Nuevos nanosistemas a base de protamina para la liberación de antígenos

UNIVERSIDAD DE SANTIAGO DE COMPOSTELA FACULTAD DE FARMACIA DEPARTAMENTO DE FARMACIA Y TECNOLOGÍA FARMACÉUTICA Tesis Doctoral NUEVOS NANOSISTEMAS A BASE DE PROTAMINA PARA LA LIBERACIÓN DE ANTÍGENOS José Vicente González Aramundiz Santiago de Compostela, 2013 DOÑA MARÍA JOSÉ ALONSO FERNÁNDEZ Y DOÑA NOÉMI STEFÁNIA CSABA, CATEDRÁTICA E INVESTIGADORA ISIDRO PARGA PONDAL, RESPECTIVAMENTE, DEL DEPARTAMENTO DE FARMACIA Y TECNOLOGÍA FARMACÉUTICA DE LA UNIVERSIDAD DE SANTIAGO DE COMPOSTELA. INFORMAN: Que la presente Memoria Experimental titulada: “Nuevos nanosistemas a base de protamina para la liberación de antígenos”, elaborada por José Vicente González Aramundiz, ha sido realizada bajo su dirección en el Departamento de Farmacia y Tecnología Farmacéutica y, hallándose concluida, autorizan su presentación a fin de que pueda ser juzgada por el tribunal correspondiente. Y para que conste, expiden y firman el presente certificado en Santiago de Compostela, el 25 de Noviembre de 2013. Fdo. María José Alonso Fdo. Noémi Csaba TABLA DE CONTENIDOS Tabla de contenidos ............................................................................................. 5 Resumen/ Abstract ............................................................................................... 7 Capítulo 1: Introducción ..................................................................................... 11 Capítulo 2: Nanovaccines: Nanocarriers for antigen delivery ........................... 31 Antecedentes, hipótesis y objetivos................................................................... 67 Capítulo 3: Protamine:polysaccharide nanoparticles as antigen delivery systems ............................................................................................................... 75 Capítulo 4: Protamine nanocapsules: a new platform for antigen delivery .... 111 Capítulo 5: Novel thermostable nanovaccines antigen-associated protamine nanocapsules .................................................................................................. 143 Discusión general ............................................................................................. 183 Conclusiones/conclusions ................................................................................ 209 Anexos .............................................................................................................. 215 RESUMEN/ABSTRACT RESUMEN En la presente tesis doctoral se plantea el diseño, desarrollo y optimización de sistemas coloidales nanométricos a base de protamina para la vehiculización y liberación de antígenos. En particular los sistemas desarrollados se dividen en (i) nanopartículas, sistemas de tipo matricial que se compone de un polisacárido como ácido hialurónico o alginato entrecruzado con protamina y (ii) nanocápsulas, sistemas de tipo reservorio, constituidas por un núcleo líquido oleoso (Miglyol®, escualeno o α tocoferol) y una cubierta de protamina. Dependiendo del tipo de estructura, se han asociado distintos antígenos modelo como son el antígeno recombinante de la hepatitis B y el antígeno de influenza H1N1. Los sistemas presentan una adecuada estabilidad en suspensión y en polvo seco a temperatura ambiente. Estudios in vitro han demostrado que ambos tipos de sistemas son eficazmente internalizados por células inmunocompetentes y estimulan la secreción de diversos tipos de citocinas. Estudios in vivo han demostrado la efectiva acción de los distintos nanosistemas con el antígeno asociado generando una respuesta inmune adecuada y prolongada. Por otra parte, se ha evaluado la posibilidad de que los diferentes sistemas sean formulaciones administradas libres de agujas, a través de la vía nasal, obteniendo respuestas inmunes protectoras frente al antígeno asociado y pudiendo modular el tipo de respuesta obtenida (humoral o celular), dependiendo de la vía de administración. Todos estos resultados ponen de manifiesto el potencial que poseen las estructuras desarrolladas a base de protamina para la vehiculización y liberación de antígenos, consiguiendo avanzar en el desarrollo de nuevos adyuvantes así como en la termo-estabilidad de las diversas formulaciones. 16 INTRODUCCIÓN El complejo de antígeno - MHC I o II presente en las membranas de las APC, son reconocidos por los receptores de células T (TCR). El complejo antígeno - MHC I es reconocido por las células de tipo T CD8+ o citotóxicas, generando una respuesta celular y diversas citoquinas como interferón gama (INF-γ) y factor de necrosis tumoral alfa (TNF-α). Por otra parte, el reconocimiento de MHC II es a través de los receptores de células T CD4+ o colaboradoras (T helper), las cuales al ser activado puede diferenciarse en células de tipo Th1 o Th2. El tipo de diferenciación se caracteriza por el tipo de citoquinas que producen, por ejemplo Th1 produce INF-γ e interleucina 2 (IL-2), mientras que Th2 secreta interleucinas 4, 5 y 10. Una respuesta Th1 permite una inmunidad celular, en cambio una respuesta Th2, permite la activación de linfocitos B, lo que genera una respuesta humoral mediada a través de anticuerpos. Los anticuerpos también conocidos como inmunoglobulinas, pueden ser de cinco clases: M (IgM), A (IgA), G (IgG), D (IgD) y E (IgE). Todas ellas tienen la propiedad de unirse específicamente al anticuerpo que indujo su formación y favorecer su destrucción. Como se puede apreciar, todo el largo proceso de generación de la inmunidad, se inicia por el reconocimiento del antígeno por una APC, por ello es indispensable que el antígeno sea reconocido por el sistema inmune. Lamentablemente, los nuevos antígenos, aunque son más seguros, no son fácilmente captados por el sistema inmune por su falta de inmunogenicidad. Esto ha obligado al estudio y al uso de un adyuvante y/o un sistema de liberación que sea capaz de favorecer el reconocimiento del antígeno y la activación y maduración de las APC. Adyuvantes y Vacunas Como mencionamos previamente, el uso de antígenos más seguros trae consigo la necesidad de emplear un adyuvante en la administración de vacunas. 17 INTRODUCCIÓN En términos generales un adyuvante es un compuesto que puede incrementar y/o modular la inmunogenicidad intrínseca de un antígeno [8]. Los únicos adyuvantes universalmente aprobados para su uso en humanos son las sales insolubles de aluminio, más conocidas como álum [9]. Su uso en la precipitación de proteínas data de 1926 por Glenny y col. Aunque el efecto adyuvante de estas sales fue descrito en la década de los 70s, hasta el día de hoy se estudia su mecanismo de acción exacto [10, 11]. El álum ha presentado una buena acción por vía parenteral sin embargo, posee algunos inconvenientes entre los que destacan: (i) reacciones de hipersensibilidad, hinchazón y/o eritemas tras ser administrado (ii) inestabilidad tras la congelación, debido a la destrucción de la estructura tipo gel que forma, disminuyendo o dejando inactiva la vacuna (iii) es un adyuvante que por otras vías distintas a la parenteral no presenta actividad adyuvante y por último (iv) su perfil de respuesta inmune está asociado principalmente a una respuesta humoral, relacionada con la producción de linfocitos Th2. Por ello, los esfuerzos de investigación se han concentrado en la búsqueda de nuevos adyuvantes que sean capaces de mejorar aquellos aspectos en los cuales el álum falla. Algunos de estos nuevos adyuvantes se resumen en la Tabla 1, en donde destacan el MF 59TM, ASO4TM y ASO3TM aprobados por la Agencia Europea de Medicamentos (EMA) para su uso en humanos. La nanomedicina y la búsqueda de nuevos adyuvantes La nanomedicina es un área de la nanotecnología que abarca diferentes aplicaciones de la biotecnología medicina, química, tecnología farmacéutica, entre otras en la prevención, tratamiento, monitorización y diagnóstico de enfermedades utilizando materiales y estructuras de escala nanométrica (1- 1000 nm) [12, 13]. Si bien, es en cáncer donde se han utilizado los mayores recursos y se pueden observar los mayores avances, otras áreas como la 18 INTRODUCCIÓN inmunización están siendo un campo de la nanomedicina que está en pleno auge. Tabla 1: Diversos adyuvantes utilizados en la administración de antígenos. MPL: monofosforil lípido A, QS21: saponina purificada de quillaja A, GSK: GlaxoSmithKline. Nombre Compañía Componentes Enfermedad Ref. Álum Varias (AlPO 4) ó (Al(OH) 3 ) Diversas: DTP (difteria, pertussis y tétanos), Neumococo, Hepatitis B, etc. [9] MF 59 Novartis Emulsión O/W de: escualeno, tween® 80 y sorbitan triolato) Influenza [14] ASO1 GSK Liposomas con MPL y QS21 Malaria [15] ASO2 Emulsión O/W de: MPL y QS21 Malaria [16] ASO3 Emulsión O/W de: escualeno, α tocoferol y tween® 80 Influenza [17] ASO4 MPL y álum Hepatitis B [18] AFO3 Sanofi - Pasteur Emulsión O/W de: escualeno, oleato de sorbitan, polioxietileno cetoestearil y manitol Influenza [19] En términos generales, la nanomedicina en inmunización se ha enfocado en el diseño y elaboración de sistemas de liberación de antígenos. Se pretende que estas estructuras de tamaño nanométrico puedan mejorar la inmunogenicidad 19 INTRODUCCIÓN del antígeno asociado, permitir una administración libre de agujas, favorecer la obtención de una formulación termoestable, ayudando con ello a una mejora consistente en la cobertura inmunológica [20, 21]. Ya desde la década de los 70s el profesor Peter Speiser, utilizó nanopartículas de poliacrilamida para la liberación controlada de vacunas [22]. Este hallazgo abrió un nuevo campo de investigación, donde diferentes tipos de estructuras y con diversos tipos de materiales comenzaron a diseñarse y desarrollarse, con una asociación eficaz de antígenos, para su aplicación en vacunas [23]. Diferentes sistemas nanoparticulados han sido estudiado para su aplicación en vacunas, entre ellos se pueden encontrar vectores virales, virosomas, liposomas, complejos inmunoestimulantes, nanopartículas de calcio, nanopartículas orgánicas e inorgánicas no biodegradables (ej: oro, látex, sílica, poliéster, etc) y nanopartículas poliméricas biodegradables, entre otras. El uso de nanosistemas para la liberación de antígenos es una interesante estrategia para el desarrollo de vacunas. Entre sus ventajas se encuentra el proteger al antígeno de su degradación y el poder controlar su liberación. Esta última característica es la que más destaca en el uso de nanosistemas en vacunas, ya que la manera eficiente de liberar los antígenos a las APC, especialmente a las células dendríticas, además de promover la internalización del antígeno y la posterior maduración de la APC determinará el desarrollo de una respuesta inmune protectora [24]. Otra ventaja es la posibilidad de coencapsular múltiples epítopes de antígenos en un solo vehículo [25], abriendo la posibilidad de vacunas multivalentes. Los diversos sistemas de liberación de antígenos de tamaño nanométrico pueden ser internalizados por las APC de diversas maneras: a través de receptores (de forma selectiva), fagocitosis, macropinocitosis y endocitosis (ya sea mediado por clatrina, caveolina o independiente de ambos) [26]. El que un 20 INTRODUCCIÓN nanosistema se internalice de una u otra manera dependerá principalmente de su tamaño, forma, propiedades y ligandos que tenga en la superficie [27]. En los últimos años, el estudio de los receptores presentes en las APCs y las diferentes vías de reconocimiento e internalización de sistemas y patógenos ha revolucionado la forma de diseñar vacunas. Diferentes receptores que reconocen señales de peligro (PRRs, del inglés “pattern-recognition receptors”), han sido identificados en donde destacan: TLR (del inglés: Toll Like Receptor), tanto de superficie como endosomal, NLR (del inglés: Nucleotide-binding oligomerization domain Receptors), CLR (del inglés C-type lectin receptor) RLH (del inglés RIG-like helicases) y Scavenger [28]. De todos estos, los que han tenido mayor impacto han sido los TLR. Diferentes moléculas han sido reconocidas como agonistas de estos receptores con interés tanto para el diseño de vacunas (principalmente como adyuvante), como para diferentes enfermedades infecciosas, cáncer, alergias y enfermedades autoinmune entre otras [29, 30]. Con estos nuevos hallazgos el uso de nanopartículas como sistemas de liberación de antígenos no podía quedar indiferente. Es por ello que se han utilizado diferentes materiales, que sean capaces de imitar señales de peligro para el sistema inmune, muchos de ellos agonistas de los receptores anteriormente mencionados y que sean capaces de modular la respuesta inmune [31]. Dentro de éstos, los biomateriales se presentan como buenos candidatos para cumplir estas funciones ya que pueden liberar el antígeno selectivamente a las células dendríticas, facilitando su presentación y por medio de ello, las células dendríticas son capaces de activarse, madurar y desencadenar una respuesta inmune eficiente [32]. Dentro de los biomateriales, los polímeros han sido ampliamente estudiados para la elaboración de nanosistemas en el área de inmunización. Las 21 INTRODUCCIÓN nanopartículas poliméricas han tenido una gran relevancia con el precedente de las micropartículas de PLGA (ácido poli-láctico-glicólico). Este material biodegradable (aprobado por la FDA en 1969 para su uso clínico) en la década de los 80s fue recomendado por la organización mundial de la salud (OMS) para su uso en vacunas monodosis y así mejorar la cobertura de inmunización [33]. La OMS sugirió como antígeno el toxoide tetánico por la elevada incidencia del tétanus neonatal producto de la escasa inmunización de las mujeres en países del tercer mundo. Es así como a partir de esta evidencia, este material en conjunto con el ácido poli-láctico (PLA), han sido dos de los polímeros más estudiado para su uso en sistemas de liberación de vacunas. Otro de los polímeros ampliamente estudiado en el ámbito de vehiculización de antígenos y también de fármacos es el quitosano. Este material, obtenido tras la desacetilación de la quitina, ha demostrado que por su alta capacidad de formar estructuras nanométricas y fácil manejo, es un material ideal para la vehiculización de macromoléculas [34]. Por otra parte, nanoestructuras de quitosano, han sido capaces de asociar eficientemente macromoléculas y antígenos y poder vehiculizarlos por vías mucosas [35]. Con la previa experiencia de partículas en quitosano cargadas con el toxoide tetánico [35, 36], recientemente nuestro grupo de investigación ha publicado el desarrollo de sistemas a base de quitosano estructurado en forma de nanopartículas (nanoesferas) [37] o nanocápsulas [38], en las que se ha demostrado su efectividad para transportar el antígeno recombinante de superficie de la Hepatitis B (rHBsAg) por vía nasal o a través de una sola dosis por vía parenteral, logrando inmunidad frente a esta enfermedad. Otros biomateriales que han comenzado a ser ampliamente estudiado en el campo de la vehiculización de fármacos y antígenos son los polipéptidos y poliaminoácidos. Si bien su uso es menos extendido que los polímeros, estudios recientes han demostrado su potencial como material para formar diferentes 22 INTRODUCCIÓN micro- y nanosistemas. Una interesante revisión de este tema se encuentra recogido en los anexos de la presente memoria. Polipéptidos y poliaminoácidos y su uso en liberación de antígenos Los polipéptidos (PP) y poliaminoácidos (PAA) son moléculas formadas por monómeros de aminoácidos a través de enlaces amidas siendo generalmente biocompatibles y no tóxicos; lo que los hacen un interesante material para su uso en liberación de fármacos y antígenos [39]. Uno de los poliaminoácidos utilizados en el desarrollo de nanopartículas para la liberación de antígeno es el ácido poli-glutámico. Este biomaterial ha demostrado que es capaz de producir una mayor liberación a las APC de la proteína vehiculizada, ayudando a activar la maduración de las células dendríticas [40]. Otro de los poliaminoácidos ampliamente estudiado en el desarrollo de nanosistemas es la polilisina. Este material, por su carga positiva a pH neutro, ha sido empleado especialmente como agente complejante en la vehiculización de material genético. En el área de inmunización se ha demostrado que nanopartículas recubiertas de este material actuarían como adyuvante en vacunas ADN [41]. Un grupo especial de péptidos, que es necesario mencionar son los CPP (del inglés: Cell-penetrating peptides). De manera general, son péptidos de no más de 30 aminoácidos, anfipáticos y poseen una carga neta positiva [42]. Tiene la propiedad de penetrar las membranas celulares y translocar diferentes moléculas dentro de las células de forma independiente de receptores y energía [43]. Por esta propiedad en las APC, es posible considerarlo en inmunización y en el diseño de nuevas vacunas como una alternativa a los adyuvantes convencionales, asociado a su fácil manufactura, sencilla manipulación y baja toxicidad [44]. 23 INTRODUCCIÓN Dentro de los CPPs, uno que se ha sido ampliamente estudiado en cuanto a su actividad adyuvante es la poliarginina (pARG). Una nueva vacuna (IC41) ha sido reportada en diversos estudios clínicos, en donde la pARG junto con 5 péptidos sintéticos, son la base de una vacuna terapéutica contra la hepatitis C [45, 46]. Otro péptido con propiedades CPP es la protamina. Éste nombre refiere a un conjunto de péptidos catiónicos ricos en arginina (Figura 2). De forma natural es sintetizada en la última etapa de la espermátide de muchos animales y plantas, siendo su rol fisiológico la condensacion del ADN [47]. Si bien su estudio comienza en el año 1868 por Friedrich Miescher, desde aquello muchos son los trabajos que se han realizado para caracterizar a este conjunto de péptidos alifáticos, fuertemente básicos, de peso molecular aproximado entre los 4 – 10 kDa [48]. La protamina es utilizada actualmente como excipiente farmacéutico siendo su principal aplicación en la formulación de liberación sostenida de insulina: NPH (del ingles: Neutral Protamine Hagedorn), aparte de tener registro sanitario como principio activo por ser el antagonista de la heparina. Debido a su alta afinidad por el material genético la protamina ha sido ampliamente estudiada en la liberación controlada de material genético (ADN, ARN y siARN). De hecho, su mezcla con oligonucleótidos forma nanopartículas de manera espontánea llamadas “Proticles”, las cuales aumentan considerablemente la internalización celular del material genético con baja toxicidad [49, 50]. Este polipéptido ha sido estudiado para diversas patologías y vías de administración, como por ejemplo formando parte de nanoparticulas por vía nasal para su administración a cerebro [51], en conjunto con liposomas y ADN (denominados LPD, del inglés liposome-polication-DNA) [52, 53], para la 24 INTRODUCCIÓN vehiculización de moléculas delicadas como el péptido vasoactivo intestinal [54], utilizando una probable sinergia en actividad antimicrobiana [55], etc. Nº A.A. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Péptido 1 P R R R R R S S S R P I R R Péptido 2 P R R R R - S S R R P V R R Péptido 3 P R R R R - S S S R P V R R Péptido 4 P R R R R - A S R R - I R R Nº A.A. 15 16 17 18 19 20 21 22 23 24 25 Péptido 1 R R R P R A S R R R R Péptido 2 R R R P R V S R R R R Péptido 3 R R R P R V S R R R R Péptido 4 R R R P R V S R R R R Nº A.A. 26 27 28 29 30 31 32 33 Total AA Péptido 1 R - G G R R R R 32 (21 R) Péptido 2 R R G G R R R R 32 (21 R) Péptido 3 R R G G R R R R 32 (20 R) Péptido 4 R - G G R R R R 30 (21 R) Figura 2: Comparación de la estructura primaria de los 4 péptidos principales de la protamina. En gris las diferencias aminoacídicas (AA). P: Prolina, R: Arginina, S: Serina, V: Valina, G: Glycina, I: Isoleucina y A: Alanina. Adaptado de [56]. En el área de la inmunización, estudios preliminares han demostrado que la protamina incrementa los efectos inmumoestimulante de oligonucleotidos CpG [57] y mRNA [58, 59]. Además, micropartículas de PLGA recubiertas con protamina para su uso en vacunas para alergia, demostraron un efecto adyuvante con el uso de este polipéptido [60, 61]. Por último, liposomas LPD cargados con el antígeno ántrax, administrado por vía nasal, evidenciaron de que nanosistemas a base de protamina puede ser una plataforma válida para la vacunación a través de mucosas [62]. 25 INTRODUCCIÓN REFERENCIAS 1. Plotkin, SA and Plotkin, SL, The development of vaccines: how the past led to the future. Nat Rev Micro, 2011. 9(12): p. 889-893. 2. Fenner, F, Handerson, DA, Arita, I, Jezek, Z, and I.D., L, Smallpox and its eradication1988: World Health Organization. 3. 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CAPÍTULO 2 NANOVACCINES: NANOCARRIERS FOR ANTIGEN DELIVERY Adapted from: “Nanovaccine: Nanocarriers for antigen delivery” González-Aramundiz JV, Cordeiro AS, Csaba N, De la Fuente M Alonso MJ. Biologie Aujourd`hui, 2012 206 (4): 249-261. 35 CAPÍTULO 2 ABSTRACT Vaccination has become one of the most important health interventions of our times, revolutionizing health care, and improving the quality of life and life expectancy of millions all over the world. In spite of this, vaccine research remains a vast field for innovation and improvement. Indeed, the shift towards the use of sub-unit antigens, much safer but less immunogenic, and the recognized need to facilitate the access to vaccines in the global framework is currently stimulating the search of safe and efficient adjuvants and delivery technologies. Within this context, nanocarriers have gained particular attention over the last years and appear as one of the most promising strategies for antigen delivery. A number of biomaterials and technologies can be used to design nanovaccines that fulfill the requirements of new vaccination approaches, such as single-dose and transmucosal immunization, critical for achieving a widespread coverage while reducing the overall costs in relation to traditional forms of vaccination. Here we present an overview of the current state of nanocarriers for antigen delivery, developed with the perspective of contributing to the global vaccination goal. 36 CAPÍTULO 2 Framework and technological needs The first scientific attempt to control an infectious disease by the deliberate use of vaccination was brought forth by Edward Jenner more than 200 years ago. Jenner’s work turned the scientific community attention towards the development of vaccines and their potential for prevention and eradication of life-threatening diseases, such as smallpox, polio, diphtheria or tetanus [1]. The implementation of vaccination has increased over the time, being nowadays recognized as a greatest milestone in health protection. Furthermore, vaccination remains an interesting and vast field for innovation [2-4]. A major goal in vaccination is to achieve worldwide coverage against highly infectious diseases, such as tuberculosis, polio, diphtheria, tetanus, cholera, pertussis and to focus research on emerging or re-emerging diseases such as HIV or malaria. Currently, the population with access to the existing vaccines is extremely reduced and some infectious diseases exhibit complex pathogenesis mechanisms, which introduce great difficulties in the development of efficacious vaccines. The benefits of achieving an effective immunization program go beyond the life-saving objective, particularly in developing countries where vaccination allows the reduction of the sickness burden, thus contributing to their global development. This crucial observation has led several public and private institutions, such as the World Health Organization (through the Global Alliance for Vaccines and Immunization) and the Bill & Melinda Gates Foundation, to commit with the challenge of improving current vaccination programs [5, 6]. Traditionally developed vaccines consist of either live attenuated or inactivated pathogenic agents. Live attenuated vaccines stand out for their unique capacity to enhance strong innate and long-lasting immune responses without needing additional adjuvant components. However, their instability and potential to revert to the virulent form represents a serious risk for the patient health. To 37 CAPÍTULO 2 overcome this problem, by the end of the 19th century, researchers developed inactivated whole organism vaccines as well as toxoid vaccines. These vaccines were shown to be safer than the previous ones, despite the limitation of yielding weaker immunity levels, thus requiring the use of an adjuvant, i.e. alum, and multiple-dose vaccination programs. More recently, vaccine research has been oriented towards the development of purer, safer and easier to produce antigens, namely 1) sub-unit vaccines in which a unique fraction of the pathogenic agent is used as antigen, 2) DNA vaccines and 3) conjugate vaccines, which consist of the presentation of the antigen covalently linked to a protein or membrane complex with stronger immunogenicity [2, 4]. Regardless of the promising features of these forms of vaccination, their use in a global perspective is still limited, essentially because of their low immunogenicity, thus making the search for new adjuvants a critical need for improving vaccination coverage [7, 8]. Besides the development of new adjuvants the current technological challenges in vaccination are aimed at improving the stability of the commercially available vaccines and decreasing the number of doses needed for an efficient immunization, altogether reducing the costs of this type of health intervention. Indeed, it is known that the assumed commitment to a cold chain protocol for the worldwide distribution of vaccines is frequently infringed with the subsequent risk of irreversible antigen damage [9, 10]. On the other hand, current schedules typically require the parenteral administration of several vaccine doses in order to reach adequate levels of protection, a fact that represents a real challenge in developing countries [9, 11]. The goals are therefore set for progress and innovation in the vaccination field with the final aim of reaching universal accessibility to vaccines that are simultaneously effective, affordable and safe. The most remarkable advances in 38 CAPÍTULO 2 vaccination reported to date, and the role of nanotechnology are reviewed in the following sections and summarized in Figure 1. Figure 1: Milestones in vaccination and evolution of vaccines towards the development of single-dose and needle-free approaches. NPs: nanoparticles; WHO: World Health Organization; PLGA: poly(D,L-lactic-co-glycolic acid). The essential role of adjuvants in vaccination The term adjuvant encloses a heterogeneous group of compounds with different activities and functions, intended to enhance the quality, length and extent of a specific immune response [12]. The discovery and rational design of new compounds with adjuvant properties is becoming a key point in vaccination research that benefits from the knowledge gathered in the field of immunology. Indeed, advances in the study of activation mechanisms of antigen presenting cells (APCs) and the discovery of pattern recognition receptors for highly conserved structures of pathogens, such as the toll-like receptors (TLRs), are having a major impact in the development of new adjuvants [13]. The first significant adjuvants developed for human vaccines are the aluminum compounds depicted for the first time in 1926 and generally referred to as alum. Despite its favorable safety profile, alum is not adequate for the recently developed sub-unit vaccines, due to the probable loss of antigenicity of these antigens when adsorbed to this adjuvant [14]. Another concern of alum is its sensitivity to damage upon freezing [10]. 39 CAPÍTULO 2 Emulsion technologies represent a second approach to the development of new adjuvants. The first attempt involved the use of water-in-oil emulsions, known as Complete and Incomplete Freund’s Adjuvant (CFA and IFA). Since then, toxicity concerns have led to the development of new oil-in-water emulsions made from highly purified emulsifiers, which have successfully led to some marketed vaccines for influenza (Fluad®, Focetria® and Prepandrix®) and human papilloma virus (HPV) (Cervarix®) [15, 16]. In parallel to the development of emulsions, vaccine research has focused on exploring alternative lipid-based antigen delivery technologies. Liposomes are the main class of delivery systems explored for drug delivery applications, and consequently, they have been widely investigated as antigen delivery systems in vaccination. In this field, it is worth highlighting the therapeutic tuberculosis vaccine RUTI® which is now undergoing clinical development [4, 15-17]. The integration of functional viral envelope glycoproteins into liposomes has led to an interesting type of antigen adjuvants named virosomes. Importantly, they retain the cell binding and membrane fusion properties of the native virus, therefore manifesting an improved capture by the APCs and antigen processing. Vaccines against influenza (Inflexal V®) and hepatitis A (Epaxal®) have already reached the market, and several are undergoing clinical trials [18]. Colloidal structures constituted by a combination of phospholipids, cholesterol and saponines, named immunostimulating complexes (ISCOMs), have been explored for their ability to associate hydrophobic and hydrophilic antigens, their good stability, and ability to induce both humoral and cellular immune responses. However, their potential use in human vaccines is hampered by toxicity concerns attributed to certain saponines as Quil A and QS-21 [19]. Other advanced lipidic formulations are the synthetic biomimetic supra molecular Biovector™ (SMBV) particles, consisting in a polysaccharide core of 40 CAPÍTULO 2 positive or negative particles, surrounded by a phospholipid layer, which allows the incorporation of distinct active compounds. These structures are particularly studied in the area of mucosal vaccine delivery and have reached the clinical development status [20]. Regardless of the interesting features of lipid-based adjuvants, there is a trend to explore the potential of biocompatible polymeric nanostructures for antigen delivery. These delivery systems present several advantages and are promising adjuvants in vaccination, as highlighted in the next sections. The potential of polymeric nanostructures as adjuvants in vaccination: nanovaccine In 1976, Kreuter and Speiser reported for the first time the potential use of polyacrilic nanoparticles as an adjuvant for an influenza vaccine [21]. Following this pioneering work there has been a significant number of reports on the development of micro- and nano-sized particulate delivery systems [4, 22, 23]. Throughout the last years, different studies have been carried out to understand the importance of particle size in vaccination. Recent works have made clear that small and large nanoparticles reach the lymph nodes by different mechanisms and interact differently with APCs. While nanoparticles with a size inferior to 100 nm have shown an improved ability to drain to the interstitial flow and be transported to the lymph nodes for antigen presentation to resident DCs, larger particles typically reach the lymph nodes in a cell-associated manner [17, 24]. Similarly, the internalization mechanisms by APCs are dependent on the size of the particle engulfed: small nanoparticles are usually taken up by DCs through receptor-mediated endocytosis into clathrin-coated pits or through caveolae, while larger particles are generally phagocytosed specially by macrophages [25, 26]. In this regard, the route of internalization is gaining interest for the understanding of immune responses, 41 CAPÍTULO 2 and receptor-mediated endocytosis has been related to cross-presentation processes and induction of combined cellular and humoral responses [27-29]. For the engineering of nanovaccines, the forming materials must be biocompatible and biodegradable and posses a good safety record. Biodegradable polymeric delivery systems exhibit a number of advantages as vaccine adjuvants: (i) they reproduce the natural particulate form of pathogenic agents, passively targeted to APCs; (ii) they can be engineered to specifically interact with certain cell populations as microfold-cells (M-cells) and dendritic cells (DCs) [30]; (iii) they can accommodate immunopotentiators as TLR agonists for an increased response; (iv) they can control the release of the antigen and prolong the exposure and duration of the immune response; (v) they can be administered by alternative non-invasive transmucosal routes; (vi) in the case of dry powder formulations generated upon freeze-drying, these adjuvants are known stabilizers of the associated antigens [9, 31-33]. Among the materials studied, the polyesters poly(D,L-lactide) (PLA) and poly(D,L-lactic-co-glycolic acid) (PLGA), as well as their PEGylated derivatives, have been widely explored, first for the preparation of microparticles, and few years later for the development of smaller nanosized particles [34-36]. Other biomaterials, such as oils, polyethylenoxides, and cationic polymers, have subsequently been incorporated to PLA/PLGA formulations in the search for vaccines with improved properties [37-40]. Further optimization of the adjuvant properties can be achieved by modification of their surface properties with immunostimulatory components such as the TLRs agonists CpG oligonucleotides [41]. Among the natural polymers that have been disclosed for the preparation of nanovaccines, special mention should be given to the biodegradable polysaccharide chitosan (CS), which has either been used as a polymeric coating or as the core forming material of nanovaccines [42-45]. Nanovaccines based 48 CAPÍTULO 2 Overall, the above-disclosed information has made evident the potential of micro and nanoparticles as single-dose vaccine formulation approaches. It is however worth noting that the most investigated delivery carriers, PLGA micro- and nanoparticles, have not reached the clinical development status yet, with only a small trial of clinical evaluation [15]. Irrespective of the commercial justifications for the implementation of these technologies, it seems reasonable to conclude on the necessity of making further progress in the development of safe, inexpensive and efficacious polymer particles, which may one day represent single-dose formulation vaccines. The promise of nanovaccines for transmucosal vaccination Regardless of the intended achievements in parenteral immunization, needlefree vaccination has been recognized as a great challenge in global health. Needle-free vaccination facilitates compliance with immunization schedules, decreases pain and suffering, requires less healthcare training for vaccination, enables faster vaccine delivery, avoids the risk of incorrect or repeated use of injection devices and allows an eventual cost reduction [11]. This section is focused on mucosal immunization, among the different needle-free delivery approaches, as this is a suitable and already established vaccination protocol for some specific vaccines, i.e. polio, rotavirus, Salmonella typhi and adenovirus type 4 and 7 vaccines for oral administration, and influenza vaccine for nasal administration. The interest in mucosal vaccines relies not only in the induction of systemic immune responses but also of mucosal responses, thus providing additional protection against pathogens even at their site of entry, since most infections start at or affect specifically mucosal surfaces. The use of antigen delivery systems in mucosal vaccination aims at the improvement of the antigen stability and the facilitation of its penetration across the mucosal surface, so that the intact and active antigen could be taken up by APCs and transported 49 CAPÍTULO 2 from the mucosal associated lymphoid tissues (MALT) to the lymph nodes or other secondary lymphoid organs [66]. As an example of this process, Figure 2 represents schematically the pathways involved in intranasal vaccine delivery. Depending on the organ or site to which it is associated, the MALT may have some differences in structure. For example, in the digestive tract this tissue is often referred to as gut-associated lymphoid tissue (GALT) and is mainly composed of Peyer's patches and M-cells, which are capable of carrying antigens in a particulate formulation from the absorption site until they reach APCs, which migrate to the regional lymph nodes and trigger the adequate immune response. In the human nasal cavity this tissue is known as “diffuse” nasal-associated lymphoid tissue (NALT) and is composed of a collection of isolated subepithelial lymphoid follicles and the lingual, palatine and nasopharyngeal tonsils (adenoids) [32, 66]. 50 CAPÍTULO 2 Figure 2: Schematic illustration of the general pathways involved in mucosal immune response following intranasal vaccination. In all cases, the first barrier to overcome in mucosal vaccination is a dense and dynamic mucus layer that covers and protects the underlying epithelium. For a successful mucosal vaccination, the vehicles should overcome the mucus barrier, adhere or penetrate the epithelium and deliver the immunologically active antigen in a controlled-release fashion. The following sections are intended to analyze information on the most relevant technologies developed for needle-free nasal and oral vaccination as well as the most remarkable immunization results obtained from these vaccination approaches. Nasal vaccine delivery systems Some relevant characteristics of the nasal cavity physiology that make it especially attractive for vaccination are its relatively reduced enzymatic activity, Vaccine Delivery System Epithelial Cells M Cell Maturation and Migration Dendritic Cell IgA Cytotoxic T Cell B Cell xic Lymph Node Systemic Immune Response IgA 51 CAPÍTULO 2 a moderately permeable epithelium, and its high amount of available immunereactive sites. For the success of needle-free nasal immunization, nanoparticles appear to be an interesting approach, as they are known to increase the residence time upon intranasal deposition and improve the interaction with the mucosal epithelium. As highlighted in this review, PLGA and PLA have been the most studied polymers for the design of antigen delivery systems, being the first to be evaluated for needle-free nasal vaccination in the early 90s. In 1993, Almeida et al demonstrated that PLA microspheres, administrated through the nasal route to guinea pigs, could effectively enhance the immune response of the adsorbed TT, when compared to the soluble antigen [67]. The first nanoparticulate compositions to be evaluated for nasal delivery of antigens were nanoparticles composed by PLA or the PEGylated derivative PLAPEG. Experiments in rats performed with nasally administered radiolabelled tetanus toxoid 125I-TT have shown that PLA-PEG nanoparticles successfully prolonged the residence time, antigen release and transport to a greater extent than conventional PLA nanoparticles [34]. The hydrophilic coating was proved to be essential for increasing the stability of the nanoparticles upon contact with the mucosal fluids, thereby enhancing their interaction with the epithelium [68]. These results were corroborated in subsequent studies showing that through the careful control of the nanoparticle size and density of PEGylation, it is possible to modulate the interaction of these nanocarriers with the epithelium and hence its mucosal transport. PLA-PEG nanoparticles with an elevated PEG coating density (20-35 %) evidenced a significantly increased transport versus the PLA control nanoparticles [49]. Optimized nanoparticles of PLGA blended with polyoxyethylene derivatives have also been studied for DNA vaccination through the nasal route [37]. These improved nanoparticles could effectively overcome the mucus layer upon 52 CAPÍTULO 2 intranasal deposition and enter epithelial cells. Most importantly, immunization studies proved the ability of pDNA-loaded nanoparticles to elicit a fast and strong immune response, with IgG antibody titers against the encoded protein significantly higher than those corresponding to the naked pDNA [37]. Further studies demonstrated the suitability of these nanostructures to associate other types of antigens as proteins and even virus-like particles [38, 59]. Besides the promise of PLA/PLGA nanoparticles for nasal delivery of antigens, CS -based nanocarriers hold a great potential for this application mainly due to the mucoadhesive properties of this polysaccharide. Vila et al reported the first studies performed with CS nanoparticles for nasal immunization and showed the positive role of CS in improving the transport of antigens across the nasal mucosa [44, 69]. With the purpose of exploring how the polysaccharide properties could influence the immunization process, nanoparticles loaded with TT were prepared from CS polymers of different molecular weights (ranging from 23 to 70 kDa). Upon intranasal administration to mice, results showed comparable levels of IgA antibody in saliva, broncho-alveolar and intestinal lavages, irrespective of the CS molecular weight, as highlighted in Figure 3. In all cases, the titers were superior to those obtained in animals immunized with the antigen solution [44]. Chitosan nanoparticles also resulted to be suitable for the encapsulation of more complex antigens such as HBsAg, without altering its antigenicity [42]. Intranasal immunization to mice was performed at two different priming HBsAg doses (10 and 20 μg) and a boost dose after 28 days. The anti-HBsAg IgG levels showed a low but increasing immune response over the time, regardless of the dose administered. In both cases, the antibody concentrations were considered to be seroprotective against hepatitis B although the rather low overall response indicates the need for further optimization of the delivery system for this specific antigen. 53 CAPÍTULO 2 Stealth PEGylated CS (PEG-CS) nanoparticles have also been studied for intranasal delivery of different antigens and the possible role of PEG in improving the observed immune response was explored [51, 70]. For example, PEG-CS and CS nanoparticles were able to induce systemic and mucosal immune responses against diphteria toxoid (DT) upon intranasal administration to mice. Besides, PEG-CS achieved significantly higher systemic antibody titers in comparison to the ones obtained for CS nanoparticles [51]. FIGURE 3: IgA responses obtained after intranasal immunization of mice with 10 μg of tetanus toxoid encapsulated into nanoparticles prepared with chitosan polymers that differ in their molecular weight: 23 KDa (Np CS 23), 38 KDa (Np CS 38) and 70 kDa (Np CS 70). As control, the antigen was administered in saline. Titres are presented as the geometric mean titre (GMT) per group. Adapted from [44] with permission. Other CS derivatives have similarly been explored for antigen delivery purposes. The most recurrent modifications involve the methylation of CS amine groups [71]. Nanoparticles made of oppositely charged methylated CS derivatives (positively charged N-trimethyl CS-TMC and negatively charged mono-N-carboxymethyl CS-MCC) were loaded with TT and intranasally administered to mice. The results have shown that positive CS and TMC 54 CAPÍTULO 2 nanoparticles, with a size ranging from 300-400 nm, induced higher serum IgG titers than negative MCC nanoparticles of 90 nm. These results emphasized the assumption that the nature of the nanoparticles surface, as well as the particle size, have a crucial role in obtaining an enhanced immune response [52, 72]. Mangal et al have recently reported the superiority of TMC nanoparticles vs. classical CS nanoparticles with regard to their ability to elicit anti-HBsAg antibody titers following intranasal administration to mice. This was attributed to the improved mucoadhesion of TMC nanoparticles, followed by an enhanced antigen uptake [73]. Interestingly, CS-coated nanocapsules, formed by an oily nanocore stabilized with phospholipids and surrounded by a CS shell, as already described in section on Crontrolled release technologies, have also shown a great capacity of association of HBsAg. The most remarkable advantage of this type of improved nanostructure is its versatility, as it is feasible to efficiently incorporate simultaneously lipophilic immunostimulants in the oily core, i.e. imiquimod, and the antigen in the polymer shell. [43]. The results of the in vivo experiments performed with this novel formulation have shown a progressive increase of the specific IgG levels over time, achieving seroprotection against the HBsAg for up to 112 days. This response was significantly higher than the one obtained with the nanocapsule formulation in the absence of imiquimod. Nanoemulsions have similarly been explored for the stabilization and nasal delivery of different molecules, in particular OVA, porcine intestinal alkaline phosphatase (AlkP), and HBsAg [74]. In vivo results show that upon intranasal administration to mice of HBsAg associated to the nanoemulsion, the reported serum anti-HBsAg IgG antibody titers were comparable to the ones obtained for the antigen adsorbed on aluminum hydroxide and given intramuscularly. These results were similarly reported in other animal species (rats and guinea pigs), in a single- or two-doses scheme [75]. 55 CAPÍTULO 2 Despite the promising examples reported here, there is no doubt about the need to further improve the design and, thus, the efficiency of intranasal delivery vehicles. Nevertheless, the accumulated knowledge and the crossdisciplinary approaches currently underway will hopefully define the way to proceed in the optimization of the design of nanostructures for intranasal antigen delivery. Oral vaccine delivery While live-attenuated vectors have shown promising results for oral immunization, problems related to their safety made it necessary to find new solutions for oral vaccination. This route is particularly challenging due to the harsh conditions of the gastrointestinal environment and the need to confront the intestinal mucosa. Nano-sized delivery systems are expected to have several functions in oral vaccine administration: (i) to improve the antigen stability in the gastric environment and increase its bioavailability, (ii) to overcome the mucus layer and interact with the underlying epithelium, (iii) to increase the uptake of these systems by the epithelial and M-cells [76]. The physicochemical properties of particulate vehicles are known to affect the uptake of the antigen along the intestine and by APCs. The smaller size of nanoparticles is considered to be a key parameter influencing the uptake and immunogenicity of these delivery systems, as nanoparticles are better taken up by intestinal cells than microparticles [77-79]. With respect to the surface properties, both the hydrophobicity and the surface charge affect colloidal stability, mucoadhesion properties and absorption of the carriers [77]. In addition, targeting specific receptors on the apical surface of M-cells may enhance the entry of antigens, triggering the immune response and leading to effective protection against mucosal pathogens [30]. Nanoparticles can be conveniently targeted to M-cells by surface modification with selective ligands such as lectins or certain bacterial surface proteins [78, 79]. 56 CAPÍTULO 2 The importance of the surface properties on the ability of particles to efficiently deliver antigen through the oral route was evidenced by Tobío et al [68]. Similarly to what observed when the nanoparticles were administered to the nasal mucosa, the coating of PLA nanoparticles with a hydrophilic and stabilizing PEG coating was found to be crucial in order to avoid nanoparticle aggregation in gastro-intestinal fluids and upon contact with the mucosa. This increased stability has been the explanation for the improved absorption of 125I- labelled TT after oral administration to rats in PLA-PEG nanoparticles [68]. Poly(anhydrides) were also used for the preparation of a specific type of polymer named Gantrez® AN [80]. Nanoparticles prepared with this polymer present interesting bioadhesion characteristics and were therefore studied for oral immunization with ovalbumin (OVA). Nanoparticles provided better immune response than that obtained for the OVA in solution. The survival rate of 100% achieved by the OVA-loaded nanoparticles versus the 40% obtained with the OVA-solution after challenge with an intraperitoneal injection of this allergen also proves that this can be an interesting alternative for oral immunization [81]. Other recent studies have aimed at exploring the possibility to target the M- cells, through the development of carriers that mimic the entry of pathogens. An example of this approach was the study of PLGA nanoparticles decorated with a specific type of lectin (Lotus tetragonolobus - LTA). These nanoparticles interacted with Peyer’s patches M-cells, as confirmed by confocal microscopy, and provided anti-HBsAg titers, which were higher than those corresponding to the control PLGA nanoparticles. This positive behavior was attributed to the lectin-mediated selective targeting [54]. The RGD peptide (a small sequence composed of L-arginine, glycine, and L- aspartic acid which is involved in cell recognition processes) was also grafted to the PEG residues of the poly(ε-caprolactone-co-ethylene glycol) (PCL–PEG) 57 CAPÍTULO 2 copolymer used to prepare nanoparticles. Unfortunately, despite the targeting ability evidenced in vivo, the improvement of the immune response achieved by any of the RGD-grafted formulations was minimal [82]. Thus, these results put in question the interest in targeting the M-cells for achieving adequate immune responses. Overall, on view of the described works, the evidence of the potential of nanostructures for oral vaccination is still scarce. Besides, the positive role of a specific targeting to M-cells needs to be confirmed. Despite of this, the reported results have paved to way towards the optimized design of nanoparticles intended for oral vaccination. Considering the whole perspective on transmucosal vaccine delivery, it becomes clear that this approach is a valid and interesting pathway for future developments. The research on new delivery vehicles for this type of administration is therefore expected to play a very important role in global health improvement and may represent a new milestone in vaccine research. CONCLUSIONS Over the past few decades, the search for new adjuvants capable of enhancing the immunogenic properties of current and developing antigens has driven the attention of the scientific community towards the design of particulate antigen delivery systems. These new adjuvants are not only able to enhance the immunogenicity of safer but poorly effective antigens but may also allow the targeting of these antigens to the adequate immune-competent cells. Beyond this adjuvant capacity polymer-based micro/nanostructures can be now presented as promising single-dose and transmucosal vaccination approaches. Giving room to new routes of administration, nanovehicles are influencing decisively the pathways of vaccinology and gaining an essential place in this field. Hopefully, these progresses in immunization strategies will help to 64 CAPÍTULO 2 by single-dose oral immunization with biodegradable microparticles containing DNA encoding HBsAg. J Gen Virol, 2005. 86(3): p. 601-610. 64. Vicente, S, Díaz-Freitas, B, Sanchéz, A, González-Fernández, A, and Alonso, MJ, Adjuvant formulations based on polysaccharidic nanocapsules as potential single-dose vaccines, in Bill & Mellinda Gates Foundation Congress2010: Seattle, USA. 65. González-Aramundiz, JV, Lozano, MV, Souza-Herves, A, Fernandez- Megia, E, and Csaba, N, Polyaminoacids and Polypeptides in Drug Delivery. Expert Opin Drug Del, 2012. 9(2): p. 183-201. 66. Holmgren, J and Czerkinsky, C, Mucosal immunity and vaccines. Nat Med, 2005. 11: p. s45 - s53. 67. Almeida, AJ, Alpar, HO, and Brown, MR, Immune Response to Nasal Delivery of Antigenically Intact Tetanus Toxoid Associated with Poly(l-lactic acid) Microspheres in Rats, Rabbits and Guinea-pigs. J Pharm Pharmacol, 1993. 45(3): p. 198-203. 68. Tobı o, M, Sánchez, A, Vila, A, Soriano, I, Evora, C, Vila-Jato, JL, and Alonso, MJ, The role of PEG on the stability in digestive fluids and in vivo fate of PEG-PLA nanoparticles following oral administration. Colloids and Surfaces B: Biointerfaces, 2000. 18(3-4): p. 315-323. 69. Vila, A, Sánchez, A, Tobı o, M, Calvo, P, and Alonso, MJ, Design of biodegradable particles for protein delivery. J Control Release, 2002. 78(1–3): p. 15-24. 70. Csaba, N, Koping-Hoggard, M, Fernandez-Megia, E, Novoa-Carballal, R, Riguera, R, and Alonso, MJ, Ionically Crosslinked Chitosan Nanoparticles as Gene Delivery Systems: Effect of PEGylation Degree on In Vitro and In Vivo Gene Transfer. J Biomed Nanotechnol, 2009. 5(2): p. 162-171. 71. Amidi, M, Mastrobattista, E, Jiskoot, W, and Hennink, WE, Chitosanbased delivery systems for protein therapeutics and antigens. Adv Drug Delivery Rev, 2010. 62(1): p. 59-82. 72. Hagenaars, N, Verheul, RJ, Mooren, I, de Jong, PH, Mastrobattista, E, Glansbeek, HL, Heldens, JG, van den Bosch, H, Hennink, WE, and Jiskoot, W, Relationship between structure and adjuvanticity of N,N,N-trimethyl chitosan (TMC) structural variants in a nasal influenza vaccine. J Control Release, 2009. 140(2): p. 126-133. 73. Mangal, S, Pawar, D, Garg, NK, Jain, AK, Vyas, SP, Rao, DS, and Jaganathan, KS, Pharmaceutical and immunological evaluation of mucoadhesive nanoparticles based delivery system(s) administered intranasally. Vaccine, 2011. 29(31): p. 4953-4962. 65 CAPÍTULO 2 74. Makidon, PE, Nigavekar, SS, Bielinska, AU, Mank, N, Shetty, AM, Suman, J, Knowlton, J, Myc, A, Rook, T, and Baker, JR, Characterization of Stability and Nasal Delivery Systems for Immunization with Nanoemulsion-Based Vaccines. J Aerosol Med Pulm D, 2010. 23(2): p. 77-89. 75. Makidon, PE, Bielinska, AU, Nigavekar, SS, Janczak, KW, Knowlton, J, Scott, AJ, Mank, N, Cao, ZY, Rathinavelu, S, Beer, MR, Wilkinson, JE, Blanco, LP, Landers, JJ, and Baker, JR, Pre-Clinical Evaluation of a Novel Nanoemulsion- Based Hepatitis B Mucosal Vaccine. PLoS ONE, 2008. 3(8). 76. Brayden, DJ, Oral vaccination in man using antigens in particles: current status. Eur J Pharm Sci, 2001. 14(3): p. 183-189. 77. des Rieux, A, Fievez, V, Garinot, M, Schneider, YJ, and Préat, V, Nanoparticles as potential oral delivery systems of proteins and vaccines: A mechanistic approach. J Control Release, 2006. 116(1): p. 1-27. 78. Florence, AT, Nanoparticle uptake by the oral route: Fulfilling its potential? Drug Discovery Today: Technologies, 2005. 2(1): p. 75-81. 79. O'Hagan, DT, The intestinal uptake of particles and the implications for drug and antigen delivery. J Anat, 1996. 189: p. 477-482. 80. Arbós, P, Wirth, M, Arangoa, MA, Gabor, F, and Irache, JM, Gantrez® AN as a new polymer for the preparation of ligand–nanoparticle conjugates. J Control Release, 2002. 83(3): p. 321-330. 81. Gómez, S, Gamazo, C, Roman, BS, Ferrer, M, Sanz, ML, and Irache, JM, Gantrez® AN nanoparticles as an adjuvant for oral immunotherapy with allergens. Vaccine, 2007. 25(29): p. 5263-5271. 82. Garinot, M, Fiévez, V, Pourcelle, V, Stoffelbach, F, des Rieux, A, Plapied, L, Theate, I, Freichels, H, Jérôme, C, Marchand-Brynaert, J, Schneider, YJ, and Préat, V, PEGylated PLGA-based nanoparticles targeting M cells for oral vaccination. J Control Release, 2007. 120(3): p. 195-204. ANTECEDENTES, HIPÓTESIS Y OBJETIVOS 69 ANTECEDENTES, HIPÓTESIS Y OBJETIVOS ANTECEDENTES 1. La nanomedicina se presenta como una estrategia prometedora en el diseño de nuevas vacunas, para aquellas enfermedades que no poseen aún cobertura o para mejorar aquellas que ya se encuentran en el mercado1. Estas “nanovacunas” permiten: (i) estabilizar el antígeno, (ii) mejorar la interacción y captación del antígeno asociado por parte de las células inmunocompetentes y (iii) aumentar el transporte del antígeno a través de barreras mucosas, protegiéndolo frente la degradación y vehiculizándolo a las células inmunes2. 2. La protamina está formada por un conjunto de polipéptidos naturales y es capaz de formar diferentes nanoestructuras mediante procedimientos sencillos, administrarse por diversas vías y vehiculizar moléculas delicadas3. Posee un gran potencial como biomaterial para la preparación de nanosistemas por promover la translocación a través de membranas4, estimulación del sistema inmune5,6 y un perfil de seguridad aceptable7; confiriendo estas propiedades a los sistemas a los que se les ha asociado o en los que forma parte. 1 González-Aramundiz, J.V. et al. Nanovaccine: nanocarriers for antigen delivery. Biologie Aujourd'hui, 2012. 206(4): p. 249-261. 2 Amorij, J.-P. et al. Towards tailored vaccine delivery: Needs, challenges and perspectives. J. Control. Release, 2012. 161(2): p. 363-376. 3 Xia, H. et al. Low molecular weight protamine-functionalized nanoparticles for drug delivery to the brain after intranasal administration. Biomaterials, 2011. 32(36): p. 9888-9898. 4 Reynolds, F. et al. Protamine as an Efficient Membrane-Translocating Peptide. Bioconjug. Chem., 2005. 16(5): p. 1240-1245. 5 Martínez Gómez, J.M. et al. Surface coating of PLGA microparticles with protamine enhances their immunological performance through facilitated phagocytosis. J. Control. Release, 2008. 130(2): p. 161-167. 6 Kerkmann, M. et al. Immunostimulatory properties of CpG-oligonucleotides are enhanced by the use of protamine nanoparticles. Oligonucleotides, 2006. 16(4): p. 313- 322. 7 Horrow, J.C. Protamine a review of its toxicity. Anesth. Analg., 1985. 64(3): p. 348- 361. 70 ANTECEDENTES, HIPÓTESIS Y OBJETIVOS 3. Las nanopartículas poliméricas permiten la encapsulación eficaz de diversos antígenos8 y son vehículos válidos para la administración libre de agujas9. 4. Las nanocápsulas constituidas por una cubierta polimérica son una estrategia innovadora en la vehiculización de antígenos y en la coadministración de aceites con capacidad adyuvante10,11. 8 Prego, C. et al. Chitosan-based nanoparticles for improving immunization against hepatitis B infection. Vaccine, 2010. 28(14): p. 2607-2614. 9 Csaba, N. et al. Nanoparticles for nasal vaccination. Adv. Drug Delivery. Rev., 2009. 61(2): p. 140-157. 10 Vicente, S. et al. A Polymer/Oil Based Nanovaccine as a Single-Dose Immunization Approach. PLoS ONE, 2013. 8(4): p. e62500. 11 Vicente, S. et al. Highly versatile immunostimulanting nanocapsules for specific immune potentiation. in press, 2013. 71 ANTECEDENTES, HIPÓTESIS Y OBJETIVOS HIPÓTESIS 1. Los sistemas constituidos por protamina pueden ser una estrategia adecuada para la asociación, vehiculización y presentación de antígenos. 2. Los sistemas a base de protamina pueden incrementar la captación del antígeno por parte de las células presentadoras de antígeno y potenciar así una respuesta inmune específica. 3. La protamina como componente de los diversos sistemas desarrollados puede favorecer el transporte de antígenos a través de barreras mucosas, induciendo una respuesta inmune eficaz y constituyendo una formulación libre de agujas. 4. Las nanopartículas y nanocápsulas pueden ser transformadas en polvo seco a través de un proceso de liofilización y dicho producto presenta una adecuada estabilidad a temperatura ambiente. 73 ANTECEDENTES, HIPÓTESIS Y OBJETIVOS OBJETIVOS Teniendo en cuenta los antecedentes expuestos y las hipótesis planteadas, el objetivo general de la presente tesis doctoral ha sido el diseño, desarrollo y optimización de nuevos vehículos nanométricos, basados en protamina, para potenciar y modular una respuesta inmune específica frente al antígeno asociado ya sea a través de su administración parenteral y/o por medio de administración nasal. Para lograr este objetivo se han planteado las siguientes etapas: 1. Desarrollo de nuevos sistemas de nanopartículas constituidos de protamina, evaluación in vitro e in vivo de su capacidad para vehiculizar antígenos. Esta parte de la memoria se ha dirigido al diseño, desarrollo y caracterización de nanopartículas de tipo matricial, que poseen protamina en su estructura. Se ha estudiado su capacidad para asociar un antígeno modelo, su interacción con células inmunes y la capacidad de promover una respuesta inmune eficaz, tanto por vía parenteral como por vía nasal. Estos resultados aparecen recogidos en el capítulo 3 de la presente memoria. 2. Desarrollo de nuevos sistemas constituidos por nanocápsulas de protamina y evaluación como sistemas transportadores de antígenos Los estudios realizados en esta etapa se han dirigido al diseño, desarrollo y optimización de sistemas de tipo nanocapsular con una cubierta de protamina y un núcleo constituido por aceites con capacidad adyuvante. Se ha estudiado su eficacia para asociar antígenos, su estabilidad a temperatura ambiente tras ser liofilizado y su capacidad para interaccionar in vitro con el sistema inmune. Por último, ha sido evaluada su capacidad para generar respuestas inmunes sistémicas tras su administración tanto por vía parenteral como nasal. Los 80 CAPÍTULO 3 1. INTRODUCTION Since their implementation in 1926, aluminum salts have had a crucial role in making vaccination the most important human intervention in global health. Despite of its value, alum suffers of important deficiencies, such as (i) insufficient adjuvant effect specially for new antigens such as subunit vaccines and peptides, (ii) lack of effect when administered by other routes than parenteral injection, (iii) associated side effects, i.e. local reactions or hypersensitization of allergic patients and (iv) it is only stable in a low and narrow temperature range [1, 2]. Due to these limitations, significant efforts have been directed towards developing new alternative adjuvants. In a broad sense, an adjuvant could be defined as a molecule or structure that can increase and/or modulate the immunogenicity of an antigen, allowing it to induce a potent and persistent immune response at low doses [3]. Some authors divided this group in immunostimulants (those interacting with specific receptors) and delivery systems [4]. Particulate delivery systems may act as adjuvants in the sense that they can modify the uptake, trafficking and processing of antigens, resulting in better and more adequate immune responses [3, 5]. In the design of novel “alum-free” vaccine particulate delivery systems, it is necessary to consider the nature of the antigen and its intrinsic immunogenicity, the administration route and the availability of biomaterials with an adequate safety profile. On the other hand, the importance of the particle size of these delivery systems has been widely discussed over the past years. Nanometric systems have raised expectancy because of their ability to control the release and increase the trans-epithelial transport of antigens, thus being considered as promising strategies for the development of single dose and needle-free vaccines [6, 7]. Different materials have been studied to develop particulate antigen delivery systems, such as lipids, polymers and polysaccharides. In particular, chitosan, 81 CAPÍTULO 3 polylactic and polylactic-co-glycolic acid have been the most widely applied polymers in the development of vaccine nanocarriers. [4, 8, 9]. More recently, polyaminoacids and polypeptides have been highlighted for their versatility to form different structures, their safe pharmacokinetic profile and the adjuvant properties presented by some of them [10]. In particular, protamine, an arginine-rich peptide with cell-penetrating properties has shown a synergistic adjuvant effect with other immunomodulatory molecules, i.e. CpG [11]. This effect was also observed when protamine was used to coat PLGA microparticles, the resulting system being able to stimulate the proliferation of antigen-specific T cells and the secretion of IFN-γ [12]. Other materials, such as hyaluronic acid (HA) and alginate (ALG) have also shown immunoadjuvant activity, measured by macrophage and dendritic cell recruitment plus activation and/or induction of cytokine production, when presented in a nanoparticulated form [13-16]. Based on this information, the aim of this work has been the development and characterization of a new nanometric antigen delivery system based on protamine to harness its adjuvants properties in association with a polysaccharide such as alginate or hyaluronic acid. In order to evaluate the ability of these systems to encapsulate and deliver antigens, recombinant hepatitis B surface antigen (rHBsAg) was used as a model antigen. Finally, the in vitro and in vivo performance of this novel vaccination approach was assessed in macrophage cell cultures and upon administration to mice by either intramuscular or nasal administration. 82 CAPÍTULO 3 2. MATERIALS AND METHODS 2.1. Chemicals Protamine sulfate used in this work was purchased from Yuki Gosei Kogyo, Ltd. (Japan) Hyaluronic acid (HA) of 162 KDa and 29 kDa were provided by Bioiberica (Spain) and by Soliance (France), respectively. Sodium alginate (ALG) (PRONOVA UP VLVG) of <75 kDa was supplied by Novamatrix (Norway). Recombinant hepatitis B surface antigen (rHBsAg) was kindly donated by Shantha Biotechnics Limited (Hyderabad, India). Enzyme linked immunosorbent assay (ELISA) kit (Murex rHBsAg Version 3) was obtained from Diasorin (United Kingdom). Chicken polyclonal antibody to hepatitis B virus surface antigen and rabbit polyclonal to chicken conjugated with horseradish peroxidase were purchased from Abcam pcl (United Kingdom). Rabbit IgG and mouse monoclonal IgG used as controls were purchased from Biokit (Spain) and Acris Antibodies GmbH (Germany), respectively, and secondary Abs conjugated to horseradish peroxidase were from Southern Biotech (USA). 5-TAMRA, 5- Carboxytetramethylrhodamine, Succinimidyl Ester (single isomer) and Alexa Fluor 488-phalloidin were obtained from Invitrogen (United Kingdom). Triton X- 100, glucose, trehalose, PBS, hyaluronidase, heparin and aluminum hydroxide gel were obtained from Sigma-Aldrich (Spain). All other chemicals used were of reagent grade or higher purity. 2.2. Preparation of protamine:polysaccharide nanoparticles and loading of rHBsAg Nanoparticles were prepared by an ionic cross-linking technique as previously described by Calvo et al [17]. Briefly, 1 ml of, either protamine, hyaluronic acid or alginate was kept under magnetic stirring at a concentration of 1 mg/ml. The counter ion was also dissolved in purified water and particles with different protamine:polysaccharide ratios ranging from 1:6 to 6:1 (w/w) were prepared 83 CAPÍTULO 3 upon mixing. Nanoparticles were instantaneously obtained by the interaction of the protamine with the polysaccharide. For the encapsulation of rHBsAg, the antigen was mixed with the polysaccharide at a theoretical loading of 2.5 and 5 % (with respect to the total amount of polymers), prior to the production of nanoparticles. 2.3. Nanoparticle characterization 2.3.1. Size and zeta potential The hydrodynamic diameter and polydispersity index of the particles were determined by photon correlation spectroscopy (PCS) after dilution of the samples with ultrapure water. The zeta potential was measured by laser- Doppler anemometry diluting the samples with KCl 1 mM. (Zetasizer®, NanoZS, Malvern Instruments, Malvern, UK), 2.3.2. Morphological analysis The morphological examination of the nanoparticles was carried out by transmission electron microscopy (TEM, CM12 Philips, The Netherlands). The samples were stained with 2% (w/v) phosphotungstic acid solution and dried on a copper grid. 2.3.3. Production yield and nanoparticle composition In order to determine the yield of production, the nanoparticles were isolated by centrifugation for 40 minutes, at 18000 x g and 15ºC (Universal 32R, Hettich Zentrifugen, Germany) and the supernatants were discarded. The sediments of the nanoparticles were freeze-dried (Genesis SQ freeze drier, Virtis, US) and weighed. The yield of production was calculated as follows (equation 1): ሺΨሻൌ  ሺሻൈ ͳͲͲ 84 CAPÍTULO 3 In order to determine the rate of incorporation of the different components, nanoparticle samples were freeze-dried and analyzed by elemental analysis (FISONS EA 1108, United Kingdom). Protamine and hyaluronic acid content in the nanoparticles samples was analyzed by the nitrogen and carbon content in comparison with protamine and hyaluronic acid as raw materials. As additional controls, nanoparticle supernatants, physical mixtures of the components and non-isolated nanoparticles were also analyzed by the same method. 2.4. Stability of the nanoparticles The stability of nanoparticles as an aqueous suspension was evaluated under storage conditions (4ºC for three months) and also under physiological conditions (PBS, 37ºC, pH: 7.4 for 7 days), by measuring nanoparticle size at different time points. 2.5. Association efficiency and structural integrity of rHBsAg Nanoparticles were loaded with the viral antigen rHBsAg as detailed in section 2.2. The efficiency of antigen association was determined by an indirect method, calculating the difference between the total amount of antigen added to the nanoparticle formulation and the amount of rHBsAg remaining in the aqueous external medium. The separation of the antigen-loaded nanoparticles from the aqueous medium was carried by centrifugation for 40 min, at 18000 x g and 15ºC. The amount of non-associated antigen was quantified in the supernatant by ELISA following manufacturer’s instructions. In order to confirm the integrity of the associated antigen, isolated nanoparticles were degraded with a mixture of hyaluronidase and heparin in acetate buffer pH 7.2 during 2 hours at 37ºC. Samples were then centrifuged at 18000 x g for 10 min and analyzed by Western blot as a qualitative tool, following the methods detailed in [18]. 85 CAPÍTULO 3 2.6 Freeze-drying studies Different concentrations of nanoparticles (0.5; 0.25; 0.125 and 0.0625 mg/ml) loaded with rHBsAg at 2.5%, either alone or in combination with cryoprotectants (trehalose or glucose at 5% w/v), were freeze dried using Labconco Corp (USA). Freezing temperature was -20ºC, and the drying process was maintained for up to 72 hours. The nanoparticle formulations were recovered by manual resuspension by adding ultrapure water to the freezedried powders. The reconstituted nanoparticles were characterized with regard to their physico-chemical characteristics (Section 2.3) and the structural integrity of the associated antigen was analyzed by Western blot. 2.6. Cell viability studies The viability studies were performed in adherent RAW 264.7 murine macrophages (ATCC, USA) by Quick cell counting solution (QCCS) (GenScript Corporation, USA). Cells were cultured in RPMI supplemented with 10% (v/v) heated-inactivated fetal bovine serum (FBS) (PAA, Austria), 2 mM glutamine and 100 U/mL of penicillin/streptomycin, at 37ºC in 5% CO2 atmosphere. Periodic tests showed that cultures were mycoplasma free. The procedures were adapted from the protocol previously described by T. Lozano et al [19]. Briefly, 20.000 cells were plated onto 96-well plates. Cells were then incubated with different concentrations of blank nanoparticles (without antigen) protamine:HA 1:4 and 4:1 (from 3.9 to 250 μg/mL) in 200 μl of RPMI 10 % FBS. After 24 or 48 hours, the plates were centrifuged at 100 x g for 5 min, 100 μl of supernatant were discarded in order to add 50 μl of Quick Cell Counting Solution and mixed gently. Subsequently, plates were incubated for 4 hours, centrifuged again and the supernatants were transferred to clean plates, to avoid possible interference due to the presence of nanoparticles. Finally, the absorbance was measured at 450 nm in an Envision multidetector 86 CAPÍTULO 3 (Perkin Elmer Inc, USA). As a positive control, cells were incubated with 5% Triton X-100. As a negative control, RPMI and nanoparticles alone were used. The results were then analyzed through the following equation (equation 2): Ψ ൌ  ൅  െ   െ  ൈ ͳͲͲ 2.7. Cellular uptake studies For these studies, nanoparticles were prepared with fluorescently labeled protamine (protamine-TAMRA). Briefly, protamine was dissolved in 0.1 M sodium bicarbonate buffer, pH 9.0 at 10 mg/ml concentration. On the other hand, 5-carboxy tetramethyl rhodamine succinimidyl ester (TAMRA) was dissolved in DMSO at 1 mg/ml. An aliquot of 100 μl of this dissolution of TAMRA was slowly added to 1 ml protamine solution under stirring. This solution was incubated for 1 hour at room temperature and finally was dialyzed for 48 h, to remove free TAMRA (Slide- A Lyzer® dialysis cassette 2000 MWCO, Thermo). Nanoparticles were then prepared with protamine-TAMRA following the same protocol described in 2.2, using protamine:HA ratios of 1:4 and 4:1. Raw 264.7 cells (5 x 105) were plated onto a 24-well plate (Falcon 3047, BD Biosciences, USA) with 1 ml of RPMI 10% FBS in the presence of labeled nanoparticles (120 μg/ml, 30 min). After washing three times with PBS to remove non internalized nanoparticles, cells were observed under an inverted fluorescent microscope (IX50, Olympus Optical Co GmbH, Germany). For flow cytometry analysis, the adherent cells were washed once with PBS and then detached by incubation with Accutase® (200 μL, 10 min, 37ºC, 5% CO2.) Finally, cells were washed with complete medium, centrifuged, and the resulting cell suspension was analyzed using a flow cytometer (FC500, Beckman-Coulter, USA). 87 CAPÍTULO 3 Internalization of nanoparticles was also examined using a confocal microscope (Leica SP5, Germany). Cells were seeded on a glass coverslip (Menzel-Gläser; Braunschweig, Germany) in a 24-well plate and incubated with nanoparticles as described above. After several washes, cellular cytoskeleton was stained with Alexa Fluor 488-phalloidin (20 min incubation). Finally, the coverslips containing the attached cells were mounted over the slides in the presence of ProLong® Gold Antifade mounting medium (Invitrogen; Eugene, OR, USA) containing DAPI as nucleus stain and were analyzed by confocal microscope. 2.8. Cytokine Secretion Studies Murine peritoneal macrophages were obtained from female BALB/c mice (6-8 weeks old). Three independent experiments were done using for each experiment a pool of peritoneal macrophages from 3 different mice. In brief, 5 mL of DMEM medium (Gibco; Grand Island, NY) containing 10% FBS were injected into the mouse peritoneal cavity and cells were then collected with a Pasteur pipette. After centrifugation at 100 x g for 5 min at 4°C, the cell pellet was resuspended in complete medium. Then 1 × 105 cells were incubated for 24 hours in the presence or absence of rHBsAg-loaded nanoparticles.. As positive and negative controls, cells were incubated with either 1 μg/mL of lipopolysaccharide (LPS) (InvivoGen, USA) or free medium, respectively. After 24 hours, supernatants were collected, centrifuged at 100 x g for 5 min at 4ºC, and stored at -20ºC before analysis. Levels of different cytokines were determined using the FlowCytomixTM assay (eBioscience, Austria) following the instructions of the manufacturer. First, Ab-coated microspheres, samples (culture supernatants) and biotin-conjugated secondary Abs were incubated for 2 hours at room temperature (RT). After several washes, streptavidin conjugated to phycoerythrin was added and incubated for 1 hour at RT. Finally, beads were studied by flow cytometry. 88 CAPÍTULO 3 2.9. Immunization studies The protocol was adapted to the guidelines of the Spanish regulations (Royal Decree 1201/2005) regarding the use of animals in scientific research and was approved by the ethical committee of the University of Vigo. Groups of 10 female BALB/c mice with an average weight of 20 g and 4 weeks of age were randomly assigned. The animals were kept conscious during immunization and subsequent sample collection. The groups receiving intramuscular injections of rHBsAg loaded protamine:HA 1:4 and 4:1 nanoparticles and the control groups receiving alum-adsorbed antigen, were immunized with a prime dose of 10 μg of rHBsAg and a boost dose at day 28. The control alum-adsorbed antigen was freshly prepared before immunization as described by Prego et al [18]. In parallel, intranasal administration with 10 μg of rHBsAg loaded in Pr:HA 1:4 and 4:1 nanoparticles was also performed, with two boost doses at days 28 and 56. Serum samples were collected from the mouse maxillary vein at days 42, 126 and 183 (weeks 6, 18 and 26). A pool of sera from mice administered with each formulation and each time point was prepared and IgG endpoint titers against rHBsAg were determined by ELISA. Briefly, rHBsAg at 5μg/mL diluted in Carbonate Buffer (pH 9.6) was incubated overnight at 4ºC in MaxiSorp 96-well plates. Then plates were blocked with PBS-BSA 1% for 1 hour at 37ºC. Serum samples and a mouse monoclonal IgG directed against rHBsAg used as control were serially diluted and incubated for 2 hours at 37ºC. Then, secondary Ab (goat anti-mouse IgG) was added to each well and incubated for 1h at 37ºC. Finally, bound antibodies were revealed with ABTS and the titers were expressed as mIU/ml concentration. For this purpose control rabbit IgG of known concentration (mIU/mL), were also serially diluted and used to transform data from μg/mL to mUI/mL. All serum samples were tested in triplicates. 89 CAPÍTULO 3 2.10 Statistical analysis The analysis of variance (ANOVA) and the Kruskal-Wallis test were performed using Statgraphic centurion XVI (Statpoint Technology). Differences were considered to be significant al a level of p<0.05. 96 CAPÍTULO 3 Figure 3: Elemental analysis studies of protamine:HA nanoparticles at 1:4 and 4:1 ratio. (n>6 ± SD). : % protamine, : % hyaluronic acid: HA. The nanoparticles yield also confirms the efficient incorporation of both components in the nanosystem and these values are not contradictory with the results obtained by elemental analysis. Additionally, the yield results might indicate that the minor component is completely incorporated in the nanostructure. These results show that it is possible to prepare similar core nanocompositions with sizes and/or different surface characteristics (as shown in Table 1). Stability of nanoparticles The stability of protamine:HA 4:1 and 1:4 nanoparticles upon incubation in biological media (PBS at 37 ºC) and during storage at 4ºC was monitored by analyzing the evolution of mean particle size with time. The results indicated that the size of the nanoparticles remains constant upon incubation in PBS at 37ºC for up to 7 days. In addition, nanoparticles stored at 4ºC remained stable for at least 3 months. Overall, from these results it maybe concluded that protamine:HA nanoparticles have a very positive stability profile. In particular, the stability profile in PBS is quite exceptional if we take into account that these nanoparticles are formed by a simple ionic cross-linking process. Other 0% 20% 40% 60% 80% 100% 120% Theorical Ratio Pr:HA 1:4 No isolated Pr:HA 1:4 Isolated Pr:HA 1:4 Theorical Ratio Pr:HA 4:1 No isolated Pr:HA 4:1 Isolated Pr:HA 4:1 percentage Content Pr (%) Content HA (%) 97 CAPÍTULO 3 protamine based carriers, such as protamine-albumin-oligonucleotide nanoparticles, have also shown an adequate stability profile upon incubation of high ionic strength media) [30]. However, this is not the case for other nanostructures combining polycations such as polyarginine and HA [31]. This underlines the necessity to properly assess the colloidal stability of nanoparticles under physiologically relevant conditions since this property cannot be effectively predicted. Antigen encapsulation efficiency For the entrapment of rHBsAg within the nanoparticles, the antigen was previously mixed with hyaluronic acid at theoretical loadings of 2.5 and 5%, with respect to the total amount of solid material (protamine + hyaluronic acid) and, then, added to protamine solution. As shown in Table 2, the encapsulation of rHBsAg inside the nanoparticles is high, irrespective of the protamine:HA polymer ratio. Nevertheless, some differences between the two different NP compositions could be observed. The particle size increased slightly in the case of nanoparticles with a high amount of hyaluronic acid, while in the case of nanoparticles protamine:HA 4:1 e the size, this increase is more remarkable, around 300 nm. This difference could be explained by the distinct structural organization of nanocarriers made of different polymer ratios. The surface charge was unmodified with respect to the nanoparticles without antigen, thus suggesting that the rHBsAg might be efficiently entrapped within the nanoparticle matrix. In order to confirm that the integrity of the antigen is not affected by the encapsulation process, rHBsAg-loaded protamine:HA 1:4 and 4:1 nanoparticles were degraded with hyaluronidase and the extracted antigen was analyzed by Western blot. Results indicate that the antigenicity of the antigen was preserved during the particle preparation process. Similar results were found for chitosan nanoparticles prepared by ionic cross-linking and the same antigen (rHBsAg) [18]. This protective effect is in fact considered 98 CAPÍTULO 3 a key advantage of these nanocarriers with regard to their ability to entrap delicate molecules, i.e. proteins. Table 2: Physicochemical properties of rHBsAg loaded nanoparticles and their encapsulation efficiency (n>3 ± SD). Freeze-drying studies Despite having no information about the nanoparticles stability at room temperature in aqueous suspension, there is no doubt that preserving this stability would represent a major advance for their use as antigen carriers. Thermostable vaccines would have a particularly great impact in developing countries, where the lack of equipment for vaccine storage at 4ºC and the maintenance of the cold chain still remains a critical limitation. Taking this into account, in the present work, we chose freeze drying as a strategy to increase the stability of vaccine formulations [32]. Nanoparticle formulations were lyophilized at different concentrations, in presence or absence of cryoprotectors (detailed in 2.6) and, then reconstituted for further particle size analysis. The results indicated that the size of the nanoparticles did not suffer significant changes upon freeze-drying and reconstitution. The fact Formulation Pr:HA mass ratio rHBsAg Theoretical Loading (%) Size (nm) PDI ] ] potential (mV) Encap. effic. (%) rHBsAg Loading (μg/ml) 1:4 - 126 ± 13 0.1 -37 ± 4 - - 2.5 146 ± 11 0.1 -31 ± 5 90 ± 3 20.0 5 197 ± 10 0.1 -31 ± 3 92 ± 1 41.1 4:1 - 265 ± 10 0.2 +21 ± 3 - - 2.5 693 ± 29 0.2 +26 ± 1 95 ± 3 21.2 5 563 ± 38 0.2 +23 ± 2 90 ± 2 40.2 99 CAPÍTULO 3 that these nanoparticles do not need any cryoprotective agent for their freezedrying represents a specific advantage of this technology. The ease of freezedrying of the nanocarriers could be attributed to the arginine rich structure of protamine, since this amino acid is used as a stabilizing excipient to prevent damage to the vaccine during the drying process [33]. In addition to these positive results, it was also possible to confirm by Western blot analysis that the antigenicity of the freeze-dried antigen associated to protamine:HA nanoparticles was preserved. Therefore, we can conclude that the association of rHBsAg to protamine:HA nanoparticles represent a useful strategy for designing a powder-based vaccine formulation. Cell viability and nanoparticles internalization studies The toxicity of protamine:HA 1:4 and 4:1 formulations was evaluated in a macrophage cell line (RAW 264.7). For this purpose, RAW 264.7 cells were incubated with blank nanoparticles and the metabolic activity of the cells was monitored by Quick cell counting solution (QCCS). As shown in Figure 4, cells maintain their viability upon incubation with nanoparticle concentration within the range of 3.9 – 62.5 μg/mL. Interestingly, despite the presence of nanoparticles, the cell viability increased over the time. This might be due to the fact that after 48-hrs exposure, cells are able to adapt to the presence of the nanoparticles and their viability is no longer affected. These results are similar to those reported for hyaluronic acid:chitosan nanoparticles, the positive features being attributed to the high biocompatibility of the polymers and its potential implications in biological process such as cellular regeneration [34]. 100 CAPÍTULO 3 Figure 4: Effect of nanoparticles formulations on the viability of Raw 264.7 cells determined by QCCS assay. (n=3 ± SD) The process of internalization of the nanostructures in antigen presenting cells is a crucial step to trigger the immune response. In fact, the recognition and consequent phagocytosis of pathogens by antigen presenting cells is the first step during innate immune responses, allowing the development of an adaptive immune response [35]. Therefore, in order to study the possible interaction of the developed formulations with immune cells, we examined their internalization by RAW 264.7 cells using confocal microscopy. For this study, nanoparticles were prepared with TAMRA conjugated fluorescent protamine. As confirmed by PCS and laser-Doppler anemometry analysis, the use of the protamine-TAMRA conjugate did not alter the physicochemical characteristics of formulations. As illustrated in Figure 5, at 30 minutes postincubation the nanoparticles were efficiently internalized by the cells. Complementary cytometry analysis showed that nanoparticles with a positive charge exhibit a higher uptake than those with a negative charge. This can be attributed to a non-specific uptake mediated by the electrostatic interaction between the positively charged nanoparticles and the negatively charged cell membranes [36] as well as to the inherent penetration properties of protamine [37]. 0 25 50 75 100 125 4 8 16 31 63 125 250 % Viability Nanoparticles concentration (μg/mL) NP Pr:HA 1:4 (24 hours) NP Pr:HA 4:1 (24hours) NP Pr:HA 1:4 (48 hours) NP Pr:HA 4:1 (48 hours) 101 CAPÍTULO 3 Figure 5: Cellular uptake profile of protamine:HA 1:4 and 4:1 nanoparticles. A and B show the flow cytometry analysis and a confocal image of Protamine:HA 1:4 nanoparticles, respectively. C and D show the same data obtained for protamine:HA 4:1 nanoparticles. In the cytometry analysis (A and C), the black curve corresponds to control cells and the red curve represents the cells treated with the internalized labeled nanoparticles. The percentages are related to the number of positive events. The systems were prepared with protamine-TAMRA conjugate (red channel). The cytoskeleton of RAW 264.7 cells was labeled with Alexa Fluor 488-phalloidin (green channel) and the nuclei with DAPI (blue channel). Cytokines secretion studies Cytokines have an important role linking innate and adaptive immune responses. Antigen presenting cells (APCs) recognize pathogens that induce their activation and maturation with the secretion of proinflammatory cytokines, among other processes [38]. These cytokines are multifunctional A D B C 102 CAPÍTULO 3 proteins that are mostly involved in the regulation of immune responses, hematopoiesis and inflammation. In particular, IL-1α has an important role in T and B cell growth and differentiation, whereas TNF α, has an important tumoricidal activity and participates in the growth and differentiation of B cells. As a consequence, cytokines secretion capacity does normally relate to the adjuvant properties of specific compounds [39, 40]. To investigate the adjuvant capacity of protamine:HA nanoparticles, we analyzed their cytokine secretion profile by immunofluorescence in murine macrophages. The results showed that rHBsAg loaded nanoparticles stimulated greater production of TNFα, IL-1α and IL-6 than the free antigen (Figure 6). Apparently, negatively charged nanoparticles, despite their lower internalization, exhibited a greater IL-1α production activity (p<0.05). This could be explained by the differences in size and structural organization of the materials, and their possible internalization through different pathways. For example, while the internalization through scavenger receptors does not always induce an inflammatory response, the internalization through other routes (Fcγ, mannose or complement receptor-mediated) in most cases produces an inflammatory response [41]. 103 CAPÍTULO 3 Figure 6: Cytokines released from macrophages after stimulation with rHBsAg alone ( ) or loaded into protamine:HA 1:4 ( ) and protamine:HA 4:1 nanoparticles ( ). : Positive control: LPS. * Significant differences between groups (p<0.05). Irrespective of this small difference in the cytokine release profile, the results suggest overall that both types of nanoparticles might exhibit potential adjuvant properties [42]. Immunization studies In order to evaluate the potential of protamine-based nanoparticles as antigen delivery carriers, we chose hepatitis B as a model antigen and monitored the IgG responses following either intramuscular or nasal administration to mice. For intramuscular immunization we administered 10 μg of rHBsAg associated to the nanoparticles, at day 1 and day 28. As a positive control, we used alumadsorbed rHBsAg administered according to the same protocol. The results in Figure 7A indicate that both protamine:HA nanoparticles 4:1 and protamine:HA 1:4 nanoparticles induce significant serum IgG antibody levels (> 100 mUI/mL). Although these antibody titers are slightly lower than those induced by the conventional adjuvant (alum), these values are all above those considered to be protective in humans (10 mUI/mL) [43]. Significant differences between the two nanoparticles formulations were only found at the first bleeding (42 days 0 250 500 750 1000 1250 IL6 (X10) IL 1alpha TNF alpha Pg/ml Cytokines * * * * ** ** * * 104 CAPÍTULO 3 post administration), being the formulation with positive zeta potential and higher internalization rate the one inducing higher specific IgG levels. This underlines the importance of the surface charge of the nanoparticles, thus confirming previous data in the literature where cationic liposomes [44] and cationic chitosan nanocapsules [45] produced stronger immune responses than the corresponding neutral nanostructures. Overall, the adjuvant effect of protamine:HA nanoparticles is in agreement with their cytokine production profile showed previously and the protective levels achieved in vivo, although the response triggered by this antigen adsorbed in alum was higher. This may be related to the well-known hyper-responsiveness of mice to this antigen when inoculated with rHBsAg using alum as adjuvant. We also investigated the potential of protamine:HA nanoparticles for nasal vaccination. In this study, we used 3 doses of 10 μg rHBsAg loaded in protamine:HA 4:1 or 1:4 (days 0, 28 and 56). The resulting antibody response is shown in Figure 7B. In contrast to what was found following i.m. administration, in this case we could see clear differences in the responses elicited by the different nanoparticle compositions. While the protamine:HA 1:4 formulation failed to elicit immune responses intranasally, the protamine:HA 4:1 , achieved relatively high specific IgG levels (with a peak of 80mUI/mL at week 18). 105 CAPÍTULO 3 Figure 7: Serum antibody titers of BALB/c mice immunized with protamine:HA 1:4 and protamine:HA 4:1 nanoparticles. (A) After 2 immunizations (0 and 28 days) with rHBsAg-loaded nanoparticles or alum-adsorbed antigen through intramuscular (i.m.) administration. (B) After 3 immunizations (0, 28 and 56 days) with rHBsAg loaded into protamine:HA 1:4 or 4:1 nanoparticles by intranasal (i.n.) administration. In both cases the rHBsAg dose administered was 10 μg/mice. Results are presented as mean ± SD (p<0.05). Similar levels of IgG have been reported with other cationic polymers such as chitosan in form of nanoparticles loaded with rHBsAg and administered by nasal route [18]. In this field, it is worth mentioning the work of Vicente et al [47], where chitosan nanocapsules loaded with the rHBsAg and imiquimod (TLR 7 agonist) trigger a higher immune response when compared with chitosan nanoparticles or the systems described in this work. Further studies loading the antigen together with other adjuvants (such as a TLR agonist) in protamine- -1 0 1 2 3 4 61826 10Log IgG against HBsAg (mUI/mL) Time (weeks) * * B * -1 0 1 2 3 4 61826 10Log IgG against HBsAg (mUI/mL) Time (weeks) HBsAg-Alum Pr:HA 4:1 Pr:HA 1:4 ** ** ** * A CAPÍTULO 4 PROTAMINE NANOCAPSULES: A NEW PLATFORM FOR ANTIGEN DELIVERY Este trabajo ha sido realizado en colaboración con Inmaculada Dalmau- Mena1, Susana Martínez-Pulgarín2, Covadonga Alonso1,2 y José Ángel Escribano1,2. 1Departamento de Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA). Autovía A6 Km. 7.5, 28040, Madrid, Spain. 2Alternative Gene Expression S.L. (ALGENEX), Centro empresarial, Parque Científico y Tecnológico de la Universidad Politécnica de Madrid, Campus de Montegancedo, 28223, Pozuelo de Alarcón, Madrid, Spain. 115 CAPÍTULO 4 ABSTRACT Here we report for the first time a new antigen-drug nanocarrier consisting of an oily core of Miglyol or α tocopherol, and a shell made of protamine, stabilized with pegylated surfactants. This nanocarrier was rationally designed with the final goal of promoting the intracellular delivery of antigens to the immunocompetent cells and to trigger an efficient and long-lasting immune response.Protamine nanocapsules were prepared by the solvent displacement technique and characterized for their size, surface charge and the amount of polypeptide forming the shell around the oily core. They were also studied for their ability to associate influenza H1N1 antigen, which was chosen as a model antigen. The nanometric systems developed presented an average size of 250 nm, positive zeta potential and a great association capacity without loss of the antigen structure. The nanocapsules were stable upon storage at 4ºC in the form of an aqueous suspension for up to 3 months. They could also be freezedried and resuspendend without the need for the use of cryoprotectants. In addition, in vitro studies showed that protamine nanocapsules were efficiently internalized by macrophages without exhibiting any signs of toxicity below the dose of 76 μg/cm2. Finally, the results of the in vivo studies indicated that protamine nanocapsules loaded with influenza antigen trigger an immune response that was comparable to the one achieved using alum as adjuvant. Their versatility for the loading of different antigens and oily immunomodulators together with their stability profile, and adjuvant properties render these nanocapsules as a novel platform for antigen or active molecules delivery. 116 CAPÍTULO 4 1. INTRODUCTION Different kinds of active molecules, either for solubility, degradation or uptake problems, need to be administered with delivery systems that can improve their properties and allow them to achieve their target for a successful therapy. The emerging of new nanosystems tries to solve the current deliver challenges of these complex active molecules. This approach has been used in the immunization area for antigen delivery. In fact, the term nanovaccine has been recently coined to describe nanometric sized carriers studied and used in vaccine development [1]. Using different strategies, it is intended that this nanovaccines can be a new generation of vaccines that act as adjuvants, administered in a single dose, thermostable and/or administered via mucosa allowing a needle-free vaccination approach Recently, our research group has published an innovative and interesting work about chitosan nanocapsules as nanovaccine. These nanocapsules consist of an oily core surrounded by a chitosan coating and allowed the efficient association of antigens in combination with immunostimulants. These chitosan nanocapsules have shown to be an adequate strategy for single-dose immunization against a hepatitis B and for improving vaccine stability through the production of a freeze-dried forme [2]. Based on these promising results and with the aim of further exploring the potential of polymeric nanocapsules for vaccine delivery, in the present work we have explored the feasibility to develop a new nanocapsules platform based on a natural cationic polypeptide, protamine. The name protamine refers to a diverse family of aliphatic and strongly basic arginine-rich proteins (Figure 1), with average molecular mass of 4.5-5.5 kDa [3]. These proteins are synthesized in nature during the late stage of spermatid development in different animals or plants with the function of condensing 117 CAPÍTULO 4 their genetic material [4]. In addition to this main physiological role, the repetitive sequence of arginine present in protamine (about 70%) also provides this polypeptide with an efficient translocation activity through biological membranes [5]. Protamine is an FDA approved drug for reverting the anticoagulant effects of heparin and is also used as an excipient in the formulation of NPH (Neutral Protamine Hagedorn) insulin to prolong its action. Additionally, it has been studied as a protein stabilizer [6], conjugated with enzymes to improve their internalization [7] and as an antibacterial agent [8]. 1)Pro-Arg-Arg-Arg-Arg-Arg-Ser-Ser-Ser-Arg-Pro-Ile-Arg-Arg-Arg-Arg- Arg-Pro-Arg-Ala-Ser-Arg-Arg-Arg-Arg-Arg-Gly-Gly-Arg-Arg-Arg-Arg. 2) Pro-Arg-Arg-Arg-Arg-Ser-Ser-Arg-Arg-Pro-Val-Arg-Arg-Arg-Arg-Arg- Pro-Arg-Val-Ser-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Gly-Arg-Arg-Arg-Arg. Figure 1: Primary structure of the two principal polypeptides of protamine from chum salmon. Protamine and its low molecular weight derivative are frequently used in gene therapy to improve intracellular the delivery of genetic material enhance gene expression [9-11]. In addition, protamine has also been investigated recently as biomaterial in drug delivery applications due to its non-toxic, biocompatible nature and its safety profile [12, 13]. For example, protamine has been used as composing polymer of innovative nanocarriers such as liposomes [14, 15], nanoparticles [16, 17] or layer-by-layer nanocapsules [18, 19]. In the immunization field, there are recent evidences that nanoparticles or microparticles associated or decorated with protamine show higher immune responses when compared with the systems without this component [20, 21]. Moreover, protamine complexes with mRNA, stimulating TNF-α and INF-α secretion in human blood cells and directly activating B cells [22] suggesting that Toll-like receptors could be recognizing this complex and can be acting as adjuvant. 118 CAPÍTULO 4 With this previous information in mind, the main goal of this work has been to design and develop an original carrier consisting of an oily core stabilized with one or more surfactants and surrounded with protamine shell. The versatility of such nanocapsules allows the incorporation of different lipidic materials and oils in their core. Taking advantage of this, the use of oils with adjuvant properties such as α tocopherol (vitamin E) combined with a protamine shell emerges as an interesting strategy to combine the properties of both structural elements (shell and core) and maximize the adjuvant capacity of the developed nanovaccine. These new carriers were characterized with respect to their physicochemical properties and their capacity to associate the model antigen (influenza haemagglutinin) was also evaluated. The optimized carriers were tested for their interaction with macrophages and an in vivo study was also performed as a proof-of-principle of their capacity to trigger immune responses against the associated antigen. In summary, this is the first report about protamine nanocapsules intended to describe the rational design of these carriers and demonstrate their potential use as a new platform for antigen or active molecules delivery. 119 CAPÍTULO 4 2. MATERIALS AND METHODS 2.1 Chemicals Protamine sulphate was kindly donated by LEO Pharma (Denmark). PEG- Stearate (Simulsol® M52) and Macrogol 15 hydroxystearate (Solutol® HS 15) were obtained of Seppic (France) and BASF (Germany) respectively. Miglyol® 812 was a kind gift from Sasol Germany GmbH (Germany) and α-tocopherol (TCPH) was supplied by Merck (Germany). 5-Carboxytetramethylrhodamine succinimidylester single isomer (TAMRA) and Alamar blue were obtained from Invitrogen (United Kingdom). Haemagglutinin from the H1N1 A/PR/8/34 influenza antigen (HI) was kindly offered by Algenex S.A (Spain). Antibodies for Western blot, mouse monoclonal antibodies against the terminal His tag domain of HI and goat anti-mouse IgG-HRP were supplied by Abcam pcl (United Kingdom). Dulbecco's Modified Eagle's Medium (DMEM), glutamine, penicillin and streptomycin were supplied by Gibco (USA). Tween® 20, Tween® 80, d-α tocopheryl polyethylene glycol 1000 succinate (TPGS), glucose, trehalose, Triton-X100, WGA (wheat germ agglutinin), PBS and sodium cholate were obtained from Sigma-Aldrich (Spain). All other products used were of high purity or reagent grade. 2.2 Preparation of protamine nanocapsules Blank protamine nanocapsules were prepared by the solvent displacement technique following the procedure described and optimized by our group [23]. Different amounts of PEG-stearate (Simulsol® M 52) (0, 12, 24 and 48 mg) and sodium cholate (0, 5, 10 and 20 mg) were dissolved in 750 μl of ethanol, followed by the addition of Miglyol® (31.25, 62.5 or 125 μl) and 4.25 ml of acetone. This organic phase was immediately poured over 10 ml of an aqueous phase with 0.05% w/v protamine (one step procedure). The elimination of organic solvents was performed by evaporation under vacuum (Rotavapor Heidolph, Germany), to a constant volume of 5 ml. For the nanocapsule 120 CAPÍTULO 4 preparation method in two steps, we used the same materials and amounts indicated but omitting protamine in the external aqueous phase. The resulting nanoemulsion (NE) was incubated in a subsequent step with a protamine solution (0.5 % w/v). The volume ratio used was 4:1 (NE:protamine solution). Through the same solvent displacement technique, nanocapsules were also prepared using different oils (Miglyol vs tocopherol) in combination with others surfactants such as Solutol® HS 15, Tween® 20, Tween® 80 or TPGS with sodium cholate. The ratio between TCPH and the main surfactant used was 12:12, 60:12 and 60:60 (mass ratio). Protamine nanocapsules were isolated by ultracentrifugation (Optima™ L-90K, Ultracentrifuge, Beckman Coulter, USA) at 30000 RPM for 1h (at 15ºC) and, then, resuspended in ultrapure water to a final theoretical protamine concentration of 1 mg/mL. 2.3 Physicochemical characterization of protamine nanocapsules The hydrodynamic diameter and polydispersity index of the systems were determined by photon correlation spectroscopy (PCS) (Zetasizer®, NanoZS, Malvern Instruments, Malvern, UK), after sample dilution with ultrapure water. Zeta potential was measured by laser-Doppler anemometry after diluting the samples with 1 mM KCl. Morphological examination of nanocapsules was carried out by transmission electron microscopy (TEM, CM12 Philips, The Netherlands). The samples were stained with 2% (w/v) phosphotungstic acid solution. 2.4 Quantification of protamine association to the nanocapsules The amount of protamine associated to the nanocapsules was indirectly measured through the quantification of the polypeptide remaining in the aqueous medium after the isolation of the blank nanocapsules by 121 CAPÍTULO 4 ultracentrifugation (described in 2.2). Protamine was quantified by Ultra Performance Liquid Chromatography (Acquity UPLC, Waters, Spain). The column Acquity UPLC® BEH C18 1.4 μm 2.1X50 mm was used with flow rate of 0.3 ml/min and the following gradient: 0-2 min, 100% A; 2 – 2.1 min 70% A; 2.1- 6 min 100 % A. The column was maintained at 35ºC. Solvent A was trifluoroacetic acid 0.1% (v/v) and solvent B was acetonitrile with 0.1%TFA (v/v). The detector wavelength was set at 214 nm. 2.5 Association of influenza antigen to the nanocapsules and determination of its structural integrity The association of influenza antigen (HI) as model antigen was performed using isolated protamine nanocapsules (section 2.2). These nanocapsules were incubated with different amounts of antigen, in 6:1, 4:1 and 2:1 mass ratios, in equal volumes. Incubation was performed at room temperature for 1 hour. The ability of protamine nanocapsules to associate the antigen was measured by an indirect method by Western blot as described Dickinson et al [24]. Loaded nanocapsules were isolated by ultracentrifugation (section 2.2) and the aqueous solution was analyzed. For this purpose, samples were reduced with βmercaptoethanol and loaded onto a 4% acrylamide stacking gel. Different concentrations of HI were treated in the same way and loaded onto the same gel. Samples were then subjected to electrophoresis on a 10 % separation gel at 200V (Bio-Rad, USA). Antigen and samples for the calibration curve were transferred to a PVDF (polyvinylidene fluoride) membrane at 90V for 90 min. Membranes were soaked subsequently for 1 hour with non-fat milk 5% (w/v) and incubated for another hour in the presence of the primary antibody. After the incubation process, the membranes were carefully washed and the secondary antibody was added, followed by incubation for an additional hour. Finally, the membranes were washed and the antibody–antigen complexes were visualized by chemiluminescence using the ECL Plus Western Blotting 128 CAPÍTULO 4 The analysis of particle size for these formulations showed that the amount of Miglyol® used in the formulation was the principal variable affecting this parameter (Figure 2A). It is possible to see a decrease in the size of the systems when the amount of oil is low, regardless of the oil:surfactans ratio. Previous studies using the same preparation method reported a decrease in particle size with the shell polymer amounts, which does not occur in protamine nanocapsules [32]. Nevertheless, these studies do not evaluate the influence of the amount of oil, which in our case is apparently the most important parameter to take in account. Component Function Miglyol® (μl) Oil 31.25 62.5 125 Peg-stearate (mg) Surfactant 0 12 24 48 Sodium Cholate (mg) 0 5 10 20 Figure 2: Influence of the amount of surfactants (PEG-stearate and sodium cholate) and Miglyol® included in protamine nanocapsules on the mean particle size and zeta potential of the formulations. (A) Size. (B) Zeta potential. With respect to the surface charge, Figure 2B shows that this physicochemical property is clearly affected by the combination of surfactants studied. A B 129 CAPÍTULO 4 Concretely, zeta potential values increase when sodium cholate concentration increases, leading to increasing negativity of the nucleus, which can therefore attract more of the positively charged protamine to form the outer shell and defines the overall positive zeta potential value. On the other hand, the formulations without sodium cholate have closely neutral surface charge, a typical characteristic of systems that include nonionic PEG-containing surfactants [33]. In order to further study the versatility of the developed nanosystems, we have also studied the feasibility of incorporating different oils as core components. Therefore we changed Miglyol® for α tocopherol (TCPH). This oil was chosen for its safety profile, its presence in the regular diet and its adjuvant and antioxidant properties [34, 35]. In Table 1, it is possible to see that the replacement of Miglyol for TCPH does not affect the nanometric size and the polydispersity index of the formulations. Also, the zeta potential remains positive, suggesting the presence of protamine in the shell. Moreover, PEG-stearate (which has PEG in a lineal form) has shown to be necessary in the development of protamine nanocapsules. For this reason, we evaluated the feasibility of using other PEG-containing surfactants such as Macrogol (15)-hydroxystearate (Solutol® HS15), Tween® 20 and Tween® 80, which have less PEG monomers than PEG-stearate and in a ramified form. A special case is d-α tocopheryl polyethylene glycol succinate (TPGS), which is a conjugate between TCPH and PEG, in a linear configuration (similar to PEG- stearate) but with less PEG monomers [36]. In Table 2 we can see the physicochemical characteristics of protamine nanocapsules depending on the different surfactants studied and with different mass ratios. It is possible to observe that all formulations have nanometric size 130 CAPÍTULO 4 with low polydispersity index and zeta potential values between 0-+30 mV. As previously noted, the particle size depends on the oil:surfactant ratio, thereby when the nanocapsules had the same amount of oil and surfactant , the size decreases below 200 nm. This decrease in particle size could be very interesting for other applications where it has been shown that this parameter is an important feature, such as for accesing the lymphatic system [37]. Table 2: Mean particle size, polydispersity index (PI), zeta potential and protamine association of nanocapsules with different surfactants and α tocopherol as oil core (n>3 ± SD).TPGS: D-α-Tocopherol polyethylene glycol succinate Surfactant Units of PEG (Form) Ratio (mg) (TCPH: Surfac.) Size (nm) PDI ] ] Potential (mV) Protamine assoc. (%) PEG - stearate 40 (linear) 12:12 181 ± 11 0.1 +6 ± 1 60:12 234 ± 5 0.2 +1 ± 2 10 ± 3 60:60 203 ± 3 0.1 -4 ± 0,5 PEG- stearate / Sodium Cholate 40 (linear) 60:12 247 ± 9 0.2 +24 ± 3 57 ± 9 Macrogol ( 15) - hydroxyste arate 23 (Ramified) 12:12 146 ± 1 0.1 -1 ± 0.4 60:12 250 ± 39 0.3 +23 ± 5 19 ± 10 60:60 176 ± 23 0.2 -2 ± 0,5 Tween® 20 20 (Ramified) 12:12 143 ± 15 0.4 +18 ± 3 60:12 232 ± 148 0.3 +11 ± 1 Tween® 80 20 (Ramified) 12:12 105 ± 25 0.2 +19 ± 2 60:12 221 ± 26 0.2 +13 ± 4 20 ± 3 TPGS / Sodium Cholate 23 (linear) 60:12 189 ± 5 0.2 +21 ± 6 When we compare the influence of PEG in the different formulations we can see that the size is higher when there are more linear PEG monomers present 131 CAPÍTULO 4 since these are is oriented towards the aqueous phase, increasing the hydrodynamic diameter of the nanocapsules. The protamine association was quantificated by UPLC, It is possible to see that surfactants with shorter PEG chains, such as Solutol® and Tween® 80 produced an increase in protamine association compared with PEG-stearate (20 % vs 10 % association respectively). In addition, the association of protamine was higher when we used a combination of surfactants that increase the negativity of the core, allowing a greater interaction between protamine and the nucleus. The best results were obtained when we used a combination of surfactants such as PEG-stearate and sodium cholate. Due to this result, we have chosen this formulation for further studies. Association of influenza antigen to the nanocapsules Hemagglutinin from the H1N1 A/PR/8/34 influenza antigen (HI) is a protein with a molecular weight of 63 KDa and an isoelectric point close to 7. The antigen was used as model in a solution at pH 9 which could guarantee negative charged and therefore facilitates its association to protamine nanocapsules by ionic interaction. This process was performed by incubation with isolated protamine nanocapsules at different nanocapsule:antigen ratios (section 2.5)., The capacity of protamine nanocapsules to associate HI was quantified by Western blot. As it can be seen in Table 1, the association of HI to isolated blank nanocapsules in a mass ratio 4:1 (protamine:HI) does not affect the nanometric size of the formulation. The surface charge decreases when HI is associated, suggesting that the antigen was effectively associated to the nanocapsules shell. This surface presentation of the antigen is expected to allow rapid recognition by immunocompetent cells and triggers an efficient immune response. 132 CAPÍTULO 4 TEM images (Figure 3) show that protamine nanocapsules loaded with HI maintain their spherical shape and distribution within a homogenous population. Western blot images show that the antigen structure is not affected by the association to the nanocarriers. This is expected since the association method (by incubation) it is a mild procedure, without use of organic solvent or other components that could damage the associated molecule. The association efficacy was near to 70%, demonstrating the high capacity of the nanocapsules to associate the antigen (HI). These results are similar to those reported recently by our group, where polysaccharidic nanocapsules (composed by highly deacetylated chitosan and a squalene core) achieved 69% association efficiency with the same antigen [38]. Figure 3: Transmission electron microscopy (TEM) images of protamine nanocapsules loaded with influenza antigen. Stability of the nanocapsules and freeze-drying studies Antigen-loaded protamine nanocapsule suspensions maintained their nanometric size for at least 3 months under storage (4ºC) and for 7 days in physiological conditions (PBS at 37ºC). This profile can be seen in other nanocapsular systems and it may be due to the polymer or polypeptide corona enclosing the oily nanodroplets and therefore giving stability to the formulation [32]. To have one stable formulation at 4°C is a positive result but the challenge is more ambitious: achieving room temperature stability. Therefore, freezedrying can be and interesting approach for the accomplishment of this purpose. 133 CAPÍTULO 4 Lyophilization is a strategy to increase the stability of colloidal systems and labile molecules, easing their shipment and increasing their storage time in adequate conditions [39]. Different concentrations of blank protamine nanocapsules were freeze-dried in presence or absence of trehalose or glucose at 5 or 10%. As it is shown in Figure 4, the reconstituted powder maintains the nanometric size even without the use of cryoprotectants. This is an excellent property which decreases the cost of the formulation for a possible scaling up. Figure 4: Particle size of protamine nanocapsules (oily core: α tocopherol) upon freeze-drying and further reconstitution in water. The nanocapsules were freeze-dried at different concentrations in the presence of trehalose at 5 or 10 % or in the absence of this cryoprotectant. These results are different from those obtained for other nanocapsules rich in arginine where cryoprotectants were found to be crucial for the recovery of the initial particle size of the formulation [26]. This leads us to speculate that the protamine could play a important role in the freeze-drying process, stabilizing the system and possibly acting as a cryoprotectant. Nor the concentration of nanocapsules neither the type of cryoprotectants showed influence on the physicochemical characteristics of the freeze dried nanocapsules. Figure 4 shows the results obtained with trehalose as a 0 100 200 300 400 500 Before Freezedrying 1.54 1 0.75 0.5 0.25 Size (nm) % NC (w/v) Trehalose 0% Trehalose 5% Trehalose 10% 134 CAPÍTULO 4 representative example from the freeze drying study. Due to its less hygroscopic character, this sugar is considered to have advantages over other cryoprotectants for the freeze drying of nanocarriers [40]. Cell viability and uptake studies Different concentrations of protamine nanocapsules loaded with influenza antigen were incubated in vitro with the macrophage cell line RAW 264.7 and Alamar blue was used for the evaluation of cell viability by measuring mitochondrial activity [41]. As shown in Figure 5, cells viability remained unaltered in a wide range of nanocapsule concentrations (9-76 μg/cm2). Figure 5: Cell viability of Raw 264.7 cells upon contact with different concentrations protamine nanocapsules loaded with influenza antigen (NC:HI 4:1) for 24 and 48 hours. Over this range, viability decreased gradually with increasing concentrations, showing minor differences between 24 and 48 hours. This decrease in cell viability may be due to the fact that polycations, such as protamine, present higher cytotoxicity than neutral or negative macromolecules at high concentration. It is nevertheless possible to control this effect by adjusting the concentration used and the exposure time [42]. Within this regard, the highest 0 50 100 150 9 19 38 76 152 303 606 1212 Cell Viability (%) Protamine Nanocapsules Concentration (μg/cm2) 24 hours 48 hours 135 CAPÍTULO 4 concentration of protamine nanocapsules without toxic effect (76 μg/cm2) is considered to be sufficient for further in vitro and in vivo studies. In addition to cellular toxicity, internalization studies were also performed since the recognition and uptake of the nanocarriers by the immune system cells is considered to be crucial step to trigger an immune response. Protamine was labeled with TAMRA and with this modified polypeptide it was possible to prepare nanocapsules without changing their physicochemical characteristics or the association of influenza antigen (Table 1). Labeled nanocapsules were incubated for 30 min with a primary culture of alveolar macrophages and a line of monkey kidney epithelial cells (Vero cells). As it is shown in Figure 6, both cell types of cells have efficiently internalized the protamine nanocapsules. This result confirms that the positive charge given by the protamine at the systems is an attractive feature for phagocytic cells such as macrophages [43]. Moreover, the ability of protamine to enhance membrane translocation has been reported and this effect has already been described as the basis of the high internalization levels observed for other protamine nanosystems [44]. Different mechanisms have been postulated for this property, although it was known that the basic guanidine group of the arginine forms stable hydrogen bonds with sulfates or phosphates present in biological membranes. This interaction is considered essential for membrane translocation activity and can consequently improve the cellular uptake of protamine-based nanosystems [45]. In vivo studies The immunization studies were performed in BALB/c mice, measuring the systemic humoral immune response elicited after subcutaneous immunization. Animals were immunized with protamine nanocapsules loaded with influenza antigen in two doses (2 and 7.5 μg/ mice) and with 7.5 μg of antigen adsorbed in alum as a control (commercially available as Fluval P). The first immunization 136 CAPÍTULO 4 was followed by two boost-doses (at weeks 3 and 7) and the blood samples were monitored for 28 weeks. Figure 6: Visualization of the internalization of TAMRA-labeled protamine nanocapsules loaded with influenza antigen (HI) by confocal microscopy. A: image of primary culture of alveolar macrophages from pig lungs, where the green channel (A1) was used to visualize the plasma membrane (labelled with fluorescent-WGA), the red channel (A2) illustrates the internalized TAMRA- labeled protamine nanocapsules loaded with HI and the overlapped image is shown in (A3). B: Vero cells (B1) transmitted image (B2) internalized TAMRA- labeled protamine nanocapsules loaded with HI and (B3) overlay. In Figure 7 shows the profile of immune response generated by the different antigen formulations and doses. Protamine nanocapsules presented an elevated peak of immune response in the 3rd week, higher than that achieved for the antigen adsorbed in alum. This immune response is still comparable with the commercial formulation in the following two points and becomes slightly lower at the last time point. On the other hand, when we used three times less antigen dose (2 μg) with the nanocapsule formulation, the immune response only showed a slight decrease, maintaining its effectiveness and protection features. In addition, the capacity of inhibition of hemagglutination induced by the protamine nanocapsules is A B B B A A 137 CAPÍTULO 4 much higher than the dilution considered protective against influenza diseases ( 1:12800 vs 1:64 dilution) [46]. These results open an option to administer low doses of antigen loaded in protamine nanocapsules and to obtain an efficient and long-lasting immune response. Further studies will allow corroboration of this hypothesis. These results are similar to the ones obtained with chitosan nanocapsules, where the immune response generated with 2 μg of the same antigen was similar to the one produced with 7.5 μg of antigen, leading the authors to conclude about the immunopotentiating effect of the carrier used [38]. Figure 7: Serum neutralizing antibody levels against influenza antigen (HI) in mice (5 for each group) elicited upon three subcutaneous injection (0,3 and 5 weeks) of HI-loaded protamine nanocapsules containing two different doses of antigen: 7.5 μg ( ) and 2 μg ( ).Alum-adsorbed HI at the dose of 7.5 μg was used as a control ( ). These in vivo results show the potential of the protamine nanocapsules as a platform for antigen delivery. Protamine as a polycation, due to its repetitive 0 1 2 3 4 100 1000 10000 100000 O.D. (450 nm) Serum Dilution Week 3 0 1 2 3 4 100 1000 10000 100000 Serum Dilution Week 5 NC Pr (7,5 μg HI) NC Pr (2 μg HI) ALUM (7,5 μg HI) 0 1 2 3 4 100 1000 10000 100000 O.D. (450 nm) Serum Dilution Week 7 0 1 2 3 4 100 1000 10000 100000 Serum Dilution Week 28 CAPÍTULO 5 NOVEL THERMOSTABLE NANOVACCINES ANTIGEN-ASSOCIATED PROTAMINE NANOCAPSULES Este trabajo ha sido realizado en colaboración con Mercedes Peleteiro1 y África González-Fernandez1. 1Immunology, Biomedical Research Center (CINBIO), University of Vigo, 36310 Campus Lagoas de Marcosende, Vigo, Spain. 147 CAPÍTULO 5 ABSTRACT One of the main challenges in the development of vaccines is their stability at room temperature, which could allow avoiding the cold chain, administering more effective and safer vaccines. In this work we describe the development and optimization of thermostable nanocarriers consisting of an oily core with immunostimulating activity containing squalene or α tocopherol, surrounded by a protamine shell. These formulations can efficiently associate the recombinant hepatitis B surface antigen (rHBsAg) without losing the antigen epitope. Freeze-dried protamine nanocapsules maintain their physicochemical characteristics for at least 12 months at room temperature and they preserve the integrity and bioactivity of the associated antigen. Nanocapsules show high internalization in immunocompetent cells and efficiently stimulate cytokine secretion and complement activation. Formulations loaded with rHBsAg achieve protective levels upon intramuscular and/or intranasal administration to mice. Overall, our data put, protamine nanocapsules forward as an innovative thermostable nanovaccine platform for improved antigen delivery. 148 CAPÍTULO 5 1. INTRODUCTION Nanoparticles are raising expectations in the antigen delivery field because they combine adjuvant properties with special abilities for overcoming biological barriers. In fact, this specific behavior has rendered nanoparticles promising strategies towards achieving single dose and needle-free vaccines [1]. Among the materials used in the development of antigen delivery nanoparticles polylactic acid (PLA) and related polymers, as well as chitosan, are probably those receiving the greatest deal of attention [2, 3]. This is mainly due to their adequate safety profile and their promising behavior in preclinical research as well as in clinical trials. More recently, the interest has also been focused on the use of biomaterials with inherent adjuvant properties; materials that are able to activate the pattern-recognition receptors (PRRs) of immunocompetent cells and modulate the cytokines secretion profile [4]. Our group has designed a new antigen delivery vehicle, named as chitosan nanocapsules and explored their potential for immunization [5]. These nanocapsules, composed of an oily core and a chitosan shell, are very versatile and may offer advantages as compared to the matrix-type nanoparticles. For example, they can associate different antigens in their shell and lipophilic immunostimulants in their core. Furthermore, the chitosan shell itself and the specific antigen localization may also contribute to their potential role as immunoactive carriers. In fact, these nanocarriers were found to enhance the immunogenic response of antigens, i.e. recombinant hepatitis B surface antigen (rHBsAg) following either, intramuscular or nasal administration [5, 6]. As an alternative, in the current work we have chosen protamine as material for the elaboration of these nanocapsules. Protamine is a rich-arginine polypeptide that has been is approved by the FDA as an excipient in insulin formulation and also as a drug used to neutralize the effect of heparin in cardiac surgery [7]. In addition to this interesting safety record, protamine 149 CAPÍTULO 5 exhibits cell penetrating properties [8] and the ability to enhance the production of cytokines and the proliferation of antigen-specific T cells [9]. There is also a preliminary report on the use of protamine to enhance the adjuvant properties of liposomes upon nasal vaccination [10]. On the other hand, in our attempt to rationally design these nanocapsules, we have selected as a core material, two different types of oils: squalene and α tocopherol (vitamin E), both of them with proven adjuvant activity [11]. In fact, squalene and α tocopherol have been included as adjuvants in some marketed influenza vaccine formulations such as MF59TM and ASO3TM [12]. Finally, the model antigen chosen to assess the potential of protamine nanocapsules for immunization was recombinant Hepatitis B surface antigen (rHBsAg). Although this antigen gives very high responses upon injection in association to alum [13], it has important limitations that remain to be solved. For example, the alum-adsorbed vaccine needs storage in a narrow range of temperature (2-8 ºC), a fact that represents an important limitation for the worldwide distribution of this vaccine [14]. On the other hand, this vaccine needs to be administered according to a multiple injection protocol, and the possibility to be administered in a needle-free format is seen as a major milestone towards improving immunization coverage, especially in developing countries. Based on these premises, in this work we report the development and the comprehensive analysis of protamine nanocapsules as antigen delivery vehicles. Namely, the resulting nanovaccines were characterized with respect to their physic-chemical properties, their capacity to load and release rHBsAg, there stability upon storage as a freeze-dried powder, their ability to stimulate cytokine production in blood mononuclear cells and their in vivo performance, following either intramuscular or intranasal administration. With this work we expect to provide a view of the potential of protamine nanocapsules in the 150 CAPÍTULO 5 development of advanced and safe vaccines that could potentially help increasing the immunization coverage worldwide. 151 CAPÍTULO 5 2. MATERIALS AND METHODS 2.1 Chemicals Protamine sulfate was produced by Yuki Gosei Kogyo, Ltd. (Japan) and offered by LEO Pharma (Denmark). Recombinant hepatitis B surface antigen (rHBsAg) antigen was kindly donated by Shantha Biotechnics Limited (Hyderabad, India). Poliethylenglicol (PEG)-Stearate (Simulsol M52) was from Seppic (France). Squalene (SQL) and α tocopherol (TCPH) were supplied by Merck (Germany). Enzyme linked immunosorbent assay (ELISA) kit (Murex rHBsAg Version 3) was from Diasorin (United Kingdom). Antibodies for Western-blot detection, chicken polyclonal antibody to hepatitis B virus surface antigen and rabbit polyclonal to chicken conjugated with horseradish peroxidase were purchased from Abcam pcl (United Kingdom). For complement analysis by Western-Blot, mouse monoclonal antibody (mAb) against human complement factor 3 (C3) was from Abcam (Cambridge, UK). Secondary polyclonal goat anti-mouse IgG conjugated to alkaline phosphatase were from Dako (Glostrup, Denmark). For ELISA, mouse and rabbit mAbs against HBsAg were purchased from Biokit (Barcelona, Spain) and Acris Antibodies GmbH (Hiddenhausen, Germany), respectively. Secondary Abs (both polyclonal goat anti-rabbit and anti-mouse IgG, IgG1, IgG2a conjugated to horseradish peroxidase) were from Southern Biotech (Birmingham, AL). 5-Carboxytetramethylrhodamine succinimidyl ester (TAMRA) was from Invitrogen (United Kingdom). Fetal bovine serum (FBS), glutamine and penicillin/streptomycin were purchased by PAA (Austria). Glucose, trehalose, PBS, sodium cholate and aluminum hydroxide gel were obtained from Sigma-Aldrich (Spain). All other chemicals used were of reagent grade or higher purity. 152 CAPÍTULO 5 2.2 Preparation of protamine nanocapsules Blank protamine nanocapsules were prepared by a solvent displacement technique [15]. Briefly, PEG-stearate (12 mg) and sodium cholate (5 mg) were dissolved in ethanol (750 μl) and mixed with a solution of the oil, either TCPH (60 mg) or SQL (62.5 μl) in acetone (4.25 ml). The organic phase was immediately poured into 10 ml of aqueous phase with protamine (0.05 % w/v). The formation of the systems was evident due to the milky appearance of the mixture. The elimination of organic solvents was performed by rotaevaporation under vacuum (Heidolph, Germany) until a constant volume of 5 ml. The isolation of the protamine nanocapsules either with SQL or with TCPH was carried out by ultracentrifugation (OptimaTM L-90K, Ultracentrifuge, Beckman Coulter; USA) at 61740 x g for 1h, at 15 ºC. The isolated nanocapsules were resuspended in ultra-pure water to a final theoretical protamine concentration of 1 mg/mL. 2.3 Characterization of protamine nanocapsules The hydrodynamic diameter and polydispersity index of the systems were determined by photon correlation spectroscopy (PCS) (Zetasizer®, NanoZS, Malvern Instruments, Malvern, UK), after sample dilution with ultrapure water. The zeta potential was measured by laser-Doppler anemometry diluting the samples with 1 mM KCl. Morphological examination was carried out by transmission electron microscopy (TEM, CM12 Philips, Netherlands). The samples were stained with 2% (w/v) phosphotungstic acid solution. 2.4 Association of rHBsAg to protamine nanocapsules and determination of its structural integrity The preparation of protamine nanocapsules loaded with rHBsAg was performed after incubation for 1h at room temperature of the isolated 153 CAPÍTULO 5 protamine nanocapsules with the model antigen, at a mass ratio of 4:1 (protamine:rHBsAg). The volume of the protamine nanocapsules suspension and the antigen solution used was the same. The ability of protamine nanocapsules to associate the rHBsAg antigen was determined indirectly, calculating the difference between the total amount of antigen used in the incubation process and the amount of free rHBsAg in the remaining aqueous medium after the isolation process. This amount of nonassociated antigen was quantified by ELISA following the manufacturer’s instructions. Western blot assay (Wb) was used as a qualitative tool for the determination of the antigen’s structural integrity. For this purpose, we followed the same procedure described by Prego et al [16] with minor modification. In our case, the primary antibody was incubated for 1 hour at room temperature and the antibody–antigen complexes were visualized using the ECL Plus Western Blotting Detection Reagents (Amersham Biosciences, UK) in UVP imaging system (EC3™ Imaging System, UVP, USA). 2.5 In vitro release studies of rHBsAg from protamine nanocapsules In vitro release studies of the rHBsAg from protamine nanocapsules were performed to assess the ability of this system to release the associated antigen in its active form. Thus, 50 μl of isolated rHBsAg-loaded protamine nanocapsules (TCPH core) were incubated with 1.95 ml of PBS (pH 7.4) with Tween 80® (0.02 % v/v) and placed in an incubator at 37ºC, under moderate shaking. Previously, we ensured that the physicochemical characteristics of protamine nanocapsules were not modified in the above conditions. At different time points (1, 4, 8 and 24 hours), the suspension was ultracentrifuged, (as described in section 2.2) and the remaining aqueous medium was analyzed for released rHBsAg by ELISA. These samples were also 14 Tal como se ha definido anteriormente, las nanocápsulas de la invención también comprenden de manera opcional al menos un principio activo. El término “principio activo” se refiere a cualquier sustancia que se utiliza en el tratamiento, cura, prevención o diagnóstico de una enfermedad o que se utiliza para mejorar el bienestar físico y mental de seres humanos y 5 animales. El principio activo podrá ser por ejemplo un fármaco, un antígeno, una vitamina, etc. Los sistemas de nanocápsulas objeto de la presente invención son adecuados para incorporar principios activos de naturaleza lipófila o hidrófila. En una realización preferida, los ingredientes activos son antígenos de superficie recombinante de hepatitis B, antígeno de la influenza (H1N1) y docetaxel.10 La proporción de principio activo incorporado dependerá en cada caso del ingrediente activo que va a incorporarse, la indicación para la que se utiliza y la eficiencia de administración. Por otro lado, los procedimientos de obtención de los sistemas de nanocápsulas de la 15 invención son métodos sencillos que evitan condiciones drásticas como altas temperaturas. Además, tampoco es necesario llevar a cabo ningún tipo de reacción química para la obtención de los mismos, ya que según se ha indicado anteriormente la obtención del sistema implica interacciones no covalentes. Por tanto, se preserva así la integridad de las moléculas incorporadas al sistema, susceptibles de ser degradadas. Para lograr la formación de 20 nanocápsulas en un rango de tamaños deseado, se procede a la formación de los núcleos oleosos que comprenden un aceite y uno o más tensoactivos, en cuya superficie se une el polímero de recubrimiento a través de diferentes tipos de interacción. Se trata, por tanto, de un proceso de difusión de solventes, que ocurre de manera controlada y proporciona estabilidad al sistema, sin que exista la necesidad de crear enlaces covalentes entre los componentes.25 Un procedimiento particular para la obtención de los sistemas de la invención (denominado en los ejemplos procedimiento de difusión de disolvente en una etapa), comprende: a) preparar una disolución acuosa que comprende protamina, y opcionalmente un tensoactivo soluble en agua; 30 b) preparar una disolución orgánica que comprende un aceite y uno o más tensoactivos, caracterizados por tener una relación hidrofílica-lipofílica superior a 8, con la condición de que el tensoactivo no es un fosfolípido; 15 c) mezclar bajo agitación las disoluciones preparadas en las etapas a) y b), obteniéndose espontáneamente las nanocápsulas; y d) opcionalmente, evaporar total o parcialmente los disolventes orgánicos de la mezcla obtenida en la etapa anterior hasta volumen constante. 5 Los sistemas de la presente invención se pueden preparar mediante un procedimiento alternativo (denominado en los ejemplos procedimiento de difusión de disolvente en dos etapas) que comprende recubrir una nanoemulsión con protamina mediante un proceso de incubación con una disolución acuosa del polímero. Asimismo, la formación de la nanoemulsión puede favorecerse mediante ultrasonidos (denominado en los ejemplos 10 procedimiento de sonicación) u homogeneización (denominado en los ejemplos procedimiento de homogeneización). En una realización particular, el proceso de incubación comprende mezclar la nanoemulsión con una disolución acuosa del polímero de recubrimiento. Dicha nanoemulsión está 15 constituida al menos por un aceite, uno o más tensoactivos, caracterizados por tener una relación hidrofílica-lipofílica superior a 8 y una fase acuosa. La fase acuosa puede contener otros agentes tensoactivos, sales, y otros agentes auxiliares. Los procedimientos de preparación de dicha nanoemulsión son conocidos en el estado de la 20 técnica, y pueden comprender un proceso de difusión, sonicación u homogeneización (Prego et al. J. Nanosci. Nanotechnol. (2006) 6:1; Tadros et al. Adv. Colloid Interface Sci. (2004) 109:303). Un procedimiento particular para la obtención de la nanoemulsión (denominado en los 25 ejemplos procedimiento de difusión de disolvente) comprende: i) preparar una disolución orgánica que comprende un aceite, uno o más tensoactivos, caracterizados por tener una relación hidrofílica-lipofílica superior a 8, con la condición de que el tensoactivo no es un fosfolípido; 16 ii) añadir la disolución obtenida en la etapa i) sobre una fase acuosa que opcionalmente contiene un tensoactivo soluble en agua y que está bajo agitación para formar una nanoemulsión; iii) opcionalmente, evaporar total o parcialmente los disolventes orgánicos hasta volumen constante.5 Otro procedimiento particular para la obtención de la nanoemulsión (denominado en los ejemplos procedimiento de sonicación) comprende: i) preparar una disolución orgánica que comprende un aceite, uno o más tensoactivos caracterizados por tener una relación hidrofílica-lipofílica superior a 10 8, con la condición de que el tensoactivo no es un fosfolípido; ii) añadir la disolución obtenida en la etapa i) sobre una fase acuosa que opcionalmente contiene un tensoactivo soluble en agua y sonicar; iii) diluir con agua la emulsión obtenida en la fase ii); iv) opcionalmente, evaporar total o parcialmente los disolventes orgánicos hasta 15 volumen constante. Aún otro procedimiento particular para la obtención de la nanoemulsión (denominado en los ejemplos procedimiento de homogeneización) comprende: i) preparar una disolución orgánica que comprende un aceite, uno o más 20 tensoactivos, caracterizados por tener una relación hidrofílica-lipofílica superior a 8, con la condición de que el tensoactivo no es un fosfolípido; ii) añadir la disolución obtenida en la etapa i) sobre una fase acuosa que opcionalmente contiene un tensoactivo soluble en agua y homogeneizar; iii) diluir con agua la emulsión obtenida en la fase ii) y homogeneizar;25 iv) opcionalmente, evaporar total o parcialmente los disolventes orgánicos hasta volumen constante. Según realizaciones particulares de los procedimientos anteriores, si el principio activo es lipofílico o anfifílico, dicho ingrediente activo se añade a la disolución orgánica de la etapa b) 30 17 o de la etapa i). Según otras realizaciones particulares, si el ingrediente activo es hidrofílico, dicho ingrediente activo se añade a la disolución de la etapa a) o de la etapa ii). De manera preferida, dicho ingrediente activo hidrófilo se añade disuelto en una disolución acuosa. También es posible incorporar el ingrediente activo hidrófilo mediante adsorción a la suspensión de nanocápsulas obtenidas en la etapa d) o tras el proceso de incubación una vez 5 formadas las nanocápsulas. En el contexto de la presente invención se entiende por lipofílico a las moléculas, sustancias, principios activos, estructuras o parte de ellas que no son capaces de interaccionar por si sola con moléculas de agua y son principalmente disueltas en solventes apolares.10 En el contexto de la presente invención se entiende por anfifílico a las moléculas, sustancias, principios activos, estructuras o parte de ellas que poseen tanto propiedades hidrofóbicas como hidrofílicas. 15 En el contexto de la presente invención se entiende por hidrofílico a las moléculas, sustancias, principios activos, estructuras o parte de ellas que son atraídas por el agua y son capaces de disolverse en ella o en solventes polares La formación de las nanocápsulas se produce al mezclar volúmenes de las soluciones 20 mencionadas que contienen la nanoemulsión con disoluciones acuosas del polímero de recubrimiento en diferentes proporciones, variando la relación de polímero de recubrimiento. El disolvente de la disolución orgánica es preferentemente una mezcla de disolventes polares tales como etanol, isopropanol y acetona pudiendo incluir además disolventes no polares 25 como por ejemplo el diclorometano. En esta fase orgánica se incorpora el aceite y el o los tensoactivos, caracterizados por tener una relación hidrofílica-lipofílica superior a 8. En una composición particular se incorpora igualmente el ingrediente activo. Un ejemplo particular para la obtención de los sistemas de nanocápsulas de la invención que 30 comprenden protamina siguiendo el primer procedimiento descrito anteriormente comprende: 18 a) preparar una disolución acuosa de 20 ml al 0,05 % p/v de Protamina; b) preparar una fase oleosa compuesta por una disolución etanol/acetona un o ambos tensoactivos (Colato de Sodio y/o PEG estearato), a la que se le adiciona Miglyol® 812. c) mezclar bajo agitación las disoluciones resultantes de las etapas a) y b), 5 obteniéndose espontáneamente las nanocápsulas; d) opcionalmente, evaporar los disolventes orgánicos de la mezcla obtenida en la etapa anterior hasta volumen constante. El procedimiento de elaboración de los sistemas de nanocápsulas puede incluir una etapa 10 adicional de liofilización, con el fin de preservarlos durante su almacenamiento para que conserven sus características iniciales. Debido a la naturaleza de la cubierta de las nanocápsulas de la presente invención, así como de las características de las mismas, no es necesario emplear crioprotectores durante la liofilización. Otra ventaja adicional, es que no es necesario diluir el sistema coloidal antes de la liofilización, ya que los sistemas 15 nanocapsulares de la invención no forman agregados durante la reconstitución del liofilizado. Alternativamente, es posible añadir uno o mas azúcares que ejerzan efecto crioprotector como por ejemplo trehalosa, glucosa, sucrosa, manitol, maltosa, polivinil pirrolidona (PVP). En forma liofilizada, las nanocápsulas pueden ser almacenadas durante largos períodos de tiempo, y ser fácilmente regeneradas, en caso necesario, simplemente añadiendo un volumen 20 de agua óptimo. De acuerdo con esta etapa adicional, la presente invención se refiere también a los sistemas de nanocápsulas que comprenden una cubierta de protamina bajo forma de liofilizado. Adicionalmente, la invención en una realización particular se refiere al sistema nanocápsular 25 de la invención para su uso en terapia. Más concretamente, a una composición farmacéutica, que comprende los sistemas de nanocápsulas de la invención, y opcionalmente uno o más excipientes farmacéuticamente aceptables. En particular, la incorporación de principios activos en las nanocápsulas de la invención origina sistemas, cuyas características en cuanto a su composición, propiedades y morfología, les convierte en excelentes candidatos para el área 30 de la terapéutica. El principio activo a incorporar en los sistemas de la invención será aquél con propiedades farmacoterapéuticas adecuadas de acuerdo con la aplicación terapéutica a la 19 cual sea destinada la formulación. En una realización particular, el principio activo se selecciona entre péptidos, proteínas, compuestos lipídicos o lipofílicos, compuestos sacarídicos, compuestos de ácidos nucleicos o nucleótidos como oligonucleótidos, polinucleótidos o bien combinaciones de las moléculas citadas. 5 En una realización preferida, el principio activo lipofílico es docetaxel. En otra realización preferida, el principio activo es de naturaleza hidrofóbica, anfifílica o hidrofílica. Los principios activos de naturaleza hidrofóbica o anfifílica preferentemente son añadidos en la etapa b) del procedimiento de preparación de nanocápsulas de la invención. 10 Los principios activos de naturaleza hidrofílica preferentemente son añadidos en la etapa a) del procedimiento o en una etapa posterior a la d) mediante un proceso de incubación. Sin embargo, la invención también contempla otras realizaciones como por ejemplo añadir en la etapa b) un principio activo hidrófilo disuelto en un pequeño volumen de fase acuosa. A diferencia de los principios activos hidrofóbicos, que son encapsulados dentro de las 15 nanocápsulas, los principio activos de naturaleza hidrofílica se pueden asociar a la superficie de las mismas mediante adsorción. En una realización preferida, los principios activos hidrofílicos son el antígeno de superficie recombinante de hepatitis B y el antígeno de la influenza (H1N1).20 Dichas composiciones farmacéuticas pueden ser administradas por diferentes vías, tales como a través de mucosas, tópica o parenteral. La proporción de principio activo incorporado en los sistemas puede llegar a ser de hasta 25 aproximadamente el 50% en peso con respecto al peso total, base seca, de los componentes del sistema de nanocápsulas. Sin embargo, la proporción adecuada dependerá en cada caso del ingrediente activo que va a incorporarse, la indicación para la que se utiliza y la eficiencia de administración. En una realización particular, la proporción de principio activo lipofílico puede llegar a ser de hasta aproximadamente el 10% en peso, preferentemente hasta 30 aproximadamente el 5%. 20 Tal como se ha descrito anteriormente, cabe la posibilidad de que los sistemas de nanocápsulas descritos en la presente invención incorporen más de un principio activo, que podrán estar disueltos en la misma disolución o por separado, dependiendo esto de la naturaleza de las moléculas a incorporar, evitando que exista ningún tipo de interacción, bien sea química o física, entre ellos. 5 Tal como se ha definido anteriormente, la invención se refiere al uso de dicho sistema en la preparación de un medicamento. En una realización particular, dicho uso está relacionado con el uso de vacunas o el tratamiento del cáncer. En este sentido, en una realización preferida de la invención, el sistema nanocapsular de la invención comprende como principio activo 10 docetaxel y se usa para el tratamiento del cáncer, preferentemente del cáncer de pulmón o páncreas. Alternativamente, la presente invención también se refiere al uso del sistema nanocapsular de la invención comprendiendo como principio activo docetaxel para la elaboración de un medicamento para el tratamiento del cáncer, preferentemente del cáncer de pulmón o páncreas.15 En otra realización preferida de la invención, el sistema nanocapsular de la invención comprende como principio activo el antígeno recombinante de la hepatitis B (rHBsAg) y se usa para el tratamiento o prevención de la hepatitis B. Alternativamente, la presente invención también se refiere al uso del sistema nanocapsular de la invención comprendiendo como 20 principio activo el antígeno recombinante de la hepatitis B (rHBsAg) para la elaboración de un medicamento para el tratamiento o prevención de la hepatitis B. En aún otra realización preferida de la invención, el sistema nanocapsular de la invención comprende como principio activo el antígeno recombinante de la influenza H1N1 (HI) y se 25 usa para el tratamiento o prevención de la influenza del tipo H1N1. Alternativamente, la presente invención también se refiere al uso del sistema nanocapsular de la invención comprendiendo como principio activo el antígeno recombinante de la influenza H1N1 (HI) para la elaboración de un medicamento para el tratamiento o prevención de la la influenza H1N1.30