Chitosan-Based Nanomedicine for Rosmarinic Acid Ocular Delivery

Chitosan-based nanomedicine for rosmarinic acid ocular delivery Thesis presented to obtain the PhD degree in Pharmaceutical Sciences, Pharmaceutical Technology Specialty, Faculty of Pharmacy of University of Porto by Sara Isabel Macedo Baptista da Silva Under supervision of Prof. Dr. Bruno Sarmento and co-supervision of Prof. Dr. Domingos Ferreira and Prof. Dr. Manuela Pintado October, 2014 iv Declaration The partial reproduction of this thesis is authorized only for research purposes by written declaration of the person concerned. (Sara Baptista da Silva) v Inspiration “Success is a journey, not a destination!” Ben Sweetland vi Dedication “There are only two ways to live life. One is as though nothing is a miracle. The other is as though everything is a miracle.” Albert Einstein I dedicate this thesis to the most important persons in my life, my family, Oscar and our daughter, Catarina Isabel. . vii Acknowledgements “Lord I can’t say it in words…can you please just listen through my heart” Unknown I would like to formally express my deep gratitude to the following people and institutions – who (and which) have meant a lot to me during my PhD program, and made it possible: Fundação para a Ciência e a Tecnologia, for financial support via a PhD fellowship (ref.: SFRH/BD/61423/2009), under the supervision of Professor Bruno Sarmento; said grant permitted timely development of my research program, as well as participation in several international scientific meetings to complement my training and sharing my results. Laboratory of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto for accepting me as a PhD student, for the hospitality and work conditions available, during my doctoral program. My most sincerely acknowledges to Escola Superior de Biotecnologia of Universidade Católica Portuguesa (ESB-UCP), for the crucial collaboration in my PhD course, the indubitable hospitality and for providing facilities and logistical to best support my studies. INEB – Instituto de Engenharia Biomédica, ISCS–N – Instituto Superior de Ciências da Saúde – Norte and IBILI-Institute for Biomedical Imaging and Life Sciences – University of Coimbra, for the acceptance, kindness and constant availability cooperation during this project. Professor Bruno Sarmento my supervisor, who I am sincerely grateful for having accepted me as a PhD student, for the wide support and comprehensive scientific guidance he continuously gave me. His consistent and integrated contribution for my growth, both as a person and as a researcher, has been by all means outstanding. I would like, in particular, to thank him for every effort made in guarantee the best research conditions, going wherever necessary to find the most appropriate support; and for his everlasting encouragement, patience and motivation, as well as his availability to discuss specific and general topics of my dissertation. I am indeed deeply grateful, for having always believed in my abilities, for all the concerns, problems, opportunities and achievements shared along this journey, for the friendship and affection, my heartfelt thanks. viii Professor Manuela Pintado my co-supervisor, I would like to thank for always accepting me as a student, having accompanied during these 12 years of academic training, for always believing in my abilities, skills and competences, for her unconditional understanding, patience, friendship and complicity. For her believed and dedication to my work, for all the conversations established and knowledge shared throughout these years and during this doctoral course, as well as for her encouragement, affection and help; and also for providing a healthy, happy and extremely professional working environment, my deepest thanks. Professor Domingos Ferreira my co-supervisor, I would like to thank for his sympathy, kindness, constant availability and willingness to help during my doctoral program. For every effort to ensure the best institutional reception conditions and every logistical that could ensure the success of my work, my sincere grateful. Professor Francisco Ambrósio, my deepest gratitude for agreeing to collaborate in this PhD project. For all the help, cooperation, understanding, by all the efforts made the investment of time and resources in better monitoring and performance of this work. For having given me the opportunity to work with his research team and by the unmatched amiability. My most sincere thanks to Professor Horacina Cavalcante and to the entire Stamford family: Professor Newton, Professor Tania, Thayza and Thathiana, for have accompanied me during my doctoral plan in every possible way, for all the help, dedication, and enormous friendship. My colegues and friends within the different research groups – José das Neves, Fernanda Andrade, Filipa Antunes, Pedro Fonte, Francisca Araújo, Rute Nunes, Carla Pereira, Manuela Amorim, Ana Oliveira, Raquel Madureira, Débora Campos, Raquel Boia, Filipe Elvas, Tiago Martins, Pedro Tralhão, Maria Madeira, Joana Martins, and so many others. By somehow helping me in developing my studies and shared by so many difficulties and achievements, which certainly has helped me grow as a person and as a professional. My closest friends which are the family that I chose for me every day: Helena Monteiro, thank you for your friendship which already makes “silver wedding”, for our perpetual oath on time, for always find yourself without seeking, for even far in distance seems like I have been with you yesterday, and always. ix Sandra Borges, words will be for sure fall short in expressing my eternal friendship. Thank you for bringing a rain-bow to my life, for the craziness shared. For every concern, worry, and happiness moments joint, for listening, advising, for the constant patience and complicity; For being always there. Manuela Amorim, I will not be able to thank you everything that you art for me and everything what you already has done for me during this project, much less in a nutshell. Above all thank you for the unconditional friendship, complicity, and confidence, for every laugh and tear shared, for being my true friend. Inês Cravo Roxo, Joana Barbosa, Franklin Costa (and the little Matilde), Ricardo Freixo, Luciana Silva and Renato Resende thank you for always accompanied my life at its best and worst for every moment spent, I appreciate and reciprocate with eternal friendship. I also have to leave my deepest gratitude to my sweet Francisca Maria, who accompanied my life forever, and thank her for the immense dedication to my family, for their undying affection and unconditional support in every moment of our life, from best to worst; My most sincere and profound thanks. I would like to thank to my brother Jorge Filipe and my sister Sofia Manuel, being my best friends, for existing and making my life so full field, so complete; For being with me in every dream, fight, step and for never live me alone. I would also have to thank to my nephews (Carlos Eduardo, Filipa Alexandra and Barbara Sofia), for being the best continuity of my brothers, for their love and for being always with me in a health madness. My Grandmother, Isabel Maria, will always be my sunshine, my companion of all hours, the example of strength and light, and her way of being and living will always inspire my life. Words will be reductive and insufficient to thank my parents for the unconditional love, for making me what I am today, for everything they have given me throughout my life, by making the development of my academic training possible and for always believed in me. Thank you for the unconditional love, strength, support and courage injected in me, today and every day. Thank you for everything, now and forever. And last, but far from least – Oscar, is my Alter Ego, my companion of dreams and struggles. Oscar is the better half of me, the more aware, responsible and realistic. I grew up with him personal and intellectually. He has accompanied every moment of my life, xvi 96,1 ± 0,2 e 98,2 ± 0,1% para as nanopartículas de ácido rosmarínico, salva e segurelha, respetivamente. Estes valores mais elevados associados à nanoencapsulação dos extratos também podem ser associados à menor quantidade de ácido rosmarínico nas nanopartículas de quitosano, uma vez que a concentração inicial é documentada como inversamente proporcional à própria eficiência de associação. O perfil de liberação in vitro do ácido rosmarínico foi avaliado em tampão fosfato (PBS), a pH 7,4 nas diferentes formulações, por um período de 60 min, e não se observaram diferenças significativas (P > 0,05). A rápida libertação do ácido rosmarínico dá indicações que estes sistemas de nanopartículas podem fornecer uma estratégia racional para o desenvolvimento de formulações de libertação imediata para administração ocular do ácido rosmarínico. A eficiência de associação e de liberação in vitro foram realizadas utilizando um método de cromatografia líquida de alta eficiência (HPLC), especialmente desenvolvido e otimizado para garantir a obtenção de resultados precisos e exatos. As análises de calorimetria diferencial de varrimento (DSC) e a espetrofotometria de infravermelho por transformada de Fourier (FTIR) permitiram concluir que não foram encontradas interações químicas entre os antioxidantes e o quitosano, depois do processo de encapsulação. A atividade antioxidante dos nanosistemas foi avaliada pelos métodos de 2,2-azinobis-(3-etilbenzotiazolin-6-ácido sulfónico) (ABTS) e de capacidade de absorção radical (ORAC), antes e depois do processo de liofilização, para garantir que a atividade antioxidante não é comprometida durante o processo de secagem das partículas. Os melhores resultados de atividade antioxidante foram obtidos pelo método de ORAC após liofilização das partículas, os resultados para as nanoparticulas de ácido rosmarinico, salva e segurelha foram: 3,6520 ± 0,1770, 0,4251 ± 0,0069 e 0,4526 ± 0,0087 µmol/eq Trolox, respetivamente. Todavia foi observada uma atividade antioxidante mais baixa nas nanopartículas do que nos compostos livres, devido ao efeito da nanoencapsulação. As partículas demonstraram propriedades mucoadesivas após incubação com mucina, pelo aumento em tamanho e consequente diminuição da carga de superfície. Os resultados indicam que pode ser expectável um aumento do tempo de retenção sobre a mucosa ocular após a instilação. Todas as formulações demonstraram ser seguras para o teste de citotoxicidade 3-(4,5-dimetiltiazol-2yl)-2,5-difenil brometo de tetrazolina (MTT) e para o teste da libertação da enzima lactato desidrogenase (LDH), sem citotoxicidade relevante (abaixo de 10%, para todas as formulações e concentrações), em linhas oculares da retina (epitélio pigmentar da retina - ARPE-19) e da córnea (linha de células da córnea humana - HCE-T). O teste da membrana corioalantóide (HET-CAM Teste) foi utilizado como alternativa aos testes biológicos em coelhos (teste de Draize) e também sugere a xvii ausência de irritação das partículas no olho. Os estudos de permeabilidade em monocamada de células da córnea (HCE) revelou um coeficiente de permeabilidade aparente (Papp) de 3,41 ± 0,99 x 10-5 e 3,24 ± 0,79 x 10-5 cm / s para as nanopartículas de ácido rosmarínico e para o ácido rosmarínico livre, respectivamente. O estudo de permeabilidade em monocamada de células da retina (ARPE-19) revelou valores de Papp de 3,39 ± 0,18 x 10-5 e 3,60 ± 0,05 x 10-5 cm/s para as nanopartículas de ácido rosmarínico e para o ácido rosmarínico livre, respectivamente. Não houve diferença significativas (P > 0,05) entre os valores de permeabilidade das nanoparticulas, composto livre e entre ambas as linhas celulares, provavelmente devido ao perfil de liberação rápido das nanopartículas acima descrito. Foram feitos testes preliminares in vivo, em que o ácido rosmarínico foi injetado na cavidade intravítrea de ratos Wistar, num modelo animal isquemia-reperfusão (I-R). Eletrorretinogramas (ERG) e ensaios imunohistoquímicos revelaram que o ácido rosmarínico (a uma concentração de: 50 µM), por injeção intravítrea, não teve um efeito protector na retina. O que poderá ser devido danos pró-inflamatórias severos no modelo I-R, difíceis de reverter com o estudo de uma única injeção. No entanto, e considerando os bons resultados obtidos neste trabalho, as partículas de quitosano contendo ácido rosmarínico demonstraram ser seguras, mucoadesivas, com elevado potencial de permeabilidade ocular e com um bom perfil de atividade antioxidante, o que permite concluir que estes nanosistemas podem ser importantes para a prevenção de doenças degenerativas oculares. Os resultados desta tese, permitem também concluir que estes nanosistemas naturais são promissores na administração tópica de antioxidantes no olho e ressalta a necessidade de se explorar novos sistemas para ultrapassar as limitações na eficiência da administração tópica de fármacos no olho. Palavras-chave: Quitosano, ácido rosmarinico, extratos, doenças oculares xviii This work was submitted as a PhD Thesis in partial fulfilment of the requirements for Philosophiæ Doctor (PhD) degree in Pharmaceutical Sciences at the Faculty of Pharmacy, University of Porto. It was conducted under the guidance of Prof. Dr. Bruno Filipe Carmelino Cardoso Sarmento, PhD, Affiliated Researcher at INEB - Instituto de Engenharia Biomédica and Assistant Professor at Instituto Superior de Ciências da Saúde-Norte (ISCS-N), and under the co-supervision of Prof. Dr. Maria Manuela Estevez Pintado, Assistant Professor at Biotechnology School of Portuguese Catholic University and Prof. Dr. Domingos Carvalho Ferreira, Full Professor of Faculty of Pharmacy, University of Porto. The research experimental work was conducted at the Laboratory of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, in collaboration with CBQF - Biotechnology School of Portuguese Catholic University, INEB - Instituto de Engenharia Biomédica, ISCS–N - Instituto Superior de Ciências da Saúde – Norte and IBILI - Institute for Biomedical Imaging and Life Sciences – University of Coimbra. xix Scope and outline This thesis was organized in 9 chapters, thus closely reflecting the development of my research work. All chapters were related to each other and the aims and methodology chosen in each chapter were indeed dependent on the conclusions brought about in previous one(s). Overall, the work described in this thesis encompasses development and characterization of chitosan nanoparticles for the rosmarinic acid, sage and savory encapsulation - to prevent and control degenerative eye diseases. Part I include Chapter 1, and entail a bibliographic review regarding chitosan biological proprieties, biomedical potential as well as chitosan-based delivery systems. A particular emphasis was put on the key factor of antioxidants in the degenerative eye diseases prophylaxis, as well as in the nanocarriers as a way to improve antioxidant activity performace and efficacy. In Part II - Chapter 2, the project aims and goals were detailed to be a guideline of the work major core. In Part III - Chapter 3 a high-performance liquid chromatography (HPLC) method was developed and optimized to be used throughout the experimental work of this thesis and to allow the best precise quantification of antioxidant content in the natural extracts, the nanoparticles association efficiency and either release and permeability profiles, developed in the following chapters. In Part IV - Chapter 4 a comprehensive development, optimization and physical-chemical characterization of antioxidant-chitosan nanoparticles was presented. The effect of rosmarinic acid content, mass correlation and pH of nanoparticle preparation were evaluated for the ionic gelation optimization process. Complementary methodologies were employed to provide a more rational understanding of the interactions between components and the success of the encapsulation, such as the particle size and zeta potential. In Chapter 5 the nanocarriers were evaluated and characterized regarding the in vitro antioxidant activity potential. In Part V - Chapter 6 the nanocarriers were then tested to guarantee their safety performance, mucoadhesion proprieties and in vitro ocular cell permeability. In Chapter 7, it was performed the first attempting efforts to prove rosmarinic acid therapeutical potential in an ischemia-reperfusion (I-R) animal model. Finally, in Part VI, the overall conclusions were presented in Chapter 8 - and future prospects, based on critical questions arising from this dissertation, were put forward in Chapter 9. xx Most information presented in the 9 chapters that constitute this dissertation has been already submitted to international peer review, via publication in scientific journals – according to the following list: Part I: Chapter 1 – State of the art Baptista da Silva S., Costa J., Pintado M., Ferreira D., Sarmento B. (2010). Antioxidants in the prevention and treatment of diabetic retinopathy – A Review. Jornal of Diabetes and Metabolism 1:111. doi:10.4172/2155-6156.1000111. Baptista da Silva S., Fernandes J., Tavira F., Pintado M., Sarmento B. (2011). The potential of chitosan in drug delivery systems. In Focus on Chitosan Research, Edited by Arthur N. Ferguson and Amy G. O'Neill, Nova Publishers, ISBN: 978-1-61324-454-8. Tavaria, F., Fernandes, J., Santos-Silva, A., Baptista da Silva, S., Sarmento, B. and Pintado, M. (2011). Biological activities of chitin, chitosan and respective oligomers. In Focus on Chitosan Research, Edited by Arthur N. Ferguson and Amy G. O'Neill, Nova Publishers, ISBN: 978-1-61324-454-8. Andrade F., Antunes F., Nascimento V., Batista da Silva S., Neves J., Ferreira D., Sarmento B. (2011). Chitosan formulations as carriers for therapeutic proteins. Current Drug Discovery Technologies. 8(3):157-172. doi: 10.2174/157016311796799035. Sarmento B., Andrade F., Baptista da Silva S., Rodrigues F., Neves J., Ferreira D. (2012). Cell-based in vitro models for predicting drug permeability. Expert Opinion on Drug Metabolism and Toxicology. 8(5):607-621. doi: 10.1517/17425255.2012.673586. Silva N., Baptista da Silva S., Sarmento B., Pintado M. (2013). Chitosan nanoparticles for daptomycin delivery in ocular treatment of bacterial endophthalmitis. Drug Delivery, doi: 10.3109/10717544.2013.858195. Baptista da Silva S., Borges S., Ramos O., Pintado M., Ferreira D., Sarmento B. (2014). Treating retinopathies: Nanotechnology as a tool in protecting antioxidants agents in xxi Systems Biology of Free Radicals and Antioxidants. Springer-Verlag (Germany), ISBN: 978-3-642-30017-2. Baptista da Silva S., Borges S., Pintado M., Sarmento B. (2014). Formulation of essential oils in pharmaceutical dosage forms - biopharmaceutics and therapeutic potentials, Pharmaceutical Biology. (Accepted for publication). Vasconcelos T., Baptista da Silva S., Ferreira D., Pintado M., Marques S. (2015). Cellbased in vitro models for ocular permeability studies. In Concepts and Models for Drug Permeability Studies: Cell and Tissue-based in vitro Culture Models. Edited by Bruno Sarmento, Elsevier. (Accepted for publication). Part III: Chapter 3 - High-performance liquid chromatographic method validation; Baptista da Silva S., Oliveira A., Ferreira D., Sarmento B., Pintado M. (2013). Development and validation method for simultaneous quantification of phenolic compounds in natural extracts and nanosystems. Phytochemical Analysis. 24(6): 638-644. doi: 10.1002/pca.2446. Part IV: Chapter 4 - Development, optimization and physical-chemical characterization of chitosan-based nanoparticles; Chapter 5 - In vitro assessment of antioxidant activity of chitosan-based nanoparticles; Baptista da Silva S., Amorim M., Fonte P., Madureira R., Ferreira D., Pintado M., Sarmento B. (2015). Natural extracts into chitosan nanocarriers for rosmarinic acid drug delivery. Pharmaceutical Biology. doi:10.3109/13880209.2014.935949. xxii Part V: Chapter 6 - In vitro evaluation of cytotoxicity, mucoadhesion and ocular permeability of rosmarinic acid into chitosan-based nanoparticles; Chapter 7 - Therapeutical potential evaluation in ischemia-reperfusion animal model of chitosan based-nanoparticles; Baptista da Silva S., Ferreira D., Pintado M., Sarmento B. Evaluation of chitosan-based nanoparticles for ocular delivery of rosmarinic acid through in vitro mucoadhesion and permeability studies, submitted for publication. xxiii List of contents DECLARATION ............................................................................................................... IV INSPIRATION ................................................................................................................... V DEDICATION ................................................................................................................... VI ACKNOWLEDGEMENTS ............................................................................................... VII ABSTRACT ..................................................................................................................... XI RESUMO ....................................................................................................................... XV SCOPE AND OUTLINE ................................................................................................ XIX LIST OF FIGURES .................................................................................................... XXVIII LIST OF TABLES ....................................................................................................... XXXI LIST OF ABBREVIATIONS ....................................................................................... XXXII PART I - INTRODUCTION ................................................................................................ 1 CHAPTER 1 - STATE OF ART ........................................................................................... 3 1. INTRODUCTION ........................................................................................................ 5 2. CHITOSAN PROPRIETIES AND BIOMEDICAL APPLICATION ............................... 8 2.1. CHITOSAN-BASED DRUG DELIVERY SYSTEMS ............................................. 10 2.1.1. CHITOSAN SOLUTIONS ..................................................................................... 11 2.1.2. FILMS ............................................................................................................. 12 2.1.3. TABLETS ......................................................................................................... 14 2.1.4. HYDROGELS.................................................................................................... 16 2.1.5. MICROPARTICLES ............................................................................................ 19 2.1.6. NANOPARTICLES ............................................................................................. 21 2.2. CLINICAL TRIAL - SAFETY AND TOLERABILITY OF CHITOSA-N-ACETYLCYSTEINE EYE DROPS IN HEALTHY YOUNG VOLUNTEERS ........................................................................ 23 3. OXIDATIVE PRODUCTS AND THE CLINICAL IMPORTANCE OF ANTIOXIDANTS 24 3.1.1. TYPES OF ANTIOXIDANTS AGENTS ..................................................................... 25 3.1.2. PHYSIOLOGY AND PATHOBIOLOGY OF REACTIVE OXYGEN SPECIES IN RETINOPATHIES ............................................................................................................. 27 xxiv 3.1.3. OXIDATIVE STRESS IMBALANCE AND RETINAL AFFECTED DISEASES ..................... 29 4. NANOTECHNOLOGY APPLIED TO ANTIOXIDANTS PROTECTION .................... 30 4.1. NANOANTIOXIDANTS PHARMACOTHERAPY ............................................................ 35 4.2. SAFETY ISSUES OF ANTIOXIDANT NANOPARTICLES ................................................ 36 5. SUMMARY .............................................................................................................. 38 PART II - AIMS AND GOALS ......................................................................................... 39 CHAPTER 2 - AIMS AND ORGANIZATION OF THE THESIS .................................................. 41 PART III .......................................................................................................................... 45 ABSTRACT .................................................................................................................... 47 CHAPTER 3 - HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD VALIDATION ....... 49 3. INTRODUCTION ...................................................................................................... 51 3.1. EXPERIMENTAL.................................................................................................. 52 3.1.1. MATERIALS ..................................................................................................... 52 3.1.2. EQUIPMENT AND CHROMATOGRAPHIC CONDITIONS ............................................ 52 3.1.3. PREPARATION OF STANDARD AND SAMPLE SOLUTIONS ....................................... 53 3.1.4. METHOD VALIDATION ....................................................................................... 53 3.1.5. METHOD APPLICABILITY ................................................................................... 54 3.2. RESULTS AND DISCUSSION ............................................................................. 54 3.2.1. APPLICATION OF THE CHROMATOGRAPHIC METHOD ........................................... 54 3.2.2. LINEARITY ....................................................................................................... 55 3.2.3. PRECISION ...................................................................................................... 56 3.2.4. ACCURACY ..................................................................................................... 58 3.2.5. SPECIFICITY .................................................................................................... 58 3.2.6. RANGE ........................................................................................................... 59 3.2.7. ROBUSTNESS .................................................................................................. 59 3.2.8. DETECTION LIMIT AND QUANTITATION LIMIT ....................................................... 59 3.2.9. METHOD APPLICABILITY ................................................................................... 60 3.3. CONCLUSION ..................................................................................................... 61 PART IV .......................................................................................................................... 63 ABSTRACT .................................................................................................................... 65 xxv CHAPTER 4 - DEVELOPMENT, OPTIMIZATION AND PHYSICAL-CHEMICAL CHARACTERIZATION OF CHITOSAN-BASED NANOPARTICLES ............................................................................ 67 4. INTRODUCTION ...................................................................................................... 69 4.1. EXPERIMENTAL.................................................................................................. 70 4.1.1. MATERIALS ..................................................................................................... 70 4.1.2. PREPARATION OF CHITOSAN-BASED NANOPARTICLES ........................................ 70 4.1.3. ENCAPSULATION OF SAGE, SAVORY AND ROSMARINIC ACID INTO CHITOSAN-BASED NANOPARTICLES ............................................................................................................ 71 4.1.4. SIZE AND SURFACE CHARGE ............................................................................. 71 4.1.5. MORPHOLOGY ................................................................................................. 72 4.1.6. ASSOCIATION EFFICIENCY ................................................................................ 72 4.1.7. IN VITRO RELEASE OF ROSMARINIC ACID FROM CHITOSAN NANOPARTICLES ......... 73 4.1.8. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ANALYSIS AND ROSMARINIC ACID QUANTIFICATION ............................................................................................................ 73 4.1.9. DIFFERENTIAL SCANNING CALORIMETRY ANALYSIS ............................................. 74 4.1.10. FOURIER-TRANSFORM INFRARED ANALYSIS ....................................................... 74 4.1.11. STATISTICAL ANALYSIS .................................................................................... 75 4.2. RESULTS AND DISCUSSION ............................................................................. 75 4.2.1. PARTICLE SIZE, POLYDISPERSITY AND ZETA POTENTIAL ...................................... 75 4.2.2. MORPHOLOGY ................................................................................................. 77 4.2.3. ASSOCIATION EFFICIENCY AND DRUG LOADING .................................................. 79 4.2.4. IN VITRO ROSMARINIC ACID RELEASE FROM CHITOSAN NANOPARTICLES .............. 80 4.2.5. THERMAL BEHAVIOR BY DIFFERENTIAL SCANNING CALORIMETRY ANALYSIS .......... 82 4.2.6. SPECTROSCOPY BY FOURIER-TRANSFORM INFRARED ANALYSIS ......................... 85 4.3. CONCLUSION ..................................................................................................... 88 CHAPTER 5 – IN VITRO ASSESSMENT OF ANTIOXIDANT ACTIVITY OF CHITOSAN-BASED NANOPARTICLES ............................................................................................................ 89 5. INTRODUCTION ...................................................................................................... 91 5.1. EXPERIMENTAL.................................................................................................. 91 5.1.1. MATERIALS ..................................................................................................... 91 5.1.2. SAMPLE PREPARATION .................................................................................... 92 5.1.3. CHITOSAN NANOPARTICLES DEVELOPMENT AND OPTIMIZATION ........................... 92 xxxii List of abbreviations ABTS - 2,2-Azinobis (3-Ethylbenzothiazoline-6-Sulphonic) Acid AE - Association Efficiency AGE - Advanced Glycation End Products AMD - Age Macular Degeneration ANOVA - One-Way Analysis of Variance ARVO - Association for Research in Vision and Ophthalmology BAB - Blood-Aqueous Barrier BRB - Blood-Retinal Barrier CAT - Catalase CBQF - Centro de Biotecnologia e Química Fina Cupper - Cu Da - Dalton DAPI - 4',6-Diamidino-2-Phenylindole DD - Deacetylation Degree DL - Detection Limit DMEM - Dulbecco’s Modified Eagle’s Medium DMSO - Dimethyl Sulfoxide DNA - Desoxyribonucleic Acid DPPH - Diphenyl-1-Picrylhydrazyl DSC - Differential Scanning Calorimetry EMA - European Medicines Agency ERG - Electroretinograms FDA - Food and Drug Administration FFUP - Faculdade de Farmácia da Universidade do Porto FTIR - Fourier Transform Infrared GCL - Ganglion Cell Layer GNP - Gold Nanoparticles GPx - Glutathione peroxidase GSH - Glutathione HBA - p – Hydroxybenzyl Alcohol HBSS - Hanks’ Balanced Salt Solution HCE-T - Human Cornea Cell Line HET-CAM - Chorioallantoic Membrane Test xxxiii HPLC - High Performance Liquid Chromatography HPOX - Hydroxybenzyl Alcohol Incorporated Copolyoxalate IBILI - Institute for Biomedical Imaging and Life Sciences INEB - Instituto de Engenharia Biomédica INL - Inner Nuclear Layer IOBA – NHC - Immortalized Epithelial Cell Line from Human Conjunctiva IOP - Intraocular Pressure IPL - Inner Plexiform Layer I-R - Ischemia-Reperfusion ISCS-N - Instituto Superior de Ciências da Saúde – Norte LCPUFA - Long-Chain Polyunsaturated Fatty Acid LDH - Lactate Dehydrogenase MNP - Magnetically Responsive Nanoparticles MnSOD - Superoxide Dismutase MSc - Master of Science MTT - Thiazolyl Blue Tetrazolium Bromide MW - Molecular Weight NR - Rosmarinic Acid Nanoparticles NSG - Satureja montana Nanoparticles NSV - Salvia officinalis Nanoparticles OCT - Optimal Cutting Temperature ORAC - Oxygen Radical Absorbance Capacity OS - Oxidative Stress Papp - Apparent Permeability Coefficient PBS - Phosphate Buffer Saline PdI - Polydispersity Index PhD - Philosophiæ Doctor PLA - Polylactic Acid PP - Polypropylene PUFA - Polyunsaturated Fatty Acid PVA - Poly (Vinyl Alcohol) QL - Quantification Limit QUEN - Quercetin Nanoparticles RGC - Retinal Ganglion Cells ROS - Reactive Oxygen Species xxxiv RPE - Retina Pigment Epithelium rpm - Rotations per Minute RSD - Relative Standard Deviation SD - Standard Deviation SEM - Scanning Electron Microscopy SiNPs - Silicate Nanoparticles SOD - Superoxide dismutase TEM - Transmission Electron Microscopy TPP - Tripolyphosphate TUNNEL - Terminal Deoxynucleotidyl Transferase (TdT)-Mediated dUTP Nick End Labeling UV - Ultra-Violet v - Volume VEGF - Vascular Endothelial Growth Factor w - Weight Zn - Zinc 1 PART I - Introduction “Somewhere, something incredible is waiting to be known” Carl Sagan 2 3 CHAPTER 1 - State of art 4 5 1. Introduction In pharmaceutical science there is a continuous blockbuster drug development, and nowadays biomolecules as active agents, are widely explored to develop new therapeutics. Nevertheless, most of these new active compounds are unstable and must be protected from degradation in the physiological environment, due to the poor absorption that constrains the transport across biological barriers. Thus, the efficacy of most drugs clearly depends on the design of appropriate carriers for their physical protection, delivery and controlled release (1). Among the different approaches explored so far, colloidal carriers are particularly interesting, especially those made of mucoadhesive polymers to assure their epithelium permanence (2, 3). For this application, chitosan has had quite impact in the association and delivery of labile macromolecular compounds (4). Chitosan carriers have an exceptional potential for drug delivery, especially for mucosal, since these systems are stable in contact with physiological fluids and barriers. They are also able to control drug release and protect against adverse conditions like mucosal enzymes and biological protective fluids. Due to its mucoadhesion, particle size, particle surface chemistries, charge and the unique absorption enhancing properties, the chitosan potential in the medical field is widely promising. Different formulations such as films, tablets, hydrogels, micro and nanosystems are expected to optimize, characterize and select the drug performance, improved properties and increase stability for great specific applications. Pharmacokinetics and toxicological relevance of chitosan systems are guaranteed by in vitro model systems in molecular, subcellular and cellular levels, as well as their therapeutic efficacy and safety performance should also be proven in vivo. One category of compounds in which these chitosan carriers may be a key for success, are the antioxidants. Considering the biology definition, antioxidants are chemical compounds or a substance that inhibits oxidation, counteracting the damage of free radicals effects in a living organism, and for this reason are reactive species (5). Antioxidants are widespread virtually in plant foods, often at high levels, and include phenols, phenolic acids and flavonoids (6). Rosmarinic acid (a-O-caffeoyl-3,4-dihydroxyphenillactic acid) (7) is a phenolic compound, which can provide protection against cancer (7) and have other multitude biological activities, namely adstringent, anti-inflammatory, anti-mutagen, antibacterial and antiviral (7, 8). The latter activity has been tested in the therapy of Herpes simplex infections with rosmarinic acid-containing extracts of Melissa officinalis (7). It is also one of the efficient natural antioxidants (9) since rosmarinic acid displays a 6 huge potential radical scavenging activity, higher than trolox (a derivative of a-tocopherol) (10-12). Rosmarinic acid has also an anti-angiogenic activity to retinal neovascularization in a mouse model of retinopathy (13). Significantly inhibited the proliferation of retinal endothelial cells in a dose-dependent manner, and inhibited in vitro angiogenesis of tube formation. Moreover, rosmarinic acid showed no retinal toxicity. These data suggest rosmarinic acid could be a potent inhibitor of retinal neovascularization and may be applied in the treatment of vasoproliferative retinopathies (13). It is the major component of Salvia officinalis (sage) and Satureja montana (savory) natural extracts. These are plants often used in traditional medicine, and which grow in the poor soils of the Mediterranean basin (14). Besides application as condiment, sage and savory have been used as an anti-diarrhea vector, digestion adjuvant, contribute to heal wounds, play an anti-inflammatory role, disinfectant, fight insomnia and decrease blood pressure. Some of these biological activities have been associated with its high contents of rosmarinic acid and the presence of other relevant phenolic compounds such as quercetin and rutin (9, 14). Beyond the biological huge benefits, antioxidants are extremely sensitive to light, oxygen, are highly reactive with other compounds, in some cases possess poor solubility, inefficient permeability, and are extremely unstable (15-17). For all these reasons their delivery using the conventional dosage forms is a challenge (18). In this context, alternative carriers are being considered, regarding the optimization of pharmacokinetics and pharmacodynamics of antioxidant molecules. Chitosan nanoparticles, due to their proper properties, are on the raw. For this concern the nanotechnology is expected to increase the ability, to retain the antioxidant activity during the preparation process, to optimize the release of the compound from the carrier system, and to ensure a good control of their physical-chemical properties increasing their stability. If these nanocarriers may improve the efficacy performance of the antioxidants, several diseases like cancer, diabetes, hypertension, arterio-sclerosis, cardiovascular disease or ocular anomalous conditions that have a clinical impairment with oxidation processes, may be prevented or better controlled (19). Considering the eye disorders, there are many types of retinopathies conditions that may have an oxidative etiology (20, 21), like retinitis pigmentosa, glaucoma, macular degeneration, retinoblastoma and diabetic retinopathy (19, 21). Multiple factors have been also proposed to explain retinopathies, including genetic disorders, infections by microbial agents, sorbitol pathway hyperactivity, accumulation of advanced glycation end products (AGEs) (22) and protein kinase C activation (23). Nevertheless the precise pathological mechanism remains to be elucidated. Which is clear is the collateral damage of these disorders, that may result on 7 reflectivity changes, bifurcations, tortuosity neovascularization as well as other patterns of blood vessels and even blindness (19, 21). In the case of ocular pathologies the oxidative stress (OS) clinical impairment has a significant impact, since the ocular globe is the organ most affected by OS. Its constantly expose to light and oxygen and its high polyunsaturated fatty acid (PUFA) content that is prone to lipid peroxidation, may be some prominent reasons (24). OS is also associated with increased vascular permeability, disruption of blood-retinal barrier, apoptotic loss of retinal capillary cells, microvascular abnormalities and neovascularization (25). High levels of OS are also usually associated with increased levels of oxidative modified desoxyribonucleic acid (DNA) and nitrosylated proteins, and antioxidant defense enzymes impair (26). When such damages are presented, without effective medical treatment, cells and tissues of the retina become malnourished and progressively degenerate, which leads to damage in cells responsible for vision, leading to its inevitable loss (21). Due to this intimate relationship between OS and the pathogenesis of retinopathies, the use of appropriate antioxidants may have potential on the metabolic and functional abnormalities in retinopathies (27). Nevertheless, for antioxidants assure these conditions they need the nanocarrier support to take them to the right place without losing their functional activity. 14 provide alternative methods of treatment to clinicians for compromised wound sites where avascular zones can prevent the delivery of antibiotics to the infected tissue. A recent study demonstrated that incorporating antibiotics in chitosan films could provide alternative methods of treating musculoskeletal infections (60). Novel chitosan based polyelectrolyte complexes were developed and optimized in order to obtain films possessing the optimal functional properties (flexibility, resistance, water vapor transmission rate and bioadhesion) to be applied on skin (61). The development was based on the combination of chitosan and two polyacrylic acid polymers with different cross-linkers and crosslinking densities. The optimized film, including adhesive property, has shown very good properties for application in the skin and represents a very promising formulation for further incorporation of drugs for topical and transdermal administration. 2.1.3. Tablets Various studies with chitosan regarding controlled release delivery systems have been conducted for oral dosage forms, from film coated pellets, tablets or capsules to more sophisticated and complicated delivery systems such as osmotically driven systems, systems controlled by ion exchange mechanism, systems using three dimensional printing technology and systems using electrostatic deposition technology (62). The most common controlled delivery system have been tablets and granules because of its effectiveness, low cost, ease of manufacturing and prolonged delivery time period, where the drug is uniformly dissolved or dispersed throughout the polymer (62). The tableting process is associated with relatively high pressure in order to form suitable compacts. However, not only the tableting excipients are deformed during the process of tablet formation, but also the tablet itself. This can lead to total or partial damage to such materials, namely loss of biological activity of proteins and enzymes, polymorphic transformation of excipients or damage of the coating material. Most recently, different excipients were tested in order to prevent such damages. Amongst others, polysaccharides like chitosan and carrageenan have shown to be advantageous because of their elastic tableting behavior (63). Several reports have been published on the use of chitosan as tablet excipients. It was applied as a carrier for sustained release tablets, a direct compressible diluent, a tablet disintegrate and a tablet binder (37). As a diluent, chitosan was used for preparation of direct compressed tablets (64, 65) where drug release was controlled. Studies using chitosan as 15 directly compressible tablet excipient showed its potential for use in modified release drug delivery systems without the need for additional adjuvants (64). Chitosan also showed higher binder efficiency than other tablet binders such as methylcellulose and sodium carboxymethylcellulose (66) and used as a binder for colon specific drug delivery tablets with slow drug release compared with other polysaccharides or synthetic polymers (67). Chitosan was utilized as tablets disintegrate (66) and showed bioadhesive properties in mixture with sodium alginate and in the form of thiolated chitosan derivative with slow drug release for intra-oral drug delivery tablets (68). Furthermore, the solubilizing and amorphizing properties of low MW chitosan toward naproxin made it an optimal carrier for developing fast release oral tablet (69). Depending mainly on ionic interaction, chitosan was also used for the preparation of tablets matrix to control drug release (70, 71). When used in a matrix-type tablet formulation, chitosan forms a gel-barrier in an acid environment that can modulate or constrain drug release. Furthermore, at acidic pH amines of chitosan are protonated and can therefore interact with oppositely charged drug ions, serving as excipient for modified release of drug delivery systems (65). Chitosan was studied as excipient in the preparation of prolonged theophylline tablets. These tablets showed higher drug bioavailability than of the commercial ones, which becomes a new potential formulation to respiratory problems (66). The biological potential of chitosan adds also clear benefits to the tablet formulation and process. In a recent research five different polysaccharides with potential antioxidant activity for extended-release matrix tablets were compared (72). The results suggest that chitosan would be potentially useful in an extended-release tablet with the higher antioxidant activity, able to catch the most diverse and natural oxidative species, usually involved in different pathologies. A new study concerning vaginal infections and inflammations were evaluated using chitosan tablets (73). Topical administration of the antibacterial metronidazole represents the most common therapy in the treatment of bacterial vaginosis caused by Trychomonas vaginalis. The formulations generally available for such therapy are creams, gels, vaginal lavages and vaginal suppositories. In this study, a new dosage form, containing metronidazole was developed with the aim to realize vaginal mucoadhesive tablets by including bioadhesive polymers as chitosan. This kind of delivery systems suitable for formulating metronidazole for topical application represents a good alternative to traditional dosage forms for vaginal topical administration in the treatment of infections or inflammations. These solutions overlap the limitations of conventional therapies that are 16 not suitable to assure drug permanence on the vaginal mucosa surface for adequate time assuring the complete elimination of bacteria and pathology eradication. Nonetheless, all applications of chitosan as tablet excipient were not in its derivative forms. However, attempts have been made to improve chitosan property by developing derivative salts. Chitosan derivatives such as glutamate, aspartate and hydrochloride salts have been used for colon-specific drug delivery and to enhance the delivery of therapeutic peptide across intestinal epithelia (37). 2.1.4. Hydrogels Hydrogels are networks of hydrophilic polymers that can absorb large quantities of water without dissolution. Due to their physical properties resembling human tissue and its excellent tissue compatibility, hydrogels have been extensively studied for biomedical applications. They can be used as soft contact lenses (74), tissue engineering scaffolds (75), drugs carriers and controlled-release systems (76). In addition, hydrogels have the potential for further healing (77). They can absorb excess wound exudates, protect the wound from secondary infection and effectively promote the healing process by providing an environment for moist wound healing (78). Even can also be removed without causing trauma to the wound (78). Several models of hydrogels have been studied, including chitosan, poly (vinyl alcohol) (PVA) that is a water-soluble polyhydroxy polymer and alginate. However, chitosan has been widely exploited in hydrogel formulation and in practical applications because of its easy manipulation, excellent chemical resistance, physical properties, biodegradability and low price (78). This polymer is used to produce hydrogels with well-known properties that are used for delivery of proteins and synthetic drugs. Since this compound is also polyelectrolyte, its ionic form produces complexes through hydrogen bonding or electrostatic interactions. Besides this, another interesting property of chitosan is its ability to gel in contact with specific polyanions. This gelation process is due to the formation of interand intramolecular bonding mediated by these polyanions (1). In the last decade, different chitosan hydrogels were produced for drug delivery in micro or nano-scale using the polyelectrolyte complexation technique. There are many factors that affect the relevant properties of the capsules of chitosan, in particular the composition, MW and DD of chitosan. Several methods have been developed in which the particle size of chitosan 17 hydrogel and its related properties are quite distinct, according to the method of preparation and the reaction conditions that are employed. One of the major factors that may influence the final properties is the method of preparing hydrogel. Moreover, few attempts were made to correlate statistically the reaction conditions with the final properties of chitosan hydrogel. Liu and his collaborators (79) evaluated the influence of chitosan MW and its concentration, along with pH, upon the swelling behavior of microcapsules of chitosan-alginate, and postulated that all factors have an effect on the behavior of the hydrogel swelling. The alginate-chitosan hydrogels are commonly prepared by ionic complexation using alginate as a gel core (80) and then characterized by the vibration modes of their main groups using FTIR. Other hydrogels formulation procedures can be performed by UV crosslinking. In this method, lactose moieties are introduced into chitosan to obtain much better water-soluble chitosan at neutral pH, and photoreactive azide groups are added to provide the ability to form a gel through crosslinking azide groups with amino groups (81). This photocrosslinkable chitosan is then exposed to UV irradiation to form an insoluble and adhesive hydrogel within 60 s. Hydrogel has the consistency of transparent and soft rubber (81). The crosslinking can also be performed by high temperature. It is based on the neutralization of a chitosan solution with a polyol counterionic dibase salt such as βglycerophosphate. Chitosan/glycerophosphate is a thermosensitive solution, which is liquid at room temperature and solidifies into a white hydrogel at body temperature (81). In addition, crosslinking can be achieved by high pH, employing the pH-sensitive property of chitosan solutions at low pH. Once injected into the body, these polymer solutions face different environmental pH conditions and form gels (81). The chitosan hydrogel formulation can also be made by, freezing, thawing or chemical methods. Irradiation has the advantages of easy control of processing, without adding initiators or cross-linkers that can be harmful and difficult to remove and also has the option of combining the hydrogel formation and sterilization in one technological step. The main disadvantage of hydrogels prepared by irradiation is its poor mechanical strength. However, hydrogels prepared by freeze-thaw for example, of aqueous solutions of PVA has good mechanical strength, are stable at room temperature and does not require initiators or cross-linkers. The main disadvantage of this type of hydrogel is its opaque appearance and limited expansion capability (78). The hydrogel yields are evaluated through the weight difference, placing the washed hydrogels into pre-weighed flasks and then into a stove at 50 ºC until dryness. The chitosan hydrogel particles can then be visualized and characterized by particle size and 18 size distribution using an inverted optical microscope (80) as well as, by solubility, X-ray diffraction, thermal analysis, and solvent uptake (82). An ophthalmic delivery system with improved mechanical and mucoadhesive properties that could provide prolonged retention time for the treatment of ocular diseases were evaluated considering chitosan hydrogel formulation. For this, an in situ forming gel was developed by the combination of a thermosetting polymer, poly (ethylene oxide)–poly (propylene oxide)–poly (ethylene oxide) with chitosan. Therefore, the final formulation presented adequate mechanical and sensory properties and remained in contact with the eye surface for a prolonged time. In conclusion, the in situ forming gel comprised of poloxamer/chitosan is a promising tool for the topical treatment of ocular diseases (83). To overlap the limitation of topical delivery of antimicrobial agents and to prolong active drug concentrations in the oral cavity, it was designed a hydrogel formulation containing chitosan for delivery of chlorhexidine gluconate to the oral cavity (84). Chitosan prolongs the adhesion time of oral gels and drug release also inhibiting the adhesion of Candida albicans to human buccal cells since it has antifungal activity (84). The antifungal agent, chlorhexidine gluconate also induces the reduction of Candida albicans adhesion to oral mucosal cells. The preparation and characterization of thiol-modified chitosan, which formed crosslinked hydrogels, was also described to characterize in vitro release kinetics of insulin encapsulated in different chitosan MW hydrogels and evaluated for their potential use as a scaffold for the culture of NIH 3T3 cells (85). The results demonstrated that insulin is not immobilized locally within the gel network. Since the incorporation into the gel has no impact on insulin stability it may be assumed that the chitosan thermogelling system is an attractive delivery system for peptides and proteins. The main goal of other study was to developed a chitosan bioadhesive gel for nasal delivery of insulin (86). The proposed gel formulation could be useful preparation for controlled delivery of insulin through the nasal route and may represent an alternative treatment to diabetes. Mucoadhesive chitosan lactate gels were developed intended for the controlled release of lactic acid onto vaginal mucosa (87). The conclusions finding makes it reasonable to envisage a complete release of lactate from the tested formulations in vaginal environment. A recent research reports an in situ gelling chitosan-based hydrogel system that sustains the release of a potential anti-cancer gene (pigment epithelium-derived factor) to the tumor site. A significant reduction of the primary osteosarcoma in a clinically relevant orthotopic model was measured. The combination of plasmid treatment and 19 chemotherapy together with the use of this delivery system led to the highest suppression of tumor growth without side effects. The results obtained from this study demonstrate the potential application of a hydrogel system as an anti-cancer drug delivery for successful chemo-gene therapy (88). Another notorious study focuses on the current use of injectable to form in situ chitosan hydrogels in cancer treatment (81). Formulation protocols for in situ hydrogel systems, their cytotoxic properties, loading and in vitro release of drugs, their in vitro effect on cell growth, the inhibition of tumor growth in vivo using mouse models, and future directions to enhance this technology were discussed. In conclusion, chitosan gelling systems due to their antibacterial, biocompatible, biodegradable and mucoadhesive properties are a potential carrier for various cancer treatments. These hydrogels may also be useful to detect the localized growth of cells (81), which can also be directed to innovative methods of diagnosis. 2.1.5. Microparticles Microparticles are defined as multiparticulate delivery systems, usually spherical with size varying from 1 to 1000 µm, containing a core active substance (48). The terms microcapsules and microspheres are often used synonymously. Spheres and spherical particles are also used for a large size and rigid morphology (48). The use of microparticles-based therapy allows drug release to be carefully tailored to the specific treatment site through the choice and formulation of various drug–polymer combinations. The total dose of medication and the kinetics of release are the variables, which can be manipulated to achieve the desired result. Using innovative microencapsulation technologies, and by varying the copolymer ratio, MW of the polymer among other parameters, microparticles can be developed into an optimal drug delivery system, which will provide the desired release profile (34). Chitosan with different MW and concentration, degradation rate of chitosan particles and drug concentration interfere on microparticle properties (1). Chitosan microparticles are used to provide controlled release of many drugs and to improve the bioavailability of degradable substances such as protein or enhance the uptake of hydrophilic substances across the epithelial layers (34). Having in mind bio/mucoadhesive properties of natural biopolymers, chitosan microparticles have potential for colon targeting. In order to achieve localization and prolonged residence time in the colon, matrices should have optimal 20 particle size, between 4 and 15 µm (89). Carrier systems in that size range are able to attach more efficiently to the mucus layer and accumulate in the affected region without the need for macrophage uptake. This novel formulation will offer efficient treatment of colon inflammatory diseases like ulcerative colitis and Chron’s disease (89), increasing therapeutic concentration (at the site of inflammation) and activity and minimizing side effects that occur by conventional systemic absorption. Chitosan microparticulate carrier systems are also efficient in the treatment of inflammatory bowel diseases (89). Budesonide is one of the most used drug substances in the treatment of active inflammatory bowel diseases. Chitosan microparticles loaded with budesonide were produced using novel one step spray-drying procedure. Coated microparticles were suitable candidates for oral delivery of budesonide with controlled release properties for local treatment of inflammatory bowel diseases. Chitosan microparticles also represents a promising polymer in nasal peptide delivery (90) prolonging the residence time of nasal drug delivery systems at the site of drug absorption. Additionally, chitosan improves the absorption of peptides by opening transiently the tight junctions. Oral administration of the nonsteroidal anti-estrogen tamoxifen is the treatment of choice for metastatic estrogen receptor-positive breast cancer. Chitosan microparticles were developed for tamoxifen delivery into the lymphatic system (91), improving tamoxifen oral bioavailability and decreasing its side effects. These data underline other potential therapies to this serious cancer condition. It was also reported the importance of chitosan microparticles in the purification of immunoglobulin G from human plasma by affinity chromatography using linoleic acid attached chitosan microparticles (92). It was concluded that the microparticles allowed just one-step purification of immunoglobulin G from human plasma. Chitosan and its derivative N-trimethyl chitosan chloride, given as microparticles associated to the non-toxic mucosal adjuvant LTK63, were evaluated for intranasal immunization with the group C meningococcal conjugated vaccine. The bactericidal activity measured in serum of mice immunized intranasally with the conjugated vaccine formulated with this delivery system and the LT mutant was superior to the activity in serum of mice immunized sub-cutaneous. Importantly, intranasal but not parenteral immunization, induced bactericidal antibodies at the nasal level, when formulated with both delivery system and adjuvant (93). In another study, it was evaluated the ability of chitosan microparticles to enhance both the systemic and local immune responses against diphtheria toxoid after oral and nasal 21 administration in mice. Significant systemic humoral immune responses were also found after nasal vaccination with diphtheria toxoid associated to chitosan microparticles. Diphtheria toxoid associated to chitosan microparticles results in protective systemic and local immune response against this toxoid after oral vaccination and in significant enhancement of immunoglobulin G production after nasal administration. Hence, these in vivo experiments demonstrate that chitosan microparticles are very promising mucosal vaccine delivery system (94). Other similar studies considered the chitosan microparticles as encapsulating agent of large amounts of antigens such as ovalbumin, or tetanus toxoid (95). Besides chitosan particles are a promising candidate for mucosal vaccine delivery, mucosal vaccination not only reduces costs and increases patient compliance, but also limits the invasion of pathogens through mucosal sites. 2.1.6. Nanoparticles Nanoparticles are defined as a microscopic particle whose size is in the nanoscale, varying from 1 to 1000 nm, able to deliver drugs (2) to the right place, at appropriate times and at the right dosage, also improving their bioavailability, efficiency and reducing cytotoxicity associated to other systemic drugs carriers, actually becomes one of the most attractive areas of research in drug delivery (2). These submicron particles containing entrapped drugs are intended for enteral or parenteral administration, which may prevent or minimize the drug degradation and metabolism as well as cellular efflux, extending the shelf-life (50). Some researchers have also observed that the number of nanoparticles, which cross the intestinal epithelium is greater than that of the microparticles and hydrophilic nanoparticles generally have longer resident time in blood then microspheres (96). With their easy accessibility in the body, nanoparticles can also be transported via the circulation to different body sites. These particulate delivery systems have been shown to enhance the immune response following mucosal application. Nanoparticles have been made of safe materials, including synthetic biodegradable polymers, natural biopolymers, lipids and polysaccharides and have the potential for overcoming important mucosal barriers, such as the intestinal, nasal and ocular barriers (50). Chitosan based nanoparticles show great potential for delivering macromolecular therapeutics (in particular drugs and genes) and control the complete release of the drugs in their native forms across biological barriers (41). Important advantages of these nanoparticles include 22 their rapid preparation under extremely mild conditions and also their ability to incorporate bioactive compounds (45). Chitosan nanoparticles topically applied into the eye has been proven to increase the residence time of drugs in the precorneal area due to their adhesive properties and, therefore, could prolong the penetration of drugs into the intraocular structures (2). An important research shows that a chitosan derivative can be used to prepare norcantharidin-associated nanoparticles by taking advantage of the ionic cross-linkage between the drug molecule and of the chitosan carrier for antihepatocarcinoma medicine (97). The potential of nanoparticles as a vaccine delivery has also been demonstrated in several studies (98-100). Nanoparticle-mediated gene delivery is an alternative to viral gene delivery. Nanoparticles offer the potential for safe, targeted and efficient gene delivery in a variety of organs (101). Borges et al. (102), recently described a delivery system that is composed of a nanoparticulate chitosan core to which the hepatitis B surface antigen (AgHBs) was adsorbed and subsequently coated with sodium alginate. The enhancement of the immune response observed with the antigen-loaded nanoparticles demonstrated that chitosan is a promising platform for parenteral delivery of this antigen, since it resulted in a mixed Th1/Th2 type immune response. Chitosan nanoparticles have been also produced as a carrier system for the nasal delivery of a monovalent influenza subunit vaccine (103). The intranasal administered antigenchitosan nanoparticles induced higher immune responses compared to the other intranasal antigen formulations, and these responses were enhanced by intranasal booster vaccinations. Moreover, among the tested formulations only intranasal administered antigen-containing chitosan nanoparticles induced significant immunoglobulin A levels in nasal washes of all mice tested, demonstrating that chitosan nanoparticles are a potent new delivery system for intranasal administered influenza antigens. Another study indicate that chitosan nanoparticles are a good carrier for DNA vaccines against tuberculosis by pulmonary delivery, which may provide an advantageous delivery route compared to intramuscular immunization, due increasing higher immunogenicity (104). Chitosan nanoparticles have been also introduced as a useful carrier for peptide oral delivery, because they can protect these compounds from degradation. Insulin, like other peptides, has low therapeutic activity when administered orally due to degradation by proteolytic enzymes (105). 23 Sarmento et al. (106) developed chitosan nanoparticles as drug carrier of insulin. Insulin was entrapped in different polyanion/chitosan nanoparticulate systems with high efficiency, to study morphologic and physical properties of resulting nanoparticulate complexes and to investigate insulin release behavior under gastrointestinal conditions (106). These nanoparticulate complexes appear to possess good properties for oral protein delivery, particularly those containing dextran sulfate/chitosan polyelectrolytes, which provided highest insulin association efficiency and retention of insulin in gastric simulated conditions. However alginate/chitosan nanoparticles also appear as promising in oral delivery system for insulin and potentially for other therapeutical proteins (107). Moreover, it was demonstrated that blood glucose levels of diabetic rats can be effectively controlled by insulin-loaded chitosan nanoparticle administration, following either single or multiple oral administration. In addition, the hypoglycemic effect was observed for more than 24 h (108). 2.2. Clinical trial - Safety and tolerability of chitosa-n-acetylcysteine eye drops in healthy young volunteers There are many definitions of clinical trials, generally are studies of biomedical and health research related to human beings following a pre-defined protocol (109). These tests can only take place when quality and safety of the test are guaranteed by the Health Authority or the Ethics Committee recognized by the country, which will run the clinical trial. The randomized controlled trial is commonly accepted as the gold standard research method for evaluating health care interventions (110). In any clinical trial it is desirable not only to achieve similar numbers of patients in each treatment group, but also to ensure that patient groups are similar with respect to prognostic factors such as age or stage of disease (110). Nowadays, many studies have come and explore the multi-potential of chitosan as a carrier for drug delivery and release (109). The "dry eye syndrome" DES is a highly prevalent ocular disease, particularly in the elderly population and that current therapy is the use of topically administered lubricants, no "ideal" formulation has yet been found. Recently, Croma Pharma has introduced chitosan-n-acetylcysteine eye drops (111), designed for treatment of symptoms related to “dry eye syndrome". The new formulation comprises n-acetylcysteine, which has been used in ophthalmology because of its mucolytic properties for several years. Based on theoretical considerations, one can hypothesize that the new chitosan derivative may 30 oxygen, environmental chemicals and physical abrasion (137, 138). Protective components like water-soluble antioxidants, lipid-soluble antioxidants and highly specialized enzymes are thought to serve as a frontline defense for the ocular surface tear film and underlying tissues (118). If the antioxidants are depleted or acting inefficiently, an imbalance between the high increased production of ROS and the sharp reduction in antioxidant defenses will alter cellular redox status (136). In retinopathy, OS has been widely involved in decreased retinal blood flow (139), increased vascular permeability, disruption of BRB (140) and the appearance of cellular capillaries from the apoptotic loss of retinal capillary cells (141). OS has also been linked to microvascular abnormalities in retinopathies, degenerative process of retinal neovascularization and the suppression of antioxidants systems (25). 4. Nanotechnology applied to antioxidants protection Nanoparticles have been made of safe materials, including synthetic bio-degradable polymers, natural biopolymers, lipids and polysaccharides and have the potential for overcoming important mucosal barriers, such as ocular barriers (50). Nanoparticles may be obtained via different preparation protocols and have been widely studied in recent years as carriers for therapeutic agents with varying degree of effectiveness (142). Different preparation of micro/nanoparticles can be considered, mainly physical methods, chemical crosslinking methods and miscellaneous. Physical methodologies involve ionotropic gelation, emulsification and ionotropic gelation, modified emulsification and ionotropic gelation, floating hollow chitosan microspheres obtained by ionic interaction with sodium dioctyl sulfosuccinate, coacervation and complex-coacervation (34). Crosslinking with other chemicals are used for emulsion crosslinking method, multiple emulsion method, precipitation–chemical crosslinking and crosslinking with a naturally occurring agent (34). Miscellaneous methods include thermal crosslinking, solvent evaporation method, spray drying and interfacial acylation (34). Nanocarriers can be applied to improve the solubility, permeability, stability of the compounds and some can even surpass the first pass metabolism (Figure 1.4). These delivery systems have been beneficial to the pharmaceutical industries as it is a strategic tool for expanding drug market and patent life. Novel drug delivery systems would make antioxidant reach site of action and improve the efficacy of therapy, generally by improving the bioavailability, which are of prime importance when antioxidants intended for prophylactic purpose (116). 31 Implication of novel delivery systems for antioxidants is ruled by physical-chemical characteristics, biopharmaceutics and pharmacokinetic parameters of the antioxidant to be formulated (116). Recently, chemical modifications, coupling agents, liposomes, microparticles, nanoparticles and gel-based systems have been explored to overcome difficulties in the development of new products for the improvement of human healthcare (18). Figure 1. 4. Classification of antioxidants and characterization of some nanostructures. Highlights to real benefits of antioxidants encapsulation considering its liberation, absorption, distribution, metabolism, elimination and response, adapted from (18). Nevertheless, it may be highlighted the huge importance of nanotechnology and nanocarriers to be applied in common delivery systems, like immunoglobulin’s deliver to 32 the retina. In particular, the injecting vascular endothelial growth factor (VEGF) to neutralize the antibodies into the vitreous. Silicate nanoparticles (SiNPs) have been demonstrated efficiency in inhibition of VEGF-induced angiogenesis. Via suppression of VEGF receptor-2 phosphorylation induced by VEGF, SiNPs blocked ERK 1/2 activation (143). Intravitreal injection of gold nanoparticle (GNP) also has shown an inhibition of retinal neovascularization in a mouse model of retinopathy of prematurity. GNP not affected the cellular viability of retinal microvascular endothelial cells and not induced retinal toxicity. GNP can be used in a variable vaso-proliferative retinopathies mediated by VEGF (144). A molecule with ideal solubility and permeability profile can be administered with a minimum effective dose and with no presystemic loss due to mucosas, physical barriers or enzymatic degradation (18). For antioxidants assure these conditions they need nanocarriers to take them to the right place without losing their functional activity. Therefore, considering the therapeutic potential of the antioxidants, there is every need to implicate novel drug delivery technologies to improve their performance. Nanoparticles are expected to develop and improve protection, stability, bioavailability and therefore the therapeutic efficacy of antioxidants, without compromising the safety performance of the drug. The Age-Related Eye Disease Study (AREDS) (145), an National Eye Institute– sponsored, multicenter, controlled, randomized clinical trial, demonstrated that the combination of oral supplements consisting of antioxidant vitamins C (500 mg), E (400 international units), and β-carotene (15 mg), and minerals, ZN (80 mg of zinc oxide) with Cu (2 mg cupric oxide). This reduced the 5-year risk of developing advanced age macular degeneration (AMD) in eyes with intermediate AMD by 25% (estimated probability of progression was 28% for placebo vs 20% for antioxidants plus zinc). The primary purpose of the Age-Related Eye Disease Study 2 (AREDS2) (145) was to evaluate the efficacy and safety of lutein plus zeaxanthin and/or ω-3 long-chain polyunsaturated fatty acid (LCPUFA) supplementation, reducing the risk of developing advanced AMD. Nevertheless and besides the promising effect of this huge antioxidant supplementary, it would be expectable that this effect could be also potentiated by the protection of these antioxidants using the nanotechnology. The protection may be crucial in order to guarantee the safe antioxidant performance, bioavailability and absorption, over the several barriers of the digestive system (such as the biological fluids and enzymatic pathways). Other studies have been performed to maintain or even increase the activity and stability of antioxidants using nanotechnology (Table 1.2). These are evidences that antioxidant nanoparticles 33 may be used for therapy of several diseases, however further studies are needed to prove their efficacy in the prevention and treatment of retinopathies. Table 1. 2. Recent published data of antioxidants nanoencapsulation for several applications. Antioxidant Method Results Application References 3,5-di-tertbutyl-4hydroxycinna mic acid A nanosilicaimmobilized antioxidant was prepared and incorporated into polypropylene (PP) by melt compounding The antioxidant efficiency of the nanosilicaimmobilized antioxidant was superior to the corresponding low molecular counterpart (AO). The thermal oxidative stability of PP/nanosilica-immobilized antioxidant was much higher than that of PP/AO compound during the long-term accelerated thermal aging Application not described (146) Antioxidant enzymes (catalase and superoxide dismutase) Magnetically responsive nanoparticles (MNP) formed by precipitation of calcium oleate in the presence of magnetite-based ferrofluid (controlled aggregation/ precipitation) Catalase stably associated with MNP was protected from proteolysis and retained 20% of its initial enzymatic activity after 24 h of exposure to pronase. Under magnetic guidance catalase-loaded MNP were rapidly taken up by cultured endothelial cells providing increased resistance to OS (62 ± 12%) cells rescued from hydrogen peroxide induced cell death vs. 10 ± 4% under non-magnetic conditions Can be highly relevant for the treatment of vascular disease (147) Antioxidants of Salvia miltiorrhiza (salvianolic acid B, cryptotanshino ne, tanshinone I and tanshinone IIA) The dried roots of S. miltiorrhiza were ground by the atomizer and further sprayed granulating with the aid of floating bed. The resulting materials were dried to form the nanoparticles under the dry processes Stronger antioxidant bioactivities were observed for the extracts prepared using nanotechnology in all tested assays. The polar active constituent in the nanoparticles samples was released faster compared to the traditionally powdered samples Application not described (116) Idebenone Co-drying with chitosan (Ch) or Ncarboxymethylchitos an (N-CMCh) crosslinked with tripolyphosphate (TPP) Nanoparticles showed a 10-fold increase of drug stability in comparison with free drug and preserved antioxidant activity in vitro. Compared with the severely irritative free form of idebenone, the nanoparticle formulation showed decreased mucous membrane irritation These nanoparticles have potential roles a carriers for hydrophobic and irritative drugs such as the antioxidant idebenone for topical or nasal use (148) 34 Natural antioxidants extracted from Ilex paraguariensis (ILE) Nanoparticles were prepared by ionic gelation of chitosan hydrochloride and sodium tripolyphosphate. The active components were added to the sodium tripolyphosphate solution and this was added dropwise to the chitosan hydrochloride solution while stirring Chitosan hydrochloride–TPP nanoparticles maintain the antioxidant activity of ILEpolyphenols. Nanoparticles released 100% of ILE-polyphenols loaded after 15 min in two buffers with different pH values (pH 5.7 and 6.5) This method is a promising technique for nutraceutical and cosmetic applications (149) pHydroxybenzyl alcohol (HBA) HBA-incorporated copolyoxalate (HPOX) HBA released from HPOX demonstrated excellent antioxidant activity, such as inhibition of nitric oxide (NO) production by suppressing iNOS (inducible nitric oxide synthases) expression in lipopolysaccharide (LPS)-activated RAW 264.7 cells. HPOX nanoparticles delivered intranasally significantly reduced pulmonary inflammation and suppressed the iNOS expression HPOX nanoparticles are highly potent for the treatment of oxidative damage-related diseases, such as asthma (150) Quercetin Nanoprecipitation technique with Eudragit® E (EE) and PVA as carriers Particle distribution with polydispersity index <0.3, and its yield and encapsulation efficiency were over 99%. The release of the drug from the quercetin-loaded nanoparticles (QUEN) was 74-fold higher compared with the pure drug. The antioxidant activity of the QUEN was more effective than pure quercetin on diphenyl-1-picrylhydrazyl (DPPH) scavenging, anti-superoxide formation, superoxide anion scavenging, and anti-lipid peroxidation These nanoparticles may be applied in clinical setting (151) Quercitrin (Albizia chinensis isolated antioxidant) Quercitrin has been encapsulated on PLA nanoparticles by solvent evaporation method The encapsulation efficiency of nanoencapsulated quercitrin evaluated by HPLC and antioxidant assay is 40%. The in vitro release kinetics of quercitrin under physiological condition reveals initial burst release followed by sustained release. Less fluorescence quenching is observed with equimolar concentration of PLA encapsulated quercitrin than free quercitrin. The presence of quercitrin specific peaks on FTIR of five times washed quercitrin loaded PLA nanoparticles provides an extra evidence for the encapsulation of quercitrin into PLA nanoparticles Potential use for therapeutic of intestinal antiinflammatory effect and nutraceutical compounds (152) α-tocopherol (vitamin E) Gold nanoparticles prepared with 2,2-DPPH Chromanol groups on gold nanoparticles could efficiently enhance the activity of the vitamin E-derived antioxidant Potential strategy for antioxidant design with several applications (153) 35 4.1. Nanoantioxidants pharmacotherapy Drugs topically administered in the eye have low probability of reaching the posterior segment in significant amounts, as they have to pass through several metabolic and physical barriers to reach the retina, namely the corneal and conjunctival epithelium, then aqueous humor, and lens (132). One of the main problems encountered with the topical administration of liquid forms is the rapid and extensive loss of drugs to the limited capacity of drug retention in the ocular surface and also as a result of the blinking process, which normally is stimulated after instillation (118). The BRB and the extra ocular epithelia represent the obstacle in the drug delivery to the choroid, retina and vitreous. Only a fraction of the drug administered orally or by subcutaneous or intramuscular routes reaches the retina, requiring large doses to be therapeutically effective (118). Presently, there are several antioxidant products on market which have been formulated into these conventional dosage forms, with vitamins leading the group. Vitamins have been formulated mainly into tablets and capsules. Generally, these agents were found in combinations rather than individual products (18). A possible approach to improve retinal drug delivery is to facilitate localized delivery to the posterior segment of the eye by using Anopore™ nanoporous filter. Catalase and vitamin C were delivered using these inorganic nanoporous filter, which is made up of aluminum oxide filter with pores of 20 nm size, as a semipermeable membrane to separate two compartments in vitro. The data shown represented the possibility of biocompatible capsules based on nanoporous filters, which are able to provide controlled delivery of antioxidant molecules (154). Several nanocarriers may be consider as nanoantioxidants transporters as self-emulsifying drug delivery systems, which offer the potential for enhancing the absorption of poorly soluble and/or poorly permeable compounds. For drugs that are poorly soluble and/or poorly permeable, a significant improvement in reproducibility in performance and bioavailability might be achieved with these nanocarriers. Lutein is a well-known antioxidant and antifree radical used in cosmetic, nutraceutical industry with potential application in pharmaceutics as supportive antioxidant in treatments. However, lutein is a lipophilic molecule which is poorly soluble in water and has a low bioavailability. The lutein nanosuspension was converted into pellets and filled into hard gelatin capsules for nutraceutical use, yielding a superior in vitro release (155). However, there are some limitations associated with these formulations, including stability, manufacturing methods, interaction of the fill with the gelatin shell and limited solubility of some drugs in lipid solvents (18). Liposomes are potential systems for drug delivery because of their size, 36 hydrophilic and hydrophobic character and biocompatibility (156). The term liposomes with antioxidants could be liposomes containing lipid-soluble, water-soluble or enzymatic antioxidants. Antioxidant liposomes hold great promise in the treatment of many diseases in which OS plays a significant role (157). A major problem associated with conventional liposomes is that they are recognized by the immune system as foreign substances and rapidly removed by phagocytic cells of the reticuloendothelial system (18). Nanoparticles have been explored as drug delivery systems for encapsulation of different drugs or incorporation either into lipid or polymeric particles. The nanoencapsulation can help deliver drugs with poor aqueous solubility and permeability. In biodegradable polymeric nanoparticles the drug is dissolved, adsorbed, attached or encapsulated in the polymeric matrix of nanometer size. Depending upon the method of preparation, nanospheres or nanocapsules are obtained with different release and surface properties (158). Nanoparticles are also being explored for targeted drug delivery (159). Polymeric nanocarriers are attractive vehicles for vascular drug delivery as well, but remained a waif technology for antioxidant enzymes due to poor loading and inactivation of proteins during formulation (159). Microparticles have also been designed and evaluated as delivery systems for antioxidants produced endogenously and exogenously. The matrix is generally a polymer which sustains the release of drug. These particles are either matrix type entrapping the active moiety or capsule type encapsulating the drugs (18). The need to improve the ocular drug bioavailability, effectiveness and higher retention time becomes an emerging field in medicine, considering ocular conditions. Although there are not any antioxidant nanoparticles agent approved by European Medicines Agency (EMA) and Food and Drug Administration (FDA) there are at least 60 agents in the clinic touted as anti-angiogenic, many more potential anti-angiogenic candidates are currently in preclinical development, with the distinct possibility of moving into ocular clinical studies (160, 161). Recent data from very important multicenter clinical trials have emphasized the importance of new and evolutional therapies. 4.2. Safety issues of antioxidant nanoparticles Despite of nanoparticles with antioxidants are known to be from natural sources, then safe, biocompatible and biodegradable with no toxicity effects or collateral damages, pharmacokinetics and toxicological analyses should not be dispensable. This safety 37 performance should guarantee the molecular, subcellular and cellular behavior of nanoparticles in ocular tissues and in the systemic circulation. The development of in vitro cell culture models for studying ocular barriers undoubtedly provides a platform to investigate the impact of pharmaceutical trafficking on ophthalmic diseases (162). The human corneal epithelium HCE-T model, formed by transfection of human corneal epithelial cells from a 47-year-old female donor with a recombinant SV40-adenovirus, represents a standard tool for drug permeation, bioavailability prescreening, and toxicity assessment (163), should be used to simulate the corneal absorption of antioxidants. The cell line has good growth characteristics and shows a cobblestone-like appearance. The IOBA-NHC cell line, a nontransfected, spontaneously immortalized epithelial cell line derived from human conjunctiva (164), should be used for predicting the conjunctival absorption of antioxidants. The IOBA-NHC cells demonstrated high proliferative ability in vitro and typical epithelial morphology. Furthermore, cytokeratins, mannose, and sialic acid residues are immunologically detected, therefore appears that this cell line can be a useful experimental tool in the field of ocular surface cell biology. Immortalized human cells ARPE-19 cells are particularly important in the retinopathies studies, since they simulate the retinal behavior and may indicate the absorption of antioxidants. These cells are fully characterized regarding their morphology, the expression of retina specific marker, and their barrier properties (162, 165). Monolayers of ARPE-19 cells have become a well-established in vitro model of the outer BRB. Moreover, monolayers of ARPE-19 cells are used by researchers for a variety of other in vitro experiments, including studies of the regulation of gene expression, polarized distribution and secretion of proteins, delivery of genes and antisense oligonucleotides, for toxicity studies, and as models of retinal diseases (162, 166, 167). The fate of nanoparticles loaded with natural antioxidants must be followed with a minimum required amount of in vivo experiments using topical routes of administration. The therapeutic efficacy and safety in vivo in rabbits should be monitored by means of physiological and behavioral parameters, complemented by toxicological and monitoring of conjunctival tissue. 38 5. Summary The physical-chemical properties of chitosan, such as interand intramolecular hydrogen bonding and the cationic charge in acidic medium in addition with its several bioactivities such as nontoxicity, biocompatibility, biodegradability and transmucosal absorption allows chitosan to become an excellent candidate for drug carrier and excipient for controlled release systems. A number of chitosan-based colloidal systems have been explored for bioactive molecules carriers, like films, tablets, hydrogel, micro and nanoparticle. In these formulations chitosan can reach the target sites with low collateral damages, toxicity and higher efficiency. The antioxidant therapy major problem in ocular treatments is the difficult to maintain an effective drug concentration at the site of action for an appropriate period of time, in order to achieve the expected pharmacological response. This suboptimal delivery can be overcome by different novel delivery strategies. Innovative nanomedicines with antioxidants pharmacotherapy could be seen as the key factor for eye diseases, since the use of topical nano-antioxidants to treat or delaying OS-related ocular manifestations is still unexplored, while current retinopathies therapy includes invasive method like laser photocoagulation or surgery, which may also increase risk of endophthalmitis, cataract formation and retinal detachment. Besides the development of laser for retinopathy, there have been no major advances in treatment for the disease, despite numerous clinical trials. Drugs applied directly to the eye represent a non-invasive and safe methodology, increasing the effectiveness of treatment and reducing toxicity associated with systemic administration. An ideal anti-angiogenic agent should be developed for neovascularization control and regression. It may inhibit and stabilize the disease, to prevent the vision loss, retinal scarring and detachment with no toxicity as well as the formulation should be for long term drug delivery. Agents should also be classified into early and late acting, specific and non-specific, and reversible and irreversible. The understanding of where a drug falls into these classes may help in the comprehension of the potential and/or limitation of the drug when used in the clinic, as well as how to predict potential serious adverse events. The nano-antioxidants are expected to be early acting, especially for prophylaxis. Considering this, there are a number of challenges associated to the treatment of ocular diseases. Thus, successful alternatives for ocular therapies are needed and they should provide non-invasive and a cost effective treatment reaching every economic status. Therefore, useful knowledge for future adaptation is provided for pharmaceutical industry, medical assistance and human health care. 39 PART II - Aims and goals “There is a world of advantages of nanotechnology application in different fields ... are all a part of its own disadvantages!” Jacques de la Palice 46 47 Abstract Introduction: Salvia officinalis and Satureja montana (sage and savory, respectively) are plants used in traditional medicine. The quality control of their herbal formulations is of paramount concern to guarantee the expected biological activity of their antioxidant compounds. Objectives: To establish a simple and effective HPLC method to evaluate simultaneously quercetin and rosmarinic acid, in a pure form, in natural extracts (sage and savory), and encapsulated into chitosan-based nanoparticles (in the next sections). Methodology: Chromatography was performed on a RP C18 column, in a gradient mode with a mobile phase comprising methanol:formic acid:water 92.5:2.5:5 (v/v) at a flow rate of 0.75 mL/min and at detection wavelength of 280 nm. Results: The method was specific, linear in the range of 0.05-1.0 mg/mL (R2 = 1.00), precise at the intra-day and inter-day levels, accurate (recovery rate percentage of 90.5 ± 0.6), and robust to changes in equipment conditions. Conclusion: The established method was effective for quercetin and rosmarinic acid characterization in natural extracts and in chitosan nanoparticles, allowing the loading capacity determination, the association efficiency as well as its in vitro release. 48 49 CHAPTER 3 - High-performance liquid chromatography method validation 50 51 3. Introduction During the last decade, scientific evidences have proved that plant phenolics are an important class of defense antioxidants. These compounds are virtually widespread in all plant foods, often at high levels, and include phenols, phenolic acids, flavonoids, tannins, and lignans (6). Sage and savory are plants used in traditional medicine, and grow in the poor soils of the Mediterranean basin (14). Besides application as condiments, sage and savory have been used in folk medicine for their anti-diarrheal, digestive, wound healing, anti-inflammatory, disinfectant, anti-hypertensive and sedative properties. Some of these activities have been associated with their high contents of rosmarinic acid and the presence of other relevant phenolic compounds such as, quercetin, rutin and rosmarinic acid (9, 14). Rosmarinic acid (α-O-caffeoyl-3,4-dihydroxyphenillactic acid) is a phenolic compound that has been claimed to provide protection against cancer (7), among other biological activities, namely astringent, anti-inflammatory, antimutagen, antibacterial and antiviral (7, 8). It is also an efficient natural antioxidant (9). Quercetin (3,3’,4’,5,7pentahydroxyflavone) is a major representative of the flavonol subclass (170). In its natural sources it exists mainly in the form of glycosides and can be found in vegetables, fruits, herbs, or red wine (151, 171). It has been demonstrated a variety of quercetin biological activities and pharmacological actions, such as dilating coronary arteries, decreasing blood lipid, anti-platelet aggregation, anti-cancer, antioxidant, anti-anemia, anti-inflammation, anti-anaphylaxis and hepatoprotective effects (151). Several studies have also reported that quercetin can inhibit the proliferation of multiple cancer cell types (lung, colon, prostate, pancreatic carcinoma cells) (171-173). Accurate identification and quantification in final formulations or plant extracts is essential to guarantee the expected biological activity. The aim of this study was to validate a HPLC method for simultaneous quantification of rosmarinic acid and quercetin in natural extracts and in polymeric nanoparticles to assess the content, the association efficiency, the in vitro release and permeability profile further evaluated in the next chapters. 52 3.1. Experimental 3.1.1. Materials Two plants were selected as extract source namely sage and savory, both provided by ERVITAL (Castro Daire, Portugal), from a previous study involving 48 medicinal plants made by our research group (115). These plants had been cultivated as organic products, and were supplied in their commercial form of dried leaves: ca. 4 g was then crushed (using a coffee mill) for 1 min, so as to obtain the corresponding powder. A fraction (ca. 1 g) was contacted, under uniform stirring, with 100 mL of boiling distilled water. After the powder deposition, samples were filtered, frozen at -80 ºC and lyophilized for further procedures (Heto Holten A/S Drywinner). Then solutions (1%) of lyophilized powder were prepared in methanol for chromatographic analyses. Before injections, samples were filtered through a 0.45 µm filter. Rosmarinic acid (96.5 w/w, HPLC), quercetin dehydrate (99.0% w/w, HPLC), methanol CHROMASOLV® (HPLC ≥ 99.9%) and formic acid (HPLC ≥ 98.0%) were purchased from Sigma-Aldrich (Missouri, USA). 3.1.2. Equipment and chromatographic conditions All HPLC runs were performed using a Waters Series 600 HPLC and results were acquired and processed with Empower® Software 2002 for data acquisition (Mildford MA, USA). HPLC analysis was conducted by using a Nova-Pack® RP C18 column (250 x 4.6 mm i.d., 5 µm particle size and 125 Å pore size) from Waters. Chromatographic analysis was performed in gradient mode. The mobile phase consisted of methanol: formic acid:ultra-pure water in the ratio of 92.5:2.5:5 (v/v). Stationary phase was made with the same components in the ratio of 5:2.5:92.5, respectively. The phases were filtered through 0.22 µm filter and degassed. Eluent was pumped at a flow rate of 0.75 mL/min, the injection volume was 20 μL and detection wavelength was set to 280 nm. All experiments occurred at room temperature and the total area of peak was used to quantify the rosmarinic acid and quercetin compounds. The conditions were investigated to provide a simple procedure with the best peak resolution regarding symmetry and tailing, reduced run time and cost-effective analysis. 53 3.1.3. Preparation of standard and sample solutions Stock standard solutions of 2 mg/mL of rosmarinic acid and quercetin were prepared with methanol. The calibration curve was made from the dilution of stock solutions in methanol of seven standards: 0.05, 0.1, 0.2, 0.3, 0.5, 0.8 and 1.0 mg/mL. 3.1.4. Method validation The HPLC method was validated according to the International Conference on Harmonization (ICH) guidelines (174), using the following analytical parameters: linearity, precision, accuracy, specificity, range, robustness, detection and quantification limits. Linearity was evaluated by calculation of a regression line using least squares method. Calibration curves were obtained from seven different concentrations analyzed three times. Precision was assessed by testing the repeatability of three different standard solutions ten times (intra-day) and by intermediate precision analyzing the same three standard solutions, three times on different days (inter-day). Accuracy was tested by percentage recovery of mean of three determinations of rosmarinic acid and quercetin at three different concentrations precisely prepared and by determination of the relative standard deviation (RSD). Specificity was determined by comparing rosmarinic acid and quercetin samples under different stress conditions that may common affect natural extracts and antioxidant activity as the temperatures of (60, 20 and 1 ºC) for 24 and 72 h. The solutions were also subjected to the effect of light and air in the same period of time. Range was derived from linearity, accuracy, and precision studies. Robustness was evaluated by testing the same chromatography conditions in different HPLC equipment (Merck-Hitachi Interface D-7000). Detection limit (DL) and quantification limit (QL) were determined based on the standard deviation of the response and on the slope of the calibration curve, using the following expressions: 𝐷𝐿= 3.3𝜎 𝑆 𝑄𝐿=10𝜎 𝑆 54 where σ is the standard deviation of the response and S is the slope of the calibration curve. 3.1.5. Method applicability Applicability of the method was governed with the simultaneous determination of two antioxidant compounds with great impact on health. The determination could be made for each independent compound or combined, since the presence of both does not affect their quantification, also it could be made in natural extracts or even in the nanoparticulate systems. This HPLC method was also developed, optimized and validated for the best precise results regarding, rosmarinic acid association efficiency in chitosan-based nanoparticles, rosmarinic acid in vitro release from the aformentioned nanoparticles and it permeability profile in cell monolayers, assays developed and characterized in the next chapters. 3.2. Results and discussion 3.2.1. Application of the chromatographic method An HPLC method for the assessment of rosmarinic acid and quercetin has been proposed. Previous experiences were exploited to provide a simple procedure with the best chromatographic peak resolution, reduced run time and cost-effective analysis. All these factors contribute to the establishment of an analytical method which permits the analysis of a large series of samples. A typical chromatogram for the proposed method was depicted in Figure 3.1. The rosmarinic acid and quercetin peak retention time was 48.9 ± 0.1 min (i) and 57.9 ± 0.6 min (ii), respectively. These retention times were not completely straight, but this method may also be useful for the identification and detection of other compounds in natural extracts, which were complexes matrixes with different compounds (latest retention time compound at 90 min). However, retention factor parameter (k’) was measured in order to evaluate the chromatographic performance 55 (175). This retention factor was often used to describe the migration rate of an analyte on a column. The retention factor for analyte A was defined as: 𝐾′𝐴=𝑡𝑅−𝑡𝑀 𝑡𝑀 where retention time of the analyte (tR) and retention time of the eluent (tM) were easily obtained from the chromatogram. When the analytic retention factor was less than one, elution was so fast that accurate determination of the retention time was very difficult. High retention factors (greater than twenty) mean that elution takes a very long time. Ideally, the retention factor for an analyte was between two and ten (175). The retention factors of both compounds were 0.918 and 0.930, for rosmarinic acid and quercetin, respectively. Both compound parameters were in the optimal range (k’ < 10), which means that the migration rate of the analytes were adequate and so was the method. A good experimental design is crucial, and this seems particular important in herbal medicines in order to obtain an optimal separation, since these natural matrixes may entail hundreds of natural compounds (176). 3.2.2. Linearity Linearity was studied in the concentration range of 0.05-1.0 mg/mL by visual inspection of a calibration curve plotting (n = 21) and by calculating the regression equation and the correlation coefficient (R2) by the method of least squares. A primary stock solution was accurately prepared followed by rigorous dilution to give secondary standard solutions. Each sample was analyzed three times. A good linearity was obtained in the range of study. The calibration curve for rosmarinic acid was: 𝐴=5.44 × 107 (±2.66 × 105 ) × 𝐶− 6.21×105 (±1.43 × 105)(𝑛=21) 𝑅2=1.00 and the calibration curve for quercetin was: 𝐴=4.48 × 107 (±2.73 × 105 ) × 𝐶− 9.75×105 (±1.47 × 105)(𝑛=21) 𝑅2=1.00 where A is the peak area, C is the standard solution concentration in mg/mL, and standard deviation values for A and C are indicated in brackets. The R2 obtained was higher than 0.999 as frequently recommended (177), indicating a good linearity in the proposed range. 62 63 PART IV “Science is a way of thinking much more than it’s a body of knowledge” Carl Sagan 64 65 Abstract Introduction: Nanotechnology can be applied to deliver and protect antioxidants in order to control the OS phenomena in several chronic pathologies. Chitosan nanoparticles are biodegradable carriers that may protect antioxidants with potent biological activity such as rosmarinic acid in Salvia officinalis (sage) and Satureja montana (savory) extracts for safe and innovative therapies. Objective: Development, optimization and characterization of chitosan-based nanoparticles as stable and protective vehicle to deliver rosmarinic acid for medical applications using natural extracts as sage and savory. Methodology: Antioxidant-chitosan based nanoparticles were prepared by ionic gelation with sodium tripolyphosphate (TPP), at pH 5.8 with mass ratio of 7:1 (chitosan:TPP), with a theoretical antioxidant-chitosan loading of 40 to 50%. The size and shape of nanoparticles were then characterized by different methods such as: photon correlation spectroscopy, laser Doppler anemometry, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Chemical interactions between antioxidants and chitosan were assessed by differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR). HPLC allowed the association efficiency and in vitro released measurements. Antioxidant activity was evaluated by 2,2-azinobis (3ethylbenzothiazoline-6-sulphonic) (ABTS) and oxygen radical absorbance capacity (ORAC) methods before and after lyophilization to assure that antioxidant activity was not compromised after the dried process. Results: Small sizing nanoparticles, around 300 nm, were obtained. SEM and TEM confirmed smooth and spherical nanoparticles. No chemical interactions were found between antioxidants and chitosan, after encapsulation, by DSC and FTIR. The association efficiency was 51.2% for rosmarinic acid (with 40% loading), 96.1 and 98.2%, for sage and savory nanoparticles, respectively (both with 50% loading). The best antioxidant activity results were obtained after nanoparticles lyophilization and by ORAC method, the values for rosmarinic acid, sage and savory nanoparticles were: 3.6520 ± 0.1770, 0.4251 ± 0.0069 and 0.4526 ± 0.0087 µmol/eq Trolox, respectively. Conclusion: The extracts under study were promising vehicles for rosmarinic acid drug delivery in chitosan nanocarriers. 66 67 CHAPTER 4 - Development, optimization and physical-chemical characterization of chitosanbased nanoparticles 68 69 4. Introduction Sage and savory, are plants often used in traditional medicine to improve digestion (179), as disinfectant (180), to decrease blood pressure (7), to prevent premature ejaculation (181), to treat neuropathy (182), urinary and pulmonary infections (183), and other diseases such as Alzheimer’s disease (184) and cancer (183). Some of these biological activities have been associated with its high contents in rosmarinic acid (9, 179, 185). Rosmarinic acid, (a-O-caffeoyl-3,4-dihydroxyphenillactic acid), is a phenolic compound generally admitted as a free radical scavenger (125). Besides its huge antioxidant activity rosmarinic acid can have many beneficial functionalities like antibacterial and antiviral activity, anti-inflammatory activity (127), anti-mutagenicity character (128), capability to reduce atopic dermatitis symptoms (129), prevention of Alzheimer’s disease (130) and apoptosis induction of colorectal cancer cells (131). Nevertheless, besides the poor absorption that constrain the transport across biological barriers, most of the natural antioxidants or other active compounds are unstable and must be protected from degradation in the physiological environment (186). Thus, the efficacy of these drugs clearly depends on the design of appropriate carriers for their delivery, protection and release (1). Among the different approaches explored so far, colloidal carriers have particular interest, especially those made of mucoadhesive polymers to assure drug time retention at the absorption site (2). For this application, chitosan has become of particularly interesting for the association and delivery of labile macromolecular compounds, due to its exceptional potential for drug delivery, especially for mucosal, control drug release and protect against adverse conditions like mucosal enzymes and biological protective fluids (187). The smart symbiosis of these chitosan nanoparticles with a high potent antioxidant could be a hope for future therapies, considering the important effect of OS in several chronic pathologies. In this study chitosan nanoparticles were used, to incorporate natural extracts of sage and savory as a stable and protective vehicle to deliver rosmarinic acid for medical applications. There were no reports in the literature that have demonstrated the good performance of chitosan nanoparticles to incorporate these extracts in order to bring the huge benefits of rosmarinic acid antioxidant, as well as, other compounds that may act synergistically. 70 4.1. Experimental 4.1.1. Materials The two selected plants sage and savory were provided by ERVITAL (Castro Daire, Portugal). The plants had been cultivated as organic products, and were supplied in their commercial form of dried leaves. The dried leaves were then kept in the dark at 20 ºC. Rosmarinic acid (purity 96.5%), methanol CHROMASOLV® (HPLC ≥ 99.9%) and formic acid (HPLC ≥ 98.0%) were purchased from Sigma-Aldrich (Missouri, USA). Chitosan low molecular weight and sodium tripolyphosphate (TPP) were also purchased from SigmaAldrich (Lisbon, Portugal). The degree of deacetylation for the low molecular weight (LMW) chitosan was 85%, with a purity grade of 85%. Pure acetic acid was purchased from Pornalab (Lisbon, Portugal). Sodium hydroxide (NaOH) and hydrochloric acid (HCl) were from Merck (Darmstadt, Germany). Ultra-pure water was obtained in the laboratory using a MilliporeTM water purification equipment (Massachusetts, USA). 4.1.2. Preparation of chitosan-based nanoparticles Optimized conditions to obtain chitosan nanoparticles were based on previously studies (188), and was schematically described in Figure 4.1. The chitosan nanoparticles were obtained by inducing the gelation of a chitosan solution with TPP. Chitosan was dissolved in acetic acid aqueous solutions at various chitosan concentrations: 0.05, 0.5, 1, 2, 3 and 5% (w/v), the pH value was adjusted to 5.8 with 1M NaOH. The concentration of acetic acid was, in all cases, 1.75 higher than chitosan. Then, TPP was dissolved in purified water at 0.05, 0.1, 0.2, 0.5, 1 and 2% (w/v). For the study of the best ratio chitosan:TPP, a volume of the TPP solution of 2 was added to 5 mL of the chitosan solution under magnetic stirring at room temperature, thus achieving a final concentration of 2 mg/mL and 0.28 mg/mL of chitosan and TPP respectively (7:1). Stock solution of chitosan (1%) and TPP (0.1%) were maintained at 4 ºC for a period of 1 month. 71 Figure 4. 1. Schematic illustration of ionic gelation process of antioxidant-chitosan based nanoparticles. 4.1.3. Encapsulation of sage, savory and rosmarinic acid into chitosan-based nanoparticles The addition of 1% extracts aqueous solution and a 1% of aqueous rosmarinic acid solution was added to chitosan previously dissolved in acetic acid at a pH value adjusted to 5.8, in different volumes in order to guarantee the best concentration ratio between chitosan and the different compounds. The encapsulation of rosmarinic acid, sage and savory were tested in different theoretical loadings (5, 10, 15, 20, 30, 40 and 50%) fairly to the initial concentration of chitosan (2 mg/mL). All the tests were made for the 7 batches, considering the two plants and the antioxidant pure. Some of the final batches were then lyophilized (Heto Holten A/S Drywinner) and maintained at -20 ºC for 1-2 months for further analysis. 4.1.4. Size and surface charge Size and polydispersity (size distribution) of freshly loaded nanoparticles were determined by photon correlation spectroscopy using ZetaPALS (Brookheaven, New York, USA). A sample of 1.6 mL was gently homogenized, placed into analyzer chamber and measured. Collective 6 readings were performed three times on a sample of particles at 25 ºC with a detection angle of 90º. The zeta potential was determined by laser Doppler anemometry, Rosmarinic acid/ Sage/Savory TPP Chitosan Antioxidantchitosan based nanoparticles stirring 78 maintained after freeze dried (dehydration process). In the SEM analysis the particles after lyophilization also confirmed smooth and spherical shape with size below 500 nm, even with some aggregation due to the dry process (196). It can be therefore affirmed that rosmarinic acid and extracts encapsulation (either fresh or lyophilized), did not considerably affect particle shape and overall size as it was described above. Figure 4.3 shows the SEM images of lyophilized chitosan nanoparticles prepared by ionic gelation under the same pH conditions, for the encapsulation of rosmarinic acid in a pure form, sage and savory. The lyophilized samples even with some aggregation of the nanoparticles formed by dispersion during freeze drying guarantee the nanoscale particles with the diameters, which was in accordance with other previous studies (197). The microstructural analysis confirmed the morphology and size of the nanoparticles. Other studies reported an increase in the particle size after lyophilization with the unmodified chitosan particles (198). This was resulted from aggregation from the strong interand intra-molecular hydrogen bonding, which was not possible to breakdown even by vortex homogenization (198). This particles size increase after lyophilization process was also reported in essentials oils encapsulation (197). Figure 4. 3. SEM micrographs of lyophilized chitosan-based nanoparticles loaded: (a). commercial rosmarinic acid; (b). sage; and (c). savory. 1µm 1µm 1µm 1µm 1µm 1µm (a) (b) (c) 79 4.2.3. Association efficiency and drug loading It is known that chitosan in acidic media (pKa 6.5) can interact with the negatively charged TPP, forming interand intra-molecular cross-linkages, yielding ionically crosslinked chitosan nanoparticles (36). This is a spontaneous method for smaller nanoparticles formation with positive charge, without using any organic solvent or surfactants (198). It is also known that the interand intra-molecular linkages created between TPP and the positively charged amino groups of chitosan are responsible for the success of the gelation process (188). In the present study it was described a nanoparticulate system able to encapsulate natural extracts. The particle size was observed to be dependent on both chitosan and TPP concentrations as described in previously studies (188), being the minimum sizes obtained for the lowest chitosan and TPP concentrations. Further experiments were conducted using the mass ratio chitosan:TPP of (7:1), where TPP final concentration was 0.28 mg/mL and chitosan final concentration was 2 mg/mL. Rosmarinic acid was selected as a model antioxidant in order to investigate the feasibility of using chitosan and chitosan nanoparticles for natural extract carriers. Association efficiency and theoretical antioxidant loading of these nanoparticles were displayed in Table 4.2. The pH of the nanoparticles formation medium was between 5.8 and 6.0, a pH value that favors the interaction of rosmarinic acid and chitosan, thus reaching a maximum leading to the entrapment of high amounts of rosmarinic acid. Among the different samples considering the encapsulation of rosmarinic acid into chitosan nanoparticles, it was observed similar association efficiency around 50% (Table 4.2), for all the different loadings no significant differences were observed (P > 0.05). Higher association efficiency was found for rosmarinic acid entrapment in extracts of sage and savory, 96 and 98% respectively with no significant differences observed (P > 0.05). This was in accordance with previous reports (190).The higher association efficiency in extracts nanoparticles may be due to the different amount of rosmarinic acid in chitosan nanoparticles and inside the extracts in chitosan nanoparticles (Table 4.2). Since competitive interaction may be happen between phenolic (OH−) of rosmarinic acid and (P3O105−) groups of TPP for protonated amino groups of chitosan resulting in low levels of particle formation compared to the chitosan nanoparticles, this may be intensified with the highest amount of rosmarinic acid and phenolic groups (198). This was in accordance with other studies that encapsulate other phenolic compound, such as cathechin, in chitosan nanoparticles (198) and in other nanoparticles (199). 80 Table 4. 2. Association efficiency, theoretical loading, and final rosmarinic acid content in chitosan nanoparticles. Nano AE (%) Theoretical Loading (%) Final content in the chitosan nanoparticles (µg/mL) Final rosmarinic acid content in chitosan nanoparticles (mg/mL) Rosmarinic acid 51.2 ± 3.0 40 800 400 Sage 96.1 ± 0.2 50 1000 100 Savory 98.2 ± 0.1 50 1000 50 The association efficiency values were higher than the results for Ilex paraguariensis entrapment in calcium alginate nanoparticles coated with chitosan, (around 50%) since active compound was lost during immersion in chitosan (200). The good results for sage and savory association efficiency may be due to the huge affinity of chitosan and this two crude extracts. The results were also higher than a study reported for quercitrin encapsulation into nanoparticles, which was only 40% (152). However the high association efficiency reported in this study, was in accordance to other previously studies with the encapsulation of natural antioxidants extracted from Ilex paraguariensis into chitosan nanoparticles (193) and for the encapsulation antioxidant idebenone-loaded into chitosan nanoparticles (201). 4.2.4. In vitro rosmarinic acid release from chitosan nanoparticles All the chitosan-based nanoparticles suspension and the PBS used as medium release were adjusted to the tear normal osmolarity (300 mOsm / L). This normal osmolarity is essential to maintain cellular volume, enzymatic activity, and cellular homeostasis (202). In this in vitro release study every effort was made in order to best fate the ocular physiological conditions to test the kinetic nanoparticles behavior in ocular surface. During the experiment a fast release was observed during the in vitro release assay which was in accordance with previous data (190). The different rosmarinic acid contents (in rosmarinic acid nanoparticles, sage and savory nanoparticles) were released in all the formulations freshly and lyophilized within 60 min with no significant differences (P > 0.05). An initial burst effect was observed in the first 30 min with approximately 80% in sage nanoparticles and almost 100% in rosmarinic acid and savory nanoparticles (Figure 4.4). The results 81 seem to demonstrate that a significant amount of rosmarinic acid or extracts were initially associated with nanoparticles on their surfaces by weak linkages to chitosan, which did not have the necessary strength to entrap all the compounds. This represents that chitosan nanoparticulate system could retain the primary structure of rosmarinic acid or extracts during encapsulation. The protection release happens only for few minutes due to the polymer network, which was in accordance with other studies, that encapsulate polyphenols from Ilex paraguariensis in chitosan nanoparticles prepared also by ionic gelation and a complete release of 100% were demonstrated in the first 15 min (193). In another study for quercetin encapsulation into other nanocarriers, the release within the first 20 min was also 95% (151). Nevertheless, this results were different from other works that demonstrated a complete release of polyphenols from chitosan nanoparticles within 4 h (198). The efficient application of these nanocarriers will certainly depend of the drug purpose. Chitosan nanoparticles maintain their characteristics for this theoretical loadings, and this confers them valuable properties, such as protective and moisturizer, for the encapsulation of active agents for cosmetic or ocular applications, since a rapid released is intentional (193). However for other applications, like oral drug delivery a slower release should be optimized. Figure 4. 4. Rosmarinic acid in vitro release from rosmarinic acid, sage and savory chitosan-based nanoparticles. 0 20 40 60 80 100 120 020 40 60 80 100 120 Rosmarinic acid (%) Time (min) Rosmarinic acid nanoparticles Sage nanoparticles Savory nanoparticles 82 4.2.5. Thermal behavior by differential scanning calorimetry analysis The DSC measurements provide quantitative and qualitative information about physical and chemical changes that involve endothermic or exothermic processes, or changes in heat capacity. Endothermic and exothermic peaks correspond to transitions that absorbs or release energy, respectively. DSC was performed to understand the behavior of rosmarinic acid, sage and savory loaded and unloaded chitosan nanoparticles and the thermograms were displayed in Table 4.3. Table 4. 3. Peak temperatures in the DSC thermograms collected from chitosan, rosmarinic acid, sage and savory, physical mixtures, and nanoparticles. T (oC) Onset Peak EndSet Nano Chitosan 102 ± 0.55 122 ± 1.20 131 ± 0.12 Rosmarinic acid 112 ± 0.61 130 ± 0.73 141 ± 0.24 Sage 83 ± 0.27 108 ± 0.71 118 ± 0.93 Savory 103 ± 1.11 124 ± 0.82 137 ± 0.44 Free Rosmarinic acid 134 ± 0.13 144 ± 0.23 149 ± 0.25 Sage 140 ± 0.21 141 ± 0.14 145 ± 1.03 Savory 141 ± 0.53 154 ± 0.51 158 ± 0.81 Physical mixture Chitosan-Rosmarinic acid 90 ± 0.38 122 ± 0.83 136 ± 0.14 147 ± 0.14 150 ± 0.70 154 ± 0.13 Chitosan-Sage 94 ± 0.91 121 ± 0.10 134 ± 0.50 147 ± 0.67 151 ± 0.63 155 ± 0.84 Chitosan-Savory 93 ± 0.70 121 ± 0.32 132 ± 0.32 145 ± 0.28 151 ± 0.64 154 ± 0.24 Note: The results were given as mean of triplicate samples. The same chitosan-based nanoparticles thermal behavior was observed in all thermograms (Figure 4.5Ia, IIa and IIIa). In all chitosan curves, an endothermic peak near 70°C can be ascribed to the loss of water as previously reported (203). The endotherm of rosmarinic acid, sage and savory nanoparticles showed different shift temperatures (Figure 4.5Id. IId and IIId, respectively). This may be accounted by the hydrophilic groups 83 incorporated due to rosmarinic acid that were in different amounts inside the extracts particles. Other previously studies also reported similar shifts in DSC plots of chitosan and chitosan nanoparticles (198, 204). It could also be seen that the peaks of the complexes were shifted from those of physical mixture. Peaks of physical mixture (Figure 4.5Ic, 4.5IIc, 4.5IIIc) appeared to be combinations of each material but they were different from those of nanoparticles, probably because complexation of polyelectrolytes, in accordance with other similar works (205). Also, comparing endothermic peak of antioxidants loaded chitosan nanoparticles to the one obtained with unloaded nanoparticles, the former started at higher temperature, which was a possible evidence of the presence of antioxidants once its decomposition started at higher temperature when comparing to unloaded nanoparticles. The rosmarinic acid, sage and savory loaded sample showed the similar shift of chitosan nanoparticles, which confirms that there were no significant covalent interactions between antioxidants and chitosan after encapsulation and cross-linking (205). 84 Figure 4. 5. Thermogram of: I.(a). chitosan empty nanoparticles; (b). free rosmarinic acid; (c). rosmarinic acid and chitosan physical mixture (mixing ratio 1:1); (d). rosmarinic acid encapsulated in chitosan nanoparticles (at a theoretical 40% loading) (d). II.(a). chitosan 85 empty nanoparticles; (b). free sage; (c). sage and chitosan physical mixture (mixing ratio 1:1); (d). sage encapsulated in chitosan nanoparticles (at a theoretical 50% loading). III.(a). chitosan empty nanoparticles; (b). free savory; (c). savory and chitosan physical mixture (mixing ratio 1:1); (d). savory encapsulated in chitosan nanoparticles (at a theoretical 50% loading). 4.2.6. Spectroscopy by Fourier-transform infrared analysis Structural features, functional groups that represent backbone produce characteristic and reproducible absorptions in the spectrum, which can be analyzed by FTIR. With these series of experiments it was intended to monitorize the complexation of contrary charged polyelectrolytes at specific pH and stoichiometric relationship between the polyelectrolytes and antioxidants in nanocarriers. For this concern and to examine this relationship between components of nanoparticulate systems, preliminary concerns were taken over polyelectrolytes interactions and antioxidants entrapment. It is well established that the carboxyl group (–COO) of the anionic polymer may interact with the amino group ð-NH3 of chitosan and form an ionic complex between the two compounds (205). Rosmarinic acid displays a typical vibrational absorption bands with the main bands located between 1800 and 700 cm–1 (206). The three bands around 1605, 1520, and 1445 cm–1 were due to the presence of aromatic rings in the molecule indicating an aromatic ring stretching (206). Other evidences for phenolic groups were delivered through the bands at 1360 and 1180 cm–1 resulting from O-H and C-O stretches (206). Therefore, the band at 1684 cm–1 and the two shoulders recognized with this band result probably from the shifted bands due to the presence of carboxylic acid groups and ester group 1725-1750 cm–1 (207). Figure 4.6 (I, II and III) showed that all the above characteristic peaks appear in the spectra of combined drugs loaded chitosan nanoparticles at the same wavenumber indicating no modification or interaction between the drug and carrier. This was in accordance to previous work (208). Nevertheless, some peaks clearly decreased in intensity, after the preparation of rosmarinic acid, sage and savory nanoparticles. Particularly evident were the phenolic group bands (1360 and 1180 cm–1) in rosmarinic acid nanoparticles and this is may be due to the highest amount of rosmarinic acid in these nanoparticles, comparing to the inside content of rosmarinic acid in extracts and into the nanoparticles. This effect was also reported by some authors that developed a new 86 FTIR method for the characterization of rosmarinic acid in Lavandula officinalis cultures (206). Nevertheless it must be underline that this characteristic bands decrease observed in the nanoparticles by FTIR analysis, and the decrease in antioxidant activity, may be due to the partial retention of antioxidant before their complete release (considering the theoretical loadings under studied). This was documented for other antioxidant nanoparticles studies, such as essential oils (197). Also no new peaks appeared in nanoencapsulation spectrum, and chitosan, rosmarinic acid, sage and savory were mixed together physically without any chemical reaction (152). 87 Figure 4. 6. Spectrum of: I. (a). chitosan empty nanoparticles; (b). rosmarinic acid in a free form; (c). physical mixture between chitosan unloaded nanoparticles and rosmarinic acid (mixing ratio 1:1); (d). rosmarinic acid encapsulation into chitosan nanoparticles. II.(a). chitosan empty nanoparticles; (b). sage in a free form; (c). physical mixture between 94 where AbsABTS•+ denotes the initial absorbance of diluted ABTS•+, and Abs sample denotes the absorbance of the sample by 6 min of reaction. Triplicates of each sample were averaged to generate each datum point (which implies a total of six replicates per sample). The final result was expressed as equivalent concentration of ascorbic acid (in g/L), using a calibration curve. 5.1.5.2. Oxygen radical absorbance capacity The oxygen radical absorbance capacity (ORAC) assay was employed to evaluate the antioxidant potential of chitosan-antioxidant nanoparticles as described in previously reports (209). All reaction mixtures were prepared in duplicate, and at least three independent measures were performed for each experiment. ORAC-fluorescein (FL) values were expressed in µmol trolox equivalent per mg hydrolyzed of antioxidant, as purposed elsewhere (210). 5.1.6. Statistical analysis Statistical analysis was performed using IBM SPSS Statistics v 19.0.0 (Illinois, USA). The one-way analysis of variance (ANOVA) was used with Scheffé post hoc test comparison of groups with normal distribution, and Mann-Whitney test for groups with non-normal distribution. Differences were considered to be significant at a level of P < 0.05. 5.2. Results and discussion 5.2.1. Antioxidant activity measurement Rosmarinic acid is phenolic compound, with many beneficial functionalities and generally admitted as a free radical scavenger (125). Its high biological activity is particularly related to its two catechol moieties. Catechol is an important sub-structure for the potent antioxidant activity of phenolic antioxidants (125). The general antioxidant mechanism of 95 phenolic compounds is thought to be divided into two stages: radical catching stage and radical conclusion stage (126). For both methods the antioxidant activity was tested according to the antioxidant activity of rosmarinic acid, either in free solution or encapsulated, both in extracts or in a free form. For both methods no significant differences were found for antioxidant activity between the all nanoformulations, before and after lyophilization processes (P > 0.05), except for savory encapsulation. For these nanoparticles a decrease in the antioxidant activity was observed after lyophilization, especially evident in ORAC method. For the loading concentrations of 5 to 15%, no antioxidant activity was found by the ABTS or ORAC methods. This means that the antioxidant activity was clearly compromised for these low theoretical loadings. Considering the both extracts and rosmarinic acid nanoparticles, the highest antioxidant activity was correlated to the highest antioxidants concentrations (or higher loading value) (Table 5.1). Comparing rosmarinic acid, sage and savory in a free form, it can be easily observed that rosmarinic acid has the highest antioxidant activity, followed by sage that has the highest content of rosmarinic acid (10%) and then savory with only (5%) of rosmarinic acid content. By ABTS method, comparing the antioxidant activity before and after the encapsulation process, it was clear a decrease in the antioxidant activity after the encapsulation. This proofs that the antioxidant activity were decrease due to the partial entrapment effect of the compounds. Nonetheless the particles still demonstrated good antioxidant activity. This was in accordance to other previously reports that have showed the same good antioxidant effect of Trolox in chitosan nanoparticles (211). Other good results were obtained for the chitosan encapsulation of idebenone antioxidant (201). Furthermore and by ORAC, which is fluorimetric assay and a more sensitive one, it can be easily observed that after the encapsulation process the antioxidant activity was even lower, than the results achieved by ABTS (Table 5.1). These results were consistent with previously studies with the encapsulation of quercetin and rutin (212). This still may be due to the nanoparticles entrapment of rosmarinic acid, in other way because the antioxidant was not completely released. This partial retention means that the compounds will take more time to build up their specific activity. Nevertheless the nanosystems with this entrapment effect still have good antioxidant activity. Although even if the nanosystem antioxidant activity was lower than the unloaded compounds, it is well known that nanocarriers protects the antioxidants for degradation by biological and enzymatic fluids, increasing their bioavailability. The drug release can also be optimized to be even longer, prolonged and controlled in time considering the purpose application of this nanoparticles (187). This makes the nanoencapsulation advantageous and necessary. 96 Table 5. 1. Antioxidant activity measurements by ABTS and ORAC, considering the 50% loading (m/m) sage and savory nanoparticles; and 40% loading (m/m) of and rosmarinic acid nanoparticles for (n = 3). ABTS x ORAC y (eq [Asc. Ac.]g/L)/g extract (µmol/eq Trolox)/g extract Fresh Nanoparticles Lyophilized Nanoparticles Fresh Nanoparticles Lyophilized Nanoparticles Nano I Rosmarinic acid 0.0348 ± 0.0050 a 0.0554 ± 0.0139 a 4.8374 ± 0.1719 b 3.6520 ± 0.1770 b Sage 0.0537 ± 0.0015 c 0.0440 ± 0.0029 c 0.6227 ± 0.0901 d 0.4251 ± 0.0069 d Savory 0.0828 ± 0.0102 e 0.0378 ± 0.0015 f 1.5315 ± 0.2784 g 0.4526 ± 0.0087 h Free II Rosmarinic acid 0.0917 ± 0.0018 34.1218 ± 2.5733 Sage 0.1621 ± 0.0470 19.5924 ± 1.9791 Savory 0.1410 ± 0.0087 16.8117 ± 1.3605 Note: The results were given as mean of triplicate samples, each with three measurements. The same letters, in the same line indicate that no significant differences were observed between the fresh and freeze-dried process (P > 0.05). The values are significantly different (P > 0.05) for the antioxidant methods (x, y) and for the encapsulation process (I, II). Considering the same loading concentrations of the rosmarinic acid and the extracts nanoparticles it was clear that rosmarinic acid in the extracts nanoparticles was in lower concentration than when was purely encapsulated. Rosmarinic acid nanoparticles (40% loading), with 50% efficiency means encapsulate, 0.4 mg/mL of rosmarinic acid. Nevertheless, sage or savory nanoparticles (50% loading), with almost 100% association efficiency (Table 4.2 – chapter 4), the nanosystem encapsulate 1 mg/mL of extract, but only 10 and 5% of rosmarinic acid (Table 4.2 – chapter 4), for sage and savory, respectively. It is also known and documented by our group (179), that these crude extracts (sage and savory) are complex natural matrixes with different antioxidant content (as Protocatechuic acid, coumaric acid, gallic acid, caffeic acid, ferulic acid, naringenin, quercetin, isorhamnetin, chlorogenic acid, prunin, isoorientin, quercitrin and rutin). Nevertheless when natural extracts were encapsulated, the pH value was to allow the great amount of rosmarinic acid interaction with chitosan solution, and then the success of the encapsulation. This means that some phenolic compounds were present in the extracts, but were not negatively charged at formation medium pH, may be lost in encapsulation procedure. However, other antioxidant compounds in extract nanoparticles that have a close pKa of rosmarinic acid were encapsulated adding a synergic antioxidant 97 activity. This justifies that with lower rosmarinic acid concentrations, the extracts have the same antioxidant activity, being good vehicles of rosmarinic acid and with good antioxidant synergic performance. Furthermore it was important to underline that rosmarinic acid-nanoparticles can encapsulate with only 50% of association efficiency, which means a huge waste of the compound. This could represent that at this moment, for all the tests made for chitosan nanoparticles with sage or savory, they seem to be good vehicles for rosmarinic acid incorporation and represent a more economically process, than nanoparticles with rosmarinic acid pure. Another advantage could be added with the inclusion of biological activities of other natural compounds that were incorporated at this pH value in the nanoparticles. However, in vitro tests must be done in order to guarantee that all the biological activities of the extract and of rosmarinic acid were maintained. 5.3. Conclusion In this study, chitosan nanoparticles incorporating rosmarinic acid, sage and savory were prepared and characterized in order to ensure the highest antioxidant activity performance. The best antioxidant activity results were obtained for rosmarinic nanoparticles by ORAC method after lyophilization, ca. 3.6520 ± 0.1770 µmol/eq Trolox. Nonethless, sage and savory showed to be good vehicles for rosmarinic acid regarding the antioxidant activity outline. After nanoparticles lyophilization the obtained values were: 0.4251 ± 0.0069 and 0.4526 ± 0.0087 µmol/eq Trolox, for both sage and savory nanoparticles, respectively. Nevertheless, it is important to underline that the crude extracts have a lower concentration of rosmarinic acid, and the good antioxidant activity results suggested that other synergic compounds may be also encapsulated. Besides a lower antioxidant activity was observed in the nanoparticles comparing to the free compounds due to the partial entrapment effect of the compounds, chitosan-based nanoparticles still maintained the rosmarinic acid good antioxidant activity performance and also allowing its protection and best control its profile release. These results underline the pharmaceutical potential of chitosan-based nanosystems for antioxidants delivery. 98 99 PART V “Science isn’t about why…it’s about why not?” Unknown 100 101 Abstract Introduction: In vitro assays are crucial to mimic biological conditions and predict nanoparticles cytotoxicity, mucoadhesion and the encapsulated bioactive permeability profiles. Neuroinflammation is a biological condition intimate related to the glaucoma pathophysiology, increasing the reactivity of microglia and the release of pro-inflammatory mediators. Objective: The goal of the study was to test the safety performance, mucoadhesiveness and permeability of rosmarinic acid encapsulated into the aforementioned nanoparticles towards ocular cell-based models, and predict their potential to prevent oxidative eye diseases. Nonetheless, it was also intend to evaluate the effect of rosmarinic acid in retinal pro-inflammatory control and infer if it may confer neuroprotection to the retina in an animal model of I-R. Methodology: Chitosan nanoparticles were evaluated considering mucoadhesiveness and their safety performance and cell permeability by in vitro tests using ARPE-19 and HCE-T monolayer cell lines. In vivo assays were performed injecting intravitreally rosmarinic acid in an I-R model, the rosmarinic acid neuroprotection was further evaluated by electroretinograms (ERG) and immunohistochemistry. Results: Nanoparticles previously characterized demonstrated to be safe without relevant cytotoxicity against ARPE-19 and HCE-T cell lines, with no irritancy to the eye. The permeability study in HCE monolayer cell line showed an apparent permeability coefficient Papp of 3.41 ± 0.99 x 10-5 and 3.24 ± 0.79 x 10-5 cm/s for rosmarinic acid loaded chitosan nanoparticles and free in solution, respectively. In ARPE-19 monolayer cell line the Papp were 3.39 ± 0.18 x 10-5 and 3.60 ± 0.05 x 10-5 cm/s for rosmarinic acid loaded chitosan nanoparticles and free in solution, respectively. Considering the mucin particle method, nanoparticles indicate mucoadhesive proprieties. ERG and immunohistochemistry showed that at this concentration, by intravitreal injection, rosmarinic acid did not have a retina protective effect in I-R studied model. 102 Conclusion: The natural nanoparticles developed in this study demonstrated to be in vitro promising drug delivery systems for ocular application. Nonetheless, the in vivo results were not effective regarding retina neuroprotection, which may be due to the acute I-R model, which leads to severe pro-inflammatory damages that antioxidants are not able to revert with a single injection. Despite, this antioxidants pharmacotherapy may be crucial to ocular diseases prophylaxis. 103 CHAPTER 6 - In vitro evaluation of cytotoxicity, mucoadhesion and ocular permeability of rosmarinic acid into chitosan-based nanoparticles 110 6.1.6.1. Cytotoxicity test using chorioallantoic membrane To evaluate the cytotoxicity and biocompatibility of extracts and rosmarinic acid-containing nanoparticles, Hen's Egg Tests (HETs) were performed on the chorioallantoic membrane (CAM) as previously described (227). HET-CAM test method was used for the detection of ocular corrosives and severe irritants, as defined by the U.S. Environmental Protection Agency (EPA 1996), the European Union (EU; EU 2001), and in the United Nations Globally Harmonized System (GHS) of Classification and Labelling of Chemicals (UN 2003). Fertile hen’s eggs at 10 days of incubation at 37 °C, obtained from Guaraves Guarabira Aves Ltda, were used in the tests. Five eggs were used for each nanoparticles solution assay. After 10 days of incubation, the egg shell above the air space was removed. The exposed membrane was moistened with a drop of 0.9% physiological saline and the saline was removed, uncovering the chick embryo chorioallantoic membrane (CAM). An aliquot of 200 μL of nanoparticles solution was applied on the CAM. All assays were repeated five times. Signs of vasoconstriction, hemorrhage and coagulation for 5 min were observed evaluate the potential for irritation according to the method of HET-CAM. The time (in seconds) at which the indicated processes began were applied in Equation (228). The time (in seconds) at which the indicated processes began were applied in Equation (228): 𝐻𝑒𝑡− 𝐶𝑎𝑚 (𝐼𝑆)=(301−ℎ)×5 300 + (301−𝑣)×7 300 + (301−𝑐)×9 300 After application of the formula above, it was possible to quantify the observed potential for irritation (irritation score-IS) and to obtain means and standard deviations for the analysis as follows: 0-0.9 no irritation, 1-4.9 slight irritation, 5-8.9 moderate irritation and 921 severe irritation (228). All procedures with chicken eggs were followed by the regulations and procedures for handling of human or animal materials. 111 6.1.7. Permeability studies 6.1.7.1. Cell monolayers culture Immortalization of human corneal epithelium (HCE) and retinal pigment epithelium (ARPE) cells have been described earlier. Polycarbonate Transwell® cell culture filters (Corning, 3 μm, 6 wells, USA) were used for permeability assays. Suspension of HCE and ARPE cells were seeded onto the filters at a concentration of 200.000 cells/cm2. The cells were grown at 37 °C in humidified air with 5% of CO2, in standard culture medium in apical chamber for 21 to 30 days until the cells were confluent. The culture DMEM medium was replaced every two days. 6.1.7.2. Transepithelial electrical resistance Transepithelial electrical resistance (TEER) was measured at different phases of cell growth (Evom; World Precision Instruments, Sarasota, FL), as an indicator of epithelial differentiation and epithelial tightness. TEER data were corrected for low-background TEER by using a blank filter containing the possible coating materials and culture medium. At the end of each permeability experiment, TEER was measured to detect the condition of the cells. 6.1.7.3. Permeation studies in cell monolayers The permeation study with different solutions was initiated by washing with HBSS (1x) liquid (without calcium and magnesium) both the basolateral and apical side one time and then adding 2.5 mL of HBSS to the basolateral side (receiver side) and 1.5 mL of HBSS to the apical side (donor side). At different time points, during 60 minutes, aliquots of 100 µL were withdrawn from the receiver chamber and replaced with an equal volume of blank medium. TEER was measure at each time and the plates were incubated with the sample 112 at 37 ºC to sum to the test times. Papp was calculated from the measurement of the flow rate of insulin from the donor to the acceptor chambers: P𝑎𝑝𝑝 (𝑐𝑚 𝑠)= 𝑑𝑄 𝑑𝑡 ⁄ (𝐴 × 𝐶0) ⁄ where, dQ is the total amount of permeated rosmarinic acid (mg), A is the diffusion area (cm2), C0 is the initial concentration of rosmarinic acid (mg/mL), and dt is the time of experiment in seconds (s). The coefficient dQ/dt represents the steady-state flux of rosmarinic acid across the monolayer. 6.1.7.4. High performance liquid chromatography analysis The HPLC method was developed and validated, as previously described in chapter 3. Briefly, aliquots of 100 µL were injected at HPLC to quantify which quantity concentration permeates to the basolateral side of the plates. All HPLC runs were performed using a Waters Series 600 HPLC and results were acquired and processed with Empower® Software 2002 for data acquisition (Mildford MA, USA). HPLC analysis was conducted by using a Nova-Pack® RP C18 column (250 x 4.6 mm i.d., 5 µm particle size and 125 Å pore size) from Waters. Chromatographic analysis was performed in gradient mode. The mobile phase consisted of methanol: formic acid: water UP in the ratio 92.5:2.5:5 (v/v). Stationary phase was made with the same components in the ratio of 5:2.5:92.5, respectively. The phases were filtrated through 0.22 µm filter and degassed. Eluent was pumped at a flow rate of 0.75 mL/min, the injection volume was 20 μL and detection wavelength was 280 nm. 6.1.8. Statistical analysis Statistical analysis was performed using IBM SPSS Statistics v 19.0.0 (Illinois, USA). The one-way analysis of variance (ANOVA) was used with Scheffé post hoc test comparison of groups with normal distribution, and Mann-Whitney test for groups with non-normal distribution. Differences were considered to be significant at a level of P < 0.05. 113 6.2. Results and discussion 6.2.1. Particle size, polydispersity and zeta potential The particle size and mean size distribution are fundamental features that influence the in vivo distribution, biological fate, toxicity and the targeting ability of nanoparticles containing therapeutic drugs (191). It is also known that the highest value of zeta potential represents the greater electrostatic repulsive interactions among the particles. Zeta potential values of ± 30 mV indicates that the colloidal systems are stable in time and that amine groups of chitosan are on the surface (193). The developed nanoparticles ranged from 200-300 nm in size and zeta potential were around 20-30 mV (Table 6.1). It is described that for ultrafine particles the induction of ROS, OS, inflammation and vasculature are a risk (229). In this sense it can be considered that the particles of this study will have no such harmful effects. Moreover, the results are in agreement with similar nanoparticles containing rosmarinic acid we recently developed (230) and with other extracts, also encapsulated in chitosan nanoparticles (193). The results did not show significant differences (P > 0.05) in size between the rosmarinic acid and the two crude extracts, as previously described (190, 196). This may be due to the high rosmarinic acid content in extracts composition 10 and 5% in sage and savory, respectively (196). Thus the extracts under study are promising vehicles for rosmarinic acid nanoincorporation, as previously documented (190, 196). The size obtained is also in agreement with ocular drug delivery demands, as particles with size ≤ 200 nm were observed to reach the retina, vitreous and trabecular meshwork (231) and have more vitreal half-life compared to huge nanoparticles (232). The obtained nanoparticles also showed values of polydispersity between 0.1 and 0.2 corresponding to a narrow distribution and monodispersed particles. These results are in accordance to other works using similar antioxidant nanoparticles (194). 114 Table 6. 1. Average hydrodynamic diameter (Z), polydispersity index (PdI) and zeta potential of chitosan nanoparticles loaded rosmarinic acid, sage and savory. Nano Z-average (nm) PdI Zeta potential (mV) Association efficiency (%) Loading capacity (%) RA 280.0 ± 16.0 a 0.201 ± 0.091 b 30.1 ± 1.8 c 60.2 ± 1.3 d 5.3 ± 0.4 f Sage 302.4 ± 18.2 a 0.288 ± 0.074 b 27.5 ± 0.9 c 96.8 ± 0.2 e 8.1 ± 0.6 g Savory 298.3 ± 20.8 a 0.214 ± 0.085 b 28.2 ± 2.2 c 98.0 ± 0.3 e 7.8 ± 0.2 g Note: Values were means of triplicate samples ± standard deviation; a,b,c,d,e,f,g means within the same column, labelled with the same letter, were not statistically different from each other (P > 0.05). 6.2.2. Association efficiency and loading capacity Chitosan can interact with the negatively charged TPP, forming interand intra-molecular cross-linkages, yielding ionically cross-linked chitosan nanoparticles in acidic media (pKa 6.5) (36). It is also known that the interand intra-molecular linkages created between TPP and the positively charged amine groups of chitosan were responsible for the success of the gelation process (188). Among the different sources of rosmarinic acid encapsulated into chitosan nanoparticles, it was observed different loading capacity between free drug and extracts (Table 6.1). This may be correlated to the lower initial amount in rosmarinic acid used to prepare nanocomplexes, as previously described for insulin encapsulation into chitosan-alginate ionotropic nanoparticles (233). The results were in line with previous data for ionic gelation chitosan nanoparticles (234, 235). Higher association efficiency was found for rosmarinic acid entrapment in extracts of sage and savory (with no significant differences observed between them (P > 0.05), than in pure rosmarinic acid entrapment in chitosan nanoparticles. The results were on the row with other chitosan idebenone and ilex paraguensis encapsulation (193, 201). Other studies showed similar association efficiency for other phenolic compound, such as cathechin (198) and epigallocatechin gallate (199). The results also exhibited higher association efficiency than the obtained for quercetin encapsulation in PLA nanoparticles (i.e. 40%) (152). Nevertheless, higher association efficiency for rosmarinic acid nanoparticles were previous described in solid-lipid-nanoparticles (230). Nevertheless, higher association efficiency for rosmarinic acid nanoparticles were previous described in solid-lipid- 115 nanoparticles (230). Therefore, the association efficiency was highly dependent on the nanosystem used, as well as from the loading and rosmarinic acid amount utilized into the nanoparticles. 6.2.3. Mucoadhesion proprieties evaluation by mucin interaction method Ocular mucosa may affect the stability chitosan nanoparticles in the presence of mucus components (2). Mucus consists in a heterogeneous tridimensional network, being basically composed of a mucin fibers network, creating an endless system of canals in which particles can diffuse and/or be retained (236, 237). Chitosan is a mucoadhesive polymer that may increase residence time and intimate contact of the delivery vehicle with the mucosa, consequently increasing the drug bioavailability, such proprieties were well documented in literature (168). The chitosan adhesion mechanism is mainly associated to the electrostatic interactions established between protonated amine groups of mucoadhesive chitosan and negatively charged groups of mucin (238). In this study, the nanoparticle-mucin interaction was determined measuring the amount of mucin that attaches the nanoparticles (239). The degree of adsorption of nanoparticles/mucin particles can be determined by the variations in size (240), zeta potential (241) or electrophoretic mobility (242) of formed complexes with mucosal fluids, in particular with mucin. Size can influence the diffusion of drug carriers through the mucin mesh that composes mucus fluids, being the optimal range between 200 and 500 nm to enhance diffusion (243). Higher or smaller diameters may decrease transport through the mucus layer. This is in contrast to the prevailing belief, demonstrate that large nanoparticles, can rapidly penetrate physiological human mucus, and that large nanoparticles can be used for mucosal drug delivery (243). For this purpose, rosmarinic acid containing nanoparticles were evaluated, since chitosan was the only responsible for the mucoadhesion results and rosmarinic acid was the principle active of all the formulations used. As depicted in Table 6.2, the rosmarinic acid nanoparticles obtained in this study were in optimal range mentioned, for the three pHs used. The influence of pH values was intended to demonstrate that the particles were mucoadhesive in different conditions, at the normal ionic gelation pH (5.8), at an inflammation pH (5.0) and in homeostasis pH (7.4). The increased size after mucin incubation suggests that nanoparticles-mucin interaction were forming microaggregates, and decrease in zeta potential values was probably due to the electrostatic interaction between positive charged of chitosan and anionic mucin (241). 116 Strong ability of those nanoparticles to interact with mucin through electrostatic forces were observed, highlighting their potential as mucoadhesive carriers (226). Other study demonstrated the effect of low, medium and high molar mass chitosan in coated polycaprolactone (PCL) nanoparticles (238). The results were according to mucoadhesive nanoparticles made of thiolated quaternary chitosan crosslinked with hyaluronan (244). Table 6. 2. Average hydrodynamic diameter (Z), polydispersity index (PdI) and zeta potential of chitosan nanoparticles loaded rosmarinic acid before and after mucin interaction (n = 3). Nanoparticles pH Z-average (nm) PdI Zeta potential (mV) Rosmarinic acid 5.8 236.0 ± 7.1a 0.719 ± 0.036 40.1 ± 0.8 c Rosmarinic acid + Mucin 5.0 488.2 ± 30.5 b 0.619 ± 0.049 22.5 ± 0.9 d Rosmarinic acid + Mucin 7.4 414.1 ± 32.3 b 0.511 ± 0.014 23.1 ± 0.6 d Note: Values were means of triplicate samples ± standard deviation; a, b, c, d means within the same column, labelled with the same letter, were not statistically different from each other (P > 0.05). 6.2.4. Cell viability studies In order to evaluate any potential cytotoxicity of rosmarinic acid, sage and savory on ARPE-19 and HCE-T cell lines, pure rosmarinic acid and extracts-loaded chitosan nanoparticles were tested for 4 and 24 h. The effect of nanoparticles on membrane integrity was measured by the LDH enzyme release assay and the effect on cell viability was measured using the MTT conversion assay. In vitro cytotoxicity results were presented in (Figure 6.1). Results showed that after 4 h the cytotoxicity was below 10% for the tested concentration range, for both cells lines. Moreover, there were no significant differences (P > 0.05) between the 4 and 24 h (testing time) considering all formulations and different cell lines. 117 Note: NR, R - rosmarinic acid loaded and unloaded chitosan nanoparticles, respectively; NSL, SL – Salvia officinalis loaded and unloaded chitosan nanoparticles, respectively; NSG, SG – Satureja montana loaded and unloaded chitosan nanoparticles, respectively. Figure 6. 1. Effect of rosmarinic acid, sage and savory-loaded chitosan nanoparticles on cell cytotoxicity of ARPE (A, C and E) and HCE (B, D and F) cell lines after 4 h (black bar) and 24 h (white bar) of incubation. DMEM+cells and DMSO were used as controls. The formulation concentration used was displayed in the tables, relatively to rosmarinic acid (A, B), sage (C, D) and savory (E, F) (results were the mean of 6 replicates, bars represent standard deviation). 118 Results of cell viability for rosmarinic acid, sage and savory loaded chitosan nanoparticles obtained from the MTT test were shown in Figure 6.2. These were in a good correlation with those from the LDH assay. Antioxidant/chitosan nanoparticles presented a good profile in terms of cell viability of ARPE-19 and HCE-T cells. The results showed that these nanoparticles were not toxic for the cells at concentrations below 1 mg/mL. The results were in line with chitosan cell safety performance regarding its use as drug delivery systems and data show that it was completely safe with no-toxicity effect upon cell lines since chitosan can interact with cell membranes and be uptake with no cytotoxicity and cell collateral damages. The results were in line with previous studies on the use of chitosan in drug delivery systems, (40) regarding its non-toxicity, good cell membrane interaction and cellular uptake with no collateral damages. Moreover, other studies demonstrated that chitosan nanoparticles besides being non-toxic, may have protective effects on cell lines (245-247). 119 Note: NR, R - rosmarinic acid loaded and unloaded chitosan nanoparticles, respectively; NSL, SL – Salvia officinalis loaded and unloaded chitosan nanoparticles, respectively; NSG, SG – Satureja montana loaded and unloaded chitosan nanoparticles, respectively. Figure 6. 2. Effect of rosmarinic acid, sage and savory-loaded chitosan nanoparticles on viability of ARPE (A, C and E) and HCE (B, D and F) cell lines after 4 h (black bar) and 24 h (white bar) of incubation. DMEM+cells and DMSO were used as controls. The formulation concentration used was displayed in the tables, relatively to rosmarinic acid