at the surface of polymeric nanoparticles as a therapeutic strategy for arthritic diseases 110 sense, intra-articular (IA) injections offers several benefits by achieving high local bioavailability, reducing systemic exposure and requiring lower doses [13, 14]. However, due to the rapid clearance of Abs by the synovium, the therapy has limited efficacy. Hence, there is an unmet need for the development of new effective approaches, with prolonged efficacy and reduced side effects. Nanoparticles (NPs) are promising approaches to solve limitations of conventional therapies [14]. The unique properties of NPs make them highly attractive for the design of novel modalities for arthritis treatment. Nanoscale features can be used to protect the therapeutic agent from degradation and to have targeted and controlled delivery, which improves drug efficacy, reduces the dosage and significantly reduces the side effects. Actually, polymeric NPs are one of the most studied strategies for nanomedicine [15]. Polymers are widely used as building blocks, due to the flexible design based on functionalization, macromolecular synthesis methods, and polymer diversity. Regarding degradation properties, biodegradable polymeric NPs are highly preferred for medical applications, providing outstanding bioavailability, stability, compatibility and controlled release [16]. In this work, natural-derived polymers were used, namely chitosan (Ch) and hyaluronic acid (HA). They possess a high level of functional groups, such as amino (NH2) and carboxylic acid (COOH) groups that can be further modified and functionalized with other polymers, crosslinkers and/or biomolecules [17, 18]. Ch, the second most abundant polysaccharide in nature, is particularly attractive due to its advantages, such as non-cytotoxicity, low immunogenicity, high stability and reasonable cost [19, 20]. Although there are some studies reporting Ch as vaccine adjuvant [21], the capacity to promote cellular immunity is related with the deacetylation degree (DD) of the polymer [22]. Indeed the DD and immunogenicity are inversely correlated in this biomaterial. Therefore, in this work, Ch with a high degree of deacetylation was used to avoid any immunogenicity. HA is a natural component of the ECM of articular cartilage and synovial fluid. Due to the interaction with CD44 receptors of the cells, especially chondrocytes, HA plays an important role on cartilage function [23]. Nonetheless, HA physicochemical and biological properties depend on its molecular weight (MW) [24]. High MW HA displays anti-inflammatory and immune-suppressive properties, whereas low MW HA is a potent pro-inflammatory molecule. It has been reported that HA with MW of 700–6000 kDa are the best suited for cartilage repair [19]. The aim of this study was to develop a carrier intended for IA administration and allowing the capture and neutralization of IL-6, a crucial pro-inflammatory mediator in arthritic joints (Figure III-1). Ch and HA were used to produce biofunctionalized NPs with anti-IL-6 Abs immobilized at NPs’ surface. The maximum immobilization of Abs and the capture capability was assessed. NPs cytocompatibility was validated by their culturing with human articular chondrocytes (hACs) and human macrophages. Our system was also Chapter III – Interleukin-6 neutralization by antibodies immobilized at the surface of polymeric nanoparticles as a therapeutic strategy for arthritic diseases 111 validated by stimulating hACs with macrophage conditioned medium, and then treating with biofunctionalized NPs or the free Abs. Considering the drawbacks of the current treatments, we hypothesized that this strategy offers a maximum therapeutic effect of the immobilized Abs, avoiding unnecessary exposure to healthy tissues and systemic side-effects. Figure III-1 – Schematic illustration of the biofunctionalized NPs role in arthritis treatment. (A) Production of the biofunctionalized polymeric NPs. (B) Inflammation reduction in the synovial fluid after the neutralization of IL6 by the NPs. Abbreviations: Ch, chitosan; HA, hyaluronic acid; EDC, ethyl-3-(3- (dimethylaminopropyl)carbodiimide; NHS, N-hydroxysuccinimide. III-2. MATERIALS AND METHODS This section provides details on the NPs preparation and characterization, the maximum Abs immobilization and their performance in capture as well as their cytocompatibility and biologic activity. III-2.1. Materials Ch with a MW of 150 kDa and 95% of DD was purchased from Heppe Medical Chitosan GmbH (Germany). HA with a MW of 750 kDa was bought from Lifecore Biomedical (USA). Mouse monoclonal anti-IL-6 Abs and human IL-6 full length protein was purchased from Abcam (UK). Alexa Fluor® 594 donkey Chapter III – Interleukin-6 neutralization by antibodies immobilized at the surface of polymeric nanoparticles as a therapeutic strategy for arthritic diseases 112 anti-mouse IgG, Roswell Park Memorial Institute (RPMI)-1640 media and Fetal Bovine Serum (FBS) were purchased from Thermo Fisher Scientific (USA). Human IL-6 Standard ABTS ELISA Development Kits and human basic Fibroblast Growth Factor (bFGF) was acquired from Peprotech (USA). CD14 MicroBeads and recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) was obtained from Miltenyi Biotec (USA). All other reagents were purchased from Sigma-Aldrich (USA). III-2.2. NPs preparation Ch-HA NPs were prepared by polyelectrolyte complexation of both natural-based polyelectrolytes. Briefly, Ch was dissolved in 1% (v/v) acetic acid, while HA was dissolved in ultrapure water, overnight at room temperature (RT). Both solutions were filtered through a 0.22 µm pore membrane. The NPs formation occurred spontaneously when the HA solution was added dropwise at 1 mL/min rate to the Ch solution under strong magnetic stirring (600 rpm). Different initial polymers concentration (0.25, 0.5 and 1 mg/mL) and pH values (3, 4, 5, 6 and 7) were used to evaluate the influence of these parameters on the properties of the NPs. During preparation, NPs were stabilized through carbodiimide chemistry. 1-ethyl-3-(3- (dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) reagents were dissolved in 0.1 M MES buffer (pH 4.7) with 0.9% (w/w) NaCl. Five different EDC/NHS ratios were tested, namely 400/100, 100/400, 200/200, 50/200 and 200/50 mM. For fluorescence biological assays, 200 μL of fluorescein isothiocyanate (FITC, 2 mg/mL in ethanol:water, 1:10) was added to the Ch solution before the NPs formation. To remove unreacted compounds, the NPs were washed twice with ultrapure water by centrifugation (30 min, 4000 rpm at 20 ºC) using Vivaspin 300 kD Filter Units (Fisher Scientific, USA). To avoid NPs aggregation, glucose at 2 mg/mL was added before centrifugation [25]. III-2.3. NPs characterization The developed NPs were characterized regarding their size distribution, surface charge, stability and morphology, as described in the following sub-sections. Chapter III – Interleukin-6 neutralization by antibodies immobilized at the surface of polymeric nanoparticles as a therapeutic strategy for arthritic diseases 113 III-2.3.1. Size distribution and zeta potential measurements The size and polydispersity index (PDI) of the produced NPs were assessed by dynamic light scattering (DLS) and the zeta potential was determined by laser Doppler micro-electrophoresis using a Zetasizer Nanoseries ZS equipment (Malvern Instruments, Portugal). The measurements were performed at 25 ºC using samples diluted in ultra-pure water (1:20; v/v). III-2.3.2. Stability studies For pH stability assessment the pH was increased until 7.4 using NaOH 1M. For storage stability, the NPs suspensions were kept at 4 ºC under static conditions. During the experimental time (6 months), it was determined the size, PDI and zeta potential, as just described. III-2.3.3. NPs morphology Morphological analyses of the developed Ch-HA NPs were performed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Prior to analysis, Ch-HA NPs were diluted in water (1:20; v/v) and disposed into the surface of a glass slide for air-dry. For SEM analyses, the NPs were sputter-coated with palladium (EM ACE600, LEICA) and analyzed using High-Resolution Field Emission Scanning Electron Microscope (Auriga Compact, ZEISS). AFM measurements were performed using a MultiMode STM microscope controlled by a NanoScope III from Digital Instruments system, operating in tapping mode at a frequency of 1 Hz. III-2.4. Abs immobilization determination The anti-IL-6 Abs were immobilized at the surface of the optimized Ch-HA NPs. In order to determine the NPs’ maximum immobilization capacity, a range of primary Abs concentrations were tested (from 5 to 20 μg/mL). First, the anti-IL-6 Abs were activated with a solution of 50/200 mM EDC/NHS in 0.1 M MES buffer for 15 min. Then, the NPs were incubated with the activated anti-IL-6 Abs, overnight at 4 ºC. To remove the unbound Abs, the biofuncionalized NPs were washed twice by centrifugation, as previously described. Chapter III – Interleukin-6 neutralization by antibodies immobilized at the surface of polymeric nanoparticles as a therapeutic strategy for arthritic diseases 114 To determine the degree of anti-IL-6 Abs immobilization, the biofunctionalized NPs were incubated with the secondary Abs Alexa Fluor® 594 solution for 1 h at RT. As negative control, NPs without immobilized Abs were used (0 μg/mL). After centrifugation, the fluorescence of the unbound secondary Abs (in supernatant) were determined using an excitation-emission wavelengths of 590/20 - 645/40 nm, respectively, in a microplate reader (Synergy HT, BioTek, USA). The concentration of the anti-IL-6 Abs at the NPs surface corresponds to the difference between the initial and unbound secondary Abs. III-2.5. IL-6 capturing Ch-HA NPs biofunctionalized with anti-IL-6 Abs (5-20 μg/mL) were incubated with 1.25 μg/mL of human IL-6 full length protein, overnight at RT. After centrifugation, the supernatants were collected and the unbound IL-6 was assessed using the ELISA Development Kit. The amount of IL-6 captured by the biofunctionalized NPs corresponds to the difference between the initial and unbound amount of the added cytokine. III-2.6. Biological Assays In order to assess the cytocompatibility and biological effects of the developed NPs, the cell isolation and culture, viability, proliferation, protein content and morphology were performed as described in the following sub-sections. III-2.6.1. Isolation and cell culture hACs were isolated from knee cartilage samples collected from arthroplasties surgeries biopsies. Samples were obtained through the cooperation agreement between Centro Hospitalar do Alto Ave, Guimarães, Portugal, and 3B’s Research Group, and after informed donor consent. Cells were isolated by enzymatic digestion, according to a previously described protocol [26]. hACs cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FBS, 10 mM HEPES buffer, Llanyl-L-glutamine, MEM Non Essential Aminoacids, 100 units/mL of penicillin, 100 μg/mL of streptomycin and 10 ng/mL human bFGF, and incubated at 37 °C in a humidified 5% CO2 atmosphere. Chapter III – Interleukin-6 neutralization by antibodies immobilized at the surface of polymeric nanoparticles as a therapeutic strategy for arthritic diseases 115 The human monocytic cell line THP-1 was maintained in complete RPMI, containing RPMI-1640 media supplemented with 2 mM L-glutamine, 100 units/mL of penicillin, 100 μg/mL of streptomycin, 10 mM HEPES buffer and 10% FBS. Human monocyte-derived macrophages were generated from peripheral blood mononuclear cell (PBMCs). Buffy coats from healthy donors were obtained after written informed consent at the Hospital de Braga, Braga, Portugal. Briefly, PBMCs were enriched from buffy coats by density gradient using Histopaque®-1077. Cells present in the enriched mononuclear fraction were washed twice in PBS and resuspended in RPMI-1640 culture medium with 2 mM glutamine and 2 g/L NaHCO3 supplemented with 10% human serum, 100 units/mL of penicillin, 100 μg/mL of streptomycin and 10 mM HEPES buffer. Monocytes were then separated by positive selection using magnetically labelled CD14 MicroBeads on a MiniMACS separator. Isolated monocytes were re-suspended in complete RPMI medium and seeded at 5×105 cells per well in 24-well plates for 7 days in the presence of 20 ng/mL of GM-CSF. Acquisition of macrophage morphology was confirmed by visualization in a BX61 microscope (Olympus, Japan). III-2.6.2. Cell seeding For hACs seeding, cells at 5×104 cells per well were added to tissue culture polystyrene (TPCS) coverslips in 24-well plates. After cell attachment during 5h, culture medium was added to a final volume of 1 mL. Different concentrations of sterilized Ch-HA NPs were added to hACs subsequently to 24 h of incubation. For the induction of THP-1 cell differentiation, cells were seeded at 5×105 cells per well in 24-well plates in cRPMI with 100 nM phorbol 12-myristate-13-acetate (PMA) for 24 h. After incubation, nonattached cells were removed by aspiration, and the adherent cells were washed three times with cRPMI. To ensure reversion of cells to a resting macrophage phenotype before stimulation, cells were incubated for an additional 48 h in cRPMI without PMA. For stimulation and retrieval of conditioned media, cells were further incubated for 24 h with 100 ng/mL of lipopolysaccharide (LPS) in fresh media and the supernatants were collected and stored at -80 °C. IL-6 production by cells was assessed in the supernatants by commercial ELISA. Then, Ch-HA NPs were added at different concentrations. Regarding the human primary macrophages, after 7 days in the presence of GM-CSF, culture medium was replaced and different concentrations of Ch-HA NPs were added. Cells cultured without NPs (only with culture medium) were used as control. After 1, 2, 3 and 7 days of culture with NPs, the different samples in triplicate were washed with sterile PBS and analyzed Chapter VII – Nanoparticle-mediated neutralization of IL-6 and TNFα for osteoarthritis treatment 218 VII-2.3.2. NPs morphology Morphological analyses of the biofunctionalized NPs were performed by atomic force microscopy (AFM). Diluted NPs (1:20; v/v) were air-dried into the surface of a glass slide. A MultiMode STM microscope controlled by a NanoScope III from Digital Instruments system was used for AFM measurements, using a tapping mode at a frequency of 1 Hz. VII-2.4. Biological assays A co-culture model of human chondrocytes and macrophages was used to assess the biologic effects of the biofunctionalized NPs. VII-2.4.1. Isolation and Cell Culture Knee cartilage samples collected from arthroplasties surgeries biopsies were used to isolate hACs. Samples were obtained through the cooperation agreement between Centro Hospitalar do Alto Ave, Guimaraes, Portugal, and I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, and after informed donor consent. Cells were isolated by enzymatic digestion, as previously described [22]. hACs cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FBS, 10 mM HEPES buffer, 10 mM L-lanyl-L-glutamine, 10 mM MEM nonessential amino acids, 100 units/mL of penicillin, 100 μg/mL of streptomycin and 10 ng/mL of human bFGF, and incubated at 37 °C in a humidified 5% CO2 atmosphere. The human monocytic cell line THP-1 was maintained in complete RPMI, containing RPMI-1640 media supplemented with 2 mM of L-glutamine, 100 units/mL of penicillin, 100 μg/mL of streptomycin, 10 mM HEPES buffer and 10% FBS. VII-2.4.2. Co-culture of hACs with macrophages For the induction of THP-1 cell differentiation, cells were seeded 2.5×105 cells per well in cRPMI with 100 nM phorbol 12-myristate-13-acetate (PMA) in 24-well cell culture inserts (pore size: 1 µm). After incubation during 24 h, non-attached cells were removed by aspiration, and the adherent cells were Chapter VII – Nanoparticle-mediated neutralization of IL-6 and TNFα for osteoarthritis treatment 219 washed three times with cRPMI. To ensure reversion of cells to a resting macrophage phenotype, cells were incubated for an additional 48 h in cRPMI without PMA. For hACs seeding, cells at a concentration of 5×104 cells per well were added to 24-well plates. After 24 h of incubation, the co-culture system was established by transferring the inserts to the hACs culture. VII-2.4.3. Biological effects of the biofunctionalized NPs Resting M0 macrophages were activated to the M1 phenotype by adding 100 ng/mL of lipopolysaccharide (LPS). Monoculture systems were used as controls. After 2 h of stimulation, different conditions were tested: (i) no treatment, (ii) treatment with anti-TNF-α Abs, (iii) treatment with anti-IL-6 Abs, (iv) treatment with anti-TNF-α and anti-IL-6 Abs, (v) treatment with biofunctionalized NPs with antiTNF-α Abs, (vi) treatment with biofunctionalized NPs with anti-IL-6 Abs, and (7) treatment with biofunctionalized NPs with anti-TNF-α and anti-IL-6 Abs. The Abs were administered at 1 µg/mL. After 1, 3, 7 and 14 days, samples were collected and evaluated regarding cell viability, proliferation and morphology, as described. The amount of IL-6 and TNF-α in the media was assessed by ELISA. During the time of experiment 300 μL of fresh media was added each 3 days, but no media was removed to keep the NPs in contact with the cells. VII-2.4.3.1 Cell viability Alamar blue (AB) reagent (Bio-Rad, USA) was used to assess the metabolic activity of cells, following the manufacturer’s instructions. Samples were incubated 4 h with medium containing 10% AB. The fluorescence was measured in a microplate reader (Synergy HT, BioTek, USA), using an excitation wavelength of 528 nm and an emission wavelength of 590 nm. VII-2.4.3.2 DNA quantification A fluorimetric dsDNA quantification kit (Quant-iTTM, PicoGreen, Molecular Probes, Invitrogen, USA) was used to assess cell proliferation, following the manufacturer’s instructions. The fluorescence was measured in a microplate reader (Synergy HT, BioTek, USA), using an excitation wavelength of 485 nm and an emission wavelength of 528 nm, being the DNA concentration of the samples inferred from the standard curve. Chapter VII – Nanoparticle-mediated neutralization of IL-6 and TNFα for osteoarthritis treatment 220 VII-2.4.3.3 SEM analyses SEM was used to analyze the hACs morphology. After fixation with 2.5% glutaraldehyde, samples were dehydrated using increasing concentrations of ethanol (10%, 20%, 40%, 60%, 80%, 90%, 95%, and 100%). In order to evaluate the samples by High-Resolution Field Emission Scanning Electron Microscope (Auriga Compact, ZEISS), they were sputter-coated (EMACE600, LEICA) with a thin layer (8−12 nm) of palladium. Microphotographs were recorded at 5 kV. VII-2.4.4. Cytokines quantification IL-6 and TNF-α cytokines were quantified using human sandwich ELISAs Kit that were performed according to the manufacturer procedure. ABTS liquid substrate was added to each well and the color development was monitored in a microplate (Synergy HT, BioTek, USA) at 405 nm, with a wavelength correction set at 650 nm. Cytokines concentration was inferred from the standard curve. VII-2.5. In Vivo Studies The experimental protocol was approved by the Institutional Ethical Commission (SECVS 109/2016) and followed the European Community Council Directive 86/609/EEC and 2010/63/EU concerning the use of animals for scientific purposes. Efforts were always made to minimize the number of animals used per experiment or test and their potential suffering. VII-2.5.1. Animal use and care Wistar rats (n=32, 8 weeks old) were housed in pairs in a limited-access rodent facility, with food and water available ad libitum . The temperature was maintained at 22.0 ± 0.5 °C with a 12/12 h light/dark cycle (starting at 8:00 a.m.). General health parameters were surveyed twice per week by the resident veterinary and the animals’ weight was recorded every week throughout the experimental period. On the day of the experiments, animals were left in the experimental room for one hour in order to get acquainted with the surroundings. Chapter VII – Nanoparticle-mediated neutralization of IL-6 and TNFα for osteoarthritis treatment 221 VII-2.5.2. Induction of arthritis The induction of arthritis was performed as described in detail elsewhere [23]. Briefly, a 3% solution of carrageenan was dissolved in sterile saline solution (0.9% NaCl) and injected into the synovial cavity of the right knee joint at a volume of 0.1 mL. Control animals (SHAM) were injected with 0.1 mL saline in the synovial cavity of the right knee joint. VII-2.5.3. Behavioral assessment and clinical parameters Behavioral and clinical parameters were assessed to analyze the edema and nociception of the animals through different parameters. VII-2.5.3.1 Evaluation of the knee perimeter Joint perimeter was assessed as an indirect indicator of the development of an inflammatory state with edema. Knee perimeters of both hind paws were measured using a paper strip with a ruler. VII-2.5.3.2 Flexion/extension test Mechanical allodynia was evaluated by the flexion/extension test, in which animals were submitted to five consecutive flexion/extension movements in both knees. The number of vocalization during each flexion/extension movement was registered. VII-2.5.3.3 Pressure application measurement A classical approach to measure mechanical hyperalgesia is the application of noxious pressure to the primary site of injury [24]. The pressure application measurement (PAM) applies a force range of 0– 1500 g and allows an accurate behavioral quantification of the mechanical hypersensitivity in rodents with chronic inflammatory joint pain [25]. PAM method was used as previously described [26]. Briefly, with the animal securely held, an increasing force was gradually applied across the joint until a behavioral response was observed (paw-withdrawal, freezing of whisker movement, wriggling or vocalization) with a cut-off of 5 s. Limb withdrawal threshold (LWT) was recorded as the peak force (in grams force - gf) Chapter VII – Nanoparticle-mediated neutralization of IL-6 and TNFα for osteoarthritis treatment 222 applied immediately prior to the behavioral response. LWT was measured twice in both paws at 1 min intervals. The mean LWTs were calculated per animal. VII-2.5.3.4 Footprint area The animals performed the catwalk gait analyses and the footprint area of both feet was measured using the Image J software. VII-2.5.4. Experimental design In vivo studies were divided in two main experiments: (i) assessment of NPs biocompatibility after IA delivery and their therapeutic potential, and (ii) evaluation of the NPs therapeutic efficacy and its comparison with the injection of both free Abs. Both experiments used a carrageenan-induced inflammatory arthritis model of OA. In the first experiment, three days after the arthritis induction through an IA injection of carrageenan into the right knee joint of adult male and female Wistar rats, the development of arthritis was verified as previously described (time point 0) and the animals were treated with NPs or NPs+Abs. The control (SHAM) animals were injected with saline during the induction and then treated with the NPs. At the end of the behavioral session, after 4 days of treatment, animals were sacrificed with a lethal dose of pentobarbital and the knee joints were removed for further histological analyses. In the second in vivo experiment, arthritis induction (after 3 days of the carrageenan injection) in male Wistar rats was assessed as previously described (time point 0), and four groups were tested: (i) saline, (ii) NPs, (iii) Abs and (iv) NPs+Abs. At time points 4 and 10, the disease progression was assessed, and in the last time point the animals were sacrificed as described and the joints removed for subsequent histological analyses. VII-2.5.5. Histological analyses Rats were sacrificed 4 or 10 days after treatment administration, in the first and second experiment, respectively. The joints were removed, fixed with 10% (v/v) of formalin, decalcified in Biodec R (BioOptica, Italy) until all the mineral part of the bone was removed. Then, samples were transferred to histological cassettes, processed and embedded in paraffin. Sagittal sections (5 μm) were cut through the knee joint Chapter VII – Nanoparticle-mediated neutralization of IL-6 and TNFα for osteoarthritis treatment 223 using a manual rotary microtome (Micron Technology, USA) and stained with hematoxylin and eosin (H&E, Thermo Scientific, USA) following a routine protocol. Briefly, sections were deparaffinized with xylene, rehydrated in ethanol and stained with Gill hematoxylin and alcoholic eosin. Afterwards, the sections were dehydrated and mounted with resinous mounting medium. The histological sections were analyzed under Leica DM750 microscope. For immunohistochemistry (IHC) analyses, after deparaffinization in xylene and rehydration, the tissue sections were subjected to heat-induced antigenretrieval with sodium citrate buffer (10 mM sodium citrate, 0.05% (v/v) tween 20 (Bio-Rad, USA), pH 6) for 20 min at 98 ºC. To block nonspecific antigen binding, sections were incubated for 30 min with 3 % (w/v) BSA. Sections were incubated with the mouse anti-IL-6 Abs and rabbit anti-TNF-α Abs at 4 ºC overnight. Then, sections were incubated with the secondary Abs Alexa Fluor 488 or 594 for 2 h at RT. After removing unbound secondary Abs, the sections were mounted using aqueous mounting medium. For negative controls, the incubation step for primary Abs was replaced with Abs diluent solution alone (data not shown). The samples were examined using a confocal laser scanning microscope (TCS SP8, Leica). VII-2.6. Statistical Analyses GraphPad software was used to perform statistical analyses. Normality was analyzed using the Shapiro-Wilk test. Since data did not follow a normal distribution, results were analyzed using the KruskalWallis test with Dunn’s Multiple Comparison post-test. Significance was set to * p <0.05, ** p <0.01; *** p <0.001. Results are presented as mean ± Standard deviation (SD). VII-3. RESULTS Biofunctionalized NPs characterization, in vitro biological effects as well as in vivo studies were performed to validate their activity in inflammatory arthritis. VII-3.1. Biofunctionalization and characterization of Ch-HA NPs After Ch-HA NPs preparation, anti-TNF-α Abs or anti-IL-6 Abs were covalently immobilized at their surface using the carbodiimide chemistry. The maximum immobilization was 11.85 µg/mL for anti-TNFα Abs and 10.81 µg/mL for anti-IL-6 Abs using an initial concentration of 15 µg/mL (Figure VII-2A). Chapter VII – Nanoparticle-mediated neutralization of IL-6 and TNFα for osteoarthritis treatment 224 Biofunctionalized NPs displayed 132.05 ± 2.58 nm of diameter, 0.12 ± 0.01 of PDI and +20.07 ± 2.10 mV of zeta potential, keeping their stability for at least 6 months (size increased less than 10%). AFM analyses revealed the spherical shape of the biofunctionalized NPs, as shown in Figure VII-2B. Moreover, the NPs size was around 130 – 140 nm, confirming the DLS measurements. Figure VII-2 – (A) Maximum immobilization capacity at the surface of polymeric NPs of anti-TNF-α Abs or anti-IL-6 Abs. (B) AFM micrographs of the biofunctionalized NPs with anti-IL-6 and anti-TNF-α Abs. Scale bar: 400 nm. VII-3.2. Biological Assays To assess the biological performance of the Abs immobilized at the NPs’ surface, a co-culture model was used. The co-culture of hACs with M1 simulated macrophages significantly decreased the cell viability and DNA concentration comparatively to the control (Figures VII-3A and B). Alternatively, the treatment with biofunctionalized NPs with anti-IL-6 Abs and biofunctionalized NPs with anti-TNF-α and anti-IL-6 Abs (NPs-IL6 and NPs-TNF+IL6) increased the cell viability and proliferation in comparison to No treat group. Moreover, no significant differences were observed when compared to the control of hACs (Figures VII3A and B). The biofunctionalized NPs with TNF-α Abs were not able to prevent the reduction of the cell viability. Remarkably, the dual targeting of the biofuntionalized NPs with anti-TNF-α and anti-IL-6 Abs had the highest effect in the cell viability. Furthermore, the dual treatment with the soluble Abs (TNF+IL6) was only able to prevent the nefarious effects of the macrophage stimulation in hACs at the first time point (Day 1; Figures VII-3A and B). These results were corroborated by the cell morphology analyses (Figure VII-4). Indeed, after 14 days of co-culture, a low density and altered morphology with shrinkage of hACs were observed. The addition of biofunctionalized NPs with anti-TNF-α and anti-IL-6 Abs (NPs-TNF+IL6) significantly prevented these features and in a higher extension than soluble Abs. Chapter VII – Nanoparticle-mediated neutralization of IL-6 and TNFα for osteoarthritis treatment 225 Regarding the amount of unbound IL-6 and TNF-α in the medium (Figures VII-3C and D, respectively), the results showed that the co-culture of hACs and activated macrophages had a huge impact on those cytokines production. For IL-6 cytokines, both hACs and LPS stimulated THP-1 produce around 10 ng/mL. After the establishment of the co-culture system, the IL-6 amount increased to a maximum ≈ 2 µg/mL. This huge production of IL-6 cytokine corroborates the susceptibility to inflammation of hACs isolated from osteoarthritic patients. The treatment with the biofunctionalized NPs containing anti-IL-6 Abs reduced more the IL-6 amount in the medium than the soluble Abs. Moreover, since these reduction was significantly higher for biofunctionalized NPs with anti-IL-6 and anti-TNF-α Abs (Figure VII-3C), it was confirmed the synergistic effect of the dual targeting and the value of the proposed approach. For TNF-α cytokines (Figure VII-3D), whereas hACs without any stimulus do not produce this cytokine, activated M1 macrophages produce around 1.2 ng/mL after 1 day and reduced to 0.1 ng/mL after 14 days. In this case, both soluble and immobilized Abs were able to effectively reduce the amount of this cytokine in the medium. Nevertheless, dual targeting with biofunctionalized NPs had a significant reduction of the TNFα quantity at all time points when compared to no treatment group. Therefore, the overall results confirmed the initial hypothesis that NPs can prolong Abs half-life and efficacy as well as the synergistic effect of the dual capture and neutralization of IL-6 and TNF-α cytokines. Chapter VII – Nanoparticle-mediated neutralization of IL-6 and TNFα for osteoarthritis treatment 226 Figure VII-3 – Biochemical performance of hACs co-cultured with activated M1 macrophages: (i) no treatment (No treat), (ii) treatment with soluble anti-TNF-α Abs (TNF), (iii) treatment with soluble anti-IL-6 Abs (IL6), (iv) treatment with soluble anti-TNF-α and anti-IL-6 Abs (TNF+IL6), (v) treatment with biofunctionalized NPs with anti-TNF-α Abs (NPs-TNF), (vi) treatment with biofunctionalized NPs with anti-IL-6 Abs (NPs-IL6), and (vii) treatment with NPs biofunctionalized with anti-TNF-α and anti-IL-6 Abs (NPs-TNF+IL6). The samples were analyzed regarding (A) cell viability, (B) cell proliferation, (C) IL-6 concentration, and (D) TNF-α concentration. hACs and/or activated THP-1 cultured alone were used as controls (Ctr). Letter “a” denotes significant difference compared to the hACs group, and “b” denotes significant difference compared to No treat group, being * p <0.05, ** p <0.01, *** p <0.001. Chapter VII – Nanoparticle-mediated neutralization of IL-6 and TNFα for osteoarthritis treatment 227 Figure VII-4 – SEM micrographs of hACs co-cultured with activated M1 macrophages after 14 days. (A) Control (hACs cultured alone), (B) THP-1 stimulation without treatment, (C) THP-1 stimulation and addition of free soluble anti-TNF-α and anti-IL-6 Abs, and (D) THP-1 stimulation and addition of biofunctionalized NPs with anti-TNF-α and anti-IL6 Abs. Scale bars: 10 µm. VII-3.3. In vivo studies Two different in vivo experiments were performed to: (i) assess the NPs biocompatibility after IA delivery and the therapeutic potential of the biofunctionalized NPs, and (ii) evaluate the therapeutic efficacy of the biofunctionalized NPs and compare with the injection of the free Abs. VII-3.3.1. NPs biocompatibility To investigate the NPs biocompatibility after IA delivery and the potential therapeutic efficacy of the biofunctionalized NPs, an inflammatory arthritis rat model was used (Figure VII-5) – Experiment 1. Clinical and behavior parameters were assessed regarding the edema (increased number of knee perimeter Chapter VII – Nanoparticle-mediated neutralization of IL-6 and TNFα for osteoarthritis treatment 234 Abs due to the blockade of different pathological mediators and pathways [27]. Indeed, many fusion Abs are been designed to treat arthritic diseases, including IL-6 and TNF-α fusion protein [28] and IL-17A and TNF-α fusion protein [29]. Beyond its higher effectiveness, the dual targeting can overcome drug resistance caused by cytokine redundancy [30]. Therefore, the aim of this work was to study the therapeutic effects of biofunctionalized NPs in OA by capturing and neutralizing two central proinflammatory cytokines, namely IL-6 and TNF-α. The IA injection of developed NPs can lead to a reduction of the systemic side effects. Moreover, the immobilization of the Abs at the surface can protect them from degradation, prolonging their half-life and increasing their efficacy [31, 32]. In a previous study [21], the cytocompatibility in contact with hACs and human macrophages as well as the capacity to capture and neutralize IL-6 by biofunctionalized NPs with anti-IL-6 Abs were clearly demonstrated in vitro . Thus, in this study the polyelectrolyte complexation of the same polysaccharides were used to prepare biodegradable polymeric NPs, as previously described [21]. Then, the maximum immobilization of each Ab was determined, being 11.85 µg/mL for anti-TNF-α Abs and 10.81 µg/mL for anti-IL-6 Abs using an initial concentration of 15 µg/mL. After this point, the values reached a plateau and no more Abs were able to bind to the NPs’ surface. In addition, the biofunctionalization of the NPs did not affect their properties. Since inflammation plays a pivotal role in the pathological processes of OA, human chondrocytes were co-cultured with activated M1 macrophages and whether the biofunctionalized NPs could avoid the harmful impact of inflammation in those cells was investigated. Indeed, in vitro models could play a vital role not only to advance research into the etiological mechanisms, but also to help in the design and assessment of the efficacy of potential treatments. An in vitro inflammation model of osteoarthritic chondrocytes and macrophages transwell co-culture system was used as it closely resembles the permeable synovial joint [33]. Considering the polarization of the macrophages into pro-inflammatory phenotype (M1) and anti-inflammatory phenotype (M2) [34], in this study they were stimulated to the inflammatory M1 phenotype using LPS. Therefore, high levels of TNF-α, IL-1, IL-6, IL-12, IL-23 and reactive oxygen species will be produced. The induction of inflammation in the co-culture system was confirmed by the higher amounts of IL-6 and TNF-α cytokines in the medium in comparison to hACs cultured alone. IL-6 production in the co-culture system was much higher than TNF-α (≈2 µg/mL and ≈1.2 ng/mL, respectively). These outcomes are also found in the plasma and synovial fluid of OA patients [10]. The co-culture of hACs with M1 macrophages led to a higher reduction of hACs viability and proliferation and significant changes in their morphology. The treatment with the biofunctionalized NPs was able to prevent more effectively those harmful effects than the free Abs. Indeed, the quantification of Chapter VII – Nanoparticle-mediated neutralization of IL-6 and TNFα for osteoarthritis treatment 235 the amount of free IL-6 and TNF-α in the medium clearly demonstrated that the Abs successfully capture those cytokines, being the biofunctionalized NPs able to reduce significantly more IL-6 and TNF-α than the soluble Abs. Importantly, they were able to maintain those effects even after 14 days of culture. Thus, this strategy could increase the Abs half-life and efficacy in the inflamed joints. Moreover, those effects were higher when both cytokines were inhibited, confirming the synergistic effects of the simultaneous blockage of IL-6 and TNF-α. Since TNF-α is responsible for the increased synthesis of other cytokines, especially IL-6 [35, 36], its inhibition could reduce IL-6 expression. In vivo studies were performed using a carrageenan-induced arthritis rat model to validate if the biofunctionalized NPs are compatible and to evaluate their therapeutic efficacy after IA administration. In the first experiment, the compatibility of the biofunctionalized NPs was clearly demonstrated, as no harmful effects were detected. Moreover, biofunctionalized NPs were able to reduce inflammation and pain, indicating their potential therapeutic efficacy. In the second experiment, this innovative approach was able to reduce the mechanical allodynia and hyperalgesia associated with this inflammatory model. Thus, the capture and inactivation of both pro-inflammatory cytokines by the biofunctionalized NPs demonstrated their clinical and behavioral beneficial therapeutic effect. In normal joints, the synovium comprises a continuous surface layer of cells, intima (20 to 40 µm thickness in cross section), and the underlying tissue, subintima (up to 5 mm in thickness), which can be fibrous, areolar or adipose depending on its structure and content [37]. Histopathological assessment of the control group (healthy joints) showed a thin intima and an adipose-like sub-intima. After arthritis induction and treatment with saline, the synovial membrane presented a marked fibrosis and high infiltration of immune cells in the synovium was observed. Outstandingly, the microscopic images showed that NPs+Abs reduced more the infiltration of immune cells than the Abs group. In addition, the IHC analyses confirmed the reduction of IL-6 and TNF-α amount by the Abs immobilized at the NPs surface. Since this effect was higher than the obtained for soluble Abs, the initial hypothesis that the primary Abs immobilized at the NPs’ surface protect the Abs from degradation, prolonging their effects, was also demonstrated. Hence, the IA injection of this advanced therapy can be a viable and effective strategy to increase the Abs efficacy and to avoid associated systemic side effects. Chapter VII – Nanoparticle-mediated neutralization of IL-6 and TNFα for osteoarthritis treatment 236 VII-5. CONCLUSION To the best of our knowledge, we are the first to develop biofunctionalized NPs to simultaneously target IL-6 and TNF-α, the most important cytokines involved in arthritic disease. Our findings suggest the inhibition of those cytokines in inflamed joints may have a therapeutic benefit in OA. Indeed, the capture and neutralization of IL-6 and TNF-α by the biofunctionalized NPs effectively reduced the inflammatory scenario in vitro and in vivo . They were able to reduce the harmful effects on the hACs provided by inflammatory macrophages and also ameliorated inflammatory arthritis more efficiently than soluble Abs. By increasing the therapeutic action of the Abs and avoiding the systemic side effects, this innovative strategy will be able to increase the therapeutic efficacy of the currently available treatments. Thus, the developed biofunctionalized NPs provide a promising strategy for the local and sustained treatment of OA and other arthritic conditions. VII-6. ACKNOWLEDGEMENTS Authors acknowledge the financial support from FCT/MCTES (Portuguese Foundation for Science and Technology/Ministry of Science, Technology and Higher Education) and the FSE/POCH (European Social Fund through the Operational Program of Human Capital), for the PhD scholarship PD/BD/11384/2015 of A. C. Lima (PD/59/2013). Authors would also like to acknowledge FCT for the project PTDC/CTM-BIO/4388/2014 – SPARTAN, the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000023-FROnTHERA), and the NORTE 2020 Structured Project within the R&D&I Structured Project, co funded by Norte2020 - Programa Operacional Regional do Norte. VII-7. REFERENCES 1. Cross, M., Smith, E., Hoy, D. , et al. , The global burden of hip and knee osteoarthritis: estimates from the Global Burden of Disease 2010 study . Ann Rheum Dis . 2014, 73 (7): p. 1323-30. 2. Glyn-Jones, S., Palmer, A. J., Agricola, R. , et al. , Osteoarthritis . Lancet . 2015, 386 (9991): p. 376-87. 3. Chen, D., Shen, J., Zhao, W. , et al. , Osteoarthritis: toward a comprehensive understanding of pathological mechanism . Bone Res . 2017, 5 : p. 1-13. 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Open Rheumatol J . 2011, 5 : p. 100-6. 239 SECTION 6 GENERAL CONCLUSIONS 240 Chapter VIII General conclusions and future perspectives Chapter VIII – General conclusions and future perspectives 241 Chapter VIII Chapter VIII - General conclusions and future perspectives VIII-1. GENERAL CONCLUSIONS The increasing burden of arthritic diseases makes their effective treatment an unmet clinical need. Despite the breakthroughs in the field of drug discovery, current treatments still present low efficiency and severe side effects. To overcome these limitations, different strategies of drug delivery have been widely investigated. One of the most promising strategies to treat arthritic diseases comprises the design of NPs with tunable properties to extend the therapeutic index of current therapeutic agents. Indeed, their unique properties enable to reduce the dose and frequency of the administration and, consequently, the systemic side effects of the drug. Therefore, an appropriate delivery system can give a new hope to overcome the limitations of the current treatments. The major goal of the work developed under the scope of the present thesis was to develop and validate in vitro and in vivo novel strategies to treat arthritic conditions. Thus, taking into consideration the nature of the drugs to be incorporated and the mechanisms that will control its therapeutic action and/or release in the inflammatory environment, different nanomedicine systems were developed in Section 3. The nanocarriers herein proposed and studied are aimed to match the main drawbacks of current treatments and overcome them. In Chapter III, biodegradable polymeric NPs were successfully developed from natural origin polysaccharides and biofunctionalized with anti-IL-6 Abs. Since the biological agents have limited efficacy, due to their short half-life and unspecific tissue targeting, their immobilization at the NPs surface will protect, extend and enhance the therapeutic efficacy after its local administration in the affected joint. Biological studies led to the conclusion that a concentration of 50 µg/mL of Ch-HA NPs was ideal for IA administration, since it was demonstrated their cytocompatibility in contact with chondrocytes and macrophages. Moreover, when the chondrocytes are stimulated with macrophage conditioned medium, the biofunctionalized NPs show the beneficial role of the capture and neutralization of IL-6, exhibiting a prolonged action and stronger efficacy than the free Ab. As the local therapy in OA may offer particular advantages over systemic therapy, the findings of this work may lead to important implications in the treatment of this debilitating condition. In RA, the systemic therapy is Chapter VIII – General conclusions and future perspectives 242 generally more indicated and appropriated. For that, in Chapter IV liposomes encapsulating AuNPs with long-circulation times were functionalized with anti-IL-23 Abs at their surface. The aim of this work was also to enhance the therapeutic efficacy of the immobilized Abs, while reducing their adverse side effects, due to the accumulation of the nanocarrier in the inflammatory sites via the EPR effect. Biological studies demonstrated their cytocompatibility when cultured with chondrocytes, macrophages and endothelial cells within a concentration of 2 mM. Importantly, PBMCs of healthy donors and RA patients that were activated though Th17 differentiation presented an efficient reduction of the IL-17A production, after the treatment with biofunctionalized liposomes. Moreover, the therapeutic effect is potentiated by the synergistic effects of IL-23 level reduction and vitamin E and AuNPs anti-oxidant activity. Thus, the results validate the biofunctionalized liposomes as a promising treatment for RA. Chapter V describes the development of enzymaticand redox-responsive polymeric micelles for targeted and controlled drug delivery in inflammatory arthritic conditions. The inadequate pharmacokinetics of GCs, with low drug bioavailability and off-targeted biodistribution profile, is a major limitation of their therapeutic efficacy and safety. Thus, polymeric micelles were designed to increase the therapeutic index of the drug and reduce the severe side effects. To establish a sensitive system, the thiol groups of the GSH were oxidized intermolecularly to retain the drug inside the micelles, providing a barrier against its blood dilution. After accumulation at the inflammatory site via the EPR effect, the drug will undergo a quick release triggered by both redox and GR activity. After demonstrating their cytocompatibility in contact with human endothelial cells, chondrocytes and macrophages until the concentration of 50 µg/mL, a co-culture system was used to show the beneficial role of encapsulating the drug into the micelles. Indeed, Dex encapsulated into the polymeric micelles, in the presence of GR and redox media, exhibited higher efficacy than the free drug. Importantly, as they were able to reduce the negative effects of Dex in normal cells, this strategy may provide important outcomes in arthritis treatment. Overall results from Section 3 highlight the importance of designing nanocarriers considering specifically their application. Hence, the positively charged NPs will enhance their retention in the joint cavity after IA administration, whereas the PEG chains of both micelles and liposomes will enhance the blood circulation time after systemic administration. Moreover, while Ch-HA NPs and LUVs reduce the inflammatory scenario by the capture and inactivation of key pro-inflammatory cytokines through Abs linked at their surface, polymeric micelles release in-situ (arthritic inflammation) the drug though the break of S-S bonds in the presence of GR and high intracellular concentrations of GSH. Importantly, all the developed formulations avoid the harmful effects of the drugs in normal cells, which will severely limit their side effects after administration. Chapter VIII – General conclusions and future perspectives 243 Despite the vital role of NPs internalization process by the cells, which determines their activity, biodistribution and toxicity, our understanding on NPs cellular uptake is rather limited. Section 4 aims to provide evidence by not only studying the role of the physicochemical characteristics of the NPs, but also taking into consideration the effects of the disease environment in this process. Accordingly, the goal of the Chapter VI was to assess the internalization and pathways used for different NPs previously developed (polymeric NPs, LUVs and micelles) in a normal and inflammatory scenario by different cells, namely endothelial cells, chondrocytes and macrophages. The results shown the important role of surface chemistry in NPs internalization by the cells. Despite some studies established a higher internalization degree of cationic particles, in the present work the results shown the opposite. Moreover, it was highlighted the complexity and interplay regarding the cell-NPs interaction and, consequently, their mechanisms of cell uptake by the different cell types. Indeed, each NP had a similar uptake level regardless the cell type, but the same NP exploited different cellular pathways depending on the cell type. As internalization route is of utmost importance for the NPs fate into the cell, it is crucial to understand their pathways of cellular uptake and intracellular trafficking. Indeed, in order to avoid the degradation of the NPs in the lysosomes, NPs should enter in the cells via specific pathways. For instance, caveolaemediated endocytosis or via energy-independent non-endocytic pathway, which avoid the degradation of the NPs in the lysosomes, can be achieved in chondrocytes and endothelial cells by the developed LUVs and in M1 macrophages by the micelles. As such, we can conclude that the micelles will provide an intracellular delivery of the inflammatory drug to the target cells (M1 macrophages). Hence, understanding the mechanism of cellular uptake by each cell type and disease state will provide in the future nanomedicines with efficiently targeted delivery of the biomolecules to a specific sub-cellular compartment of the cell and, consequently, enhanced therapeutic efficacy. Section 5 provides the in vivo evaluation of the safety and therapeutic efficacy of the biofunctionalized Ch-HA NPs immobilizing anti-TNF-α and anti-IL-6 Abs at their surface. The synergistic effects of neutralizing two key pro-inflammatory cytokines were confirmed in Chapter VII though their in vitro investigation using a co-culture model of inflammation and in vivo using an experimental carrageenaninduced arthritis rat model. In vitro studies demonstrated a higher inhibition of the harmful effects on the chondrocytes provided by inflammatory macrophages after the treatment with biofunctionalized NPs in comparison with soluble Abs. Moreover, those effects were higher when both cytokines were inhibited, confirming the synergistic effects of the simultaneous blockage of IL-6 and TNF-α. After IA administration, biofunctionalized NPs demonstrated a safe profile and stronger efficacy on reducing arthritic symptoms