Nanocápsulas de quitosano : nuevos vehículos para el transporte de péptidos a través de la mucosa nasal e instestinal

UNIVERSIDAD DE SANTIAGO DE COMPOSTELA FACULTAD DE FARMACIA Departamento de Farmacia y Tecnología Farmacéutica Nanocápsulas de quitosano: nuevos vehículos para el transporte de péptidos a través de la mucosa nasal e intestinal CECILIA PREGO RODRÍGUEZ Santiago de Compostela, 2005 DÑA MARÍA JOSÉ ALONSO FERNÁNDEZ Y DÑA DOLORES TORRES LÓPEZ CATEDRÁTICA Y PROFESORA TITULAR DEL DEPARTAMENTO DE FARMACIA Y TECNOLOGÍA FARMACÉUTICA DE LA UNIVERSIDAD DE SANTIAGO DE COMPOSTELA CERTIFICAN: Que la presente memoria titulada “Nanocápsulas de quitosano: nuevos vehículos para el transporte de péptidos a través de la mucosa nasal e intestinal” ha sido elaborada bajo su dirección por la Licenciada en Farmacia Dña. Cecilia Prego Rodríguez 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 así conste, expiden y firman la presente certificación en Santiago de Compostela a 11 de Mayo de 2005. Fdo. M.J. Alonso Fernández Fdo.: D. Torres López Resumen El objetivo global de la presente memoria experimental ha sido investigar el potencial de las nanocápsulas de quitosano como nuevos vehículos para la administración de péptidos a través de la mucosa nasal e intestinal. Estas nanoestrucutras, constituidas por un núcleo oleoso y una cubierta de quitosano con distintas propiedades (peso molecular, tipo de sal y grado de peguilación del polímero), se prepararon mediante la técnica de desplazamiento del disolvente. Todas las formulaciones presentaron un tamaño de partícula en rango nanométrico, una carga superficial positiva y la capacidad para encapsular eficazmente el péptido modelo calcitonina. Las nanocápsulas de quitosano mostraron una estabilidad adecuada en fluido gástrico. Asimismo, la peguilación del quitosano permitió obtener un sistema estable en los fluidos gastrointestinales. Los estudios llevados a cabo en cultivo celular Caco-2 revelaron que las distintas nanocápsulas de quitosano presentan una baja citotoxicidad, la cual se redujo como consecuencia de la peguilación del quitosano. Además, tras evaluar la interacción de las nanocápsulas de quitosano con un co-cultivo constituido por células Caco-2 y células secretoras de mucus, se observó una asociación muy importante del sistema a las células secretoras de mucus, resultado que constata las propiedades mucoadhesivas de dichas nanoestructuras. Finalmente, la evaluación in vivo de las nanocápsulas de quitosano permitió observar un importante efecto farmacológico tanto tras su administración por vía nasal como por vía oral. Además se observó que modulando el grado de peguilación del quitosano es posible obtener un sistema estable en los fluidos gastrointestinales, de toxicidad muy reducida y con capacidad para promover la absorción oral de péptidos. En definitiva, el trabajo en su conjunto permite pronosticar el interés de las nanocápsulas de quitosano para mejorar la absorción nasal e intestinal de péptidos. Abstract The main goal of the present work has been to investigate the potential of chitosan nanocapsules as new carriers for nasal and oral administration of peptides. These nanostructures, composed of an oily core and a chitosan coating with different properties (molecular weight, type of salt and pegylation degree of chitosan), were prepared by the solvent displacement technique. All the formulations presented a particle size in nanometer range, a positive zeta potential and the ability to encapsulate salmon calcitonin as a model peptide. The nanocapsules were stable in the gastric fluid. In addition, the pegylation of chitosan improved the stability of the nanocapsules in the gastrointestinal fluids. The studies performed in Caco-2 cells revealed that chitosan nanocapsules have a low citotoxicity, which could be further reduced as a consequence of the chitosan pegylation. Moreover, the evaluation of the interaction of chitosan nanocapsules with the co-culture Caco-2:HT29-M6 (enterocytes and mucus-secreting cells), evidenced an important association of the system with mucus-secreting cells, thus corroborating the mucoadhesive properties of these nanocapsules. Finally, the studies performed in vivo indicated that chitosan nanocapsules are able to elicit a pharmacological effect after nasal or oral administration to rats. Additionally, it was observed that modulating the pegylation degree of chitosan it was possible to achieve a stable carrier with a very low toxicity and the ability to promote the oral absorption of peptides. Briefly, altogether these results allowed us to suggest the interest of chitosan nanocapsules for improving the nasal and intestinal absorption of peptides. Índice Página Introducción 1 Artículo 1 de revisión: The potential of chitosan for the oral administration of peptides 19 Antecedentes, hipótesis y objetivos 55 Trabajo experimental 63 Parte 1: Evaluación de las nanocápsulas de quitosano como nuevos sistemas ara la administración nasal de péptidos 65 Artículo 2: Chitosan nanocapsules: a new carrier for nasal peptide delivery 67 Parte 2: Evaluación del potencial de las nanocápsulas de quitosano como sistemas para la administración oral de péptidos 93 Artículo 3: Transmucosal macromolecular drug delivery 95 Artículo 4: Chitosan nanocapsules as carriers for oral peptide delivery: Effect of chitosan molecular weight and type of salt on their in vitro behaviour and in vivo effectiveness 123 Parte 3: Evaluación del potencial de las nanocápsulas de quitosano-PEG como sistemas de administración de péptidos por vía oral 149 Artículo 5: Chitosan-PEG nanocapsules as new carriers for oral peptide delivery. Effect of chitosan pegylation degree 151 Parte 4: Estudio del mecanismo de interacción de las nanocápsulas de quitosano con células Caco-2 y con células goblet (HT29-M6) 181 Artículo 6: Efficacy and mechanism of action of chitosan nanocapsules for oral peptide delivery 183 Discusión general 209 Conclusiones 239 Bibliografía 245 Anexo: Trabajo científico relacionado 265 INTRODUCCIÓN Introducción 3 Introducción Los avances logrados en el campo de la biotecnología y de la ingeniería genética han permitido el creciente desarrollo de macromoléculas activas de naturaleza peptídica y proteica. A pesar del interesante potencial terapéutico de estas macromoléculas, su aplicación en la práctica clínica se ve notablemente restringida debido a la necesidad de ser administradas por vía parenteral. Esta necesidad deriva de la alta susceptibilidad de estas macromoléculas frente a la degradación y a su dificultad para atravesar barreras biológicas. Como consecuencia, en la actualidad, uno de los principales retos en el campo de los nuevos sistemas de liberación de fármacos, lo constituye el diseño de estrategias dirigidas a la administración de estas macromoléculas por vías alternativas a la parenteral, como son la vía nasal y la vía oral. Interés de la vía nasal para la administración de péptidos Tradicionalmente, la vía nasal se ha utilizado para el tratamiento local de distintas enfermedades como son la congestión nasal, infecciones o alergias nasales. 10 Nanocápsulas de quitosano: nuevos vehículos… el epitelio intestinal y de liberar la insulina de forma sostenida33. Por otro lado, las nanocápsulas han mostrado poseer un efecto protector del péptido encapsulado frente a la degradación enzimática33. Estudios posteriores realizados en perros han permitido constatar el potencial de estas formulaciones, si bien los resultados del efecto hipoglucémico tanto en intensidad como en extensión han sido menos llamativos que los obtenidos en ratas34. Dentro de la gama de polímeros acrílicos utilizados para mejorar la absorción oral de insulina cabe destacar los interesantes resultados obtenidos para las nanopartículas elaboradas a partir de poli(ácido metacrílico-etilén glicol) P(MAA-g-EG)35, 36, 37. La eficacia de estos vehículos se debe a su capacidad para responder a cambios de pH, lo que permite proteger al péptido atrapado en la matriz del entorno gástrico y liberarlo en el fluido intestinal35. Además, se ha demostrado que estos vehículos poseen propiedades mucoadhesivas38, así como también la facultad de inducir una reducción de la resistencia transepitelial en cultivos Caco-2, dando lugar a un aumento en el transporte de la insulina a través de las células39. 33 Damge C., Michel C., Aprahamian M., Couvreur P., Devissaguet J.P. Nanocapsules as carriers for oral peptide delivery. J. Control. Release (1990) 13: 233-239. 34 Damgé C., Hillaire-Buys D., Puech R., Hoeltzel A., Michel C., Ribes G.Effects of orally administered insulin nanocapsules in normal and diabetic dogs. Diabetes, Nutrition & Metabolism (1995) 8: 3-9. 35 Lowman A.M., Morishita M., Kajita M., Nagai T., Peppas N.A. Oral delivery of insulin using pH-responsive complexation gels. J. Pharm. Sci. (1999) 88: 933-937. 36 Nakamura K., Murray R.J., Joseph, J.I., Peppas N.A. Morishita, Mariko; Lowman, Anthony M. Oral insulin delivery using P(MAA-g-EG) hydrogels: effects of network morphology on insulin delivery characteristics. J. Control. Release (2004) 95: 589-599. 37 Morishita M., Goto T., Peppas N.A., Joseph J.I., Torjman M.C., Munsick C., Nakamura K., Yamagata T., Takayama K., Lowman A.M. Mucosal insulin delivery systems based on complexation polymer hydrogels: effect of particle size on insulin enteral absorption. J. Control. Release (2004) 97: 115-124. 38 Ascentiis A., deGrazia J.L., Bowman C.N., Colombo P., Peppas N.A. Mucoadhesion of poly(2-hydroxyethyl methacrylate) is improved when linear poly(ethylene oxide) chains are added to the polymer network. J. Control. Release (1995) 33: 197-201. 39 Ichikawa H., Peppas N.A. Novel complexation hydrogels for oral peptide delivery: In vitro evaluation of their cytocompatibility and insulin-transport enhancing effects using Caco-2 cell monolayers. J. Biomed. Mat. Res. Part A (2003) 67: 609-617. Introducción 11 Las nanopartículas de poli(ácido láctico-glicólico) (PLGA) en asociación con polianhídridos, ácido fumárico y sebácico, también han resultado exitosas para mejorar la absorción oral de la insulina40 41. Los autores atribuyen este resultado positivo a las propiedades mucoadhesivas de los polianhídridos y a la capacidad de las nanopartículas de ser internalizadas. No obstante también señalan la necesidad de incorporar PLGA a las nanoestructuras ya que las formulaciones preparadas sólo con polianhídridos o con PLGA no resultaron exitosas. En cuanto a las formulaciones coloidales destinadas a mejorar la absorción intestinal de calcitonina, cabe destacar las nanopartículas de poliestireno recubiertas con derivados polivinílicos, las nanopartículas de PLGA conteniendo complejos de ácidos grasos y los liposomas de doble capa. La asociación de calcitonina a nanopartículas constituidas a base de núcleos de poliestireno recubiertos por cadenas de derivados de poli-ácido metacrílico, poliacrilamida, poli-vinilamida y poli-vinilacetamida permitió lograr un efecto hipocalcémico dependiente de la estructura química del material de recubrimiento42, 43, 44. Así pues, los mejores resultados se obtuvieron tras la administración de estas nanopartículas recubiertas por cadenas hidrofílicas catiónicas (cadenas de poli-Nisopropilacrilamida copolimerizadas con poli-vinilacetamida). El descenso del nivel de calcio observado con estas nanopartículas se atribuyó a sus propiedades 40 Mathiowitz E., Jacob J.S., Jong Y.S., Carino G.P., Chickering D.E., Chaturvedi P., Santos C.A., Vijayaraghavan K., Montgomery S., Bassett M., Morrell C. Biologically erodable microspheres as potential oral drug delivery systems. Nature (1997) 386: 410-414. 41 Carino G.P., Jacob J.S., Mathiowitz E. Nanosphere based oral insulin delivery. J. Control. Release (2000) 65: 261-269. 42 Sakuma S., Suzuki N., Kikuchi H., Hiwatari K., Arikawa K., Kishida A., Akashi M. Oral peptide delivery using nanoparticles composed of novel graft copolymers having hydrophobic backbone and hydrophilic branches. Int. J. Pharm. (1997) 149: 93-106. 43 Sakuma S., Suzuki N., Kikuchi H., Hiwatari K., Arikawa K., Kishida A., Akashi M. Absorption enhancement of orally administered salmon calcitonin by polystyrene nanoparticles having poly(N-isopropylacrylamide) branches on their surfaces. Int. J. Pharm. (1997) 158: 69-78. 44 Sakuma S., Suzuki N., Sudo R., Hiwatari K., Kishida A., Akashi M. Optimized chemical structure of nanoparticles as carriers for oral delivery of salmon calcitonin. Int. J. Pharm. (2002) 239: 185-195. 12 Nanocápsulas de quitosano: nuevos vehículos… mucoadhesivas45 y a la capacidad de inhibir la degradación enzimática del péptido asociado46. En cuanto al éxito de las nanopartículas de PLGA para la administración oral de calcitonina (en forma de complejo con oleato sódico) cabe destacar un aumento importante de la concentración del péptido en sangre47. Este resultado fue atribuido al efecto protector de las nanopartículas frente a la actividad enzimática. Por otro lado, estudios realizados en el cultivo celular Caco-2 mostraron que las nanopartículas poseen capacidad para transportar la calcitonina a través del epitelio intestinal mediante transcitosis. Por último, los estudios realizados tras la asociación de calcitonina a liposomas de doble capa con carga positiva, negativa y neutra mostraron que el máximo efecto hipocalcémico ocurría con los liposomas dobles cargados positivamente48, 49. Este efecto se atribuyó a la interacción entre el liposoma cargado positivamente y la mucosa intestinal. Hipotéticamente, dicha interacción daría lugar a un aumento del tiempo de residencia del sistema en el intestino, traduciéndose en una mejora en la absorción del péptido. En definitiva, el conjunto de los resultados recogidos en la literatura relativos a la utilización de nanosistemas para mejorar la absorción de péptidos indican que el éxito de los mismos está directamente relacionado con su capacidad protectora del péptido encapsulado, así como también con su facilidad para 45 Sakuma S., Sudo R., Suzuki N., Kikuchi H., Akashi M., Ishida Y., Hayashi M. Behavior of mucoadhesive nanoparticles having hydrophilic polymeric chains in the intestine. J. Control. Release. (2002) 81: 281-290. 46 Sakuma S., Ishida Y., Sudo R., Suzuki N., Kikuchi H., Hiwatari K., Kishida A., Akashi M., Hayashi M. Stabilization of salmon calcitonin by polystyrene nanoparticles having surface hydrophilic polymeric chains, against enzymatic degradation. Int. J. Pharm. (1997) 159: 181189. 47 Yoo H.S., Park T.G. Biodegradable nanoparticles containing protein-fatty acid complexes for oral delivery of salmon calcitonin. J. Pharm. Sci. (2004) 93: 488-495. 48 Ebato Y., Kato Y., Onishi H., Nagai T., Machida Y. In vivo efficacy a novel double form of salmon calcitonin. Drug Dev. Res. (2003) 58: 253-257. 49 Yamabe K., Kato Y., Onishi H., Machida Y. Potentiality of double liposomes containing salmon calcitonin as an oral dosage form. J. Control. Release (2003) 89: 429-436. Introducción 13 interaccionar con la mucosa intestinal. Asimismo, en algunos casos, se señala el efecto de los polímeros constitutivos de los nanosistemas en las uniones estrechas intercelulares. No obstante, además de estos factores, un aspecto determinante, aunque no suficientemente discutido, de la eficacia de los nanosistemas reside en su estabilidad en los fluidos gastrointestinales y en su capacidad de control de la liberación. En efecto, estos nanosistemas no han de liberar el péptido asociado de forma prematura en los fluidos gastrointestinales; sin embargo han de procurar su liberación una vez en contacto con la mucosa intestinal. Por tanto, se podría concluir resaltando el interesante potencial de los nanosistemas a la vez que llamando la atención acerca de la necesidad de su optimización de forma individualizada, en función de las características del péptido a asociar. Tabla 1: Estudios basados en la administración oral de péptidos encapsulados en nanoestructuras. Sistema Composición Fármaco Parámetro analizado Ref. Nanopartículas PECL Ciclosporina Biodisponibilidad 50 Nanopartículas Ácido esteárico Ciclosporina Biodisponibilidad 51 Nanopartículas Lipídicas Ciclosporina Biodisponibilidad 52 Nanopartículas Eudragit® Ciclosporina Biodisponibilidad 53 Nanopartículas Eudragit® y ácidos grasos Ciclosporina Biodisponibilidad. 54 Nanopartículas HPMCP Ciclosporina Biodisponibilidad 55 Nanocápsulas PIBCA Insulina Respuesta farmacológica 56, 57, 58 Nanocápsulas PIBCA Insulina Biodisponibilidad 59 Nanopartículas PIBCA Insulina Respuesta farmacológica 60, 61 Nanopartículas PECA Insulina Respuesta farmacológica 62 Hidrogeles P(MAA-g-EG) Insulina Respuesta farmacológica Biodisponibilidad 63, , 64 65 Nanopartículas P(FA:SA) Insulina Respuesta farmacológica 66 Nanopartículas FAO:PLGA Insulina Respuesta farmacológica 67 Nanopartículas PS-g-PNIPAA/ PS-g-PNIPAAg-PVA Calcitonina Respuesta farmacológica 68, , 69 70 Nanopartículas PLGA/ ácidos grasos Calcitonina Biodisponibilidad 71 14 Nanocápsulas de quitosano: nuevos vehículos… 50 Varela M.C., Guzman M., Molpeceres J., Aberturas M.R., Rodriguez-Puyol D., RodriguezPuyol M. Cyclosporine-loaded polycaprolactone nanoparticles: immunosuppression and nephrotoxicity in rats. Eur. J. Pharm. Sci. (2001) 12: 471-478. 51 Zhang Q., Yie G., Li Y., Yang Q., Nagai T. Studies on the cyclosporin A loaded stearic acid nanoparticles. Int. J. Pharm. (2000) 200: 153-159. 52 Bekerman T., Golenser J., Domb A. Cyclosporin nanoparticulate lipospheres for oral administration. J. Pharm. Sci. (2004) 93: 1264-1270. 53 Dai J., Nagai T., Wang X., Zhang T., Meng M., Zhang Q. pH-sensitive nanoparticles for improving the oral bioavailability of cyclosporine A. Int. J. Pharm. (2004) 280: 229-240. 54 Ubrich N., Schmidt C., Bodmeier R., Hoffman M., Maincent P. Oral evaluation in rabbits of cyclosporin-loaded Eudragit RS or RL nanoparticles. Int. J. Pharm. (2005) 288: 169-175. 55 Wang X., Dai J., Chen Z., Zhang T., Xia G., Nagai T., Zhang Q. Bioavailability and pharmacokinetics of cyclosporine A-loaded pH-sensitive nanoparticles for oral administration. J. Control. Release (2004) 97: 421-429. 56 Damge C., Michel C., Aprahamian M., Couvreur P. New approach for oral administration of insulin with poly(alkyl cyanoacrylate) nanocapsules as drug carrier. Diabetes (1988) 37: 246-251. 57 Damge C., Michel C., Aprahamian M., Couvreur P., Devissaguet J.P. Nanocapsules as carriers for oral peptide delivery. J. Control. Release (1990) 13: 233-239. 58 Damgé C., Hillaire-Buys D., Puech R., Hoeltzel A., Michel C., Ribes G.Effects of orally administered insulin nanocapsules in normal and diabetic dogs. Diabetes, Nutrition & Metabolism (1995) 8: 3-9. 59 Cournarie F., Auchere D., Chevenne D., Lacour B., Seiller M., Vauthier C. Absorption and efficiency of insulin after oral administration of insulin-loaded nanocapsules in diabetic rats. Int. J. Pharm. (2002) 242: 325-328. 60 Damge C., Vranckx H., Balschmidt P., Couvreur P. Poly(alkyl cyanoacrylate) nanospheres for oral administration of insulin. J. Pharm. Sci. (1997) 86: 1403-1409. 61 Radwan M.A. Enhancement of absorption of insulin-loaded poly(isobutyl cyanoacrylate) nanospheres by sodium cholate after oral and subcutaneous administration in diabetic rats. Drug Develop. Ind. Pharm. (2001) 27: 981-989. 62 Radwan M.A., Aboul-Enein H.Y. The effect of oral absorption enhancers on the in vivo performance of insulin-loaded poly(ethyl cyanoacrylate) nanospheres in diabetic rats. J. Microencapsulation (2002) 19: 225-235. 63 Lowman A.M., Morishita M., Kajita M., Nagai T., Peppas N.A. Oral delivery of insulin using pH-responsive complexation gels. J. Pharm. Sci. (1999) 88: 933-937. 64 Nakamura K., Murray R.J., Joseph, J.I., Peppas N.A. Morishita, Mariko; Lowman, Anthony M. Oral insulin delivery using P(MAA-g-EG) hydrogels: effects of network morphology on insulin delivery characteristics. J. Control. Release (2004) 95: 589-599. 65 Morishita M., Goto T., Peppas N.A., Joseph J.I., Torjman M.C., Munsick C., Nakamura K., Yamagata T., Takayama K., Lowman A.M. Mucosal insulin delivery systems based on complexation polymer hydrogels: effect of particle size on insulin enteral absorption. J. Control. Release (2004) 97: 115-124. Introducción 15 66 Mathiowitz E., Jacob J.S., Jong Y.S., Carino G.P., Chickering D.E., Chaturvedi P., Santos C.A., Vijayaraghavan K., Montgomery S., Bassett M., Morrell C. Biologically erodable microspheres as potential oral drug delivery systems. Nature (1997) 386: 410-414. 67 Carino G.P., Jacob J.S., Mathiowitz E. Nanosphere based oral insulin delivery. J. Control. Release (2000) 65: 261-269. 68 Sakuma S., Suzuki N., Kikuchi H., Hiwatari K., Arikawa K., Kishida A., Akashi M. Oral peptide delivery using nanoparticles composed of novel graft copolymers having hydrophobic backbone and hydrophilic branches. Int. J. Pharm. (1997) 149: 93-106. 69 Sakuma S., Suzuki N., Kikuchi H., Hiwatari K., Arikawa K., Kishida A., Akashi M. Absorption enhancement of orally administered salmon calcitonin by polystyrene nanoparticles having poly(N-isopropylacrylamide) branches on their surfaces. Int. J. Pharm. (1997) 158: 69-78. 70 Sakuma S., Suzuki N., Sudo R., Hiwatari K., Kishida A., Akashi M. Optimized chemical structure of nanoparticles as carriers for oral delivery of salmon calcitonin. Int. J. Pharm. (2002) 239: 185-195. 71 Yoo H.S., Park T.G. Biodegradable nanoparticles containing protein-fatty acid complexes for oral delivery of salmon calcitonin. J. Pharm. Sci. (2004) 93: 488-495. Abreviaturas DMPC: Dimiristoilfosfatidilcolina DPPC: dipalmitoil-fosfatidilcolina FAO:PLGA: Oligómeros de ácido fumárico: ácido poliláctico HPMCP: Ftalato de hidroxipropil metilcelulosa P(FA:SA): poli(ácido fumárico y sebácico) P(MAA-g-EG): poli(ácido metacrílico-etilén glicol) PECA: poli(etilcianoacrilato) PECL: poli-ε-caprolactona PIBCA: poli(isobutilcianoacrilato) PIHCA: poli(isohexilcianoacrilato) PLGA: poli(ácido láctico-glicólico) PS-g-PNIPAA: poliestireno-poli-N-isopropilacrilamida PS-g-PNIPAA-g-PVA: poliestireno-poli-N-isopropilacrilamida-g-polivinilamina SA: estearilamina Artículo de revisión: El potencial del quitosano para la administración oral de péptidos. 26 Nanocápsulas de quitosano: nuevos vehículos… From a pharmaceutical perspective, chitosan has also attracted significant attention for a variety of applications and modalities of administration. One area in which interest is growing is related to the use of chitosan as a material for transmucosal drug delivery. This is justified by some interesting characteristics of chitosan, such as bioadhesiveness and absorption promoting characteristics. These two properties have been exploited for achieving either, local or systemic drug delivery. For example, chitosan has become particularly well known because of its ability to increase the systemic absorption of drugs and vaccines administered intranasally [8-13]. A clear proof of its efficacy and acceptability is the fact that there are several formulations in different stages of clinical trials. For example, at this moment a chitosan-based liquid formulation is in clinical evaluation for nasal administration of several drugs and vaccines [14, 15]. Chitosan has also shown interesting potential as an ocular drug delivery agent [16]. For this specific modality of administration, the interest relies on the ability of chitosan-based systems to enhance the intensity and time of retention of topically applied drugs [17-19]. A wide range of applications have also been identified for the use of chitosan in oral drug delivery. A simple one would be its use as a hydrophilic excipient to increase the solubility of poorly soluble drugs [20]. Chitosan can also be presented in the form of microspheres (cross-linked or reacetylated) which have the ability to control the release of therapeutic agents along the intestinal tract [21, 22] or to deliver drugs, i.e. antibiotics, locally to the gastric wall [23]. Finally, a number of chitosan derivatives and chitosan-based carriers have been proposed to increase the systemic absorption of peptides by providing the polymer with a better solubility at the absorption intestinal pH or an enhanced mucoadhesive and permeation enhancing properties [24-26]. There are already several general reviews on the general potential of chitosan in the field of drug delivery [26-28] as well as specific reviews covering the application of chitosan for nasal [29] and ocular drug delivery [16]. Therefore, in this article we aimed to review a specific application of chitosan with a great market potential: the enhancement of the oral absorption of peptides. This is with no doubt one of the greatest challenges confronted by the pharmaceutical scientists in the last decade. A challenge that, on the other hand, is clearly justified by the increasing Artículo 1: The potential of chitosan for oral… 27 number of macromolecular drugs approved and which have to be administered parenterally. Keeping this goal in mind, we will first describe the physicochemical and the biopharmaceutical properties of chitosan that are critical for the oral application of chitosan. Then, in the second part, we will present the features and efficacy of the different chitosan delivery systems evaluated so far for oral peptide delivery, making the distinction between solutions, microspheres and nanostructures. Finally, we will analyze the mechanisms suggested until now in order to explain the way these systems are able to enhance the bioavailability of orally administered peptides. Chitosan properties Bearing in mind that this review is focused on the use of chitosan as a material for oral peptide delivery, in the next paragraphs we will discuss about some physicochemical and biological properties which may affect this specific application of chitosan. In this sense, it is important to keep in mind that chitosan comprises a series of polymers, which vary in the percentage of N-acetyl groups, the degree of deacetylation, and in the molecular weight. Both characteristics are critical, not only from a physicochemical point of view, but also from a biological perspective. Solubility properties A property that affects greatly the solubility of chitosan is its deacetylation degree. Highly deacetylated chitosans (85%) are readily soluble up to a pH value of 6.5. However, their solubility decreases significantly with the deacetylation degree [30]. Consequently, most of the studies regarding the pharmaceutical use of chitosan have been performed with highly deacetylated chitosan. Highly deacetylated and purified chitosans are commercially available, in a broad range of molecular weights, in the form of a base and also as a salt. The chitosan base form is soluble in acidic solutions such as hydrochloric, glutamic, 28 Nanocápsulas de quitosano: nuevos vehículos… acetic and lactic acid solutions, in which the amino groups of chitosan become protonated leading to a positively charged polymer. Obviously, chitosan salts do not require the use of acids and are readily soluble in water. However, irrespective of the initial form, the solubility of chitosan decreases significantly when raising the pH to neutral or basic values. In addition to the pH, the ionic strength affects the solubility of chitosan. The higher the ionic strength is, the lower the solubility. In fact, a higher electrolyte concentration results in a salting-out effect which leads to the precipitation of the polymer [31]. This solubility behaviour is important from the perspective of the use of chitosan for oral administration. Indeed, chitosans administered orally in the form of an aqueous solution are expected to precipitate upon reaching the intestinal region due to the increase in the pH up to values in the range 6.5-7.5. On the other hand, chitosans administered as powders are supposed to dissolve in the acidic pH of the gastric cavity and, then, precipitate in the intestinal compartment. Consequently, the behaviour of classical solutions and powders justifies the search of optimized presentations of chitosan, either in the form of a chemical derivative that is soluble at the intestinal pH or, in the form of a device (nanoparticles/ microspheres) that is stable in the physiological conditions of the gastrointestinal tract. Penetration enhancement properties It is well known that chitosan can enhance the permeability of different compounds through the intestinal monolayer. This has been very clearly shown using the Caco-2 cell line for a variety of compounds such as buserelin, inulin, mannitol, and horseradish peroxidase [32-36]. The increase in the permeability has been generally related to a decrease in the transepithelial electric resistance (TEER) which was attributed to a partial disruption of the tight junctions. Moreover, the results of these studies have shown that the increase in the permeability of the monolayer and, hence, on the transport of drugs is dependent on a number of factors, such as the chitosan dose as well as the chitosan molecular weight and deacetylation degree [33-35]. More specifically, Schipper et al. [34] observed, using mannitol as a Artículo 1: The potential of chitosan for oral… 29 model molecule, that chitosans with low acetylation degree (1-15%) showed a clear effect on the transport of the molecule, regardless the molecular weight. In contrast, for high acetylation degree of chitosan (>35%), a permeability enhancement of mannitol across the monolayers was only achieved with high molecular weight chitosans. Despite the evidence of the penetration enhancement ability of chitosan, the mechanism of action remains unclear. Studies performed at the beginning nineties concluded that the exposure of the cell surface to chitosan solutions induces clear changes in the F-actin distribution [32]. Some years later, it was observed that chitosan induces not only a redistribution of F-actin but also that of the tight junction proteins zona occludens 1 (ZO-1) [33, 35]. In addition, it was found that the content of ZO-1 and occludin in the cytoskeleton increase, revealing the ability of chitosan to disrupt the epithelial cell tight junctions involving the translocation of the proteins ZO-1 and occludin from the membrane to the cytoskeleton [35]. Very recently, the same authors found that chitosan acts, at least in part, via an activation of protein kinase C (PKC). [37] Interestingly, the results of the previous studies [33-35] also indicated that the effect of chitosan on the Caco-2 cell monolayer is reversible and, hence, that the opening of the cellular barrier is transient. This specific behavior makes a great difference, in terms of toxicity, between chitosan and the classical penetration enhancers which are known to cause irreversible epithelial damage. Within the context of this review article, we found it important to analyze this mechanism of action of chitosan with regard to its ability to enhance the absorption of peptides. However, as will be discussed later, the relevance of this mechanism on the efficacy of a specific chitosan-based oral drug delivery system will be obviously dependent on the characteristics of the device and, obviously, on the physical state of chitosan. 30 Nanocápsulas de quitosano: nuevos vehículos… Mucoadhesive properties One of the limitations of the oral route is the rapid transit that reduces the chances for the drug to interact with the absorptive epithelium. One of the approaches to deal with this limitation has been based upon the use of materials which favour the interaction of the drug with the mucus layer that covers the intestinal epithelium. Chitosan belongs to the category of these so-called mucoadhesive materials. Indeed, due to its positive charge, chitosan is able to interact with the negatively charged mucus components [38, 39]. This property has attracted significant attention to the use of chitosan for transmucosal drug delivery and, in particular, for nasal drug delivery. For example, in this case, the chitosanmucus interaction leads to the formation of a viscous gel that reduces the mucociliary clearance and increases the residence time of the drug in the absorptive mucosa [29]. However, while the advantage of this property is clear for nasal application, its contribution to the potential of chitosan for oral drug delivery is uncertain. This is due to the fact that the mucus covering the intestinal wall undergoes a rapid turnover and, consequently, the mucoadhesion of chitosan does not necessarily imply a more intense and prolonged contact of the co-administered drug with the absorptive epithelium. Therefore, a critical point in the design of a chitosan-based mucoadhesive delivery device would be to achieve, first, a facilitated interaction with the mucus and, second, an adequate diffusion through the mucus layer towards the underlying epithelium. In addition, as mentioned above, classical chitosan solutions and powders are expected to precipitate upon reaching the intestinal tract and this uncontrolled precipitation may logically affect the inherent mucoadhesive properties of chitosan. Irrespective of the importance of the role of mucoadhesion in the efficacy of chitosan-based delivery systems, there are some characteristics of chitosan that have been found to affect the intensity of the mucoadhesion phenomenon. Logically, as expected from the mechanism of mucoadhesion, the mucoadhesive character of chitosan is dependent on its acetylation degree. Indeed, a higher deacetylation degree of chitosan leads to a more important number of positive charges and, hence, to a more marked adhesiveness [39]. Artículo 1: The potential of chitosan for oral… 31 On the other hand, the molecular weight of chitosan has also been regarded as a parameter that affects the mucoadhesive properties of chitosan. For example, Kawashima et al. [40] observed, using a rat everted intestinal sac, that the mucoadhesion of chitosan increased with the molecular weight. The same conclusion was derived from a rheological study aimed at predicting the mucoadhesive properties of chitosan. The results of this study indicated that the mucoadhesion forces could be modulated by adjusting the chitosan molecular weight and concentration [41]. As in the case of the permeability enhancing property, it is worthwhile to mention that, despite the evidence of the chitosan inherent properties on its mucoadhesive character, the consequence that these properties may have on the behavior of chitosan-based delivery devices will obviously be highly dependent on the specific characteristics of the device. Chitosan toxicity issues and regulatory status The safety of chitosan has been investigated, showing its low toxicity and biocompatibility [5, 6, 42]. In fact, the oral LD50 of chitosan in rodents has been reported to be over 16 g/kg [42], showing that chitosan is safe following oral administration. On the other hand, the results in humans indicate that it is necessary to consume several grams of chitosan a day in order to observe signs of constipation or diarrhea [43, 44]. These amounts are far beyond those needed in pharmaceutical formulations and, consequently, it is accepted that the risk of side effects following oral administration of chitosan formulations is negligible. With regard to the regulatory aspects, there is a monograph of chitosan hydrochloride in the European Pharmacopoeia (EP1774). In addition, The American Society for Testing and Materials (ASTM) has published a guideline (ASTM F 2103) for the characterization of chitosans for use in Tissue Engineered Medical Products (TEMPs). Finally, according to the information provided by the company 32 Nanocápsulas de quitosano: nuevos vehículos… Novamatrix, a Drug Master File covering the chitosan salts and bases have been submitted to the US FDA in July 2004. Chitosan-based systems for oral peptide delivery Over the last years, a number of chitosan-based formulations (i.e. solutions, microspheres and nanostructures) have been developed for improving the oral administration of peptides and proteins. In this section we describe the performance of these vehicles in terms of their ability to enhance the intestinal absorption as well as the mechanistic details. The pharmacological efficacy obtained for the two model peptides, insulin and calcitonin, that have received the greatest attention as candidates for these chitosan-based carriers are summarized in Tables 1 and 2. Table 1: Pharmacological efficacy obtained after oral administration to rats of insulin encapsulated in chitosan-based carriers. Chitosanbased carrier Drug Dose (IU/kg) R maxa (%) tmaxb (h) Duration of effect Reference Chitosan nanoparticlescInsulin 7, 14, and 21 60 10 From 8 until 24 h Pan et al., 2002 [68] Chitosan nanoparticlescInsulin 50 and 100 50 19 From 13 h until 24 hours Ma et al., 2001 [69] Chitosan/glucomannan nanoparticles Insulin 50 50 14 From 14h until 24 h AlonsoSande et al., 2004 [73] Chitosancoated liposomes Insulin 100 30 3 From 30 min until 12 h Takeuchi et al., 1996 [82] a: Maximum pharmacological effect. b: Time of the maximum pharmacological effect. c: In vivo study performed in diabetic rats. Artículo 1: The potential of chitosan for oral… 33 Table 2: Pharmacological efficacy obtained after oral administration to rats of calcitonin encapsulated in chitosan-based carriers. Chitosan-based carrier Drug Dose (IU/kg) R maxa (%) tmaxb (h) Duration of effect Reference Enteric-coated chitosan-based microspheres Calcitonin 500c15 10 From 8 h until 12 h Lamprecht et al., 2004 [59] Chitosan nanocapsules Calcitonin 500 30 1 More than 24 h Prego et al., 2005 [76] Chitosan-coated solid nanoparticles Calcitonin 500 30 1 More than 24 h GarciaFuentes et al., 2005 [82] Chitosan-coated PLGA nanoparticles Calcitonin 125, 250 and 500 25 8 36 h Kawashima et al., 2000 [40] Chitosan-coated liposomes Calcitonin 500 22 2 8 h Takeuchi et al., 2003 [84] a: Maximum pharmacological effect. b: Time of the maximum pharmacological effect. c: Expressed by the authors in mg/kg and converted to IU taking the relation 1mg=5000IU. Chitosan solutions Most of the studies intended to evaluate the ability of chitosan solutions to improve the absorption of drugs across the intestinal epithelium were performed in vitro, either in cell culture [45] or in rat intestinal [46]. The in vivo evaluation of the effectiveness of chitosan solutions for oral peptide delivery is limited. This is understandable if we take into account that, as indicated above, chitosan precipitates at the pH of the intestinal tract (~6.5-7.5). In fact, from our literature search, we only found one study reporting the in vivo absorption enhancing effects of chitosan hydrochloride [47]. More concretely, the results showed an increase in the bioavailability of a nonapeptide following intraduodenal injection to rats. This positive result was attributed to the inherent capacity of chitosan to open the intercellular junctions. 34 Nanocápsulas de quitosano: nuevos vehículos… The problem associated to the low solubility of chitosan at neutral and high pH values could be overcome by chemical modification of the chitosan molecule. For example, the derivative, N-trimethyl chitosan, was found to be readily soluble at neutral and basic pH values [48]. Unfortunately, the studies performed in Caco-2 cells showed that the mucoadhesive [49] and the promoting enhancing [46] properties of trimethylated chitosan were not as remarkable as those of the parent chitosan molecule. According to the authors [46, 49], this phenomenon could be related to a change in the conformation of the trimethylated chitosan that reduces the flexibility of the polymer molecules and, therefore, the interpenetration into the mucus layer. This reduced mucoadhesion could also be due to a decrease in the density of amino groups available for protonation subsequent to the chemical modification. Nevertheless, despite the reduced mucoadhesion of the chemically modified polymers in the in vitro cell line, the in vivo studies performed in rats or pigs revealed that trimethylated chitosan was significantly more efficient than chitosan hydrochloride at increasing the absorption of the peptide octreotide [50, 51]. This greater efficacy was attributed to the more important absorption enhancing effect of trimethylated chitosan at neutral pH values as compared to chitosan hydrochloride [52]. A quite successful approach has been the chemical modification of chitosan by the introduction of a thiol group [53]. Thiolated chitosans exhibited improved mucoadhesive properties in vitro [54] as well as an enhancement in the epithelial drug permeability [55]. Their in vitro behavior correlated well with their ability to increase the absorption of calcitonin or insulin following oral administration to rats [53, 56]. It should be, however, clarified that, in these studies, chitosan was presented in the form of a solid matrix (1.5 mm minitablets). Chitosan-based microspheres A different strategy towards increasing the systemic absorption of peptides administered orally has been designed specifically to deliver drugs in the colonic region. The strategy was proposed to take advantage of two critical facts: the limited Artículo 1: The potential of chitosan for oral… 35 peptide enzymatic activity, as compared to that of the small intestine, and the markedly slower rate of colonic transit. Chitosan-based microspheres were chosen as candidate vehicles to achieve this goal due to the specific degradation of chitosan in the colonic microflora [57, 58], and to its mucoadhesive/absorption enhancing effects. Indeed, if conveniently designed, chitosan-based microspheres can travel intact along the gastrointestinal tract and reach the colonic region. Once in this region, the polymer matrix degrades and releases the peptide which, at this level, is free to cross the colonic mucosa. The technological approach, adopted to prevent the alteration of chitosanbased microspheres during their gastrointestinal transit, was their entrapment or coating with pH-sensitive polymers (acrylic or cellulosic) [59-61]. Using fluorescent markers, i.e. carboxyfluorescein, or the peptide insulin, it has been shown that the pH-sensitive microspheres containing chitosan exhibit a pH-dependent release behavior. More specifically, when exposed to a pH-gradient they provide a negligible release until the colonic pH was reached (~6.5-7), and then, a continuous and controlled drug release [59-61]. Unfortunately, the limited number of in vivo studies intended to evidence the efficacy of these pH-sensitive chitosan systems do not permit us to extract clear conclusions regarding the efficacy of this approach. For example in the study performed by Lamprecht et al. [59], aimed at evaluating the performance of entericcoated chitosan-based microspheres containing calcitonin, it was observed that the pharmacological response was not affected by the presence of chitosan. Consequently, the authors concluded that the success of the formulation was due to the enteric coating and that the role of chitosan was negligible in this type of formulation. These results differ from those observed by Tozaki et al. [62] who studied the efficacy of a large chitosan capsule, administered orally to rats. The chitosan capsule had an enteric polymer coating and contained insulin in association with an absorption enhancer and an enzyme inhibitor. A hypoglycemic response was noted at 6 hours post-administration, when the capsule reached the colon, giving an insulin bioavailability of 5.73%. The authors justified the success of this formulation to the specific disintegration of the capsules in the colonic region and to the 42 Nanocápsulas de quitosano: nuevos vehículos… systems. More recently, we tested chitosan nanocapsules using a co-culture of Caco2 cells and mucus secreting cells (HT29-M6) and observed the fluorescence signals by confocal microscopy [78]. The images indicated that the level of interaction of chitosan nanocapsules was greatly enhanced by the presence of the mucus secreting cells. In addition, no transport of the particles across the monolayer was observed [78]. Therefore, overall, these results led us to suggest that the expected mucoadhesion of the chitosan-coated system could be responsible for the facilitated access of the drug to the underlying epithelium, and, hence for the pronounced and long-lasting hypocalcemic effect. An illustration of this mechanism is presented in figure 4. Cell monolayer Mucus layer Nanostructures Peptide Cell monolayer Mucus layer Nanostructures Peptide Figure 4: Schematic illustration of the interaction of chitosan-based nanostructures with the intestinal mucosa and the transport of the associated peptide Chitosan-coated solid nanoparticles In order to investigate the importance of the nature of the lipid core in the performance of chitosan-coated lipid systems, in our laboratory, we have also produced chitosan-coated tripalmitin nanoparticles [79]. First, calcitonin-loaded tripalmitin nanoparticles were prepared by the double emulsion-solvent emulsification method [80] and, then, coated with chitosan by simple incubation in a chitosan aqueous solution (Fig. 2C). After oral administration of calcitonin-loaded chitosan-coated tripalmitin nanoparticles to rats, the hypocalcemic effect was Artículo 1: The potential of chitosan for oral… 43 evaluated. A great and long-lasting reduction of the serum calcium levels was obtained [81, 82]. This response was similar to the one observed for calcitonincontaining chitosan nanocapsules [76]. However, as in the case of non-coated nanoemulsion, tripalmitin cores were inefficient at reducing the serum calcium levels. An obvious conclusion from these observations was that the chitosan coating was critical for the success of the formulation. As in the case of the chitosan nanocapsules, our interpretation was that the presence of chitosan could facilitate the interaction with the overlying mucus layer leading to a prolonged site-specific delivery of calcitonin and, thus, an extended pharmacological response. These results agree with those previously reported for chitosan-coated poly(lactic acid/glycolic acid) (PLGA) nanoparticles [40]. In this study, elcatoninloaded PLGA nanoparticles were prepared by the emulsion solvent diffusion method. Then, the nanoparticles were isolated and incubated with a chitosan solution, forming chitosan-coated PLGA nanoparticles. The effectiveness of chitosan-coated PLGA nanoparticles was assayed in rats, showing a reduction of the serum calcium levels as compared to the peptide solution and the uncoated nanoparticles. As in the case of the chitosan-coated lipid nanoparticles, this positive in vivo behavior was attributed to the mucoadhesive character of the carrier and its intimate contact with the intestine. This explanation was justified by the observed mucoadhesion of the carrier using the everted intestinal sac model. Chitosan-coated liposomes Liposomes have been considered as candidate vehicles for oral peptide delivery due to their capacity to encapsulate peptides and to protect them from enzymatic degradation. One of approaches to improve their interaction with the intestinal mucosa and, hence, to increase the absorption of the associated peptide has been their coating with mucoadhesive polymers such as chitosan. For example, Takeuchi et al. prepared multillamelar liposomes coated with chitosan by hydration the lipid film with an aqueous solution of the polymer [83, 84]. As expected, these chitosan-coated liposomes exhibited a mucoadhesive character whose degree was 44 Nanocápsulas de quitosano: nuevos vehículos… dependent on the amount of chitosan attached to their surface [83]. The efficacy of the coated liposomes as carriers for oral peptide delivery was tested in vivo for two model peptides, insulin and salmon calcitonin [83-85]. The results of these studies indicated that the chitosan-coated liposomes were more effective than the uncoated ones in terms of improving the pharmacological effect of the peptides administered orally to rats. This improved response was related to the mucoadhesive properties of the chitosan-coated liposomes. Moreover, the same authors tested the performance of the chitosan solutions for improving the absorption of calcitonin and observed that the simple presence of chitosan in solution did not help the absorption of this peptide. As a consequence, they concluded that the protection of the peptide in the liposomal core as well as their coating with the mucoadhesive polymer were critical for the success of the formulation. Overall, the results obtained from chitosan-coated nanostructures underline the efficacy of this type of colloidal carrier. In general, the chitosan coating around the carriers showed a positive effect at improving the pharmacological response of the peptide, which was mainly attributed to the mucoadhesive properties of the polymer. However, the differences in the core material could also influence the level of protection of the associated peptide and also in its release from the carrier. As a consequence, these differences could have an impact on the intensity and duration of the pharmacological response. Conclusion Different chitosan-based drug delivery systems were revealed as promising peptide carriers. Among them, those based on chemically modified chitosan and also nanoparticulate carriers (nanoparticles and nanocapsules) have been particularly successful. Moreover, there is a clear evidence of the greater performance of nanoparticulate chitosan carriers, as compared to the solutions of non-modified chitosan, in terms of enhancing the absorption of peptides such as insulin and salmon calcitonin. The explanation to this phenomenon is that the particulate carriers are able not only to increase the peptide absorption due to the mucoadhesive Artículo 1: The potential of chitosan for oral… 45 properties of chitosan, but also to offer protection from enzymatic degradation. Furthermore, while more work is needed to fully understanding the mechanism of action and the efficacy of these carriers, we can certainly conclude that chitosanbased systems have a promising future in oral drug delivery. Expert opinion At present, only the hydrophobic peptide cyclosporine A can be administered orally in the form of a microemulsion (Neoral®). However, the progress made over the last years, towards making feasible the oral administration of peptides, offers an optimistic perspective. Indeed, the Eligen® technology (based on using a low molecular weight delivery agent) has reached phase II-clinical trials for insulin and salmon calcitonin [86, 87]. Similarly, hexyl –insulin monoconjugate 2 (HIM2), a modified insulin conjugated to an amphiphilic polymer [88], as well as oral formulations of calcitonin and parathyroid hormone consisting of a combination of enzyme inhibitors, absorption enhancers, and enteric coating, have been evaluated in humans [89]. On the other hand, a number of particulate polymer and bioadhesive systems have given evidence of their effectiveness in large scale animals [90, 91]. Therefore, these delivery-based strategies are opening the way for future great developments, preferably based on nanosystems and polymers. This article presents the value of a more immature, but promising, strategy based upon the use of the bioadhesive polysaccharide chitosan. Chitosan as such (in solution or powder) can not be used because it is soluble in the gastric fluids but precipitates at the intestinal pH. However, a number of delivery approaches based on chitosan have shown a degree of success in small scale animals: (i) the use of chemically modified chitosan which is soluble at the intestinal pH; (ii) the use of nanosystems that protect the peptide and facilitate its interaction with the absorptive epithelium; and (iii) the design of devices that specifically deliver the peptide together with chitosan in the colonic region. Despite the difficulties to compare the results of these approaches, the specific characteristics of the nanosystems and the documented information about their efficacy lead us to the consideration of their special potential for oral 46 Nanocápsulas de quitosano: nuevos vehículos… peptide administration. More detailed studies about their mechanism of action will help designing the way to proceed for their further optimization. Five-year view At present, there is the proof of concept that chitosan-based nanocarriers can enhance the absorption of model peptides such as insulin and salmon calcitonin in small scale animal models. It could be anticipated that these initial results will stimulate the optimization of oral peptide formulations based on chitosan delivery nanosystems. Further, this accumulated information is expected to lead to the evaluation of the efficacy of these nanosystems in large-scale animals in fed and fasting conditions. 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Release (2004) 95: 547–555. 57. Yamamoto A, Tozaki H, Okada N, Fujita T: Colon-specific delivery of peptide drugs and anti-inflammatory drugs using chitosan capsules. S.T.P. Pharma Sci. (2000) 10: 23-34. 58. Sinha VR, Kumria R: Microbially triggered drug delivery to the colon. Eur. J. Pharm. Sci. (2003) 18: 3-18. 59. Lamprecht A, Yamamoto H, Takeuchi H, Kawashima Y: pH-sensitive microsphere delivery increases oral bioavailability of calcitonin. J. Control. Release (2004) 98: 1-9. 60. Torres D, Rodríguez M, Cuña M: Microencapsulated lipid cores for site-specific delivery of corticosteroid and peptide drugs to the colonic region. S.T.P. Pharma Sci. (2003) 13: 49-56. 58 Nanocápsulas de quitosano: nuevos vehículos… 3. La modificación de la superficie de los sistemas coloidales con PEG ha permitido mejorar la estabilidad de los vehículos en los fluidos biológicos5, 6 y la absorción sistémica de las proteínas asociadas a los mismos, tras su administración por vía nasal y oral 5, 7. Hipótesis 1. Nanocápsulas constituidas por un núcleo oleoso y una cubierta de quitosano, pueden proteger el fármaco encapsulado de su degradación en fluidos biológicos así como, mejorar la absorción del fármaco a través de la mucosa nasal e intestinal. 2. La modificación de la cubierta polimérica mediante la peguilación del quitosano puede mejorar la estabilidad de los vehículos en los fluidos biológicos y, posiblemente, la absorción sistémica del péptido asociado. Objetivos Teniendo en cuenta lo previamente expuesto, el objetivo global del trabajo se ha dirigido a la evaluación del potencial que ofrecen las nanocápsulas de quitosano como sistemas para la administración de péptidos a través de la mucosa 5 Tobío M., Sánchez A., Vila A., Soriano I., Evora C., Vila-Jato J.L., Alonso M.J. The role of PEG on the stability in digestive fluids and in vivo fate of PEG-PLA nanoparticles following oral administration. Colloids and Surfaces. (2000) 18: 315-323. 6 Garcia-Fuentes M., Torres D., Alonso M.J. Design of lipid nanoparticles for the oral delivery of hydrophilic macromolecules. Colloids and Surfaces, B: Biointerfaces (2003) 27: 159-168. 7 Tobío M., Gref R,. Sánchez A., Langer R., Alonso M.J. Stealth PLA-PEG nanoparticles as protein carriers for nasal administration. Pharm. Res. (1998) 15: 270-275. Antecedentes, hipótesis y objetivos 59 nasal e intestinal. A fin de facilitar el seguimiento de esta memoria, hemos desglosado este objetivo global en los siguientes objetivos parciales: Desarrollo y evaluación del comportamiento in vitro de nanocápsulas de quitosano para la administración de péptidos por vía nasal Este objetivo ha consistido en la optimización del proceso de preparación de las nanocápsulas de quitosano, evaluando la influencia de distintas variables tecnológicas sobre las características finales de los sistemas desarrollados. Asimismo, el objetivo se ha centrado en la incorporación de un péptido modelo, la calcitonina, a las nanocápsulas de quitosano para, posteriormente, explorar el potencial de dichos vehículos con el fin de mejorar la biodisponibilidad de la calcitonina por vía nasal. Los resultados del trabajo correspondiente a este objetivo se recogen el artículo 2 titulado: “Chitosan nanocapsules: a new carrier for nasal peptide delivery” (sometido a evaluación). Evaluación del potencial de las nanocápsulas de quitosano como sistemas para la administración oral de péptidos Este objetivo ha consistido en evaluar si las nanocápsulas de quitosano pueden o no favorecer la absorción sistémica de calcitonina administrada por vía oral. Para ello se analizó la influencia de distintas variables como son el tipo de sal de quitosano y su peso molecular. Los resultados correspondientes a este objetivo aparecen recogidos en los artículos titulados: “Transmucosal macromolecular drug delivery”; Journal of Controlled Release 101 (2005) 151–162. 60 Nanocápsulas de quitosano: nuevos vehículos… “Chitosan nanocapsules as carriers for oral peptide delivery: Effect of chitosan molecular weight and type of salt on their in vitro behaviour and in vivo effectiveness” (sometido a evaluación). “Efficacy and mechanism of action of chitosan nanocapsules for oral peptide delivery” (sometido a evaluación). Evaluación del potencial de las nanocápsulas de quitosano-PEG como sistemas de administración de péptidos por vía oral El objetivo de esta etapa de trabajo ha sido el de evaluar si la peguilación del quitosano podría mejorar la estabilidad de las nanocápsulas en los fluidos gastrointestinales y, como consecuencia, su eficacia desde el punto de vista de mejorar la absorción intestinal de la calcitonina. Asimismo, se ha considerado como objetivo adicional la evaluación de la toxicidad y del mecanismo de interacción de las nanocápsulas de quitosano-PEG con la línea celular Caco-2. Los resultados del trabajo correspondiente a este objetivo se recogen el artículo 5 titulado: “Chitosan-PEG nanocapsules as new carriers for oral peptide delivery. Effect of chitosan pegylation degree” (sometido a evaluación). Estudio del mecanismo de interacción de las nanocápsulas de quitosano con células Caco-2 y con células goblet (HT29-M6) El objetivo de este estudio ha sido el de investigar el papel del recubrimiento con quitosano en la interacción con la línea celular Caco-2 y con el cocultivo Caco-2:HT29-M6. Para ello se analizó el efecto de las nanocápsulas en la viabilidad celular, en la modificación de las uniones intercelulares, así como, en la interacción de las nanocápsulas con las células. Finalmente, con la finalidad de conocer la influencia de la presencia de mucus en la interacción de los sistemas con las células, se evaluó el comportamiento de las nanocápsulas de quitosano en el Antecedentes, hipótesis y objetivos 61 cocultivo Caco-2:HT29-M6, constituido por enterocitos (Caco-2) y células secretoras de mucus (HT29-M6). Los resultados correspondientes a este objetivo aparecen recogidos en los artículos ya mencionados: “Chitosan nanocapsules as carriers for oral peptide delivery: Effect of chitosan molecular weight and type of salt on their in vitro behaviour and in vivo effectiveness” (sometido a evaluación). “Efficacy and mechanism of action of chitosan nanocapsules for oral peptide delivery” (sometido a evaluación). TRABAJO EXPERIMENTAL Parte 1 Evaluación de las nanocápsulas de quitosano como nuevos sistemas para la administración nasal de péptidos. Artículo 2 Chitosan nanocapsules: a new carrier for nasal peptide delivery Cecilia Prego, Dolores Torres and María José Alonso Sometido a evaluación 74 Nanocápsulas de quitosano: nuevos vehículos… The formulations variables investigated for the association of sCT to CS nanocapsules were: the concentration of lecithin (0.4 and 1.2% w/v) and the concentration of CS (0 and 0.1% w/v). Characterization of CS nanocapsules The particle size and polydispersion index of the nanocapsules were determined by photon correlation spectroscopy (PCS). Samples were diluted to the appropriate concentration with filtered ultrapure water. Each analysis was performed at 25ºC with an angle detection of 90º. The zeta potential was calculated from the mean electrophoretic mobility values, which were determined by laser Doppler anemometry (LDA). Samples were diluted with KCl 1mM and placed in the electrophoretic cell where a potential of ±150 mV was established. The PCS and LDA analysis were performed using a Zetasizer® 3000 HS (Malvern Instruments, Malvern, UK). Each batch was analyzed in triplicate. The morphological examination of CS nanocapsules and nanoemulsions was performed using a transmission electron microscopy (TEM), (Philips CM12, Eindhoven, Netherlands), following staining with a 2% w/v phosphotungstic acid solution. sCT encapsulation efficiency Due to the important affinity of sCT for the lipids, the attempts directed to extract or separate the encapsulated sCT from the nanocapsules and nanoemulsions were unsuccessful. Consequently, the encapsulation efficiency of sCT in the nanocapsules was calculated by the difference between the total amount of sCT used in the encapsulation process and the amount of free sCT that remained in the aqueous suspending medium. The amount of free sCT was determined in the supernatant following separation of nanocapsules from the aqueous medium by a combined ultrafiltration-centrifugation technique (Centricon YM-100, Millipore, Artículo 2: Chitosan nanocapsules: a new carrier… 75 USA) at 1000 x g for 1h. The supernatant was diluted with pH=4 acetate buffer and assayed for sCT content by HPLC at 220 nm (Agilent Technologies, Germany) as described in the British Pharmacopoeia, 1998 (column: Vidac 218TP). A calibration curve was made with solutions of sCT in pH=4 acetate buffer at concentrations ranging from 5 to 100 μg/ml. Each sample was assayed in triplicate. In vitro release of sCT from CS nanocapsules In vitro release studies of sCT from CS nanocapsules and control nanoemulsions were performed by incubating the formulations in acetate buffer (pH= 4.0) in a shaking water bath at 37°C. At appropriate time intervals, individual samples were ultrafiltered at 1,000x g for 1 h. The amount of sCT released at each time point was determined by HPLC. Nasal administration of sCT-loaded CS nanocapsules Male Sprague-Dawley rats (225-275 g), from the Central Animals House of the University of Santiago de Compostela (Spain), were fasted for 12 h before experiments, but allowed water ad libitum. Animals were kept conscious during the experiments. These experiments were approved by the Ethical Committee of the University of Santiago de Compostela. The following formulations were instilled intranasally to rats by means of a micropipette inserted into the nostrils: (1) isolated sCT-loaded CS nanocapsules and (2) non-isolated sCT-loaded CS nanocapsules (3) sCT-loaded nanoemulsion, (4) sCT aqueous solution and (5) sCT aqueous solution containing CS. The dose of sCT administered was, in all cases, 15 UI/kg in a volume of 20 µl (10µl in each nostril). Blood samples were collected from the tail vein 30 min prior to the nasal administration, in order to establish the baseline calcium levels, and at different times after dosing. The serum was separated by centrifugation at 3,000x g for 5 min. Hypocalcemic effects were determined in serum samples by a colorimetric method at 570 nm (Kit OR-cresolphtalein v/v, Spinreact, Spain). The area above the hypocalcemic effect-time curve (AAC0-12h) 76 Nanocápsulas de quitosano: nuevos vehículos… was calculated by means of the trapezoidal method. Results are shown as the mean values of serum calcium levels (± s.e.) of 6 animals. Statistical analysis Particle size and zeta potential were statistically analyzed by the analysis of variance (ANOVA) combined with a lineal regression using the program SPSS 11.5. Differences were considered to be significant at a level of p <0.01. The mean serum calcium levels determined in samples collected before sCT administration were taken as the baseline levels. Using these values, the statistical comparison of the percentages of calcium reduction at each time was performed by the ANOVA test followed by the Student-Newman-Keuls test for multiple comparisons between treatments. Differences were considered to be significant at a level of p <0.05. Results and discussion The main goal of the present work was to investigate some formulation factors involved in the preparation of CS-coated oily nanodroplets and to evaluate their potential for the association and delivery of peptides following nasal administration. With this purpose in mind, sCT was selected as a model peptide. The rational behind this work was that the combination of an oily core with a CS coating, in the form of a colloidal system, would help protect the associated peptide from degradation while favoring its interaction with the nasal mucosa. Development and characterization of CS nanocapsules The supporting hypothesis for the preparation of CS nanocapsules was that the ultradispersion of a lipid solution in a CS aqueous solution would facilitate the Artículo 2: Chitosan nanocapsules: a new carrier… 77 ionic interaction between the negatively charged lipids and the positively charged CS molecules. This hypothesis was verified a number of years ago (22). In the present work, we aimed to elucidate the factors that were determinant in the formation of these CS nanocapsules. Hence, the variables evaluated were: the concentration of lecithin in the organic phase, the concentration of CS in the aqueous phase and the way CS was incorporated into the aqueous phase, either before or after mixing the organic and the aqueous phase. The analysis of the particle size by PCS indicated that all formulations displayed a size in the nanometer range, between 200 and 570 nm, depending on the values of the variables selected (Figure 1 and 2). In addition, the results indicated that, irrespective of the way CS was incorporated, both lecithin and CS concentration had a statistically significant influence on the particle size, the presence of CS being the main factor affecting the particle size. More specifically, figure 1 depicts the surface response showing the simultaneous influence of the variables selected on the size of CS nanocapsules formed by incorporating CS prior to the oil-in-water mixing process. It can be noted that the addition of a small amount of CS to the external aqueous phase (0.1-0.2%) did not significantly change the size of the oily droplets, however, a further increase in the CS concentration (0.3%), led to a significant enlargement in the particle size (from 200 to 570 nm). On the other hand, in figure 1 it can also be noted that the influence of the lecithin concentration was determined by the CS concentration. Concretely, for the lowest CS concentration, an increase in the lecithin concentration did not lead to a significant change in the particle size. However, for the upper limit of the CS concentration, the particle size decreased significantly when the lecithin concentration increased. This size decrease was attributed to the presence of a population of CS-lecithin nanocomplexes that accompanied the formation of the nanocapsules. In order to corroborate this hypothesis, these high lecithin-content formulations were ultracentrifuged at 120,000 x g for 1 hour. The result of this process was a three-phase system consisting of a foam caused by the association of nanocapsules, a precipitate attributed to the formation of complexes between lecithin and CS and the aqueous suspending medium. In contrast, for the low lecithin content formulations only a-two phase system consisting of a foam and aqueous suspending medium was observed. Consequently, these results indicate that there is a critical 78 Nanocápsulas de quitosano: nuevos vehículos… value for the parameters investigated, lecithin and CS concentration, in order to obtain a unique population of CS nanocapsules. Figure 1: Response surface showing the effect of lecithin and CS concentrations on the particle size of CS nanocapsules obtained by adding CS to the external aqueous medium prior to the mixing with the organic solution of the lipids. Mean particle size = 211.231 + 181.462 X1 – 1426.453 X2 + 9581.111 X22 – 1269.295 X1X2; r = 0.966. X1 = Lecithin concentration in the organic phase. X2 = CS concentration in the aqueous phase. Figure 2 shows the influence of the concentration of both CS and lecithin, on the size of CS nanocapsules formed by incorporating CS after the dispersion of the lipids in the aqueous medium. The size of the nanosystems was affected by the variables investigated, CS and lecithin concentration, although to a different extent. As observed in the previous method, the increase in the CS concentration led to a significant enlargement in the particle size. In both cases, this effect, which was slightly affected by the concentration of lecithin, was attributed to the deposition of increasing amounts of CS around the oily nanodroplets. In fact, as shown in figure 3, the size enlargement occurred simultaneously with a progressive inversion in the zeta potential. A similar effect was previously observed when increasing either the amount of CS or its molecular weight (22, 23). In contrast, the lecithin concentration had an opposite effect on the size as compared to that observed in the previous Artículo 2: Chitosan nanocapsules: a new carrier… 79 method: for the highest CS concentration, the size became larger as the lecithin concentration increased. This could be simply attributed to a more important deposition of CS onto the lipid cores. However, as occurred with the previous method, the presence of lecithin-CS complexes was detected upon ultracentrifugation of the formulations prepared with the highest lecithin concentration. Consequently, the increase in the size could be attributed not only to the more important deposition of CS but also probably to some lecithin-CS complexes attached to the nanoemulsion during the incubation step. Figure 2: Response surface showing the effect of lecithin and CS concentration on the particle size of CS nanocapsules prepared by adding CS to the external aqueous medium after the formation of the submicron emulsion. Mean particle size = 122.624 + 173.027 X1 + 217.725 X2 – 60.299 X12 + 420.139 X22 + 5.163 X1 X2; r = 0.994. X1 = Lecithin concentration in the organic phase. X2 = CS concentration in the aqueous phase. Finally, irrespective of the moment of addition of CS, the particle size distributions were close to a monomodal distribution for the lowest CS concentration (polydispersion index < 0.2), however for higher CS concentration values, a less homogeneous population was obtained, as confirmed by the polydispersion index values which evolved to a value of 0.4. The increase in the polydispersion index could also be related to the presence of different populations corresponding to the nanocapsules and the nanocomplexes. 80 Nanocápsulas de quitosano: nuevos vehículos… The appearance of CS nanocapsules and the corresponding control nanoemulsion is presented in figure 3. In the case of the nanoemulsion, we could only appreciate a spherical morphology. However, for CS nanocapsules we visualised the presence of an oily core surrounded by a CS coating, regardless the concentration of lecithin. AA BB CC Figure 3: Transmission electron micrographs of: (A) uncoated nanoemulsion, (B) CS nanocapsules prepared with 0.4% w/v of lecithin and (C) CS nanocapsules prepared with 1.2% w/v of lecithin. In addition to the results of the particle size analysis, the evidence of the CS coating around the oily nanodroplets was corroborated by the measurement of the zeta potential of the nanocapsules. As shown in figure 4, the uncoated nanoemulsions (0% CS) exhibited a high negative charge that was inverted upon coating with CS. Moreover, it can be noted that the concentration of CS was the most relevant factor on the evolution of the surface charge of the nanocapsules. This inversion from highly negatives values (about -60 mV) to highly positive values (about +50 mV) was certainly due to the presence of the cationic polymer on the surface of the nanodroplets. On the other hand, the lecithin concentration did not have a significant effect on the surface charge. As observed in a previous study (22), a minimum amount of lecithin is enough to facilitate the formation of the CS coating, and thus to confer the nanocapsules with a positive charge. After the inversion of the charge, neither the lecithin concentration, nor the CS concentration produced significant changes on the surface charge. Artículo 2: Chitosan nanocapsules: a new carrier… 81 Figure 4: Response surface showing the effect of lecithin and CS concentration on the zeta potential of CS nanocapsules prepared by adding CS to the external aqueous medium after the formation of the submicron emulsion. ζ Potential = -54.340 + 923.767 X2 – 1981.667 X22; r = 0.977. X2 = Chitosan concentration in the aqueous phase. Characterization of sCT-loaded CS nanocapsules An important goal of this work was to associate the peptide sCT to CS nanocapsules. To achieve this goal the peptide was dissolved in 50 µl of water and then added to the organic phase containing the lipids. Using this approach, it was thought that the interaction between the peptide and the lipids would be facilitated due to the positive charge of sCT at the selected pH (pKa= 10.4). As describe in the Materials and Methods section, these peptide-loaded formulations were prepared by the method involving the addition of CS upon mixture of the oily and water phases. The results presented in table 1 indicate that more than 90% of sCT was associated to the control nanoemulsion, regardless of the lecithin concentration. The reason for this high association could be related to the strong electrostatic interaction between the negatively charged lecithin and the positively charged sCT, as previously described for other lipid systems containing sCT (24, 25). Moreover, sCT has some hydrophobic regions in their structure enabling them to interact with lipidic structures by means of some specific interactions (26). On the other hand, as can be noted in table 1 that the association of sCT to the nanocapsules was reduced 82 Nanocápsulas de quitosano: nuevos vehículos… by the presence of CS and that this reduction was affected by the lecithin concentration (values of encapsulation efficiency between 44 and 52%). This effect was ascribed to the positive character of both sCT and CS, which may be a result of a competition between the polymer and the peptide for the anionic binding sites in the oily core. Theoretically, this displacement effect should affect the sCT molecules which are associated to the surface of the oily droplets, since the attachment of CS occurred upon encapsulation of sCT in the lipid droplets. In order to verify the displacement of sCT molecules due to the presence of CS, some control experiments were performed. The experiment involved the incubation sCT-loaded nanoemulsions in the presence of a cation (0.1% w/v calcium chloride) and the evaluation of the sCT displacement. The results indicated that, as in the case of CS, calcium competed with sCT leading to its displacement from the surface of the oily nanodroplets. An additional control experiment consisted of incubating an uncoated nanoemulsion with sCT and subsequently with CS (0.1% w/v). The results showed that sCT was greatly adsorbed onto the nanoemulsion and further displaced following incubation with CS. Therefore, overall these results indicate that the surface association of the cationic peptide is reduced upon coating of the system with CS. These results agree well with those previously reported by Kawashima et al. (27) who observed that the coating of PLGA nanospheres with CS reduced the elcatonin encapsulation. Nevertheless, despite the above mentioned displacement effect, it must be noted that the association efficiency of sCT to the nanocapsules remains sufficiently important. Table 1: Physicochemical properties and encapsulation efficiency of chitosan nanocapsules and uncoated nanoemulsions containing sCT (mean ± s.d.; n=3). % Lecithin (w/v) % Chitosan (w/v) Particle Size (nm) ζ Potential (mV) Encapsulation efficiency (%) 0 193.3±1.2 -52.0±1.1 > 90 0.4 0.1 224.7±0.6 +20.2±0.4 44.1±3.2 0 243.7±2.1 -59.9±2.0 > 90 1.2 0.1 333.0±6.1 +28.3±2.1 51.9±1.9 Artículo 2: Chitosan nanocapsules: a new carrier… 83 With regard to the physicochemical properties (size and zeta potential) of the sCT-loaded systems, the results in table 1 show that, as expected, the coating with CS increased the particle size of the oily nanodroplets and led to an inversion of the surface charge. These changes were more pronounced for the highest lecithin concentration. In vitro release of sCT from CS nanocapsules Figure 5 displays the release profiles of sCT from CS nanocapsules prepared with different lecithin concentration (0.4 and 1.2% w/v). These profiles indicate that a certain amount of sCT was rapidly released in the first 30 minutes and then, no further release was observed in the 6 hours of the study. The initial release was attributed to the surface-associated peptide whereas the second slow phase would correspond to the release of the well entrapped peptide molecules showing a high affinity for the oily core. Interestingly, the initial burst effect observed for CS nanocapsules was higher for those prepared with 1.2% lecithin compared to those prepared with 0.4% lecithin (15% vs 50%). This difference may be related to the presence of complexes lecithin-CS in those prepared with the highest concentration of lecithin. Indeed, some sCT molecules could be associated to these complexes and be rapidly released upon incubation in the release medium. In contrast, the release of sCT from lecithin nanoemulsions was negligible irrespective of the lecithin concentration, probably due to the peptide binding affinity to the lipid. Similarly, the leakage of calcitonin from positively liposomes was greater than for negative or neutral ones, suggesting that the charge repulsion played an important role in the leakage of calcitonin from positive liposomes (24). 90 Nanocápsulas de quitosano: nuevos vehículos… 13. P. Sinswat, P. Tengamnuay, Enhancing effect of chitosan on nasal absorption of salmon calcitonin in rats: comparison with hydroxypropyland dimethyl-ßcyclodextrins, Int. J. Pharm., 257 (2003) 15-22. 14. L. Illum, Nasal drug delivery-possibilities, problems and solutions, J. Control. Release, 87 (2003) 187-198. 15. S.L. Law, K.J. Huang, V.H.Y. Chou, J.Y. Cherng, Enhancement of nasal absorption of calcitonin loaded in liposomes, J. Liposome Res., 11 (2001) 165-174. 16. P. Tengamnuay, A.K. Mitra, Bile salt-fatty acid mixed micelles as nasal absorption promoters of peptides. II. In vivo nasal absorption of insulin in rats and effects of mixed micelles on the morphological integrity of the nasal mucosa. Pharmaceutical Research 7 (1990) 370-375. 17. R. Mitra, I. Pezron, A. Chuw, A.K. Mitra, Lipid emulsions as vehicles for enhanced nasal delivery of insulin, Int. J. Pharm., 205 (2000) 127-134. 18. R. Fernández-Urrusuno, P.Calvo, C. Remuñán-López, J.L. Vila-Jato, M.J. Alonso, Enhancement of nasal absorption of insulin using chitosan nanoparticles, Pharm. Res., 16 (1999) 1576-1581. 19. Vila, A. Sánchez, K.A. Janes, I. Behrens, T. Kissel, J.L. Vila Jato, M.J. Alonso, Low molecular weight chitosan nanoparticles as new carriers for nasal vaccine delivery, Eur. J. Pharm. Biopharm., 57 (2004) 123-132. 20. P.J. Lowe and C.S. Temple, Calcitonin and insulin in isobutylcyanoacrylate nanocapsules: Protection against proteases and effect on intestinal absorption in rats, J. Pharm. Pharmacol., 46 (1994) 547-552. 21. S. Muranishi, Absorption enhancers, Crit. Rev. Therap. Drug Carrier Systems, 7 (1990) 1-33. 22. P. Calvo, C. Remuñán-López, J.L. Vila-Jato, M.J. Alonso, Development of positively charged colloidal drug carriers: chitosan-coated polyester nanocapsules and submicron-emulsions, Colloid Polym Sci., 275 (1997) 46-53. 23. S. Ogawa, E.A. Decker, D.J. McClements, Production and characterization of O/W emulsions containing cationic droplets stabilized by lecithin-chitosan membranes, J. Agric. Food Chem., 51 (2003) 2806-2812. 24. S.L. Law, C.L. Shih, Characterization of calcitonin-containing liposome formulations for intranasal delivery, J. Microencapsulation, 18 (2001) 201-211. Artículo 2: Chitosan nanocapsules: a new carrier… 91 25. M. Garcia-Fuentes, D. Torres, M.J. Alonso, New surface-modified lipid nanoparticles as delivery vehicles for salmon calcitonin, Int. J. Pharm., 296 (2005) 122-132. 26. R.M. Epand, R.F. Epand, R.C. Orlowski, R.J. Schlueter, L.T. Boni, S.W. Hui, Amphipathic helix and its relationship to the interaction of calcitonin with phospholipids, Biochemistry, 22 (1983) 5074-5084 27. Y. Kawashima, H. Yamamoto, H. Takeuchi, Y. Kuno, Mucoadhesive DLLactide/ Glycolide copolymer nanospheres coated with chitosan to improve oral delivery of elcatonin, Pharm. Develop. Technol., 5 (2000) 77-85. 28. H.S. Yoo, T.G. Park, Biodegradable nanoparticles containing protein-fatty acid complexes for oral delivery of salmon calcitonin, J. Pharm. Sci., 93 (2004) 488-495. Parte 2 Evaluación del potencial de las nanocápsulas de quitosano como sistemas para la administración oral de péptidos. Artículo 3 Transmucosal macromolecular drug delivery Cecilia Prego, Marcos García, Dolores Torres and María José Alonso Adaptado de: Journal of Controlled Release (2005) 101: 151–162 Artículo 3: Transmucosal macromolecular drug … 97 Abstract Mucosal surfaces are the most common and convenient routes for delivering drugs to the body. However, macromolecular drugs such as peptides and proteins are unable to overcome the mucosal barriers and/or are degraded before reaching the blood stream. Among the approaches explored so far in order to optimize the transport of these macromolecules across mucosal barriers, the use of nanoparticulate carriers represents a challenging but promising strategy. The present paper aims to compare the characteristics and potential of nanostructures based on the mucoadhesive polysaccharide chitosan (CS). These are CS nanoparticles, CScoated oil nanodroplets (nanocapsules) and CS-coated lipid nanoparticles. The characteristics and behaviour of CS nanoparticles and CS-coated lipid nanoparticles already reported [A. Vila, A. Sanchez, M. Tobio, P. Calvo, M.J. Alonso, Design of biodegradable particles for protein delivery, J. Control. Release 78 (2002) 15–24; R. Fernandez-Urrusuno, P. Calvo, C. Remuñan-Lopez, J.L. Vila-Jato, M.J. Alonso, Enhancement of nasal absorption of insulin using chitosan nanoparticles, Pharm. Res. 16 (1999) 1576–1581; M. Garcia-Fuentes, D. Torres, M.J. Alonso, New surface-modified lipid nanoparticles as delivery vehicles for salmon calcitonin (submitted for publication).] are compared with those of CS nanocapsules originally reported here. The three types of systems have a size in the nanometer range and a positive zeta potential that was attributed to the presence of CS on their surface. They showed an important capacity for the association of peptides such as insulin, salmon calcitonin and proteins, such as tetanus toxoid. Their mechanism of interaction with epithelia was investigated using the Caco-2 model cell line. The results showed that CS-coated systems caused a concentration-dependent reduction in the transepithelial resistance of the cell monolayer. Moreover, within the range of concentrations investigated, these systems were internalized in the monolayer in a concentration-dependent manner. This uptake was slightly enhanced by the presence of the CS coating but, as compared with previously published results [M. GarciaFuentes, C. Prego, D. Torres, M.J. Alonso, Triglyceride-chitosan nanostructures for oral calcitonin delivery: evaluation in the Caco-2 cell model and in vivo (submitted for publication)], highly dependent on the nature of the lipid core. Nevertheless, 98 Nanocápsulas de quitosano: nuevos vehículos… these differences in the uptake of the CS-coated systems (solid lipid core or oily core) by the Caco-2 cells did not have a consequence in the in vivo behaviour. Indeed, both CS-coated systems (nanocapsules and CS-coated nanoparticles) showed an important capacity to enhance the intestinal absorption of the model peptide, salmon calcitonin, as shown by the important and long-lasting decrease in the calcemia levels observed in rats. Keywords: chitosan; nanoparticles; nanocapsules; transmucosal transport; peptide/protein carriers. Artículo 3: Transmucosal macromolecular drug… 99 Introduction Over the last decades significant efforts have been dedicated to explore new routes, alternative to injection, for the administration of macromolecules such as peptides and proteins. Among them, the transmucosal routes such as the nasal, pulmonary and oral routes, are those which have received the most important deal of attention [1–5]. As a consequence of this activity, the pulmonary administration of the peptide insulin has recently become a reality in clinical practice [5]. In addition, the number of peptides that are being administered intranasally increases year by year. However, the possibility to administer large macromolecules by the oral route needs still further investigations. The harsh conditions of the gastrointestinal tract and the limited permeability of the intestinal barrier make the situation very complex, and the solution to these problems a great challenge [6]. Nevertheless, despite the past pessimistic view, the knowledge accumulated during recent years regarding the mechanisms of interaction between nanostructured biomaterials and biological surfaces, as well as the discovery of new nanotechnologies and characterization approaches, has led to a new promising perspective on the use of nanocarriers for transmucosal macromolecular drug delivery [7–9]. The hypothesis behind this strategy has been that nanosystems, due to their colloidal size, are able to cross and, hence, transport the associated drug through the mucosal barrier, thus acting as transmucosal macromolecular nanocarriers. Indeed, at present, there is no doubt on the fact that the size is a critical parameter for the nanosystems to cross biological barriers [10,11]. However, the question that remains to be answered is how the ability of these systems to overcome these barriers can be optimized. By accepting the premise that not only the size but also the surface properties and composition of the nanocarriers may affect their stability in biological fluids and their interaction with mucosal surfaces, we have focused our activity to the design of new types of nanocarriers especially adapted for transmucosal drug delivery [12–16]. The first relevant conclusion from this work was that a hydrophilic polyethylene glycol (PEG) coating around the nanosystem has a very positive effect on its in vivo success as a macromolecular drug carrier. More specifically, using tetanus toxoid as a model of a large protein, we showed that its transport across the 106 Nanocápsulas de quitosano: nuevos vehículos… either an oil (Miglyol® 812) or a solid lipid (tripalmitin). Here, we describe the physicochemical properties of these systems (size and zeta potential) as well as their ability to associate and release macromolecules. In addition, we provide information about the in vitro viability of Caco-2 cells exposed to CS nanocapsules and about their mechanism of interaction with this cell model and compared with that reported for CS-coated lipid nanoparticles and CS nanoparticles. Finally, we present the efficacy of the CS-based systems as transmucosal macromolecular drug carriers. ChitosanChitosan corona Oil core Chitosan corona Solid lipid core Chitosan Nanocapsules Chitosan-coated Lipid Nanoparticles Chitosan Nanoparticles ChitosanChitosan corona Oil core Chitosan corona Solid lipid core Chitosan Nanocapsules Chitosan-coated Lipid Nanoparticles Chitosan Nanoparticles Figure 1: Illustration of the different chitosan-based nanosystems described in the present report. Preparation and physicochemical characterization of the CS-based nanosystems We have previously reported [14,27–30] the optimal conditions for the formation of CS nanoparticles and CS nanocapsules. The formation of the nanoparticles was achieved according to an ionic gelation process of CS upon contact with a counter anion, i.e. sodium tripolyphosphate. On the other hand, the formation of CS-coated oily droplets (nanocapsules) was achieved by means of an ionic complexation process between the negatively charged lecithin and the positively charged CS. This polymer layer was found to be responsible for the stabilization of the oily core of the nanoemulsions [27,28]. The same approach was recently applied to the formation of CS coated-lipid nanoparticles [25,26]. In this latter case, the coating was formed by simple incubation of the lipid nanoparticles with an aqueous solution of CS. The attachment of CS was due to its interaction Artículo 3: Transmucosal macromolecular drug… 107 with lecithin and probably with other negative lipids that are present in the core lipid nanoparticles. Table 1 shows the physicochemical properties (size and zeta potential) of the different systems we have designed and those of the corresponding control nanoemulsion. In addition, the table presents some macromolecules that have been associated to the systems and the modality of administration for which we have tested them as carriers for transmucosal macromolecular drugs. These results indicate that, despite their different composition, all CS-based systems have a colloidal size and a positive zeta potential. Moreover, it can be noted that the zeta potential was inverted from highly negative values for the uncoated nanoemulsion (about -52 mV) to highly positive values for CS nanocapsules (about +34 mV). The positive zeta potential can be logically explained by the presence of the cationic polysaccharide CS on the surface of these nanosystems. Simultaneously to the inversion of the zeta potential, we observed an increase in the size that was attributed to the presence of a CS coating. Table 1: Physicochemical properties and encapsulation efficiency of different macromolecules into a nanoemulsion, chitosan-coated lipid nanoparticles chitosan nanocapsules and chitosan nanoparticles as well as the route of administration of the nanosystems (mean ± s.d.; n=3). Carrier Size (nm) ζ Potential (mV) Encapsulated peptide Administration route Encapsulation efficiency (%) Nanoemulsion 195.8±1.1 -52.0±1.1 Salmon calcitonin Oral Nasal >98 Chitosan nanocapsules 266.6±7.6 +34.8±0.6 Salmon calcitonin Oral Nasal 44.1±3.2 Chitosancoated lipid nanoparticles* 537±16 +29.2±6.2 Salmon calcitonin Oral 30.7±2.3 Chitosan nanoparticles** 337±14 +36.9±0.3 Insulin Nasal 94.7±2.1 * Data from Garcia-Fuentes et al, (ref. 26, 33). ** Insulin/chitosan ratio (w/w): 0.4. Data from Urrusuno et al, 1999 (ref. 23). The presence of this coating could also be visualized by transmission electron microscopy (Fig. 2). It can be seen that both, the control nanoemulsion and 108 Nanocápsulas de quitosano: nuevos vehículos… CS nanocapsules, have a spherical shape. However, the nanocapsules show a corona that was attributed to the CS molecules attached to the surface of the oily droplets. Interestingly, besides the potential mucoadhesive characteristics that CS confers to the nanosystems, it was recently observed that this CS corona enhanced the stability of the oily nanodroplets in the gastric fluids [31]. AA BB Figure 2: Transmission electron micrographs of (A) the control nanoemulsion and (B) chitosan nanocapsules. The three CS-based systems showed a great capacity to associate macromolecules. For example, CS nanoparticles have a high loading capacity for acidic proteins such as insulin (I.P.=5.4) and tetanus toxoid (I.P.=4.4–5.9), reaching association efficiencies as high as 97% and 55%, respectively [14,23]. The reason for this high association was related to the affinity of these negatively charged molecules with the positively charged CS molecules. The association of other types of macromolecules such as the lipophilic peptide cyclosporin A has also been possible through the adjustment of the formulation conditions. On the other hand, the CS-coated lipid systems have also shown an ability to associate insulin as well as the cationic peptide sCT (I.P.=10.4) [25,26,29]. The association of insulin was attributed to its affinity for lipid materials and also for CS, whereas the association of sCT was facilitated by the lipids but slightly inhibited by CS. This inhibition of sCT association was ascribed to the positive character of both sCT and CS, that may compete for the anionic binding sites of the lipid cores. Overall, these results indicated that a variety of macromolecules, either hydrophilic or lipophilic, can be efficiently associated to these CS-based nanosystems. Artículo 3: Transmucosal macromolecular drug… 109 With respect to the in vitro release behaviour, our previous work on CS nanoparticles led us to the conclusion that the chemical structure of the protein, and its interaction with CS in the release medium conditions, were major factors governing the release process. For example, in the case of insulin, we observed a fast release that was associated to its easy detachment from CS in buffered conditions [23]. In contrast, in the case of tetanus toxoid-loaded CS nanoparticles, we noted an initial burst of release followed by a slow release that was dependent on the CS molecular weight [32]. This slow release was justified by the affinity of the toxoid for CS and the characteristics of the release medium. Similarly, the release of the peptide sCT from the CS-coated lipid systems followed a biphasic pattern consisting of an initial burst followed by a continuous and slow release [26,29]. The burst of release was attributed to the surface-associated peptide whereas the second slow phase corresponds to the release of the well-entrapped peptide molecules. These results suggest that more studies are needed in order to fully understand the factors that govern the release from the different nanostructures. However, we must keep in mind that the physiological conditions under which these nanosystems release their content in vivo are expected to be quite different from those in vitro and, consequently, in vitro–in vivo correlations should not be expected. Cytotoxicity of CS-based nanosystems in Caco-2 cells In order to investigate the cytotoxicity of CS associated to a nanosystem, we determined the intracellular dehydrogenase activity in Caco-2 cells exposed to different concentrations of CS nanocapsules and to the control nanoemulsion. Fig. 3 shows that, as it was the case for the lipid nanoparticles [33], the control nanoemulsion did not affect cell viability in the range of concentrations assayed. On the other hand, following exposure of Caco-2 cells to CS nanocapsules, we observed a dose-dependent cell viability [34]. More specifically, the cellular viability decreased when the concentration varied between 250 μg/ml (152 μg/cm2) and 1000 μg/ml (606 μg/cm2), being the media lethal concentration (LC50) around 1000 μg/ml of nanocapsules suspension (606 μg/cm2), a value that corresponds to approximately 80 μg of chitosan/ml (48.49 μg/cm2). A similar dependence of cell viability with CS dose was observed for CS-coated lipid nanoparticles [33] and CS 110 Nanocápsulas de quitosano: nuevos vehículos… [35]. A conclusion that can be drawn from this study is that the toxicity values are not clearly dependent on the nanocarrier structure (polymer solution, or CS-coated nanosystems). This conclusion is also corroborated by a recent study that showed similar cellular toxicity for CS nanoparticles and CS solutions [36]. On the other hand, from the perspective of the potential in vivo toxicity, it is worthwhile to mention that the toxicity of these new formulations is low if we take into account the surface area of the intestinal barrier (1 m2 for rats, 200 m2 for humans) [37]. 0 0,2 0,4 0,6 0,8 1 1,2 1,4 0 6 30 61 152 303 455 606 1212 Concentration (µg/cm 2 ) Cellular Viability Nanoemulsion Chitosan Nanocapsules * * *** Figure 3: Cytotoxicity profiles of chitosan nanocapsules and the control nanoemulsion in Caco-2 cell monolayer (Mean±SD, n=4–8). *Statistically significant differences from the nanoemulsion ( p<0.01). Interaction of CS-based nanosystems with the Caco-2 cells monolayer: measurement of the transepithelial electrical resistance (TEER) It is well known that CS solutions cause a significant and dose-dependent decrease of the TEER of the Caco-2 cell monolayers. The first evidence of this phenomenon was reported in the early 1990s [20]. These initial studies intended to explore the mechanism of interaction between CS and the intestinal epithelium concluded that the loss of tight junction integrity was caused by a reorganization of the actin rings [20]. However, a more recent hypothesis points to an interaction between chitosan and the tight junction protein ZO-1, leading to its translocation to Artículo 3: Transmucosal macromolecular drug… 111 the cytoskeleton [38]. In addition to these mechanistic details, some studies have shown that the ability of CS to modify the TEER of Caco-2 monolayers is dosedependent [38,39]. Unfortunately, quantitative comparisons between studies are difficult due to the different experimental conditions used and also to the different types of CS tested (different deacetylation degrees and molecular weights). For example, very marked TEER reductions (up to 80%) were observed for high CS concentrations (5 mg/ml) [38]. However, significant reductions in the TEER values after prolonged exposure to low CS concentrations 0.5 mg/ml were also reported [39]. Based on this information, we found it important to evaluate if the association of CS to a colloidal carrier would modify its interaction with the intestinal epithelial cells. With this purpose in mind, we have measured the TEER of the monolayer exposed to different doses of CS nanocapsules and to the corresponding controls. In Fig. 4 we can notice that neither the controls (HBSS and HBSS pH 6) nor the control nanoemulsion influenced the TEER values after incubation for 100 minutes. On the other hand, following exposure of the cells to CS nanocapsules, we detected a reduction in the TEER values of the monolayer, however, the extent of this decrease was remarkable only for high concentrations of CS nanocapsules. More precisely, when the dose of CS nanocapsules was 80 μg/cm2 the TEER value was slightly reduced (10%), and a decrease of around 35% was only observed for a high dose of CS nanocapsules (204.5 μg/cm2). Higher doses were not tested since the dose of 204.5 μg/cm2 led to a decrease in cell viability of 20–30%. An additional observation was that in all the cases, the TEER values returned gradually to the normal values (total recuperation after 24 h in MEM). A comparable decrease in TEER was observed with similar CS doses in the form of CS-coated lipid nanoparticles [33]. Despite the difficulties to compare experiments only on the basis of the polymer concentrations, globally CS nanosystems and solutions seem to have similar doseTEER reduction profiles [39]. This indicates that CS-coated lipid systems mostly maintain the intrinsic permeabilizing properties of the CS polymer solutions. In contrast, preliminary data using CS nanoparticles suggested that these nanosystems do not display this permeabilizing property at least at concentrations below 250 μg/ml. At this point it is tempting to speculate that the 112 Nanocápsulas de quitosano: nuevos vehículos… different presentation of CS, in the form of a soluble coating or as solid CS nanoparticles, may be responsible from this different behaviour. 50 70 90 110 0 255075100 Time (min) TEER (% of initial) *** Figure 4: Transepithelial electric resistance (TEER) of Caco-2 monolayer exposed to chitosan nanocapsules with two doses of formulations per surface area, 80 μg/cm2 (solid black line with open circles, o) and 204.5 μg/cm2 (solid black line with close circles, ●), the nanoemulsion (solid grey line, ─), or their respective controls of Hank´s balanced salt solution (HBSS) (HBSS pH 7.4; dotted grey line, - - -, and HBSS pH 6; dotted black line, - - -). (Mean±SD, n=3). *Statistically significant differences from chitosan nanocapsules with the lowest dose per surface area, the nanoemulsion and both HBSS (p<0.01). Interaction of chitosan-based nanosystems with the Caco-2 cells monolayer: quantitative uptake studies We have previously investigated the mechanism of interaction of CS nanoparticles with the Caco-2 cells [40]. The results of this study made clear that CS nanoparticles were internalized by the cells and also that this internalization process was saturable (30 min approx.) as well as energy and temperature-dependent. Moreover, these previous results showed that the presence of mucus in the monolayers (MTX-E12cells) strongly increased the association of the nanoparticles. In the study reported here, we aimed to investigate the behaviour of CS nanocapsules and, hence, to elucidate if the composition of the carrier would affect the interaction of CS-based systems with the Caco-2 cells monolayer (Fig. 5). For Artículo 3: Transmucosal macromolecular drug… 113 this purpose, we used fluorescent rhodamine-loaded CS nanocapsules and quantified the percentage of internalized fluorescence as a function of the dose per area of nanocapsules added to the incubation medium. The results showed a linear dosedependent uptake for the range of concentrations between 250 and 1000 μg/ml (71– 286 μg/cm2). It was also noted that the uptake was low and similar for the nanocapsules (maximum value of uptake of 7 μg/cm2) than for the corresponding nanoemulsion (5 μg/cm2). Interestingly, a much higher association was observed for both CS-coated lipid nanoparticles and the corresponding control lipid nanoparticles as compared to that observed for the nanocapsules [33]. Consequently, these results suggest that, not only the CS coating but mainly the nature of the core (either oil or tripalmitin) influences the systems uptake. On the other hand, we observed that the low association values of CS nanocapsules to the Caco-2 cells monolayer are also comparable to those we have observed for CS nanoparticles [40]. The differences observed for the various nanosystems in the Caco-2 cells association profiles, as well as the suggested dependence on the structure of the system, underline the necessity of additional studies in order to gain a better understanding about the role of CS in the interaction of nanosystems with the intestinal epithelium. 0 1 2 3 4 5 6 7 8 0 50 100 150 200 250 300 Amount of nanosystem (µg/cm2) Associated nanosystem (µg/cm2) Nanoemulsion Chitosan Nanocapsules * * Figure 5: Percentages of fluorescent nanoemulsions and chitosan nanocapsules associated to the Caco-2 monolayer following their incubation for 1 hour at different concentrations (Mean±SD, n=3). The dose of the incubated formulations per surface area values were: 0, 71.43, 142.86 and 286.71 μg/cm2. *Statistically significant differences from the nanoemulsion (p<0.01). 114 Nanocápsulas de quitosano: nuevos vehículos… In vivo efficacy of CS-based nanosystems as transmucosal macromolecular drug carriers Bearing in mind that the main goal of our work has been to design new transmucosal macromolecular drug carriers, we have tested different CS-based nanosystems for their ability to increase the transport of peptides and proteins across the nasal and intestinal mucosa. We showed the first evidence of this capacity for CS nanoparticles containing insulin. Indeed, these nanoparticles led to a significant decrease of the glycemia levels, as compared to a control solution of insulin and CS, following intranasal administration to rabbits [23]. Moreover, we observed that this increased absorption was maintained after freeze-drying and further resuspension of the nanoparticles [41]. Recently, we also showed that chitosan nanoparticles were able to increase the nasal transport of antigens, i.e. tetanus toxoid [32]. More importantly, the results of the increasing and long-lasting IgG levels generated following intranasal administration of tetanus toxoid-loaded CS nanoparticles led us to hypothesize that the nanoparticles facilitated the delivery of the antigen to the immunocompetent cells. As an alternative vehicle for nasal administration, we have also evaluated the efficacy of CS nanocapsules for enhancing the systemic absorption of sCT [29]. With this purpose in mind, we determined the hypocalcemic effect of sCT in aqueous solution or sCT associated to a control nanoemulsion and to CS nanocapsules after nasal administration to rats. The results showed a negligible response for the controls (either the aqueous sCT solution or the nanoemulsion) but a significant reduction in the calcemia levels for the nanocapsules. The potential of CS nanoparticles for oral peptide administration has also been reported [42]. The results of this previous study, which was performed in rats, showed that CS nanoparticles were efficient in improving the response of insulin administered orally. The authors speculated that the hypoglycaemic response obtained with CS nanoparticles could be attributed to the protection of the peptide by the formulation and to the potential mucoadhesive and absorption enhancing properties of CS. Artículo 3: Transmucosal macromolecular drug… 115 Very recently, we have evaluated the potential of CS-coated lipid systems (CS nanocapsules and CS-coated lipid nanoparticles) for oral administration of sCT, using a rat animal model [25,28,33]. In order to elucidate the role of the CS coating we have compared the behaviour of the CS-coated systems with that of the control formulations (nanoemulsion and lipid nanoparticles). In Fig. 6 it is shown that the reduction of the serum calcium levels following oral administration of sCT in the form of an aqueous solution or a nanoemulsion was insignificant. However, the hypocalcemic response was greatly enhanced for CS nanocapsules. More importantly, the percentage of reduction of the serum calcium levels (27% for CS nanocapsules reached at 1 h post-administration) was maintained for 24 h. This long-lasting hypocalcemic response was similar to the one observed for sCTcontaining CS-coated lipid nanoparticles [25,33]. Given the lack of efficacy of the controls (non-coated nanoemulsion and lipid nanoparticles), the important hypocalcemic effect elicited by the new formulations was, logically, attributed to the presence of the CS corona around the nanosystems. These results agree with those previously reported for CS-coated poly(lactic acid/glycolic acid) nanoparticles and CS-coated liposomes containing sCT [43,44] and underline the positive effect of a CS coating around the colloidal peptide carriers. 60 70 80 90 100 110 0 4 8 12162024 Time (hours) Serum calcium levels (% of basal) sCT Solution Nanoemulsion Chitosan Nanocapsules * * ** * * * * Figure 6: Hypocalcemic effect observed following oral administration of chitosan nanocapsules, a control nanoemulsion and an aqueous solution of sCT to rats (mean±s.e.; n=6). The dose of sCT administered was 500 IU/kg. *Statistically significant differences from sCT solution ( p<0.05). Artículo 4 Chitosan nanocapsules as carriers for oral peptide delivery: Effect of chitosan molecular weight and type of salt on their in vitro behaviour and in vivo effectiveness Cecilia Prego, Dolores Torres and María José Alonso Sometido a evaluación Artículo 4: Chitosan nanocapsules as carriers for oral … 125 Abstract Very recently, we reported preliminary data that showed the efficacy of chitosan nanocapsules as carriers for oral peptide delivery. In the present work, our aim was to investigate the influence of some chitosan properties, such as molecular weight and type of salt, on the in vitro behaviour of these nanocapsules in terms of their interaction with the Caco-2 cells and also on their in vivo effectiveness. Chitosan nanocapsules were prepared by the solvent displacement technique using high (450 kDa) and medium (160 kDa) molecular weight chitosan glutamate as well as high molecular weight chitosan hydrochloride (270 kDa). The results of their in vitro characterization indicated that the size of the nanocapsules was dependent on the chitosan molecular weight, varying between 250 and 344 nm, whereas the zeta potential (around +30 mV) and the association efficiency of salmon calcitonin (40% approx.) were not affected by the chitosan properties. Upon incubation with the Caco-2 cells, chitosan nanocapsules exhibited a dose-dependent cellular viability, which was hardly affected by, either the chitosan molecular weight or, the type of salt. In addition, it was observed that the transepithelial resistance of the Caco-2 monolayer was not significantly modified upon their exposure to a dose of chitosan nanocapsules which does not compromise the cellular viability. The results of the in vivo response achieved following oral administration to rats indicated that chitosan nanocapsules were able to reduce significantly the serum calcium levels, in comparison to the uncoated nanoemulsion, and to prolong this reduction for at least 24 hours. Moreover, this response was not affected by the type of chitosan salt nor by its molecular weight. In conclusion, this study corroborates the efficacy of chitosan nanocapsules for improving the oral absorption of salmon calcitonin. Moreover, it indicates that changes in chitosan properties such as chitosan type of salt and molecular weight did not affect either the interaction or the permeability of the Caco-2 monolayer or their in vivo efficacy. 126 Nanocápsulas de quitosano: nuevos vehículos… Keywords: chitosan nanocapsules, chitosan properties, Caco-2 cell culture, oral efficacy. Artículo 4: Chitosan nanocapsules as carriers for oral … 127 Introduction There is no doubt that the oral route is the more convenient way to deliver drugs to the body. Unfortunately, up until now the oral administration of peptides and proteins has not been feasible due to their instability in the gastrointestinal tract and also to their low permeability across the epithelium. However, the important efforts that many researchers have dedicated over the last few years to the design of approaches intended to overcome these problems have crystallized in very positive results. In fact, nowadays there are a number of peptide formulations in clinical trials which hold great promise and open optimistic prospects towards the oral peptide administration (1-3). These technologies include the chemical modification of peptides, but also the use of carriers that facilitate the transepithelial transport of macromolecules (Eligen technology) as well as the use of a combination of enzyme inhibitors, absorption enhancers and enteric coating. Apart from the technologies undergoing clinical trials, one of the approaches that has led to great expectations is one based upon the use of nanosystems (4-7). Indeed, in the late 80s it was shown for the first time that poly(alkylcyanoacrylate) nanocapsules were able to protect insulin from degradation and to facilitate its transport across the intestinal barrier (8). Following this original study, a number of reports have made it clear that the size is critical for the interaction of the delivery carrier with the absorptive site (9, 10), and also that the biological behaviour of the constituent of the carrier is a determining factor for its efficacy. More specifically, it has been found that polymers which exhibit mucoadhesive properties are very good candidates for the design of a trasmucosal drug nanocarrier (11-13). The efficacy of this approach is well illustrated with the results reported for the nanosystems based on the polysaccharide chitosan (14, 15). Indeed, this hydrophilic and cationic polymer has been presented in the form of nanomatrices (16, 17) and also as a coating of different types of nanosystems (12, 18, 19). In all cases, the nanostructural presentation of chitosan has resulted in a very positive behaviour in terms of their application for transmucosal drug delivery and, in particular for ocular (20), nasal (21) or intestinal (19, 22) drug delivery. While the mechanistic details need to be further investigated, the hypothesis is that the known 128 Nanocápsulas de quitosano: nuevos vehículos… mucoadhesive and penetration enhancing properties inherent to chitosan may play a significant role in their performance (23, 24). Chitosan is commercially available in the form of different types of salt and with different molecular weights. Some authors have investigated the influence of these properties, using chitosan as an aqueous solution, on its in vitro cell toxicity and penetration enhancing properties (25-29). Unfortunately, the analysis of these results makes it difficult to extract clear conclusions. For example, Schipper et al, 1996 (25) observed that soluble chitosans are able to promote the drug transport across the Caco-2 monolayer, regardless the molecular weight of the polymer (ranging between 4.7 and 19 kDa). However, Chae et al, have shown an effect of the molecular weight of chitosan on the ability to modify the tight junctions, chitosan olygomers being (molecular weigh <10 kDa) those that produced the lowest decrease in the transepithelial resistance values (26). With respect to the effect of the type of salt on the penetration enhancing properties, the in vitro transport study performed by Kotze et al. (27) indicated the superiority of chitosan hydrochloride with respect to glutamate. On the other hand, from the analysis of the influence of chitosan molecular weight on its cytotoxicity in Caco-2 cells, it can be deduced that there is a tendency for the cytotoxicity to increase with the molecular weight (25, 26, 28). However, despite the difficulties of comparing these data, the differences in the toxicity values do not appear to be important. Similarly, there is preliminary evidence that the type of salt in which chitosan is presented may affect its cytotoxicity, chitosan hydrochloride being more toxic than glutamate, glycol or lactate (29). Finally, with respect to the influence of chitosan properties on its in vivo performance, the results reported up until now, either for chitosan solutions or chitosan nanoparticles have not made such influence evident (21, 30, 31). For example, we have previously shown that the ability of chitosan nanoparticles in enhancing the nasal absorption of insulin (21) or eliciting an immune response against tetanus toxoid administered intranasally was not or hardly affected by the molecular weight of chitosan (31). Artículo 4: Chitosan nanocapsules as carriers for oral … 129 Besides these previous reports, the influence of the above indicated parameters on the toxicity and efficacy of chitosan nanocapsules has never been reported. Therefore, taking into account this information, the aim of the present work was to investigate the effect of chitosan molecular weight and chitosan salt on the behaviour of chitosan nanocapsules upon contact with the Caco-2 cell monolayer. In addition, we studied the influence of these properties on the potential of chitosan nanocapsules as carriers for oral peptide administration using salmon calcitonin as a model peptide. Materials and Methods Materials Salmon calcitonin (sCT) was kindly donated by Almirall Prodesfarma, S.A. (Spain). Miglyol 812®, a triglyceride formed from medium chain fatty acids was supplied by Lemmel (Spain). The surfactant soybean L-α-lecithin and Poloxamer 188 (Pluronic F-68®) were supplied from Sigma-Aldrich (Spain). Chitosans with an acetylation degree of 15% and different salts (glutamate and chloride) and viscosities (<20 mPa and 20-200 mPa) were purchased from FMC Biopolymer/ Novamatrix (Norway). The molecular weight of these different polymers is presented in table 1. For the purpose of this work, we consider low a molecular weight <100 kDa, medium, a molecular weight between 100-200 kDa and high, a molecular weight >200 kDa. Table 1: Physicochemical characterization of different types of chitosan. Chitosan brand name Salt form Viscosity (mPa) Molecular weight (kDa) Protasan® UP Cl 213 Chloride 71 270 Protasan® UP G113 Glutamate 16 160 Protasan® UP G213 Glutamate 133 450 130 Nanocápsulas de quitosano: nuevos vehículos… The Caco-2 cell line was obtained from the European Collection of Cell Cultures (ECACC, UK). Minimum Eagle Medium (MEM), fetal bovine serum (FBS), non-essential amino acids, L-glutamine, 100 UI/ml penicillin/ 100 μg/ml streptomycin solution, 0.05% trypsin/0.02% EDTA solution and Hank’s balanced salt solution (HBSS) were purchased from Sigma (Spain). Preparation of chitosan nanocapsules Chitosan nanocapsules were prepared according to the procedure previously described by our group (22, 32), in two steps. First, we prepared a nanoemulsion (33) as a reference formulation by the solvent displacement technique. Secondly, this colloidal carrier was incubated with chitosan solutions leading to the formation of chitosan nanocapsules. Briefly, 125 µl de Miglyol were added to an organic phase composed of 40 mg of lecithin dissolved in 0.5ml of ethanol and 9.5 ml of acetone. This organic solution was poured into 20 ml of an aqueous phase containing Poloxamer 188 (0.25% w/v). The mixture immediately turned milky due to the diffusion of the acetone towards the aqueous phase and the consequent formation of colloidal particles. Then, the solvent was evaporated under vacuum and the nanoemulsion was concentrated to a final volume of 10 ml. Finally, this nanoemulsion was coated by different types of chitosan (See Table 1) by simple incubation in polymer solutions. More specifically, 4 ml of the control nanoemulsion were incubated with 1 ml of chitosan aqueous solution (0.5% w/v) for 1 h, leading to the formation of chitosan nanocapsules. Characterization of chitosan nanocapsules The mean particle size of the colloidal systems was analyzed by photon correlation spectroscopy (PCS). For the determination, samples were diluted to the appropriate concentration with filtered ultrapure water. Each analysis was performed at 25ºC with an angle detection of 90º. Artículo 4: Chitosan nanocapsules as carriers for oral … 131 The zeta potential was calculated from the mean electrophoretic mobility values, which were determined by laser Doppler anemometry (LDA). Nanocapsule suspensions were diluted with KCl 1mM and placed in the electrophoretic cell where a potential of ±150 mV was established. The PCS and LDA analysis were performed using a Zetasizer® 3000 HS (Malvern Instruments, Malvern, UK). Each batch was analyzed in triplicate. Salmon calcitonin encapsulation efficiency The encapsulation of sCT in chitosan nanocapsules made with different types of chitosan (see Table 1) as well as the control nanoemulsion was performed by incorporating 50 μl of an aqueous solution of sCT to the organic phase. The amount of sCT associated to the nanocarriers was indirectly calculated from the amount of free sCT in the supernatant of the nanocarriers. The non-encapsulated sCT was determined following separation of nanocapsules from the aqueous medium by a combined ultrafiltration-centrifugation technique (Centricon YM-100, Millipore, USA) at 1000 x g for 1h. The supernatant was diluted with pH=4 acetate buffer and assayed for sCT content by HPLC at 220 nm (Agilent Technologies, Germany), as described in the British Pharmacopoeia, 1998 (column: Vidac 218TP). A calibration curve was made with solutions of sCT in pH=4 acetate buffer at concentrations ranging from 5 to 100 μg/ml. Each sample was assayed in triplicate. In vitro release studies In vitro release studies of sCT from nanocapsules made of chitosan chloride and chitosan glutamate of different molecular weight were performed by incubating 3 ml of the loaded nanocapsules in 3 ml of acetate buffer (pH= 4.0) at 37°C, under horizontal shaking (100-110 cycles min-1). At appropriate time intervals, the supernatant of chitosan nanocapsules was collected by ultrafiltration at 1000 xg for 1 h. The amount of sCT released in each time-interval was determined by HPLC. 138 Nanocápsulas de quitosano: nuevos vehículos… Cytotoxicity studies An important aspect of the investigation of the potential of chitosan nanocapsules for oral delivery is the analysis of their toxicity upon interaction with the intestinal epithelium. It has been previously shown that the viability of Caco-2 cells decreases in a dose-dependent manner after incubation with chitosan (25, 26, 28). Additionally, it has been reported that specific properties of chitosan, such as salt form and molecular weight may influence this cytotoxic effect (25, 26, 28, 29). However, as indicated in the introduction, the influence of these properties has not been clearly elucidated. This is due, on the one hand, to the difficulties in comparing published data; and, on the other hand, to the fact that these differences are not remarkable. Moreover, it is important to keep in mind that the relevance of these properties may be dependent on the physical presentation of chitosan (as a solution, as a coating material or as a nanomatrice). Thus, in the present work we have evaluated the cellular viability of Caco-2 monolayer after exposure to chitosan nanocapsules for up to two hours (Fig. 2). More specifically, we studied the effect of chitosan coating properties, salt form and molecular weight, on the intracellular dehydrogenase activity in Caco-2 cells. As shown in Figure 2, the first observation was that chitosan nanocapsules presented concentration-dependent cytotoxicity. Upon exposure of Caco-2 cells to concentrations of nanocapsules higher than 0.25 mg/ml (152 µg/cm2), the cellular viability started to decrease, reaching the 50% lethal concentration (LC50) at a concentration between 1 and 2 mg/ml. In addition, it can be observed that either the salt form of chitosan or its molecular weight have only a minor effect on the cell viability. These results agree with those previously reported for chitosan solutions and chitosan nanoparticles prepared with chitosan of molecular weights ranging between 4.7 and 213 kDa (25, 28). However, these results should not lead to the conclusion that chitosan molecular weight does not influence the cellular viability, but only that this effect was not seen within the range of chitosan molecular weight investigated (between 160-450 kDa). In fact, it has been recently reported that chitosan olygomers (molecular weight <10 kDa) have less cytotoxicity effects than high molecular weight chitosans (molecular weight 230 kDa) (26). Artículo 4: Chitosan nanocapsules as carriers for oral … 139 Figure 2: Effect of the different formulations of chitosan nanocapsules on the cellular viability of Caco-2 cells (mean ±d.e., n≥4). Transepithelial electrical resistance studies Chitosan is known for its ability to enhance the penetration of drugs across the epithelium by a mechanism involving the interaction of the positively charged amino groups of chitosan with the negatively charged sites on the cell surfaces and tight junctions (24, 25, 36, 37). A way to study the intensity of this effect is the measurement of the transepithelial electrical resistance of the monolayer. For example, in a previous work, we have shown that the exposure of the Caco-2 monolayer to chitosan nanocapsules may lead to a reduction of the TEER (22). However, the results of this work also indicated that it was necessary to use a high concentration of nanocapsules (which are at the toxic level) in order to observe a significant reduction of the TEER value. Unfortunately, the comparison of these results with those obtained for chitosan solutions is unviable due to the different experimental conditions among experiments and to the absence of meticulous information concerning these conditions. Thus, in this work, our objective was to evaluate whether or not chitosan properties (molecular weight and salt form) affect the interaction (TEER value) of the nanocapsules with the monolayer. Additionally, we have analyzed the effect of the dose of chitosan nanocapsules on the TEER values, up to the limit to the 140 Nanocápsulas de quitosano: nuevos vehículos… maximum dose which does not compromise cell viability. In figure 3, we can appreciate the evolution of TEER values after exposing the cells to chitosan Cl213 nanocapsules and the previously assayed chitosan Cl110 nanocapsules (22). We can observe that the TEER values of the Caco-2 monolayer decreased slowly, over the time, upon exposure to high molecular weight chitosan hydrochloride (Protasan® Cl 213; Mw: 270 kDa) nanocapsules, reaching a 10% reduction after 75 min. This slight reduction was similar to the one observed for medium molecular weight chitosan (Protasan® Cl 113; Mw: 140 kDa) nanocapsules (22). 70 80 90 100 110 0255075100 Time (min) TEER (% of initial) HBSS pH6 Chitosan Cl110 nanocapsules (80µg/cm2) Chitosan Cl213 nanocapsules (80µg/cm2) Figure 3: Transepithelial electrical resistance (TEER) of Caco-2 cells after incubation with 250µg/ml of chitosan Cl 213 nanocapsules, chitosan Cl 110 nanocapsules* and the control HBSS pH 6 in a surface area of 4.5 cm2 (mean ± s.d.; n= 3). *Data from Prego et al 2005 (22). The effect of chitosan glutamate nanocapsules, with two different molecular weights of chitosan (160 and 450 kDa), on their ability to modify TEER values is shown in figure 4. Additionally, this graph shows the influence of the dose of chitosan G113 nanocapsules (80 and 150 µg/cm2) on the TEER values. As it occurred in the case of chitosan hydrochloride nanocapsules, the different chitosan glutamate molecular weights (160 and 450 kDa) used to prepare chitosan nanocapsules led to a slight and similar modification of the TEER values at the dose Artículo 4: Chitosan nanocapsules as carriers for oral … 141 per surface area of 80 µg/cm2. An additional observation from this figure is that an increase in the dose of chitosan G113 nanocapsules from 80 to 150 µg/cm2 led to a significant and progressive reduction of the TEER values, reaching the 20% of reduction after 50 min of exposure to the cells. 70 80 90 100 110 0255075100 Time (min) TEER (% of initial) HBSS pH6 Chitosan G213 nanocapsules (80µg/cm2) Chitosan G113 nanocapsules (80µg/cm2) Chitosan G113 nanocapsules (150µg/cm2) *** Figure 4: Transepithelial electrical resistance (TEER) of Caco-2 cells after incubation with of chitosan G 113 nanocapsules, chitosan G 213 nanocapsules, at two does per surface area, 80 and 150 μg/cm2, as well as the control HBSS pH 6 (mean ± s.d.; n= 3). * Significantly different from HBSS pH6 (p < 0.01). Consequently, these results led us to conclude that, within the chitosan molecular weight range of 140450 kDa and the salt form of chitosan chloride or glutamate, chitosan nanocapsules have a minor effect on the TEER values of the Caco-2 monolayer. Only when the dose of nanocapsules was increased up to the limit which does not compromise cell viability, we could observe a significant reduction (20%) in the TEER values. Despite the difficulties for a strict comparison of these results with those previously reported for chitosan solutions, there is an agreement in the irrelevance of chitosan properties on the transepithelial resistance values (38). It is also worthwhile to mention that, in all cases, there is a gradual recuperation of the original TEER values. 142 Nanocápsulas de quitosano: nuevos vehículos… In vivo efficacy of chitosan nanocapsules As indicated above, we have already performed some preliminary studies, which showed that chitosan Cl110 nanocapsules are able to improve the pharmacological effect of sCT administered orally (22). In this study, we have attempted to evaluate this effect further by studying the behaviour of nanocapsules prepared with different types of chitosan (different salts and molecular weight). Fig. 5 depicts the hypocalcemic profiles following oral administration of sCT associated to different formulations of chitosan nanocapsules. As reference formulations, we have also tested a sCT solution and a nanoemulsion (22). The results showed that the oral administration of sCT alone or associated to the nanoemulsion did not lead to a change in the serum calcium levels. On the contrary, chitosan nanocapsules showed an important hypocalcemic effect which was prolonged for, at least, 24 h. Moreover, it can be noted that the chitosan molecular weight did not affect this capacity of the nanocapsules to enhance the absorption of the associated peptide. 60 70 80 90 100 110 0 4 8 12 16 20 24 Time (min) Serum calcium levels (% of basal) sCT Sol NE CS NC G113 CS NC G213 Figure 5: Hypocalcemic effect observed for salmon calcitonin-loaded chitosan nanocapsules prepared with chitosan glutamate of different molecular weight (CS G113 NC and CS G213 NC) as well as for a control nanoemulsion (NE) and for an aqueous solution of salmon calcitonin (sCT Sol), following oral administration to rats (mean ± s.e.; n=6). The dose of salmon calcitonin administered was 500UI. Finally, the comparison of areas above the hypocalcemic effect curve (AAC 0-24h) obtained following administration of the different formulations (Fig. 6) confirms the significantly greater pharmacological effect of chitosan nanocapsules Artículo 4: Chitosan nanocapsules as carriers for oral … 143 with different chitosan molecular weights in comparison to the sCT aqueous solution and the nanoemulsion. Figure 6: Area above hypocalcemic effect curve (AAC0-24 hr) after oral administration to rats of salmon calcitonin in solution or incorporated in the nanoemulsions and chitosan nanocapsules made of chitosan glutamate of different molecular weight.* Significantly different from salmon calcitonin solution and the control nanoemulsion (p < 0.05). Therefore, a general conclusion from these in vivo studies is that the presence of chitosan was essential to obtain a pharmacological response. These results are in accordance with those previously found for other chitosan-coated based nanosystems such as chitosan-coated PLGA nanoparticles (12), chitosancoated liposomes (18) and chitosan-coated lipid nanoparticles (19) in the sense that all these colloidal systems led to an increase in the systemic absorption of sCT. However, an interesting observation from this comparative analysis is that the high response attained for nanostructures composed of a lipid core and a chitosan coating is more sustained and prolonged than that corresponding to chitosan-coated PLGA nanoparticles (12), chitosan-coated liposomes (18). Regarding the mechanism of action of these nanocapsules, we could speculate about their penetration enhancing effect. However, this hypothetic mechanism does not appear to be relevant given the minor effect in the TEER observed in vitro. A more probable mechanism would be the one related to the mucoadhesive properties of chitosan and, hence, to the efficient interaction of the 144 Nanocápsulas de quitosano: nuevos vehículos… nanocapsules with the intestinal mucosa. In this sense, it is worthwhile to mention the fact that chitosan forming a coating around a colloidal carrier is critical. Indeed, previous studies performed by other groups (18) as well as by our own group have shown that chitosan in the form of a solution or in the form of spray-dried microparticles were not efficient at increasing the systemic absorption of sCT. It should also be taken into account that the fact that the peptide is entrapped in an oily core may help protect the peptide from hydrolytic and enzymatic degradation in the gastro-intestinal tract. Conclusions The results of this work confirm the potential of chitosan nanocapsules as carriers for improving the oral absorption of sCT. 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A comparative study of the potential of solid triglyceride nanostructures coated with chitosan or poly(ethylene glycol) as carriers for oral calcitonin delivery M. Garcia-Fuentes, C. Prego, D. Torres and M.J. Alonso Adaptado de Eur. J. Pharm. Sci. (2005) 25: 133-143. Anexo 269 Abstract We have previously reported the formation and characterization of poly(ethylene glycol) (PEG)-coated and chitosan (CS)-coated lipid nanoparticles. In the present work our goal was to study the interaction of these surface-modified lipid nanoparticles with Caco-2 cells and to evaluate the potential of these nanostructures as oral delivery systems for salmon calcitonin (sCT). The interaction of rhodamine®-loaded nanoparticles with the Caco-2 cell monolayers was evaluated quantitatively and qualitatively by confocal laser scanning microscopy and fluorimetry, respectively. The ability of these nanoparticles to reversibly enhance the transport of hydrophilic macromolecules through the monolayers was investigated by measuring the transepithelial electric resistance and the permeability to Texas Red®-dextran. Finally, in vivo studies of the response to sCT-loaded nanoparticles were performed in rats. The results showed that the association of rhodamine®-loaded nanoparticles to the Caco-2 cell monolayer was independent of the surface coating of the nanoparticles (CS-coated versus PEG-coated nanoparticles). However, while PEG-coated nanoparticles did not affect the permeability of Caco-2 monolayers, CScoated nanoparticles produced a dose-dependent reduction in the transepithelial electric resistance, simultaneously to an enhanced dextran transport. The results obtained following oral administration of sCT-loaded CS-coated nanoparticles to rats showed a significant and prolonged reduction in the serum calcium levels as compared to those obtained for control (sCT solution). In contrast, the hypocalcemic response of sCT-loaded PEG-coated nanoparticles was not significantly different of that provided by the control (sCT solution). Therefore, these results indicate that the surface composition of the particles is a key factor in the improvement of the efficiency of oral sCT formulations. Moreover, the encouraging results obtained for CS-coated nanoparticles underline their potential as carriers for peptide delivery. Keywords: Nanoparticles; Chitosan; PEG; Peptide delivery; Transepithelial transport. 270 Nanocápsulas de quitosano: nuevos vehículos…. Introduction The low transepithelial transport together with the instability in the gastrointestinal tract, are the main obstacles for making the oral administration of peptides feasible. Among the alternatives explored so far to overcome these limitations, the design of submicrometric carriers appears to be a promising approach (Chen and Langer, 1998). For example, it has already been shown that the nanoencapsulation of proteins in colloidal particles protects them against the harsh environment of the gastrointestinal tract (Lowe and Temple, 1994), and enhances their transmucosal transport (Mathiowitz et al., 1997 and Tobío et al., 1998). This ability of the colloidal carriers to enhance the transport of the associated macromolecules has been attributed to different mechanisms depending on the nanocarrier composition. These mechanisms are (i) mucoadhesion, (ii) particle internalization phenomenon and (iii) permeation enhancing effect. The adhesion of a carrier system to the mucus may improve the residence time and contact of the drug with the underlying epithelium, thus increasing the drug concentration in the site of absorption (Mikos et al., 1991). Such mechanism of transport enhancement has led to the idea of modifying the surface of colloids in order to optimize their mucoadhesion. Hydrophilic polymers such as polyacrylate derivatives and chitosan are examples of mucoadhesive materials used to coat colloidal carriers. For example, polyacrylate derivatives in the form of nanoparticles with mucoadhesive characteristics have recently been proposed for the oral administration of sCT (Torres-Lugo et al., 2002). Similarly, Sakuma et al. (1997) reported an enhancement of the oral absorption of sCT following its association to poly(N-isopropylacrylamide) nanoparticles. Moreover, the formation of a mucoadhesive CS coating around PLGA nanoparticles has shown a positive effect in improving the oral absorption of sCT (Kawashima et al., 2000). Moreover, in our group, we have shown that CS-based colloidal particles, in which CS is in the form of a coating or a nanomatrice, are able to improve the transport of drugs across the nasal (Vila et al., 2002) and the ocular mucosa (Calvo et al., 1997 and De Campos et al., 2003). Anexo 271 In contrast with the well-defined role of mucoadhesion in drug absorption, the factors that affect the interaction and internalization of colloidal particles with epithelia remain unclear. In fact, despite the deep understanding achieved of the transport of non-degradable polystyrene particles (Florence and Hussain, 2001), little is still known about the behaviour of colloids of pharmaceutical interest. For a number of years it was broadly accepted that nanoparticles with hydrophobic surfaces are taken up more extensively by the intestinal epithelium than those with hydrophilic surfaces (Eldridge et al., 1990). However, some recent results have suggested that the presence of hydrophilic polymers such as PEG (Tobío et al., 2000) or CS (Vila et al., 2002) on the surface of nanoparticles can increase the transport of these systems through mucosal surfaces. Overall, these results indicated that the composition of the nanoparticle could affect not only the intensity but also the mechanism of transport. Indeed, mechanistic studies performed with different types of nanoparticles indicated that hydrophobic nanoparticles are preferentially transported through the gut associated lymphoid tissue, whereas particles with a more hydrophilic nature are transported across the regular enterocytes (Mathiowitz et al., 2000). Finally, with respect to the permeation enhancing effect, this has been extensively investigated for polymer solutions, however, whether or not this effect remains unaltered when the polymer is in the form of nanoparticles or attached to them, needs to be further clarified. For example, in the case of poly(methacrylic) derivatives (Torres-Lugo et al., 2002), it has been shown that their typical permeation enhancing effect is maintained when they are in the form of nanogels. However, in the case of CS based colloidal systems, the maintenance of the inherent capacity of CS to open the tight junctions (Lueben et al., 1997) has not been fully identified (Behrens et al., 2002). In the present work our goal was to investigate the in vitro and in vivo behaviour of two new drug nanocarriers consisting of a lipid nanoparticles coated by a hydrophilic coating (either PEG or CS). More specifically, we studied the interaction of polymer-coated lipid nanoparticles with the Caco-2 cell culture model and, then, we evaluated the pharmacological response of sCT associated to both 272 Nanocápsulas de quitosano: nuevos vehículos…. types of nanoparticles following oral administration to rats. The preparation of the lipid nanoparticles made of tripalmitin and coated with PEG or CS was previously reported by our group (Garcia-Fuentes et al., 2002; Garcia-Fuentes et al., in press). Moreover, we have already shown that these systems are stable in simulated gastrointestinal media and capable of effectively associating and releasing sCT. Therefore, the purpose of the studies reported here were to identify the role of the polymer coating on the efficacy and mechanism of action of the nanosystems as carriers for the oral administration of sCT. Materials and methods Materials The Caco-2 cell line was obtained from the European Collection of Cell Cultures (ECACC, UK). Minimum Essential Medium Eagle (MEM), fetal bovine serum (FBS), non-essential amino acids, l-glutamine, 100 UI/ml penicillin/100 μg/ml streptomycin solution, 0.05% trypsin/0.02% EDTA solution, Dulbecco's phosphate buffered saline (DPBS) and Hank's balanced salt solution (HBSS) were purchased from Sigma (Sigma, Spain). Tripalmitin [Dynasan® 116] (Condea, Germany) was the lipid used for nanoparticle core formation. l-α-lecithin (Sigma, Spain), poloxamer 188 [Symperonic® F68] (ICI, Spain) and poly(ethylene glycol)-stearate (PEG, Mw≈2000 Da) [Simulsol® M52], a gift from Seppic (France), were used as surfactants. Chitosan (CS) (specifications: 75–85% of deacetylation degree, viscosity 20–200 cP at 1% in 1% acetic acid) in basic form was purchased from Aldrich (Spain). Rhodamine® was from Sigma (Spain) and Texas Red®-dextran and Bodipy® 650/665-Phalloidin from Molecular Probes Europe BV (The Netherlands). Salmon calcitonin (sCT) was a kind donation from Almirall Prodesfarma S.A. (Spain). Other reagents were analytical grade or better. Anexo 273 Nanoparticle preparation Prior to the preparation of nanoparticles, CS was dissolved in 0.1 M hydrochloric acid and purified. More specifically, the CS solution was filtered and washed by dialysis for a period of three days. Then, the filtrate was centrifuged at 30,000×g for 1h to eliminate non-dissolved impurities. The supernatant from the centrifugation was freeze-dried, leading to a powder readily soluble in water. CS-coated nanoparticles were prepared following a two-step procedure: first, tripalmitin nanoparticles, used as cores for their further coating with CS, were prepared by a double emulsion solvent evaporation method, as previously described (Garcia-Fuentes et al., 2002). This procedure was adopted because of its effectiveness for the encapsulation of peptides. Briefly, 50 μl of an aqueous solution of sCT (10 mg/ml) were added to a 0.5 ml solution of 25 mg of lecithin and 50 mg of tripalmitin in methylene chloride. A water-in-oil emulsion was formed upon 15 s of sonication (20 W). Then, 1 ml of water was added to the first emulsion, and sonicated again for 60 s (20 W). The solvent was eliminated by evaporation, first 30 min at room temperature and afterwards 30 min under vacuum. In the second step, tripalmitin nanoparticles at 1% (w/v) final concentration were incubated for 10 min in a 0.05% (w/v) CS/1.5% poloxamer 188 solution in order to allow the formation of a CS coating by simple interaction of the positively charged CS molecules and the negatively charged lipid cores. Nanoparticles were isolated by ultracentrifugation for 1 h at 50,000 × g and resuspended in purified water. PEG-coated tripalmitin nanoparticles were prepared by an analogous procedure to that used to obtain tripalmitin nanoparticles, plus 25 mg of PEGstearate added to the organic phase. Nanoparticles were isolated by ultracentrifugation for 1 h at 85,000 × g and then, resuspended in purified water. For the preparation of rhodamine®-labeled nanoparticles, a 10 mg/ml methanol stock solution was prepared. Five microliter from this stock solution were diluted in the organic phase of the double emulsion method described. 274 Nanocápsulas de quitosano: nuevos vehículos…. Blank nanoparticles (non-loaded with sCT) were obtained following the procedure described above but substituting the aqueous solution of the peptide by water. Physicochemical characterization of the nanoparticles and sCT association efficiency The size and polydispersity of the nanoparticle suspensions were determined by Photon Correlation Spectroscopy (PCS) (Zetasizer® 3000HS, Malvern, UK), following appropriate dilution in purified water. The zeta potential of the nanoparticles was measured by Laser Doppler Anemometry (Zetasizer® 3000HS, Malvern, UK) upon dilution in a 1 mM NaCl solution. The amount of rhodamine® encapsulated and released from the rhodamine®-loaded nanoparticles was determined from the supernatant after ultracentrifugation and measured by fluorimetry (λex = 525 nm; λem = 550 nm). The association efficiency of sCT to the nanoparticles was calculated from the difference between the total amount of peptide added in the encapsulation process and quantity of non-encapsulated peptide. The separation of the nanoparticles from the non-encapsulated peptide was performed by ultrafiltration (Contricom® YM-100, Millipore, Spain) for PEG-coated nanoparticles or by ultracentrifugation for CS-coated nanoparticles (50,000 × g). Ultrafiltration was found to be more suitable to completely isolate sCT from PEG-coated nanoparticles, due to the high centrifugation speed required for their sedimentation. In contrast, CS-coated nanoparticles could be isolated by ultracentrifugation due to their adequate sedimentation characteristics. The concentration of sCT was determined by HPLC as described (British Pharmacopoeia (BP) 1998). For the cell culture experiments, nanoparticles were isolated by ultracentrifugation and resuspended at proper concentrations in HBSS (pH 7.4). CScoated nanoparticles were resuspended in HBSS with a pH value of 6.5 in order to Anexo 275 avoid particle aggregation. Control experiments for this formulation were also performed with the same HBSS solution. Cell culture experiments Caco-2 cells were cultivated on 80 cm2 flasks (Nunc, Denmark) using MEM supplemented with 10% FBS, 1% l-glutamine, 1% nonessential amino acids and penicillin/streptomycin solution. Cells were maintained on a controlled atmosphere at 37ºC with 95% of relative humidity and 5% CO2. The culture medium was changed every other day for approximately 5–6 days until cells reached approximately 80–90% confluency. After the passage operation, cells were seeded approximately at 2.5×105 cells per flask. For the experiments, cells with passage numbers between 25 and 35 were used. Cytotoxicity studies Cells were cultivated on 96-well plates (0.33 cm2/well) (Nunc, Denmark) with a cell density of 1.4×104cells/cm2 for approximately 7 days until a homogeneous cell monolayer was obtained. Cytotoxicity experiments were conducted for PEGand CS-coated nanoparticles at concentrations between 0.1 and 20 mg/ml (0.2 ml/well). Monolayers were in contact with the suspension of nanoparticles for 2.5 h. After this time, the nanoparticle suspensions were removed and cell viability was determined using a colorimetric method (Cell Titer 96®, Promega, Madison, WI) where the reagents are bioreduced by the cells to a colored formazan product (readable at 490 nm) through a pathway where NADPH or NADH are involved. HBSS was used as a negative control, whereas a 2% sodium dodecyl sulfate (SDS) solution in HBSS as a positive control. 282 Nanocápsulas de quitosano: nuevos vehículos…. and Behrens et al., 2002). On the other hand, indirect observations by Gasco and coworkers (Bargoni et al., 1998) have led to the conclusion that lipid nanoparticles are able to cross the intestinal epithelium. However, despite the value of this previous information, no studies have been reported so far aimed at investigating the factors that affect the interaction of lipid nanoparticles with the intestinal epithelium. Consequently, one of the primary goals of this work was to study the interaction of these lipid nanostructures with the Caco-2 cells and to investigate whether or not the polymer coating affects this interaction. Before these transport studies, the cellular toxicity of the different types of particles was also evaluated. Cytotoxicity of the nanoparticles The evaluation of the cytotoxicity of the nanoparticles had a double objective: (i) to determine the concentrations that could interfere with the cellular metabolism in further studies in Caco-2 and (ii) to obtain a preliminary estimation of the safety of these new formulations. The toxicity of the lipid core was expected to be very low given the fact that tripalmitin is a physiological triglyceride. In addition, the low toxicity of lipid nanoparticles has already been shown in human promyelotic cells (HL60) and human granulocytes (Müller et al., 1997). Therefore, the evaluation of the toxicity of the lipid cores was excluded from this study. As shown in Fig. 1, nanoparticles coated with PEG, a polymer of very low toxicity, showed high viabilities even at concentrations as high as 20 mg/ml. On the other hand, CS-coated nanoparticles exhibited certain toxicity at concentrations above 1 mg/ml, having an IC50 of 3.3 mg/ml. These values, indicative of a toxic effect of the CS coating are in agreement with those previously reported for CS solutions (Schipper et al., 1996). Consequently, from these studies we concluded that PEG-coated nanoparticles showed an extremely low cytotoxicity while CS-coated nanoparticles appeared to have a low toxicity. Therefore, a concentration of 1 mg/ml was selected as the threshold nanoparticles concentration for further Caco-2 experiments. Anexo 283 Fig. 1: Relative viability of Caco-2 cells following incubation with different concentrations of PEG-coated nanoparticles ( ) and CS-coated nanoparticles ( ) for 2.5 h (mean±S.D., n=8). Qualitative and quantitative analysis of the interaction of nanoparticles with the Caco-2 cells In order to visualize the limits of the cellular compartments and, thus, to help the localization of the nanoparticles associated to the monolayers we used Bodipy® 650/665-Phalloidin. This fluorescent dye binds selectively to F-actin, which is a major component of the cytoskeleton (Artursson et al., 1994). Control experiments showed that cells had low self-fluorescence and that no crossfluorescence between the fluorochromes occurred. Confocal laser scanning microscopy images show that both, CS and PEG, surface-modified nanoparticles were able to penetrate through the Caco-2 monolayers (Figs. 2 and 3, supplementary material). More specifically, after 1 h of incubation, both nanoparticles formulations were seen in consecutive confocal sections of the monolayer. The disposition and appearance of the fluorescence spots varied depending on the depth of the section. In the apical side (top images of Fig. 2 284 Nanocápsulas de quitosano: nuevos vehículos…. and Fig. 3) the nanoparticles are randomly distributed. However, in the underlying sections, the nanoparticles are preferentially co-localized with Bodipy®-Phalloidin in the cytoskeleton. In addition, as the sections are taken deeper into the cell, the fluorescent spots appear less aggregated and with a smaller size. Under the conditions of the present study, the observed localisation of the nanoparticles, does not allow us to discriminate between paracellular and/or transcellular internalization. Therefore, we could hypothesize that nanoparticles can enter the epithelium by either or both paracellular and transcellular mechanisms of transport. Despite these possible routes, previous reports on the internalization of CS nanoparticles by the Caco-2 monolayers, led us to accept the transcellular internalization as the most plausible mechanism (Ma and Lim, 2003 and Behrens et al., 2002). Fig. 2: Confocal scanning microscopy images of Caco-2 cell monolayers exposed to PEG-coated nanoparticles (0.25 mg/ml) and Bodipy® 650/665-Phalloidin for 1 h. The left column shows four sections collected at the Bodipy® 650/665-Phalloidin emission wavelength. The right column shows the same sections collected at the rhodamine® emission wavelength. The images are sections separated 3 μm each starting from the apical side (top). Anexo 285 Fig. 3: Confocal scanning microscopy images of Caco-2 cell monolayers exposed to CS-coated nanoparticles (0.25 mg/ml) and Bodipy® 650/665-Phalloidin for 1 h. The left column shows four sections collected at the Bodipy® 650/665-Phalloidin emission wavelength. The right column shows the same sections collected at the rhodamine® emission wavelength. The images are sections separated 3 μm each starting from the apical side (top). Therefore, the analysis of this previous information and the results of the present study led us to suggest that the polymer-coated lipid systems may enter the Caco-2 monolayer. Nevertheless, a more detailed study using different fluorescence markers would be necessary in order to understand the determinants of the mechanism of transport of the nanoparticles. The quantitative analysis indicated that both nanoparticles formulations have an important interaction with the Caco-2 cells (Fig. 4). Interestingly, irrespective of the nature of the coating, the amount of nanoparticles bound to the cells increased linearly with the concentration of nanoparticles, reaching values of 286 Nanocápsulas de quitosano: nuevos vehículos…. around 15% of the dose. The similarity of the results obtained for CS-coated nanoparticles and PEG-coated nanoparticles was surprising for us, given the different nature of both coatings. In fact, the association values of PEG-coated PLA nanoparticles to the Caco-2 cells were much less important than those observed in the present work for PEG-coated lipid nanoparticles (Behrens et al., 2002). Consequently, more comparative studies are needed in order to identify the importance of the core and the coating in these mechanistic studies. Fig. 4: Quantity of fluorescent nanoparticles (PEG-coated nanoparticles ( ), CScoated nanoparticles ( )) bound to Caco-2 monolayers after 1 h of incubation as a function of the concentration (mean±S.D., n=3). Transepithelial electrical resistance and Texas Red®-dextran transport A number of polymers, CS among them, have shown the ability to permeabilize the intestinal epithelium (Artursson et al., 1994). However, whether or not particles prepared from these polymers maintain their intrinsic capacity to modify the epithelial permeability remains unknown. Consequently, in the present work, we found it important to determine if this association has a consequence in the epithelial permeability. The measurement of the transepithelial electrical resistance (TEER) is a common way to determine paracellular permeability to ions and their changes upon exposure to a permeation enhancer. This information is relevant as reductions in the Anexo 287 TEER can be correlated with partial disruption of the tight junction complex that restricts the paracellular route for hydrophilic compounds. In the present study, no significant changes were appreciated in the control wells during the experiment (Fig. 5). In addition, the TEER values of the monolayers exposed to PEG-coated nanoparticles exhibited a profile that was not significantly different than that of the control experiment: a small initial drop in TEER followed by a progressive recuperation of the initial TEER value. On the other hand, CS-coated nanoparticles presented a different behaviour compared to that of the corresponding control experiment. More specifically, the TEER values of the monolayers exposed to CScoated nanoparticles decreased continuously during the experiment (2.5 h). Then, after removal of the nanoparticles formulations from the apical side, the monolayers were washed and incubated with MEM for the next 24 h. During this 24 h period, a slow recuperation of the TEER values was observed, reaching half of the initial TEER value after 6 h, and total recuperation in 24 h. Fig. 5: Transepithelial electric resistance (TEER) of Caco-2 monolayers exposed to PEG-coated nanoparticles (1 mg/ml) (◊), CS-coated nanoparticles (1 mg/ml) (▲) or their respective controls (HBSS (□), HBSS pH 6.5 (●)) (mean±S.D., n= 3–6). The apparent permeability (Papp) of the fluorescent marker Texas Red®- dextran (3000 Mw) was in good agreement with the TEER data (Table 2). The results showed that PEG-coated nanoparticles do not have a permeability enhancing effect. In contrast, CS-coated nanoparticles elicited a very marked transport 288 Nanocápsulas de quitosano: nuevos vehículos…. enhancing effect compared with their control. Indeed, the permeability values of the monolayers incubated with CS-coated nanoparticles were almost four-fold those of the controls. This transport enhancing effect is in good agreement with that previously reported for CS solutions (twoto four-fold enhance in manitol permeability with respect to the control) (Artursson et al., 1994 and Schipper et al., 1996). Table 2. Apparent permeability coefficient (Papp) of Texas Red®-dextran (3000 Mw) determined in Caco-2 cell monolayers exposed to PEG-coated nanoparticles, CS-coated nanoparticles and their controls (mean±S.D., n= 3–6). Treatment Papp (×10−7 cm/s) Relative Papp (carrier/control) HBSS 1.70±0.86 – PEG-coated nanoparticles 1.83±0.18 1.08 CS-coated nanoparticles 6.12±1.45 3.6 Overall, these results indicate that CS-coated nanoparticles produce a transient increase in the transepithelial permeability. This effect could be related to an opening of the paracellular route due to the interactions of CS with the proteins involved in the tight junctions between epithelial cells (Artursson et al., 1994 and Smith et al., 2004). Nevertheless, in the interpretation of these results we should be conscious of the fact that in these cell culture experiments an important amount of nanoparticles (0.333 mg/cm2) is forced to be directly in contact with the cells, a situation that differs substantially from that expected following oral in vivo administration. Anexo 289 Oral administration of sCT-loaded surface modified nanoparticles As a final step of the present work, and in order to elucidate the role of the polymer coating around the lipid nanoparticles in their ability to enhance the intestinal absorption of peptides, we determined the calcemia levels following the oral administration of the nanoparticles and the corresponding control (sCT solution). Two very distinct profiles were observed for PEGand CS-coated nanoparticles (Fig. 6). PEG-coated nanoparticles did not produce any significant effect on serum calcium levels. In contrast, CS-coated nanoparticles showed a rapid and drastic reduction in serum calcium levels. Moreover, these low calcemia levels were maintained for at least 24 h. This hypocalcemic effect was significantly lower than that of the control for all the time points (α < 0.01). This important extent of reduction of the serum calcium levels is similar to that previously reported for other systems, such as CS-coated poly(lactic-co-glycolide) nanoparticles and poly(Nisopropyl acrylamide)-coated polystyrene nanoparticles (Kawashima et al., 2000 and Sakuma et al., 2002). However, to our knowledge, the ability of CS-coated nanoparticles to prolong the pharmacological response is superior to that of the previously reported systems. Fig. 6: Serum calcium levels after oral administration to conscious rats of sCT in solution (□), or associated to PEG-coated nanoparticles (◊) or CS-coated nanoparticles (▲) (mean±S.D., n= 6). *Significant differences from the sCT solution (α<0.01). 290 Nanocápsulas de quitosano: nuevos vehículos…. At the present stage, our hypothesis to explain the success of the CS-coated nanoparticles is that they interact favourably with the mucus covering the intestinal mucosa (Takeuchi et al., 2001 and Behrens et al., 2002) and then, diffuse through this mucus layer reaching the underlying epithelium. These interactions would permit a site-specific delivery of sCT for prolonged times and, consequently, a prolonged pharmacological response. Moreover, it could be expected that CS, because of its ability to interact with the tight junctions, could enhance the paracellular transport of the peptide released at the epithelial level. In contrast, PEGcoated nanoparticles do not have a favourable interaction with the mucus layer covering the intestinal epithelium and, thus, have a limited access to the underlying epithelium. In addition, PEG-coated nanoparticles did not produce an enhancement of the epithelial permeability, which could also be responsible for the absorption of the peptide. These differences could justify the lack of pharmacological response observed for the PEG-coated nanoparticles despite their ability to enter the Caco-2 cells monolayer. In summary, due to the extreme complexity inherent to the study of the particle uptake phenomenon and of the mechanisms of interaction with the epithelium (particularly for biodegradable particles), more detailed studies will have to be performed in order to fully understand the in vivo behaviour of these novel nanocarriers. Nevertheless, the results presented in this work are relevant as they show that particle uptake may not be the main mechanism behind the enhanced bioavailability of sCT in chitosan-coated nanoparticles. Conclusions Overall, the results of this work underline the importance of the surface composition of the nanoparticles in their ability to enhance the intestinal absorption of peptides. More specifically, the results showed that lipid nanoparticles coated with PEG or CS were able to enter the Caco-2 cell monolayers. However, only those coated with CS were able to open the tight junctions, thereby increasing the permeability of the model epithelium. Moreover, CS-coated nanoparticles were able Anexo 291 to enhance the oral absorption of the peptide sCT leading to a prolonged hypocalcemic response, whereas, PEG-coated nanoparticles were unsuccessful at increasing the absorption of the peptide. Consequently, the results suggest that the favourable interaction of the CS-coated nanoparticles with intestinal mucosa together with their permeation enhancing characteristics might be responsible for the improved oral absorption of the associated sCT. Acknowledgements This work was supported by grants from the Spanish Ministry of Science and Technology (SAF 2000-0145) and Almirall Prodesfarma S.A. We thank María Isabel Loza for her help with the cell culture experiments. 298 Nanocápsulas de quitosano: nuevos vehículos…. Introduction Overcoming the low bioavailability of drugs thathave low permeability through biological membranes is one of the most prominent pharmaceutical problems [1]. The advent of the biotech revolution has stressed even more this limitation as an increasing number of molecules with high therapeutic potential are dumped because of their poor biopharmaceutical properties [2]. Peptides and proteins are the molecules that best exemplify these biopharmaceutical properties: together with their poor permeability, thesemolecules are extremely labile to the action of proteolytic enzymes [3]. The combination of these two biopharmaceutical barriers (low permeability through the epithelia and degradation) constrainsthe oral bioavailability of peptides to values usually below 1%. One of the most appealing strategies to improve the oral absorption of these delicate macromolecules has been the association or inclusion of peptides in submicrometer-size carriers [4,5,6]. The challenge, however, has been the definition of the characteristics of the carrier that will lead to an optimized transport and, an adequate delivery of the associated peptide. Among the materials chosen for the design of these nanocarriers, the lipids have played an important role. From lipid excipients to nanostructured lipid carriers Lipids comprise a rather heterogeneous group of molecules that are characterized by the presence of aliphatic chains. Lipids have been used for pharmaceutical preparations due to their excellent biocompatibility and their wide spectrum of physicochemical characteristics, which make them useful for multiple applications [7]. To this date, waxes, mono-, diand triglycerides, fatty acids, cholesterol derivatives or phospholipids are the most common classes of lipids used in traditional pharmaceutical formulations such as lipid emulsions, intramuscular depot injections, matrix tablets or suppositories. In the last, a better understanding of the interactions of lipids at the cellular level has considerably increased the interest for these materials as drug transmucosal Anexo 299 carriers. In fact, electron spin resonance and NMR studies have shown that monoglycerides and fatty acids interact causing a disorder in the lipid membranesthat is supposed to be related to their capacity to permeate the epithelia [8]. Moreover, some lipidshave exhibited an affinity for the SH-proteins, which are known to play a role in transmembrane transport and tight junction permeability [9]. More recently, it has also been shown that some lipids can also interact with P glycoprotein and cytocrome P 450, thereby facilitating intracellular drug delivery [10]. However, maybe the most unique characteristic of lipid based formulations is their ability to be digested in the intestine, thus producing micelles that can enter the organism through the intestinal lipid transport system [7]. Drugs incorporated in the micelles resulting from lipid digestion may be internalized through this pathway [11]. The absorption enhancing properties of lipid excipients have already been tested for oral insulin delivery [12,13]. This early works provided some proofofconcept of the possibility to achieve higher oral bioavailability of peptides if administered with lipid promoters. The new challenges faced by lipid formulations have stimulated the design of more advanced drug delivery systems, particularly submicrometric carriers, whose high surface/volume ratio maximizes the interactions with the biological environment. Moreover, some of these nanocarriers, i.e. liposomes and solid lipid nanoparticles, have shown their ability to cross the intestinal epithelium, probably though an endocytic pathway [14,15]. Liposomes in oral peptide delivery The capacity of liposomes to encapsulate hydrophilic macromolecules has been suggested as a way to protect them in the gastrointestinalract [16]. However, in order to achieve this protective effect, it is critical to preserve the liposome integrity in this hazardous environment. Since the early works from Dapergolas et al. [17] 300 Nanocápsulas de quitosano: nuevos vehículos…. it is well known that liposomes made of phospholipids with a phase transition temperature above 37ºC are more stable in the GI tract than those composed of lipids in a liquidcrystalline state (such as natural phospholipids). A step further in the development of oral liposomes has been based upon the optimization of their surface characteristics. For example, the modification of the surface of liposomes with protective coatings such as mucin or PEG resulted in enhanced oral insulin effect compared to uncoated liposomes. In vitro experiments confirmed that these liposome formulations were able to protect the peptide more efficiently against its degradation in intestinal fluids [16]. Nevertheless, the beneficial effect of these coatings should not only be attributed to its protective capability, as a subsequent study has also shown that these polymers increased the residence time of the liposomes in the GI tract. More concretely, mucin-coated liposomes displayed an extended gastric retention and PEGcoated liposomes an increase in the residence time in the small intestine [18]. Liposomes coated with other type of mucoadhesive polymers such as Carbopol® or chitosan have also shown to enhance the absorption of orally administered peptides [19,20]. The surface charge of liposomes also seems to have an important role in the capacity of liposome formulations to promote the oral peptide absorption, however, the optimal value of that parameter remains unclear. Some papers have indicated that the presence of anionic lipids improves the intestinal absorption of insulin [21,22], while others have reported the same effect, either for insulin or calcitonin for cationic liposomes [16,23]. Microemulsions and nanoemulsions in oral peptide delivery Microemulsions and nanoemulsions are dispersed systems of two immiscible liquids. Microemulsions can also be defined as colloidal dispersions of liquids thermodynamically stabilized by a layer of surface-active molecules. Nanoemulsions are not thermodynamically stable, however, their coalescence can be hindered for prolonged periods of time if they are properly stabilized. With an Anexo 301 already marketed oral Cyclosporine A formulation (Sadimmune Neoral®), increased attention has been paid to the possibility of formulating peptides orally by including them in submicrometric emulsions. Nevertheless, up to date, the most successful results have been obtained with small peptides such as desmopressin [24], vasopressin [25] or thefibrinogen antagonist peptide SK&F 106760 [26]. This positive behaviour has been understood as a consequence of the lipid absorption promoting effect. This hypothesis has been further supported by the fact that composition, rather than emulsion droplet size was found to be the main factor responsible for the enhanced peptide absorption. Indeed, the presence of a polyoxyethylene derivative (Cremophor EL) was considered essential to achieve those high peptide oral bioavailabilities [27]. Despite the encouraging data obtained for Cyclosporine A and the SK&F 106760 peptide, the success of the submicron emulsion for oral delivery of high molecular weight peptides remains to be a challenge. Some improvements in the absorption of peptides, i.e. salmon calcitonin, have been observed for submicron emulsions which were coated with a mucoadhesive polymer such as Carbopol® [24]. This improved absorption of the associated peptide was attributed to the enzyme inhibitory action of this polymer [28]. More recent studies [29] have confirmed that the presence of Carbopol® as a coating was critical for the efficacy of the formulation. Polymeric nanocapsules in oral peptide delivery Nanocapsules are carriers comprising an inner lipid reservoir and an outer polymeric wall. Insulin-loaded polycyanoacrylate nanocapsules have render, so far, some of the most outstanding results in animal models. Significant reductions in the glycemia of diabetic rats were produced and maintained for prolonged periods of time, when that nanocapsule formulation was administered orally [30]. The extent and duration of the pharmacological effect was explained by the penetration of the nanocapsules through the mucosa and the further release of the peptide from the 302 Nanocápsulas de quitosano: nuevos vehículos…. internalized formulation [31]. Unfortunately, the results obtained in the dog model, although positive, were not as promising as those previously reported with rats [32]. New polymer-lipid nanostructures for oral peptide delivery Although over the last years some breakthrough technologies have led to a new perspective of oral peptide delivery, renewed efforts are still needed to obtain a clinically useful carrier system. Lipid-based carrier systems are not an exception and still have to face important challenges to optimize their performance. First, lipids are labile materials, very susceptible to enzymatic degradation in the intestine. As a consequence, lipid drug delivery systems are usually destroyed before reaching the adequate site for peptide absorption, thus loosing their ability to protect and enhance the transport of the peptide. Finally, it is generally accepted that the capacity of transport of lipid carriers, even though significant, may be insufficient at this stage to achieve a clinically useful effect. In order to advance a further step in the development of a useful peptide delivery system for the oral route, we have designed new carrier systems based on a lipid core-polymer coat structure. The polymeric coating was intended to optimize the interaction of the delivery system with the biological environment present in the gastrointestinal tract. Hence, we selected polymer coatings that were supposed to prevent aggregation and destruction of the delivery systems, to confer mucoadhesive properties to the carriers or to enhance transepithelial permeability. A schematic illustration of the nanostructures prepared is depicted in Figure 1. Since the composition of the core is supposed to affect drug incorporation and release [33], we have proposed three different cores for the nanostructures: a solid lipid (tripalmitin), a liquid lipid (Miglyol® 812) or a solid-liquid lipid mixture (tripalmitin/Miglyol® 812). Anexo 303 Lipid core (Miglyol, tripalmitin or tripalmitin/ Miglyol mixtures) Poly mer s hell ( PEG or c hitos an) Lipid core (Miglyol, tripalmitin or tripalmitin/ Miglyol mixtures) Poly mer s hell ( PEG or c hitos an) Figure 1: Illustration of the proposed architecture of the designed polymer-coated lipid nanostructures. Regarding the polymer coating, two hydrophilic polymers have been selected: PEG and chitosan. The selection of PEG is based on the results of previous studies performed by our group, which have shown that a PEG coating around PLA nanoparticles greatly enhanced their stability in simulated gastrointestinal fluids [6]. Similarly, studies performed by Olbrich et al. have shown that a coating with ethylene oxide derivatives (Poloxamers) around the lipid nanoparticles protected them from degradation by pancreatic enzymes [34]. Moreover, it has been indicated that a PEG coating may favour the interaction of colloidal carriers with mucosal surfaces [18,35]. However, while the protective effect of PEG is well understood, its possible role in enhancing the interaction of nanosystems with epithelia remains unclear. Nevertheless, whether this improved interaction is simply a consequence of the enhanced stability of the nanoparticles in contact with biological fluids, or is additionally helped by some specific mechanisms, the clear observation is that the PEG-coated PLA nanoparticles are able to cross the nasal mucosa up to a more important extent than those non-coated [36,37]. On the other hand, the selection of the polysaccharide chitosan as a coating for transmucosal nanocarriers is justified by its interesting biopharmaceutical behaviour. From the point of view of drug delivery, some important features of this material are its mucoadhesiveness [38] and its capacity to increase the permeability of epithelia [39]. In addition, previous studies from our group have shown that chitosan-based nanosystems are able to interact with mucosal surfaces, thus facilitating the interaction and transport of the associated active compound across these surfaces. More specifically, chitosan nanoparticles were able to enhance the 304 Nanocápsulas de quitosano: nuevos vehículos…. transport of insulin across the rabbit nasal mucosa up to a much greater extent than chitosan solutions [40]. Similarly, low molecular weight chitosan nanoparticles elicited an interesting capacity for the transport of antigens across the nasal mucosa [41]. In addition, besides their favorable behaviour for the nasal transport of macromolecules, chitosan nanoparticles exhibit an affinity for the ocular mucosa. Recent studies aimed at studying the residence time of these nanoparticles in the ocular mucosa, following topical administration, indicated that the interaction of chitosan with the cornea and conjunctiva was enhanced and prolonged when presented in a nanoparticulate form [42]. All these studies provided evidence of the benefits of chitosan nanosystems as compared to other forms of presentation of chitosan. Taking this previous information into account the aim of the present paper was not simply to review the state-of-the-art of the potential of lipid-based colloidal systems for oral peptide administration but to specifically emphasize the latest advances from our work regarding the design and evaluation of polymer-coated lipid nanosystems. Preparation and characterization of polymer-coated lipid nanostructures PEG-coated tripalmitin (or PEG-coated tripalmitin/Miglyol®) nanoparticles were prepared in a single step by a modified double emulsionsolvent evaporation method [43]. In this technique, hydrophilic drugs such as peptides may be incorporated to the nanoparticles in the inner aqueous phase. The PEG coating was formed due to the addition of the modified fatty acid PEG-stearate to the organic phase. On the other hand, chitosan-coated nanocarriers have been prepared following different procedures depending on the nature of the core. Chitosan-coated solid lipid nanostructures were prepared by the double emulsion-solvent evaporation technique, indicated above [43]. When using this procedure, chitosan can be either added to the external aqueous phase of the double emulsion or once the lipid cores Anexo 305 were precipitated [44]. Alternatively, chitosan-coated submicron emulsions were prepared according to the solvent displacement-solvent evaporation technique, as previously reported [45]. As in the case of the solid cores, chitosan can be added before or after the solvent evaporation takes place. Irrespective of the technique, a critical ingredient for the formation of the chitosan-coated systems was the presence of lecithin, which was added to the organic phase in both procedures. Indeed, an ionic complex between lecithin and chitosan is formed at the interface of the colloidal system, thus facilitating the attachment of chitosan onto the surface of the system. Lipid cores (non-coated) showed particle sizes around 200 nm upon preparation and strongly negative surfaces (Table 1). The coating of the tripalmitin cores with PEG produced a negligible effect on the particle size whereas the zeta potential shifted towards more neutral values [43]. As described for other PEGcoated systems, this change is a consequence of the extension of the shear plane of the colloidal system [46]. The formation of a chitosan coating around the lipid cores led to an increase in the particle size, accompanied by an inversion of the zeta potential from negative to positive values (Table 1) [47]. Both, the size increase and the inversion of the zeta potential, support the successful formation of a chitosan layer surrounding the lipid cores. Table 1: Hydrodynamic diameter (analyzed by photon correlation spectroscopy) and zeta potential (analyzed by laser Doppler anemometry) of the studied nanostructures (Mean ± SD., n=3). Data from: 1Prego et al. [54] and 2GarcíaFuentes et al. [47]. Formulation Diameter (nm) Zeta Potential (mV) Miglyol® cores1195.8 ± 1.1 -52.0 ± 1.1 Chitosan Miglyol® nanocapsules1266.6 ± 7.6 +34.8 ± 0.6 Tripalmitin cores2200.0 ± 2.3 -50.3 ± 1.8 PEG-coated tripalmitin nanoparticles2226.4 ± 7.5 -34.8 ± 2.8 PEG-coated tripalmitin/ Miglyol® nanoparticles2207.4 ± 19.1 -36.6 ± 2.5 Chitosan-coated tripalmitin nanoparticles2537 ± 16 +29.2 ± 6.2 306 Nanocápsulas de quitosano: nuevos vehículos…. The size and the morphology of the PEGand chitosan-coated systems were visualized by transmission electron microscopy (Figure 2). All the nanostructures presented a spherical shape. In the case of PEG-coated nanoparticles, a difference in the staining was appreciated between the inner and the outer part of the nanoparticles, being this a possible indication of the core-coat structure of the systems. Unfortunately, this difference in staining could not be observed for chitosan-coated tripalmitin nanoparticles although the change in particle size was clearly visible in the micrographs. This dissimilarity between micrographs could be related not only to the different nature of the coating polymers but also to the high chitosan molecular weight as compared to that of PEG. This observation led us to suggest that the chitosan may be forming a more compact polymer layer compared to that of the PEG. Figure 2: Transmission electron microscopy pictures of different lipid nanostructures: PEG-coated tripalmitin nanoparticles (left), PEG-coated tripalmitin/Miglyol® nanoparticles (center) and chitosan-coated tripalmitin nanoparticles (right). NMR analysis has allowed us to confirm that the PEG-coated nanostructures are mainly composed of a triglyceride core and a small amount of surfactants and surface modifying molecules (i.e. lecithin, PEG-stearate) [48]. On the other hand, the core-coat model proposed for these structures was fully confirmed in the case of PEGtripalmitin nanoparticles. NMR experiments aimed to analyze the relaxation of the chemical groups of nanoparticle suspensions and their spin-population effects with the water protons confirmed the presence of PEG protruding towards the external phase and the presence of a solid lipid core comprising mainly the triglycerides. Moreover, quantitative NMR analysis has Anexo 307 confirmed the possibility of modulating the PEG coating density of these carriers by controlling the amount of PEGstearate added in the preparation procedure [48]. The NMR characterization of nanoparticles having a tripalmitin/Miglyol® core indicated that there were significant amounts of the oil incorporated within the nanoparticles matrix. Moreover, the presence of Miglyol® as separated liquid domains associated to the nanoparticles was confirmed by NMR relaxation analysis and the observed restricted diffusion dynamics of the entrapped oil. A further confirmation of this structure was obtained from differential scanning calorimetry (DSC) and x-ray diffraction spectroscopy [49]. Stability in simulated gastrointestinalfluids Taking into account that the designed lipid nanostructures were intended for oral drug delivery, the assessment of their stability in gastrointestinal fluids was critical. Indeed, lipid nanoparticles have shown important particle aggregation in gastric media and a marked degradation in simulated intestinal fluid with enzymes [50,51]. Interestingly, the aggregation of the lipid cores in gastric medium has been found to be dependent on the mechanism of particle stabilization. Thus, we previously prepared lipid cores that were stabilized only with lecithin (uncoated cores), a surfactant that confers on the system a distinctly negative charge at neutral pH. At low pH, the surface charge of the lipid nanostructures was significantly reduced due to the less important ionization of the negatively charged phospholipids, thus leading to the aggregation of the nanoparticles in simulated gastric medium (Figure 3) [43]. The importance of the pH of gastric medium in the aggregation processes was also assessed by incubating the formulations in an inorganic acid solution (pH 1.2), without enzymes. As can be appreciated in Figure 3, the addition of a second ionic stabilizing surfactant (sodium cholate) did not enhance the stability of tripalmitin cores neither in inorganic medium nor in simulated gastric medium. On the contrary, by coating the lipid cores with polymers with steric stabilizing properties such as the PEG-stearate or the poloxamer 188, completely stabilized the formulations in both media [43,52]. As indicated before, previous experiments 314 Nanocápsulas de quitosano: nuevos vehículos…. established from the in vitro experiments. First, the enhanced stability provided by the polymeric coatings seems to be a relevant issue for peptide delivery. Chitosan, a polymer with well defined mucoadhesive properties, not only provides better colloid stability but possibly enhances the interaction of the nanostructures with the mucosal surfaces [19,38]. At this stage, increasing evidence points to the process of mucoadhesion as a way of gaining better access to the underlying epithelium [56]. Finally, taking into account the results from the Caco-2 experiments, one could attribute the success of the chitosan-coated lipid nanostructures to their ability to reduce the TEER. However, we should underline that sCT co-administered with chitosan solutions that show similar reduction in the TEER do not produce any significant hypocalcemic effect. In addition, it should be noted that the marked reductions in the TEER were observed for high doses of chitosan-coated lipid nanostructures while doses in animal where far below this limit. In conclusion, the positive behaviour of chitosan-coated lipid nanostructures seems to be determined by a combination of functions from the carrier that may include better peptide protection, improved carrier stability and enhanced interaction between drug and the mucosal surfaces. Conclusions This article reports the design and characterization of new polymer-coated lipid-based nanostructures for oral drug delivery. Among the systems prepared, chitosan-coated lipid nanostructures have shown the capacity to improve the oral efficacy of sCT. 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