Nanomedicamentos para el tratamiento localizado de patologías pulmonares

UNIVERSIDADE DE SANTIAGO DE COMPOSTELA FACULTAD DE FARMACIA Departamento de Farmacia e Tecnoloxía Farmacéutica. NANOMEDICAMENTOS PARA EL TRATAMIENTO LOCALIZADO DE PATOLOGÍAS PULMONARES Felipe Andrés Oyarzún Ampuero Santiago de Compostela, 2011 DOÑA DOLORES TORRES LÓPEZ Y DOÑA MARÍA JOSÉ ALONSO FERNÁNDEZ, PROFESORA TITULAR Y CATEDRÁTICA, RESPECTIVAMENTE, DEL DEPARTAMENTO DE FARMACIA Y TECNOLOGÍA FARMACÉUTICA DE LA UNIVERSIDAD DE SANTIAGO DE COMPOSTELA. INFORMAN: Que la presente Memoria Experimental titulada: “Nanomedicamentos para el tratamiento localizado de patologías pulmonares”, elaborada por el Licenciado en Farmacia Felipe Andrés Oyarzún Ampuero, ha sido realizada bajo su dirección en el Departamento de Farmacia y Tecnología Farmacéutica y, hallándose concluida, autorizan su presentación a fin de que pueda ser juzgada por el tribunal correspondiente. Y para que conste, expiden y firman el presente certificado en Santiago de Compostela, el 20 de Julio de 2011. Fdo. Dolores Torres Fdo. María José Alonso A mi familia...  "Lo escuché y lo olvidé, lo vi y lo entendí, lo hice y lo aprendí." Confucio.  AGRADECIMENTOS Quiero expresar mi agradecimiento a todas las personas que me han prestado su apoyo, tanto profesional como personal, durante todo el camino recorrido para finalizar esta tesis doctoral. A mis directoras de tesis, las profesoras Dolores Torres y María José Alonso, por haberme recibido y dado la gran oportunidad de realizar este trabajo de investigación, por sus consejos, orientaciones, apoyo y gran comprensión (incluso en importantes aspectos personales) durante todo el tiempo que duró la parte práctica y escrita de esta tesis. Es importantísimo hacer notar que un trabajo de similares características habría sido muy difícil de desarrollar en mi país. Al Ministerio de Educación de Chile y a la Xunta de Galicia, por facilitarme el apoyo económico necesario para realizar esta tesis. A Gustavo Rivera, mi gran amigo, por su valiosísima colaboración, tanto teórica como práctica, desde su llegada al laboratorio. A Begoña Seijo, por toda la ayuda prestada en su calidad de profesora y Coordinadora del programa de doctorado. Ésta se extiende desde antes de mi ingreso oficial al doctorado y posiblemente perdurará luego de finalizar éste. A todos los demás profesores, investigadores y colaboraderes del grupo: Carmen Remuñán, Alejandro Sánchez, Marcos García, Noemí Csaba, Francisco Goycoolea, Purificación Domínguez, Rafael Romero, etc. Todos, absolutamente todos, tienen, además de grandes cualidades profesionales,   Resumen, Abstract  17 Resumen El objetivo de la presente memoria se ha dirigido al diseño y evaluación de nanoestructuras para el tratamiento localizado de patologías pulmonares. En una primera etapa, se han desarrollado nanopartículas de quitosano, en combinación con ácido hialurónico o con carboximetil-β- ciclodextrina, conteniendo la macromolécula hidrofílica heparina. Dichos sistemas fueron evaluados en relación a su capacidad de mejora de eficacia de la heparina sobre mastocitos, en el tratamiento del asma bronquial. Se demostró por microscopía confocal de fluorescencia que los nanosistemas eran internalizados por mastocitos de rata y, en el caso de los nanosistemas con ciclodextrinas, se consiguió mejorar de manera significativa el efecto de la heparina sobre la inhibición de la liberación de histamina en mastocitos. La segunda parte del trabajo se orientó al diseño de un nuevo nanosistema, consistente en nanocápulas de ácido hialurónico, con el fin último de dirigirlo al tratamiento del cáncer de pulmón. Los nanosistemas incrementaron significativamente el efecto citotóxico del antitumoral hidrofóbico docetaxel, sobre la línea celular de cáncer de pulmón NCI-H460, hecho que se atribuyó a la internalización de las nanocápsulas y la liberación intracelular del docetaxel. Estos resultados resaltan el enorme interés de los nanosistemas desarrollados para la liberación intracelular de fármacos en el tratamiento de enfermedades pulmonares.  Resumen, Abstract 19 Abstract The purpose of this work has been the design and evaluation of targeted nanostructures for the treatment of lung diseases. In a first stage, we developed nanoparticles made of chitosan combined with hyaluronic acid or carboxymethyl-β-cyclodextrin, containing the hydrophilic macromolecule heparin. The final aim was to explore the potential of these nanocarriers to treat asthma. It was demonstrated that the systems were able to get inside the mast cells and, in the case of nanosystems prepared with cyclodextrins, it was obtained a significantly greater effect to prevent histamine release in mast cells compared with the heparin alone. In a second stage, we developed a new nanocarrier, named as hyaluronic acid nanocapsules, for the intracellular delivery of hydrophobic anticancer drugs, with potential application in lung cancer. It was shown that these systems significantly improved the cytotoxic effect of docetaxel in the lung cancer cell-line NCI-H460. This result was attributed to the internalization of nanocapsules and the intracellular delivery of docetaxel. In summary, these nanostructures hold promise as intracellular drug delivery systems for the treatment of lung diseases.    LISTADO DE ABREVIATURAS   Listado de abreviaturas 23 Listado de abreviaturas: AFM: Atomic force microscopy. ANOVA: Analysis of variance. BKC: Benzalkonium chloride, cloruro de benzalconio. CMβCD: Carboxymethyl-β- cyclodextrin, carboximetil-β- ciclodextrina. CS: Chitosan, quitosano. CTAB: Hexadeciltrymethylammonium bromide, bromuro de hexadeciltrimetilamonio. DCX: Docetaxel EGF: Epidermal growth factor, factor de crecimiento epidermal. EPR: Enhanced permeability and retention effect. HA: hyaluronic acid, ácido hialurónico. HBSS: Hanks´ balanced salt solution. HPLC: High performance liquid chromatography. IP3: Inositoltrisphosphate, trifosfato de inositol. LMWH: Low molecular weight heparin, heparina de bajo peso molecular. LNC: Lipid nanocapsules. MDR: Multidrug resistance. MTT: Tretazolium salt 3-(4,5- dimewthylthiazol-2-yl)2,5 diphenyltetrazolium bromide. NCs: Nanocapsules. NMR: Nuclear magnetic resonance. ODN: Oligodeoxynucleotide. PACA: Poly(alkylcyanoacrylate). PBCA: Poly(isobutylcyanoacrylate). PBS: Phosphate buffered saline. PCL: Poly-ε-caprolactone. PEG: Polyethyleneglycol, polietilenglicol. PEI: Polietilenimina. SEM: Scanning electron microscopy. siRNA: Small interfering RNA, ARN pequeño de interferencia. TEM: Transmission electron microscopy. TPP: Pentasodium tripolyphosphate, tripolifosfato pentasódico. UFH: Unfractioned heparin, heparina no fraccionada.     INTRODUCCIÓN Introducción 32 Otro sistema que podemos destacar es el constituído por nanopartículas poliméricas de poli (ácido glutámico)-dextrano recubiertas por alcohol cetílico-tripalmitina, cargadas con 5-fluorouracilo. Dichos sistemas se atomizaron y administraron a hámsters para evaluar su eficacia sobre tumores de células escamosas, obteniéndose niveles efectivos de fármaco de un modo prolongado24. En el caso de la administración de material genético para el tratamiento del cáncer, un trabajo interesante se refiere al tratamiento de un modelo de metástasis pulmonar con nanosistemas constituídos por polietilenimina y el plásmido del gen p53. Se ha encontrado que la mutación/deleción de dicho gen está presente en la mayoría de los cánceres de pulmón de células pequeñas y no pequeñas, por lo que transfectar dichas células con el plásmido en cuestión resultaría favorable. Dichos sistemas fueron nebulizados y administrados a ratas, obteniéndose reducciones muy significativas en el tamaño y número de tumores25. También destaca el trabajo realizado Xu y col. (2008), que diseñaron nanosistemas constituídos por policaprolactona y polietilenimina conteniendo el RNA de interferencia akt1 (siRNA). La proteína Akt (proteína kinasa B) es un importante regulador de la supervivencia y proliferación celular y la amplificación de los genes que la codifican ha sido evidenciada en varios tumores. La nebulización de estos sistemas en ratas demostró una significativa disminución de la progresión del tumor de pulmón, a través de la inhibición de las señales celulares dependientes del gen Akt26.  24 Hitzman CJ, Wattenberg LW, Wiedmann TS. (2006). J. Pharm. Sci. 95(6):1196-211. 25 Densmore CL, Kleinerman ES, Gautam A, Jia SF, Xu B, Worth LL, Waldrep JC, Fung YK, T'Ang A. (2001). Cancer Gene Ther. 8(9):619-27. 26 Xu CX, Jere D, Jin H, Chang SH, Chung YS, Shin JY, Kim JE, Park SJ, Lee YH, Chae CH, Lee KH, Beck GR Jr, Cho CS, Cho MH. (2008). Am. J. Respir. Crit. Care Med. 178(1):60-73. Introducción   33 Tabla 1: Ejemplos de sistemas nanoparticulares administrados por vía pulmonar para el tratamiento de patologías pulmonares. Composición nanosistema Tamaño (nm) Fármaco(s) Especie animal Forma de administración Respuesta biológica y referencia PLGA, PLGA modificado con aglutinina de germen de trigo 186-400 Rifampicina, isoniazida, pirazinamida Cobayas Nebulización Mejora la biodisponiblidad y permite reducir la frecuencia de dosis en tuberculosis16;17. Quitosano-Alginato 235 Rifampicina, isoniazida, pirazinamida Cobayas Nebulización Mejora la biodisponiblidad y permite reducir la frecuencia de dosis en tuberculosis18. PLGA 213 Rifampicina Rata Nanopartículas incluidas en microesferas de manitol administradas en inhalador de polvo seco Retención pulmonar prolongada del nanosistema y localización en macrófagos alveolares27. Quitosano 376 pDNA Ratón Instilación intratraqueal Eficacia inmunogénica contra tuberculosis28. Gelatina modicada con EGF 230 Cisplatino Ratón Nebulización Alta concentración del fármaco en tumores localizados en pulmón23. Poli(ácido glutámico)- dextrano; recubrimiento con alcohol cetílicotripalmitina 800 5-fluorouracilo Hámsters Nebulización Se demuestra retención pulmonar y efecto prolongado del fármaco24. PEI No disponible pDNA Ratón Nebulización Reducción significativa en número y tamaño de tumores23.  27 Ohashi K, Kabasawa T, Ozeki T, Okada H. (2009). J. Control Release. 135(1):19-24. 28 Bivas-Benita M, van Meijgaarden KE, Franken KL, Junginger HE, Borchard G, Ottenhoff TH, Geluk A. (2004). Vaccine. 22(13-14):1609-15. Introducción 34 Policaprolactona y PEI 150 siRNA Ratón Nebulización Mayor eficacia en la supresión tumoral23. Quitosano modificado con ácido urocánico No disponible Proteína de programación de muerte celular 4 (PPMC4) Ratón Nebulización Facilita la apoptosis, inhibe importantes vías de proliferación celular y suprime eficientemente las vías de angiogénesis tumoral29. PLGA 201-240 TAS-103 (fármaco antineoplásico) Rata Nanopartículas incluidas en microesferas de trealosa administradas en inhalador de polvo seco Concentración del fármaco en pulmón superior a la detectada en plasma y superior que tras administración IV30. PLGA-PEG 44 Oligonucleótido secuestrador del factor nuclear κB (regula la expresión de importantes citoquinas inflamatorias) Rata Instilación intratraqueal Atenúa el desarrollo de hipertensión pulmonar arterial y de la remodelación arterial31. PLGA: poli(ácido láctico-ácido glicólico); PEI:polietilenimina; PEG:polietilenglicol; EGF: factor de crecimiento epidermal; pDNA:ADN plasmídico; siRNA:ARN pequeño de interferencia.   29 Jin H, Kim TH, Hwang SK, Chang SH, Kim HW, Anderson HK, Lee HW, Lee KH, Colburn NH, Yang HS, Cho MH, Cho CS. (2006). Mol. Cancer Ther. 5(4):1041-9. 30 Tomoda K, Ohkoshi T, Hirota K, Sonavane GS, Nakajima T, Terada H, Komuro M, Kitazato K, Makino K. (2009). Colloids Surf. B Biointerfaces. 71(2):177-82. 31 Kimura S.; Egashira K.; Chen L.; Nakano K.; Iwata E.; Miyagawa.; Tsujimoto H.; Hara K.; Morishita R.; Sueishi K.; Tominaga R.; Sunagawa K. (2009).Hypertension. 53(5):877-83. Introducción   35 2.1. Asma bronquial y heparina La heparina es una macromolécula que pertenece a la compleja familia de los glucosaminoglucanos. Está compuesta por unidades disacarídicas altamente sulfatadas (Figura 2), de tal manera que su naturaleza polianiónica favorece la interacción con una gran variedad de proteínas que poseen aminoácidos cargados positivamente32. Figura 2: Estructura molecular de heparina. De estas interacciones, la mejor caracterizada es la que lleva a la formación del complejo heparina-antitrombina III, que posee importantes repercusiones en el proceso de coagulación sanguínea33;34. Adicionalmente a su actividad antiacoagulante, la heparina ha demostrado poseer interesantes propiedades antiinflamatorias en enfermedades alérgicas. Ejemplo de ello, es la demostrada interacción entre la heparina y el receptor intracelular de trifosfato de inositol (IP3) en mastocitos35;36. El resultado de dicha interacción es la inhibición de la movilización intracelular de calcio, lo que impide la activación de mediadores intracelulares y la contracción del citoesqueleto, previniéndose finalmente la liberación de histamina (Figura 3). Este efecto   32 Wong WS, Koh DS. (2000). Biochem. Pharmacol. 59(11):1323-35. 33 Jaques LB. (1980). Pharmacol. Rev. 31: 99-166. 34 Lindhal U, Backstrom G, Thundberg L. (1983). J. Biol. Chem. 258: 9826-9830. 35 Lucio J, D'Brot J, Guo CB, Abraham WM, Lichtenstein LM, Kagey-Sobotka A, Ahmed T. (1992). Appl. Physiol. 73(3):1093-101. 36 Ahmed T, Syriste T, Mendelssohn R, Sorace D, Mansour E, Lansing M, Abraham WM, Robinson M.J. (1994). J. Appl. Physiol. 76(2):893-901. Introducción 36 induce a pensar el interesante rol que puede tener esta molécula en el tratamiento del asma alérgico37;38. Figura 3: Mecanismo de acción de heparina para prevenir la degranulación de los mastocitos. Confirmando la información anterior, se ha demostrado en distintos estudios que la inhalación de heparina de pesos moleculares diferentes (con y sin actividad antiacoagulante) es efectiva para prevenir las respuestas de broncoconstricción aguda y de hipersensibilidad bronquial, típicas de la enfermedad asmática39;40;41;42. De hecho, se llegó a demostrar que la potencia de las heparinas para prevenir dichas respuestas es inversamente proporcional a su peso molecular. Otro hallazgo interesante de la heparina, y que complementa su utilidad para el tratamiento del asma, es su potencial para prevenir la remodelación de las vías aéreas. Esta remodelación, mediada principalmente   37 Tyrrel DJ, Kilfeather S, Page CP. (1995). TiPS. 16:198-204. 38 Diamant Z, Page CP. (2000). Pulm. Pharmacol. Ther. 13(1):1-4. 39 Martinez-Salas J, Mendelssohn R, Abraham WM, Hsiao B, Ahmed T. (1998). J. Appl. Physiol. 84:222–228. 40 Molinari JF, Campo C, Shahida S, Ahmed T. (1998). Am. J. Respir. Crit. Care Med. 157: 887–893. 41 Campo C, Molinari JF, Ungo J, Ahmed T. (1999). J. Appl. Physiol. 86: 549–557. 42 Ahmed T, Ungo J, Zhou M, Campo C. (2000). J. Appl. Physiol. 88, 1721–1729. Introducción 37 por la acumulación de células musculares lisas, induce un estrechamiento excesivo de las vía aéreas y conduce a hipersensibilidad y dificultad para respirar43. La heparina ha demostrado una alta eficacia al inhibir la proliferación de células musculares lisas extraídas de las vías aéreas de humanos44, bovinos45 y perros46. 2.2. Docetaxel y su vehiculización tumoral Los taxanos (paclitaxel y docetaxel; Figura 4) son potentes agentes quimioterápicos cuyo núcleo químico fundamental es de origen natural y se extrae a partir de árboles del género “Taxus”. Su mecanismo de acción consiste en promover el ensamblaje intracelular de tubulina y en inhibir la despolimerización de los microtúbulos, impidiendo el crecimiento celular47. Estas moléculas han contribuido de manera trascendental a la supervivencia de pacientes con cáncer, siendo eficaces frente a un amplio rango de tumores sólidos como el cáncer avanzado de mama, de ovarios y de células no pequeñas de pulmón48;49;50, entre otros. Algunos estudios, como el publicado por Jones y col. (2005), indican que el docetaxel resulta más eficaz que el paclitaxel cuando se evalúa la sobrevida en pacientes con cáncer metastásico de mama51.   43 Kanabar V, Hirst SJ, OʼConnor BJ, Page CP. (2005). Br. J. Pharmacol. 146, 370–377. 44 Johnson PR, Armour CL, Carey D, Black JL. (1995). Am. J. Physiol. 269, L514–L519. 45 Kilfeather SA, Tagoe S, Perez AC, Okona-Mensa K, Matin R, Page C.P. (1995). Br. J. Pharmacol. 114, 1442–1446. 46 Halayko AJ, Recto E, Sthepens NL. (1997). Can. J. Physiol. Pharmacol. 75, 917–919. 47 Abal M.; Andreu J.M.; Barasoain I. (2003). Curr Cancer Drug Targets. 3(3):193-203. 48 Rowinsky RK, Donehower RCN. (1995). Engl. J. Med. 332: 1004-1014. 49 Trudeau ME, Eisenhauer EA, Higgins BP, Letendre F, Lofters WS, Norris BD, Vandenberg TA, Delorme F, Muldal AM. (1996). J. Clin. Oncol. 14: 422-428. 50 Piccart MJ, Gore M, Huinink WTB, Vanoosterom A, Verweij J, Wanders J, Franklin H, Bayssas M, Kaye S. (1995). J. Natl. Cancer Inst. 87: 676-681. 51 Jones SE, Erban J, Overmoyer B, Budd GT, Hutchins L, Lower E, Laufman L, Sundaram S, Urba W J, Pritchard KI, Mennel R, Richards D, Olsen S, Meyers ML, Ravdin PM. (2005). J. Clin. Oncol. 2005, 23, 5542–5551. Introducción 38 Figura 4: Estructura molecular de los taxanos (en azul y rojo se destaca la diferencia estructural entre las moléculas). Independientemente de su potencia, ambas moléculas se caracterizan por su elevado carácter hidrofóbico, lo que obliga a incluir agentes solubilizantes como el Cremophor EL y Tween 80, ambos en combinación con etanol, en las formulaciones endovenosas de paclitaxel y docetaxel, respectivamente. Desgraciadamente, estos vehículos son responsables de efectos secundarios severos, lo que limita la cantidad de fármaco que puede ser administrada al paciente de modo seguro. Entre estos efectos podemos mencionar: hipersensibilidad anafilactoide severa, neuropatía periférica, agregación de eritrocitos y patrones anormales de lipoproteínas52;53;54. Además de este problema de toxicidad, los taxanos son fármacos que comparten los problemas asociados a los antitumorales, es decir, su baja permanencia plasmática, y su biodistribución indiscriminada. Para superar estas limitaciones, se han propuesto nuevas formulaciones que no requieren   52 Gelderblom H, Verweij J, Nooter K, Sparreboom A. (2001). Eur J Cancer. 37(13):1590- 8. 53 Van Zuylen L, Verweij J, Sparreboom A. (2001).Invest. New Drugs. 19(2):125-41. 54 Engels FK, Mathot RA, Verweij J. (2007). Anticancer Drugs. 18(2):95-103. Introducción 39 la hidrosolubilización de los fármacos, y modifican su perfil farmacocinético. Entre ellas, destacan las basadas en nanoestructuras poliméricas, como por ejemplo, el sistema denominado Abraxane®, constituído por nanopartículas de albúmina conteniendo paclitaxel, la primera formulación de nanopartículas en clínica, introducida en el año 200555. Es importante recalcar que este hito impulsó de manera importante el desarrollo de nuevas formulaciones que contienen taxanos, entre ellas podemos destacar: liposomas56, lipoplejos57, nanopartículas58;59;60;61, nanocápsulas62;63 y conjugados64;65;66. En general, todos estos nanosistemas proveen al fármaco de una mayor citoespecificidad lo que puede verse traducido en menos efectos no deseados y una mayor eficacia en el tratamiento. Los sistemas nanocapsulares basados en estructuras de tipo reservorio (núcleo de aceite recubierto por una capa polimérica), son vehículos muy adecuados para el transporte de fármacos hidrofóbicos como el docetaxel pues, además de obviar la necesidad de utilizar solubilizantes en la formulación, permiten obtener una elevada eficacia de encapsulación y   55 Sparreboom A, Scripture CD, Trieu V, Williams PJ, De T, Yang A, Beals B, Figg WD, Hawkins M, Desai N. (2005). Clin. Cancer Res. 11, 4136-43. 56 Eliaz RE, Szoka FCJr. (2001). Cancer Res. 61(6):2592-601. 57 Surace C, Arpicco S, Dufaÿ-Wojcicki A, Marsaud V, Bouclier C, Clay D, Cattel L, Renoir JM, Fattal E. (2009). Mol. Pharm. 6(4):1062-73. 58 Hyung W, Ko H, Park J, Lim E, Park SB, Park YJ, Yoon HG, Suh JS, Haam S, Huh Y.M. (2008). Biotechnol. Bioeng. 99(2):442-54. 59 Pandita D, Ahuja A, Lather V, Dutta T, Velpandian T, Khar RK. (2011). Pharmazie. 66(3):171-7. 60 Hong GY, Jeong YI, Lee SJ, Lee E, Oh JS, Lee HC. (2011). Arch Pharm Res. 34(3):407- 17. 61 Liu D, Wang L, Liu Z, Zhang C, Zhang N. (2010). J Biomed Nanotechnol. 6(6):675-82. 62 Lozano MV, Torrecilla D, Torres D, Vidal A, Domínguez F, Alonso MJ. (2008) Biomacromol. 9(8):2186-93. 63 Hureaux J, Lagarce F, Gagnadoux F, Rousselet MC, Moal V, Urban T, Benoit J. (2010). Pharm Res. 27(3):421-30. 64 Luo Y, Bernshaw NJ, Lu ZR, Kopecek J, Prestwich GD. (2002). Pharm Res. 19(4):396- 402. 65 Rosato A, Banzato A, De Luca G, Renier D, Bettella F, Pagano C, Esposito G, Zanovello P, Bassi P. (2006). Urol. Oncol. 24(3):207-15. 66 Xin D, Wang Y, Xiang J. (2010). Pharm. Res. 27(2):380-9. Introducción 40 modificar su perfil de distribución67;68. El Capítulo 1 de la presente tesis doctoral es una revisión acerca de los citados sistemas, en la que se puede obtener una visión más general y detallada de las nanocápsulas en aspectos relacionados con su elaboración, caracterización y evaluación in vitro/in vivo. En distintas investigaciones, se ha propuesto la utilización de nanocápsulas para la vehiculización tumoral de docetaxel. Un ejemplo, es el sistema propuesto por Khalid y col. (2006)69, quienes desarrollaron nanocápsulas lipídicas recubiertas de polietilenglicol para obtener tiempos prolongados de circulación en sangre. Este trabajo fue el primero que demostró que la encapsulación de docetaxel en sistemas coloidales podía ser utilizada para dirigir pasivamente el fármaco a los tejidos neoplásicos. De hecho, las nanocápsulas demostraron una mejora en la acumulación del fármaco en el tumor cuando se comparaban con la formulación convencional (Taxotere®). Lozano y col. (2010)62 demostraron que la inclusión de docetaxel en nanocápsulas recubiertas con quitosano da lugar a una rápida captura de los sistemas en líneas celulares de cáncer de mama (MCF-7) y pulmón (A-549) y que, tras 24 horas, el efecto sobre la viabilidad celular obtenido con las nanocápsulas cargadas con docetaxel fue significativamente mejor que el obtenido con el fármaco solo. Se demostró igualmente que las nanocápsulas de quitosano mostraban, tras inyección intratumoral, un efecto similar en la reducción del volumen tumoral al de la formulación comercial de docetaxel, siendo un efecto más lento, pero más duradero en el período de seguimiento del proceso70.   67 Mora-Huertas CE, Fessi H, Elaissari A. (2010). Int. J. Pharm. 385(1-2):113-42. 68 Huynh NT, Passirani C, Saulnier P, Benoit JP. (2009) Int. J. Pharm. 379(2):201-9. 69 Khalid M N, Simard P, Hoarau D, Dragomir A, Leroux J C. (2006). Pharm. Res. 23, 752–758. 70 Lozano M.V.; Torrecilla D.; Lallana E.; Vidal A.; Fernández-Megía R.; Riguera R.; Dominguez F.; Alonso M. J. and Torres D. Chitosan nanocapsules for active tumor targeting, 7th World Meeting on Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology, Malta, 2010.   Capitulo 1 Nanocapsules as carries for the transport and targeted delicery os bioactive molecules Nanocapsules as carriers for the transport and targeted delivery of bioactive molecules 48 Figure 3: Preparation of nanocapsules by interfacial polymer deposition following solvent displacement Other interesting polyacrylates, also used to prepare nanocapsules, are polymethacrylates (Eudragit®). These nanocapsules have been obtained by interfacial deposition of the preformed Eudragit®. The interest of these systems relies in their pH sensitive character that can be employed to improve the stability and bioavailability of therapeutic drugs after oral administration 31. Polyester nanocapsules Polyesters such as poly-ε-caprolactone (PCL), poly lactic acid (PLA) and its copolymer poly(lactic-co-glycolic) acid (PLGA) have also been used for the preparation of nanocapsules. To date, all polyester nanocapsules have been prepared by the interfacial deposition of a preformed polymer following solvent displacement 5, 13, 14. This effective and reproducible method allows the production of polyester nanocapsules with size ranges between 100-350 nm and wall thickness of 1 to 20 nm15, 32, 33. Capitulo 1 49 The surface properties of polyester nanocapsules can be modified in order to reach the therapeutical purpose. For example, chitosan, a bioadhesive polymer, can be attached to the surface of polyester nanocapsules by incubation 16. In addition, it is possible to obtain PEG-coated polyester nanocapsules by using the amphiphilic PEGylated copolymer, i.e. PEG-PCL, PEG-PLA or PEG-PLGA34-37. The polymer deposition technique leads to the orientation of the hydrophobic segment towards the oily phase whereas the PEG portion protrudes towards the external aqueous medium. Nanocapsules made of natural polymers Naturally occurring polymers such as polysaccharides have also been used for the formation of nanocapsules. Among these, chitosan has received increasing attention for a number of years as a biomaterial for transmucosal drug delivery. Our group described for the first time the preparation of chitosan nanocapsules according to an interfacial deposition method slightly modified when compared to that used for PACA or polyester nanocapsules described above (Figure ). In this case, chitosan is incorporated into the external aqueous phase and its deposition at the oil/water interphase occurs because of its electrostatic interaction with the negatively charged phosphatidylcholine, which is used as a stabilizer of the nanodroplets. 7, 8, 34 We have also proposed an alternative method which involves first, the formation of a nanoemulsion and, the incubation of the nanoemulsion in an aqueous solution of chitosan 7-9. This method has also been employed for the formation of PEG-chitosan nanocapsules38. In this case, the PEG molecule gets oriented towards the external phase due to the cationic nature of chitosan and its natural tendency to associate to the negatively charged nanodroplets. Nanocapsules as carriers for the transport and targeted delivery of bioactive molecules 50  Figure 4: Preparation of nanocapsules by polymer adsorption following solvent displacement  The incubation approach has been recently proposed for the formation of nanocapsules with a double polysaccharic wall consisting of chitosan and lambda-carrageenan 39. In this case, the nanoemulsion was formed by high pressure homogenization using a modified starch as negatively charged stabilizer; then, it was incubated first in a chitosan solution and afterwards in a lambda-carrageenan solution. Capitulo 1 51 Lipid nanocapsules A new generation of nanocapsules, named lipid nanocapsules, were first prepared by the group of Benoit6, 40-42. These systems consists of an oil core surrounded by a thick polymeric shell, made of PEG-hydroxystearate and phosphatidylcholine. These nanocapsules can be prepared via a novedous, solvent-free, phase inversion process (Figure ). In this process, all the components of the system are mixed together with the aqueous phase and, then, exposed to several cycles of heating and cooling (usually between temperatures around 65 and 85ºC). The size and polydispersity of the nanocapsules decrease as a function of the number and temperature cycles and a thick interfacial layer is created with this cycling process, since the surfactant is forced to overconcentrate at the interface of the oily droplets.43 Finally, the process is quenched at a temperature below the phase inversion temperature (o/w emulsion), followed by addition of cold water. This fast cooling-dilution process led to the formation of lipid nanocapsules with particle sizes between 20 and 100 nm42. These nanocapsules showed a rigid shell surrounding the oily core and were physically stable for at least 18 months without fusion of the dispersed oily phase41. These nanocapsules are very versatile as they can be produced using different types of oils and lipids, thus exhibiting high drug encapsulation efficiency values 44, 45. Nanocapsules as carriers for the transport and targeted delivery of bioactive molecules 52 Figure 5: Preparation of nanocapsules by phase inversion temperature. Therapeutical applications of nanocapsules The use of nanocapsules has been reported as a promising strategy for improving the oral bioavailability of therapeutic molecules 46. It has been shown that due to their colloidal size, nanocapsules are able to interact favorably with the mucosal barrier and, simultaneously, protect the encapsulated drug from the harsh environment of the gastrointestinal tract 46, 47. For these reasons, nanocapsules have been extensively studied as vehicles for improving the oral bioavailability of poorly absorbed drugs such as peptides or some lipophilic compounds, as well as for obtaining drug controlled release48. Capitulo 1 53 Nanocapsules for oral peptide delivery The oral administration of peptides and proteins continue to be a challenge because of their susceptibility to the enzymatic degradation and their low permeability across the intestinal ephitelium. The encapsulation of these macromolecules into polymeric nanocapsules is nowadays considered a promising approach towards this ambitious goal49,50. An example is represented by the nanocapsules made of mucoadhesive polymers, such as chitosan, as described for transmucosal absorption of calcitonin 51. Chitosan nanocapsules loaded with calcitonin were able to enhance and prolong the systemic absorption of the drug, thus leading to an improvement of the hypocalcemic effect (Figure ). The in vitro studies performed in the Caco-2 cells cocultured with the a model of mucus-secreting cells (HT29-M6) suggested that chitosan nanocapsules do not cross the monolayer, but rather they remain at the apical side of the cells 51. Figure 6: Serum calcium levels in rats after oral administration of salmon calcitonin in an aqueous solution (sCT Sol) or encapsulated in chitosan nanocapsules (CS NC) at two different doses (250 and 500 IU/Kg), (mean ± SE; n = 6).51 Reproduced by permission of Springer. Promising results have also been obtained with poly (isobutylcyanoacrylate) (PBCA) nanocapsules containing calcitonin. The Nanocapsules as carriers for the transport and targeted delivery of bioactive molecules 54 results of the in vivo studies performed on rats indicated that these nanocapsules allowed a great decrease of calcium levels to occur29. On the other hand, Damgé et al. investigated the potential of PACA nanocapsules for the oral administration of insulin52,53. Following intragastric administration of insulin loaded nanocapsules (12.5, 25 and 50 IU of insulin per kg) to diabetic rats (diabetes induced by the administration of 65 mg/kg of spreptozocin), the authors observed that the nanocapsules remained intact in simulated gastric fluid, thus ensuring a good protection of the peptide. Moreover, the new formulation produced a significant reduction of the glycemia (50-60%), a response that was maintained for up to 20 days. The authors attributed this long-term effect to the adsorption of the nanocapsules across the intestinal epithelium and the subsequent release of the encapsulated peptide54. More recently, other authors studied the bioavailability of orally administered insulin loaded PBCA nanocapsules (50 IU of insulin per kg) in diabetic rats (diabetes induced by the administration of 65 mg/kg of spreptozocin 55. These results showed that the oral administration of nanocapsules allows the delivery of noticeable levels of insulin into the bloodstream in diabetic rats, however decrease in glycemia could not be observed. The low reproducibility of the results in animal models hampered the comprehensive analysis of the results from the different studies. Nanocapsules were also investigated for the delivery of hydrophobic peptides such as cyclosporine. The oral absorption of this peptide was tested using nanocapsules made of an oily core consisting of Cremophor® or Maisine® and surrounded by Eudragit RL® or RS®. Unfortunately, the absolute bioavailability achieved with the nanocapsules ranged from 4 to 7.5 %, a result that is far below that observed with the marketed Neoral® premicroemulsion (about 22%) 56. The authors related the lower cyclosporine bioavailability to the size of the nanocapsules, more than to the constituents of the systems, however, the low bioadhesion of the polymers used to the Capitulo 1 55 intestinal epithelium could also play an important role in the absorption of the drug. Nanocapsules for oral delivery of lipophilic low molecular weight drugs Nanocapsules have also been used for oral delivery of low molecular weight compounds. Anti-inflammatory agents are known to exhibit important gastrointestinal side effects such as irritation and mucosal damage. Moreover, they are characterized by very low water solubility, a property which makes these good candidates for the encapsulation within oily core nanocapsules 57. Nanocapsules made of PLA were investigated for their potential of improving the gastrointestinal tolerance to indometacin and diclofenac57. The encapsulation of these drugs into PLA nanocapsules led to a great reduction of the irritation of the gastrointestinal mucosa. The diuretic drug, spironolactone, used in premature infants to reduce lung congestion, has also been efficiently encapsulated in PCL nanocapsules58. Nowadays there is no commercially available oral liquid preparation of spironolactone due to its poor water solubility and its dissolution rate. Its incorporation into nanocapsules solved these problems, although further pharmacokinetics studies are needed in order to fully demonstrate their in vivo effectiveness. The use of nanocapsules has also been proposed for the oral administration of drugs which suffer the efflux transport, mediated by P- glycoprotein (P-gp), across the apical membrane of the intestinal ephitelium. This transport is known to drastically reduce the absorption of antibiotics, antivirals, antitumorals and other drugs. Recently, it was shown that the encapsulation of tacrolimus, an immunosuppresor agent substrate of P-gp, into Eudragit® nanocapsules, protect the drug from the efflux transports and increase the concentration of the drug within the cell and therefore its bioavailability59. This evidence was observed in two animal models, rats and Nanocapsules as carriers for the transport and targeted delivery of bioactive molecules 56 minipigs. In addition, in these studies it was also observed that the small lipophilic oil cores were able to enter the enterocytes and reach the lamina propria behind the P-gp. Similar results were obtained with the encapsulation of the antitumor drug, paclitaxel, into lipid nanocapsules. Due to the effect of P-gp and its low water solubility, paclitaxel is currently admistered intravenously. Following in vivo administration of lipidic nanocapsules containing placlitaxel to rats, an increase in its absorption when compared to that of the control was observed (Taxol®, paclitaxel dissolved in Cremophor® and ethanol). The positive role of the nanocapsules was attributed to two mechanisms: first, as could be expected, the presence of lipids in the formulation increased the intestinal lymphatic transport and, second, the entrapment of the molecule in the nanocapsules could reduce the P-gp-mediated transport of the drug. Nevertheless, these promising results should be taken into account cautiously due to the high interindividual variability and need to be confirmed by further experimentation60. Overall, nanocapsules can be considered as potential vehicles for promoting the oral absorption of peptides and lipophilic low molecular weight drugs. Particularly noticeable is their capacity to overcome multidrug resistance (MDR) mechanisms, such as the P-gp efflux transport. Despite this evidence, the validation of the efficacy of these nanosystems in large-scale animals, in fed and fasted conditions will have to be proved in order to make sure of their potential for clinical use. Capitulo 1 57 Nanocapsules as nasal drug carriers The intranasal delivery is an attractive non-invasive route which offers several unique advantages for peptide drugs, such as the ease of administration, the looseness of the epithelium and the avoidance of the hepatic first-pass metabolism. Our group has explored the potential of chitosan nanocapsules for increasing the nasal absorption of the peptide salmon calcitonin7. The results observed in the rat model indicated that, as expected, the response of this peptide could be significantly enhanced and prolonged following its association to the nanocapsules (Figure ). These results highlight the critical role of the polymer in enhancing the transport of the associated peptide and consequently the potential of chitosan nanocapsules for nasal peptide delivery. Figure 7: Serum calcium levels in rats after nasal administration of salmon calcitonin (sCT, dose: 15 IU/kg) in aqueous solution (with or without CS) or encapsulated in the control nanoemulsion (NE) or in chitosan nanocapsules (CS NC); (mean ± SE; n = 6). *Significantly different from salmon calcitonin solutions (p < 0.05). #Significantly different from nanoemulsion (p < 0.05).7 Reproduced by permission of Ed. Sante. Nanocapsules as carriers for the transport and targeted delivery of bioactive molecules 64 increased their half-life in serum in comparison with the naked molecules or those adsorbed onto nanospheres86. Moreover, the ODNs cell uptake was significantly improved when the molecule was included in the nanocapsules87. The encapsulation into the aqueous core of PBCA nanocapsules of an antisense-siRNA (siRNA-AS) against a fusion oncogen (Fli1) overexpressed in Ewing sarcoma, resulted in an important inhibition of tumor growth tested in a murine model of Ewing sarcoma-related tumor (Figure )88, 89. Figure 10: Inhibition of Erwing sarcoma fusion oncogen (EWSFli1)-expressing tumor growth in nude mice by: ○ siRNA-antisense (siRNA-AS) loaded NCs; ▲siRNA-control loaded NCs; ■, siRNA-AS naked; ♦, siRNA-control naked; ●, saline.89 Reproduced by permission of Springer. Hillaireau et al.19 described the incorporation of ODNs to PBCA nanocapsules. They observed that the association could be significantly improved when ODN is associated first to a cationic polymer, such as chitosan or poly(ethylenimine), and afterwards this complex being encapsulated into a water containing nanocapsule. In a different work, Bouclier et al.90 reported the encapsulation of a specific siRNA (target to estrogen receptor alfa [ERα-siRNA]) in three different systems: PBCA nanocapsules, PEG-PLGA nanoparticles and PEG-PCL-malic acid nanoparticles. The in vitro studies indicated that PBCA nanocapsules showed Capitulo 1 65 a high efficiency in MCF-7 cancer cells, whereas the other systems showed no antiproliferative effect in the same cancer cell lines. In a preliminary in vivo study, these nanocapsules showed a slight decrease in tumor growth in comparison to scramble-siRNA loaded nanocapsules or the siRNA naked90, showing the benefits of the nanocapsules over other nanosystems for the encapsulation of siRNA. Conclusions The liquid nature of nanocapsules and, thus, their fluidity and elasticity make them ideal nanovehicles able to facilitate the contact with the epithelia and target cells, as well as to enter intracellularly. They have unique properties as their simplicity and their capacity of obtaining great loadings of either lipophilic or hydrophilic drugs. Moreover, nanocapsules have shown to be capable of inhibiting multidrug resistance cellular mechanisms, specially important in cancer therapy. In conclusion, polymeric or lipid nanocapsules are a promising tool for transmucosal drug delivery as well as for cancer therapeutics, particularly for drugs which are water-insoluble and that, until recently, have required solvents to be formulated. Concerning gene therapy, nanocapsules emerge as an interesting approach, due to the high affinity of nucleic acids for their water core and to the possibility of adapting these systems to the requirements of this novel therapy. In addition, the use of reservoir structures composed by inorganic nanoparticles (iron, silica or gold nanoparticles, quantum dots, carbon nanotubes, etc.) surronded by a polymer and, optionally, a targeting ligand, represents a promising and powerful tool to enhance the biocompatibility and the biodistribution of these nanostructures widely used in the diagnostics and threatment of several diseases. This composite nanocapsules will be discussed widely in following chapters. 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Staniscuaski Guterres, S., Fessi, H., Barratt, G., Puisieux, F., and Devissaguet, J.P. (1995).Poly(D,L-Lactide) nanocapsules containing non-steroidal anti-inflammatory drugs: Gastrointestinal tolerance following intravenous and oral administration. Pharm Res.12.pp 1545- 1547. 58. Limayem Blouza, I., Charcosset, C., Sfar, S., and Fessi, H. (2006).Preparation and characterization of spironolactone-loaded nanocapsules for paediatric use. Int J Pharm.325.pp 124-131. 59. Nassar, T., Rom, A., Nyska, A., and Benita, S. (2009).Novel double coated nanocapsules for intestinal delivery and enhanced oral bioavailability of tacrolimus, a P-gp substrate drug. J Control Release.133.pp 77-84. 60. Peltier, S., Oger, J.M., Lagarce, F., Couet, W., and Benoit, J.P. (2006).Enhanced oral paclitaxel bioavailability after administration of paclitaxel-loaded lipid nanocapsules. Pharm Res.23.pp 1243-1250. 61. Barar, J., Javadzadeh, A.R., and Omidi, Y. (2008).Ocular novel drug delivery: Impacts of membranes and barriers. Expert Opin Drug Deliv.5.pp 567-581. 62. Lang, J.C. (1995).Ocular drug delivery conventional ocular formulations. Adv Drug Deliv Rev.16.pp 39-43. 63. Losa, C., Marchal-Heussler, L., Orallo, F., Vila-Jato, J.L., and Alonso, M.J. (1993).Design of new formulations for topical ocular   Antecedentes, hipótesis y objetivos 81 ANTECEDENTES 1. Las nanoestructuras poliméricas administradas por inhalación, han demostrado ser capaces de proporcionar niveles terapéuticos de fármacos durante períodos prolongados, al potenciar el acceso de éstos hacia las dianas localizadas a nivel pulmonar71;72;73;74;75. 2. Algunos polisacáridos mucoadhesivos, como el quitosano y el ácido hialurónico, ofrecen interesantes posibilidades como constituyentes de nanoestructuras capaces de interaccionar con el epitelio pulmonar y maximizar su período de contacto76;77. Ambos polímeros pueden, además, formar nanoestructuras híbridas gracias a la interación iónica de sus cargas opuestas78;79, lo que aporta ventajas en cuanto al perfil de seguridad ofrecido por el quitosano79. 3. La combinación del quitosano con ciclodextrinas en nanopartículas, aporta mejoras a las propiedades de los nanosistemas constituídos únicamente por el polisacárido, al disminuir posibles alteraciones de la función barrera del epitelio y ofrecer mayor protección a las macromoléculas encapsuladas80;81.   71 Pandey R, Sharma A, Zahoor A, Sharma S, Khuller GK, Prasad B. (2003). J. Antimicrob. Chemother. 52(6): 981-6. 72 Sharma A, Sharma S, Khuller GK. (2004). J. Antimicrob. Chemother. 54(4): 761-6. 73 Ahmad Z, Sharma S, Khuller GK. (2005). Int .J. Antimicrob. Agents. 26(4): 298:303. 74 Hitzman CJ, Wattenberg LW, Wiedmann TS. (2006). J Pharm Sci. 95(6):1196-211. 75 Ohashi K, Kabasawa T, Ozeki T, Okada H. (2009). J. Control Release. 135(1):19-24. 76 Yamamoto H, Kuno Y, Sugimoto S, Takeuchi H, Kawashima Y. (2005). J Control Release. 102(2):373-81. 77 Liu XB, Ye JX, Quan LH, Liu CY, Deng XL, Yang M, Liao YH. (2008). Eur J Pharm Biopharm. 78 de la Fuente M, Seijo B, Alonso MJ. (2008). Macromol Biosci. 8(5):441-50. 79 de la Fuente M, Seijo B, Alonso MJ. (2008). Invest. Ophthalmol. Vis. Sci. 49(5):2016-24. 80 Teijeiro-Osorio D, Remuñán-López C, Alonso MJ. (2009). Biomacromol. 10(2):243-9. 81 Chen Y, Siddalingappa B, Chan PH, Benson HA. (2008). Biopolymers. 90(5):663-70. Antecedentes, hipótesis y objetivos 82 4. La heparina es una macromolécula que ofrece un potencial terapéutico para el tratamiento del asma bronquial, al prevenir la desgranulación de los mastocitos82;83. Este potencial se ve limitado por su acceso restringido al interior celular84, donde se encuentran localizados los receptores. 5. El docetaxel constituye un tratamiento antitumoral de elección, sin embargo su extremada hidrofobicidad obliga a la inclusión de disolventes en su formulación endovenosa, que dan lugar a importantes reacciones de sensibilización que se suman a los efectos secundarios provocados por el propio citostático85;86. 6. Nuestro grupo de investigación ha demostrado el potencial de nanocápsulas de polisacáridos y poliaminoácidos catiónicos como transportadores intracelulares de antitumorales hidrofóbicos, como el docetaxel, consiguiendo vehículos eficaces sin necesidad de la inclusión de solventes tóxicos87;88. 7. Se ha demostrado que en distintos tumores sólidos, entre ellos varios de pulmón, hay sobreexpresión del receptor CD-44, que es la diana endógena del ácido hialurónico89;90. La incorporación de ácido hialurónico en diferentes   82 Tyrrel DJ, Kilfeather S, Page CP. (1995). TiPS. 16:198-204. 83 Diamant Z, Page CP. (2000). Pulm. Pharmacol. Ther. 13(1):1-4. 84 Motlekar NA, Youan BB. (2006). J. Control Release. 113(2):91-101. 85 Engels FK, Mathot RA, Verweij J. (2007). Anticancer Drugs. 18(2):95-103. 86 Ten Tije AJ, Verweij J, Loos WJ, Sparreboom A. (2003). Clin. Pharmacokinet. 42(7), 665-85. 87 Lozano MV, Torecilla D, Torres D, Vidal A, Dominguez F, Alonso MJ. (2008). Biomacromol. 9, 2186-2193. 88 Lozano MV, Lollo G, Brea J, Torres D, Loza MI, Alonso MJ. Polyarginine nanocapsules: a new platform for intracellular drug delivery (submitted). 89 Penno MB, August JT, Baylin SB, Mabry M, Linnoila RI, Lee VS, Croteau D, Yang XL, Rosada C. (1994). Cancer Res. 54(5):1381-7. 90 Tran TA, Kallakury BV, Sheehan CE, Ross JS. (1997). Hum Pathol. 28(7):809-14. Antecedentes, hipótesis y objetivos 83 tipos de transportadores ha confirmado la especificidad del targeting hacia el receptor CD-4491;92;93;94. HIPÓTESIS 1.- El desarrollo de nanoestructuras constituídas por polisacáridos mucoadhesivos como el quitosano y/o el ácido hialurónico, puede constituir una estrategia adecuada para tratar localmente patologías que cursan a nivel pulmonar, como el asma bronquial o el cáncer de pulmón. El éxito de esta estrategia residirá fundamentalmente en favorecer la accesibilidad de los fármacos a las células diana, a la vez que prolongar su contacto con las mismas y potenciar su captura intracelular. 2.- La combinación del quitosano con ciclodextrinas en sistemas nanoparticulares es una alternativa que puede aportar ventajas al sistema puro en su administración pulmonar, en lo que se refiere a mejora de su perfil de seguridad, promoción de su captura intracelular, y protección de la macromolécula encapsulada. 3.- El diseño de un nuevo sistema constituído por nanocápsulas de ácido hialurónico puede resultar de interés para la vehiculización pulmonar del antitumorales hidrofóbicos. El nuevo nanosistema contendrá en su núcleo oleoso la molécula activa, mientras que el ácido hialurónico potenciará la interacción con células tumorales que sobreexpresan receptores CD-44 en su superficie.   91Akima K, Ito H, Iwata Y, Matsuo K, Watari N, Yanagi M, Hagi H, Oshima K, Yagita A, Atomi Y, Tatekawa I. (1996). J Drug Target. 1996;4(1):1-8. 92 Auzenne E, Ghosh SC, Khodadadian M, Rivera B, Farquhar D, Price RE, Ravoori M, Kundra V, Freedman RS, Klostergaard J. (2007). Neoplasia. 9(6):479-86. 93 Eliaz RE, Szoka FCJr. (2001). 61(6), 2592-601. 94 Peer D, Margalit R. (2004). Neoplasia 6, 343-353. Antecedentes, hipótesis y objetivos 84 OBJETIVOS El objetivo de esta Tesis se ha dirigido a evaluar el potencial que presentan distintos nanotransportadores para vehiculizar y promover el efecto intracelular de fármacos tan distintos, como la macromolécula hidrofílica heparina o el antitumoral hidrofóbico docetaxel, en células diana pulmonares. Este objetivo se cubrirá a través de las siguientes etapas: 1.- Desarrollo de nanosistemas híbridos de quitosano conteniendo heparina y evaluación ex vivo de su interacción y de su actividad antiinflamatoria sobre mastocitos. Esta parte de la memoria se ha dirigido en primer lugar a optimizar los nanosistemas de quitosano-ácido hialurónico y quitosano-ciclodextrinas en cuanto a su contenido en heparina, para asegurar la producción del efecto antiasmático. En segundo lugar, se estudió la capacidad de los nanosistemas para interaccionar y ser internalizados en mastocitos extraídos de rata. Finalmente, se ha evaluado el potencial de estos vehículos para inhibir la liberación de histamina por parte de los mastocitos. Los resultados de este apartado se recogen en los capítulos experimentales 2 y 3. 2.- Desarrollo de un nuevo sistema constituído por nanocápsulas de ácido hialurónico conteniendo docetaxel y evaluación de su eficacia antitumoral sobre cultivos celulares de cáncer de pulmón. Antecedentes, hipótesis y objetivos 85 Para poner a punto el nuevo nanosistema, se llevó a cabo la optimización del proceso de recubrimiento con el polímero aniónico, evaluando sus características tras la incorporación de distintos tensoactivos catiónicos en el núcleo oleoso. Se determinó finalmente la eficacia antitumoral de las nanocápsulas conteniendo docetaxel sobre el modelo celular de cáncer de pulmón NCI-H460. Los resultados de este apartado se recogen en el capítulo experimental 4.  PARTE I: DESARROLLO DE NANOSISTEMAS HÍBRIDOS DE QUITOSANO CONTENIENDO HEPARINA Y EVALUACIÓN EX VIVO DE SU INTERACCIÓN Y DE SU ACTIVIDAD ANTIINFLAMATORIA SOBRE MASTOCITOS.   Capitulo 2  Chitosan-hyaluronic acid nanoparticles loaded with heparin for the treatment of asthma Chitosan-hyaluronic acid nanoparticles loaded with heparin for the treatment of asthma 96 The production yield of the systems was obtained by centrifugation of fixed volumes of the nanoparticle suspensions (16000×g, 30 min, 25° C), without a glycerol bed. The supernatants were discarded and the sediments were freeze-dried. The yield was calculated as follows: 100 components theofamount Total lesnanopartic ofWeight Yield ×= Physicochemical characterization of heparin-loaded CS-HA nanoparticles The size and zeta potential of the colloidal systems were determined by photon correlation spectroscopy and laser Doppler anemometry, with a Zetasizer Nano-ZS (Malvern Instruments, United Kingdom). Each batch was analyzed in triplicate. Morphological examination of the nanoparticles was performed by transmission electron microscopy (TEM) (CM12 Philips, Netherlands). The samples were stained with 1% w/v phosphotungstic acid for 10 sec., immobilized on copper grids with Formvar® and dried overnight for viewing by TEM. Association efficiency and drug loading of heparin-loaded CS-HA nanoparticles The association efficiencies of the selected formulations were determined after isolation of nanoparticles by centrifugation, as described in Section 2.2. The amount of unbound heparin in the supernatant was determined by a colorimetric method (Stachrom® Heparin, Diagnostica Stago, France). Capitulo 2  97 The association efficiency of heparin and the drug loading were calculated as follows: 100 drug ofamount Total drug unbound ofAmount - drug ofamount Total efficiencyn Associatio ×= 100 lesnanopartic ofWeight drug unbound ofAmount - drug ofamount Total Loading Drug ×= Stability study of heparin-loaded CS-HA nanoparticles in different media Selected nanoparticle formulations were prepared and centrifuged in the presence of glycerol. Nanoparticles were tested for their stability taking into account the change in size of nanoparticles and possible precipitations in different media at 37º C, including: Hanks´ balanced salt solution (HBSS) at pH 6.4 and 7.4, and phosphate buffered saline (PBS), at pH 7.4 (for composition of these solutions, see below). Nanoparticles were incubated in these media and samples were collected at several time intervals (0, 1, 3, 5, 10 and 24 h), and the size distribution of the nanoparticles was measured by photon correlation spectroscopy. The composition of HBSS was: 137 mM NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4 and 4.2 mM NaHCO3. The composition of PBS was: 137 mM NaCl, 2.7 mM KCl, 1.4 mM NaH2PO4 and 1.3 mM Na2HPO4. In vitro heparin release studies from CS-HA nanoparticles Heparin release studies were performed by incubating 0.1 mg of the selected nanoparticles in 1 mL of PBS (pH 7.4) at 37º C. The samples were Chitosan-hyaluronic acid nanoparticles loaded with heparin for the treatment of asthma 98 centrifuged at appropriate time intervals (1, 5 and 12 h), and the amount of heparin released was evaluated with the heparin kit described above. The concentration of heparin was quantified and calculated by interpolation from the corresponding standard curve. Study of interaction of fluorescent heparin-loaded CS-HA nanoparticles with rat mast cells by confocal microscopy Fluorescein labelling of CS Chitosan was labelled with fluorescein following a slight modification of the method described by De Campos et al. (2004). The covalent attachment of fluorescein to CS was by the formation of amide bonds between primary amino groups of the polymer and the carboxylic acid groups of fluorescein. Briefly, 250 mg of CS was dissolved in 25 mL of water, and 10 mg of fluorescein (Sigma Aldrich, Spain) was dissolved in 1 mL of ethanol. These solutions were then mixed, and EDAC (1-ethyl-3- (dimethylaminopropyl) carbodiimide hydrochloride) (Sigma Aldrich, Spain) was added to a final concentration of 0.05 M, to catalyze the formation of amide bonds. The reactive mixture was incubated under permanent magnetic stirring for 12 h in the dark, at room temperature. The resulting conjugate was finally isolated by dialysis for 72 h (cellulose dialysis tubing, pore size 12400 Da; Sigma Aldrich, Spain) against demineralised water, and freezedried. The pH of fluorescent CS was adjusted to the same value as the raw CS solution (pH 4.9) with HCl, for the preparation of fluorescent nanoparticles. Capitulo 2  99 Preparation of fluorescent heparin-loaded CS-HA nanoparticles Fluorescent nanoparticles were prepared according to the same procedure described in 2.2. The selected mass distribution for the preparation of fluorescent nanoparticles was: 4 mg of fluorescent CS, 0.6 mg of HA, 0.21 mg of TPP and 1.4 mg of UFH. Confocal laser scanning microscopy study An aqueous solution (50 μL) containing 0.3 mg of isolated fluorescent UFH-loaded CS-HA nanoparticles was incubated with 450 μL of a suspension of mast cells (10x103 cells/100 μL) in Umbreit (for composition, see below) containing 0.05% w/v of BSA. The mixture was incubated for 2 h at 37 °C, and the cells were then separated by centrifugation (10 min, 200xg) and discarding the supernatants. Two hundred μL of Umbreit+BSA solution (at 4 °C) were then added to the cell pellet. The pellet was resuspended and centrifuged again to extract the non-internalized nanoparticles. This procedure was repeated once more. Mast cells were fixed for 5 minutes in paraformaldehyde (2% w/v, 100 μL) and washed 3 times with the Umbreit+BSA solution, by centrifugation. Two hundred μL of a Bodipi® phalloidin solution (Invitrogen, USA) were added to the cell pellet and the cells were incubated for 30 min at room temperature. The cells were washed 3 times (Umbreit+BSA) by centrifugation, the supernatant was discarded, and the pellet was resuspended in 20 μL of the Umbreit+BSA solution. The resuspended sample was placed on the surface of a positively charged microscope slide (Superfrost Ultra Plus, Menzel-Glaser, Irland) and dried at room temperature overnight. The sample was prepared in Vectashield medium (Vector, USA) for visualization by confocal microscopy (CLSM, Zeiss 501, Germany) (all of the described procedures were carried Chitosan-hyaluronic acid nanoparticles loaded with heparin for the treatment of asthma 100 out in darkness to prevent the loss of the fluorescent signal from the nanoparticles and mast cells). The composition of Umbreit saline solution was: 1.2 mM MgSO4, 1.2 mM NaPO4H2, 22.85 mM NaHCO3, 5.94 mM KCl, 1 mM CaCl2, 119 mM NaCl and 0.1% glucose. Ex vivo studies with rat mast cells: Inhibition of histamine release by heparin-loaded CS-HA nanoparticles Rat mast cell purification and viability Mast cells were obtained by lavage of pleural and peritoneal cavities of female Sprague–Dawley rats (400–800 g) with Umbreit saline solution, following procedures similar to those described in other studies (Lago et al., 2001; Buceta et al., 2008). The suspension obtained from each rat was centrifuged at 100xg for 5 min (4 ºC) and suspended in a final volume of 1 mL of Umbreit containing 0.05% w/v of BSA. Purification was carried by centrifugation on 4 mL of an isotonic Percoll gradient at 600 g for 10 min (4 ºC). The mast cells were washed twice with the Umbreit+BSA solution and maintained at 4 °C in this solution until use. Mast cells were quantified by toluidin blue staining (95% purity) and the viability assessed by trypan blue staining (the procedure is described below). Trypan blue staining procedure: Mast cell viability studies were carried out by trypan blue staining in an inverted microscope, as described by Lago et al. (2001). This involved visual counting of the stained cells in the five fields of a counting chamber. The percentage of viability was calculated with the following formula: 100 cellsmast ofnumber Total fields five thefrommean Arithmetic cells stained blueTrypan ×= Capitulo 2  101 In order to test the mast cell viability after contact with heparinloaded nanoparticles, the same procedure was used, and the UFH or LMWH- loaded CS-HA nanoparticles added to the rat mast cell suspension (1x105 cells per test tube). The tested dose of nanoparticles was equivalent to 200 μg/mL of UFH or LMWH. Measurement of histamine release in rat mast cells Rat mast cells (1x105 cells per test tube) were pre-warmed at 37 ºC (10 min) in BSA-free Umbreit saline solution containing the UFH or LMWH solutions or the nanoparticles loaded with UFH or LMWH. Histamine release from mast cells was then initiated by incubating the cells with 100 µM of compound 48/80 (Sigma Aldrich, Spain) for 20 min at 37 ºC. The cells were then centrifuged at 1100xg for 3 min at 4 ºC, and two aliquots (100 µl) of the supernatants were collected in a 96-well microplate. The rest of the supernatants were discarded and the pellets were resuspended in 500 μl of 0.1 mM HCl, sonicated for 1 min and centrifuged at 1100 x g for 6 min. Two aliquots of 100 μL of the supernatants were collected for residual histamine determination. Histamine was assayed fluorometrically, as described by Lago et al. (2001); briefly, 80 µL NaOH 1 M were added to 100 µL of the sample, then 50 µl phthaldialdehyde 0.04% w/v were added to each well and plate was incubated for 4 min at 25° C. After this time, 50 µl of 3 M HCl were added and fluorescence was measured within 20 min, at excitation and emission wavelengths of 360 nm and 465 nm respectively, in a Tecan Ultra Evolution reader (Tecan, Switzerland). Data analysis for measurement of histamine release in rat mast cells Chitosan-hyaluronic acid nanoparticles loaded with heparin for the treatment of asthma 102 Results were expressed as percentage of the total histamine released after stimulation with compound 48/80. The results were corrected for spontaneous histamine release in the absence of any chemical and under the same conditions. The equation used for the calculation was HR=[(S−ER)/(S+P−ER)]×100, where HR is the percentage histamine release; S, supernatant fluorescence; ER, fluorescence of spontaneous release supernatants and P, pellet fluorescence. IC50 values were obtained by fitting the data with non-linear regression, with Prism 2.1 software (GraphPad, San Diego, CA). Statistical analysis The statistical significance of the differences between formulations was determined by application of two-way analysis of variance (ANOVA) followed by a two-tailed paired Student’s test. Differences were considered significant at p<0.05. Results and discussion Preparation and characterization of heparin-loaded CS-HA nanoparticles Nanoparticles loaded with heparin were prepared by the ionotropic gelation technique. The ability of CS to form a gel after contact with polyanions by promoting inter and intramolecular linkages (Calvo et al., 1997) enables the formation of the nanoparticles. In this case, an ionic interaction occurs between the positively charged CS and the negatively charged HA, heparin and the polyanion TPP. The ionic gelation process is extremely simple and involves mixing two aqueous phases at room temperature. Capitulo 2  103 When the nanoparticles loaded with UFH were prepared, it was necessary to establish the best ratio between components that enabled formation and also adequate isolation of the nanosystems. The size, polydispersity index, zeta potential and appearance of the tested formulations are shown in Table 1. In general, it is possible to argue that when the amount of polyanions was too low (relative to CS), nanoparticles could not be formed, or that the quantity of the formed nanoparticles was too low. Nanoparticles with different characteristics were obtained when greater amounts of polyanions were used. However, if the amount of polyanions was too high, it was impossible to isolate the particles, because the nanosystems were not resuspendable or, in extreme cases, precipitation occurred. In addition, when the amount of polyanions was higher, slight decreases in the positive zeta potential values were observed. This may be caused by increased shielding of free positively charged groups of CS. All the resulting nanosystems ranged in size from 162 to 217 nm; polydispersity values were between 0.11-0.45 and the positive zeta potential ranged from +28.1 to +34.6 (Table 1). This positive zeta potential indicates that the surface of the nanosystems is preferably composed by CS. Among the tested formulations, we selected those formed by the following mass distribution for subsequent studies: 4 mg of CS, 0.6 mg of HA, 0.21 mg of TPP and 1.4 mg of UFH. This formulation was able to encapsulate the highest amount of UFH tested, showed reasonable polydispersity, and high turbidity (related to a higher production yield). Chitosan-hyaluronic acid nanoparticles loaded with heparin for the treatment of asthma 104 Table 1: Physicochemical properties of the nanoparticles prepared with different ratios of CSHA-TPP-heparin (mean ± S.D., n=3). Amount (mg) CS-HA-TPP- heparin Size (nm) Polydispersity Index Zeta potential (mV) Appearance 4-1.2-0.21-1.0a 201 ± 24 0.22 – 0.36 +32.1 ± 1.6 Medium turbidity 4-1.2-0.21-1.2a217 ± 30 0.23 – 0.35 +28.1 ± 0.9 High turbidity 4-1.2-0.21-1.4aNot resuspendable --- --- --- 4-1.2-0.21-1.6aPrecipitation --- --- --- 4-0.6-0.21-1.2a 162 ± 17 0.11 – 0.30 +34.6 ± 0.6 Medium turbidity 4-0.6-0.21-1.4a193 ± 32 0.24 – 0.45 +32.5 ± 1.7 High turbidity 4-0.6-0.21-1.5aNot resuspendable --- --- --- 4-0.6-0.21-1.4 b 152 ± 10 0.17 – 0.27 +33.0 ±1.3 Low turbidity a= UFH; b= LMWH The loading capacity, association efficiency and yield of the selected formulation are shown in Table 2. The association efficiency was 72.3% and therefore 1.01 mg of UFH (of the initial 1.4 mg) formed nanoparticles. The drug loading was about 34%, with the remaining mass corresponding to CS, HA and TPP. The same formulation prepared with LMWH was similar in size, zeta potential and association efficiency, but showed lower values of polydispersity and yield and higher drug loading (see Tables 1 and 2). Interestingly, the drug loading of the formulation with LMWH is approximately twice that of the formulation with UFH, and it is possible that LMWH may induce greater displacement of the anionic molecules (HA and TPP) from the nanoparticles and, consequently, lead to a lower yield. Table 2: Loading characteristics and yield of selected CS-HA nanoparticles containing UFH or LMWH (mean ± S.D., n=3). Amount (mg) CS-HA-TPP-heparin Loading capacity (%) Association Efficiency (%) Yield (%) 4-0.6-0.21-1.4a33.6 ±1.2 72.3 ± 2.7 49.0 ± 1.2 4-0.6-0.21-1.4 b 60.6 ± 0.3 69.7 ± 7.6 24.9 ± 4.3 a = UFH; b = LMWH Capitulo 2  105 Considering that the only difference between the prepared formulations was the type of heparin, these changes should be attributed, on one hand, to the different molecular weight (~18 KDa for UFH and ~4 KDa for LMWH) and, on the other hand, to possible chemical differences between UFH and LMWH, associated with very different values of anticoagulant activities (202 and 53 USP units/mg, respectively). The TEM micrographs shown in Figures 1a and 1b indicate that the selected nanosystems loaded with UFH or LMWH were spherical. Interestingly, the systems with UFH appeared denser in the center than at the surface, and differed from those containing LMWH, which appeared more homogeneous. Figure 1: Electron transmission micrographs of selected CS-HA nanoparticles containing UFH (a) or LMWH (b). Stability studies of heparin-loaded CS-HA nanoparticles Determination of nanoparticle colloidal stability under conditions similar to those used for cell culture is crucial for future studies. Therefore, the stability of the selected systems was investigated in media usually used for cell culture studies. These media included: HBSS (pH 7.4), HBSS (pH 6.4) and PBS (pH 7.4). The stability of the selected nanoparticles was better in media of pH 7.4, and was maximal for PBS, where the size was maintained for up to 24 h (Fig. 2) (the stability in HBSS (pH 6.4), is not shown because the nanosystems aggregated immediately). The explanation Chitosan-hyaluronic acid nanoparticles loaded with heparin for the treatment of asthma 112 Figure 6: Effect of heparin solutions and heparin-loaded CS-HA nanoparticles on histamine release from rat mast cells. Histamine release was initiated by incubating the cells with a 100 µM solution of compound 48/80 (black bar) and preincubating different concentrations of (a) UFH solution (vertical-line bars), UFH-loaded CS-HA nanoparticles (horizontal-line bars) or (b) LMWH solution (vertical-line bars), LMWH-loaded CS-HA nanoparticles (horizontal-line bars), before the addition of compound 48/80. As a control, the cells were preincubated with a fixed concentration of blank CS-HA nanoparticles (squared bars) before the addition of compound 48/80 (n=3, p<0.05). The results obtained with heparin-loaded nanoparticles are not so promising if they are compared with those obtained with heparin solutions. However, the experimental conditions do not reflect physiological barriers in airways such as mucociliary clearance (via the mucociliary escalator) and enzymatic activity. These barriers may be better overcome by the described b a Capitulo 2  113 polysaccharide nanosystems because of the mucoadhesive-properties of CS (Aspden et al., 1997; Lim et al., 2000) and HA (Prichtard et al., 1996; Lim et al., 2000) and because of the intrinsic capacity of nanoparticles to protect the loaded drug from the enzymatic attack. Additionally, the nanoparticulate formulations may improve the effect of a conventional heparin formulation because of slow drug release, thus prolonging the antiasthmatic effect. Unfortunately, the experimental ex-vivo conditions do not allow long-term experiments to be carried out. Whether CS-HA nanoparticles loaded with heparin can really improve the effect of heparin in preventing mast cell degranulation can only be answered by conducting in-vivo experiments. This is the next challenge in validating our hypothesis. Conclusions Nanosystems were produced from CS and HA and their suitability as heparin carriers for the treatment of asthma was investigated. Confocal microscopy revealed that heparin-loaded CS-HA nanoparticles were internalized by rat mast cells. However, the capacity of free heparin and of heparin encapsulated in the nanosystems to prevent histamine release was very similar, and showed the same dose-response dependence. Acknowledgements The authors acknowledge financial support from the Spanish Government (SAF 2004-08319-C02-01 and Consolider-Ingenio CSD 2006- 00012); Felipe Oyarzun-Ampuero was in receipt of a CONICYT scholarship. J.B. received financial support from the Programa Isabel Barreto (Xunta de Galicia). We also thank Mr. Salvador Arines for technical assistance with the mast cells assays. Chitosan-hyaluronic acid nanoparticles loaded with heparin for the treatment of asthma 114 References 1. Ahmed, T., Ungo, J., Zhou, M., Campo, C., 2000. Inhibition of allergic late airway responses by inhaled heparin-derived oligosaccharides. J. Appl. Physiol., 88, 1721-1729. 2. Aspden, T.J., Mason, J.D., Jones, N.S., Lowe, J., Skaugrud, O., Illum, L., 1997. Chitosan as a nasal delivery system: the effect of chitosan solutions on in vitro and in vivo mucociliary transport rates in human turbinates and volunteers. J. Pharm. Sci. 86(4), 509-13. 3. Buceta, M., Dominguez, E., Castro, M., Brea, J., Alvarez, D., Barcala, J., Valdes, L., Alvarez-Calderon, P., Dominguez, F., Vidal, B., Diaz, J.L., Miralpeix, M., Beleta, J., Cadavid, M.I., Loza, M.I., 2008. A new chemical tool (C0036E08) supports the role of adenosine A(2B) receptors in mediating human mast cell activation. Biochem. Pharmacol., 76(7), 912-21. 4. Calvo, P., Remuñan-Lopez, C., Vila-Jato, J.L., Alonso, M.J., 1997. Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers. J. Appl. Pol. Sci., 63, 125-132. 5. Campo, C., Molinari, J.F., Ungo, J., Ahmed, T., 1999. Molecularweight-dependent effects of nonanticoagulant heparins on allergic airway responses. J. Appl. Physiol., 86(2), 549-557. 6. De Campos, A.M., Diebold, Y., Carvalho, E.L., Sanchez, A., Alonso, M.J., 2004. Chitosan nanoparticles as new ocular drug delivery systems: in vitro stability, in vivo fate, and cellular toxicity. Pharm. Res., 21(5), 803-10. 7. De la Fuente, M., Seijo, B., Alonso, M.J., 2008a. Bioadhesive hyaluronan-chitosan nanoparticles can transport genes across the ocular mucosa and transfect ocular tissue. Gene Ther., 15(9), 668-76. Capitulo 2  115 8. De la Fuente, M., Seijo, B., Alonso, M.J., 2008b. Novel hyaluronanbased nanocarriers for transmucosal delivery of macromolecules. Macromol. Biosci., 8(5), 441-50. 9. Garcia-Fuentes, M., Prego, C., Torres, D., Alonso, M.J., 2005. A comparative study of the potential of solid triglyceride nanostructures coated with chitosan or poly(ethylene glycol) as carriers for oral calcitonin delivery. Eur. J. Pharm. Sci., 25(1), 133-43. 10. Green, W.F., Konnaris, K., Woolcock, A.J., 1993. Effect of salbutamol, fenoterol, and sodium cromoglicate on the release of heparin from sensitized human lung fragments challenged with Dermatophagoides pteronyssinus allergen. Am. J. Respir. Cell Mol. Biol., 8, 518-521. 11. Halayko, A.J., Recto, E., Sthepens, N.L., 1997. Characterization of molecular determinants of smooth muscle cells heterogeneity. Can. J. Physiol. Pharmacol., 75, 917-919. 12. Johnson, P.R., Armour, C.L., Carey, D., Black, J.L., 1995. Heparin and PGE2 inhibit DNA synthesis in human airway smooth muscle cells in culture. Am. J. Physiol., 269, L514-L519. 13. Kanabar, V., Hirst, S.J., O´Connor, B.J., Page, C.P., 2005. Some structural determinants of the antiproproliferative effect of heparin-like molecules on human airway smooth muscle. Br. J. Pharmacol., 146, 370-377. 14. Kilfeather, S.A., Tagoe, S., Perez, A.C., Okona-Mensa, K., Matin, R., Page, C.P., 1995. Inhibition of serum-induced proliferation of bovine tracheal smooth muscle cells in culture by heparin and related glycosaminoglycans. Br. J. Pharmacol., 114, 1442-1446. 15. Köping-Höggård, M., Sanchez, A., Alonso, M.J., 2005. Nanoparticles as carriers for nasal vaccine delivery. Expert Rev. Vaccines., 4(2),185-96. 16. Lago, J., Alfonso, A., Vieytes, M.R., Botana, L.M., 201. Ouabaininduced enhancement of rat mast cells response. Modulation by protein phosphorylation and intracellular pH. Cell Signal., 13(7), 515-24. Chitosan-hyaluronic acid nanoparticles loaded with heparin for the treatment of asthma 116 18. Lim, S.T., Martin, G.P., Berry, D.J., Brown, M.B., 2000. Preparation and evaluation of the in vitro drug release properties and mucoadhesion of novel microspheres of hyaluronic acid and chitosan. J. Control. Rel., 66, 281-292. 18. Lopez-Leon, T., Carvalho, E.L., Seijo, B., Ortega-Vinuesa, J.L., Bastos- Gonzalez, D., 2005. Physicochemical characterization of chitosan nanoparticles: electrokinetic and stability behavior. J. Colloid Interface Sci., 283(2), 344-51. 19. Martinez-Salas, J., Mendelssohn, R., Abraham, W.M., Hsiao, B., Ahmed, T., 1998. Inhibition of allergic airway responses by inhaled low-molecular-weight heparins: molecular-weight dependence. J. Appl. Physiol., 84(1), 222-228. 20. Molinari, J.F., Campo, C., Shahida, S., Ahmed, T., 1998. Inhibition of antigen-induced airway hyperresponsiveness by ultralow molecularweight heparin. Am. J. Respir. Crit. Care Med., 157, 887-893. 21. Niven, A.S., Argyros, G., 2003. Alternate treatments in asthma. Chest., 123(4), 1254-65. 22. Ortner, M.J., Chignell, C.F., 1981. The effect of concentration on the binding of compound 48/80 to rat mast cells: a fluorescence microscopy study. Immunopharmacology, 3(3), 187-91. 23. Page, C.P., 1991. One explanation of the asthma paradox: inhibition of natural anti-inflammatory mechanism by beta 2-agonist. Lancet, 337, 717-720. 24. Page, S., Ammit, A.J., Black, J.L., Armour, C.L., 2001. Human mast cell and airway smooth muscle cell interactions: implications for asthma. Am. J. Physiol. Lung Cell. Mol. Physiol., 281, L1313-L1323. 25. Prego, C., García, M., Torres, D., Alonso, M.J., 2005. Transmucosal macromolecular drug delivery. J. Control Release., 101(1-3), 151-62. 26. Prichtard, K., Lansley, A.B., Martin, G.P., Helliwell, M., Marriot, C., Benedetti, L.M., 1996. Evaluation of the bioadhesive properties of Capitulo 2  117 hyaluronan derivates: detachment weight and mucocilliary transport studies. Int. J. Pharm., 129, 137-145. 27. Robinson, D.S., 2004. The role of the mast cell in asthma: induction of airway hyperresponsiveness by interaction with smooth muscle? J. Allergy Clin. Immunol., 114, 58-65. 28. Wong, W.S., Koh, D.S., 2000. Advances in immunopharmacology of asthma. Biochem Pharmacol., 59(11), 1323-35.       Capitulo 3 A potential nanomedicine consisting in heparin-loaded polysaccharide nanocarriers for the treatment of asthma    Capitulo 3 121 Abstract The aim of this study is to produce and characterize a new nanomedicine consisting of chitosan (CS)/carboxymethyl-β-cyclodextrin (CMβCD) loaded with unfractioned or low-molecular-weight heparin (UFH or LMWH, respectively), and evaluate its potential in asthma treatment. The nanoparticles are prepared by ionotropic gelation showing a size ranged between 221 and 729 nm with a positive zeta potential. The drug association efficiency is higher than 70%. Developed nanosystems are stable in Hank's balanced salt solution pH 6.4, releasing slowly the drug. Ex vivo assays, show that nanocarriers led to an improvement of heparin at preventing mast cell degranulation. These results agree with the effective cellular internalization of the fluorescently-labelled nanocarriers, and postulate these nanomedicines as promising formulations for asthma treatment. Keywords: chitosan; cyclodextrins; heparin delivery; mast cells; nanoparticles. A potential nanomedicine consisting in heparin-loaded polysaccharide nanocarriers for the treatment of asthma 128 was finally isolated by dialysis for 72 h (cellulose dialysis tubing, pore size 12400 Da; Sigma Aldrich, Spain) against demineralised water, and freezedried. The pH of fluorescent CS was adjusted to the same value as the raw CS solution (pH 4.9) with HCl, for the preparation of fluorescent nanoparticles. Preparation of Fluorescent Heparin-Loaded CS-CMβCD Nanoparticles: Fluorescent nanoparticles were prepared according to the same procedure explained in 2.2. The selected mass distribution for the preparation of fluorescent nanoparticles was: 6 mg of fluorescent CS, 0.85 mg of CMβCD, 0.34 mg of TPP and 1.6 mg of UFH. Confocal Laser Scanning Microscopy Study: An aqueous solution (50 μL) containing 0.3 mg of isolated fluorescent UFH-loaded CS-CMβCD nanoparticles was incubated with 450 μL of a suspension of mast cells (10x103 cells/100 μL) in Umbreit (for composition, see below) containing 0.05% w/v of BSA. The mixture was incubated for 2 h at 37 °C, and the cells were then separated by centrifugation (10 min, 200xg) and discarding the supernatants. Two hundred μL of Umbreit+BSA solution (at 4 °C) were then added to the cell pellet. The pellet was resuspended and centrifuged again to extract the non-internalized nanoparticles. This procedure was repeated once more. Mast cells were fixed for 5 minutes in paraformaldehyde (2% w/v, 100 μL) and washed 3 times with the Umbreit+BSA solution, by centrifugation. Two hundred μL of a Bodipi® phalloidin solution (Invitrogen, USA) were added to the cell pellet and the cells were incubated for 30 min at room temperature. The cells were washed 3 times (Umbreit+BSA) by centrifugation, the supernatant was discarded, and the pellet was resuspended in 20 μL of the Umbreit+BSA solution. The resuspended sample was placed on the surface of a positively charged microscope slide (Superfrost Ultra Plus, Menzel-Glaser, Irland) and dried at room temperature overnight. The Capitulo 3 129 sample was prepared in Vectashield medium (Vector, USA) for visualization by confocal microscopy (CLSM, Zeiss 501, Germany) (all of the described procedures were carried out in darkness to prevent the loss of the fluorescent signal from the nanoparticles and mast cells). The composition of Umbreit saline solution was: 1.2 mM MgSO4, 1.2 mM NaPO4H2, 22.85 mM NaHCO3, 5.94 mMKCl, 1 mM CaCl2, 119 mMNaCl and 0.1% glucose. Ex Vivo Studies With Rat Mast Cells: Inhibition of Histamine Release by Heparin-Loaded CS-CMβCD Nanoparticles Animal procedures were conducted in accordance with the standard ethical guidelines (National Institutes of Health, 1995; Council of Europe, 1996) and approved by the local ethical committees. Rat Mast Cell Purification and Viability: Mast cells were obtained by lavage of pleural and peritoneal cavities of female Sprague - Dawley rats (400–800 g) with Umbreit saline solution, following procedures similar to those described in other studies.[29-30] The suspension obtained from each rat was centrifuged at 100xg for 5 min (4 ºC) and suspended in a final volume of 1 mL of Umbreit containing 0.05% w/v of BSA. Purification was carried by centrifugation on 4 mL of an isotonic Percoll gradient at 600 g for 10 min (4 ºC). The mast cells were washed twice with the Umbreit+BSA solution and maintained at 4 °C in this solution until use. Mast cells were quantified by toluidin blue staining (95% purity) and the viability (90%) assessed by trypan blue staining (the procedure is described below).  A potential nanomedicine consisting in heparin-loaded polysaccharide nanocarriers for the treatment of asthma 130 Trypan blue staining procedure: Mast cell viability studies were carried out by trypan blue staining in an inverted microscope, as described by Lago et al.[29] This involved visual counting of the stained cells in the five fields of a counting chamber. The percentage of viability was calculated with the following formula: 100 CellsMast ofNumber Total Fields Five theFromMean Aritmetic Cells Stained BlueTrypan ×= (4) In order to test the mast cell viability after contact with heparinloaded nanoparticles, the same procedure was used, and the UFH or LMWH- loaded CS-CMβCD nanoparticles added to the rat mast cell suspension (1x105 cells per test tube). The tested dose of nanoparticles was equivalent to 200 μg/mL of UFH or LMWH. Measurement of Histamine Release in Rat Mast Cells: Rat mast cells (1x105 cells per test tube) were pre-warmed at 37 ºC (10 min) in BSA-free Umbreit saline solution containing the UFH or LMWH solutions or the nanoparticles loaded with UFH or LMWH. Histamine release from mast cells was then initiated by incubating the cells with 100 µM of compound 48/80 (Sigma Aldrich, Spain) for 20 min at 37 ºC. The cells were then centrifuged at 1100xg for 3 min at 4 ºC, and two aliquots (100 µl) of the supernatants were collected in a 96-well microplate. The rest of the supernatants were discarded and the pellets were resuspended in 500 μL of HCl 0.1 M, sonicated for 1 min and centrifuged at 1100 xg for 6 min. Two aliquots of 100 μL of the supernatants were collected for residual histamine determination. Histamine was assayed fluorometrically, as described by Lago et al.[29]; briefly, 80 µL NaOH 1 M were added to 100 µL of the sample, then 50 µl phthaldialdehyde 0.04% w/v were added to each well and plate was incubated for 4 min at 25° C. After this time, 50 µl of 3 M HCl were added and fluorescence was measured within 20 min, at excitation and emission wavelengths of 360 nm Capitulo 3 131 and 465 nm respectively, in a Tecan Ultra Evolution reader (Tecan, Switzerland). Data Analysis for Measurement of Histamine Release in Rat Mast Cells: Results were expressed as percentage of the total histamine released after stimulation with compound 48/80. The results were corrected for spontaneous histamine release in the absence of any chemical and under the same conditions. The equation used for the calculation was HR=[(S−ER)/(S+P−ER)]×100, where HR is the percentage histamine release; S, supernatant fluorescence; ER, fluorescence of spontaneous release supernatants and P, pellet fluorescence. IC50 values were obtained by fitting the data with non-linear regression, with Prism 2.1 software (GraphPad, San Diego, CA). Statistical Analysis The statistical significance of the differences between formulations was determined by application of two-way analysis of variance (ANOVA) followed by a two-tailed paired Student’s test. Differences were considered significant at p<0.05. Results and Discussion It has been reported that heparin could potentially be used for the treatment of asthma. However, this potential use is constrained by its limited access into the mast cells where target receptors for preventing degranulation are located.[31-32] This limited access could be related to the electrostatic repulsion between this highly negative macromolecule and the mastocyte membranes. Therefore, the hypothesis of this work was that the incorporation A potential nanomedicine consisting in heparin-loaded polysaccharide nanocarriers for the treatment of asthma 132 of heparin into nanocarriers could neutralize its charge and facilitate the internalization and controlled release of the drug into the mast cells. Preparation and Characterization of Heparin-Loaded CS-CMβCD Nanoparticles Nanoparticles loaded with heparin were prepared by the ionotropic gelation technique. The ability of CS to form a gel after contact with polyanions by promoting inter and intramolecular linkages enables the formation of the nanoparticles.[33] In this case, an ionic interaction occurs between the positively charged CS and the negatively charged CMβCD, heparin and the polyanion TPP. The ionic gelation process is extremely simple and involves mixing two aqueous phases at room temperature. As a first approximation for the formation of adequate nanoparticle formulations, we assayed different ratios of the three anionic components of the nanomedicines. Then, we identified the ratio between components that enabled the formation and also the adequate isolation of nanosystems. Table 1 shows size, polydispersity index, and zeta potential of a variety of formulations tested with UFH. Valuable information extracted from these experiments is that when the amount of polyanions was too low (relative to CS), nanoparticles could not be formed. However, if the amount of polyanions was too high, the particles precipitated or aggregated during the isolation process. This behavior could be attributed to the gradual counterionization of the positively charged CS, as noted by the reduction in the positive zeta potential values of the nanosystems. All the resulting nanosystems loaded with UFH exhibited a size in the range of 350-730 nm; polydispersity values were between 0.2-1 and the positive zeta potential ranged from +33.2 to +40.7 (Table 1). These ranges of values are similar to those previously presented by Krauland et. al.[27] who developed different formulations of UFH loaded in CS-CMβCD or CS-TPP Capitulo 3 133 nanoparticles. Among the formulation developed in this study, the one comprising 6 mg of CS, 0.85 mg of CMβCD, 0.34 mg of TPP and 1.6 mg of UFH was selected for further studies including in vitro characterization and ex vivo efficacy. Table 1: Physicochemical properties of the nanoparticles prepared with different ratios of CS- CMβCD-TPP-heparin (mean ± S.D., n=3). Amount [mg] CS-CMβCD-TPP- heparin Size [nm] Polydispersity Index Zeta potential [mV] 6-1-0-1a359 ± 21 0.3 – 0.4 +40.7 ± 1.0 6-2-0-2a729 ± 54 0.8 – 1 +33.2 ± 0.8 6-2-0-2.1aNon resuspendable --- --- 6-0.85-0.34-1.6a375±69 0.3 – 0.5 +37.0 ± 1.6 6-1-0.34-1.6a473 ± 24 0.6 – 0.9 +34.7 ± 1.2 6-1.15-0.34-1.6aNon resuspendable --- --- 6-1.3-0.34-1.6aPrecipitation --- --- 6-0.85-0.34-1.6 b 221± 26 0.2 – 0.3 +36.8 ± 0.7 a= UFH; b = LMWH Importantly, the association efficiency of UFH in the selected formulation was 77.0%. This high association value is related to the capacity of the polyanion to interact avidly with the polycationic CS as previously shown for other CS-based nanosystems.[27,30] With the aim of elucidating the influence of the heparin Mw in the in vitro and ex vivo behavior of the heparin-loaded nanoparticles, we also used a low-molecular-weight-heparin (LMWH). This new formulation exhibited similar values of zeta potential and association efficiency, however the size and polydispersity values were smaller when compared to those of high Mw heparin-loaded nanoparticles (Table 1). This could be attributed to a tighter assembling of the components forming the nanoparticles. The differences between the two formulations are also illustrated in the TEM micrographs presented in Figure 1a and b. It should be noted that the nanosystems containing LMWH form a smaller and denser structure than those containing A potential nanomedicine consisting in heparin-loaded polysaccharide nanocarriers for the treatment of asthma 134 UFH. The photographs also show the spherical shape of nanosystems, this is in agreement with previous works describing nanoparticles prepared following the same method.[27,30] Figure 1: Electron transmission micrographs of CS-CMβCD nanoparticles containing UFH (a) or LMWH (b). Stability Studies The stability of the selected systems was investigated in media that mimics biological conditions and that are usually used in cell culture studies. These media included: HBSS (pH 6.4 and 7.4) and PBS (pH 7.4). The stability of the selected nanoparticles loaded with UFH and LMWH was maintained for up to 24 h in HBSS pH 6.4 (Figure 2) while in the other media the nanoparticles aggregated immediately (data not shown). The zeta values of the selected UFH/LMWH-loaded nanoparticles in HBSS pH 6.4 were approximately +14 mV, due to the presence of ionizable groups of CS in this medium. In contrast, the zeta values of the systems in HBSS pH 7.4 and PBS 7.4 were neutral (≈0 mV), thus, making the systems vulnerable to aggregation. The stability of the nanoparticles was also assayed in water at 4° C, where formulations maintained stable for up three months (data not shown). Capitulo 3 135 Figure 2: Stability of heparin-loaded CS-CMβCD nanosystems in HBSS pH 6.4: UFH-loaded nanoparticles (■) and LMWH-loaded nanoparticles (×) (mean ± S.D., n=3). Release Studies From Selected Nanosystems As shown in Figure 3, the release kinetics of heparins in HBSS pH 6.4 was slow and clearly dependent on their molecular weight. In the case of the systems comprising LMWH, the drug was released very slowly during the first hour (approximately 10% of the encapsulated drug) followed by a plateau phase with little further change up to 12 h of incubation. Otherwise, the UFH was released in a faster, continuous manner, with a final release of approximately 50% in the same period of incubation. The slower release rate observed for LMWH could be attributed to the more packed structure of these nanosystems as we previously argument. The slow release rate observed for nanosystems loaded with UFH or LMWH agrees with the results obtained for other formulations comprising CS and heparin and is attributed to the strong ionic interaction among the anionic drug and the cationic polymer.[30] A potential nanomedicine consisting in heparin-loaded polysaccharide nanocarriers for the treatment of asthma 136 Figure 3: Heparin release from UFH-loaded CS-CMβCD nanoparticles (□) and LMWH- loaded CS-CMβCD nanoparticles (■) in HBSS pH 6.4 (means ± S.D., n=3). Study of Interaction of Heparin-Loaded CS-CMβCD Nanoparticles with Rat Mast Cells by Confocal Microscopy Taking into account that the antiasthmatic effect of heparin is attributed to its capacity of preventing the mast cell degranulation via the intracellular receptor of IP3,[34-35] the capacity of the selected nanosystems to gain intracellular access is a fundamental requisite. Thus, we performed confocal microscopy experiments for visualizing the interaction-transport of fluorescent heparin-loaded CS-CMβCD nanoparticles in rat-mast cells. In Figure 4.2, it can be seen that the overlapping of the fluorescent signal from the incubated UFH-loaded CS-CMβCD nanoparticles (green) with that corresponding to the mast cells (red), resulted in an orange color. This means that the fluorescent nanoparticles effectively interacted with the mast cells after a period of contact of two hours. We confirmed that this interaction enables the nanoparticles to be internalized in mast cells by observing the fluorescent nanoparticles in sequential slides from the “z” axis of mast cells (Fig. 4.3). The positive control (Fig. 4.1) indicates that fluorescent mastocytes did not emit the signal of fluorescent nanoparticles. The confocal images obtained upon treatment of cells with fluorescent LMWH-loaded CS- CMβCD nanoparticles, were similar to those reported in Figure 4.3. Overall, Capitulo 3 137 this capacity of heparin-loaded CS-CMβCD nanoparticles for entering in the rat mast cells is similar to that previously observed for heparin-loaded CS- hyaluronic acid nanoparticles.[30] This capacity could be attributed to the nanometric characteristic of the systems and also to the capacity CS for interacting with cellular membranes and promoting the intracellular access of the nanosystems.[20, 36] Figure 4: Confocal laser scanning microscopy images of fluorescent mastocytes and fluorescent UFH-loaded CS-CMβCD nanoparticles. (1) Mastocytes not incubated with nanoparticles (positive control): (a) excitation signal for mastocytes (red); (b) excitation signal for nanoparticles (no signal); (c) overlapping of both signals (red), and (d) optical signal. (2) Mastocytes after incubation with nanoparticles: (a) excitation signal for mastocytes (red); (b) excitation signal for nanoparticles (green); (c) overlapping of both signals (orange), and (d) optical signal. (3) Slides of mastocytes taken every 1.5 microns in the “z” axis, after incubation with nanoparticles. First line: excitation signal for mastocytes (red); second line: excitation signal for nanoparticles (green). A potential nanomedicine consisting in heparin-loaded polysaccharide nanocarriers for the treatment of asthma 144 32. T. Ahmed, T Syriste, R. Mendelssohn, D. Sorace, E. Mansour, M. Lansing, W.M. Abraham, M.J. Robinson, J. Appl. Physiol. 1994, 76(2), 893. 33. P. Calvo, C. Remuñan-Lopez, J.L. Vila-Jato, M.J. Alonso, J. Appl. Pol. Sci. 1997, 63, 125. 34. W.S. Wong, D.S. Koh, Biochem. Pharmacol. 2000, 59(11), 1323. 35. A.S. Niven, G. Argyros, Chest 2003, 123(4), 1254. 36. M. de la Fuente, N. Csaba, M. Garcia-Fuentes, M.J. Alonso, Nanomedicine (Lond.). 2008, 3(6), 845. 37. M.J. Ortner, C.F. Chignell, Immunopharmacology 1981, 3(3), 187. 38. T.J. Aspden, J.D. Mason, N.S. Jones, J. Lowe, O. Skaugrud, L. Illum, J. Pharm. Sci. 1997, 86(4), 509. 39. S.T. Lim, G.P. Martin, D.J. Berry, M.B. Brown, J. Control. Rel. 2000, 66, 281. 40. M. Bivas-Benita, K.E. van Meijgaarden, K.L. Franken, H.E. Junginger, G. Borchard, T.H. Ottenhoff, A. Geluk, Vaccine 2004b, 22(13-14), 1609. 41. Z. Ahmad, S. Sharma, G.K. Khuller, Int. J. Antimicrob. Agents 2005, 26(4), 298. 42. H. Jin, T.H. Kim, S.K. Hwang, S.H. Chang, H.W. Kim, H.K. Anderson, H.W. Lee, K.H. Lee, N.H. Colburn, H.S. Yang, M.H. Cho, C.S. Cho, Mol. Cancer Ther. 2006, 5(4), 1041. Discusión 145 DISCUSIÓN Como se ha mencionado en la sección de Introducción, la heparina posee un interesante potencial como tratamiento del asma bronquial. Sin embargo, este potencial se ve notablemente limitado por su acceso restringido al interior de los mastocitos, donde se encuentran localizados los receptores que pueden prevenir la desgranulación de estas células95;96. La dificultad en el acceso intracelular de la macromolécula se relaciona con la repulsión electrostática entre la heparina, que posee una gran densidad de carga negativa, y las membranas celulares de los mastocitos. Por lo tanto, la incorporación de la heparina en sistemas nanoparticulares se abre como una posibilidad de ocultar su carga y, con ello, facilitar su internalización, además de ofrecer la posibilidad de modular su liberación en el interior de los mastocitos. Para la elaboración de los nanosistemas, se seleccionó el polisacárido quitosano, en combinación con el ácido hialurónico (HA), o con el oligosacárido carboximetil-β-ciclodextrina (CMβCD). La técnica elegida para la elaboración de los nanosistemas fue la gelificación ionotrópica y las principales variables de formulación investigadas en la formación de las nanopartículas, fueron la proporción de los componentes en cada tipo de sistema y el peso molecular de la heparina.  95 Lucio J, D'Brot J, Guo CB, Abraham WM, Lichtenstein LM, Kagey-Sobotka A, Ahmed T. (1992). Appl. Physiol. 73(3):1093-101. 96 Ahmed T, Syriste T, Mendelssohn R, Sorace D, Mansour E, Lansing M, Abraham WM, Robinson MJ. (1994). J. Appl. Physiol. 76(2):893-901. Discusión 146 Preparación de las nanopartículas Las nanopartículas se prepararon de acuerdo con el procedimiento de gelificación iónica, previamente desarrollado en nuestro grupo de investigación97. Esta técnica consiste en mezclar dos fases que contienen disoluciones acuosas con moléculas de carga positiva y negativa (Fig. 1). Las nanopartículas se obtienen espontáneamente tras la nanogelificación que se produce por la interacción entre los grupos amino cargados positivamente del quitosano y las cargas negativas de los polímeros u oligómeros, en presencia del agente reticulante (tripolifosfato; TPP). Es un procedimiento extremadamente simple y suave que se realiza a temperatura ambiente. Figura 1. Esquema de preparación de los nanosistemas mediante el procedimiento de gelificación iónica (CS:quitosano).  97 Calvo P, Remuñan-Lopez C, Vila-Jato JL, Alonso MJ. (1997) J. Appl. Polymer Sci. 63, 125-132. Discusión 147 Para la preparación de los nanosistemas de quitosano-CMβCD y quitosano-HA, conteniendo heparina, fue necesario establecer las proporciones más adecuadas de los componentes que permitían su apropiada formación y aislamiento. En general, independientemente de la composición específica de cada sistema, las condiciones de formación se ajustaron a una pauta similar. En las Tablas 1a y 1b se puede apreciar que, en la medida en que se añadieron cantidades crecientes de los polianiones, se observó un aumento de tamaño en los sistemas junto con una disminución en el valor absoluto del potencial zeta. Esto es explicable en términos de la contraionización que el policatión (quitosano) va sufriendo producto de la adición de cantidades crecientes de polianiones. Cuando los sistemas se vuelven irresuspendibles, o precipitan, se supone una la completa contraionización del policatión, que impide a los sistemas tener una carga eléctrica superficial suficientemente alta que les permita repelerse electrostáticamente y mantener su estabilidad coloidal98. Tabla 1. Características físico-químicas de las nanopartículas preparadas usando diferentes proporciones de (1) CS-HA-TPP-heparina y (2) CS-CMβCD-TPP-heparina (media ± d.e.; n=3). Cantidad (mg) CS-HA-TPP- heparina Tamaño (nm) Índice de polidispersión Potential Z (mV) 4-1.2-0.21-1.0 a 201 ± 24 0.2 – 0.4 +32.1 ± 1.6 4-1.2-0.21-1.2 a 217 ± 30 0.2 – 0.4 +28.1 ± 0.9 4-1.2-0.21-1.4 a No resuspendible --- --- 4-1.2-0.21-1.6 a Precipitación --- --- 4-0.6-0.21-1.2 a 162 ± 17 0.1 – 0.3 +34.6 ± 0.6 4-0.6-0.21-1.4 a 193 ± 32 0.2 – 0.5 +32.5 ± 1.7 4-0.6-0.21-1.5 a No resuspendible --- --- 4-0.6-0.21-1.4 b 152 ± 10 0.2 – 0.3 +33.0 ±1.3 CS= quitosano; a= UFH; b= LMWH.  98 Krauland AH, Alonso MJ. (2007). Int. J. Pharm. 340:134-42. 1) Discusión 148 Cantidad (mg) CS-CMβCD- TPP-heparina Tamaño (nm) Índice de polidispersión Potencial Z (mV) 6-1-0-1a 359 ± 21 0.3 – 0.4 +40.7 ± 1.0 6-2-0-2a 729 ± 54 0.8 – 1 +33.2 ± 0.8 6-2-0-2.1a No resuspendible --- --- 6-0.85-0.34-1.6a 375±69 0.3 – 0.5 +37.0 ± 1.6 6-1-0.34-1.6a 473 ± 24 0.6 – 0.9 +34.7 ± 1.2 6-1.15-0.34-1.6a No resuspendible --- --- 6-1.3-0.34-1.6a Precipitación --- --- 6-0.85-0.34-1.6b 221± 26 0.2 – 0.3 +36.8 ± 0.7 Caracterización de las nanopartículas Como se puede apreciar en la Tabla 1.1, todos los sistemas de quitosano-HA presentaron un rango de valores de tamaño, polidispersión y potencial zeta relativamente estrecho (152-217 nm, 0.1-0.5, y 33.2-40.7 mV, respectivamente). Por otro lado, los sistemas de quitosano-CMβCD (Tabla 1.2) presentaron una mayor dispersión en los citados parámetros (221-729 nm, 0.2-1, y +33-+41 mV), siendo estos valores similares a los presentados por Krauland y col. (2007) para diferentes formulaciones nanopartículas de quitosano-TPP y quitosano-CMβCD conteniendo heparina no fraccionada (UFH)98. En las formulaciones en las que sustituyó la UFH por heparina de bajo peso molecular (LMWH), los parámetros de tamaño y polidispersión fueron los más bajos de todos los sistemas ensayados, lo que, evidentemente, se relaciona con el menor peso molecular del fármaco. En el caso del potencial zeta, se puede apreciar que, en todas las formulaciones, se obtienen valores altamente positivos, lo cual es indicativo de que la superficie de los nanosistemas está compuesta principalmente por quitosano. Para las siguientes etapas de caracterización y evaluación, se seleccionaron las formulaciones destacadas en negrita en las Tablas 1.1 y 1.2. El criterio utilizado para dicha selección se basó principalmente en la menor 2) Discusión 149 polidispersión de estas formulaciones y en el hecho de que éstas están elaboradas con una adecuada cantidad de heparina. En las Tablas 2.1 y 2.2 se presentan los valores de contenido en heparina, eficacia de asociación y rendimiento de las formulaciones de quitosano-HA y quitosano-CMβCD. La eficacia de asociación fue, en todos los casos, similar (~70%), lo que coincide con otros trabajos en los que se describen nanosistemas de quitosano y heparina 4;99. Esta elevada asociación se atribuye al alto potencial para interaccionar iónicamente que presentan dos macromoléculas con carga complementaria, como quitosano y heparina. En cuanto al contenido de heparina encapsulada, se puede apreciar que este valor fue siempre mayor para los sistemas que contienen LMWH y, especialmente alto para las nanopartículas de quitosano-HA (~61%). Ello se puede atribuir a un mejor ensamblaje de la LMWH en la matriz nanopartícular, lo que puede inducir un mayor desplazamiento de los demás polianiones (HA, CMβCD, TPP) que interaccionan con el quitosano y que explicaría también el menor rendimiento obtenido para los sistemas con LMWH. Tabla 2. Contenido en heparina, eficacia de encapsulación y de las nanopartículas de a) CSHA y b) CS-CMβCD (media ± d.e.; n=3). Cantidad CS-HA-TPP- heparina (mg) Contenido en heparina (%) Eficacia de encapsulación (%) Rendimiento (%) 4-0.6-0.21-1.4a 33.6 ±1.2 72.3 ± 2.7 49.0 ± 1.2 4-0.6-0.21-1.4b 60.6 ± 0.3 69.7 ± 7.6 24.9 ± 4.3 CS= quitosano; a= UFH; b= LMWH. Cantidad CS-CMβCD-TPP- heparina (mg) Contenido en heparina (%) Eficacia de encapsulación (%) Rendimiento (%) 6-0.85-0.34-1.6a 38.7 ± 2.5 77.0 ± 2.1 39.4 ± 2.5 6-0.85-0.34-1.6b 44.0 ± 1.1 70.6 ± 2.5 29.3 ± 0.6  99 Chen MC, Wong HS, Lin KJ, Chen HL, Wey SP, Sonaje K, Lin YH, Chu CY, Sung HW. (2009). Biomaterials. 30(34):6629-37. 1) 2) Discusión 150 Las imágenes de microscopía electrónica (Figura 2) indican que las formulaciones son esféricas, independientemente del tipo de contraión (HA o CMβCD) o de heparina utilizados. Es interesante hacer notar que los sistemas desarrollados con LMWH se ven aparentemente mejor ensamblados que los que contienen UFH, lo que concuerda con el argumento que acabamos de exponer. (1) (2) Figura 2. Imágenes de microscopía electrónica de transmisión de las nanopartículas de (1) CS(quitosano)-HA y (2) CS-CMβCD conteniendo a) UFH y b) LMWH. Estudios de estabilidad de las nanopartículas Evaluar la estabilidad coloidal de los nanosistemas es un punto clave para la planificación de estudios posteriores. Para ello se realizaron estudios de estabilidad con las formulaciones seleccionadas en condiciones similares a ab ba Discusión 151 las utilizadas en cultivos celulares, esto es, a 37°C y utilizando como medios HBSS a pH 6.4 y 7.4 y PBS a pH 7.4. La estabilidad de los nanosistemas se valoró a través del seguimiento de su tamaño en las diferentes condiciones. La composición de los nanosistemas y de los medios utilizados fueron factores determinantes en su estabilidad. En el caso de las nanopartículas de quitosano-HA, su tamaño no experimentó cambios significativos en 24 horas cuando se incubó en PBS a pH 7.4 (Figura 3), mientras que en HBSS a pH 7.4 sí experimentó un incremento significativo. A pH 6.4, se agregaron de inmediato, hecho que se relaciona con el cambio en el potencial zeta de las formulaciones, que presentó valores cercanos a la neutralidad. A pH 7.4, el potencial zeta de los sistemas se mantuvo en torno a -10 mV. Esta inversión en la carga superficial es consecuencia del bajo grado de ionización del quitosano (pKa ~ 6.2) en medios a pH 7.4. La diferencia entre los perfiles de estabilidad para HBSS pH 7.4 y PBS pH 7.4 tiene que ver con la distinta composición de estos tampones (HBSS posee iones CO32- y una concentración de iones PO42- 8 veces superior). En el caso de los sistemas de quitosano-CMβCD, la estabilidad se mantuvo hasta 24 h en HBSS pH 6.4 (Figura 4), mientras que en los otros medios las nanopartículas se agregaron inmediatamente. En HBSS pH 6.4 el potencial zeta de las formulaciones era cercano a +14 mV, mientras que a pH 7.4, los valores fueron neutros (~0 mV), lo que confirma la desestabilización. Discusión 152 Figura 3: Evolución del tamaño de las nanopartículas de quitosano-HA conteniendo heparina, en medios a pH 7.4: UFH (▲) y LMWH (×) en PBS; UFH (■) y LMWH (□) en HBSS (media ± d.e., n=3). Figura 4: Evolución del tamaño de las nanopartículas de quitosano-CMβCD conteniendo UFH (■) y LMWH (×) en medio HBSS pH 6.4 (media ± d.e., n=3). Finalmente, todas las formulaciones seleccionadas mantuvieron su tamaño sin alteraciones apreciables durante 3 meses en suspensión acuosa a 4 °C (resultados no mostrados). Estudios de liberación de heparina a partir de las nanopartículas Se evaluó la liberación de UFH y LMWH a partir de las formulaciones seleccionadas, en los medios en que éstas alcanzaron la Discusión 153 máxima estabilidad, esto es, en PBS pH 7.4 para los sistemas de quitosano- HA y en HBSS pH 6.4 para los sistemas de quitosano-CMβCD. Considerando la alta densidad de carga negativa que posee la heparina y la carga positiva del quitosano, se espera que la liberación del fármaco a partir de la matriz de los nanosistemas sea lenta, como consecuencia de una fuerte interacción electrostática entre las macromoléculas con carga opuesta98. Como se puede apreciar en las Figuras 6a y 6b, si bien la liberación de las heparinas fue lenta en todos los casos, su perfil varió significativamente dependiendo de su peso molecular y de la composición de cada nanosistema. En el caso de los sistemas de quitosano-HA, la UFH se liberó muy lentamente alcanzándose una liberación final de un 10% tras 12 h de incubación. La LMWH se liberó de una manera más rápida y continua alcanzándose una liberación final de aproximadamente un 80% (Figura 6a). a) Chitosan-coated lipid nanocarriers for therapeutic applications  256 mucoadhesivity and biocompatibility. Indeed, several authors had reported low or absent signs of toxicity upon CS administration by different routes [12-13]. Our first attempts conducted with the newly developed CS-coated lipid nanocarriers focused on the ocular route, in which several problems such as removal mechanisms, rapid nasolachrymal drainage and nonproductive drug absorption into the systemic circulation constrains the effects of conventional formulations. With the benefits obtained with these CS- coated lipid nanocarriers, we can achieve a higher drug concentration in ocular tissues and improve most pharmacokinetic parameters when compared to other cationiccoated carriers or to commercial formulations [14]. This information together with the low ocular toxicity of the nanosystems [14], inspired us to extend applications of these novel formulations to other administration routes. This work aims to review and to summarize our group’s contribution in aspects concerning the design and in vivo fate of the developed CS-coated lipid nanocarriers. Design and characterization of CS-coated lipid nanocarriers CS nanocapsules The initially designed CS-coated nanosystems were formed by promoting the ionic interaction of CS onto the surface of the negatively charged poly-ε-caprolactone (PεCL) nanocapsules or onto the negatively charged submicron nanoemulsion [6]. In the first case, CScoated PεCL nanocapsules were obtained by modifying the method of interfacial deposition of polyester polymers, proposed by Al Khouri et al. [15]. PεCL nanocapsules were formed by means of the spontaneous emulsification of a lipid core (Mygliol and lecithin) in water due to the diffusion of the organic solvent, in which the polymer and lipids were dissolved [15, 16]. To obtain CS-coated polyester nanocapsules, the polysaccharide CS was incorporated Anexo 1 257 in the aqueous phase during the manufacturing process, thus allowing the adsorption of CS onto the negatively charged surface of the systems [6]. In the second case, CS nanocapsules were prepared using a similar procedure to the one described above, the only difference being that the PεCL was eliminated from the formulation. Thus, the method of preparation was not a polymeric interfacial deposition, but rather a direct electrostatic interaction between CS and lecithin located in the inner core of the systems [6]. We also described the possibility of incubating the polysaccharide CS with the preformed lecithin-containing nanoemulsions for obtaining these nanocapsules [17]. Characterization by photon correlation spectroscopy of CS-coated polyester and CS nanocapsules indicated that nanocarriers between 150-300 nm were obtained, showing a monomodal dispersity. Laser doppler anemometry showed that all systems without CS displayed negative zeta potential values, while the addition of CS to the aqueous phase resulted in highly positive values, supporting the successful formation of a CS layer surrounding the lipid cores. Micrographs obtained by transmission electron microscopy (TEM), showed spherical structures in all cases (Figure 1). Figure 1 - Transmission electron micrographs of (a) CS-coated polyester nanocapsules and (b) CS nanocapsules (reproduced with permission from Springer and Editions de Santé, respectively). Chitosan-coated lipid nanocarriers for therapeutic applications  258 Considering the hydrophobic nature of the inner components in CS-coated nanosystems, it was reasonable to postulate that a variety of molecules with low water solubility could be efficiently encapsulated. The drugs that we effectively encapsulated in the CS-coated nanosystems included: diazepam [6], triclosan [18] and docetaxel [19]. Additionally, the cationic CS coating allowed the adsorption of negatively charged hydrophilic macromolecules such as the recombinant hepatitis B surface antigen [20]. The presence of the negatively charged lecithin in the oily core of the nanocapsules further allowed the incorporation of positively charged hydrophilic molecules such as salmon calcitonin [21]. The peptide was encapsulated into CS nanocapsules with an efficiency of 44 %, which was significantly lower than that shown by the uncoated nanoemulsion (> 90 %). This effect was attributed to a competition between the peptide and the CS, both positively charged, in their association with the surface of the nanoemulsion. The encapsulation efficiency was also dependent on the CS molecular weight. In fact, CS oligomer (~10 kDa) nanocapsules showed higher encapsulation efficiency (60 %) than medium molecular weight (~100 kDa) CS nanocapsules. This indicates that the CS oligomer coating was not able to displace salmon calcitonin to the same extent as medium molecular weight CS. CS-coated lipid nanoparticles Solid triglycerides are interesting biomaterials for the production of nanocarriers due to: i) their excellent biocompatibility [22]; ii) their capacity to form highly hydrophobic matrices capable of restricting the penetration of peptidases and other degradative enzymes present in biological fluids or in the intestinal epithelia, thus protecting the loaded cargo; iii) their capacity to enhance intestinal drug transport, which has been observed even in the case Anexo 1 259 of peptidic drugs [23]. Taking these reasons into account, we decided to prepare CS-coated solid triglyceride nanoparticles as alternative formulations to CS nanocapsules. More concretely, our aim was to compare the suitability of the different core structures, based on liquid or solid lipids, for protecting and promoting the intestinal absorption of peptide drugs. Solid triglyceride nanoparticles are typically prepared by high-pressure homogenization of molten lipid mixtures, by formation of microemulsions above the melting temperature of the triglycerides or by solventemulsification techniques [24]. These preparation techniques allow simple microencapsulation of hydrophobic molecules such as doxorubicin, paclitaxel, prednisolone or progesterone, among others [25]. On the other hand, solid triglyceride matrices present low affinity for hydrophilic macromolecules. Indeed, early attempts to include proteins and peptides into nanomatrices of solid lipids were undertaken by solubilizing these molecules in molten lipid mixtures that were subsequently dispersed as nanometric matrices [26, 27]. These methods frequently resulted in low protein loadings and potential peptide denaturation processes arising from contact with the molten lipids [24]. To avoid the use of high temperatures, we decided to adapt a solvent casting method and more specifically, a w/o/w doubleemulsion solvent-evaporation technique [28]. The double-emulsion solvent-evaporation method has been applied to the encapsulation of insulin and salmon calcitonin, leading to moderate/high encapsulation efficiencies and drug loadings close to 1 % (w/w) [28, 29]. Such encapsulation values represented a significant improvement over previous attempts based on peptide solubilization in the triglyceride mixture [27]. Notably, double-emulsion solvent-evaporation techniques have found application in the encapsulation of other biopharmaceutics such as antigenic proteins [30]. CS-coated lipid nanoparticles can be prepared by the deposition of CS on the nanoparticle surface, a process that is triggered by electrostatic forces. Presence of CS in the external phase of the solvent emulsification method used for lipid nanoparticle preparation destabilizes the Chitosan-coated lipid nanocarriers for therapeutic applications  260 colloidal system. For that reason, our method of CS-coated lipid nanoparticles preparation comprises first the preparation of the lipid nanoparticle cores and secondly, the formation of the CS coating by electrostatic interactions between the anionic cores and polycationic CS [29]. Typical CS-coated nanoparticles prepared by this method present a particle size between 400 and 600 nm and a positive zeta potential, as confirmed by photon correlation spectroscopy and laser doppler anemometry. Lipid nanoparticle composition and nanostructure can be further characterized by liquid-state NMR techniques, taking advantage of the different relaxation constants of the triglyceride core and polymeric shell and of the possibility of performing the analysis over the lipid mixture melting temperature [31, 32]. CS-coated nanostructures for the transmucosal delivery of macromolecules The beginning of this decade witnessed large research efforts dedicated to finding the “holy grail” of transmucosal delivery, a carrier capable of promoting the oral absorption of protein and DNA-based medicines [33]. The expectation of realizing such groundbreaking technology was fuelled by major advances from the late 80s and 90s, that demonstrated the capacity of nanometric matter to interact with the intestinal epithelium, ultimately leading to the first proofs-of-principle of their capacity to enhance the bioavailability of macromolecules delivered by transmucosal routes. Some of these first key contributions came from the Florence group, which in a systematic work demonstrated the capacity of submicrometric matter to be taken up by the intestinal epithelium and to a lesser extent, to be internalized [34-36]. Simultaneously, the Couvreur group presented the first evidence of efficient peptide delivery by the oral route using polycyanoacrylate nanocapsules [37]. This concept of oral delivery of therapeutic Anexo 1 261 macromolecules was further explored by the Mathiowitz group, this time using biodegradable, solid-core nanoparticles [38]. During the 90s, our group also studied the capacity of polymeric nanocarriers to interact with several epithelial barriers. Our results indicated the capacity of nanocarriers to tightly interact with the corneal, nasal and intestinal epithelia and even more importantly, that this interaction can lead to significant improvements in the transmucosal absorption of drugs [39-41]. All of this collected evidence pointed us to a potential opportunity to apply specifically optimized nanostructures for the transmucosal delivery of macromolecules. The blueprint of an ideal transmucosal nanocarrier would be a system capable of protecting the macromolecule of interest from enzymatic degradation in the administration route [42], but also capable of enhancing macromolecule transport through biological barriers. CS is a biocompatible polymer with mucoadhesive and penetrationenhancing properties, which makes it a promising candidate for nanocarrier surface modification [43]. Additionally, studies performed by our group have confirmed that CS-coated nanostructures present improved stability in biological fluids compared to lipid nanocores [29]. The modification of nanocarriers with CS applied as a mucoadhesive polymer coating for transmucosal delivery was reported simultaneously by the Kawashima group and our own. These works comprised CScoating strategies implemented into PεCL nanocapsules, poly(lactic-co-glycolic acid) nanoparticles and liposomes as transmucosal peptide carriers [6, 44-46]. In the next part of the review, we will summarize our main findings in the field of transmucosal delivery using the proposed CS-coated lipid nanocarriers. The interaction of the nanocarriers with mucosal surfaces and the administration of peptidic drugs by oral and nasal routes will be reviewed. As an alternative application, the utilization of CS nanocapsules in cancer therapy will be presented. Interaction of CS-coated nanostructures with mucosal surfaces Chitosan-coated lipid nanocarriers for therapeutic applications  262 We had previously reported a study showing the preferential internalization of CS nanoparticles compared with poly(ethylene glycol) (PEG)-coated nanoparticles in the Caco-2 cell model [47]. These results were even more remarkable when the formulations were assayed in the mucussecreting cells MTX-E12, suggesting that mucoadhesivity of CS nanoparticles is an important factor for interacting with mucosal surfaces. The above findings contrast with those obtained with CS-coated lipid nanostructures in Caco-2 cells. Here, the amount of associated CS-coated lipid nanoparticles or CS nanocapsules was similar to those obtained with PEG-coated nanoparticles or with a nanoemulsion, respectively [17, 48]. A further goal was to elucidate the possible internalization together with the intracellular localization of CS-coated lipid nanostructures. For this, CS nanocapsules were visualized after their incubation with Caco-2 cells and with a coculture of enterocytes and mucus-secreting cells using confocal laser scanning microscopy [21]. The results indicated that i) while in Caco-2 cells nanosystems interacted with a random distribution, in the coculture they interacted highly and preferentially with the mucus secreting cells; ii) the systems were not capable of crossing the monolayer and were preferentially located in the apical region of the cells (Figure 2). The higher interaction with mucus secreting cells of CS-coated nanosystems was attributed to the strong mucoadhesive character of the polymer which is related to the formation of hydrogen and ionic bonds between the positively charged amino groups of CS and the negatively charged sialic acid residues of mucin glycoproteins [49]. Consequently, we can infer from this study that the mucoadhesive character of CS nanocapsules is a determinant factor of their ability to interact with the intestinal mucosa. Theoretically, this mechanistic behavior would also be applicable to other CS-coated nanostructures such as lipid nanoparticles [29]. We have also checked the capacity of our polymercoated lipid nanostructures to modify the permeability of the cell monolayers. Anexo 1 263 In this case, it is known that CS has a capacity to cause a dose-dependent decrease in transepithelial electric resistance (TEER) [50] while PEG is assumed to be inert in terms of cellular interaction. In agreement with this, we observed that PEG-coated lipid cores did not cause a reduction in the TEER of Caco-2 monolayers in the range of concentrations investigated (220-330 μg/cm2). In contrast, CS-coated lipid nanostructures induced a dose-dependent drop in the TEER of Caco-2 monolayers, reaching significant reductions for the concentration range of 83-330 μg/cm2 [17, 48] (Figure 3). The values of the TEER reduction were additionally supported by the observation of enhanced paracellular transport of the macromolecular marker dextran-Texas Red (Mw = 3000 Da) [48]. However, it should be added that these values were, in the case of nanocapsules, close to those that comprised cell viability (220 μg/cm2) [17]. Therefore, it could be expected that this change in epithelium permeability could only be seen when an important amount of the nanocarriers accumulate in the epithelium. Interestingly, we observed that the normal TEER values slowly recuperated after the exposure of CS-coated nanostructures. Chitosan-coated lipid nanocarriers for therapeutic applications  264 Figure 2 - Confocal scanning microscopy images showing the association of fluorescent nanocapsules (green) with Caco-2 cells and coculture Caco-2/HT29-M6 (E-cadherin in red and nucleus in blue). Caco-2: (A1) Montage of 24 horizontal cross sections illustrating the interaction of fluorescent CS nanocapsules to the cells (step size in z axis of 0.5 2 m.); (A2) confocal xz section showing the accumulation of fluorescent CS nanocapsules in the apical side of the monolayer. Caco-2/HT29-M6 coculture: (B1) Montage of microscopy images showing the association of fluorescent CS nanocapsules with the coculture Caco-2:HT29-M6. (B2) Confocal scanning microscopy xz section showing the accumulation of fluorescent CS nanocapsules (green) in the apical side of the HT29-M6 cells (reprinted with permission from Springer). Anexo 1 265 Figure 3 - Transepithelial electric resistance (TEER) of Caco-2 monolayers exposed to PEG- coated nanoparticles (1 mg/mL) (◊), CS-coated nanoparticles (1 mg/mL) (s) or their respective controls (HBSS (Hanks’ balanced salt solution) (p); HBSS pH 6.5 (l)) (mean ± SD, n = 3-6) (reprinted with permission from Elsevier). Application of CS-coated nanocarriers for oral peptide delivery – case of study: salmon calcitonin The efficacy of CS nanocapsules and CS-coated lipid nanoparticles as oral carriers for peptide delivery was investigated using salmon calcitonin (sCT) as a model [17, 48]. The reduction of the serum calcium levels after oral administration of peptide-loaded CS nanocapsules was monitored in rats, using an aqueous solution and a nanoemulsion containing sCT as controls. Importantly, as shown in Figure 4, a marked hypocalcemic response was noted when the peptide was associated with the nanocapsules. The importance of the CS coating was evident because sCT was ineffective when administered in the uncoated nanoemulsion. Moreover, this reduction in the serum calcium levels was maintained for more than 24 h following administration of the CS-coated nanocapsules. This pronounced and longlasting hypocalcemic effect led us to speculate that the mucoadhesive properties of CS might be determinant in facilitating the intestinal absorption of sCT and in assuring the sustained release of the peptide from the absorptive epithelium towards the bloodstream. In the same way, sCT-loaded Chitosan-coated lipid nanocarriers for therapeutic applications  272 5. Main remarks CS-coated lipid nanostructures are a versatile tool suitable for encapsulating lipophilic or hydrophilic drugs with a considerable efficiency and capable of exhibiting sustained-release functions at the site of delivery or action. These CS lipid nanocarriers have shownpotential as transmucosal carriers for the delivery of large complex molecules and also as intracellular drug delivery vehicles. Besides the capacity of these nanocarriers to protect sensitive molecules in their lipid core, the presence of the CS coating has been identified as a critical parameter for their efficacy. This coating is responsible for the interaction of the nanostructures with the epithelial and cellular barriers. More specifically, these interactions are known to favor the residence of the nanostructures in the intestinal epithelium and facilitate the rapid uptake into the cancer cell lines. A proof-of-principle has been described for drugs such as salmon calcitonin and docetaxel. 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J. - Abraxane, a novel Cremophorfree, albuminbound particle form of paclitaxel for the treatment of advanced nonsmall-cell lung cancer. - Ann. Oncol., 17, 1263-1268, 2006. 70. De Campos A.M., Sanchez A., Gref R., Calvo P., Alonso M.J. - The effect of a PEG versus a chitosan coating on the interaction of drug coloidal carriers with the ocular mucosa. - Eur. J. Pharm. Sci., 20, 73-81, 2003. 71. Lozano M.V., Torecilla D., Lallana E., Vidal A., Fernandez-Megía E., Riguera R., Dominguez F., Alonso M.J., Torres D. – Chitosan Nanocapsules for active tumor targeting. - Proceedings of the 7th World Meeting on Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology, Malta, 8-11 March 2010. Acknowledgements The studies reported in this review were performed in our laboratory and have been supported by grants from the Ministry of Science and Technology (Consolider Nanobiomed, CSD 2006-00012) and the Xunta de Galicia (PGIDIT 08CSA045209PR), Spain. Felipe Oyarzun-Ampuero was granted a CONICYT scholarship. Manuscript Received 7 April 2010, accepted for publication 28 May 2010.   Anexo 2: Artículo relacionado A new drug nanocarrier consisting of polyarginine and hyaluronic acid F. A. Oyarzun-Ampueroa, F.M. Goycooleaa, D. Torresa, M.J. Alonsoa Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy 15782, University of Santiago de Compostela, Spain. Adapted from: European Journal of Pharmaceutics and Biopharmaceutics. (2011). 79(1): 54-57. A new drug nanocarrier consisting of polyarginine and hyaluronic acid  288 Netherlands). The samples were stained with 1% (w/v) phosphotungstic acid for 10 s, immobilized on copper grids with Formvar® and dried overnight before TEM analysis. Stability of nanoparticles Nanoparticle formulations were prepared and centrifuged in the presence of glycerol. The stability of the nanoparticles was evaluated according to size and precipitation in phosphate-buffered saline (PBS, pH 7.4) at 37 °C and in MilliQ water at 4 °C. The composition of PBS was as follows: 137 mM NaCl, 2.7 mM KCl, 1.4 mM NaH2PO4 and 1.3 mM Na2HPO4. Results and discussion Table 1a and b show the mass ratio, charge [HA]/[PArg] ratio, size, polydispersity index and zeta potential of the tested formulations prepared with either HMWHA or LMWHA, respectively. Our data show that when the [HA]/[PArg] charge ratio was lower than 0.975, nanostructures with a positive zeta potential were obtained, indicating that the surface of these systems is mainly composed of positively charged PArg. This feature is attributed to excess of PArg (relative to that of HA) in the formulation. Consequently, when the [HA]/[PArg] charge ratio increased to 0.975 and higher, inversion of the zeta potential values was observed, indicating that the nanocarrier surface was now shielded by excess of HA, which bears a negative charge. It can be appreciated that the zeta potential values (and average size) are not further modified beyond this ratio. This indicates that HA is incorporated into the nanoparticles up to saturation limit while surplus HA remains unassociated in solution and is in agreement with yield studies (data not shown). Anexo 2  289 Table 1: Physicochemical properties of the nanocarriers prepared with different ratios of HMWHA-PArg (a) or LMWHA-PArg (b) and evaluated in MilliQ water. (mean ± S.D., n=3). a) Mass ratio HMWHA- PArg Charge ratio [HA]/[PArg] Size (nm) Polydispersity index Zeta potential (mV) 2-2.4 0.325 128 ± 8 0.2-0.3 +31.3 ± 1 4-2.4 0.65 136 ± 16 0.1-0.2 +25.3 ± 4 6-2.4 0.975 154 ± 7 0.1-0.2 -32.5 ± 5 8-2.4 1.3 150 ± 7 0.1-0.2 -33.4 ± 4 10-2.4 1.625 147 ± 7 0.1-0.2 -35.9 ± 5 12-2.4 1.95 155 ± 7 0.1-0.2 -33.8 ± 4 b) Mass ratio LMWHA- PArg Charge ratio [HA]/[PArg] Size (nm) Polydispersity index Zeta potential (mV) 2-2.4 0.325 Not formed Not formed Not formed 4-2.4 0.65 131 ± 26 0.1-0.2 +25 ± 1 6-2.4 0.975 172 ± 18 0.1-0.2 -19 ± 1 8-2.4 1.3 139 ± 9 0.1-0.2 -27 ± 3 10-2.4 1.625 137 ± 13 0.1-0.2 -31 ± 3 12-2.4 1.95 146 ± 13 0.1-0.2 -33 ± 3 Alteration of the surface charge of the nanocarriers as a function of the polymer ratio allows optimization of the surface composition (and, presumably, the biological behavior) to interact with targets that have affinity for PArg or HA. Additionally, knowledge of the relative contribution of each charged species to the nanosystems may allow nanoparticle customization for the incorporation of positively or negatively charged drug molecules. A new drug nanocarrier consisting of polyarginine and hyaluronic acid  290 The transmission electron micrographs indicated that each formulation was reasonable spherical, which is in agreement with previous works describing a spherical shape of nanoparticles made with either natural or synthetic polymers [3]. Figure 1 shows a micrograph of a formulation with a [HMWHA]/[PArg] charge ratio of 1.3. Figure 1: Transmission electron micrograph of HMWHA-PArg nanocarriers; HA-PArg charge ratio= 1.3. Importantly, characterization of HMWHA-containing systems was conducted after isolation of the nanocarriers by centrifugation, whereas characterization of LMWHA-containing systems was performed without isolation. The reason for that was because the latter nanoparticles were unstable during the centrifugation process (the lower tested conditions of centrifugation were 500g during 30 min) as evidenced by the disappearance of the turbidity of the system and by photon correlation spectroscopy (PCS) size measurements. The systems containing HMWHA were effectively isolated at 16000g during 30 min, maintaining the same characteristics that the non-isolated formulations had. As shown in Table 1a and b, at a charge ratio of 0.325, nanosystems were obtained that incorporated HMWHA, whereas those containing LMWHA were not formed. In addition, the ϛ values Anexo 2  291 of the LMWHA-containing systems were generally lower than those containing HMWHA systems. Thus, fewer charged species were located on the surface of the low-molecular-weight polymer surface. Greater charge compensation for systems that have low-molecular-weight species may also account for the lower ϛ values. Hence, we postulate that the observed differences among nanostructures comprising HMWHA or LMWHA are probably due to the preferential organization of the polymers, particularly at the nanoparticle surface. Variance in particle assembly may also account for the instability of LMWHA nanosystems during centrifugation. The formation of local regions with a large number of consecutive associated residues (i.e., greater cooperativity) of both polyelectrolytes in the HMWHA-containing systems may explain its superior stability during centrifugation in comparison with LMWHA-containing systems, which presumably have lower cooperativity. These regions, in which charges are compensated, become more hydrophobic due to their neutral character and, hence, they are expected to lie within the inner core of the nanostructure. This distribution would provide the systems with more stability. A similar interpretation was offered in a previous work studying the behavior of hybrid nanoparticles of chitosan and alginate (of various molecular weight) cross-linked with tripolyphosphate [10]. It is important to point out that the ability to isolate formulations by simple centrifugation avoids tedious time-consuming procedures, such as those performed by Kim et al. [9], whose developed nanosystems were isolated by dialysis over 2 days. Colloidal stability under physiological conditions is a crucial characteristic for successful biomedical application of the nanosystems. Therefore, we investigated the stability of the systems composed of HMWHA and LMWHA in PBS at pH 7.4 and 37 °C. Figure 2 shows the stability profiles of the HMWHA-containing systems. The majority of the formulations were stable for at least 2 h, indicating that the stability of the A new drug nanocarrier consisting of polyarginine and hyaluronic acid  292 nanosystems was maintained independent of their surface composition (PArg or HMWHA). The stability profiles of each LMWHA-containing formulation and those of 0.65 charge-ratio containing HMWHA are not shown in the plot because they either immediately aggregated after addition to the medium or were larger than ~1000 nm. Differences in the stability of HMWHA- and LMWHA-containing nanostructures may also be related to the specific organization of HA and PArg on the nanoparticle surface. This would affect the colloidal stability conferred by charge repulsion and steric hindrance. In the case of the formulation of charge ratio 0.65 with HMWHA, its instability could be attributed that this formulation shows the lowest magnitude in zeta potential before the point of charge inversion, thus effectively perturbing the stability of the systems mediated by charge repulsion. Importantly, in Figure 2 it is possible to appreciate that, from the beginning of the experiment (0 h), all the formulations showed an increased size compared with the original one evaluated in MilliQ water (Table 1). Considering that PBS presents a significant quantity of salt, this can weaken and dissociate the anionic interactions between the oppositely charged polymers leading to swelling of the systems. Additionally, the salt ions could diffuse inside the nanosystems and attracting, by osmotic forces, water inside the nanosystems also exerting a possible swelling of the formulations. Anexo 2  293 Figure 2: Stability profiles of HMWHA-PArg nanocarriers in phosphate-buffered saline pH 7.4 at 37°C; HA-PArg charge ratios: 0.325 (x), 0.975 (+), 1.3 (S), 1.625 ({) and 1.95 () (mean ± S.D., n=3). Finally, the stability of nanocarriers during long-term storage is an important step for the adequate handling of the formulations. Figure 3 shows the stability profiles of each formulation composed of HMWHA at 4 °C for 3 months. Every formulation was stable and showed no significant change in particle size. Each system composed of LMWHA was also stable during storage (data not shown). Figure 3: Stability profiles of HMWHA-PArg nanocarriers in water during storage at 4°C; HA-PArg charge ratios: 0.325 (x), 0.65 (), 0.975 (+), 1.3 (S), 1.625 ({) and 1.95 () (mean ± S.D., n=3). A new drug nanocarrier consisting of polyarginine and hyaluronic acid  294 As future work, we are planning to introduce macromolecular hydrophilic drugs into the nanocarriers whose final targets could be solid tumor cells (where CD-44 receptors are overexpressed), or mucosal surfaces (that can avidly interact with both hyaluronic acid and polyarginine). More concretely, oral peptide delivery, in which polyarginine has demonstrated to be very promising, will be explored. Conclusions In conclusion, a nanoparticulate system composed of PArg and HA was successfully prepared using an extremely mild process. Negatively and positively charged nanoparticle formulations with surfaces composed preferentially of HA or PArg, were obtained. Importantly, we demonstrated that the molecular weight of HA is a crucial determinant of formulation stability during mechanical isolation and in physiological conditions. This knowledge is useful not only for systems comprising polyarginine and hyaluronic acid but also for systems composed by other polymers. Further studies testing the potential of these systems as mucoadhesive nanocarriers for targeted drug delivery will be carried out, and the in vitro-in vivo behavior of these systems will be also evaluated. Acknowledgements The authors acknowledge the Spanish Government for financial support (Consolider-Ingenio CSD 2006-00012 and Xunta de Galicia PGIDIT 08CSA045209PR) and CONICYT for a scholarship to F.A. Oyarzun- Ampuero. Anexo 2  295 References 1. M. de la Fuente, N. Csaba, M. Garcia-Fuentes, M.J. Alonso, Nanoparticles as protein and gene carriers to mucosal surfaces, Nanomed. 3 (2008) 845-57. 2. V.M. Platt, F.C.Jr. Szoka, Anticancer therapeutics: targeting macromolecules and nanocarriers to hyaluronan or CD44, a hyaluronan receptor, Mol. Pharm. 5 (2008) 474-86. 3. F.A. Oyarzun-Ampuero, J. Brea, M.I. Loza, D. Torres, M.J. Alonso, Chitosan-hyaluronic acid nanoparticles loaded with heparin for the treatment of asthma, Int. J. Pharm. 381 (2009) 122-129. 4. Lundberg M.; Wikström S.; Johansson M. (2003). Cell Surface Adherence and Endocytosis of Protein Transduction Domains. Mol. Ther. 8 (2003) 143-150. 5. V.P. Torchilin, TAT peptide-mediated intracelular delivery of pharmaceutical nanocarriers, Adv. Drug Deliv. Rev. 60 (2008) 548-58. 6. K. Lingnau, K. Riedl, A. von Gabain, IC31® and IC30, novel types of vaccine adjuvant based on peptide delivery systems, Expert Rev. Vaccines. 6 (2007) 741-746. 7. Z. Miklán, E. Orbán, G. Csík, G. Schlosser, A. Magyar, F. Hudecz, New daunomycin-oligoarginine conjugates: synthesis, characterization, and effect on human leukemia and human hepatoma cells, Biopolymers. 92 (2009) 489-501. 8. M. Miyamoto, H. Natsume, S. Iwata, K. Ohtake, M. Yamaguchi, D. Kobayashi, K. Sugibayashi, M. Yamashina, Y. Morimoto, Improved nasal absorption of drugs using poly-L-arginine: effects of concentration and molecular weight of poly-L-arginine on the nasal absorption of fluorescein isothiocyanate-dextran in rats, Eur. J. Pharm. Biopharm. 52 (2001) 21-30. A new drug nanocarrier consisting of polyarginine and hyaluronic acid  296 9. E.J. Kim, G. Shim, K. Kim, I.C. Kwon, Y.K. Oh, C.K. Shim, Hyaluronic acid complexed to biodegradable poly-L-arginine for targeted delivery of siRNA, J. Gene Med. 11 (2009) 791-803. 10. F.M. Goycoolea, G. Lollo, C. Remuñán-López, F. Quaglia, M.J. Alonso, Chitosan-alginate blended nanoparticles as carriers for the transmucosal delivery of macromolecules. Biomacromolecules. 10 (2009) 1736-1743. Anexo 3  297 Anexo 3 Patentes Solicitadas: M.J. Alonso, D. Torres, G. Rivera-Rodríguez, F. Oyarzún-Ampuero, G. Lollo, T. Gonzalo-Lázaro, M. García-Fuentes. Nanocápsulas con cubierta polimérica. Número de solicitud P201130015 (Oficina Española de Patentes y Marcas), fecha de recepción 10-01-2011. 