Protamine nanocapsules as carriers for oral peptide delivery

1 UNIVERSIDAD DE SANTIAGO DE COMPOSTELA FACULTAD DE FARMACIA DEPARTMENT OF PHARMACY AND PHARMACEUTICAL TECHNOLOGY UNIVERSITÉ CATHOLIQUE DE LOUVAIN FACULTY OF PHARMACY AND BIOMEDICAL SCIENCES Doctoral Thesis PROTAMINE NANOCAPSULES AS CARRIERS FOR ORAL PEPTIDE DELIVERY Lungile Nomcebo Thwala Santiago de Compostela, 2016 2 3 UNIVERSIDAD DE SANTIAGO DE COMPOSTELA FACULTAD DE FARMACIA DEPARTMENT OF PHARMACY AND PHARMACEUTICAL TECHNOLOGY UNIVERSITÉ CATHOLIQUE DE LOUVAIN FACULTY OF PHARMACY AND BIOMEDICAL SCIENCES Doctoral Thesis PROTAMINE NANOCAPSULES AS CARRIERS FOR ORAL PEPTIDE DELIVERY Lungile Nomcebo Thwala Santiago de Compostela, 2016 4 5 Dra. María José Alonso Fernández, Full Professor at the Department of Pharmaceutical Technology at the University of Santiago de Compostela, Spain Dra. Noémi Stefania Csaba, Assistant Professor at the Department of Pharmaceutical Technology at the University of Santiago de Compostela, Spain Dra. Véronique Préat, Full Professor at the Department of Pharmacy at the Université Catholique de Louvain, Belgium. Report: That the experimental dissertation entitled: “Protamine nanocapsules as carriers for oral peptide delivery” presented by Lungile Nomcebo Thwala was conducted under their supervision in the Department of Pharmacy and Pharmaceutical Technology at the University of Santiago de Compostela and in the Department of Pharmacy at the Université Catholique de Louvain, Belgium. Being completed, they authorize its presentation and evaluation by the assigned jury members. And for the record, they issue and sign the present certificate in Brussels, March 29th and Santiago de Compostela, March 31th 2016. Prof. María José Alonso Fernández Dr. Noemi Stefania Csaba Prof. Véronique Préa t 6 7 For my angels…..my parents “Simakadze ngumelusi wami, ngeke ngeswele lutfo” 8 9 Acknowledgements My deepest gratitude goes to my supervisors; Prof. Maria Jose Alonso, Prof. Veronique Préat, Prof. Noemi Csaba, and Dr. Ana Beloqui. Your patient guidance, encouragement and advice has been beyond sufficient to see me through this amazing but challenging journey. The great efforts and sleepless nights you put at improving this manuscript humbles me and I will forever be grateful. To my TRANSINT team; Irene, Niu, Matilde, Tamara (yes you will always be a part of us), Inma, Desiree and our latest member Lena, you guys are awesome, the moments when we spoke “siSwati” are just unforgettable. My labmates, Sara, Jorge, José Vincente, Ana Gonzalez, Natalia, Jose, Sonia, Ana Cadette, Carmen, Ana Oliveria, “Howl” (Hsu Wei-Hsin), Belen, Sofia, Carla, Fernando, Marta, Mariajo, Surasa and Andrea you guys carried me through each day; the laughter, the “Spanglish” and the Spanish food we shared and when it came to work and solving problems, you all are just geniuses and fierce. To Puri, Belen Cuesta and Rafa, your jobs made mine a lot more easy and above and beyond that you were extremely patient and kind to me, always went out of your way to make my days “facil”. You helped me tirelessly just to make sure my stay in Santiago was a pleasant one. You´re fantastic! I am indebted to my friend Belen Lopez and her kind family. Thank you for taking me in and welcoming me into your home, “my home”. You filled my days with warmth and joy and the weekends in Orense were just heaven. The sleepless nights Belen spent helping me with this manuscript are not taken lightly. I appreciate you and may God bless you abundantly. For my labmates at UCL, the adorable ADDB group; Tian, Laure, Kiran, Oriane, Aiswraya, Kevin, Alessander, Chiara, Dario, Pallavi, Bernard and our sweet Murielle. You guys made my “longish” stay in Brussels a total bliss. The work was intense but 16 17 Resumen in extenso 18 19 1. Introducción Según la Federación Internacional de Diabetes, 415 millones de personas en todo el mundo padecen diabetes y se espera que este número aumente hasta 642 millones en el año 2040 [1]. Se distinguen dos tipos principales de diabetes, denominadas tipo I y tipo II en función del origen y características de la enfermedad. Por un lado, la diabetes tipo I tiene su origen en una reacción autoinmune que provoca que el sistema de defensa del organismo ataque a las células beta pancreáticas productoras de insulina y como consecuencia, el organismo no produce la insulina necesaria. En el caso de la diabetes tipo II, el organismo es capaz de producir insulina, pero con el tiempo va desarrollando resistencia a esta hormona. A la larga, esta situación puede acabar desembocando en unos niveles de insulina insuficientes. Ambas causas, tanto la deficiencia de insulina como la resistencia a la misma, generan niveles elevados de glucosa en sangre, que generalmente son controlados mediante terapia de sustitución con insulina [2]. La insulina humana es una hormona (peso molecular de 5808 Daltons) compuesta por 51 aminoácidos organizados en dos cadenas polipeptídicas (A y B), unidas entre sí por puentes disulfuro. La estructura primaria de la insulina humana, con 21 aminoácidos en la cadena A y 30 en la cadena B, se representa en la Figura 1A [3]. Esta hormona juega un papel fundamental en la regulación de los niveles de glucosa en sangre. La elevación de la glucemia se produce como consecuencia de la absorción intestinal de glucosa a partir de la degradación de hidratos de carbono de la dieta, como el almidón o la sacarosa. Dicho aumento estimula la secreción de insulina del páncreas hacia el torrente sanguíneo y la actividad biológica de la misma se inicia cuando ésta se une a sus receptores celulares de membrana, desencadenando así distintas cascadas de activación proteica (Figura 1B). A través de ellas, esta hormona promueve la absorción, la utilización, el almacenamiento y el transporte de glucosa desde la sangre hacia las células de los tejidos-diana, como hígado, músculo esquelético o tejido adiposo (Figura 1B) [4]. La terapia actual, a base de insulina, es eficaz en cuanto a que consigue una reducción de la glucemia en pacientes diabéticos, pero su utilización se ve restringida debido a la necesidad de ser administrada mediante inyección. Además, la administración 20 parenteral de insulina evita su paso por el hígado introduciéndola directamente en el torrente sanguíneo, lo que a la larga puede provocar un estado de hiperinsulinemia asociada a hipertensión periférica, que finalmente puede traducirse en el desarrollo de aterosclerosis, hipoglucemia y otros efectos metabólicos adversos [5]. Asimismo, los pacientes diabéticos tienen que soportar múltiples inyecciones diarias, con los consiguientes efectos indeseados tales como dolor, invasión de tejidos, posibles infecciones y daños nerviosos, lo que conduce a una baja adherencia al tratamiento. Como consecuencia, el uso de la insulina inyectable es claramente inferior al deseable y se cree queel desarrollo de terapias alternativas, menos invasivas, ha de ser un objetivo prioritario en la innovación farmacéutica [6]. Figura 1: A) Secuencia de aminoácidos de la insulina humana, B) Ilustración esquemática de los efectos de la insuficiente producción de insulina en la diabetes tipo I; ilustración de la función fisiológica de la insulina en la absorción y metabolismo de la glucosa (la insulina se une a su receptor, iniciando diversas cascadas de activación proteica incluyendo la translocación de GLUT-2 hacia la membrana plasmática, influjo de glucosa y síntesis de glucógeno). 21 1.2 El reto de administrar insulina por vía oral A pesar de los grandes avances en la tecnología asociada aldesarrollo de formulaciones inyectables, se estima que más del 5% de la población mundial continúa teniendo fobia a estos dispositivos. Asimismo, de entre todas las vías de administración no invasivas (oral, nasal, pulmonar, bucal y transdérmica), la oral es la preferida. Por ello, el desarrollo de formulaciones de péptidos que permitan la administración oral de los mismos es muy importante ya que cuando se trata de una terapia crónica, la administración parenteral desemboca en una baja adherencia al tratamiento, reduciendo así su eficacia [7]. La administración oral de insulina permitiría, además, el acceso de este fármaco a la circulación portal, alcanzando el hígado antes de llegar a circulación sistémica, de forma similar a lo que ocurre con la insulina secretada fisiológicamente por el cuerpo humano [8]. Sin embargo, la administración de insulina por vía oral sigue siendo un gran reto debido a las dificultades inherentes al desarrollo de una formulación destinada a este propósito. Los fármacos administrados por vía oral deben soportar las condiciones agresivas del estómago, estando expuestos a la degradación por parte de ácidos y de enzimas. En el entorno gastrointestinal el fármaco queda expuesto a la degradación por enzimas como las proteasas pancreáticas, entre las que se incluyen la tripsina, αquimotripsina, elastasa, exopeptidasas, carboxipeptidasas A y B. Los fármacos que resisten estas condiciones deben posteriormente atravesar la capa de mucus que recubre la superficie de absorción del tracto gastrointestinal (TGI) y pasar a través de las células epiteliales para lograr acceder al torrente sanguíneo. Como la insulina es una molécula peptídica, puede ser fácilmente degradada a su paso por el TGI debido a las condiciones hostiles del mismo, lo que conlleva que su biodisponibilidad oral se vea ampliamente disminuida. Además, su elevado peso molecular (sobre 6 kDa), su carga y su hidrofilicidad limitan su absorción por las vías paracelular y transcelular [9]. Se han explorado distintas estrategias para mejorar la biodisponibilidad oral de la insulina, entre las que se incluyen su modificación química, su co-administración con promotores de la absorción y/o inhibidores enzimáticos y su incorporación en sistemas de liberación [10]. En esta línea, la nanotecnología ha mostrado resultados prometedores en el diseño de nanotransportadores con potencial para la administración oral de péptidos, por lo que se espera que la investigación en este área 22 consiga mejorar sustancialmente el diagnóstico y tratamiento de la diabetes en el futuro [11]. 1.3. Nanotransportadores para la administración oral de insulina La principal estrategia en el área de la nanotecnología para lograr la administración oral de péptidos y proteínas va dirigida a conseguir la encapsulación de los mismos en vehículos de tamaño nanométrico (10-1000 nm) que los protejan frente a la degradación, controlen su liberación en los tejidos diana y faciliten su transporte transepitelial. Los nanosistemas utilizados para el transporte de insulina incluyen nanopartículas, nanocápsulas, micelas y liposomas (Figura 2) [13], entre otros. Algunos de estos nanotransportadores han sido ampliamente estudiados para la administración oral de insulina, llegando incluso a ensayos clínicos, tema analizado en detalle en el capítulo 1. Características como el tamaño, la carga superficial y la hidrofobicidad/hidrofilicidad de las partículas han sido identificadas como factores determinantes para lograr su internalización en las células del organismo [13, 14]. La modificación de estas propiedades de superficie puede conseguirse, bien a través de un recubrimiento de los nanosistemas con polímeros biodegradables hidrofílicos, o bien a través de la inclusión de surfactantes adicionales en la formulación. Por ejemplo, debido a sus propiedades estabilizantes, se ha utilizado ampliamente el polietilenglicol (PEG) como material de recubrimiento o como componente de la matriz en nanosistemas cargados con insulina. Esto se debe a que las cadenas de PEG forman una barrera estérica en la superficie de las nanopartículas que incrementa su estabilidad frente a proteasas, previene su opsonización y reduce su inmunogenicidad [15]. También ha sido demostrado que en algunos casos la combinación de ambas estrategias (el uso de polímeros y de surfactantes en el mismo sistema) podría llevar a una potenciación de sus propiedades y, consecuentemente, a un aumento de la biodisponibilidad oral del péptido encapsulado [7]. 23 Figura 2: Diferentes tipos de nanotransportadores para liberación de insulina. 1.4 La protamina como biomaterial en el desarrollo de nanotransportadores de fármacos De entre los diferentes biomateriales explorados, los poliaminoácidos con capacidad de penetrar en las células constituyen una estrategia prometedora para la administración oral de insulina [16].Las principales razones son su capacidad para interactuar con estas moléculas, reducir su degradación enzimática y promover su absorción oral [17], propiedades que comparten con los péptidos de penetración celular (CPPs). Morishita et al. fueron pioneros a la hora de mostrar el potencial de las oligoargininas como CPPs, para mejorar la absorción de insulina a través de la pared intestinal [18] través de membranas mucosas, logrando así alcanzar niveles muy efectivos de biodisponibilidad [19]. La adsorción electrostática de la octarginina a los proteoglicanos presentes en la superficie celular y su subsecuente internalización han sido asociadas con su efecto promotor en la absorción de insulina en mucosas. Por otro lado, la protamina es una proteína nuclear de pequeño tamaño rica en arginina, que juega un papel esencial en la condensación nuclear de los espermatozoides y en la estabilización del ADN [20]. Una vez combinada con la insulina, la protamina retrasa el inicio la actividad biológica de esta hormona, además de aumentar la duración de su actividad (ver insulina NPH). Debido a su capacidad de natural para condensar ácidos nucleicos, la protamina también es un biomaterial atractivo y ampliamente utilizado en aplicaciones de terapia génica [22], [23]. Nuestro grupo de investigación tiene experiencia en el empleo de biomateriales ricos en arginina, como poliarginina [24] y protamina [25] en el diseño de sistemas de liberación de fármacos con capacidad para transportar distintas moléculas terapéuticas. En particular, la protamina ha sido inicialmente utilizada en la preparación de 24 nanopartículas y nanocápsulas, demostrando gran potencial para interactuar con células y promover la internalización de antígenos [25]. Asimismo, nanopartículas multicapa (técnica “layer-by-layer”) han sido también desarrolladas para la coencapsulación y liberación de agentes inmunoestimulantes y antígenos, utilizando una combinación de poliarginina y protamina con polisacáridos como el sulfato de dextrano o el alginato [26]. Nuestro grupo también ha diseñado recientemente nanocápsulas de protamina con estructura lipídica de tipo núcleo-cubierta, con núcleo oleoso de vitamina E, para la encapsulación de curcumina como molécula antiinflamatoria. Con este sistema se logró aumentar de forma significativa la permeabilidad de este fármaco en células Caco-2 [27]. En todos los casos, los efectos positivos observados con la utilización de protamina se atribuyen a la presencia de una secuencia repetitiva de arginina en su estructura (sobre el 70%) (Figura 3), la cual otorga a este polipéptido una eficiente capacidad de translocación a través de membranas biológicas, incluyendo las células epiteliales intestinales [28]. Se sabe que esta capacidad de translocación se produce principalmente por endocitosis, como consecuencia de la interacción entre las unidades de arginina cargadas positivamente y las cargas negativas presentes en la superficie de las membranas [29]. Figura 3: Representación esquemática de la estructura convencional de la protamina, conteniendo los aminoácidos prolina, arginina, serina, valina y glicina. 2. Hipótesis En base a las evidencias aportadas por estudios anteriores, se han formulado las siguientes hipótesis: 1. La protamina es un biomaterial prometedor a la hora de desarrollar 25 nanocápsulas para una eficaz administración oral de macromoléculas como la insulina. La capacidad de este polipéptido para translocarse a través de membranas celulares podría verse potenciada al combinarlo con otros promotores de la penetración celular (lípidos y surfactantes), aumentando así la absorción intestinal de la insulina. 2. La combinación racional de protamina con polímeros estabilizadores y mucopenetrantes, como el poliácido siálico (PSA) y derivados del PEG, podría conducir al desarrollo de un nanosistema optimizado capaz de asociar el péptido, protegerlo de la degradación enzimática y transportarlo a través de la barrera epitelial. 3. Objetivos El principal objetivo de este trabajo es el diseño racional y desarrollo de un nuevo nanosistema basado en la combinación de protamina, con otros biomateriales seleccionados para incrementar sus propiedades promotoras de la absorción y mejorar su estabilidad. Tomando la insulina como péptido modelo, el objetivo último de este nanosistema es conseguir una eficaz administración oral de péptidos. Con este fin, el objetivo global se divide en los siguientes objetivos específicos: 1. Desarrollo y caracterización in vitro de nanocápsulas de protamina cargadas con insulina. 2. Evaluación de los mecanismos de acción in vitro y de la toxicidad de las nanocápsulas de protamina en células epiteliales. 3. Evaluación de la interacción de las nanocápsulas de protamina con la barrera epitelial, incluyendo su biodistribución y eficacia in vivo. 4. Resultados y discusión Las nanocápsulas de protamina fueron preparadas mediante la técnica de desplazamiento de disolvente [8]. Se incorporó ácido polisiálico (PSA) como cubierta externa a través de interacciones iónicas con la protamina, confirmando su presencia con la inversión de la carga superficial de los nanosistemas (Tabla 1, Figura 4). Las nanocápsulas de protamina, recubiertas o no con PSA, presentaron un tamaño de 300400 nm y una buena capacidad para asociar insulina. 32 [18] M. Morishita, N. Kamei, J. Ehara, K. Isowa, and K. Takayama, “A novel approach using functional peptides for efficient intestinal absorption of insulin,” J. Control. Release, vol. 118, no. 2, pp. 177–184, 2007. [19] N. Kamei, M. Morishita, J. Ehara, and K. Takayama, “Permeation characteristics of oligoarginine through intestinal epithelium and its usefulness for intestinal peptide drug delivery.,” J. Control. Release, vol. 131, no. 2, pp. 94–9, Oct. 2008. [20] F. Reynolds, R. Weissleder, and L. Josephson, “Protamine as an efficient membrane-translocating peptide,” Bioconjug. Chem., vol. 16, no. 5, pp. 1240– 1245, 2005. [21] E. Pharmacopoeia, I. Units, L. The, C. Add, D. Heat, T. S. S. Dissolve, D. Prepare, and D. Protamine, “PROTAMINE SULPHATE Protamini sulfas Protirelinum,” Test, vol. 85, pp. 2334–2335. [22] K. Cornetta and W. F. Anderson, “Protamine sulfate as an effective alternative to polybrene in retroviral-mediated implications for human gene therapy,” October, vol. 23, pp. 187–194, 1989. [23] F. L. Sorgi, S. Bhattacharya, and L. Huang, “Protamine sulfate enhances lipidmediated gene transfer.,” Gene Ther., vol. 4, no. 9, pp. 961–968, 1997. [24] F. a Oyarzun-Ampuero, F. M. Goycoolea, D. Torres, and M. J. Alonso, “A new drug nanocarrier consisting of polyarginine and hyaluronic acid.,” Eur. J. Pharm. Biopharm., vol. 79, no. 1, pp. 54–7, Sep. 2011. [25] J. V. González-Aramundiz, M. P. Olmedo, Á. González-Fernández, M. J. A. Fernández, and N. S. Csaba, “Protamine-based nanoparticles as new antigen delivery systems.,” Eur. J. Pharm. Biopharm., vol. 97, pp. 51–59, 2015. [26] J. F. Correia-Pinto, M. Peleteiro, N. Csaba, Á. González-Fernández, and M. J. Alonso, “Multi-enveloping of particulated antigens with biopolymers and immunostimulant polynucleotides,” J. Drug Deliv. Sci. Technol., vol. 30, pp. 424–434, 2015. [27] V. P. Ana Beloqui1, Patrick B. Memvanga1, 2, Régis Coco1, Sonia Reimondez-Troitiño3, 4, Mireille Alhouayek5, Giulio G. Muccioli5, María José Alonso3, Noemí Csaba3, María de la Fuente4, “A comparative study of curcumin-loaded lipid-based nanocarriers in the treatment of inflammatory bowel disease.,” Colloids Surfaces B Biointerfaces, 2016. [28] F. Reynolds, R. Weissleder, and L. Josephson, “Protamine as an efficient membrane-translocating peptide,” Bioconjug. Chem., vol. 16, no. 5, pp. 1240– 1245, 2005. [29] Y.-S. Choi, J. Y. Lee, J. S. Suh, Y.-M. Kwon, S.-J. Lee, J.-K. Chung, D.-S. Lee, V. C. Yang, C.-P. Chung, and Y.-J. Park, “The systemic delivery of siRNAs by a cell penetrating peptide, low molecular weight protamine.,” Biomaterials, vol. 31, no. 6, pp. 1429–43, Feb. 2010. [30] M. Morishita, T. Goto, N. A. Peppas, J. I. Joseph, M. C. Torjman, C. Munsick, K. Nakamura, T. Yamagata, K. Takayama, and A. M. Lowman, “Mucosal insulin delivery systems based on complexation polymer hydrogels: Effect of particle size on insulin enteral absorption,” J. Control. Release, vol. 97, no. 1, pp. 115–124, 2004. [31] N. Reix, A. Parat, E. Seyfritz, R. Van Der Werf, V. Epure, N. Ebel, L. Danicher, E. Marchioni, N. Jeandidier, M. Pinget, Y. Frère, and S. Sigrist, “In vitro uptake evaluation in Caco-2 cells and in vivo results in diabetic rats of insulin-loaded PLGA nanoparticles,” Int. J. Pharm., vol. 437, no. 1–2, pp. 213– 220, 2012. 33 [32] C. Damgé, C. Michel, M. Aprahamian, P. Couvreur, and J. P. Devissaguet, “Nanocapsules as carriers for oral peptide delivery,” J. Control. Release, vol. 13, no. 2–3, pp. 233–239, 1990. [33] C. Damgé, P. Maincent, and N. Ubrich, “Oral delivery of insulin associated to polymeric nanoparticles in diabetic rats.,” J. Control. Release, vol. 117, no. 2, pp. 163–70, Feb. 2007. [34] C. Damge, C. Michel, M. Aprahamian, and P. Couvreur, “New approach for oral administration of insulin with polyalkylcyanoacrylate nanocapsules as drug carrier,” Diabetes, vol. 37, no. 2, pp. 246–251, 1988. 34 35 INTRODUCTION Oral nanomedicines for effective insulin delivery 36 37 1. Diabetes therapy Diabetes mellitus (DM) is a chronic condition that occurs when the body cannot produce enough insulin or cannot use insulin. DM is considered a metabolic disorder since reduced or inactive insulin results in imbalanced food metabolism, characterized by the accumulation of glucose in the blood (known as hyperglycaemia). The resulting high levels of glucose in the blood causes damage to many tissues in the body, and with progression of the disease pathological changes like nephropathy, retinopathy and cardiovascular complications start occurring in the body [1, 2]. According to the International Diabetes Federation, 415 million people worldwide are diagnosed with diabetes and this number is expected to rise to 642 million by 2040 [1]. There are two main types of diabetes: type I and type II. Type 1 diabetes is caused by an autoimmune reaction, in which the body’s defense system attacks the insulinproducing beta cells in the pancreas. As a result, the body does not produce the insulin it needs. In type II diabetes, the body is able to produce insulin but develops resistance over time so that insulin levels may subsequently become insufficient. Both the insulin resistance and deficiency lead to high blood glucose levels. The primary goal for the treatment of type I and type II diabetes is to cure the symptoms related to hyperglycemia. Type 1 is generally monitored through insulin replacement therapy [3]. 2. Advances in insulin therapy Human Insulin is a hormone (molecular weight of 5808 Daltons) composed of 51 amino acids arranged into two polypeptide chains (an A chain and a B chain), which are linked by disulfide bonds. The primary structure of human insulin, which contains 21 amino acid residues in chain A and 30 amino acid residues in chain B, is shown in (Figure 1A) [4]. Insulin plays a key role in the regulation of blood glucose levels. The intestinal absorption of glucose generated from the degradation of dietary carbohydrates including starch or sucrose, leads to elevated blood glucose levels, which stimulates the secretion of insulin from the pancreas into the blood stream. The biological actions of insulin are initiated when insulin binds to its cell surface receptor, which is followed by many protein activation cascades (Figure 1B) through which, insulin promotes the transport, intake, utilization and storage of glucose from the blood to the cells in the target tissues, such as liver, skeletal muscles and adipose tissue (Figure 1B) [5]. 38 Figure 1: a) Amino acid sequence of human insulin, b) Schematic illustration of the physiological function of insulin on glucose uptake and metabolism. [Insulin binds to its receptor (1), which starts protein activation cascades (2) including translocation of GLUT-2 to the plasma membrane and influx of glucose (3), and glycogen synthesis (4)], c) Schematic illustration of the effects of insufficient insulin production in type 1 diabetes. 2.1. Injectable insulin Current administration of insulin for diabetes treatment is almost entirely via subcutaneous injection and different insulin formulations have their unique pharmacokinetics. Human insulin preparations on the market can classified as (i) rapid-acting insulin: insulin lispro (Humalog®), insulin aspart (Novolog®), and insulin glulisine (Apidra®), (ii) regular or short-acting insulin: Humulin®R and Novolin®R (iii) intermediate-acting insulin: Neutral Protamine Hagedorn (NPH) insulin (Humulin N®), and Lente insulin, (iv) long-acting insulin: Ultralente, insulin glargine (Lantus®) and insulin detemir (Levemir®) (Figure 2) [4]. Rapid-acting insulins are inherited from the human insulin and characterised by a modification in the B chain e.g. for insulin lispro, the B28 (proline), B29 (lysine) amino acid sequence is reversed to be lysine-proline. In insulin aspart, the B28 amino acid proline is substituted with aspartic acid and finally for insulin glulisine (Apidra®) the amino acid aspargine at position B3 is replaced by lysine and the lysine in position B29 is replaced by glutamic acid. These modifications induce rapid dissociation of hexamers into dimers and monomers after injection. This rapid dissociation leads to rapid absorption after administration. Their onset peaks are sooner and they have 39 short durations compared to regular human insulin. Regular or short-acting insulin, on the other hand, is the basic human insulin without any modifications, (depicted in Figure 1A). It reaches the bloodstream within 30 minutes after injection, peaks at 2 - 3 hours after injection and is effective for approximately 3 to 6 hours [6]. The formulation Neutral Protamine Hagedorn (NPH) on the other hand, is a suspension of crystalline zinc-insulin combined at neutral pH with a positively charged, arginine-rich polypeptide, protamine. Its duration of action is intermediate due to delayed absorption of the insulin because of its conjugation with protamine, forming a less soluble complex. NPH blood absorption begins 1.5 h after subcutaneous injection; it has a peak plasma concentration at 4 to 12 h and disappears within 24 h (Figure 2) [4]. Long-acting insulin analogs attempt to replicate the body's basal insulin secretion. Insulin glargine consists of two modifications to human insulin. First, two arginines are added to the C-terminus of the B chain shifting the isoelectric point of the insulin from a pH or 5.4 to 6.7, making the insulin more soluble at an acidic pH. Secondly, the asparagine at position A21 is replaced by glycine. This substitution prevents deamidation and dimerisation that would occur with acid-sensitive asparagine. Insulin glargine is formulated at a pH of 4.0 and when it is injected into subcutaneous tissue (pH 6.8) the acidic solution is neutralized and microprecipitates of insulin glargine are formed, from which small amounts of insulin are released throughout a 24-hour period, resulting in a low level of insulin throughout the day (Figure 2) [3]. Insulin detemir, on the other hand, is a long acting insulin analog in which the B30 amino acid is omitted and a C14 fatty acid chain (myristic acid) is bound to the B29 lysine amino acid. It is slowly absorbed due to its strong association with albumin in the subcutaneous tissue and when it reaches the bloodstream it binds to albumin again, delaying its distribution to the peripheral tissues [7]. 40 Figure 2: Idealized activity profiles of human insulin and analogues. Adapted with permission from [7]. The current therapy with insulin is effective at lowering blood glucose in patients with diabetes, but its use is constrained by the need to be injected subcutaneously and because of the concerns regarding interference with patients’ lifestyle, risk of hypoglycemia. Parenteral administration bypasses the liver and goes directly into blood circulation leading over time to peripheral hyperinsulinemia, associated with peripheral hypertension, the development of atherosclerosis, cancer, hypoglycaemia and other adverse metabolic effects [2]. Moreover, diabetic patients have to endure multiple injections a day i.e. rapid acting insulin before meals to meet prandial insulin secretion and a supplemental injection of long acting insulin to mimic basal secretion levels. These repeated injections may bring about pain, tissue invasion, infections and nerve damage, which lead to poor patient compliance. Therefore, less invasive options for insulin therapy (and other similar peptides) are highly desirable and represent a priority objective in pharmaceutical innovation [8]. 2.2. Oral insulin Despite great improvements in needle technology, it is still estimated that >5% of the population are needle-phobic. Amongst all the alternative, noninvasive routes of administration (nasal, pulmonary, buccal and transdermal), oral insulin delivery remains the most preferred. Developing oral peptide formulations is important because parenteral administration by patients over a chronic period results in poor compliance thereby curtailing efficacy [9]. For patients, oral insulin would be pain 41 and stress free and this route would allow for the insulin to enter the portal circulation, reaching the liver before entering the systemic circulation, which resembles the pathway of physiologically secreted insulin [10]. Nevertheless, this ideal insulin delivery route has so far been elusive due to the difficulties faced in designing such a formulation. Orally administered drugs need to survive harsh gastric conditions in which they are exposed to acidic and enzymatic degradation. Subsequently, intestinal conditions further expose the drugs to a myriad of digestive enzymes; pancreatic proteases consisting of the serine endopeptidase (trypsin, α-chymotrypsin, elastase and exopeptidases, carboxypeptidases A and B), responsible for the degradation of proteins. The drugs that survive these conditions then need to penetrate the mucus layer overlying the absorptive surfaces of the gastrointestinal tract (GIT) and pass through the epithelial cells to enter the blood stream. Insulin, being a peptide molecule, is easily degraded by the conditions in the GIT, which significantly delimitates its oral bioavailability. Furthermore, the large molecular size (about 6 KDa), its charge and its hydrophilicity all preclude insulin absorption by paracellular and transcellular route [11]. To improve the bioavailability of insulin, different approaches have been explored, including chemical modification, co-administration with absorption enhancers and/or enzyme inhibitors and incorporation into carriers [12]. Nanotechnology has shown promising results in the design of potential oral peptide nanocarriers, and it is expected to lead to further improvement in the diagnosis and treatment of diabetes [13]. Nanoparticles have been engineered for the oral delivery of labile proteins and peptides for release into the systemic circulation [14]. 3. Nanocarriers for oral insulin delivery For insulin delivery the general idea is to encapsulate the peptide into carriers at a nanoscale range (10 – 1000 nm), allowing its protection from degradation, controlled release at target sites and transepithelial transport. Nanocarriers used for insulin delivery could broadly be categorized into: nanoparticles [15] (matrix-type particles), nanocapsules [16, 17], micelles [18] and liposomes [19] (Figure 3). Recently, a number of insulin nanocarriers for oral delivery have been investigated and a few have undergone clinical trials. This issue has been analyzed in detail in chapter 1. 48 118:177–184. 27. Kamei N, Morishita M, Ehara J, Takayama K (2008) Permeation characteristics of oligoarginine through intestinal epithelium and its usefulness for intestinal peptide drug delivery. J. Control Release 131:94–9. 28. Reynolds F, Weissleder R, Josephson L (2005) Protamine as an efficient membrane-translocating peptide. Bioconjug. Chem 16:1240–1245. 29. Pharmacopoeia E, Units I, The L, et al PROTAMINE SULPHATE Protamini sulfas Protirelinum. Test 85:2334–2335. 30. Cornetta K, Anderson WF (1989) Protamine sulfate as an effective alternative to polybrene in retroviral-mediated implications for human gene therapy. October 23:187–194. 31. Sorgi FL, Bhattacharya S, Huang L (1997) Protamine sulfate enhances lipidmediated gene transfer. Gene Ther. 4:961–968. 32. Oyarzun-Ampuero F a, Goycoolea FM, Torres D, Alonso MJ (2011) A new drug nanocarrier consisting of polyarginine and hyaluronic acid. Eur. J. Pharm. Biopharm. 79:54–7. 33. González-Aramundiz JV, Olmedo MP, González-Fernández Á, et al (2015) Protamine-based nanoparticles as new antigen delivery systems. Eur. J. Pharm. Biopharm. 97:51–59. 34. Correia-Pinto JF, Peleteiro M, Csaba N, et al (2015) Multi-enveloping of particulated antigens with biopolymers and immunostimulant polynucleotides. J. Drug Deliv. Sci. Technol. 30:424–434. 35. Beloqui A, Memvanga P, Coco R, et al (2016) A comparative study of curcumin-loaded lipid-based nanocarriers in the treatment of inflammatory bowel disease. Colloids Surfaces Biointerfaces. 36. Reynolds F, Weissleder R, Josephson L (2005) Protamine as an efficient membrane-translocating peptide. Bioconjug Chem 16:1240–1245. 37. Choi Y-S, Lee JY, Suh JS, et al (2010) The systemic delivery of siRNAs by a cell penetrating peptide, low molecular weight protamine. Biomaterials 31:1429–43. 49 CHAPTER 1 Emerging delivery platforms for mucosal administration of biopharmaceuticals 50 51 Abstract Protein and peptide based therapeutics are typically administered by injection due to their poor uptake when administered via non-parenteral routes. Despite the low frequency of clinical breakthroughs with non-invasive protein drug delivery this far, it remains an active research area with renewed interest not only due to its improved therapeutic potential, but also due to the attractive commercial outcomes it offers. Currently, a number of technologies are adopted, including mixtures of penetration enhancers with protease inhibitors and/or nanotechnology-based products are under clinical development. This review provides a critical overview of current strategies of non-invasive mucosal delivery routes for therapeutic proteins, with emphasis on their advantages and limitations. Selected new trends and interesting novel formulations in advanced preclinical and clinical development stages for the pulmonary, nasal and the oral route are discussed for the most relevant peptide and protein drugs in function of their specific requirements and intended therapeutic applications. 52 53 1. Introduction Proteins and peptides are building blocks of life and are now evolving as a very promising brand of therapeutic drugs. Once a rarely used subset of medical treatments, therapeutic proteins have increased dramatically in number and frequency of use since the introduction of the first recombinant protein therapeutic; human insulin, more than 33 years ago [1]. Peptides can bind large macromolecular targets with high potency and great selectivity, which translates into fewer off-target side effects and less potential for toxicity than conventional low molecular weight drugs [2]. Unlike other drug molecules, which often trigger side effects by producing toxic metabolites that accumulate in different organs, peptides simply degrade into amino acids, which minimizes the risk of toxicity [3]. These features arise from their macromolecular nature, which provides the structural complexity that is often required for specificity. However, this structural complexity also makes them some of the most challenging molecules to formulate and deliver. Loss of activity due to environmental factors such as moisture or temperature, during storage or in the body, puts a substantial burden on peptide formulation technologies. Whilst, the high molecular mass and hydrophilicity of these peptide/protein drugs creates delivery challenges, including substantial reduction in permeability across biological barriers and mucosal membranes. 2. Therapeutic proteins and peptides Since the 1980s, peptides have been studied as treatments for a wide variety of problematic diseases [4]. Recombinant technology has allowed the production of many potential peptide drugs at an acceptable cost, paving the way for the treatment of severe, chronic and life threatening diseases such as cancer, hypertension, arthritis, and metabolic diseases e.g. diabetes and obesity. Most of currently available peptide drugs are delivered by injection, but several biopharmaceuticals have undergone extensive research towards alternative administration routes, particularly peptide hormones, such as insulin, vasopressin, calcitonin and luteinizing hormone-releasing hormone (LHRH), and small proteins, such as human growth hormone and interferon α [5]. Peptide therapeutics research and development is dynamic, with increasing numbers of candidates entering clinical evaluation in a wide variety of therapeutic categories. 54 [1]. Overall, there is a notable increase in the number of successfully marketed peptides as compared to the 1980s and 1990s when only 2 % and 11 % of peptide therapeutics were marketed, respectively [8]. Peptides approved in 2013 alone, were notable for their number and diversity of use, with the top two therapeutic areas being oncology (21%) and metabolic diseases (15%) [2]. The clinical pipeline was composed of 128-peptide therapeutics, with 40 in phase I, 74 in phase II and 14 in phase III studies. The number of peptides and diversity of therapeutics areas represented was higher in phase II and I compared to phase III, which indicates an increase and expansion for therapeutic peptide research, with a steady growth of marketed peptides [7]. More than 15 candidates are currently in Phase 3 clinical studies or under regulatory review, which suggests that peptide therapeutic products will continue to be approved at an increasing rate in the near future [7]. This steady increase is an indication of the progress in the use of peptides as therapeutic drug. Injections (i.e. intravenous, intramuscular or subcutaneous route) remain the most common means for administering protein and peptide drugs. Patient compliance with drug administration regimens by any of these parenteral routes is generally poor and severely restricts the therapeutic value of the drug. Although, direct systemic delivery of these therapeutics overcomes the issue of absorption associated with other routes, other factors limit the chemical potency of peptide and protein therapeutics including: systemic proteases; rapid metabolism; opsonization; conformational changes; dissociation of subunit proteins; and non-covalent complexation with blood products [8]. Moreover, in the specific case of insulin delivery for example, diabetic patients, have to endure multiple injections a day i.e. rapid acting insulin before meals to meet prandial insulin secretion and a supplemental injection of long acting insulin to mimic basal secretion levels. These repeated injections may bring about pain, allergic reactions, infections and nerve damage, which lead to poor patient compliance. Therefore, less invasive options for insulin therapy (and other similar peptides) are highly desirable and represent a priority objective in pharmaceutical innovation [9]. 3. Mucosal delivery of biopharmaceuticals Owing to the above detailed limitations of injections, alternative delivery routes including; pulmonary, nasal, oral, vaginal and ocular, for biopharmaceuticals have been explored to increase patient compliance. These specific routes are collectively known as noninvasive mucosal routes of administration and each route offers unique 55 advantages and limitations [5]. Mucosal delivery of therapeutic peptides and proteins faces formidable barriers. These obstacles to efficient delivery can broadly be categorized as either the enzymatic barriers, which the peptide drug encounters after administration and the physical barriers, which prevent efficient transport of proteins and peptides across epithelial surfaces. Overall these barriers inevitably lead to low bioavailability [10, 11]. The primary barrier (chemical barrier) to mucosal absorption of peptide drugs is the enzymatic barrier in the form of degradation enzymes. Proteolytic enzymes are present all over the human body and they act by hydrolyzing the peptide bonds in polypeptides [12]. Hence, the peptide drugs may degrade, loose their functionality or aggregate prior to reaching the absorption membrane. These peptides are further subjected to digestion by aminopeptidases located in the cell membranes during their transportation through the epithelial layer of cells [13, 14]. The mucosa comprises the epithelial layer covered by a firmly adherent viscous layer of mucus and a less viscous, loosely adherent mucus layer on the surface. The mucus layer covering the epithelium represents the first physical barrier to be encountered by a drug prior to absorption and the thickness of the layer varies greatly throughout the body [15, 16]. Regardless of the localization, the function of the mucus is to lubricate and protect the underlying tissue [11]. The mucus clearance rate also varies between tissues and it has been estimated to be less than 30 minutes at different sites in the respiratory system and around 5 hours in the GI-tract. The detailed composition of mucus is dependent on the specific mucosal site, disease state as well as influenced by variance between individuals. Mucus contains approximately 95 % (w/w) water, 2-5 % (w/w) mucin proteins and a small amount of lipids and electrolytes [15, 16]. In addition to the physical diffusion barrier provided by the mucus network, the hydrophilic nature and the presence of a strong negative charge allow for drug interactions with the mucus components, which have also shown to retard or hinder diffusion and thereby limit drug absorption [17]. 56 Table 1: Physical properties and surface area of the different mucosal surfaces Nasal Pulmonary Oral Surface area 150 cm2 80-140 m2 >200 m2 Histological properties Thin mucus layer Ciliated, pseudostratified epithelium (10 μm) Thin mucus layer Bronchioles: Ciliated columnar pseudostratified epithelium (10-60 μm) Alveoli: squamous epithelial monolayer (<1μm) Thick mucus layer Columnar epithelial monolayer (10 μm) The morphology of the epithelial absorption barrier varies according to the specific anatomical site (Table 1). The nasal, lung and intestinal epithelia are commonly comprised of a monolayer of cells interconnected by tight junctions (Figure 1) [10]. The mechanism of transport through the epithelial barrier depends mainly on the physiochemical properties of peptide molecules. Common transport mechanisms in all sites include active and passive transport, which can be described by a combination of two processes; i) Paracellular transport: involves the transport of molecules via water filled pores or channels between cells. The paracellular route is restricted to relatively small hydrophilic molecules of <100-200 Da, by the presence of tight junctions or zonula occludins between epithelial cells [18]. Unfortunately, most therapeutic proteins have molecular weights much greater than 500 Da and hence exhibit low permeability. ii) Transcellular transport: involves the diffusion of drug molecules through the apical and basolateral membrane. This route is ideal for lipophilic drugs, which express relatively high affinity for the lipid bilayer of cell membranes. On the contrary, the transport of hydrophilic proteins/peptides through this path poses a significant challenge [19]. Transcytosis, is a specific type of transcellular transport i.e. the manner by which macromolecules or particles are taken up and transported across the cell to the basolateral side and it occurs through different energy dependent endocytic pathways (Figure 1) [22], [23]. Phagocytosis is used for uptake of large particles such as bacteria, and is generally the first step in the uptake and degradation of particles larger than 0.5 μm [22]. Clathrin-mediated endocytosis (CME) is the uptake and transport of particles from the cell 57 surface mediated by clathrin-coated vesicles (100 to 120 nm in diameter) to the basolateral side. Clathrin-coated vesicles (CCVs) are found in virtually all cells and cause the endocytosed material to end up in degradative lysosomes. Whilst caveolae-mediated endocytosis (CvME), involves the formation of small (50 to 100 nm) lipid raft-enriched, flask-shaped pits in the cell membrane surface. Unlike CME, CvME is a highly regulated process involving complex signaling, which may be driven by the cargo itself and it bypasses lysosomes [23]. Figure 1: Schematic representation of the different pathways employed by molecules to penetrate cells and cross the cell barrier into the systemic circulation. Tremendous efforts have been dedicated to improving the permeation of the proteins/peptides through the epithelial membranes whilst protecting them against enzymatic degradation. Hence the principal approaches for mucosal delivery include; the use of absorption enhancers [24] and protease inhibitors [25], modification or conjugation of peptides with biological entities that show stabilizing [25–27] and cellpenetrating capabilities [28, 29] and the design of multifunctional [30] and nanoparticulate drug delivery systems [31, 32] that help peptide trafficking across the fore-mentioned epithelial barriers and these strategies have been extensively review elsewhere. In recent years new delivery strategies including alternative administration routes integrated with emerging nanotechnologies have attracted increased attention in quest of shifting from the parenteral route of administration [33]. Alternative routes for 64 3.1.2. Nose-to-brain delivery Intranasal delivery provides a practical, non-invasive method of bypassing the bloodbrain barrier (BBB) to deliver therapeutic agents to the brain and spinal cord. This route allows drugs that do not cross the BBB to be delivered to the central nervous system within minutes. This is possible because of the unique connections that the olfactory and trigeminal nerves provide between the brain and external environment. A growing body of evidence suggests that insulin plays a role in cognitive processes and that insulin abnormalities may contribute to memory and brain changes associated with Alzheimer disease. Interestingly, the university of Washington recently demonstrated in phase II clinical trials, that regular insulin administered to the nasal cavity is transported within a few minutes into the brain, without affecting blood sugar or insulin levels [57]. A similar study is on going with insulin in phase II trials by Wake Forest School of Medicine [52]. Oxytocin is a small, naturally occurring peptide that acts mainly as a neuromodulator in the brain. Recently the effect of intranasal oxytocin on pain sensitivity and threshold has been evaluated. Participants received nasal sprays into each nostril of 4 Units up to 32 units of oxytocin prior to Thermal Evaluation System Testing and they reported lower pain intensity. This study (currently on phase IV clinical trials, Northwestern University) suggests that oxytocin might represent a novel, safe, and effective analgesic for acutely painful procedures [50, 51]. The university of Alabama at Birmingham is currently undergoing clinical trials for intranasal oxytocin for the treatment of pain associated with interstitial cystitis [58]. Although intranasal delivery does not necessarily require any modification to therapeutic agents the location of deposition of the drug product inside the nasal cavity may affect absorption rate. Hence modification of the administration technique to optimize droplet size, deposition fraction and, thus, volume of administration is crucial in nasal drug delivery [59]. Novel nasal delivery technologies suitable for both liquid and powder peptide drugs are currently being developed. These novel concepts combine knowledge of both functional nasal anatomy and aerodynamics. On this regard, OptiNose undertook a Phase I trial in late 2013 to investigate “nose-to-brain” transport of oxytocin via the patented OptiNose Bi-Directional™ Breath Powered 65 delivery technology. The OptiNose device offers the potential for a more efficient and consistent direct transport of oxytocin into the brain itself, using relatively low doses, which will significantly reduce drug levels in the rest of the body, reducing the risk of side-effects [51]. Other companies such as Kurve technology recently developed the ViaNase electronic atomizer, based on Controlled Particle Dispersion (CPD)® designed to deliver most formulations with efficient nasal cavity saturation and minimal deposition to the lungs and stomach [60]. Other similar devices include; the Teleflex VaxINator™ [61], and the LMA® MAD NasalTM Device [62]. Intranasal vaccines The nasal route has also been proposed as an option for mucosal vaccination, with the possibility to induce both systemic and mucosal immune responses. Endocine is a delivery system specifically developed for intranasal vaccines (by Eurocine Vaccines, Sweden). It is a lipid-based (mono-olein and oleic acid) dispersion with particles less than 100 nm. Intranasal administration of Vacc-4x with Endocine (as an adjuvant) was safe and induced dose-dependent T cell responses and both mucosal and systemic humoral responses, in patients on effective antiretroviral therapy (Phase I clinical trials) [56]. 3.2. Pulmonary delivery Aerosol administration of therapeutics to the pulmonary epithelium for systemic delivery represents a significant opportunity in the delivery of macromolecules since it allows: (i) rapid absorption into the systemic circulation (this may be especially important for drugs where fast onset of action is critical), and (ii) higher bioavailability than with other non-invasive modes of administration [63]. This can be attributed to the considerable absorptive surface area at the air interface, covered by an extremely small volume of fluid (10–20 ml) and with the entire cardiac output flowing through the underlying capillary network [64]. Due to this physiological and anatomical peculiarity, an inhaled aerosol drug can be widely dispersed and deposited in high concentrations in close proximity to the blood stream (Figure 3). The fate of drugs following inhalation depends on their site of deposition within the lungs. Aerosol particles deposited in the tracheobronchial tree come into contact with 66 the mucus, and the peptide or protein within the particle dissolves in the mucus. The macromolecules can either be eliminated by the mucociliary clearance towards the gastrointestinal tract or diffuse in the mucus and cross the airway epithelium. On the other hand, particles deposited in the alveolar region initially come into contact with the thin layer of fluid lining of the alveolar epithelium and are able to cross much more efficiently [63–65]. Figure 3: Schematic representation of the lung a description of the absorption process in the alveolus. The presence of proteases in the lung and the barrier between capillary blood and alveolar air (air-blood barrier) and clearance by alveolar macrophages impede pulmonary delivery of peptide drugs. The use of absorption enhancers such as oleic acid, oleyl alcohol, Span 85 and protease inhibitors e.g. sodium glycocholate and surfactin have been reported to be useful approaches for improving pulmonary absorption of biologically active drugs like peptides [11, 66]. Pulmonary technology is the process of making drugs inhalable (e.g. conversion into dry-powder) in order to deliver them to and through the lungs for both systemic and local lung applications whilst PEGylation is their chemical modification to enhance peptide/protein drug performance i.e. improve stability and solubility, increase half-life and reduce immune responses. These technologies form the basis for most industrial products 67 that have received FDA approval [67]. 3.2.1. Systemic delivery via the pulmonary route AFREZZA™ is an FDA approved (Mannkind corporation, 2014) ultra-rapid acting insulin comprising Technosphere® insulin powder in unit-dose inhaler cartridges. Technosphere® is a drug delivery system made up of fumaryl diketopiperazine (FDKP), which forms microspheres with 2–5μm sizes. The powder formulation is prepared by precipitating insulin from solution onto the preformed particles, which readily dissolve once in the lung environment, releasing insulin. It rapidly reaches systemic circulation and attains maximum plasma concentration in 15 minutes, which is much earlier compared to injectable insulin (i.e. 1h). AFREZZA® has a relative bioavailability of 21–25% compared to SC regular insulin and is also eliminated quickly (within 2h) from the blood circulation. This pharmacokinetic/dynamic profile mimics what happens with endogenous insulin released in a person without diabetes and the result is significantly less hypoglycemia [68]. Nektar´s PEGylation technology™ used in nine approved partnered products for other administration routes, in the U.S. including UCB's Cimzia® for Crohn's Disease, Roche's PEGASYS® for hepatitis C and Amgen's Neulasta® for neutropenia, has also been investigated for lung delivery. This conjugation technology improves peptide stability and reduces immune responses [47]. Nektar also showed that the administration of liposome-encapsulated drugs by aerosols seems to be a feasible way to deliver them via the lungs. Leuprolide acetate, is a potent agonist of luteinizing hormone-releasing hormone (LHRH) currently used for the treatment of prostatic cancer, endometriosis, and precocious puberty, administered solely by injection. In an exploratory study, leuprolide peptide was entrapped in liposomes (3.5 μm in size) containing sorbitan monooleate (Span 80) and administered as a dry powder resulting in a relative bioavailability of 18 %, compared to injectable systemic delivery. The results suggested that clinical doses of leuprolide could be given as once-a-day single inhalation dose, thanks to the liposomes that increased residence time and promoted absorption of the hormone. [55]. However, no further updates have been published on this formulation since 2009. 68 Baxter Healthcare Corporation, recently completed phase I clinical trials where they investigated the PK and PD properties of recombinant human insulin inhalation powder (RHIIP), manufactured with PROMAXX® technology, which allows the formation of protein microspheres of uniform size and shape suitable for inhalation by a temperature-controlled precipitation from an aqueous insulin solution in the presence of polyethylene glycol without the use of absorption enhancers. This microparticulate powder formulation showed a faster onset of action than SC Insulin [69]. In recent years the development of new and improved devices for administration of peptides and proteins has intensified since the site of deposition is crucial for the efficacy of inhaled drugs. Prominent examples of such devices are the AERx® from Aradigm (CA, USA) [70], Respimat® from Boehringer Ingelheim (Ingelheim, Germany) [71] and AeroDose® from Aerogen Inc. (Galway, Ireland) [72]. Some of the most recent developments with these devices are included in Table 3. The Aerodose® insulin inhaler for example, resulted in a dose-dependent increase in serum insulin concentration and a corresponding glucose lowering effect that were similar in proportion to that obtained for increasing doses of subcutaneously injected insulin. This novel device is a small hand-held breath-actuated inhaler that contains Aerogen’s electronic aerosol generator. When liquid is placed onto the aerosol generator, a micropumping action creates a fine-droplet low-velocity aerosol that is suited for deep lung delivery [72]. 3.2.2. Local delivery to the lung Although the efforts made in the field of pulmonary delivery of peptides and proteins have been tremendous, there are still a very limited number of inhaled macromolecules available on the market. As expect, inhaled peptide drugs developed for local therapy are the most promising and the fastest growing group under development [73]. One of the candidates worth mentioning is being developed by the University of Maryland, a new liposomal cyclosporine A (L-CsA) solution consisting of unilamellar liposomes of about 50 nm with a narrow size distribution inhaled through a nebulizer, in phase 1 clinical trials completed in 2015, for the treatment of bronchiolitis obliterans syndrome after lung transplantation. The treatment was well 69 tolerated, and no drug-related side effects were observed. Once or twice daily dosing of 10 mg aerosol L-CsA would result in a sufficient peripheral lung deposition of approximately 14 and 28 mg/week, respectively signifying the improvement of pharmacokinetics through encapsulation in liposomes [74]. 70 Table 3: Inhaled peptide formulations on the market or under clinical development. Brand name Drug Technology Indication Phase of development Company Ref. Systemic AFREZZA™ Insulin Technosphere® Diabetes Market, 2015 Mannkind corporation [68] Leuprolide Leuprolide Inhaler Prostate cancer and endometriosis Phase I Nektar Therapeutics [75] Recombinant human insulin inhalation powder (RHIIP) Insulin PROMAXX® in a dry powder inhaler Diabetes mellitus Phase I Baxter Healthcare Corporation [69] Alveair® Insulin Alveair™ (Liquid inhaler) Diabetes Preclinical trials Coremed Inc [76] Aerodose ® Insulin Aerodose inhaler Diabetes Preclinical trails Aerogen [72] Local Sargramostim Granulocytemacrophage colony stimulating factor (GM-CSF) Nebulizer Osteaosarcoma (Pulmonary recurrence) Phase II Children’s Oncology Group & National Cancer Institute (NCI) [77] Interferongamma Interferon-gamma Nebulizer Cavitary pulmonary tuberculosis Phase II New York University School of Medicine [78] IL-4R Recombinant human interleukin4 receptor (IL-4R) Aerosol inhaler Asthma Phase II National Institute of Allergy and Infectious Diseases [79] 71 (NIAID) AVONEX® Interferon β-1a ANOVEX inhaler Multiple sclerosis Phase I Trio Medicines Ltd [47] COLI-VLM Colimycin Aerosol inhaler Cystic Fibrosis Phase I Poitiers University Hospital [80] CR002 Alpha1 Proteinase Inhibitor (API) Inhalation solution Cystic Fibrosis Phase I CSL Behring [81] MVA85A Antigenic peptide Aerosol inhaler Tuberculosis Vaccine Phase I University of Oxford [82] Liposomal Cyclosporine (LCsA) Cyclosporine A Aerosol inhaler Bronchiolitis Obliterans Syndrome Phase I University of Maryland [83] 72 3.3. Oral delivery Systemic delivery of drugs via the nasal and pulmonary routes, circumvents the liver and, thus, avoid hepatic first-pass metabolism, which may be advantageously exploited for many peptides drugs [84]. However, the oral route of administration is often preferred due to the easy accessibility of the large surface area available for absorption to the systemic circulation. Furthermore, the pharmacological rationale for administering specific peptides such as insulin via the oral route supports this approach, as the oral delivery of insulin results in absorption directly to the liver (hepatic portal circulation), before it reaches the peripheral tissues (Figure 4) [85, 86]. Figure 4: Schematic illustration of the large surface area for absorption and first pass metabolism associated with the oral route of administration. Indeed, when the drug of interest must be used repeatedly administered, the oral route inevitably becomes the route of choice for patients since it is painless and convenient [87]. To achieve successful and satisfactory therapeutic results by the oral route, there must be minimal degradation and significant absorption of the protein molecules in the GIT [32]. Most of current approaches target the GIT barrier in an attempt to overcome its restrictive nature and subsequently increase oral bioavailability [88]. The main challenge is to improve the oral bioavailability to be higher than 1%. New approaches are mostly driven by the necessity to design an approach that not only 73 protects the protein/peptide from enzymatic degradation but also aids in enhancing its absorption without altering its biological activity [89]. The following tables portrays recent efforts of various pharmaceutical companies in making the transition towards oral peptide delivery by developing innovative technologies and testing multiple drug candidates based on these technologies (Table 3). The tables are intended to give a broad overview of the numerous strategies and applications currently in evaluation and are organized from a practical point of view as a function of developmental stage and intended site of action (i.e. systemic and local action). Given the large body of information, the discussion is limited to the most promising and interesting examples. 3.3.1. Systemic delivery using the oral route Desmopressin, is one of the marketed oral peptides. Its oral bioavailability is low (< 1%) but only a very low dose is required to elicit its therapeutic effect [90]. Desmopressin and cyclosporine (another marketed oral peptide), have cyclic structures, which render them stable and less prone to degradation in the GIT. Moreover their small molecular weight allows for faster and less constrained intestinal absorption. Astellas Pharma Inc, also developed new cyclic peptides with anti-hepatitis C virus action, which take advantage of this strategy [91]. The most promising and basic approaches reaching clinical development for oral delivery are enteric-coated formulations that include muco-interactive polymers encapsulating a peptide-drug with enzyme inhibitors and absorption enhancers, capable of rapidly and reversibly increasing epithelial permeability. In this strategy, excipients (chemicals that are added to the formulation that make up capsules, tablets or liquids) are used to shield the native drug chemically or physically and promote absorption. This strategy has been in existence for a while now in pharmaceutical industries and it is being improved progressively by the addition of novel enhancing excipients and polymers [92]. Enteris BioPharma Inc (formerly known as Unigene) developed the clinically proven Peptelligence™ technology that enables the oral delivery of peptides. This technology entails an enteric-coated tablet with two key components; an organic acid enzyme inhibitor (citric acid in the form of coated beads) and a permeation enhancer (acylcarnitine), which penetrates the mucus layer. The enteric coating prevents the tablet from disintegration within the stomach and enables dissolution within the intestinal pH. The permeation enhancer opens tight junctions and promotes paracellular transport whilst the citric acid transiently decreases the 80 in partnership with Novartis is currently employing this technology to develop an oral insulin product. Preclinical studies showed very promising results with oral bioavailability over 50 %. Traverso et al also recently demonstrated proof-of-concept experiments in swine that microneedle-based delivery has the capacity for improved bioavailability of macromolecules (e.g. insulin). The pill’s needles are initially coated by a pH-responsive coating which dissolves in the desired location in the GI tract, revealing the microneedles (solid, drug-containing microneedles fabricated from biocompatible polymers). These detach from the capsule and become lodged in the GI tissue, where they slowly release their payload. Moreover, the authors showed that microneedle-containing devices can be passed and excreted from the GI tract safely [116]. These findings strongly support the potential of implementation of microneedle technology for use in the GI tract. The Nanoparticle Oral Delivery technology, developed by NOD Pharmaceuticals, together with its subsidiary, Biolaxy, comprises enteric coated and bioadhesive calcium phosphate nanoparticles with sizes between 5 – 200 nm in the final dosage form of a capsule. The formulation is obtained by combining the peptide with calcium phosphate in the presence of PEG salts of fatty acids (e.g. caprylate, sodium caprate) and bile salts as precipitating agents. The obtained calcium phosphate nanoparticles are then enteric-coated using cellulose acetate phthalate and a bioadhesive polymer, carbomer. Nodlin is an oral insulin formulation for basal insulin supplementation currently in Phase II trials in China whilst Nodexen (phase I) is an oral exenatide formulation. Both formulations are developed by Shangai Biolaxy using the NOD technology platform [117]. TNT Pharma utilizes the company´s proprietary cell-targeted, bio-nanotechnologybased system called Nanoves® for the oral delivery of HDV-IFN and Parathyroid hormone currently in phase I and preclinical trials, respectively. The NanoVes System is composed of 20 – 50 nanometer sized phospholipid-based bio-nanoparticles that are compatible with both subcutaneous (SC) injection and oral delivery. The active pharmaceutical ingredients are incorporated into the carrier by electrostatic and hydrophobic interactions and are delivered to the desired cellular targets. This level of cell targeting and delivery is supported by a large database of histopathological results that show clear endocytosis of the NanoVes System, in vitro and in vivo, including human pharmacological data [109]. Oshadi Drug administration developed the Oshadi ICP an oral formulation consisting of: insulin, proinsulin and C-peptide in the Oshadi 81 carrier. This invention comprises, pharmacologically inert silica nanoparticles having a hydrophobic surface, a polysaccharide, and a biologically active protein or peptide suspended in an oil. The company carried out Phase II clinical trials in type 1 diabetes patients in 2014. With regard to peptide modification, a polyethylene glycol side chain at position B29 improves the stability and increases the solubility of insulin (IN-105, by Biocon), promoting its rapid absorption. IN-105 is currently under Phase III clinical trials which have showed that timing of the IN-105 administration before meals is crucial to achieve a substantial glucose lowering effect [118]. 3.3.2. Local delivery via the oral route Most of the oral peptides in the market or in advanced developmental stage are characterized by local GIT activity. For instance, Constella oral capsules used for the treatment of irritable bowel syndrome (IBS) with constipation (produced by Allergan and Iron wood Pharmaceuticals) contain the peptide linaclotide, which works locally in the gut, where it binds to receptors called guanylate cyclase C receptors, found on the walls of the intestine. It reduces abdominal pain and increase the amount of fluid in the gut, which helps loosen the stools and speed up their movement through the bowel [119]. Taken as a tablet once-a-day, plecanatide designed by Synergy pharmaceuticals, mimics the function of natural uroguanylin by working locally in the upper GI tract to activate and regulate fluid movement required for normal bowel function. Whilst dolcanatide, is designed to be highly stable and resistant to proteolysis in gastric and intestinal fluids but still operate in the same manner as natural uroguanylin. Phase III clinical trials were completed in 2015 with exceptionally good results and NDA were filled in early 2016 [120]. Enzymes such as pancreatic lipases have been routinely administered orally for decades (such as Creon), they are adapted by nature to the GIT environment and there has been a misconception that they do not need any strategic delivery. However, there is also innovation in this area. For example, Sollpura® (Liprotamase, Phase III) and Zenpep®, (marketed, 2009) are formulations intended for pancreatic enzyme replacement therapy (PERT) and for low digestive enzyme levels developed by Anthera and Forest pharmaceuticals, respectively [121]. 82 Table 4 (b): Selected oral peptide formulations under clinical development (local GIT action). Brand name Drug Technology Indication Phase of development Company Reference Constella® Linaclotide Delayed-release capsule Irritable-bowel syndrome (IBS), Market, 2012 Allergan/Iron wood Pharmaceuticals [119] ZENPEP Pancrelipase Delayed-release capsule Low digestive enzyme levels, Market, 2009 Forest Pharmaceuticals,Inc [119] Sollpura® Liprotamase Delayed-release capsule Exocrine pancreatic deficiency Phase III Anthera Pharmaceuticals [121] Plecanatide & Dolcanatide Uroguanylin peptide analogs Modified peptide for resistant against proteolysis Chronic idiopathic constipation (CIC) & Ulcerative colitis (UC) Phase III Synergy pharmaceuticals [122] Macrilen™ Ghrelin antagonist Chemical modification of peptide Adult growth hormone deficiency (AGHD) Phase III Aeterna Zentaris [123] VEN 120 Recombinant lactoferrin Modified peptide Inflammatory Bowel Disease Phase II Ventria Bioscience [124] ALV003 Cysteine protease (EPB2) Delayed-released capsule Colitis Phase IIb Alvine Pharmaceuticals [125] 83 Prolyl endopeptidase (PEP) PLD-116 Undisclosed peptide Delayed-release capsule Inflammatory bowel disease Phase II PLIVA [126] IL-23R IL-23 receptor Protagonist´s platform Crohn´s disease Phase I Protagonist Therapeutics Inc [127] 84 4. General remarks and conclusions As outlined in this article, it is clear that several pharmaceutical companies have led the efforts to develop “needle free” protein/peptide therapeutics and the most popular route being investigated is the oral route, more especially for metabolic disorders. This is mainly influenced by the fact that from a patient perspective, the oral route is simpler, cost effective and more convenient. General setbacks, which are common across all mucosal delivery routes, arise due to the excipients added in most of the formulations including penetration enhancers and enzyme inhibitors. Many of these delivery strategies may compromise the barrier function of the epithelia to incoming toxins, and implications of their long-term use in managing chronic disease such as diabetes should be considered. These possible implications raise concerns and cause both the general public and the FDA to be cautious with these innovations; hence their development and advancement into the market are hindered. Mucosal systemic bioavailabilities should be reproducible and reliable to achieve a regulatory filing, but incompatibility of hydrophilic peptide and protein drugs with hydrophobic delivery carriers can produce uncontrolled drug release which also handicapped the advancement to the market. Moreover, mucosal bioavailabilities are still much lower than those of injected doses, and therefore much higher doses must be administered to have the same effect. For more expensive peptide and protein drugs, this could preclude their mucosal delivery and the toxicity implications of administering relatively large doses of peptide and protein drugs remain a concern. Needle phobia and stress leading to low patient compliance plus the inconvenience and side effects associated with injecting drugs have encouraged scientists to investigate and exploit all promising noninvasive routes for peptide/protein delivery. In this review we presented the most common and highly sought-after, ‘noninvasive’ mucosal routes, specifically; nasal, pulmonary and oral. Many approaches have been used to study various strategies to overcome the inherent barriers to peptide uptake across mucosal routes. Each of the various routes has its own set of favorable and unfavorable properties and these were outlined. Amongst the three routes discussed here, tremendous work has been done for oral peptide delivery (including insulin). Although extensive human clinical studies are still required for most of the peptides presented here, especially of long-term clinical applications, taken together, the 85 technologies and developments described here are likely to increase the number of marketed needle free delivered peptides and proteins. 86 References: 1. Lax R (2010) The Future of Peptide Development in the Pharmaceutical Industry. Pharm. Manufacturing Int Pept Rev 10–15. 2. Tsomaia N (2015) Peptide therapeutics: Targeting the undruggable space. Eur. J. Med. Chem. 94:459–470. 3. Lien S, Lowman HB (2003) Therapeutic peptides. Trends Biotechnol 21:556– 562. 4. Ratnaparkhi MP, Chaudhari SP, Pandya V a (2011) Peptides and proteins in pharmaceuticals. Int. J. Curr. Pharm. Res. 3:1–9. 5. Ibraheem D, Elaissari a, Fessi H (2014) Administration strategies for proteins and peptides. Int. J. Pharm. 477:578–589. 6. Kaspar A a., Reichert JM (2013) Future directions for peptide therapeutics development. Drug Discov. Today 18:807–817. 7. Reichert JM (2010) The Peptide Therapeutics Foundation : mapping the future of peptide therapeutics. 8. Uhlig T, Kyprianou T, Martinelli FG, et al (2014) The emergence of peptides in the pharmaceutical business: From exploration to exploitation. EuPA Open Proteomics 4:1–12. 9. Lax R, Meenan C (2012) Challenges for therapeutic peptides part 2: Delivery systems. Innov. Pharm. Technol. 42–46. 10. Moeller EH, Jorgensen L (2008) Alternative routes of administration for systemic delivery of protein pharmaceuticals. Drug Discov. Today Technol 5:e89–e94. 11. Lassmann-Vague V, Raccah D (2006) Alternatives routes of insulin delivery. Diabetes Metab 32:513–522. 12. Khafagy E-S, Morishita M, Onuki Y, Takayama K (2007) Current challenges in non-invasive insulin delivery systems: a comparative review. Adv. Drug Deliv Rev 59:1521–46. 13. Vlieghe P, Lisowski V, Martinez J, Khrestchatisky M (2010) Synthetic therapeutic peptides: science and market. Drug Discov. Today 15:40–56. 14. Sohi H, Ahuja A, Ahmad FJ, Khar RK (2010) Critical evaluation of permeation enhancers for oral mucosal drug delivery. Drug Dev. Ind. Pharm. 36:254–282. 15. Ensign LM, Cone R, Hanes J (2012) Oral drug delivery with polymeric nanoparticles: the gastrointestinal mucus barriers. Adv. Drug Deliv. Rev. 64:557–70. 16. Cone R A. (2009) Barrier properties of mucus. Adv. Drug Deliv. Rev. 61:75– 85. 17. Maisel K, Ensign L, Reddy M, et al (2015) Effect of surface chemistry on nanoparticle interaction with gastrointestinal mucus and distribution in the gastrointestinal tract following oral and rectal administration in the mouse. J. Control Release 197:48–57. 87 18. Antunes F, Andrade F, Araújo F, et al (2013) Establishment of a triple coculture in vitro cell models to study intestinal absorption of peptide drugs. Eur. J. Pharm. Biopharm. 83:427–435. 19. Muheem A, Shakeel F, Jahangir MA, et al (2014) A review on the strategies for oral delivery of proteins and peptides and their clinical perspectives. Saudi Pharm. J. 20. des Rieux A, Fievez V, Garinot M, et al (2006) Nanoparticles as potential oral delivery systems of proteins and vaccines: a mechanistic approach. J. Control Release 116:1–27. 21. Fan T, Chen C, Guo H, et al (2014) Design and evaluation of solid lipid nanoparticles modified with peptide ligand for oral delivery of protein drugs. Eur. J. Pharm. Biopharm. 88:518–528. 22. Iversen TG, Skotland T, Sandvig K (2011) Endocytosis and intracellular transport of nanoparticles: Present knowledge and need for future studies. Nano Today 6:176–185. 23. Hillaireau H, Couvreur P (2009) Nanocarriers’ entry into the cell: relevance to drug delivery. Cell Mol Life Sci 66:2873–96. doi: 10.1007/s00018-009-0053-z 24. Renukuntla J, Vadlapudi AD, Patel A, et al (2013) Approaches for enhancing oral bioavailability of peptides and proteins. Int J Pharm 447:75–93. doi: 10.1016/j.ijpharm.2013.02.030 25. Grover GN, Maynard HD (2010) Protein-polymer conjugates: Synthetic approaches by controlled radical polymerizations and interesting applications. Curr. Opin. Chem. Biol. 14:818–827. 26. Gregoriadis G, Jain S, Papaioannou I, Laing P (2005) Improving the therapeutic efficacy of peptides and proteins : A role for polysialic acids. 300:125–130. 27. Zhang L, Bulaj G (2012) Converting Peptides into Drug Leads by Lipidation. 18:1602–1618. 28. Bechara C, Sagan S (2013) Cell-penetrating peptides: 20 years later, where do we stand? FEBS Lett 587:1693–1702. 29. Koren E, Torchilin VP (2012) Cell-penetrating peptides: breaking through to the other side. Trends Mol. Med. 18:385–93. 30. Su F-Y, Lin K-J, Sonaje K, et al (2012) Protease inhibition and absorption enhancement by functional nanoparticles for effective oral insulin delivery. Biomaterials 33:2801–11. 31. Tan ML, Choong PFM, Dass CR (2010) Recent developments in liposomes, microparticles and nanoparticles for protein and peptide drug delivery. Peptides 31:184–193. 32. Choonara BF, Choonara YE, Kumar P, et al (2014) A review of advanced oral drug delivery technologies facilitating the protection and absorption of protein and peptide molecules. Biotechnol Adv 32:1269–1282. 33. Rekha MR, Sharma CP (2013) Oral delivery of therapeutic protein/peptide for 88 diabetes-Future perspectives. Int J Pharm 440:48–62. 34. Maggio ET (2006) Intravail: highly effective intranasal delivery of peptide and protein drugs. Expert Opin Drug Deliv 3:529–539. 35. Pires A, Fortuna A, Alves G, Falcão A (2009) Intranasal drug delivery: How, why and what for? J. Pharm. Pharm. Sci. 12:288–311. 36. Meredith ME, Salameh TS, Banks W a. (2015) Intranasal Delivery of Proteins and Peptides in the Treatment of Neurodegenerative Diseases. AAPS J 17:780– 787. 37. Ozsoy Y, Gungor S, Cevher E (2009) Nasal delivery of high molecular weight drugs. Molecules 14:3754–3779. 38. Ozsoy Y, Gungor S, Cevher E (2009) Nasal delivery of high molecular weight drugs. Molecules 14:3754–3779. 39. Tarsa Therapeutics’ NDA for TBRIA (TM), The First Oral Calcitonin for the Treatment of Postmenopausal Osteoporosis, Accepted for Filing. http://tarsatherapeutics.com/tarsa-therapeutics-nda-for-tbriatm-the-first-oralcalcitonin-for-the-treatment-of-postmenopausal-osteoporosis-accepted-forfiling/. Accessed 1 Mar 2016 40. Peichl P, Marteau R, Griesmacher A, et al (2005) Salmon calcitonin nasal spray treatment for postmenopausal women after hip fracture with total hip arthroplasty. J. Bone Miner. Metab. 23:243–252. 41. Calcitonin for Treating X-linked Hypophosphatemia. https://clinicaltrials.gov/ct2/show/NCT01652573?term=nasal+and+peptide&ra nk=19. Accessed 16 Feb 2016 42. Allon DM, Anavi Y, Calderon S (2009) Central giant cell lesion of the jaw: Nonsurgical treatment with calcitonin nasal spray. Oral Surgery, Oral Med Oral Pathol Oral Radiol Endodontology 107:811–818. 43. (2016) Intravail ® Octreotide Metered Nasal Spray – Product Profile Offered for Exclusive Worldwide License. 1400. 44. Ueno H, Mizuta M, Shiiya T, et al (2014) Exploratory trial of intranasal administration of glucagon-like peptide-1 in Japanese patients with type 2 diabetes. Diabetes Care 37:2024–2027. 45. Intranasal Human Growth Hormone (hGH) Induces IGF-1 Levels Comparable With Subcutaneous Injection With Lower Systemic Exposure to hGH in Healthy Volunteers. http://press.endocrine.org/doi/10.1210/jc.20144146?url_ver=Z39.882003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpu bmed#sthash.kdSdfU8K.dpuf. Accessed 6 Feb 2016 46. Pharmaceuticals C CRITICALSORB ABSORPTION PROMOTER. http://www.criticalpharmaceuticals.com/technology/criticalsorb. Accessed 9 Mar 2016 47. Andrade F, Videira M, Ferreira D, Sarmento B (2011) Nanocarriers for pulmonary administration of peptides and therapeutic proteins. Nanomedicine (Lond) 6:123–141. 89 48. Aegis Therapeutics Ll Stabilizing alkylglycoside compositions and methods thereof. 49. CP046 Nasal Teriparatide (Parathyroid Hormone PTH 1-34). http://www.criticalpharmaceuticals.com/products/cp046. Accessed 6 Feb 2016 50. The Effect of Intranasal Oxytocin on Pain Sensitivity and Threshold. https://clinicaltrials.gov/ct2/show/NCT02550093?term=nasal+and+peptide&ra nk=26. Accessed 16 Feb 2016 51. Rash JA, Campbell TS (2014) The Effect of Intranasal Oxytocin Administration on Acute Cold Pressor Pain. Psychosom Med 76:422–429. 52. Study of Nasal Insulin to Fight Forgetfulness - Long-acting Insulin Detemir - 120 Days (SL120) (SL120). https://clinicaltrials.gov/ct2/show/NCT01595646?term=NCT01595646&rank= 1. 53. Efficacy of Nasal Spray Calcitonin on Recurrence of Aggressive Central Giant Cell Granuloma. https://clinicaltrials.gov/ct2/show/NCT02358304?term=nasal+and+peptide&ra nk=20. Accessed 16 Feb 2016 54. (2013) LTX-109. 55. Randomized, Double-blind, Placebo-controlled, Phase I Dose-escalation Study of Single Dose GHB01L1 in Healthy Volunteers (GHBCS-01). https://clinicaltrials.gov/ct2/show/NCT00724997?term=aerosol&rank=122. Accessed 16 Feb 2016 56. Intranasal Modified Vacc-4x Gag Peptides With Endocine as Adjuvant. https://clinicaltrials.gov/ct2/show/NCT01473810?term=nasal+and+peptide&ra nk=1. Accessed 16 Feb 2016 57. Washington U of SNIFF 120: Study of Nasal Insulin to Fight Forgetfulness (120 Days) (SNIFF 120). https://clinicaltrials.gov/ct2/show/NCT00438568?term=Nasal+Insulin&rank=8. 58. Robbins M Intranasal Oxytocin for the Treatment of Pain Associated With Interstitial Cystitis. https://clinicaltrials.gov/ct2/show/NCT00919802. Accessed 9 Mar 2016 59. Djupesland PG (2013) Nasal drug delivery devices: Characteristics and performance in a clinical perspective-a review. Drug Deliv Transl Res 3:42–62. doi: 10.1007/s13346-012-0108-9 60. Drug Delivery Technology: Controlled Particle Dispersion® Technology Platform. http://www.kurvetech.com/clinicalstudies.asp. Accessed 1 Mar 2016 61. Teleflex VaxINatorTM Intranasal Drug Delivery Device. http://www.vaxinator.com. Accessed 1 Mar 2016 62. Therapeutic intranasal drug delivery. http://intranasal.net/deliverytechniques/default.htm. Accessed 1 Mar 2016 63. Taylor P, Koussoroplis S, Vanbever R, Koussoroplis S (2013) Peptides and Proteins : Pulmonary Absorption. Encycl Pharm Sci Technol , Fourth Ed Pept 96 97 Background Nanocarriers can overcome critical barriers associated with the oral route of administration including: (i) the harsh gastrointestinal environment (pH range 1.27.4) with high concentrations of enzymes specialized in digestion, (ii) the thick mucus barrier (100 μm) subjected to continuous turn-over, and (iii) the intestinal epithelium, which acts as a gatekeeper and is poorly permeable to macromolecules [1, 2]. Our group pioneered the development of a delivery platform, named as polymer nanocapsules, consisting of an oily core surrounded by one or more layers a hydrosoluble polymers [3]. Specific prototypes including chitosan nanocapsules and polyarginine nanocapsules have been found efficient for the delivery of peptides and anti-cancer drugs [4]. These nanosystems portrayed remarkable penetration enhancing properties and stability in biological media, which was further enhanced by introducing PEG-stearate into the shell composition [4, 5]. Protamine is a natural cationic polymer with the intrinsic capacity to translocate through mammalian cell membranes. This ability has been attributed to its arginine-rich sequence, which promotes direct interaction with cell surface domains and subsequently facilitates cellular internalization [6–8]. Protamine is an FDA approved drug indicated for reverting the anticoagulant effects of heparin. It is also present as an excipient, in a parenteral insulin formulation (NPH, Neutral Protamine Hagedorn) [9–12]. Our group has designed a variety of nanocarriers involving protamine. Namely, Protamine nanoparticles and nanocapsules were designed for the delivery of peptides and antigens, whereby they improved their cell internalization[13, 14]. More recently, the group developed multilayered nanoparticles made of protamine and polyarginine in combination with polysaccharides, dextran sulphate and alginate, also intended for intracellular antigen delivery [15]. 98 References: 1. Oriane Bouttefeux AB and VP (2015) Delivery of peptides via the oral route: diabetes treatment by peptide-loaded nanoparticles. Curr. Pharm. Des. 22:1–30. 2. Sonia TA, Sharma CP (2014) Oral Delivery of Insulin. Oral Deliv. Insul. 1–57. 3. Prego C, Torres D, Alonso MJ (2006) Chitosan nanocapsules as carriers for oral peptide delivery: effect of chitosan molecular weight and type of salt on the in vitro behaviour and in vivo effectiveness. J Nanosci. Nanotechnol. 6:2921–2928. 4. Lozano M V., Lollo G, Alonso-Nocelo M, et al (2013) Polyarginine nanocapsules: A new platform for intracellular drug delivery. J Nanoparticle Res. 5. Prego C, Torres D, Fernandez-Megia E, et al (2006) Chitosan-PEG nanocapsules as new carriers for oral peptide delivery. Effect of chitosan pegylation degree. J. Control Release 111:299–308. 6. Reynolds F, Weissleder R, Josephson L (2005) Protamine as an efficient membrane-translocating peptide. Bioconjug. Chem. 16:1240–1245. 7. Elumalai R, Patil S, Maliyakkal N, et al (2015) Protamine-carboxymethyl cellulose magnetic nanocapsules for enhanced delivery of anticancer drugs against drug resistant cancers. Nanomedicine Nanotechnology, Biol. Med. 11:969–981. 8. He H, Sheng J, David AE, et al (2013) The use of low molecular weight protamine chemical chimera to enhance monomeric insulin intestinal absorption. Biomaterials 34:7733–7743. 9. Liang JF, Zhen L, Chang L-C, Yang VC (2003) A less toxic heparin antagonist--low molecular weight protamine. Biochemistry (Mosc) 68:116–120. 10. Sorgi FL, Bhattacharya S, Huang L (1997) Protamine sulfate enhances lipidmediated gene transfer. Gene Ther. 4:961–968. 11. Byun Y, Singh VK, Yang VC (1999) Low molecular weight protamine: a potential nontoxic heparin antagonist. Thromb. Res. 94:53–61. 12. Horrow JC (1985) Protamine: a review of its toxicity. Anesth. Analg. 64:348– 361. 13. González-Aramundiz JV (2013) NUEVOS NANOSISTEMAS A BASE DE PROTAMINA PARA LA LIBERACIÓN DE ANTÍGENOS. Universidad de santiago de compostela 99 14. González-Aramundiz JV, Olmedo MP, González-Fernández Á, et al (2015) Protamine-based nanoparticles as new antigen delivery systems. Eur. J. Pharm. Biopharm. 97:51–59. 15. Correia-Pinto JF, Peleteiro M, Csaba N, et al (2015) Multi-enveloping of particulated antigens with biopolymers and immunostimulant polynucleotides. J. Drug Deliv. Sci. Technol. 30:424–434. 100 101 Hypothesis 3. Protamine could be a promising biomaterial for the development of nanocapsules for the efficient oral delivery of macromolecules such as insulin. The ability of protamine to translocate through biological cell membranes could be combined with the capacity of lipids and surfactants to enhance the intestinal permeation of drugs. 4. The rational selection and combination of protamine with other polymers with stabilizing and muco-penetration properties, including PEG and other polysaccharides, could result in an optimized nanocarrier system able to associate the peptide, protect it from degradation and deliver it across the epithelial barrier. Objectives Based on the background evidence and hypothesis outlined, the main goal of the present work is to rationally design and develop a new protamine based nanocarrier system for the efficient oral delivery of peptides using insulin as a model peptide drug. For this purpose the overall aim is split into the following objectives with their specific tasks; 1) Development of insulin loaded protamine nanocapsules 1. To prepare and characterise protamine nanocapsules (blank and insulin loaded) with regard to their physicochemical properties, mucodiffusion, stability upon incubation in intestinal biological media and stability during storage. 2. To evaluate their ability to load insulin, prevent the degradation of the peptide by pancreatic enzymes and study the in vitro release profile. 3. To demonstrate the stability of insulin loaded protamine nanocapsules in different simulated intestinal media and confirm the conformational structure of insulin after encapsulation. 102 The results corresponding to the work described here are presented in Chapter 2: Rational design and in vitro characterization of protamine-based nanocapsules for oral peptide delivery. 2) Assessment of the in vitro mechanistic and toxicity of protamine nanocapsules 4. To determine the toxicity, penetration and mechanism of action of the nanocapsules using in vitro cell models simulating the different mucosal barriers (e.g. enterocytes and mucus) of the intestinal epithelium. 5. To evaluate the toxicity and permeation enhancing property of the nanocapsules ex vivo in human intestinal tissue. The results corresponding to the work described here are presented in Chapter 3: The interaction of protamine nanocapsules with the intestinal epithelium 3) Evaluation of particle interaction with the epithelial barrier including biodistribution and in vivo efficacy of the nanocapsules 6. To elucidate the in vitro immunological response of protamine and polysialic acid coated protamine nanocapsules in dendritic cells 7. To study the biodistribution of the fluorescent protamine nanocapsules after their oral administration to mice 8. To determine the in vivo efficacy bioactivity of the new nanocarrier in nondiabetic rats. The results corresponding to the work described here are presented in Chapter 4: Protamine nanocapsules: a versatile vehicle for oral insulin delivery. In addition to these major objectives we have also aimed to explore new technologies for the production of protamine nanocapsules, i.e. the self-emulsifying technique which could exclude the use of organic solvents and consequently simplify the preparation method. However, because this technology was used as a proof of concept 103 and only applied for a different model peptide (cyclosporine), the corresponding data will be presented in Annex 1: Development and characterization of solvent-free protamine nanocapsules as carriers for mucosal delivery of therapeutics. 104 105 CHAPTER 2 Rational design of protamine nanocapsules as carriers for oral peptide delivery 112 2.3. Physicochemical and morphological properties of protamine NCs The average hydrodynamic diameter, polydispersity index (PDI) and zeta potential of protamine NCs were measured by a Zetasizer® Nano-ZS, Malvern instruments, (Worcestershire, UK) after dilution (50x) with ultrapure water. The transmission electron microscopy (TEM, Joel 2010, 80kV, Philips, Netherlands) was used to analyze the shape and surface properties of the NCs. For TEM analysis samples were deposited on a copper grid and stained with 2% (w/v) ammonium molybdate solution, allowed to dry and then viewed under a the TEM. 2.4 Mucodiffusion studies on protamine nanocapsules To study mucodiffusion, a capillary (0.6 square internal diameter (ID) mm x 0.12 wall (mm), VitroCom, New Jersey), as filled up to three quarters from one end with mucin (5% (w/v) mucin in SIF). Then, rhodamine 6G-labeled protamine nanocapsules were introduced through the opposite end of the capillary using a syringe pump (flow rate = 14 µL.m-1) in a vertical position without disturbing the mucin-nanocarrier interface. The capillary was then incubated for 10 minutes at 37 °C, after which it was analysed under a fluorescence microscope (Leica AF6000, Leica microsystems, Germany). The diffusion of the fluorescent nanocarriers through the mucin was monitored by taking snap-shots every 5 seconds. This allowed us to determine the time taken by the nanocarriers to diffuse across 1 mm of the mucin gel. Chitosan (CS) NCs containing exactly the same core as protamine NCs were used as a control since CS is well known for its mucoadhesion property. 2.5. Association efficiency (AE%) and loading capacity (LC) The association efficiency (%) was determined using the direct method, which involves the extraction of insulin from the NCs by the complete disruption of the NCs. Briefly; insulin loaded NCs (0.1 mL) were digested using a combination of acetonitrile (0.1 mL), Triton™ X-100 (0.05 mL) and 0.1 % TFA (0.75 mL). Then the formulation was vortexed at a high speed to obtain a clear aliquot. To corroborate this data, free insulin in the aqueous phase (undernatant) was quantified after isolating the NCs (indirect method). The concentration of insulin in both cases was determined by reverse phase liquid chromatography (HPLC). A 25 μL aliquot of each sample was injected in triplicate onto the HPLC column. Chromatography was performed using a 113 Agilent 1100 series auto-sampler, equipped with an Agilent 1100 series controller and Dual Absorbance Spectrometer (Agilent 1100 series, Agilent technologies, Germany) with an ATRP11 column (Symmetry C8 5 µm (3.9 x 20 mm)) (Waters, Spain) and a UV detector set at 227 nm. The flow rate was set to 2 mL/min. The gradient was obtained by mixing proportion of phase A (0.1 % TFA) and phase B (acetonitrile:0.1 % TFA, 8:2). Initially, the mobile-phase composition was 25% B; a linear gradient was applied to reach a composition of 25% B after 4min. Quantification was achieved by comparing observed peak area ratios of insulin in the samples to a calibration curve obtained under the same experimental conditions. Linearity was observed in the range from 0.005 to 0.5mg/mL with a correlation coefficient above 0.9995. The detection limit (LOD) was 0.001mg/mL and the quantification limit (LOQ) was 0.005 mg/mL. The association efficiency (AE%) and loading capacity (LC%) were calculated using Equation 1 and 2: Equation 1 𝑨𝑬 % = 𝑰𝒏𝒔𝒖𝒍𝒊𝒏 𝒊𝒏 𝒅𝒆𝒔𝒕𝒓𝒖𝒄𝒕𝒆𝒅 𝒏𝒂𝒏𝒐𝒄𝒂𝒑𝒔𝒖𝒍𝒆𝒔 𝑻𝒐𝒕𝒂𝒍 𝒊𝒏𝒔𝒖𝒍𝒊𝒏 × 𝟏𝟎𝟎 Equation 2 𝑳𝑪 % = 𝑻𝒐𝒕𝒂𝒍 𝒊𝒏𝒔𝒖𝒍𝒊𝒏 × 𝑨𝑬 𝑻𝒐𝒕𝒂𝒍 𝒘𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑵𝑪𝒔 × 𝟏𝟎𝟎 2.6. Conformational stability of loaded insulin To evaluate the conformational stability of insulin against preparative stress, circular dichroism (CD) spectroscopy was used. For this study, the destruction method used in section 2.4 above, could not be applied since the presence of Triton™ X-100 interfered with the spectrum reading during analysis. For this purpose, protamine and PSA-protamine NCs were completely disrupted using a combination of chloroform, methanol and acidified milliQ water at pH 3.46 to obtain a final concentration of insulin at 0.125 mg/ml. The aliquots were then centrifuged at 14000 rpm for 10 minutes at 25 °C to obtain a clear supernatant, which was collected and analysed for insulin conformation by CD. A solution of insulin in the same medium at a concentration of 0.125 mg/ml was used as a control. Spectra were collected at 20°C, using a 0.5 nm step size, over a wavelength range of 180–280 nm, using a 1 mm quartz cylindrical cell; a band width of 1 nm and a scanning speed of 500 nm/minute, 114 with a 0.25 second response time, were applied (Jasco J-1100; Jasco Corp, Tokyo, Japan). The α-helical content of the protein was estimated according to the following equation: Equation 3 % 𝜶 − 𝒉𝒆𝒍𝒊𝒄𝒂𝒍 𝒄𝒐𝒏𝒕𝒆𝒏𝒕 = 𝜽𝒎𝒓𝒅−𝟒𝟎𝟎𝟎 𝟑𝟑𝟎𝟎𝟎−𝟒𝟎𝟎𝟎 Where, θmrd is the mean molar ellipticity per residue at 208 nm (deg cm2 d/mol). Raw data from the experiment, expressed in terms of θd (the ellipticity in units of mdeg) were converted to mean molar ellipticity (θmrd) per residue, using the following equation: Equation 4 𝜽𝒎𝒓𝒅 = 𝜽𝒅 𝑴 𝟏𝟎𝑪𝑳𝑵 Where, M is the insulin molecular weight (Da), C is the insulin concentration (mg/ mL), L is the sample cell path length (cm), and N is the number of amino residues [25]. 2.7. Colloidal stability of protamine/polysialic acid NCs and release studies in simulated biological media The colloidal stability of the NCs was assessed by monitoring the size, PDI and count rate (particle concentration). Different simulated intestinal media were used (prepared according to the USP XXIV) in order to mimic the intestinal environment after oral delivery. Simulated intestinal fluid (SIF) at pH 6.8 and biorelevant media simulating preprandial and postprandial conditions in the upper small intestine, including fastedstate simulated intestinal fluid (FaSSIF) and fed-state simulated intestinal fluid (FeSSIF), were also employed in this study according to the updated (V2) versions described by Jantratid et al. in order to better mimic in vivo conditions. FaSSIF-V2 has a lower amount of lecithin, whereas FeSSIF-V2 combines the postprandial changes in pH, buffer capacity, osmolality, and bile component concentrations, in addition to lipolysis products; glyceryl monooleate and sodium oleate, with in vivo correlating concentrations (Table 1) [26, 27]. 115 Table 1: Compositions of simulated intestinal fluid (SIF), fasted state, simulated intestinal fluid (FaSSIF-V2) and fed state simulated intestinal fluid (FeSSIF-V2). Adapted from Jantratid et al. [27]. Composition/medium SIF FaSSIF-V2 FeSSIF-V2 Sodium taurocholate 3 mM 10 mM Lecithin 0.2 mM 2 mM Maleic acid 19.12 mM 55.02 mM Glyceryl monooleate - 5 mM Sodium oleate - 0.8 mM Sodium hydroxide 15.4 mM 34.8 mM 81.65 mM Sodium chloride 68.62 mM 125.5 mM Pancreatin - 100 units/mL Calcium chloride - 5 mM Monobasic potassium 50 mM - - pH 6.5 5.8 For the stability studies, insulin loaded protamine NCs were diluted 50x and incubated under moderate shaking at 37 °C in the different media: SIF, pH 6.8, FaSSIF-V2, pH 6.5 and FeSSIF-V2, pH 5.8. Samples were collected at times 0, 0.5, 1, 3 and 6 h. The FeSSIF-V2 samples were centrifuged at 5000xg for 5 minutes to eliminate pancreatin aggregates before analysis. The evolution of size distribution, PDI and count rate of the NCs were monitored by dynamic light scattering on a Zetasizer® Nano-ZS, Malvern, UK. For in vitro release studies, insulin loaded NCs were incubated in SIF, pH 6.8 and FaSSIF-V2, pH 6.5 at a ratio of 1:20, NCs to the medium and shaken at 100 rpm at 37°C using a constant-temperature shaker. At specified time intervals (0, 0.25, 0.5, 1, 3 and 6 h); the supernatant was collected by ultracentrifugation (section 2.2). The concentration of insulin in the supernatant was determined by reverse phase HPLC method (section 2.4). 2.8. Insulin stability in simulated intestinal fluids containing enzymes 116 The ability of protamine NCs to protect the associated insulin was evaluated by quantifying the amount of insulin remaining in the NCs after proteolysis. For this purpose, insulin-loaded protamine NCs, PSA-protamine NCs and free insulin solution (as control) were diluted by 1:1 v/v with FeSSIF-V2 then incubated in a 37 °C incubator and shaken at 100 rpm for up to 2 hours. At predetermined time intervals (0, 0.25, 0.5, 1 and 2 h), 500 μL aliquots were withdrawn and the enzyme activity of pancreatin in the samples was terminated by the addition of 300 μL ice-cold 0.1 M HCl. The samples were subsequently treated with acetonitrile (0.01 mL) and Triton x100 (0.01 mL) and vortexed, to disrupt the NCs and extract insulin, which was then analyzed by HPLC to determine the amount of insulin remaining in the NCs. 2.9. Stability during storage The colloidal stability of insulin-loaded NCs was followed during a period of 6 months at different temperatures (4 °C, 25 ºC and 40 °C) and RH conditions, as recommended by ICH guidelines. Samples of the three different batches were withdrawn at predetermined time intervals, followed by determining particle size, zeta potential and insulin leakage coupled with observing the suspension appearance to ensure continued stability. 2.10. Freeze-drying studies Insulin-loaded protamine NCs and PSA-protamine NCs (1% w/v) were lyophilized (Labconco Corp, USA) in presence/absence of trehalose or sucrose at 5 % (w/v) as cryoprotectors. Samples were frozen at -20 ºC and then subjected to an initial drying step at -40 ºC followed by a secondary drying at 0 ºC, both steps lasting for 43 hours at a high vacuum atmosphere (200 mTorr). Finally the temperature was increased slowly up to room temperature (+22 ºC) till the end of the process. The freeze-dried formulations were resuspended in ultrapure water by manual shaking and their physicochemical characteristics were evaluated as mentioned in section 2.3. 2.11. Statistical Statistical analysis was performed using GraphPad Prism 5 program (CA, USA). Oneway ANOVA in multiple comparisons was applied for comparisons. Differences were considered statistically significant at *p<0.05 and all results are expressed as mean ± SD. 117 3. RESULTS AND DISCUSSION In this study, protamine NCs were rationally designed taking into account different criteria: (i) their capacity to load and control the release of insulin, while protecting it from degradation in intestinal fluids; (ii) their colloidal stability in intestinal fluids; (iii) their penetration across the mucus layer and, (iii) their ability to interact with the underlying epithelium. 3.1. Preparation and characterization of protamine and protamine/polysialic nanocapsules Protamine NCs are reservoir-type systems composed of an oily core and a protaminecoating layer. As indicated earlier, all ingredients and their organization into the nanostructure were rationally selected. The selection of protamine was motivated by its cell penetrating properties [14] and its already shown capacity to enhance the intestinal insulin absorption [16]. On the other hand, a second layer around protamine was formed with PSA, with the idea of enhancing the colloidal stability in the presence of enzymes [21]. The lipid core, i.e. Miglyol (a medium chain Caprylic/capric triglyceride), of the NCs was expected to help protecting the peptide cargo from degradation as well as to enhance its intestinal permeability [28, 29]. The surfactants selected to disperse the oil in the external water phase were PEG stearate and bile salts. The presence of the PEG molecules oriented towards the external aqueous phase was thought to enhance the stability of the NCs in the presence of enzymes [30, 31], as well as promote their diffusion across the mucus [32, 33]. Two types of PEG-stearates with PEG chain lengths of 40 and 100 units (PEGst-40 and PEGst-100) were initially selected and compared. On the other hand, two different bile salts sodium cholate (SC) and sodium glycocholate (SGC), known for the penetration enhancing properties were also selected and compared. The structural organization of these nanocapsules is illustrated in Figure 1. The results of the physicochemical characterization of prototypes selected from an extensive screening of ingredients in different quantities are presented in Table 2. The chosen prototypes emanated from stability studies (discussed later on) and their acceptable physicochemical properties. The results indicate that for the compositions selected (described in the Materials and Methods section 2.2), the size of the NCs is 118 within the 200-300 nm range (PDI < 0.3) irrespective of their composition. On the other hand, the zeta potential values varied between -6 mV/+30 mV depending on the type of polymer shell (PSA vs. protamine) and also on the type of PEG stearate used (PEGst-40 vs. PEGst-100), (Table 2). The positive charge on protamine NCs, as compared to the negatively charged control nanoemulsions, is an indicator of the presence of the protamine shell around the lipid core. On the other hand, the type of surfactant also influenced the resultant surface charge. Namely, the use of SGC resulted in NCs with a higher negative charge, as compared to that of those produced with SC (p<0.05). This result may be attributed to the glycol moiety, which is present in SGC molecule but not in the SC molecule. Surprisingly, the presence of PEGst-100 (Mw 2KDa) gave a higher negative charge in the nanoemulsions and a higher positive charge in the nanocapsules compared to PEGst-40 (Mw 4KDa) (p<0.05). This result led us to hypothesize that, as the amount of PEGst added to the formulation was the same, the longer PEGst-100 chains are sparsely dispersed on the surface of the oily cores, thus leaving more bile salt acid molecules exposed to the surface. In a second step, nanocapsules containing PEGst-40 were provided with a second coating layer of PSA. The positive charge of the nanocapsules was supposed to enable the ionic interaction with PSA. The presence of this extra layer was confirmed by the charge inversion of the NCs containing PSA and protamine, as compared to those having only the protamine layer (Table 2). The chemical structure of PSA and the schematic representation of the components and their arrangement in the NCs are illustrated in Figure 1 and 2, respectively. The double layer PSA/protamine nanocapsules was expected to help improving the stability of the nanocapsules in the presence of proteolytic enzymes. This expected role, could be explained by two different mechanistic perspectives. First, it is known that both, macromolecules and nanocarriers coated/linked to hydrophilic polymers, i.e. PSA, are shielded from opsonisation [19, 20, 35], and this shielding results in their greater stability in the blood circulation. On the other hand, it has been reported that the presence of negatively charged functional groups, i.e. carboxylic moieties, in polymeric materials may result on the inhibition of the proteolytic enzymatic activity by chelating the Ca2+ ions required for normal enzymatic activity [35]. Despite these attractive properties of PSA, to our knowledge, there is no work reported, on the use of PSAbased nanocarriers for oral peptide delivery. 119 Figure 1: Chemical structure of polysialic acid (PSA), also named as colominic acid Table 2: Physicochemical properties of protamine and PSA-coated protamine NCs with their corresponding nanoemulsions, (mean ± SD, n=3, #*p<0.05 significant difference between nanoemulsions containing PEGst40 and PEGst100, and SC and SGC, respectively). Control nanoemulsions Protamine NCs PSA-Protamine NCs PEG Bile salt Size (nm) PdI ζ-pot (mV) Size (nm) PdI ζ-pot (mV) Size (nm) PdI ζ-pot (mV) 40 SC 198 ± 7 0.1 -13 ± 5 207 ± 18 0.1 +6 ± 5 200 ± 16 0.2 -4 ± 1* SGC 214 ± 15 0.2 -21 ± 2* 288 ± 20 0.2 +8 ± 4 261 ± 24 0.2 -6 ± 4* #100 SC 205 ± 16 0.2 -25 ± 2 210 ± 25 0.2 +28 ± 5 - - - SGC 221 ± 13 0.2 -32 ± 1* 238 ± 16 0.3 +30 ± 2 - - - The size of the nanocapsules was confirmed by TEM analysis. The images presented in Figure 3, also illustrate the spherical shape of the NCs and different appearance of the single layer protamine nanocapsules as compared to the double layer PSA/protamine nanocapsules. 120 Figure 2: Schematic representation of protamine NCs and PSA coated protamine NCs. Figure 3: TEM images of blank protamine NCs (A) and PSA-coated protamine NCs (B). 3.2. Mucodiffusion studies on protamine nanocapsules To check if the NCs are able to penetrate through mucus, their diffusion rate in mucin (SIF + 5% mucin) was studied in a preliminary mucodiffusion study where the 121 mucodiffusion enhancing property of PEGst-40 was compared to that of PEGst-100. Herein, protamine NCs containing PEGst-40 were found to diffuse more rapidly through the mucin gel compared to chitosan NCs. Interestingly, the substitution of PEGst-40 with PEGst-100 in the nanostructure led to a clear reduction in the mucodiffusion rate of the NCs. Protamine NCs containing PEGst-100 (Mw 5KDa) took twice the time (300 seconds) taken by protamine NCs containing PEGst-40 (Mw 2KDa) (150 seconds) to diffuse across 1 mm of the mucin gel. These results are in agreement with the higher positive charge observed for protamine nanocapsules containing PEGst 100, which may hamper the diffusion of these nanocapsules. As explained, the hypothesis is that PGEst-40-containing nanocapsules would have a more dense coating of low molecular weight PEG, as compared to those containing PEGst-100, and, would, thereby facilitate the mucodiffusion of the nanocapsules [36]. Similar results have been reported for polystyrene particles (200 and 500 nm). Those coated with 2 KDa PEG were found to diffuse faster through mucus compared to the same NPs with higher MW PEG [37]. 3.3. Insulin association and stability Taking into account that insulin is a hydrosoluble polypeptide with and IP of 4.75, the strategy for its entrapment within the oily core was based on tuning its solubility and ionization degree using different pH media. As expected, the results shown in Table 3, indicate that both, the pH of the insulin solution and the presence of cholate bile salts influence the insulin association efficiency to the NCs (*p<0.05). The highest AE % values were obtained when insulin was dissolved in 0.01M NaOH, pH 9.2 in the absence of a bile salt and also when insulin was dissolved in 0.01M HCl, pH 2 These, apparently contradictory results could be explained as follows. At pH 9.2, insulin has an important net negative charge, which may help its interaction with the positively charged protamine and, thus its encapsulation is favoured in the absence of a bile salt. On the contrary, association to the NCs was highly dependent on the type of bile salt. In this case, the positively charged insulin was supposed to interact with the negatively charged bile salt surfactants in the lipid core. The higher association observed for the nanocapsules containing SGC as compared to those containing SC, might be due to the presence of the glycol moiety in the SGC molecule. In fact, the longer hydrophilic side chain has been associated with higher interaction and activity of SGC compared to SC. [38]. 128 additional coating layer of PSA. It should also be taken into account that some of the observed insulin degradation could be attributed to its release in the medium as observed in section 3.4. Figure 9: Stability profiles of the associated insulin after incubation of (A) Protamine NCs and (B) PSA coated Protamine NCs in FeSSIF-V2 (mean ± SD, n=3, 0 20 40 60 80 100 120 0 15 30 45 60 75 90 105 120 Insulin reatained % Time (minutes) FeSSIF v2, pH 5.6 PrNC no Cholate PrNC + SC PrNC + SGC Free insulin #* * 0 20 40 60 80 100 120 0 15 30 45 60 75 90 105 120 Insulin retained % Time (minutes) FeSSIF v2, pH 5.6 PSA-PrNCs no Cholate PSA-PrNCs + SC PSA-PrNCs + SGC Free insulin #* #* * * 129 p<0.05 significant difference of PSA-PrNC+SGC compared to #PSA-PrNC+SC and *PSA-PrNC no Cholate). 3.7. Stability during storage To determine the stability of the NCs under storage conditions, insulin-loaded protamine NC suspensions were stored at 4 ºC and 25 ºC and size and insulin leakage (AE %), were monitored for a period of 6 months. Table 4 below shows the variations of size and AE of protamine NCs and PSA-protamine NCs over a period of 6 months. Both formulations maintained their nanometric size (and count rate) for the duration of the study at 4 ºC. However, both formulations were only stable for 7 days at room temperature (25 ºC). Table 4: Physicochemical properties and insulin leakage from protamine NCs and PSA-protamine NCs during storage at 4 °C, (mean ± SD, n=3). Initial 1 month 3 months 6 months Size ±SD nm AE± SD % Size ±SD nm AE± SD % Size ±SD nm AE± SD % Size ±SD nm AE± SD % PrNCs 396±24 68±8 394±41 52±5 344±63 41±4 374±47 31±4 PSA-PrNCs 341±54 59±6 314±32 50±3 278±45 43±2 277±21 37±5 3.8. Freeze drying studies The conversion of the colloidal NCs into a dried state was considered as a strategy to further incorporate the nanocapsules into a solid dosage form [42]. It has been previously reported that the addition of cryoprotectants to nanoformulations is necessary in order to protect them during the freezing process. In this study, different concentrations of insulin-loaded protamine NCs were freeze-dried in presence and/or absence of cryoprotectants (trehalose or sucrose at 5 %). As shown in Figure 10A and B, the reconstituted powders of both formulations protamine NCs and PSAprotamine NCs, maintain their initial nanometric properties even without the use of cryoprotectants. Therefore, the presence of the protamine coat and the combined PSA-protamine coat around the oily core of the NCs play an important protective role during freeze-drying [43]. This is a quite unique behaviour for the nanocapsules with important technological consequences as it avoids the dry dilution and, thus, the final drug loading of insulin in the powder. 130 Figure 10: Physicochemical properties (Particle size and zeta potential) of protamine NCs and PSA-protamine NCs after lyophilization at different NC concentrations (a) and at 11.3 mg/ml NC concentration in the presence of 5% sucrose or 5 % trehalose (b), after resuspension in water (mean ± SD, n=3). The results of the in vitro release of insulin from freeze-dried NCs was also determined. It is evident from Figure 11 that converting protamine based NCs into powder also improved their in vitro release profile by promoting more controlled release and reducing the amount of insulin initially release in the first 30 minutes (compared to NCs in suspension). This change in the release behaviour could be associated to the compaction of the NC’s shell during the freeze-drying process. This is a very promising feature for the nanocarrier system as it shows that converting the NCs into powder enhances the properties of the NCs by promoting controlled release and stability, which are advantages sought by employing the concept of nanomedicine. 18.66 (Before lyophilization) 18.66 11.3 0 100 200 300 400 500 NC concentration (mg/ml) Size (nm) Protamine NCs PSA-Protamine NCs No cryprotector Sucrose 5% Trehalose 5% 0 100 200 300 400 500 Size (nm) Protamine NCs PSA-Protamine NCs -8 -6 -4 -2 0 2 4 6 8 18.66 Before lyophilization 18.66 11.3 Z-potential (mV) NC concentration (mg/ml) PrNC PSA-PrNC -8 -6 -4 -2 0 2 4 6 8 No cryprotector Sucrose 5% Trehalose 5% Z-potential (mV) NC concentration (11.3 mg/ml) PrNC PSA-PrNC a) b) 131 Figure 11: In vitro release profiles in FaSSIF-V2 (B) of lyophilized Protamine NCs and PSA-Protamine NCs, resuspended in milliQ water to the initial volume. (Mean ± SD, n=3, *p<0.05). Finally, the effect of storage temperature and relative humidity on the stability of freeze-dried NCs was tested through an accelerated stability study, according to ICH conditions and guidelines. Freeze-dried insulin loaded protamine NCs and PSAprotamine NCs were kept at 25ºC ± 2ºC / 60% ± 5% RH, and their physicochemical properties were monitored and recorded over a period of 6 months. Figure 12 shows that the physicochemical properties of the NCs did not change during a period of the study. These results affirm the concept that freeze drying improves the stability of colloidal systems since the NCs stability which was initially compromised at room temperature for the NC suspensions was preserved by converting the NCs into dry powder. Moreover, the freeze-dried NCs were also stable in simulated intestinal media. 0 20 40 60 80 100 0123456 Insulin released % Time (hours) FaSSIF v2, pH 6.5 PrNC-Powder PSA-PrNC Powder * * 132 Figure 12: Physicochemical properties of freeze-dried protamine NCs and PSAprotamine NCs stored at 25ºC ± 2ºC / 60% ± 5% RH over a period of 6 months, (mean ± SD, n=3, *p<0.05). 4. CONCLUSIONS In this work we report for the first time NCs consisting of an oily core and double layer PSA/protamine as a candidate delivery technology for the oral administration of peptides. These NCs, which were rationally designed in order to confer them with the capacity to withstand the multiple barriers associated to the intestinal tract, were also able to load a significant amount of insulin and control its release in simulated intestinal media containing bile salts and proteolytic enzymes. Moreover, the conversion of the NCs into a powder led to an improvement of their stability at room temperature and of their controlled release capacity. Therefore, this novel nanocarrier system could be considered as a promising candidate for improving the oral delivery of peptide drugs, such as insulin. In vitro and in vivo mechanistic studies presented in the next chapter were aimed to elucidate the interaction of these nanocapsules with the intestinal epithelial barrier. 133 REFERENCES: 1. Choonara BF, Choonara YE, Kumar P, et al (2014) A review of advanced oral drug delivery technologies facilitating the protection and absorption of protein and peptide molecules. Biotechnol Adv. 32:1269–1282. 2. Du AW, Stenzel MH (2014) Drug carriers for the delivery of therapeutic peptides. Biomacromolecules 15:1097–114. 3. Niu M, Lu Y, Hovgaard L, et al (2012) Hypoglycemic activity and oral bioavailability of insulin-loaded liposomes containing bile salts in rats: the effect of cholate type, particle size and administered dose. Eur. J. Pharm. Biopharm. 81:265–72. 4. Mudassir J, Darwis Y, Khiang PK (2015) Prerequisite Characteristics of Nanocarriers Favoring Oral Insulin Delivery: Nanogels as an Opportunity. Int. J. Polym. Mater. Polym. Biomater. 64:155–167. 5. Fonte P, Nogueira T, Gehm C, et al (2011) Chitosan-coated solid lipid nanoparticles enhance the oral absorption of insulin. Drug Deliv. Transl. Res. 1:299–308. 6. Fonte P, Araújo F, Silva C, et al (2015) Polymer-based nanoparticles for oral insulin delivery: Revisited approaches. Biotechnol Adv. 7. Yoncheva K, Guembe L, Campanero MA, Irache JM (2007) Evaluation of bioadhesive potential and intestinal transport of pegylated poly(anhydride) nanoparticles. Int. J. Pharm. 334:156–165. 8. Makhlof A, Werle M, Tozuka Y, Takeuchi H (2011) A mucoadhesive nanoparticulate system for the simultaneous delivery of macromolecules and permeation enhancers to the intestinal mucosa. J. Control Release 149:81–88. 9. Damge C, Michel C, Aprahamian M, Couvreur P (1988) New approach for oral administration of insulin with polyalkylcyanoacrylate nanocapsules as drug carrier. Diabetes 37:246–251. 10. Prego C, Torres D, Alonso MJ (2005) The potential of chitosan for the oral administration of peptides. Expert Opin. Drug Deliv. 2:843–854. 11. Damgé C, Michel C, Aprahamian M, et al (1990) Nanocapsules as carriers for oral peptide delivery. J. Control Release 13:233–239. 12. Shan W, Zhu X, Liu M, et al (2015) Overcoming the Diffusion Barrier of Mucus and Absorption Barrier of Epithelium by Self-Assembled Nanoparticles 134 for Oral Delivery of Insulin. ACS Nano 9:2345–2356. 13. González-Aramundiz JV, Olmedo MP, González-Fernández Á, et al (2015) Protamine-based nanoparticles as new antigen delivery systems. Eur. J. Pharm. Biopharm. 97:51–59. 14. Reynolds F, Weissleder R, Josephson L (2005) Protamine as an efficient membrane-translocating peptide. Bioconjug. Chem. 16:1240–1245. 15. Xia H, Gao X, Gu G, et al (2011) Low molecular weight protaminefunctionalized nanoparticles for drug delivery to the brain after intranasal administration. Biomaterials 32:9888–98. 16. He H, Sheng J, David AE, et al (2013) The use of low molecular weight protamine chemical chimera to enhance monomeric insulin intestinal absorption. Biomaterials 34:7733–7743. 17. Pharmacopoeia E, Units I, The L, et al PROTAMINE SULPHATE Protamini sulfas Protirelinum. Test 85:2334–2335. 18. Liang JF, Zhen L, Chang L-C, Yang VC (2003) A less toxic heparin antagonist--low molecular weight protamine. Biochemistry (Mosc) 68:116–120. 19. Yin T, Yang L, Liu Y, et al (2015) Sialic acid (SA)-modified selenium nanoparticles coated with a high blood-brain barrier permeability peptide-B6 peptide for potential use in Alzheimer’s disease. Acta Biomater. 25:172–183. 20. Zhang T, She Z, Huang Z, et al (2014) Application of sialic acid/polysialic acid in the drug delivery systems. Asian J. Pharm Sci. 9:75–81. 21. Gregoriadis G, Jain S, Papaioannou I, Laing P (2005) Improving the therapeutic efficacy of peptides and proteins : A role for polysialic acids. 300:125–130. 22. García-Fuentes M, Torres D, Alonso MJ (2002) Design of lipid nanoparticles for the oral deli v ery of hydrophilic macromolecules. Colloids Surfaces B. Biointerfaces 27:159–168. 23. Moghimipour E, Jalali A, Abolghassem S, Tabassi S (2004) The Enhancing Effect of Sodium Glycocholate and Sodium Salicylate on Rats Gastro-intestinal Permeability to Insulin. 87–91. 24. Calvo P, Remunan-Lopez C (1997) Development of positively charged colloidal drug carriers:chitosan-coated polyester nanocapsules and submicronemulsions. Colloid Polym. Sci. 275:46–53. 25. Shu S, Zhang X, Teng D, et al (2009) Polyelectrolyte nanoparticles based on 135 water-soluble chitosan-poly(L-aspartic acid)-polyethylene glycol for controlled protein release. Carbohydr. Res. 344:1197–204. 26. Jantratid E, Janssen N, Reppas C, Dressman JB (2008) Dissolution Media Simulating Conditions in the Proximal Human Gastrointestinal Tract: An Update. Pharm. Res. 25:1663–1676. 27. Jantratid E, Dressman J (2009) Simulating the Proximal Human Gastrointestinal Tract : An Update. Dissolution Technol. 21–25. 28. Marten B, Pfeuffer M, Schrezenmeir J (2006) Medium-chain triglycerides. Int. Dairy J. 16:1374–1382. 29. Traul KA, Driedger A, Ingle DL, Nakhasi D (2000) Review of the toxicologic properties of medium-chain triglycerides. Food Chem. Toxicol. 38:79–98. 30. Taylor MJ, Tanna S, Sahota T (2010) In vivo study of a polymeric glucosesensitive insulin delivery system using a rat model. J. Pharm Sci. 99:4215– 4227. 31. Parveen S, Sahoo SK (2011) Long circulating chitosan/PEG blended PLGA nanoparticle for tumor drug delivery. Eur. J. Pharmacol. 670:372–83. 32. Ensign LM, Cone R, Hanes J (2012) Oral drug delivery with polymeric nanoparticles: The gastrointestinal mucus barriers. Adv. Drug Deliv. Rev. 64:557–570. 33. Vila A, Gill H, McCallion O, Alonso MJ (2004) Transport of PLA-PEG particles across the nasal mucosa: Effect of particle size and PEG coating density. J. Control Release 98:231–244. 34. Bader R a, Wardwell PR (2014) Polysialic acid: overcoming the hurdles of drug delivery. Ther. Deliv. 5:235–7. 35. Pisal DS, Kosloski MP, Balu-Iyler S V. (2011) Delivery of Therapeutic Proteins. NIH Public Access 99:1–33. 36. Inchaurraga L, Martín-Arbella N, Zabaleta V, et al (2015) In vivo study of the mucus-permeating properties of PEG-coated nanoparticles following oral administration. Eur. J. Pharm. Biopharm. 97:280–289. 37. Lai SK, Wang YY, Hanes J (2009) Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues. Adv. Drug Deliv. Rev. 61:158–171. 38. Holm R, Mullertz A, Mu H (2013) Bile salts and their importance for drug absorption. Int. J. Pharm. 453:44–55. 39. Liu J, Gong T, Wang C, et al (2007) Solid lipid nanoparticles loaded with 136 insulin by sodium cholate-phosphatidylcholine-based mixed micelles: Preparation and characterization. Int. J. Pharm. 340:153–162. 40. Prego C, Torres D, Fernandez-Megia E, et al (2006) Chitosan–PEG nanocapsules as new carriers for oral peptide delivery. J. Control Release 111:299–308. 41. Yamamoto a, Taniguchi T, Rikyuu K, et al (1994) Effects of various protease inhibitors on the intestinal absorption and degradation of insulin in rats. Pharm. Res. 11:1496–500. 42. Abdelwahed W, Degobert G, Fessi H (2006) Investigation of nanocapsules stabilization by amorphous excipients during freeze-drying and storage. Eur. J. Pharm. Biopharm. 63:87–94. 43. Prego C, García M, Torres D, Alonso MJ (2005) Transmucosal macromolecular drug delivery. J. Control Release 101:151–162. 137 CHAPTER 3 The interaction of protamine nanocapsules with the intestinal epithelium This work was done in collaboration with: 1 Veneto Nanotech, Italy (Dr. F. Bennetti). 2 Uppsala University, Sweden (Dr. P. Lundquist, Prof. P. Artursson). 144 sodium bicarbonate buffer (1 mL, pH 8,60) and TAMRA (10 mg/mL in DMSO) was slowly added under mild stirring. After 1 h incubation (mild stirring) at room temperature, the labelled protamine was dialyzed for 72 h to remove free TAMRA (SnakeSkin, cellulose membrane MW 3.5 KDa, Thermo, Spain). The obtained polymer conjugate (TAMRA-protamine) was freeze-dried and NCs were prepared according to the procedure described in 2.2. 2.2.3. Determination of particle size and zeta potential distribution The mean size and polydispersity index (PDI) of the particle size distribution of protamine NCs were measured after dilution with ultrapure water by dynamic light scattering using a Zetasizer Nano series DTS 1060 (Malvern instruments, Malvern, UK). The zeta potential was measured by laser-Doppler anemometry after diluting the samples in water (Zetasizer®, NanoZS, Malvern Instruments, Malvern, UK). 2.2.4. Insulin association efficiency (AE %) and loading capacity (LC %) of the nanocapsules The association efficiency (AE %) of insulin was determined after isolation of the NCs. The amount of free insulin in the aqueous phase (indirect method) and also that associated to the NCs (direct method) was determined by HPLC (described below). For the extraction of insulin from the NCs, 0.1 mL of the NCs suspension (concentration: 18.6 mg/mL) were mixed with acetonitrile (0.1 mL), 0.1 % trifluoroacetic acid (TFA) (0.75 mL) and Triton™ X-100 (0.05 mL). The mixture was vortexed at a high speed to obtain a clear solution. Quantification of insulin by HPLC The amount of insulin loaded into the NCs was quantified by HPLC (Agilent model 1100 series LC and a diode-array detector set at 214 nm). The chromatographic system was equipped with a reversed-phase 125 × 4 mm Supersphere®100RP-18e125-4 column (particle size 4 µm). The mobile phase, eluted at 1 mL/min, was a mixture of phosphate buffer (0.1 M, pH 2.3) and acetonitrile (44:56, v/v). The column was set at 35°C and the injection volume was 10 µL. Calibration curves ranging from 5 µg/mL up to 1,050 µg/mL (r2=0.999) were obtained. The limit of quantification (LOQ) and limit of detection (LOD) were 200 µg/mL and 80 μg/mL, respectively. 145 Samples were transferred into auto-sampler vials, capped and placed in the HPLC auto-sampler. Each sample was assayed in triplicate. The concentration of insulin in the aliquots was used to calculate the association efficiency (AE %) using equation 1 Equation 5 𝐴𝐸 % = 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑖𝑛𝑠𝑢𝑙𝑖𝑛 𝑖𝑛 𝑑𝑒𝑠𝑡𝑟𝑢𝑐𝑡𝑒𝑑 𝑁𝐶𝑠 𝑇𝑜𝑡𝑎𝑙 𝑖𝑛𝑠𝑢𝑙𝑖𝑛 × 100 2.2.5. Assessment of in vitro behaviour of nanocapsules in cell culture media: insulin release, TAMRA release and colloidal stability Insulin loaded TAMRA-labelled NCs were incubated in Hank´s Balanced Solution Salt (HBSS), used for in vitro cellular assays, at a dilution ratio of 1:50, NCs:HBSS (v/v), a final NC concentration of 0.37 mg/mL, and shaken at 100 rpm at 37 °C. At specified time intervals (0, 0.5, 1, 2, 4 and 6 h), insulin released was evaluated after isolating the NCs by ultracentrifugation (Avanti® J-E, Ultracentrifuge, Beckman Coulter, USA) at 30,000 rpm for 1 h (at 15 ºC). The concentrations of insulin in the aqueous medium were determined by reverse phase HPLC method (section 2.2.3.). The stability of the NCs was determined in cell culture medium DMEM and transport buffer HBSS. Briefly, the NCs were diluted 50x with the relevant medium, DMEM or HBSS (NC concentration: 0.37 mg/mL) and shaken at 100 rpm at 37 °C. The size and count rate (number of particles) of the NCs were monitored at specific time intervals (0, 0.5, 1, 2, 4 and 6 h). The absence of leakage of TAMRA from the NCs during the transport studies was also evaluated. Insulin loaded TAMRA-labelled NCs (1 mL of 2 mg/mL) were placed in dialysis devices (Spectra/Por Float-A-Lyzer G2, MWCO: 5KD, Spectrum Laboratories, USA) and introduced into 50 mL of HBSS, maintained under magnetic stirring at 37 °C. At pre-determined time points (0, 0.5, 1, 2 and 4 h), samples were withdrawn from the medium and analysed using the fluorescence reader, (SpectraMax M3, Molecular devices) at 583 nm wavelength. The free TAMRA and TAMRAprotamine conjugate were also used as controls. 146 2.3. In vitro cell culture studies 2.3.1. Cell cultures Caco-2 cells (clone 1) were kindly provided by Dr Federico Benetti (Veneto nanotech, Venice, Italy) and used from passage 18 to 28. Human Burkitt's lymphoma Raji B cell line was purchased from American Type Culture Collection (Manassas, VA, USA) and used between passages x+5 to x+10. Caco-2 cells were grown in DMEM supplemented with 10% (v/v) inactivated fetal bovine serum, 1% (v/v) non-essential amino-acids (NEAA), 1% (v/v) L-glutamine, and 1% (v/v) of penicillin–streptomycin (PEST) at 37 °C under a 10% CO2/90% air atmosphere. Raji cells were grown in suspension, cultivated in RPMI medium supplemented with 10% (v/v) inactivated fetal bovine serum, 1% (v/v) NEAA, 1% (v/v) L-glutamine, and 1% (v/v) PEST, at 37 °C in a 5% CO2/95% air atmosphere. HT29-MTX cells were kindly provided by Dr T Lessufleur (INSERM UMR S 938, Paris, FR) and used between passages x+16 and x+20 [20]. HT29-MTX cells were grown in DMEM supplemented with 10 % (v/v) heat inactivated fetal bovine serum, 1% (v/v) NEAA, 1% (v/v) L-glutamine, and 1% (v/v) PEST in a humidified incubator with 5 % CO2 atmosphere at 37 °C. 2.3.2. Cytotoxicity studies Cell viability was assessed after the co-incubation of 20,000 Caco-2 cells/well on a 96-well tissue culture plate (Costar® Corning® CellBIND Surface) with the formulations, protamine NCs and PSA-protamine NCs in dispersion in culture medium (concentration range: 0.5 to 8 mg/mL). After 2, 6 and 24 h of incubation, the cells were rinsed with PBS and incubated at 37 °C for 3 h with 100 μL 0.5 mg/mL 3- (4,5-dimethylthiazol-2-yl)-(2,5-diphenyltetrazolium bromide) solution (SigmaAldrich, Belgium) (MTS assay). Cellular supernatants were then transferred into a new 96-well plate and the amount of soluble formazan produced by cellular reduction of MTS was determined by recording absorbance at 490 nm with Synergy 4 microplate reader (BioTek Instruments, Inc., Winooski, USA). Before performing MTS assay, 50 µL of cell culture media were transferred into a new 96-well plate, mixed with 50 µL of working reagent for LDH detection (LDH assay) (Roche Diagnostics Belgium, Vilvoorde, Belgium), and incubated for 20 min at room temperature in the dark. The reaction was stopped by adding 25 µL of stop solution, and the amount of produced formazan was measured by recording 147 absorbance at 500 nm with Synergy 4 microplate reader [21]. The IC50s for the different formulations were calculated using the GraphPad Prism 5 program (California, USA). 2.3.3. Interaction of TAMRA-Protamine nanocapsules with Caco2 cell monolayers The interaction of TAMRA-protamine NCs and TAMRA-PSA-protamine NCs with Caco-2 cells was quantitatively and qualitatively studied using flow cytometry (FACS) and confocal laser scanning microscopy (CLSM), respectively. For the flow cytometry study, Caco-2 cells were seeded in 24-well cell culture plates at a density of 5x105 cells per well and allowed to adhere for 48 h until confluency. For the transport studies, cells were co-incubated with a-400 μL of a TAMRA-protamine NCs or TAMRA-PSA-protamine NCs suspension in transport buffer (NC concentration 1, 1.5 or 2 mg/mL) corresponding to a final insulin concentration of 4, 8 and 12 g/mL, respectively. After 2 h of co-incubation, cells were washed three times with PBS and detached from the plates by trypsinization. Cells were then centrifuged at 1,500 x g (Eppendorf centrifuge 5804 R, Daigger scientific Inc., USA) the supernatant was discarded and the cells were resuspended in PBS. Fluorescence was measured using a BD FACS Verse flow cytometer and BD CellQuest software (Becton Dickinson Biosciences, San Jose, CA, US). Cell fluorescence was quantified by measuring the fluorescence of TAMRA (red) at 583 nm. For cell viability measurements, the TOPRO-3 reagent was employed (blue). The reagent was added to each sample at a final concentration of 10 µg/mL, and, after 10 min of incubation, the fluorescence corresponding to dead cells was measured at 642 nm. For each sample, 10,000 events were collected. The data were subsequently analysed using the FlowJo data analysis software package (TreeStar, USA). For subsequent imaging, transwell inserts were fixed in paraformaldehyde (PFA) 4 % were gently washed in HBSS. Actin was stained with 200 μL of Alexafluor® 488 phalloidin (1:50) in buffered HBSS + 0.2 % (v/v) Triton X-100 for 10 min in the dark to reveal cell borders as described by des Rieux et al. [22]. Cell nuclei were stained with DAPI (1:20). Subsequently, inserts were washed in HBSS, cut and mounted on glass slides. Images were captured using a ZeissTM confocal microscope (LSM 150). 148 Data were analysed by the Axio Vision Software (version 4.8) to obtain y-z, x-z and xy views of the cell monolayers. 2.4. Transport across intestinal cell monolayers Cell culture models 2.4.1. Caco-2 and Raji cell monolayers (Follicle-associated epithelium model) Caco-2 cells were seeded at a density of 5 x 105 cells/well onto Matrigel® (BD Biosciences, Belgium) (10 μL/mL in DMEM)-coated Transwell® polycarbonate inserts (12 mm insert diameter, 3 μm pore size) (Corning Costar, Cambridge, U.K.) and cultivated over 21 days. The medium was replaced every second day. The inverted follicle-associated epithelium (FAE) model was obtained by co-culturing Raji and Caco-2 cells as previously reported by des Rieux et al. and Beloqui et al. [1, 23]. Briefly, after 3 to 5 days of Caco-2 seeding, inserts were inverted, a piece of silicone tube was placed into the inserts and maintained until day 21 in large Petri dishes. The medium was replaced every other day, until day 9 to 11 when Raji cells were then added on top of the Caco-2 cells for the conversion of the Caco-2 cells into M cells at a density of 2.5 x 105 cells/well [23]. For the assessment of FAE model functionality, in each experiment transport studies were conducted under the aforementioned conditions with commercial fluorescent carboxylated nanoparticles (0.2 μm) (Gentaur, Belgium). A nanoparticle suspension (400 μL at a concentration of 4.5x109 NPs/mL) was added on the apical side and inserts were incubated at 37 °C for 2 h. After this incubation time, basolateral solutions were then sampled and the number of transported nanoparticles was measured by FACS (BD FACS Verse). Nanoparticle transport was expressed as mean ± SD. 2.4.2. Caco-2 and Caco-2/HT29-MTXcell monolayers (mucus model) The influence of the mucus was studied by co-culturing Caco-2 and HT29-MTX, seeded at a density of 5 x 105 cells/well in a 3:1 ratio (Caco-2:HT29-MTX) and maintained until day 21 [24]. The medium was replaced every after two days. The Alcian blue staining technique was employed to demonstrate the presence of acid mucins and sulfated mucosubstances on the surface of the Caco-2/HT29-MTX co- 149 culture [19]. For this purpose, both the Caco-2 monoculture and the Caco-2/HT29MTX co-culture were grown and seeded as described above. After confirming the integrity of the monolayer by measuring the TEER, cells were washed 2 x with HBSS and fixed with 4 % Paraformaldehyde (PFA) for 30 min, then rinsed 3x with PBS and stained with 1 % Alcian blue at pH 2.5 for 1 h. They were then washed 3x with PBS, mounted on slides and viewed under a microscope (Axioskop 40, Carl Zeiss Microscopy, LLC, New York, United States) equipped with the Zen lite 2012 software for capturing images. In all cases the integrity of the monolayers was corroborated by measuring the transepithelial electrical resistance (TEER), before and after the transport studies on day 21. These measurements were carried out at 37 °C using an epithelial voltohm meter (EVOM, World Precision Instruments, Berlin, DE). TEER values over 250 Ωcm2 for both Caco-2 and Caco-2/HT29-MTX monolayers and values above 160 Ωcm2 for Caco-2/Raji monolayers were used. TEER values after transport studies were not significantly different to initial values unless otherwise stated. All transport experiments were conducted at 37 °C (or 4 °C) by adding a volume of 400 μL at 12 μg/mL insulin concentration in HBSS on the apical side and 1 mL of HBSS on the basolateral side. After the specific incubation time (1, 2 or 4 h), samples were collected from the basolateral side and the amount of fluorescence (TAMRA) was measured using a fluorescence plate reader (λEx=540 and λEm=570). The absence of cytotoxicity in the presence of the NCs was assessed by measuring the LDH activity released from the cytosol of damaged cells in the apical medium after each transport experiment (procedure in section 2.3.2). The cell monolayers were then washed twice in cold HBSS to stop endocytosis or uptake of NCs and fixed in PFA 4% for subsequent staining and imaging. 2.5. Mechanism of interaction of insulin–loaded protamine NCs with Caco-2 and Caco-2/Raji monolayers To evaluate the mechanism of interaction of protamine and PSA-protamine NCs with Caco-2 cells and Caco-2/Raji cells, the monolayers were pre-incubated for 1h at 37 °C with 400 μL of a solution endocytosis inhibitors in HBSS. Namely, 150 chlorpromazine at 10 μg/mL was used as an inhibitor of receptor-mediated and clathrin-mediated endocytosis [25, 26] and Filipin III (1 μg/mL), was used for the inhibition of caveolae and clathrin-mediated pathways by cholesterol depletion [27, 28]. After 1 h of incubation with the inhibitors formulations were added onto the apical side of the monolayer and co-incubated for 2 h. Control transport studies were carried out in transport buffer without inhibitor solutions. The absence of cytotoxicity in the presence of inhibitors was assessed by measuring the LDH activity released from the cytosol of damaged cells into the apical medium after the interaction experiments following the same procedure in section 2.3.2. 2.6. Interaction of TAMRA-protamine nanocapsules with human intestinal tissue Intestinal tissues collected immediately after the surgery, were immediately transferred into a vessel containing ice cold, oxygenated Krebs-Ringer buffer (KRB) and quickly transported to the laboratory. Upon arrival, the epithelium was dissected away from subepithelial tissues and mounted in horizontally as well as vertically oriented Ussing chambers with 9 mm openings between the two chambers. The basolateral chamber was filled with glucose containing KRB, while the glucose in the apical chamber was substituted with mannitol in order to avoid SGLT-induced tight junction opening. The chambers were kept at 37C and bubbled with 95% O2 /5% CO2 for the duration of the experiment. To assure continued tissue viability, the electrophysiology of the tissues was monitored throughout the experiment. After mounting the tissues were allowed to equilibrate for 40 min with two medium exchanges [29]. Protamine NCs and PSA-protamine NCs (5 mg/mL) were then added to the donor chambers. Aliquots of 20-100 µL, were withdrawn at 0, 30, 60, 90 and 120 min from the receiver and donor compartments. The sample volume in the receiver compartment was replaced with fresh KBR. At the end of the experiment continued viability of the tissues was tested by addition of the cAMP-agonist forskolin. Viable tissue with oxidative metabolism will form cAMP in response to forskolin leading to an opening of CFTR Cli channels, the response was monitored as changes in potential difference and short-circuit current over the epithelium. Permeability of fluorescently 151 labelled NCs was analysed by quantifying the amount of the fluorescence dye found in the receiver chamber using a plate fluorescence reader (λEx=540 and λEm=570) [29]. For imaging, the tissue specimens were rinsed and fixed in 4% formalin for 24h. Then they were embedded in paraffin and sectioned to a thickness of 5μm. Nanocapsules in each section were visualized by LSCM using the fluorescent marker TAMRA. 2.7. Statistics Statistical analysis was performed using the GraphPad Prism 5 program (CA, USA). Normal distribution was assessed with the Shapiro–Wilk normality test. One-way ANOVA in multiple comparisons followed by Tukey's post-hoc test was applied according to the result of the Bartlett's test of homogeneity of variances for the 37 °C and 4 °C transport comparisons. All other analyses were performed using a Student's t-test. Differences were considered statistically significant at *p<0.05. Results are expressed as mean ± SD. 3. Results and discussion In this work, we studied the interaction of protamine-based NCs with the intestinal epithelial cells and the mucus layer as important barriers to successful oral peptide delivery. Preceding work showed that NCs consisting of an oily core and a poly(alkyl cyanoacrylate) coating are potential carriers for improving insulin absorption [9]. The NCs studied here present the same structure, although the components were rationally selected and organized in order to overcome specific biological barriers, i.e. the mucus layer and the underlying epithelium. The selection of protamine was based on its its ability to penetrate cell membranes, as it is an arginine rich polypeptide [30, 31]. On the other hand, the formation of a double layer with PSA was aimed at increasing stability and enhance the penetration of the NCs through the mucus layer by avoiding the interaction with the extracellular matrix including proteins [32]. Finally, the surfactants, SGC and PEG-stearate were added to the nano-composition, with the objective of enhancing the penetration properties of the nanocarrier as well as their colloidal stability and mucodiffusion properties. 3.1. Physicochemical properties, stability and release behavior of protamine nanocapsules Insulin-loaded protamine NCs were successfully prepared using the simple solvent 152 displacement technique, a mild and easily scalable preparation technique. The particle size, zeta potential and association efficiency of the two formulations are summarized in Table 1. The particle size range for both formulations is between 300 – 400 nm. The zeta potential for protamine NCs was +6 ± 3 mV whilst PSA coated protamine NCs showed a slightly negative zeta potential (-4 ± 1 mV), confirming the presence of PSA on the outer shell. The insulin association efficiency was 62 ± 16 % and 51 ± 9 % for protamine and PSA-protamine NCs respectively. Table 1: Characteristics of protamine NCs and PSA-protamine NCs (mean ± SD, n=9, *p<0.05) Formulation Size (nm) ζ-potential (mV) AE % LC% Stability in HBSS Protamine NCs 382 ± 69 +6 ± 3 62 ± 16 1.0 ± 0.03 Stable (6h) PSA protamine NCs 301 ± 84 -4 ± 1 51 ± 9 0.8 ± 0.01 Stable (6h) In vitro stability and insulin release studies were performed to ensure that the transport buffer, HBSS, does not compromise the stability of the NCs and does not cause a burst release of the associated insulin. The results shown in table 1, indicate that the stability of the NCs was not compromised upon incubation in the transport buffer medium (HBSS) during 6 h at 37 °C. On the other hand, for both, protamine and PSA-protamine NCs, less than 30 % of the associated insulin was released in the same period of time (6 h). Comparable data were obtained when the NCs were incubated in SIF and SGF for 6 h, confirming their stability in simulated gastrointestinal media. Moreover, no TAMRA leakage from the NCs was observed over the period of 0 to 6h in HBSS. 3.2. Cytotoxicity of protamine nanocapsules in Caco-2 cells The cytotoxicity of the different types of NCs was studied in order to estimate the highest safe concentration of NCs that can be used for subsequent mechanistic studies. Two different incubation times, 2 h and 6 h were tested as they fall within the average transit time in the intestinal tract. As shown in Figure 1, protamine NCs were found to induce no significant cytotoxicity (p>0.05) after 6 h incubation at concentration range of 0.5 to 2 mg/mL. However, the highest concentration tested, 8 mg/ml, significantly decreased cell viability of the Caco-2 cells for both formulations 153 regardless of the incubation time, implying that cytotoxicity of protamine NCs is concentration dependent. Figure 1: Cytotoxicity of insulin-loaded protamine (PrNCs) and polysialic acid coated PSA-PrNCs on Caco-2 cell viability measured by MTS assay (mean ± SD, n=3, N=2, *p>0.05). 3.3. In vitro evaluation of nanocapsule interaction with the intestinal barrier 3.3.1. Intracellular uptake in Caco-2 and Caco-2/Raji monolayers The adherence and internalization of the fluorescent NCs into the Caco-2 monolayers was studied using confocal microscopy and flow cytometry. Figure 2(a) represents the y-z, x-z and x-y view of Caco-2 and FAE monolayers after 2 h incubation with TAMRA-labelled protamine NCs and TAMRA-labelled PSA-protamine NCs. The images show that the two formulations were internalized by the cells up to a certain extent, although a significant amount was also localized on the surface of the cell monolayers. Based on this, it could be expected that the NCs would release the associated insulin on the surface or into the enterocytes The interaction of the nanocapsules with the Caco-2 monolayers was also quantitatively analyzed by flow cytometry. In this study, the TOPRO-3 reagent was employed to measure cell viability and untreated cells were used as controls. As shown Figure 2(b), the cellular uptake of both, protamine NCs and PSA-protamine 0 0.5 0.75 1 1.5 2 8 0 20 40 60 80 100 120 Concentration of NCs (mg/mL) Cell viability (%) Pr NCs PSA-PrNCs *