New designs of peptidic and polymeric vectors for protein and gene delivery

! TESIS DOCTORAL NEW DESIGNS OF PEPTIDIC AND POLYMERIC VECTORS FOR PROTEIN AND GENE DELIVERY Marisa Juanes Carrasco ESCOLA DE DOUTORAMENTO INTERNACIONAL PROGRAMA DE DOUTORAMENTO EN CIENCIA E TECNOLOXÍA QUÍMICA SANTIAGO DE COMPOSTELA 2019 AUTORIZACIÓN DEL DIRECTOR / TUTOR DE LA TESIS New designs of peptidic and polymeric vectors for protein and gene delivery D. ....Juan Ramón Granja Guillán.................................................................................... D. ....Javier Montenegro García........................................................................................ INFORMAN: Que la presente tesis, se corresponde con el trabajo realizado por Dª. María Luisa Juanes Carrasco, bajo mi dirección, y a utorizo su presentación , considerando que reúne l os r equisitos exigidos en el R eglamento de Estudios de Doctorado de la USC, y que como director de esta no incurre en las causas de abstención establecidas en la Ley 40/2015. De acuerdo con el artículo 41 del Reglamento de Estudios de Doctorado, declara también que la presente tesis doctoral es idónea para ser defendida en base a la modalidad de COMPENDIO DE PUBLICACIONES , en los que la participación del doctorando/a fue decisiva para su elaboración. La utilización de estos artículos en esta memoria, está en conocimiento de los coautores, tanto doctores como no doctores. Además, estos últimos tienen conocimiento de que ninguno de los trabajos aquí reunidos podrá ser presentado en ninguna otra tesis doctoral. En Santiago de Compostela, ... de Septiembre de 2019 Fdo. Juan Ramón Granja Guillán Fdo. Javier Montenegro García ! DECLARACIÓN DE LA AUTORA DE LA TESIS New designs of peptidic and polymeric vectors for protein and gene delivery Dña. ...Marisa Juanes Carrasco.......................................................................................... Presento(mi(tesis,(siguiendo(el(procedemento(adecuado(al(Regulamento,(y(declaro(que:( 1) La(tesis(abarca(los(resultados(de(la(elaboración(de(mi(trabajo.( 2) De(ser(el(caso,(en(la(tesis(se(hace(referencia(a(las(colaboraciones(que(tuvo(este( trabajo.(( 3) (La(tesis(es(la(versión(definitiva(presentada(para(su(defensa(y(coincide(con(la(versión( enviada(en(formato(electrónico.( 4) Confirmo(que(la(tesis(no(incurre(en(ningún(tipo(de(plagio(de(otros(autores(ni(de( trabajos(presentados(por(mi(para(la(obteción(de(otros(títulos.( ( En Santiago de Compostela, ... de Septiembre de 2019 Fdo. Marisa Juanes Carrasco AGRADECIMIENTOS En numerosas ocasiones, la gente me pregunta si me he arrepentido alguna vez de haber hecho la tesis doctoral, y mi respuesta siempre es la misma, un NO rotundo. Ha sido una de las mejores decisiones que he tomado a lo largo de mi etapa de estudiante, primero porque me ha convertido en la química que soy hoy en día, y segundo, porque me ha permitido conocer a grandes personas, sin ellas, estos cinco años de tesis no habrían sido lo mismo. Por tanto, me gustaría mostrar mi agradecimiento a todos los que habéis hecho esta etapa posible. En primer lugar, me gustaría dar las gracias a uno de mis directores, Juan Granja, gracias por tus sabios consejos, tu cercanía, tu ayuda y tu confianza en mí a la hora de tomar alguna decisión relacionada con el día a día del laboratorio. A mi segundo director, Javier Montenegro, que sin conocerme, decidiste confiar en mí para empezar con vosotros la tesis doctoral, es algo por lo que siempre te estaré agradecida. Todos los logros que he podido alcanzar a lo largo de estos años, han sido gracias a que has conseguido contagiarme tu pasión por la química y tu positividad para poder afrontar nuevos proyectos, haciendo que haya ganado seguridad en mí misma. A todos los organismos e instituciones nacionales e internacionales que han financiado esta tesis doctoral y todos los proyectos de investigación en los que he podido participar. Al Ministerio de Economía y Competitividad por haberme concedido una beca FPI que me ha ayudado a desempeñar mi trabajo a lo largo de estos cinco años, además de haberme permitido realizar una breve estancia en el Instituto Curie de Paris, mi ciudad preferida en el mundo. A la Prof. Patricia Bassereau, por acogerme durante tres meses en su grupo de investigación en el Instituto Curie y darme la oportunidad de aprender y conocer cómo se trabaja en centros profesionales fuera de España. Al Dr. Ajay Mahalka por enseñarme tanto en tan poco tiempo. A los doctores Manuel Amorín, Rebeca García-Fandiño, Julián Bergueiro y al Prof. Luis Castedo por vuestros consejos y discusiones científicas en los seminarios de grupo, que sin duda me han ayudado en el avance de mis proyectos. A Patricia Lago, por tu amabilidad y por tu ayuda a la hora de resolver cualquier gestión relacionada con el progreso de mi tesis doctoral. A Irene, por enseñarme toda la biología que sé hoy en día, por ayudarme siempre que lo he necesitado, incluso encontrando un hueco que dedicarme cuando más ocupada podías estar, por escucharme y aconsejarme, por sorprenderme cada día con tu sabiduría y por grandes momentos de risas en el laboratorio, GRACIAS. A Iván, quien a lo largo de estos años, además de haber sido compañero de laboratorio y compinche de numerosas aventuras fuera de él, has conseguido ser una persona muy importante para mí y la amistad que ha nacido y ha ido creciendo entre nosotros no tiene igual. A Marta, Giulia, Eva, Victoria (Vicky para nosotros), Fede y Alicia, las locuras, las salidas y las conversaciones con vosotras serán inolvidables, gracias por escucharme y comprenderme y por estar a mi lado en los buenos y en los malos momentos. A Héctor y Alfonso, mis farmacéuticos favoritos, gracias por hacer que los días dentro y fuera del laboratorio hayan sido más llevaderos y alegres con vuestro exquisito sentido del humor y múltiples bromas, no cambiéis nunca. A Nacho y Lamas, los tenistas del grupo, me siento muy afortunada por haber compartido mis últimos años de tesis con vosotros, estoy segura al 100%, para Nacho al 99%, que sin vosotros no habrían sido tan especiales. A Jose, Ale y Ángel, por los buenos momentos que me habéis hecho pasar, siempre dispuestos a ayudarme cuando lo he necesitado. A Geert, Richard y Ghibom, gracias por darme la oportunidad de conoceros y poder aprender de vosotros. A Juanillo, Lionel, Arcadio y Nuria, mis compañeros de inicio de tesis, gracias por haber hecho que mi aterrizaje en el laboratorio fuese tan agradable, gracias por grandes momentos de risas y canciones y por animarme en numerosas ocasiones. A los que empezáis ahora la tesis, Sandra, Rebeca, Marcos y Ezequiel, mucho ánimo y no desesperéis si las cosas no salen a la primera, a veces un fracaso te abre la puerta hacia algo mejor. Tal vez cada uno de nosotros por separado seamos capaces de alcanzar alguna meta importante, sin embargo, hemos demostrado que trabajando juntos sumamos, habiendo sido capaces de crear lo que para mi es una gran familia. A David, Renata, Roberto, Damián y Borja, compañeros desde que empecé la carrera hace ya 10 años, cómo pasa el tiempo, gracias por vuestra amistad y amabilidad. A mis amigas de toda la vida Natalia y Julia, las risas, las alegrías, los lloros y las penas, han estado presentes durante casi 15 años de amistad, años que sin vosotras habrían sido mucho más difíciles de llevar. A Alberto, que aunque no lo sabías, nuestros caminos ya se habían cruzado mucho antes de haber empezado la carrera, y la química, quiso que de nuevo se volviesen a cruzar. Contigo he compartido momentos que me han hecho crecer como persona, gracias por escucharme siempre, por estar ahí, y por ser el motor que a veces necesito. Ya por último, pero no por ello menos importante, me gustaría dedicarle unas palabras de agradecimiento a mi familia, sin ella, haber llegado hasta aquí habría sido imposible. Gracias por enseñarme que con esfuerzo y dedicación se puede conseguir todo lo que uno se proponga. Gracias por todo el apoyo y por todos los consejos que me han enseñado cuál era el mejor camino a seguir y que han hecho de mí la persona que soy hoy en día. Gracias porque sé que cuando algo pueda salir mal, siempre vais a estar ahí para mí. 9 ABBREVIATIONS!..........................................................................................................................................!13! SUMMARY!.......................................................................................................................................................!17! RESUMEN!........................................................................................................................................................!21! GENERAL!OBJECTIVES!................................................................................................................................!25! CHAPTER!1:!THERAPEUTIC!PROTEINS!................................................................................................!29! 1"Introduction"........................................................................................................................................................"31" 1.1"Therapeutic"interest"of"direct"protein"delivery"..................................................................................."33" 1.1.1!General!interest!of!protein!delivery!.................................................................................................................................!33! 1.1.2!Potential!therapeutic!protein!targets!..............................................................................................................................!35! 1.1.2.1!Enzymes!and!regulatory!therapeutic!proteins!..................................................................................................!35! 1.1.2.2!Targeted!therapeutic!proteins!..................................................................................................................................!36! 1.1.3!Examples!of!therapeutic!proteins!.....................................................................................................................................!37! 1.1.4!Methods!for!therapeutic!protein!delivery!.....................................................................................................................!40! 1.1.4.1!Mechanical!methods!.....................................................................................................................................................!41! 1.1.4.2!Covalent!modification!methods!...............................................................................................................................!43! 1.1.4.3!Supramolecular!methods!............................................................................................................................................!46! 2!SUPRAMOLECULAR"RECOGNITION"AND"SELECTIVE"PROTEIN"UPTAKE"BY"PEPTIDE"HYBRIDS!.....................!51! 2.1"Precedents"and"Objectives"............................................................................................................................"53" 2.2"Results"..................................................................................................................................................................."55" 2.3"Conclusions"........................................................................................................................................................."65" CHAPTER!2:!GENE!EDITION!......................................................................................................................!67! 1!INTRODUCTION!.........................................................................................................................................................!69! 1.1"Therapeutic"interest"of"gene"edition"by"direct"protein"delivery".................................................."71" 1.1.1!Introduction!of!CRISPR!systems!........................................................................................................................................!71! 1.1.2!CRISPR/Cas9!technology!applications!............................................................................................................................!73! 1.1.3!Advantages!in!the!direct!delivery!of!CRISPR/Cas9!system!....................................................................................!74! 1.1.4!Previous!delivery!methods!for!CRISPR/Cas9!...............................................................................................................!76! 2!PEPTIDE/CAS9"NANOSTRUCTURES"FOR"RIBONUCLEOPROTEIN"CELL"MEMBRANE"TRANSPORT"AND"GENE" EDITION!..............................................................................................................................................................................!79! 2.1"Precedents"and"Objectives"............................................................................................................................"81" 2.2"Results"..................................................................................................................................................................."83" 2.3"Conclusions"........................................................................................................................................................."93" CHAPTER!3:!GENE!THERAPY!...................................................................................................................!95! 1!INTRODUCTION!.........................................................................................................................................................!97! 1.1"Therapeutic"interest"of"gene"delivery"......................................................................................................"99" 1.2"Types"of"nucleic"acids"used"in"gene"delivery"......................................................................................"100" 1.3"Gene"delivery"vectors"..................................................................................................................................."102" 1.4"Gene"delivery"barriers"................................................................................................................................."103" 1.5"Overcoming"gene"delivery"barriers"with"non-viral"vectors"........................................................"103" 1.5.1!Cargo/carrier!particle!formation!.....................................................................................................................................!103! 1.5.2!Increasing!the!circulation!time!of!nucleic!acids!........................................................................................................!105! 1.5.3!Targeting!approaches!...........................................................................................................................................................!106! 1.5.4!Improving!plasma!membrane!crossing!........................................................................................................................!109! 1.5.5!Endosomal!escape!and!cargo!release!............................................................................................................................!111! 1.5.6!Nuclear!import!.........................................................................................................................................................................!116! 2!MESSENGER"RNA"DELIVERY"BY"HYDRAZONE-ACTIVATED"POLYMERS!............................................................!119! 2.1"Precedents"and"Objectives"........................................................................................................................."121" 2.2"Results"................................................................................................................................................................"123" 2.3"Conclusions"......................................................................................................................................................"131" Summary 17 SUMMARY The expansion of new therapeutic technologies is limited by the effective, selective and controlled transport and release of the corresponding biomolecules to the different tissues, cells and organelles. Therefore, there is a need to develop conceptually new strategies to transport and deliver proteins or nucleic acids inside cells. There are multiple synthetic materials for the intracellular delivery of exogenous biomolecules, such as, liposomes, nanoparticles, etc., but, the use of cell-penetrating peptides (CPPs) and polymers, has become a promising alternative for the protection, effective internalization and controlled release of biomacromolecules such as large protein complexes or nucleic acids. Due to the high compartmentalization of biological systems, cells require a very efficient way to communicate and respond to external stimuli. This process is generally carried out by carbohydrate ligands and their specific cellular receptors, which are membrane proteins known as lectins. Therefore, membrane proteins, particularly lectins, and their carbohydrate ligands are optimal candidates for the selective cell targeting of drugs and probes. Among the group of carbohydrate-binding proteins, lectins are particularly relevant because they generally display multiple binding sites, which allow cooperativity and multivalent effects. They are considered to be the key to enter into cells and they are always overexpressed during tissue inflammation and cellular metastasis. The multivalent interactions of lectins with glycoconjugates strongly depend on the topological arrangement of the glycan moiety. Therefore, there is an opportunity for the development of molecular vehicles that selectively recognize lectins and that can be subsequently transported across the cell membrane. In the first chapter of this thesis, we present the synthesis of biocompatible peptide scaffolds that incorporate specific protein ligands in their structure such as glycans, for the recognition and cellular internalization of lectins. The designed peptide is characterized for having a helical structure and an amphipathic behavior, defined by one hydrophilic cationic domain (arginines) and a hydrophobic domain (leucines). In orthogonal disposition the peptide contains two alkoxyamine residues for the attachment of the specific protein ligand (mannose for Concanavalin A (ConA) recognition). Interaction studies (SPR and fluorescence anisotropy) revealed a high affinity of the mannosylated peptide with its target protein, with values of dissociation constant in the range of 10 µM. After peptide/protein complexation, internalization experiments were performed in cell lines such as HeLa or HepG2. Moreover, in this chapter we present a wide variety of control peptide hybrids, which demonstrated that the presence of two protein ligands and the helical behavior are both required for ConA interaction and internalization. Moreover, selectivity studies of the intracellular delivery of a mixture of proteins (ConA and Streptavidin), where the mannosyl peptide is compared with Lipofectamine2000, revealed that only the peptide was able to discriminate between both proteins. Finally, the substitution of the glycan moiety of the peptide for a biotin ligand allowed the intracellular delivery of Streptavidin. Based on these results, this work exhibits the high versatility of our strategy, as the concept presented here could be adapted with the correct selection of ligands for the delivery of other proteins with the appropriate supramolecular binding motifs. MARISA JUANES CARRASCO 18 Having confirmed the versatility of CPPs for the intracellular delivery of proteins, we decided to go one step forward and start a more challenging project focused in gene therapy. Gene-based therapy is one of the most promising tools for the future of human health. Nucleic acids (siRNA, pDNA, mRNA, etc.) and gene-editing nucleases (CRISPR/Cas9, etc.) can be applied to repair the malfunction or absence of an essential protein inside cells. The discovery of RNA guided endonucleases has emerged as one of the most important tools for gene edition and biotechnology. The selectivity and simplicity of the CRISPR/Cas9 strategy allow the straightforward targeting and edition of particular loci in the cell genome without the requirement of protein engineering. CRISPR is a family of DNA sequences in bacteria whose recognition forms the basis of the genome editing technology known as CRISPR/Cas9. This technology allowed scientists to make permanent modification to genes within organisms. However, the transfection of Cas9 is a challenging task due to its large size. CRISPR scientists typically transfect cells with plasmids containing instructions to make Cas9, which could lead to permanent DNA recombination and persistent expression, a situation that enhances potential off target effects and immunogenic responses. Therefore, the direct delivery of Cas9 ribonucleoprotein (RNP) constitutes an advantageous strategy for gene edition and other potential therapeutic applications of the CRISPR/Cas9 system. In recent reports, Lipofectamine and cell-penetrating peptides have been used for the direct delivery of Cas9, however, these strategies required Cas9 engineering and covalent fusion of the CPP to the protein, respectively. Considering these precedents, in the second chapter of this memory, we have developed a supramolecular strategy for the direct delivery of Cas9 RNP, based on amphiphilic scaffolds prepared by hydrazone bond formation between a cationic peptide core and hydrophobic aldehyde tails. HeLa cells expressing EGFP were incubated with peptide/Cas9 complexes using a guide RNA against EGFP. The results of this experiment revealed that oleic aldehyde was one of the best candidates for the direct delivery of Cas9 RNP. Therefore, we decided to further study the potential of this technology using this particular aldehyde in three different cell lines (HeLa, A549 and DF1). Edition efficiency of the peptide was compared with lipofectamine, observing comparable efficiencies but lower toxicity when using the peptide. After the peptide/protein interaction, non-covalent nanoparticles of around 200 nm were formed. Mechanistic studies demonstrated that peptide/Cas9 complexes were internalized in cells via macropinocytosis. To the best of our knowledge, this was the first supramolecular strategy for the direct delivery of Cas9 using a penetrating peptide vehicle. In the third chapter of this thesis we study the potential of previously described methodology of the group, for the delivery of messenger RNA (mRNA). The transfection of mRNA represents a challenge due to its low stability in cell media but, at the same time, it presents a great relevance because of the recent development of therapies based on the use of this biopolymer. In this direction, in collaboration with Prof. Francisco Fernández Trillo, polyhydrazide polymers functionalized with cationic aldehydes (t-guanidinium) and different hydrophobic aldehydes were used for mRNA internalization in Hek293 cells. For mRNA, it was necessary to use a higher molecular weight polymer than in our previous studies of Summary 19 siRNA and pDNA transfection. Initially, internalization experiments using different hydrophobic tails revealed that myristoleic aldehyde presented the most satisfactory results. DLS studies showed nanoparticles of 50-100 nm in size, in addition to an increase of the net charge when the concentration of polyhydrazone was augmented. This methodology presented similar transfection efficiencies when compared with other polymers such as PEI, but it significantly improved the activity of peptides such as octaarginine and GALA, as well as the cationic lipid DOTAP. These results, in addition to the lack of toxicity, confirmed the excellent properties of polyhydrazones for the delivery of mRNA. Resumen 21 RESUMEN La expansión de nuevas tecnologías terapéuticas está limitada por el transporte efectivo, selectivo y controlado de biomoléculas de interés, a los diferentes tejidos, células y orgánulos. Por tanto, existe la necesidad de desarrollar conceptualmente nuevas estrategias para el transporte y entrega celular de proteínas y ácidos nucleicos al interior de las células. Existen múltiples materiales sintéticos para la entrega intracelular de biomoléculas exógenas como los liposomas, nanopartículas, etc., sin embargo, el uso de los péptidos penetrantes en células y los polímeros, ha surgido como una alternativa prometedora para la protección, la efectiva internalización y la controlada liberación de biomacromoléculas como proteínas complejas de gran tamaño o ácidos nucleicos. Debido a la alta compartimentalización de los sistemas biológicos, las células requieren mecanismos eficientes para comunicarse y responder a estímulos externos. Este proceso es llevado a cabo generalmente por ligandos que contienen carbohidratos y sus receptores específicos celulares, que son proteínas de membrana conocidas con el nombre de lectinas. Por tanto, estas proteínas de membrana y sus respectivos ligandos son candidatos óptimos para la focalización selectiva de fármacos y sondas hacia el interior celular. Entre el grupo de proteínas de unión a carbohidratos, las lectinas son particularmente relevantes debido a que generalmente disponen de múltiples sitios de unión. Se consideran la llave para entrar a las células y siempre se sobreexpresan durante la inflamación de tejidos y la metástasis celular. Las interacciones multivalentes de las lectinas con glicoconjugados dependen en gran medida de la disposición del derivado de glicano. Por tanto, existe una oportunidad para el desarrollo de vehículos moleculares que selectivamente reconozcan las lectinas y por consiguiente sean capaces de transportarlas a través de la membrana celular. En el primer capítulo de esta tesis doctoral, se plantea la síntesis de péptidos biocompatibles con ligandos específicos de proteínas como los carbohidratos para el reconocimiento e internalización celular de lectinas. El péptido diseñado se caracteriza por tener una estructura helicoidal y un carácter anfipático, definido por un dominio hidrofílico y catiónico (argininas) y un dominio hidrofóbico (leucinas). En disposición ortogonal el péptido presenta dos residuos de alcoxiamina para el anclaje específico de los ligandos de proteína (manosa para el reconocimiento de la Concanavalina A (ConA)). Estudios de interacción (SPR y anisotropía de fluorescencia) revelaron una elevada afinidad en la unión del péptido con manosas por su proteína objetivo, con valores de constante de disociación en el orden de 10 µM. Una vez formados los complejos, se estudió su internalización en líneas celulares como las células HeLa o HepG2. En este capítulo se presentan además una gran variedad de péptidos híbridos control, que demostraron que tanto la presencia de los dos ligandos de manosa, así como el carácter helicoidal, son requisitos indispensables tanto para la interacción como para la internalización de la ConA. Se presentan además estudios de selectividad en el transporte de una mezcla de proteínas (ConA y estreptavidina) al interior celular, donde se compara el péptido con dos ligandos de manosa con la Lipofectamina2000, que demostraron que el péptido de manosa era capaz de discriminar entre las diferentes proteínas. MARISA JUANES CARRASCO 22 Una vez confirmada la versatilidad de los péptidos penetrantes para el transporte intracelular de proteínas, se decidió ir un paso más allá y empezar a trabajar en un proyecto más complicado centrado en terapia génica. La terapia basada en genes es una de las herramientas más prometedoras para el futuro de la salud humana. Los ácidos nucleicos (ARNi, ADN, ARNm, etc.) y las nucleasas de edición genética (CRISPR/Cas9, etc.) pueden ser aplicados para la reparación del mal funcionamiento o ausencia de una proteína esencial en el interior celular. El descubrimiento de las endonucleasas guiadas por ARN se ha convertido en una de las herramientas más importantes para la edición de genes y la biotecnología. La selectividad y la simplicidad de la estrategia CRISPR/Cas9 permite el transporte sencillo y la edición de lugares particulares en el genoma celular sin el requerimiento de la ingeniería de proteínas. CRISPR es una familia de secuencias de ADN en las bacterias cuyo reconocimiento forma la base de la tecnología de edición génica conocida como CRISPR/Cas9. Esta tecnología ha permitido a los científicos hacer modificaciones permanentes en los genes en el interior de los organismos. Sin embargo, la transfección de Cas9 es una tarea complicada debido a su gran tamaño. Los científicos que trabajan con CRISPR normalmente transfectan las células con plásmidos que contienen instrucciones para hacer Cas9 pero que podrían dar lugar a una recombinación de ADN permanente y a una expresión persistente del mismo, lo que aumenta los posibles efectos secundarios y las respuestas del sistema inmune. Por tanto, el transporte directo de la ribonucleoproteína (RNP) Cas9 constituye una estrategia ventajosa para la edición génica y otras aplicaciones potenciales terapéuticas del sistema CRISPR/Cas9. En publicaciones recientes, tanto la lipofectamina como los péptidos penetrantes han sido utilizados para el transporte directo de Cas9, sin embargo, estas estrategias requieren la ingeniería de Cas9 y la fusión covalente de los péptidos penetrantes a la proteína, respectivamente. En el segundo capítulo, se desarrolló una estrategia supramolecular para el transporte directo de la RNP Cas9, basada en estructuras anfifílicas formadas por enlaces hidrazona entre un péptido catiónico y colas de aldehídos hidrofóbicos. Las células HeLa, que expresaban EGFP, se incubaron con complejos péptido/Cas9 utilizando una guía de ARN contra la EGFP. Los resultados de este experimento revelaron que el aldehído oleico era uno de los mejores candidatos para el transporte de la RNP Cas9, por lo que se procedió al estudio de la edición génica de Cas9 utilizando este vector particular en tres líneas celulares diferentes (Hela, A549 y DF1). La eficiencia de la edición se comparó con la de la lipofectamina mediante estudios de dosis respuesta, en los que se observó que las eficiencias eran comparables, sin embargo el péptido resultó ser menos tóxico. Después de la interacción péptido/proteína, se formaron nanopartículas no covalentes de aproximadamente 200 nm de tamaño. Estudios mecanísticos demostraron que los complejos de péptido/Cas9 se internalizaban en la célula por macropinocitosis. Esta es la primera estrategia supramolecular para el transporte directo de Cas9 usando péptidos penetrantes. En el tercer capítulo de esta tesis, nos centramos en transporte de ARNm utilizando la metodología descrita con anterioridad por el grupo de investigación. La transfección de Resumen 23 ARNm representa un reto muy importante debido a su baja estabilidad en el medio celular, pero a la vez presenta una gran relevancia debido al reciente desarrollo de terapias basadas en la utilización de este biopolímero. En esta línea, en colaboración con el grupo de Francisco Fernández Trillo, se utilizaron polímeros de polihidrazida funcionalizados con aldehídos catiónicos (t-guanidinio) y diferentes aldehídos hidrofóbicos, para llevar a cabo la internalización de ARNm en células Hek293. Para este nucleótido fue necesario emplear un polímero con un peso molecular mucho más elevado que los utilizados en estudios previos de transfección de ARNi y plásmidos de ADN. Inicialmente se llevaron a cabo estudios de internalización utilizando diferentes colas hidrofóbicas, que revelaron que el aldehído miristoleico presentaba los resultados más satisfactorios. Estudios de DLS revelaron tamaños de partícula entorno a 50 y 100 nm, además de un aumento en la carga neta de los agregados al incrementar la concentración de polihidrazona en la mezcla. Esta metodología presentó una eficiencia similar a la de otros vehículos poliméricos como el PEI, pero superó notablemente el rendimiento de péptidos como la octaarginina y GALA, y el lípido catiónico DOTAP. Todos estos resultados sumados a la nula toxicidad confirmaron las excelentes propiedades de las polihidrazonas para el transporte de ARNm. GENERAL OBJECTIVES Chapter 1: Introduction 33 1.1 Therapeutic interest of direct protein delivery 1.1.1 General interest of protein delivery Proteins are one of the most physiological relevant biomolecules as they are key players in the regulation of numerous cellular activities, such as enzyme catalysis, signal transduction, gene regulation and apoptosis.9 Their dynamic and diverse functions in the cellular machinery offer interesting challenges and opportunities in biology and medicine. Many diseases are consequence of the malfunction, mutation, reduced expression or any other abnormalities of cellular proteins. Thus, the direct delivery of functional proteins inside cells to fight diseases might be a key step in many medical applications, such as cancer therapy,10 vaccination,11 regenerative medicine, 12 etc. Protein based therapeutics, such as antibodies, enzyme replacement therapy,13 receptor traps and cell surface ligands have changed the field of drug development and have the potential to impact all areas of medicine.14 Protein-based therapy, due to their structural complexity, offers advantages over low molecular weight drugs, such as high specificity and complexity functions that cannot be mimicked by simple chemical compounds. Additionally, there is a reduced possibility of causing adverse effects, as long as the body can naturally produce many of these therapeutic proteins and, therefore, they are well tolerated. Finally, they can provide effective gene replacement treatment avoiding the risk of insertional mutagenesis.15,16 From a financial point of view, the clinical development of protein therapeutics is faster than that of small-molecule drugs and, because of its exclusivity in form and function, companies currently pursue extensive patent protections.16 Furthermore, some of the proteins that have been targeted with this therapeutic approach are essential players in signaling pathways and, therefore, they are inaccessible to smallmolecule inhibitors, rendering them “undruggable” targets. In many cases, they work facilitating protein-protein interactions (PPIs),15 which are based on the contact of highly hydrophobic and relatively flat interfaces, making difficult the attachment and blocking of small molecules to these large surfaces. However, macromolecules such as other proteins or peptides could efficiently inhibit PPIs.17 This is where protein-based therapy becomes relevant, connecting the gap between small molecules inhibitors and large protein targets. The field of protein therapeutics has grown considerably since the introduction of the first therapeutic protein other than antibodies, the human insulin, which derived from recombinant 9 Z. Gu, A. Biswas, M. Zhao and Y. Tang, Chem. Soc. Rev., 2011, 40, 3638–3655. 10 X. Liu, F. Wu, Y. Ji and L. Yin, Bioconjug. Chem., 2019, 30, 305–324. 11 H. Nakagami, J. Cardiol., 2017, 70, 201–205. 12 E. M. Green and R. T. Lee, Physiol. Rev., 2013, 93, 311–325. 13 M. Solomon and S. Muro, Adv. Drug Deliv. Rev., 2017, 118, 109–134. 14 Y. Zhang, J. J. Røise, K. Lee, J. Li and N. Murthy, Curr. Opin. Biotechnol., 2018, 52, 25–31. 15 S. Du, S. S. Liew, L. Li and S. Q. Yao, J. Am. Chem. Soc., 2018, 140, 15986–15996. 16 B. Leader, Q. J. Baca and D. E. Golan, Nat. Rev. Drug Discov., 2008, 7, 21–39. 17 D. E. Scott, A. R. Bayly, C. Abell and J. Skidmore, Nat. Rev. Drug Discov., 2016, 15, 533–550. MARISA JUANES CARRASCO 34 DNA in 1982.18 In recent years, transcription factors,19 gene editing enzymes,20 metabolic enzymes21 and protein antigens22 have shown potential to provide treatments for an extensive number of permanent diseases. Unfortunately, they still have some drawbacks that might be overcome before being developed into therapeutics. One of the main challenges is their delivery into the cytoplasm. The key barriers of delivering target proteins inside cells are related with some of their own properties, such as large sizes, variety in surface properties and unstable tertiary structures. In addition, there are other major challenges for the direct delivery of therapeutic proteins into mammalian cells. First of all, native proteins can be degraded or inactivated when administered into serum, leading to immunogenicity effects.9 Then, the cell membrane can effectively block the entrance of macromolecules, requiring the modification of the protein cargo in order to make them permeable and selective to the correct tissue, something that is a very high cost process.23 Most of these proteins are delivered into the cell by endocytosis and, therefore, once inside the cell, most of proteins stay trapped in endosomes suffering protease-mediated degradation and exocytosis. Consequently, their interaction with their cytosolic targets is restricted. Thus, the therapeutic efficacy is limited due to their poor ability to escape from the endosomes.24 Finally, to transduce protein into cells it is also required, in addition to some genetic or chemical modifications of proteins to expose them to undesirable conditions (e.g. high temperature, pH, chemicals or organic solvents). All these modifications might affect protein stability and/or function.25 Recently, a strategy that allows the chemoselective remodeling of protein surfaces has been reported, with which it is possible to reduce the anionic character while increasing the hydrophobic properties.26 The protein surfaces are decorated with cationic (i.e. guanidinium, ammonium and imidazolium) and anionic groups (carboxylates) to increase protein stability. The strategy is based on the esterification of protein-carboxyl groups using diazo compounds providing a protein with the ability to access the cytosol.26 An alternative strategy for protein delivery into cells involves the use of a transfecting reagent capable of transporting them without the requirement of protein modification.27 The use of carriers presents its own set of challenges such as, the formation of electrostatic and hydrophobic interactions with the carrier to achieve the optimal efficiency with minimal 18 I. S. Johnson, Science, 1983, 219, 632–637. 19 M. G. Peterson and V. R. Baichwal, Trends Biotechnol., 1993, 11, 11–18. 20 D. A. Scott and F. Zhang, Nat. Med., 2017, 23, 1095–1101. 21 P. Mane and V. Tale, Int.J.Curr.Microbiol.App.Sci., 2015, 4, 17–26. 22 P. J. Tacken, B. Joosten, A. Reddy, D. Wu, A. Eek, P. Laverman, A. Kretz-Rommel, G. J. Adema, R. Torensma and C. G. Figdor, J. Immunol., 2014, 180, 7687–7696. 23 N. J. Yang and M. J. Hinner, Methods Mol. Biol., 2015, 1266, 29–53. 24 M. P. Stewart, A. Sharei, X. Ding, G. Sahay, R. Langer and K. F. Jensen, Nature, 2016, 538, 183–192. 25 A. Fu, R. Tang, J. Hardie, M. E. Farkas and V. M. Rotello, Bioconjug. Chem., 2014, 25, 1602–1608. 26 K. A. Mix, J. E. Lomax and R. T. Raines, J. Am. Chem. Soc., 2017, 139, 14396–14398. 27 A. Bolhassani, B. S. Jafarzade and G. Mardani, Peptides, 2017, 87, 50–63. 9 Z. Gu, A. Biswas, M. Zhao and Y. Tang, Chem. Soc. Rev., 2011, 40, 3638–3655. Chapter 1: Introduction 35 toxicity, the correct amount of protein, and the type of cells used for the internalization and their release into the cytoplasm. 1.1.2 Potential therapeutic protein targets Proteins have the most active and varied role of any macromolecule in the body. They catalyze reactions, display recognition and molecular transport functions at the membrane, offer intracellular and extracellular scaffolding support and carry molecules within a cell or from one cell to another.16 Among of the functions, it is estimated that the human genome has between 25000 and 40000 different genes, though the gene splicing and post-translational modification of proteins and their oligomerization increase even further this functional repertory.16 From the mechanistic point of view, the protein-related diseases represent an immense challenge to modern medicine, as they emerge when some of the functional proteins mutate changing their folding, aggregation and then tune their function. In addition, the up or down regulation of protein expression also represents a major challenge. However, these abnormalities exemplify a great opportunity to mitigate, up today, intractable diseases through exploiting new therapeutic approaches. The use of protein therapeutics can be classified in two main groups depending on their mechanism of action: enzymes and regulatory proteins, and special targeted proteins. 1.1.2.1 Enzymes and regulatory therapeutic proteins In this group, protein therapeutics operates by a classic paradigm in which a specific endogenous protein is down expressed, and therefore its deficit can be treated by the administration of exogenous proteins. These proteins can be classified in three categories depending on their function: a) Replacing a protein that is deficient or abnormally produced: these proteins are used in a range of conditions, for example the use of insulin for the treatment of diabetes. Insulin acts through insulin-receptor binding, outside the cell, and it does not require entering the cytosol. However, the administration of insulin is principally limited to parenteral routes, which could cause problems such as safety issues. The oral bioavailability of these biomolecules is very limited (<1%) due to their characteristic low permeability across the epithelium and the rapid degradation. Thus, the development of protein carriers to achieve both excellent mucus permeation and transepithelial absorption is needed.28 Moreover, due to their key function and broad distribution, lysosomes are central components of cells and play a relevant role in the maintenance of cellular and body-level homeostasis.13 Genetic deficiencies of lysosomal components, most commonly enzymes, known as “lysosomal diseases”, points to aberrant accumulations of undigested substrates, leading to multi-system pathologies, which are often fatal. Enzyme replacement therapy offers an opportunity to 28 W. Shan, X. Zhu, M. Liu, L. Li, J. Zhong, W. Sun, Z. Zhang and Y. Huang, ACS Nano, 2015, 9, 2345–2356. 16 B. Leader, Q. J. Baca and D. E. Golan, Nat. Rev. Drug Discov., 2008, 7, 21–39. 13 M. Solomon and S. Muro, Adv. Drug Deliv. Rev., 2017, 118, 109–134. MARISA JUANES CARRASCO 36 improve the quality of life and survival rate of patients. Yet, its therapeutic value is obstructed by side effects, resistance and, mainly the inability of recombinant enzymes to reach the central nervous system. b) Augmenting an existing pathway: the normal protein activity is enhanced in magnitude or timing. These techniques are successfully displayed in the treatment of haematopoietic defects. One important example of these is the use of recombinant erythropoietin (EPO) and its receptor (EPOR), which are indispensable for the survival, proliferation and differentiation of erythroid progenitor cells.29 EPO is a protein hormone that functions as the primary mediator of a general protective response to tissue hypoxia. This hormone is secreted by the kidney, which stimulates erythrocyte production in the bone marrow. Recently, it has been recognized that other tissues and organs also express EPO and its receptor, including the brain and the heart, independently of its effects on red blood cell mass.30 Thus, besides its hematopoietic activity, the protein also displays non-erythropoietic actions. Therefore new drug delivery systems are being thoroughly investigated in order to fulfill the specific release of EPO required for each therapeutic approach.29,30 c) Providing a novel function or activity: this group includes the use of exogenous proteins to display novel functions and the use of endogenous proteins to act at a different time or place in the body. One remarkable example is the use of human recombinant deoxyribonuclease I (DNASE1). This protein is normally found inside human cells and can be used to degrade the DNA of dying neutrophils in the respiratory tract of patients with cystic fibrosis, pneumonia or bronchitis.31 1.1.2.2 Targeted therapeutic proteins In the last years, it has been shown that the binding specificity of monoclonal antibodies and immunoadhesins can be exploited in numerous ways by recombinant DNA technology.16 In this group, most of the protein therapeutics take advantage of the antigen recognition sites of immunoglobulin (Ig) antibodies or the receptor-binding domains of native protein ligands, to induce an immune response and therefore, destroy specifically targeted molecules or cells. Alternatively, the combination of the receptor-binding domains of protein ligands with the fragment crystallizable (Fc) region of an Ig has also been used successfully.32 This region can specifically recognize and target molecules on the cell surface, enabling the destruction by the immune system by triggering the cell death. Moreover, the Fc region can target, endocytose and break down chemically and enzymatically a soluble molecule for its destruction, because 29 L. Calvillo, R. Latini, J. Kajstura, A. Leri, P. Anversa, P. Ghezzi, M. Salio, A. Cerami and M. Brines, Proc. Natl. Acad. Sci., 2003, 100, 4802–4806. 30 A. Murua, G. Orive, R. M. Hernández and J. L. Pedraz, Med. Res. Rev., 2011, 31, 284–309. 31 S. Shak, D. J. Capon, R. Hellmiss, S. A. Marsters and C. L. Baker, Proc. Natl. Acad. Sci., 1990, 87, 9188– 9192. 32 H. I. Park, H. W. Yoon and S. T. Jung, Trends Biotechnol., 2016, 34, 895–908. 16 B. Leader, Q. J. Baca and D. E. Golan, Nat. Rev. Drug Discov., 2008, 7, 21–39. Chapter 1: Introduction 37 of the recognition of the Fc region by the immune system. Protein therapeutics of this group have been used for the treatment of inflammatory diseases, such as arthritis and psoriasis.33 1.1.3 Examples of therapeutic proteins Protein therapeutics has appeared as a key strategy for the treatment of cancer, immunological diseases and metabolic disorders. Protein drugs have starting to be used in cancer therapy because of their high pharmacological potency, molecular specificity and low toxicity.10 Therapeutic proteins exploited for cancer treatment mainly includes cytokines, antibodies, enzymes, tumor antigens and pro-apoptotic proteins among others. Cytokines are a class of secreted or membrane-bound proteins that play an important role in the growth, differentiation and activation of immune cells via cell signaling.34 A variety of cytokines, such as tumor necrosis factors (TNFs), interleukins (ILs) and interferons (IFNs), have been intensively applied in clinical cancer treatment and Hepatitis C virus infections in the case of IFNs,35 due to their direct apoptosis-inducing action in tumor cells, and their indirect antitumor effects in the regulation of immune responses. Furthermore, the synergistic effects obtained from the combination of cytokines with chemotherapeutic drugs enhance the anti-cancer efficiency and the reduction of the side effects.36 Monoclonal antibodies (mAbs) are being developed as therapeutics to complement or to fill the gap that drugs or vaccines cannot reach.37 Therapeutic antibodies (ThAb) have emerged as the fastest-growing biopharmaceutical approach for the treatment of human diseases. They are especially important for emerging pathogens or for infectious diseases where antibiotic resistance or toxin-mediated pathogenesis are present. Antibodies have a unique structure, which determines the specific nature of the effector function, which need to be considered and integrated into the design when developing ThAb, to ensure the maximum efficacy and safety. The development of mAb technology38 provided the capability to generate antibodies with required specificities, which had a deep impact on medicine. A wide variety of monoclonal ThAb are currently licensed, with hundreds more in preclinical and clinical development. These therapeutic antibodies are administered for a varied range of conditions. However, the majority are used for cancer treatment, autoimmune disorders and transplantation.37 Interestingly, almost all these ThAb used an immunoglobulin G (IgG) backbone, which consists of two different fragments: the fragment antigen binding (Fab) region and the fragment crystallizable (Fc) region. The Fab region contains the 33 X. Cui, L. Chang, Y. Li, Q. Lv, F. Wang, Y. Lin, W. Li, J. D. Meade, J. C. Walden and P. Liang, Sci. Rep., 2018, 8, 7327. 34 G. Dranoff, Nat. Rev. Cancer, 2004, 4, 11–22. 35 P. S. Sung, E. C. Shin and S. K. Yoon, Int. J. Mol. Sci., 2015, 16, 23683–23694. 36 C. He, Z. Tang, H. Tian and X. Chen, Adv. Drug Deliv. Rev., 2016, 98, 64–76. 37 V. Irani, A. J. Guy, D. Andrew, J. G. Beeson, P. A. Ramsland and J. S. Richards, Mol. Immunol., 2015, 67, 171–182. 38 G. Köhler and C. Milstein, Nature, 1975, 256, 495–497. 10 X. Liu, F. Wu, Y. Ji and L. Yin, Bioconjug. Chem., 2019, 30, 305–324. MARISA JUANES CARRASCO 38 paratope, also called the antigen-binding site, and exerts direct effects through binding interactions with antigens. The Fc region interacts with a variety of auxiliary molecules to mediate indirect effector functions, which are significant against infectious diseases where cell responses are important for the efficient elimination of pathogens. An essential area of the antibody research is the expansion of the application of this therapeutic tool for the specific detection of molecules for diagnostics, visualization, and activity blocking.39 Despite the ability to generate antibodies against different proteins, numerous applications in basic research fields, clinical practice, and biotechnology are limited to cell membrane receptors or to extracellular antigens, such as membrane or secreted proteins. Except for small groups of autoantibodies, natural antibodies against intracellular targets cannot be used within living cells. This excludes the extent of a major class of intracellular targets, including cancerassociated molecules.39 Some of these targets are “undruggable” by small molecules because of their large flat contact areas and the absence of deep hydrophobic pockets, where small molecules can insert and disrupt their activity. Thus, the development of technologies, from direct physical methods to sophisticated delivery vehicles, for the targeted intracellular delivery of antibodies, their fragments, or antibody-like molecules is extremely important. Protein toxins constitute a defense mechanism against predation or a superior pathogenic competence upon the producing organism.40 They have been improved through evolution in poisonous animals/plants and pathogenic bacteria. During decades, a big effort has been invested in studying their mechanism of action, the way they contribute to pathogenicity and in the development of antidotes to neutralize their action. Furthermore, many research groups started to explore the pharmaceutical potential of these toxins when they are used to efficiently destroy essential cellular routes and/or damage the integrity of their target cells.40 Saporin-S6, also known as saporin, is a positively-charged plant toxin that belongs to the ribosome-inactivating protein (RIP) family, a class of enzymatic toxins that inactivates the ribosomes.41 Consequently, protein synthesis is shut down, which results in cell death.42 RIPs can be classified into type 1, consisting of a single-chain protein with enzymatic activity, and type 2, which can enter the cell through the interaction between their lectin moiety and the saccharide receptors present on the cell membrane.43 While type 2 RIPs can be extremely toxic due to the presence of lectin moiety and their ability to promote entry into target cells, the type 1 RIPs are much less harmful. Saporin belongs to type 1 RIPs, a family of proteins that enter the cell less efficiently than type 2,42 which in combination with a method to cross the cell membrane, becomes a very potent toxin, as its enzymatic activity is one of the highest of all RIPs. Their highly specific RNA N-glycosidase activity cleaves the glycosidic bond of a single adenine from the ribosomal RNA. This is the “Achilles’ heel” of the ribosome and the 39 T. A. Slastnikova, A. V. Ulasov, A. A. Rosenkranz and A. S. Sobolev, Front. Pharmacol., 2018, 9, 1–21. 40 A. Shapira and I. Benhar, Toxins, 2010, 2, 2519–2583. 41 G. Bergamaschi, V. Perfetti, L. Tonon, A. Novella, C. Lucotti, M. Danova, M. J. Glennie, G. Merlini and M. Cazzola, Br. J. Haematol., 1996, 93, 789–794. 42 Polito, L.; Bortolotti, M.; Mercatelli, D.; Battelli, M. G.; Bolognesi, A. Toxins, 2013, 5, 1698–1722. 43 Stirpe, F.; Barbieri, L.; Battelli, M. G.; Soria, M.; Lappi, D. A. Bio/Technology, 1992, 10, 405–412. Chapter 1: Introduction 39 complete removal of this base inhibits the ability of ribosomes to participate in the synthesis of proteins. Saporin-S6 is extremely resistant to high temperature, denaturation by urea or guanidine and to attack by proteolytic enzymes. It is also stable in response to the chemical modifications needed for its modification and conjugation procedures. All together these characteristics make saporin a very interesting candidate for therapeutic applications. RIPs have been extensively studied as the toxic moiety of a conjugate, due to their therapeutic potential, in a variety of human diseases. The conjugation of a cytotoxic RIP to a target-specific vehicle, as for example monoclonal antibodies (mAb), allows the selective killing of target cells.42 Several mAbs are now approved for clinical use and showed excellent cytotoxic activities against certain types of cancer resistant cells, in comparison to the treatment with mAbs alone.44 However, if the antibody is attached to a cytotoxic agent, its activity is increased and cancer cells can be killed.45 This kind of antibody/toxin conjugates is known as immunotoxin, which combine the potency from the toxin to kill cells and the specificity from the antibody.46 First-generation immunotoxins, prepared by the chemical conjugation of toxins to antibodies, showed a low efficiency in animal models because of lack of selectivity.40 The removal of the cell-binding domain from the toxin and its subsequent attachment to antibodies produced immunotoxins with a higher animal tolerance.44 Several of these second-generation immunotoxins have been evaluated in phase I trials in cancer patients, observing some anti-tumor activity. However, they were expensive to produce, chemically heterogeneous and their ability to penetrate into bulky tumors was reduced due to their large size. For all these reasons, a third-generation of immunotoxins was synthesized by using recombinant DNA techniques.44 The resulting immunotoxins were designed to contain only those portions of the antibody and toxin needed to recognize and kill a target cell. In Figure 2, three different generations of immunotoxins are represented. Figure 2. Schematic representation of the three different generations of immunotoxins. 44 Pastan, I.; Hassan, R.; FitzGerald, D. J.; Kreitman, R. J. Nat. Rev. Cancer, 2006, 6, 559–565. 45 Ayyar, B. V.; Arora, S.; O’Kennedy, R. Trends Pharmacol. Sci., 2016, 37, 1009–1028. 46 Alewine, C.; Hassan, R.; Pastan, I. Oncologist, 2015, 20, 176–185. 40 A. Shapira and I. Benhar, Toxins, 2010, 2, 2519–2583. 42 Polito, L.; Bortolotti, M.; Mercatelli, D.; Battelli, M. G.; Bolognesi, A. Toxins, 2013, 5, 1698–1722. BT C TT ss FIRST GENERATION SECOND GENERATION THIRD GENERATION single chain immunotoxin disulphide-linked immunotoxin variable Ab fragments B T Cimaginary toxin: B: binding domain T: translocation domain C: catalytic domain chemical linker T C CC MARISA JUANES CARRASCO 40 1.1.4 Methods for therapeutic protein delivery The direct intracellular delivery of proteins is not an easy task because each protein is challenging due to the unique properties of each protein such as size, surface charge, function and fragility. Additionally, production of proteins in sufficient quantity and quality is not always viable. Moreover, the cell membrane constitutes an effective barrier to hydrophilic macromolecules that could be damaged initiating irreversible cell death mechanisms.47 Most of the proteins are delivered inside cells following endocytic pathways.25 These methods generally trigger their entrapment in endosomes reducing the possibility of reaching the cytosolic targets and therefore, perform their biological activity. During the last years, a variety of different methods have been developed for delivering proteins inside cells. They can be classified in three broad categories: mechanical methods, covalently-modified proteins and supramolecular delivery approaches (Figure 3). Figure 3. Schematic representation of different classical protein delivery methods. 47 Chiper, M.; Niederreither, K.; Zuber, G. Adv. Healthc. Mater., 2018, 7, 1–21. 25 A. Fu, R. Tang, J. Hardie, M. E. Farkas and V. M. Rotello, Bioconjug. Chem., 2014, 25, 1602–1608. Microinjection Electroporation Pore formation Nucleus Endosome Endocytosis Endosomal escape Lysosome Therapeutic protein Protein carrier ++ + + ++ MECHANICAL METHODS COVALENT PROTEIN MODIFICATION SUPRAMOLECULAR DELIVERY SYSTEMS cell-penetrating peptides virus-like particles supercharged proteins covalent nanoconjugates polymer conjugates Endocytosis Difficuties for escaping from the endosomes Chapter 1: Introduction 41 1.1.4.1 Mechanical methods Mechanical methods are considered the most traditional procedures to achieve direct cytosolic delivery of proteins.25 This method is especially useful for in vitro investigations. Several techniques have been developed such as microinjection, electroporation, sonoporation, and more recently mechanical deformation and microfluidics electroporation. All these methods have in common their dependence on specific instrumentation and that can not be implemented in vivo very easily.47,15 Microinjection of protein solutions across the cell membrane with a syringe is a typical powerful method.39 Solutions can be injected either into the cytosol or within the nucleus. This method is preferable when temporal and quantitative precisions are required for success, but it cannot be used if the number of cells required for protein delivery is large. Electroporation is another method for transduction of macromolecules.47 This method consists on placing host cells between two electrodes in a solution containing the protein to be delivered. A brief, few microseconds to a millisecond, high voltage electric pulse is applied to the cell suspension creating temporary pores in the plasma membrane. Soluble proteins and other solutes can then diffuse in both directions through the cell membrane holes according to the Fick’s law of diffusion.48 Electroporation effectiveness depends on the robustness of the cell, protein parameters, transduction medium, and electrical pulse voltage. Fluorescence resonance energy transfer (FRET), based on fluorescent protein biosensors have been widely used for molecular activity visualization in living cells in real time with high spatiotemporal resolution. The delivery into cells of this FRET biosensor, in its protein form and using electroporation, has been reported.49 The principal advantage of electroporation is its applicability for transitory and stable transfection of all cell types. Furthermore, it is able to transfect a large number of cells in a short time, once optimal conditions are determined. However, the major drawback of conventional electroporation procedures is the substantial cell death caused by high voltage pulses, and the only partially successful membrane repair, which require the use of higher amounts of cells as compared to other transfection methods. Additionally, a novel vector-free method that uses reversible membrane permeabilization to achieve rapid intracellular delivery of cargos with varied composition, properties and size, has been reported.50 A delivery solution was developed containing low levels of ethanol as the permeabilizing agent. After a short incubation time, permeabilization is stopped by the incubation of the cells in a phosphate buffer saline solution that dilutes the ethanol and that is non-toxic to cells. With this strategy, proteins, messenger RNA, plasmid 48 Fick, A. J. Memb. Sci., 1995, 100, 33–38. 49 Sun, C.; Ouyang, M.; Cao, Z.; Ma, S.; Alqublan, H.; Sriranganathan, N.; Wang, Y.; Lu, C. Chem. Commun., 2014, 50, 11536–11539. 50 O’Dea, S.; Annibaldi, V.; Gallagher, L.; Mulholland, J.; Molloy, E. L.; Breen, C. J.; Gilbert, J. L.; Martin, D. S.; Maguire, M.; Curry, F.-R. PLoS One, 2017, 12, e0174779. 47 Chiper, M.; Niederreither, K.; Zuber, G. Adv. Healthc. Mater., 2018, 7, 1–21. 15 S. Du, S. S. Liew, L. Li and S. Q. Yao, J. Am. Chem. Soc., 2018, 140, 15986–15996. 25 A. Fu, R. Tang, J. Hardie, M. E. Farkas and V. M. Rotello, Bioconjug. Chem., 2014, 25, 1602–1608. MARISA JUANES CARRASCO 48 cell lines. In addition, this peptide carrier presents several advantages for protein therapy, including stability in physiological buffer, low toxicity, and reduced sensitivity to serum.81,82 Polymers are attractive protein drug delivery carriers, as their physical and chemical properties can be easily modified. Functional supramolecular polymers have the ability to undergo reversible switching of structure, shape and function in response to certain external stimuli, making them exceptional candidates for a wide variety of biomedical applications, such as drug and protein delivery, gene transfection, bioimaging and diagnosis, tissue engineering and biomimetic chemistry.83 Self-assembled hydrogels with extremely high water content (up to 50%) and highly tunable mechanical properties, have been used for the sustained release of proteins under in vitro conditions.84 These hydrogels, prepared from renewable cellulose derivatives, are easily processed and their simple preparation, availability from low-cost renewable resources, and the tunability of their mechanical properties are unique for important biomedical applications. Dextran, a complex and branched polysaccharide synthesized from sucrose by enzymes or produced by bacteria and yeast, has many applications for plasma volume expansion, thrombosis prophylaxis, peripheral blood flow enhancement and for the rheological improvement of, for instance, artificial tears.85 An example of dextran applications is the in situ formation of degradable hydrogels between dextran vinyl sulfones and multifunctional mercapto-PEG, which leads to the release of model proteins with different sizes, such as immunoglobulin G (IgG), bovine serum albumin (BSA) and lysozyme and basic fibroblast growth factor.86 Additionally, nanocarriers prepared from biocompatible polymers have the potential for effectively delivering proteins to subcellular sites by exploiting the advantages of polymer shielding, which can protect proteins, such as antibodies, from protein interaction, loss of affinity and decomposition.87 For example, polyion complex (PIC) micelles, which are formed when a block copolymer, with a neutral hydrophilic and anionic blocks, is mixed with counter-charged compounds, have been extensively used for intracellular protein delivery (Figure 7).88 In a recent report, PIC micelles have been prepared by charge-converted IgG antibody derivatives and (PEG)-poly [N-{N’-(2-aminoethyl)-2-aminoethyl}aspartamide] copolymers, whose contiguous 1,2-diaminoethane units provide selective destabilization of 81 Morris, M. C.; Depollier, J.; Mery, J.; Heitz, F.; Divita, G. Nat. Biotechnol., 2001, 19, 1173–1176. 82 Deshayes, S.; Morris, M.; Heitz, F.; Divita, G. Adv. Drug Deliv. Rev., 2008, 60, 537–547. 83 Dong, R.; Zhou, Y.; Huang, X.; Zhu, X.; Lu, Y.; Shen, J. Adv. Mater., 2015, 27, 498–526. 84 Appel, E. A.; Loh, X. J.; Jones, S. T.; Dreiss, C. A.; Scherman, O. A. Biomaterials, 2012, 33, 4646–4652. 85 Van Tomme, S. R.; Hennink, W. E. Expert Rev. Med. Devices, 2007, 4, 147–164. 86 Hiemstra, C.; Zhong, Z.; van Steenbergen, M. J.; Hennink, W. E.; Feijen, J. J. Control. Release, 2007, 122, 71–78. 87 Kim, A.; Miura, Y.; Ishii, T.; Mutaf, O. F.; Nishiyama, N.; Cabral, H.; Kataoka, K. Biomacromolecules, 2016, 17, 446–453. 88 Lee, Y.; Kataoka, K. Soft Matter, 2009, 5, 3810–3817. Chapter 1: Introduction 49 endosomal membranes, facilitating the endosomal escape and delivery of charge-restored IgG antibodies into the cytosol.87 Figure 7. Polymeric structures for PIC micelles formation and biomolecule encapsulation. Although great progress has been made in the field of functional supramolecular polymers for biomedical applications, this area still faces several significant challenges such as,74 their physicochemical heterogeneity, which results in polydisperse polymer mixtures with different protein loadings, low functionalization for drug conjugation in polymers like polyesters and challenges in achieving oral bioavailability and crossing mucosal barriers. Liposomes or lipid vesicles are one of the most traditional nanocarriers showing excellent modularity and easy preparation. Phospholipids form spherical vesicles that consist of an aqueous core surrounded by a lipid bilayer or multilayer. Phospholipids have a polar head group and two hydrophobic hydrocarbon tails, which usually are fatty acids with one tail having an unsaturated bond.89 Due to the aqueous core, a huge amount of water-soluble proteins can be encapsulated inside liposomes, maintaining its native structure. Lipids with long hydrocarbon chains and a low degree of unsaturation and branching have been used to form strongly packed liposomes. Furthermore, to minimize membrane defects and improve their stability, cholesterol has sometimes been incorporated to the liposomes.89,90 Lipid vesicles have been used for medical applications because of their ability to protect and deliver hydrophilic and hydrophobic cargos. Additionally, they have been used as carriers for drug delivery due to their biocompatibility with cell membranes and their capacity to add specific ligands to their surface. Although many protein drugs are becoming available with rapid advances in recombinant DNA technology, their poor stability for physiological conditions and their difficulty to cross the cell membrane have attracted attention for the development of artificial liposomal carriers for effective protein therapeutic delivery.91 89 Lee, K. Y.; Yuk, S. H. Prog. Polym. Sci., 2007, 32, 669–697. 90 Briuglia, M. L.; Rotella, C.; McFarlane, A.; Lamprou, D. A. Drug Deliv. Transl. Res., 2015, 5, 231–242. 91 Tan, M. L.; Choong, P. F. M.; Dass, C. R. Peptides, 2010, 31, 184–193. 74 Ekladious, I.; Colson, Y. L.; Grinstaff, M. W. Nat. Rev. Drug Discov., 2019, 18, 273–294. 87 Kim, A.; Miura, Y.; Ishii, T.; Mutaf, O. F.; Nishiyama, N.; Cabral, H.; Kataoka, K. Biomacromolecules, 2016, 17, 446–453. neutral hydrophilic block ionic block O n O O OH n HN O n HO O O n HO HO HN O n Nn PEG H2NO n PAAm PiPrOx O PHEA PHPMA PNIPAAM PGMA H N O NH3 n H N O HN NH3 n O m H N O O n m O NH3 O n O H N O O n O PLL poly (aminoalkyl aspartamide) poly (aminoalkyl methacrylate) PMAA PAsp PIC micelle Charged biomolecule MARISA JUANES CARRASCO 50 Cationic lipid formulations with the ability of delivering two different types of proteins into cells, enzyme and antibody, have been reported.92 A guanidinium-cholesterol cationic lipid bis (guanidinium)-tren-cholesterol was combined with dioleoyl phosphatidylethanolamine to efficiently transport the β-galactosidase enzyme intracellularly, confirming that protein structure and function were not altered as a result of complexation. Moreover, the aminoglycoside lipid dioleyl succinyl paromomycin (DOSP) associated with an imidazolebased helper lipid was shown to achieve intracellular delivery of biologically active anticytokeratin 8 antibody. The traditional unilamellar and multilamellar liposome systems present mechanical instability in physiological conditions, resulting in a fast release of the internally loaded content. As consequence, multivesicular liposomes have been developed to overcome this limitation. These multivesicular liposomes are formed by several internal aqueous chambers linked by a continuous and non-concentric network of lipid membranes, which results in a high ratio between aqueous volumes to lipids (Figure 8). This particular disposition provides a sequential release of the encapsulated proteins due to a unique disruption site in the external liposome membrane.89 Despite the fact that liposomes are typically made from natural, biodegradable, non-toxic and non-immunogenic lipid molecules, its clinical use, usually administered intravenously, presents some inconveniences as a result of their interaction with lipoproteins presented in blood, which leads to a premature release of the encapsulated drug.93 Figure 8. Morphology of multivesicular liposomes for the sequential release of therapeutic proteins. 92 Chatin, B.; Mével, M.; Devallière, J.; Dallet, L.; Haudebourg, T.; Peuziat, P.; Colombani, T.; Berchel, M.; Lambert, O.; Edelman, A.; Pitard, B. Mol. Ther. Nucleic Acids, 2015, 4, e244. 93 Lombardo, D.; Calandra, P.; Barreca, D.; Magazù, S.; Kiselev, M. Nanomaterials, 2016, 6, 125. 89 Lee, K. Y.; Yuk, S. H. Prog. Polym. Sci., 2007, 32, 669–697. outer lipid membrane internal aqueous chambers released protein inner lipid membrane network 2 SUPRAMOLECULAR RECOGNITION AND SELECTIVE PROTEIN UPTAKE BY PEPTIDE HYBRIDS Chapter 1: Supramolecular recognition and selective protein uptake by peptide hybrids 53 2.1 Precedents and Objectives The interactions between lectins and carbohydrates play an important role in a large variety of biological processes, such as cell-cell communication, cell adhesion, cell recognition, cell differentiation, host-pathogen interactions, signal transduction, intracellular trafficking of proteins, inflammation, metastasis and development of the neuronal network.94 Therefore, the delivery of exogenous lectins inside cells has been extensively studied in the fields of cell targeting and cancer therapy.95 Moreover, covalent binding between CPPs and large cargos has been extensively used for cell internalization of a huge variety of proteins.61 However, the conjugation of CPPs to proteins might often lead to a loss of biological activity, requiring the development of new non-covalent approaches. In the first chapter of this thesis, we will study amphiphilic cell-penetrating peptides with controlled secondary structure (α-helices), which will be modified with glycan ligands to recognize lectins and trigger their uptake into cells (Figure 9). The designed CPP will present two well-defined domains, one of them being hydrophilic and cationic while the other one will be hydrophobic. In the interface of these two domains and in orthogonal disposition, hydroxylamine moieties will be introduced, for the subsequent attachment of the corresponding protein recognition unit. Secondary structure of the peptide scaffold will be determined by circular dichroism. After the attachment of the corresponding ligand (i.e. mannose for Concanavalin A) protein interaction and uptake in two different cell lines will be studied using fluorescence anisotropy, surface plasmon resonance (SPR) and fluorescence microscopy. Moreover, we will perform studies of the uptake mechanism using different inhibitors. Selective protein transport assays will be performed using the mannosyl-modified CPP as compared with Lipofectamine2000, the non-viral transporter typically used in the literature. Finally, the glycan unit will be exchanged for a different protein ligand (i.e. biotin) to explore the extension of this methodology to different protein targets. Figure 9. Steps for the selective protein cell internalization using a CPP. Cationic domain (blue), hydrophobic domain (orange), protein recognition unit (manose, red). 94 Cecioni, S.; Faure, S.; Darbost, U.; Bonnamour, I.; Parrot-Lopez, H.; Roy, O.; Taillefumier, C.; Wimmerová, M.; Praly, J. P.; Imberty, A.; Vidal, S. Chem. - A Eur. J., 2011, 17, 2146–2159. 95 Liu, B.; Bian, H. J.; Bao, J. K. Cancer Lett., 2010, 287, 1–12. 61 Mäe, M.; Langel, Ü. Curr. Opin. Pharmacol., 2006, 6, 509–514. Protein Recognition Clustering Binding Transport Release ConA https://onlinelibrary.wiley.com/doi/full/10.1002/chem.201800706 CHAPTER 2: GENE EDITION MARISA JUANES CARRASCO 74 only to increase yield, improve drought tolerance and increase growth in poor nutrient conditions, but also to produce crops with improved nutritional properties,111 as has been reported the efficient simultaneous multiple gene knockouts, native gene editing, and sitespecific gene integration in corn.112 From the therapeutic point of view, Cas9 has been developed as an antimicrobial agent that can be used to specifically target antibiotic-resistant and/or highly virulent strains of bacteria. For example, Cas9 reprogrammed to target virulence genes, kills only virulent Staphylococcus aureus.113 Recent reports about gene therapy applications showed that CRISPR/Cas9 systems corrected the genetic defect in mouse models of Duchenne muscular dystrophy (DMD),114 a monogenic disease caused by mutations in the gene encoding the protein known as dystrophin, necessary for muscle cell integrity. CRISPR/Cas9 system also presents potential applications for the treatment of viral infections such as HIV115 and hepatitis B,116 where the efficient identification and mutation of a specific region in the DNA sequences inhibits the viral gene expression and replication. Another important revolutionary application was the achievement of the precise genetic modifications in primates resulting from gene editing in embryos by the injection of Cas9-mRNA within gRNAs into one-cellstage embryos.117 A similar approach could be used to alter DNA in human embryos to prevent non-complex hereditary diseases, but also to attempt alteration of complex features, which has provoked an extensive ethical discussion, such as the recent controversial case of the twin Chinese girls with edited genomes.118 1.1.3 Advantages in the direct delivery of CRISPR/Cas9 system The S. pyogenes Cas9 (SpCas9) is a large protein (>1300 amino acids, 160 kDa) and as a consequence, its intracellular delivery is a challenging task.119 One of the main concerns when editing mammalian cells is the Cas9 format in a way that minimizes the size for more efficient delivery and at the same time preserves Cas9 activity, restricts immunological activation and limits off-target cleavage. For in vivo genome editing, three different possibilities of 111 Belhaj, K.; Chaparro-Garcia, A.; Kamoun, S.; Patron, N. J.; Nekrasov, V. Curr. Opin. Biotechnol., 2015, 32, 76–84. 112 Svitashev, S.; Young, J. K.; Schwartz, C.; Gao, H.; Falco, S. C.; Cigan, A. M. Plant Physiol., 2015, 169, 931– 945. 113 Bikard, D.; Euler, C.; Jiang, W.; Nussenzweig, P. M.; Goldberg, G. W.; Duportet, X.; Fischetti, V. A.; Marraffini, L. A.; Biotechnol, N. Nat Biotechnol, 2014, 32, 1146–1150. 114 Long, C.; McAnally, J. R.; Shelton, J. M.; Mireault, A. A.; Bassel-Duby, R.; Olson, E. N. Science, 2014, 345, 1184–1188. 115 Hu, W.; Kaminski, R.; Yang, F.; Zhang, Y.; Cosentino, L.; Li, F.; Luo, B.; Alvarez-Carbonell, D.; GarciaMesa, Y.; Karn, J.; Mo, X.; Khalili, K. Proc. Natl. Acad. Sci., 2014, 111, 11461–11466. 116 Zhen, S.; Hua, L.; Liu, Y. H.; Gao, L. C.; Fu, J.; Wan, D. Y.; Dong, L. H.; Song, H. F.; Gao, X. Gene Ther., 2015, 22, 404–412. 117 Niu, Y.; Shen, B.; Cui, Y.; Chen, Y.; Wang, J.; Wang, L.; Kang, Y.; Zhao, X.; Si, W.; Li, W.; Xiang, A. P.; Zhou, J.; Guo, X.; Bi, Y.; Si, C.; Hu, B.; Dong, G.; Wang, H.; Zhou, Z.; Li, T.; Tan, T.; Pu, X.; Wang, F.; Ji, S.; Zhou, Q.; Huang, X.; Ji, W.; Sha, J. Cell, 2014, 156, 836–843. 118 Cyranoski, D. Nature, 2019, 566, 440–442. 119 Kelton, W. J.; Pesch, T.; Matile, S.; Reddy, S. T. Chim. Int. J. Chem., 2016, 70, 439–442. Chapter 2: Introduction 75 delivering Cas9 and the gRNA are available (Figure 12): viral or plasmid DNA (pDNA), mRNA and ribonucleoprotein (RNP, complex formed by the protein Cas9 and the gRNA).120 Figure 12. Delivery strategies for the CRISPR/Cas9 system. The delivery of recombinant plasmids that encode the Cas9 protein and the guide RNA sequences under separate promoters is the most stable and cost-effective of all the aforementioned approaches. This strategy has attractive advantages due to its simplicity and the possibility of carrying out massive multiplex gene editing by the inclusion of multiple gRNAs expressed from the same plasmid. However, for therapeutic engineering of mammalian cells with CRISPR/Cas9, the use of plasmids is usually limited due to different restrictions. First of all, the delivery of pDNA could possibly increase off-target effects and induce undesired side effects due to the continued expression of Cas9 and the persistence of the protein in cells for several days post-transfection.121 Secondly, another obstacle for pDNA transfection is the necessity of nuclear internalization. Moreover, the random integration of all or a part of the pDNA into the host genome is difficult to detect and can result in permanent DNA recombination and persistent expression, a situation that enhances potential off target effects and immunogenic responses. Finally, pDNA application is often damaging to cells, which would activate cyclic GMP-AMP synthase activation.122 120 Wan, T.; Niu, D.; Wu, C.; Xu, F.-J.; Church, G.; Ping, Y. Mater. Today, 2019, 26, 40–66. 121 Kim, S.; Kim, D.; Cho, S. W.; Kim, J.-S.; Kim, J.-S. Genome Res., 2014, 24, 1012–1019. 122 Gao, D.; Li, T.; Li, X.-D.; Chen, X.; Li, Q.-Z.; Wight-Carter, M.; Chen, Z. J. Proc. Natl. Acad. Sci., 2015, 112, E5699–E5705. gRNA Cas9 gRNA Cas9 Cas9 Cas9 Cas9 mRNA mRNA/gRNA mix gRNA Cas9 mRNA DNA DNA CRISPR plasmid RNP Cas9/gRNA complex Nucleus Target cleavage Transcription RNP complex formation MARISA JUANES CARRASCO 76 In the case of mRNA delivery, the translation of mRNA into Cas9 protein happens in the ribosomes found in the cytoplasm, avoiding the difficult challenge of crossing the nuclear membrane.123 However, mRNA is relatively unstable, and it has to be combined with gRNA separately when mRNA is considered as a choice of delivery form. This suggests that a delivery vehicle should be capable of loading both mRNA and gRNA simultaneously for the genome editing application, thus promoting the difficulties in terms of loading and release. All these disadvantages could be overcome by the direct delivery of RNP. In this situation the Cas9 protein and gRNA can be complexed as one cargo that can be directly delivered into the target cells. One of the advantages of the direct delivery of Cas9 RNP by non-viral vectors is the expectation that off-target editing effects are reduced as compared with Cas9 pDNA delivery. Therefore, the direct delivery of the Cas9 RNP inside cells would be beneficial as it functions as a transitory effector that could be subsequently degraded. Cas9 direct delivery enables the transient accumulation of the protein in the nucleus avoiding the problems of plasmid integration, prolonged residual activity, off target effects and immune responses. 1.1.4 Previous delivery methods for CRISPR/Cas9 To date, most of the existing delivery methods of CRISPR/Cas9 have relied on either physical methods or viral vectors. A variety of physical methods, such as electroporation,124,125 hydrodynamic injection126 and microinjection127 have been successfully applied for CRISPR/Cas9 delivery. However, they are considered to be less suitable for in vitro and in vivo delivery due to the difficulties of cell function maintenance as well as for practical applications. A more complex alternative is the packaging of the Cas9 DNA in a single-stranded form into a non-integrating virus such as the adeno-associated virus (AAV).128 However, the maximum loading capacity for AAV is around 4.5 kb (~160 kDa), which makes combination of both Cas9 and gRNA into a single capsid challenging, as the SpCas9 gene is almost at the loading size limit of viral vectors. Thus, smaller Cas9 variants have been developed, but they suffer from a reduction in their cleavage efficiency. Moreover, safety issues, such as carcinogenesis, insertional mutagenesis and immunogenicity, also limit the clinical translation of these viral vectors. Therefore, chemical methods of delivering Cas9 RNP system via non-viral vectors have the potential to address most of these limitations related with biosafety, loading and packaging capacities. 123 Glass, Z.; Lee, M.; Li, Y.; Xu, Q. Trends Biotechnol., 2018, 36, 173–185. 124 Qin, W.; Dion, S. L.; Kutny, P. M.; Zhang, Y.; Cheng, A. W.; Jillette, N. L.; Malhotra, A.; Geurts, A. M.; Chen, Y. G.; Wang, H. Genetics, 2015, 200, 423–430. 125 Fei, J.-F.; Knapp, D.; Schuez, M.; Murawala, P.; Zou, Y.; Pal Singh, S.; Drechsel, D.; Tanaka, E. M. npj Regen. Med., 2016, 1, 16002. 126 Yin, H.; Xue, W.; Chen, S.; Bogorad, R. L.; Benedetti, E.; Grompe, M.; Koteliansky, V.; Sharp, P. A.; Jacks, T.; Anderson, D. G. Nat. Biotechnol., 2014, 32, 551–553. 127 Horii, T.; Arai, Y.; Yamazaki, M.; Morita, S.; Kimura, M.; Itoh, M.; Abe, Y.; Hatada, I. Sci. Rep., 2015, 4, 4513. 128 Senís, E.; Fatouros, C.; Große, S.; Wiedtke, E.; Niopek, D.; Mueller, A.-K.; Börner, K.; Grimm, D. Biotechnol. J., 2014, 9, 1402–1412. Chapter 2: Introduction 77 Non-viral vectors have been successfully used for CRISPR/Cas9 delivery, both in vivo and in vitro, and innovative protocols about their complexation have been recently investigated and reported.129 Lipid nanoparticles are one of the most extensively used delivery carriers, which combine negatively charged nucleic acids with positively charged lipids.120 Cas9 nuclease protein complexed with polyanionic single guide RNA can be efficiently delivered in functional form into mammalian cells using cationic lipid formulations.130 Similar strategies using bioreducible lipid-like materials have been developed to load and deliver the anionic Cas9/gRNA complexes, 131 where the electrostatic assembly of nanoparticles mediates potent protein delivery and genome editing features (Figure 13). These bioreducible lipids deliver protein cargos into cells with high efficiency, facilitating their endosomal escape and directing proteins to their intracellular target site. Figure 13. Schematic representation of protein delivery and genome editing using a bioreducible lipid-like material and negatively charged Cas9/gRNA complex. Apart from lipid nanoparticles, polymers represent another appealing class of materials for designing efficient non-viral vectors for CRISPR/Cas9 systems.120 Recently, the direct conjugation of a cationic polymer branched PEI (bPEI) with SpCas9 protein through covalent bonding has been reported (Figure 14).132 The resulting bPEI-Cas9 conjugate was further attached with gRNA to form nanocomplexes to combat antibiotic resistance in bacteria. This polymer-conjugated Cas9 showed significant uptake and therefore higher editing efficiency as compared with the delivery of native Cas9 protein. Figure 14. Activation of bPEI using sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1carboxylate (SMCC) and reaction with the free sulfhydryl groups of SpCas9. 129 Givens, B. E.; Naguib, Y. W.; Geary, S. M.; Devor, E. J.; Salem, A. K. AAPS J., 2018, 20, 108. 130 Zuris, J. A.; Thompson, D. B.; Shu, Y.; Guilinger, J. P.; Bessen, J. L.; Hu, J. H.; Maeder, M. L.; Joung, J. K.; Chen, Z.; Liu, D. R. Nat. Biotechnol., 2015, 33, 73–80. 131 Wang, M. et al. Proc. Natl. Acad. Sci., 2016, 113, 2868–2873. 132 Kang, Y. K.; Kwon, K.; Ryu, J. S.; Lee, H. N.; Park, C.; Chung, H. J. Bioconjug. Chem., 2017, 28, 957–967. 120 Wan, T.; Niu, D.; Wu, C.; Xu, F.-J.; Church, G.; Ping, Y. Mater. Today, 2019, 26, 40–66. HO N OH NO O SS OO SSgRNA Negatively charged Cas9/gRNA complex bioreducible lipid Electrostatic assembly nanoparticle Bioreduction: lipid degradation and Cas9/gRNA release CRISPR/Cas9 genome editing genomic DNA H 2 NN NH 2 N H NH 2 n N O OO ON O O SO 3 Na H 2 NN NH 2 N H H N n N O O O + SH S H 2 NN NH 2 N H H N n N O O O bPEI sulfo-SMCC SpCas9 SpCas9-bPEI MARISA JUANES CARRASCO 78 Gold nanoparticles have also been explored as promising synthetic carriers in the field of gene delivery as they possess good biocompatibility and excellent chemical stability.120 The first example of an editing nanostrategy was based on the co-delivery of Cas9 and gRNA into cells using gold nanoparticles.133 These cationic arginine-decorated gold nanoparticles were used to complex engineered Cas9 protein modified with a negatively charged glutamate peptide tag and gRNA (Figure 15). The resulting nanoassemblies could then be fused into the cell membrane to deliver encapsulated Cas9/gRNA into the cytoplasm. Figure 15. Engineering of the Cas9 protein and arginine gold nanoparticles (ArgAuNPs) for the intracellular delivery of Cas9 RNP via membrane fusion. Furthermore, CPPs have been extensively used for the delivery of multiple cargoes inside cells due to their capability to cross the cell membrane.120 In the initial studies, CPPs were directly conjugated with the anionic Cas9 RNP by covalent bonds and confirmed to target the loci inside living cells (Figure 16).134 The delivery of CPP-Cas9 RNP resulted in less off-target effects, weaker immune responses and lower cytotoxicity. However, the covalent fusion of the CPP with the Cas9 protein required multiple incubation steps with the cells to achieve low levels of edition. Figure 16. Schematic representation of Cas9 protein conjugated to a CPP by a covalent bond. Spheres in green are glycine amino acids, in blue arginine amino acids and in red leucine amino acids. 133 Mout, R.; Ray, M.; Yesilbag Tonga, G.; Lee, Y.; Tay, T.; Sasaki, K.; Rotello, V. M. ACS Nano, 2017, 11, 2452–2458. 134 Ramakrishna, S.; Kwaku Dad, A. B.; Beloor, J.; Gopalappa, R.; Lee, S. K.; Kim, H. Genome Res., 2014, 24, 1020–1027. 120 Wan, T.; Niu, D.; Wu, C.; Xu, F.-J.; Church, G.; Ping, Y. Mater. Today, 2019, 26, 40–66. Cas9 glutamate (E) peptide tag E= 0-20 NLS (PKKKRKV) SON H O NH2 N H NH NH2 Au 44 = ArgAuNPs membrane fusion Cas9En:ArgAuNPs nanoassembly Cas9En/gRNA Nucleus Cas9 His tag Hemagglutinin tag S O O G4R9L4 peptide Thioether bond 2 PEPTIDE/CAS9 NANOSTRUCTURES FOR RIBONUCLEOPROTEIN CELL MEMBRANE TRANSPORT AND GENE EDITION Chapter 2: Peptide/Cas9 nanostructures for ribonucleoprotein cell membrane transport and gene edition 81 2.1 Precedents and Objectives There is an emerging interest in the direct delivery of Cas9 RNP inside cells by using nonviral vectors. Although it has been reported that CPPs can efficiently deliver Cas9 inside cells,134 this strategy required the covalent fusion of the protein to the CPP and several rounds of transfection with the fused protein for reaching adequate levels of edition. These limitations hinder the application of gene edition technology in cell repairing and genetic treatments. The general objective of this chapter is to develop a new strategy for the direct delivery of Cas9 RNP using cell-penetrating peptides in a fully supramolecular manner (Figure 17). As in chapter 1, the proposed peptide scaffold will be based on an amphipathic backbone with potential α-helical folding. In addition, dynamic covalent bonds such as hydrazides will be inserted in the sequence allowing its modification with appropriate aldehydes. The active amphiphilic peptide will be prepared by a hydrazone bond formation between the cationic peptide scaffold and a hydrophobic aldehyde. A preliminary screening using a variety of hydrophobic aldehydes will be performed for the fast identification of amphiphilic peptides capable of delivering Cas9 with good efficiencies. Therefore, we plan to characterize in detail the formulations of the best peptide candidate obtained in the initial screening. The potential of the delivery and gene edition of Cas9 RNP will be characterized and compared with Lipofectamine2000 by a T7-E1 endonuclease assay by fixing carrier concentration and increasing RNP amount in different cell lines. The uptake mechanism of the complexes will be studied in the presence of different endocytic inhibitors by following the fluorescence of a TAMRA-labeled peptide derivative. Furthermore, the size and the stability of the peptide/protein nanoparticles will be determined by using dynamic light scattering at different ratios and at physiological pH. Electron microscopy techniques such as TEM, SEM and STEM will be used for the morphological characterization of the best functional particles. The presence and the position of the protein will be studied by exploiting the histidine tag present in the Cas9 RNP combined with gold nanoparticles coordinated to a nickel (II) cation. Figure 17. Schematic representation of the amphiphilic peptide for the delivery of Cas9 RNP. 134 Ramakrishna, S.; Kwaku Dad, A. B.; Beloor, J.; Gopalappa, R.; Lee, S. K.; Kim, H. Genome Res., 2014, 24, 1020–1027. H O H+ Nanoparticle Transfection Edition Cas9 PTn https://pubs.rsc.org/en/content/articlepdf/2017/sc/c7sc03918b Chapter 3: Introduction 99 1.1 Therapeutic interest of gene delivery Gene therapy is a novel form of molecular medicine, which has had a big impact on human health since the end of last century. The recognition of the central role of DNA in cell biology and its fundamental importance in the control of cellular processes has promoted the exponential growth of the medical applications in this field.135 Although the advent of recombinant DNA technology in modern medicine will allow prenatal genetic screening and treatment, the vast majority of those born with a certain disease are likely to be helped by gene therapy approaches. The concept of gene therapy involves the insertion of functional genes into specific cells of a patient for replacement or supplementation of mutated or missing genes, which results in the production of a therapeutic protein or the inhibition of a malfunctioning protein.136 Thus, in addition to the possibility of correcting inherited genetic disorders like cystic fibrosis, hemophilia and familial hypercholesterolemia and so on, gene therapy approaches are being studied to be used to combat acquired diseases, like cancer, AIDS, infectious diseases and Parkinson’s or Alzheimer’s disease.135 Conceptually, gene therapy involves identifying appropriate DNA sequences and cell types and is followed by the development of suitable ways to get the corresponding nucleotides or ribonucleoproteins into the targeted cells. In 1990 the first human gene therapy trial was begun and involved the transfer of adenosine deaminase (ADA) gene into lymphocytes of a patient having an otherwise lethal defect in this enzyme, which produces immune deficiency.137 The results of this initial trial have been very encouraging and have helped to stimulate further clinical tests. Antisense therapy, first reported in the late 70s,138 can also be used to turn off a diseasecausative gene by the action of an antisense oligodeoxynucleotide that inactivates the encoded mRNA. However, inadequate target engagement, insufficient biological activity and off-target toxic effects have obstructed progress translating antisense oligonucleotides (ASOs) to the clinic.139 Over the years, novel chemical modifications of ASOs have been employed to address these issues, which, in combination with the elucidation of the mechanism of action and improved clinical trials, have fueled the translation of ASO-based strategies into therapies.140 Although many neurological conditions lack an effective treatment, as research progressively disentangles the pathogenic mechanisms of these diseases, they provide an ideal platform to test ASO-based strategies. This steady progress reached a highpoint in the past few years with approvals of ASOs for the treatment of, for example, Duchenne muscular dystrophy.141 This, represented a landmark in a field were disease-modifying therapies were 135 Wang, T.; Upponi, J. R.; Torchilin, V. P. Int. J. Pharm., 2012, 427, 3–20. 136 Mulligan, R. Science, 1993, 260, 926–932. 137 Miller, A. D. Nature, 1992, 357, 455–460. 138 Stephenson, M. L.; Zamecnik, P. C. Proc. Natl. Acad. Sci., 1978, 75, 285–288. 139 Rinaldi, C.; Wood, M. J. A. Nat. Rev. Neurol., 2018, 14, 9–22. 140 Juliano, R. L. Nucleic Acids Res., 2016, 44, 6518–6548. 141 Koo, T.; Wood, M. J. Hum. Gene Ther., 2013, 24, 479–488. MARISA JUANES CARRASCO 100 practically non-existent. This technology holds the potential to dramatically change the therapeutic treatment of neurological and non-neurological conditions in the near future. The discovery of RNA interference constituted a breakthrough for gene therapy.142 Synthetic small interfering RNA (siRNA duplex) therapeutics, have emerged as one of the most promising, potential drugs in gene therapy. siRNA specifically binds to its targeted mRNA, resulting in subsequent catalytic silencing of gene expression.143 Recently, US regulators have approved the first therapy based on RNAi, which can be used to silence specific genes linked to disease.144 Patisiran is a RNA interference therapeutic agent that targets a rare condition impairing heart and nerve function.144 Its recent approval consitutes a landmark for a field that has resisted for nearly two decades to prove its worth in the clinic. 1.2 Types of nucleic acids used in gene delivery The use of nucleic acids as therapeutic agents has great potential for the treatment of heritable and acquired diseases.145 Beyond therapeutic applications, DNA delivery, especially via the non-viral route (i.e. transfection), has become a powerful and popular research tool for elucidating gene structure, regulation, and function.146 Plasmid DNA (pDNA) is commonly found as large circular double-stranded DNA molecules of several kilobases (kb) in bacteria. Their site of action inside the cell is the nucleus, where pDNA must enter to be transcribed.147 These plasmids contain several basic components such as the transgene expression system (i.e. promoter, gene of interest and terminator), regulatory signals, antibiotic resistance marker, origin of replication and the remaining bacterially-derived plasmid backbone (BB).148 The removal of unnecessary pDNA sequences is recommended to maintain the pDNA molecule small and easy to manipulate.149 Thus, minicircles in which the BB has been removed may offer advantages over conventional plasmids. The native plasmid systems are only active in vivo for only 1 or 2 months, whereas some other derivative constructs, have demonstrated improved expression durations in cells for months or years.148 Although the use of pDNA could result in insertional mutagenesis by recombination with cellular DNA, its delivery has been essential for both the expression of a protein to restore a function or to develop an immune response against it, as well as the expression of regulatory RNAs.147 142 Fire, A.; Xu, S.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.; Mello, C. C. Nature, 1998, 391, 806–811. 143 Lam, J. K. W.; Chow, M. Y. T.; Zhang, Y.; Leung, S. W. S. Mol. Ther.-Nucleic Acids, 2015, 4, e252. 144 Adams, D.; Gonzalez-Duarte, A.; O’Riordan, W. D.; Yang, C.-C.; Ueda, M.; Kristen, A. V.; Tournev, I.; Schmidt, H. H.; Coelho, T.; Berk, J. L.; Lin, K.-P.; Vita, G.; Attarian, S.; Planté-Bordeneuve, V.; Mezei, M. M.; Campistol, J. M.; Buades, J.; Brannagan, T. H.; Kim, B. J.; Oh, J.; Parman, Y.; Sekijima, Y.; Hawkins, P. N.; Solomon, S. D.; Polydefkis, M.; Dyck, P. J.; Gandhi, P. J.; Goyal, S.; Chen, J.; Strahs, A. L.; Nochur, S. V.; Sweetser, M. T.; Garg, P. P.; Vaishnaw, A. K.; Gollob, J. A.; Suhr, O. B. N. Engl. J. Med., 2018, 379, 11–21. 145 Dunbar, C. E.; High, K. A.; Joung, J. K.; Kohn, D. B.; Ozawa, K.; Sadelain, M. Science 2018, 359, eaan4672. 146 Luo, D.; Mark, W, S. Nat. Biotechnol., 2000, 18, 33–37. 147 Lostalé-Seijo, I.; Montenegro, J. Nat. Rev. Chem., 2018, 2, 258–277. 148 Hill, A. B.; Chen, M.; Chen, C.-K.; Pfeifer, B. A.; Jones, C. H. Trends Biotechnol., 2016, 34, 91–105. 149 Gill, D. R.; Pringle, I. A.; Hyde, S. C. Gene Ther., 2009, 16, 165–171. Chapter 3: Introduction 101 Messenger RNAs (mRNA) are long single-stranded RNA molecules, which encode the information for one protein. They are composed of several kb, as long as their length depends on the size of the encoded protein. They have been investigated for therapeutic applications as an alternative to DNA-based gene therapy. Although it is less stable than DNA, the advantages of mRNA are clear.150 mRNA is easily produced by an in vitro process without the need of living organisms. The manufacturing process is general and can be quickly applied for numerous different applications and genes of interest. Naturally, the protein expression can only be transient, which is sufficient for several applications and even favorable for some therapeutic approaches to better control pharmacokinetics and dosing. In addition, the risk of recombination with the cell’s genome and insertional mutagenesis are excluded when using mRNA.151 Another benefit of mRNA is that it performs its function in the cytoplasm, without needing to cross the nuclear membrane, which results in almost immediately protein translation after its delivery. The simplest application of mRNA is based on its incorporation in the protein synthesis machinery of the target cell to induce expression of a desired protein restoring a function or developing an immune response against it.152 Antisense oligonucleotides (ASOs) are short, synthetic, single-stranded oligonucleotides typically 20-25 bases in length, which bind RNA by complementary base pairing and that can alter RNA and reduce, restore or modify protein expression through several different mechanisms.139 ASOs are emerging as a therapeutic alternative to treat diseases with known genetic origin.139 They can be occasionally designed to target and bypass or overcome a patient’s genetic mutation, in particular those lesions that compromise normal pre-mRNA processing. ASOs serve as highly selective sequence pairs to specific regions of mRNA and regulate the translation of genetic material into functional proteins. Short interfering RNAs (siRNAs) and microRNAs (miRNAs), have emerged as critical regulators in the expression and function of eukaryotic genomes.153 siRNAs and miRNAs share many similarities, both are duplex RNA molecules of around 22 nucleotides that exert gene silencing effects at the post-transcriptional level by targeting mRNA.143 However, their mechanisms of action are different, which results in siRNA being highly specific with only one mRNA target, whereas miRNA comprises two approaches: miRNA inhibition and miRNA replacement.143 The inhibition approach resembles antisense therapy, with synthetic single stranded RNAs acting as miRNA antagonists to inhibit the action of the endogenous miRNA, while in the replacement approach, synthetic miRNAs are used to mimic the function of the endogenous miRNAs. Thus, it leads to mRNA degradation or inhibition producing a gene silencing effect. Single-stranded forms of both RNAs associate with effector assemblies known as RNA-induced silencing complexes (RISCs), located in the cytosol, 150 Vallazza, B. et al. Wiley Interdiscip. Rev. RNA, 2015, 6, 471–499. 151 McIvor, R. S. Mol. Ther., 2011, 19, 822–823. 152 Hajj, K. A.; Whitehead, K. A. Nat. Rev. Mater., 2017, 2, 1–17. 153 Carthew, R. W.; Sontheimer, E. J. Cell, 2009, 136, 642–655. 139 Rinaldi, C.; Wood, M. J. A. Nat. Rev. Neurol., 2018, 14, 9–22. 143 Lam, J. K. W.; Chow, M. Y. T.; Zhang, Y.; Leung, S. W. S. Mol. Ther.-Nucleic Acids, 2015, 4, e252. MARISA JUANES CARRASCO 102 being directed to specific mRNAs for degradation by siRNAs or regulating mRNA stability or translation by miRNAs. When in the nucleus, siRNAs induce long-term silencing by DNA methylation and miRNAs induce chromatin reorganization.153 1.3 Gene delivery vectors The key challenge for oligonucleotide-based therapeutics is the delivery of active oligonucleotides to their site of action inside the cells within tissues.140 Successful gene delivery depends on the ability of the vector of choice to target a specific cell type, enter the cell and obtain sufficient levels of gene expression. This is not an easy task due to the presence of several barriers that viral and non-viral vectors have to tackle. Consequently, many delivery vectors have been developed to overcome both extracellular and intracellular gene delivery obstacles. Viral vectors are the most widely used systems for gene delivery, as they are the most effective and easiest methods for transferring genes of interest into a cell. However, their use as delivery vehicles has many drawbacks, such as storage difficulties, gene carrying capacity and residual viral elements, which can potentially cause insertional mutagenesis, cytotoxicity and immunogenicity.154 These limitations of viral vectors have been avoided with the development of synthetic non-viral vectors, which include liposomes, polymers, nanoparticles, etc.155 A comparison between both carriers is depicted in Table 1. Table 1. Advantages and disadvantages of viral and non-viral vectors for gene delivery. ADVANTAGES DISADVANTAGES VIRAL -High transduction efficiency. -Natural tropism confers the capability for infection of many cell types. -Intrinsic mechanism for endosomal escape. -Natural mechanism for nuclear import of genes. -Immune response is strong and multipleinjections are limited. -Can cause chromosomal insertion. -Difficult to produce in large scale. -Can only carry limited sized genes. -Can cause toxicity and may be contaminated with live virus. NON-VIRAL -Low immunogenicity. -Easy to make and with quality control in mass production. -Can carry large-sized DNA. -Can be functionalized for targeting, endosomal escape and nuclear import. -Transfection efficiency is low. -Can be toxic at high concentrations. -Lack of intrinsic tropism. -Lack of intrinsic mechanism for endosomal escape and for nuclear import of genes. 154 Sheridan, C. Nat. Biotechnol., 2011, 29, 121–128. 155 Gottfried, L. F.; Dean, D. A. In Novel Gene Therapy Approaches; InTech, 2013; 75–88. 140 Juliano, R. L. Nucleic Acids Res., 2016, 44, 6518–6548. 153 Carthew, R. W.; Sontheimer, E. J. Cell, 2009, 136, 642–655. Chapter 3: Introduction 103 1.4 Gene delivery barriers There has been a rapid progress in the development of therapeutic nucleic acids based on DNA and RNA for the treatment of genetic and acquired disorders.156 However, nucleic acids are highly hydrophilic negatively charged macromolecules, properties that restrict their binding to cell membranes and their diffusion across them. Their internalization depends on the endocytic pathway, which can result in endosomal entrapment and lysosomal degradation, issues that reduce their availability at their sites of action. Moreover, nucleic acids that are able to escape from the endosome, have to fight with the cytosolic viscosity and dense organelles, which might prevent their movement towards target sites. As it has already been mentioned, in the case of pDNA, the nuclear envelope represents an extra barrier, since it must enter the nucleus to be transcribed.157 The most important and difficult challenge in gene therapy is the problem of in vivo delivery. In many cases, systemic administration of genes is needed due to the fact that many disease sites are not easy to access. Under these circumstances, bioactive nucleotides have to penetrate through a series of systemic barriers in order to achieve the desirable efficiency and to reduce side effects. In the blood circulation, nucleic acids must avoid uptake by macrophages, clearance by renal filtration and degradation by endogenous nucleases.158 Furthermore, undesired interactions with blood components and non-target cells such as the reticuloendothelial system,140 are important obstacles in targeting genes in vivo. The extracellular matrix is another important barrier for gene delivery as it resists the movement of genetic material to target cells due to its dense polysaccharides and fibrous proteins.159 1.5 Overcoming gene delivery barriers with non-viral vectors 1.5.1 Cargo/carrier particle formation Nucleic acids are exposed to a variety of environmental factors such as, pH or enzymes (i.e. nucleases) that can degrade or destroy them. Thus, their complexation by cationic polymers or lipids is a method widely used to prevent their destruction by nucleases. Polyethylenimine (PEI) is one such polymers, positively charged and with a molecular weight of around 22-25 kDa, which can be linear or branched, and combined with nucleic acids via electrostatic interactions.160 PEI has been used for non-viral transfection in vitro and in vivo and has an advantage over other polycations as it strongly compacts DNA with an intrinsic endosomolytic activity.161 At high N/P ratios (ratio of the nitrogen atoms of PEI to 156 Read, M. L.; Logan, A.; Seymour, L. W. Adv. Genet., 2005, 53, 19–46. 157 Lam, A. P.; Dean, D. A. Gene Ther., 2010, 17, 439–447. 158 Ogris, M.; Brunner, S.; Schüller, S.; Kircheis, R.; Wagner, E. Gene Ther., 1999, 6, 595–605. 159 Dhaliwal, A.; Lam, J.; Maldonado, M.; Lin, C.; Segura, T. Soft Matter, 2012, 8, 1451–1459. 160 Boussif, O.; Lezoualc’h, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. Proc. Natl. Acad. Sci., 1995, 92, 7297–7301. 161 Lungwitz, U.; Breunig, M.; Blunk, T.; Göpferich, A. Eur. J. Pharm. Biopharm., 2005, 60, 247–266. 140 Juliano, R. L. Nucleic Acids Res., 2016, 44, 6518–6548. MARISA JUANES CARRASCO 104 DNA phosphates), the positive net charge of the corresponding complexes increases, thus the electrostatic interaction between these positively charged complexes and negatively charged cell surface, facilitates cell binding and consequently, high levels of gene expression. Thus, PEI combines a high membrane destabilizing potential with a high DNA packaging activity, which protects DNA from degradation and increases the probability that pDNA can reach the nucleus intact.161 After endocytic internalization, the buffering capacity of amine groups of PEI in the acidic pH of endosomes, results in endosomal rupture and release of the complexes into the cytoplasm.162 This phenomenon called “proton-sponge effect” could explain why polyplexes are able to escape from the endosomes. However, PEI shows toxicity, which depends on the MW and branching degree of PEI, particle size and zeta pontential.162 Toxicity can be also be related with free PEI, which results in cell membrane destabilization before transfection, or related with the proceeding of PEI/DNA complexes after getting into cell. Liposomal vectors have also been used to encapsulate DNA. A primary demonstration of in vivo gene expression was performed through the use of pH sensitive liposomes.163 However, their application for gene therapy resulted in a low entrapment efficiency and high serum sensitivity164 until the introduction of lipofection methodology, which used the cationic lipid DOTMA (2,3-dioleyloxypropyl-1-trimethylammonium chloride).165 Cationic lipids with amphipathic behavior, interact with the negatively charged phosphate backbone of nucleic acids to form a complex capable of crossing the cell membrane. However, cationic liposomes, especially those constituted by monovalent cationic lipids, are not able to condense DNA efficiently, resulting in complexes with very heterogeneous size distribution. A strategy to improve complexation, adds a previous step of DNA condensation with cationic polypeptides. Such is the case of cationic liposome-entrapped polycation-condensed DNA type 1,164 composed by DNA, DOTAP (1,2-dioleoyl-3-trimethylammonium propane) and protamine sulfate, which substitutes the poly-L-lysine (PLL) of previous formulations.166 This protamine sulfate is smaller and highly positively charged than PLL, providing a high protection of DNA from nuclease degradation. Gold nanoparticles (Au NPs) have also been developed for the efficient package and delivery of nucleic acids combined with low generation polypropylenimine dendrimers.167 The limited surface charges of these low generation dendrimers lead to an inefficient complexation of DNA and low cellular uptake efficacy. Consequently, the support on Au NPs of low generation dendrimers, allowed the packaging of nucleic acids into discrete nanoparticles, but Au NPs are not encapsulated inside the final DNA/siRNA complexes 162 Kazemi Oskuee, R. et al. Life Sci., 2018, 197, 101–108. 163 Wang, C. Y.; Huang, L. Proc. Natl. Acad. Sci., 1987, 84, 7851–7855. 164 Liu, F.; Huang, L. J. Control. Release, 2002, 78, 259–266. 165 Felgner, P. L.; Gadek, T. R.; Holm, M.; Roman, R.; Chan, H. W.; Wenz, M.; Northrop, J. P.; Ringold, G. M.; Danielsen, M. Proc. Natl. Acad. Sci., 1987, 84, 7413–7417. 166 Gao, X.; Huang, L. Biochemistry, 1996, 35, 1027–1036. 167 Chen, A. M.; Taratula, O.; Wei, D.; Yen, H. I.; Thomas, T.; Thomas, T. J.; Minko, T.; He, H. ACS Nano, 2010, 4, 3679–3688. 161 Lungwitz, U.; Breunig, M.; Blunk, T.; Göpferich, A. Eur. J. Pharm. Biopharm., 2005, 60, 247–266. Chapter 3: Introduction 105 (Figure 18). Therefore, it becomes possible to eliminate the potential toxicity problems associated with Au NPs by selectively removing them from the resulting nucleic acid complexes before their delivery to target cells. Figure 18. Au NPs (yellow ball) anchored with several low generation dendrimers through Au-amine coordination bonds. Addition of DNA/siRNA leads to an increase of local acidity protonating the tertiary amines and therefore, weakening the Au-amine interactions. As a result, the Au NPs are released from the dendrimers and are not included in the final DNA/siRNA nanoparticles. 1.5.2 Increasing the circulation time of nucleic acids Both naked DNA and lipoplexes have shown rapid hepatic clearance during systemic administration. Absence of hydrophilic groups on DNA particles’ surface, may lead to their interaction with plasma proteins and their removal from the circulation. However, this binding, when directed to specific plasma proteins, can improve pharmacokinetics, as large protein cargo complexes can avoid renal clearance, and their half-life in the circulation can be extended. Human Serum Albumin (HSA) is a natural transport protein, involved in trafficking of a wide variety of molecules, due to its multiple ligand binding sites and extended halflife.168 The non-covalent binding of lipid-modified oligonucleotides to albumin, which increases the bioavailability of siRNAs has been recently published.168 For instance, the incorporation of cholesterol modifications into siRNA, facilitates specific binding with recombinant HSA with an affinity dependent on the number of modifications. The aim of this study was to use this finding, along with the intrinsic transport properties of albumin, to tune siRNA serum half-life, hepatic accumulation, and gene silencing. Moreover, antibodies combine selective targeting with improved bioavailability. The design of a protamine168 Bienk, K.; Hvam, M. L.; Pakula, M. M.; Dagnæs-Hansen, F.; Wengel, J.; Malle, B. M.; Kragh-Hansen, U.; Cameron, J.; Bukrinski, J. T.; Howard, K. A. J. Control. Release, 2016, 232, 143–151. NH3 NNH3 N N N NH3 NH3 N H3N H3N N N H3N H3N N NH3 NH3 NH3 N NH3 NN N NH3 NH3 N NH3 NH3 N N NH3 NH3 N NH3 H3N NH3 N NH3 NN N NH3 NH3 N NH3 NH3 N N NH3 NH3 N NH3 H3N N H3N H3N N H3N H3NH TERTIARY AMINES PROTONATION AND Au NPs RELEASE MARISA JUANES CARRASCO 106 antibody fused protein to deliver siRNA to HIV-infected cells has also been reported.169 The fusion protein (F105-P) was designed with the protamine coding sequence linked to the Cterminus of the heavy chain Fab fragment of an HIV-1 envelope antibody. siRNAs were bound to the resulting fusion protein by using electrostatic interactions. The final conjugate allowed the effective delivery, and induced silencing only in cells expressing the HIV-1 envelope glycoprotein.169 Furthermore, modification of non-viral vectors with hydrophilic molecules of PEG also decreases interaction with plasma proteins and increases transfection efficiency.156 Examples in literature have demonstrated that PEG grafted PLL (PEG-g-PLL) can be used to enhance transfection efficiency by formulation with the fusogenic peptide KALA.170 The ionic binding of the positively charged KALA with the negatively charged complexes of DNA/PEG-g-PLL, resulted in improved transfection efficiency and very low cytotoxicity (Figure 19). Figure 19. Schematic representation of the formulation of DNA/PLL and DNA/PEG-g-PLL complexes with KALA peptide. (PLL, PEG and KALA structures are represented in the right side). 1.5.3 Targeting approaches The efficient and specific delivery of therapeutic genes to target cells is a challenge that will need to be overcome in order to take advantage of the promise and potential of genetic drugs avoiding non-specific adsorption and off-target effects. The development of aptamer-siRNA chimeric RNAs capable of binding and delivering functional siRNAs into specific cell types has been reported.171 One aptamer portion of a chimera protein mediates binding to PSMA, a cell-surface receptor overexpressed in prostate cancer cells and tumor vascular endothelium, while the siRNA portion targets the expression of survival genes. Therefore, these chimeras do not bind to, or function in cells that do not express PSMA, an approach that presents numerous applications including cancer 169 Song, E.; Zhu, P.; Lee, S. K.; Chowdhury, D.; Kussman, S.; Dykxhoorn, D. M.; Feng, Y.; Palliser, D.; Weiner, D. B.; Shankar, P.; Marasco, W. A.; Lieberman, J. Nat. Biotechnol., 2005, 23, 709–717. 170 Lee, H.; Jeong, J. H.; Park, T. G. J. Control. Release, 2002, 79, 283–291. 171 McNamara, J. O.; Andrechek, E. R.; Wang, Y.; Viles, K. D.; Rempel, R. E.; Gilboa, E.; Sullenger, B. A.; Giangrande, P. H. Nat. Biotechnol., 2006, 24, 1005–1015. 156 Read, M. L.; Logan, A.; Seymour, L. W. Adv. Genet., 2005, 53, 19–46. pDNA ON H NH3 OH N NH3 HO On n KALA coating KALA coating PEG-PLL PEG-PLL-DNA complex PLL-DNA complex PLL - Aggregation - Reduced transfection - No aggregation - Enhanced transfection - Repulsion of serum problems PLL: polylisne WEAKLAKALAKALAKHLAKALAKALKACEA PEG: polyethylene glycol KALA peptide sequence Chapter 3: Introduction 107 therapeutics.171 Additionally, the programmable sequential recognition of a siRNA-loaded DNA nanovehicle by two aptamers capable of binding to two different cell receptors, has also been used to improve targeting in gene delivery.172 The siRNA is self-assembled in an oligonucleotide nanovehicle modified with a hairpin structure to act as both the “smart key” and the delivery carrier. The auto-cleavable hairpin structure can be activated on site at the target cell membrane, by reacting sequentially with two aptamers as “dual locks”, which leads to cell-subtype discrimination and precise siRNA delivery for high efficient gene silencing.162 Furthermore, aptamers conjugated to lipids can also be assembled into lipid nanoparticles and used for targeted delivery of siRNA to different tissues, such as delivery to bone tissue to improve osteogenesis.173 Thus, nucleic acid aptamers offer significant potential as convenient and evolvable targeting groups for drug delivery. The attachment of aptamers to the surface of a genome-free viral capsid carrier by an efficient oxidative coupling strategy, has been developed.174 The method involves the periodate-mediated reaction of phenylenediaminesubstituted oligonucleotides with aniline groups installed on the outer surface of the capsid shells (Figure 20). Up to 60 strands of DNA can be linked to each viral capsid without apparent loss of base pairing capabilities or protein stability. Figure 20. A) Surface modification of capsids for targeted delivery using aptamers. B) For interior surface modification, a mutation on the capsid coated with proteins allows site-specific alkylation. Up to 180 cargo molecules can be installed in these locations. C) For exterior modification, the aptamer is modified with phenylene diamine group by a periodate-mediated reaction. 172 Ren, K.; Liu, Y.; Wu, J.; Zhang, Y.; Zhu, J.; Yang, M.; Ju, H. Nat. Commun., 2016, 7, 1–10. 173 Liang, C.; Guo, B.; Wu, H.; Shao, N.; Li, D.; Liu, J.; Dang, L.; Wang, C.; Li, H.; Li, S.; Lau, W. K.; Cao, Y.; Yang, Z.; Lu, C.; He, X.; Au, D. W. T.; Pan, X.; Zhang, B.-T.; Lu, C.; Zhang, H.; Yue, K.; Qian, A.; Shang, P.; Xu, J.; Xiao, L.; Bian, Z.; Tan, W.; Liang, Z.; He, F.; Zhang, L.; Lu, A.; Zhang, G. Nat. Med. 2015, 21, 288–294. 174 Tong, G. J.; Hsiao, S. C.; Carrico, Z. M.; Francis, M. B. J. Am. Chem. Soc., 2009, 131, 11174–11178. 171 McNamara, J. O.; Andrechek, E. R.; Wang, Y.; Viles, K. D.; Rempel, R. E.; Gilboa, E.; Sullenger, B. A.; Giangrande, P. H. Nat. Biotechnol., 2006, 24, 1005–1015. SH N O O R N O O R S NH2 Et2N N H O N H O OP O O O aptamer NEt2 HN O N H O OP O O O aptamer O N phosphate buffer pH 7, RT 5 mM NaIO4 phosphate buffer pH 7, 150 mM NaCl, RT 33 5 5 INTERIOR MODIFICATION EXTERIOR MODIFICATION R: AlexaFluor 488 = A R cell-specific aptamer A) B) cell targeting delivery vehicle C) protein MARISA JUANES CARRASCO 114 Several other strategies have been developed to trigger nucleic acid release using polymeric formulations. Disulfide functionalization of polymers can also be exploited for gene delivery applications. In this regard, the high concentrations of glutathione (1-10 mM) inside the eukaryotic cell cytosol generate a reductive environment, which can be exploited to trigger a response and reducing the disulfide bonds into their individual components.203 The design and synthesis of a dipicolyamine-based disulfide-containing zinc (II) coordinative module, used for low-molecular-weight (~1800 Da) PEI (Zn-PD) functionalization has been reported.204 This non-viral vector has high affinity for DNA and can be cleaved by glutathione in the cytoplasm, facilitating DNA release post internalization in primary and stem cells, and diminishing the cytotoxicity (Figure 28). Figure 28. A) Synthesis of Zn-PD non-viral vectors and B) schematic representation of DNA transfection. In a different approach, phenylboronic acid (PBA) functionalized polyion complexes (PIC) micelles have been used for siRNA internalization and release (Figure 29).205 PBA is a synthetic molecule capable of forming reversible covalent esters with 1,2or 1,3-cis-diols included on a ribose ring, a structure present at the 3’ end of RNAs and several types of 203 Méndez-Ardoy, A.; Lostalé-Seijo, I.; Montenegro, J. ChemBioChem, 2019, 20, 488–498. 204 Liu, S.; Zhou, D.; Yang, J.; Zhou, H.; Chen, J.; Guo, T. J. Am. Chem. Soc., 2017, 139, 5102–5109 205 Naito, M.; Ishii, T.; Matsumoto, A.; Miyata, K.; Miyahara, Y.; Kataoka, K. Angew. Chemie - Int. Ed., 2012, 51, 10751–10755. Cl Cl HN N N NN N Cl HO NH2 HO N H N N N + NH SS HN HO N N N N H NSSN H O O NH SS HN HO N N N N Zn2+ H2N n PEI H N H2N n RT DCM, K2CO3EtOH, K2CO3 60ºC MeOH, 40ºC Zn(NO3)2 . 6H2O Zn-PD Zn-PD pDNA Zn coordinative ligand S-S polyplex N N N Zn P O OH O O Enhanced endocytosis Endosomal escape Glutatione triggered DNA release Nucleus Nucleus entry Zn-phosphorylated components coordination O O O O A) B) Chapter 3: Introduction 115 ribonucleotides. Consequently, this binding property offers an easy way for chemical conjugation of siRNA to the pendant PBA groups. After internalization, the high cytosolic levels of ATP can disrupt these boronate-stabilized particles, resulting in siRNA release (Figure 29). In a similar approach, this PBA-functionalized polyion strategy has been used for pDNA internalization by combining a PBA decorated polymer with a polyol decorated polymer, which will entrap pDNA until reaching the cytosol.206 Furthermore, instead of a stimulus, timed degradation can be used to protect the cargo until cytosolic delivery, as in the case of a cationic polymer, PDMAEA, that self-degrades into a negatively charged and nontoxic polymer, which repels its cargo facilitating siRNA release.207 Figure 29. Schematic representation of the PBA based strategy for siRNA delivery: chemical structure of the polymer, stability of the micelle, and mechanism of selective intracellular release. Polymers and CPPs containing dynamic bonds such as oximes and hydrazones, are promising non-viral vectors for gene delivery, as they can be formed in mild aqueous conditions with good yields and short reaction times, while being fully bioorthogonal.147 The formation of these kind of bonds is reversible leading to the endosomal escape and release of the cargo, as long as they can be hydrolyzed at the endosomal pH.208 The screening of cationic dendronised amphiphiles, combined with different hydrophobic aldehydes, allowed the fast identification of new and simple formulations for the delivery of siRNAs and 206 Yoshinaga, N.; Ishii, T.; Naito, M.; Endo, T.; Uchida, S.; Cabral, H.; Osada, K.; Kataoka, K. J. Am. Chem. Soc., 2017, 139, 18567–18575. 207 Truong, N. P.; Gu, W.; Prasadam, I.; Jia, Z.; Crawford, R.; Xiao, Y.; Monteiro, M. J. Nat. Commun., 2013, 4, 1902–1907. 208 Gasparini, G.; Bang, E. K.; Montenegro, J.; Matile, S. Chem. Commun., 2015, 51, 10389–10402. 147 Lostalé-Seijo, I.; Montenegro, J. Nat. Rev. Chem., 2018, 2, 258–277. O HO OH O OH HO ON H H N 272 PEG-b-P(Lys/FPBA) O NH3 OH N HN O BOH OH F H abm O OO O O O BOH B HO O HO OH O OH HO O O O N N H2N NN P O OOHOH OP O OH OP O OH BOH OH HO O N N H2N NN P O OOHOH OP O OH OP O OH siRNA PBA: phenilboronic acid Binding Cross-linked micelle core Extracellular: micelle stabilization ATP Intracellular: siRNA release B HO OH X Y B HO OH X Y OH O OH HO Phosphodiester Base BOH X Y O O O Phosphodiester Base R’ OH HO R OH 3’ end of siRNA BOH X Y R ’R O O siRNA EQUILIBRIUM BETWEEN PBA DERIVAYIVE BOUND TO siRNA AND OTHER DIOLS MARISA JUANES CARRASCO 116 plasmids.209,6 Following up on this strategy, our research group has reported hydrazonemodulated polymers as promising candidates for nucleic acid delivery. In this approach, polyhydrazides have been modified with different combinations of cationic and hydrophobic aldehydes to generate a library of amphiphilic polymers to complex and deliver siRNA and pDNA into living cells.7,8 1.5.6 Nuclear import The nuclear envelope that separates the cell’s genetic material from the surrounding cytoplasm, represents an extra physical barrier for nuclear importation of macromolecules such as pDNA. In 1980, the direct microinjection of pDNA into the cultured mammalian cells achieved a 50% of gene expression when injecting at the nucleus as compared with the inexistent expression when injecting the same amount of pDNA into the cytoplasm.210 However, this methodology is inefficient when the number of cells for transfection is large. The transport of therapeutic DNA from the cytoplasm into the nucleus is an inefficient process that is considered as the major limiting step in non-dividing cells. One of the strategies to improve nuclear uptake of DNA is taking advantage of the cellular nuclear import machinery. Synthetic peptides containing a nuclear localization signal (NLS, PKKKRKV)211 are bound to the DNA, resulting in a DNA-NLS complex that can be recognized as a nuclear import substrate by specific intracellular receptor proteins, such as importins and facilitate transfer through the nuclear pore.212,213 A strategy for the effective nucleic acid delivery into T cells, where gene carriers must be taken on by T cells and import their DNA cargo into the nucleus, has been reported.214 The methodology consists on using biodegradable poly(β-amino ester)-based nanoparticles (PBAE) containing peptides with microtubule-associated sequences and nuclear localization signals, as a means to facilitate fast track nuclear import of the genetic cargo via the microtubule transport machinery. In an extraordinarily promising approach, a pDNA packed with this PBAE polymer was coated with poly(glutamic) acid attached to a targeting antibody and, the resulting polyplex allowed the in vivo reprogramming of T cells to express chimeric antigen receptors for treating leukemia in a mouse model (Figure 30).214 209 Gehin, C.; Montenegro, J.; Bang, E. K.; Cajaraville, A.; Takayama, S.; Hirose, H.; Futaki, S.; Matile, S.; Riezman, H. J. Am. Chem. Soc,. 2013, 135, 9295–9298. 210 Capecchi, M. R. Cell 1980, 22, 479–488. 211 Kalderon, D.; Roberts, B. L.; Richardson, W. D.; Smith, A. E. Cell, 1984, 39, 499–509. 212 Cartier, R.; Reszka, R. Gene Ther., 2002, 9, 157–167. 213 Bremner, K. H.; Seymour, L. W.; Logan, A.; Read, M. L. Bioconjug. Chem., 2004, 15, 152–161. 214 Smith, T. T.; Stephan, S. B.; Moffett, H. F.; McKnight, L. E.; Ji, W.; Reiman, D.; Bonagofski, E.; Wohlfahrt, M. E.; Pillai, S. P. S.; Stephan, M. T. Nat. Nanotechnol., 2017, 12, 813–820. 6 Louzao, I.; García-Fandiño, R.; Montenegro, J. J. Mater. Chem. B, 2017, 5, 4426–4434. 7 Priegue, J. M.; Crisan, D. N.; Martínez-Costas, J.; Granja, J. R.; Fernandez-Trillo, F.; Montenegro, J. Angew. Chemie Int. Ed., 2016, 55, 7492–7495. 8 Priegue, J. M.; Lostalé-Seijo, I.; Crisan, D.; Granja, J. R.; Fernández-Trillo, F.; Montenegro, J. Biomacromolecules, 2018, 19, 2638–2649. Chapter 3: Introduction 117 Figure 30. Design and manufacture of lymphocyte-programming nanoparticles. Diagram of the fabrication of the PBAE nanoparticles. (Adapted from Nat. Nanotechnol., 2017, 12, 813–820). Recently, a combination of electroporation and microfluidic cell deformation has been described for nuclear pDNA direct delivery.215 This methodology consists on passing cells at high speed through microfluidic constrictions, smaller than the cell diameter, which mechanically disrupts the cell membrane, combined with subsequent electric field to further disrupt the nuclear envelope, allowing DNA molecules to be driven into the cytoplasm and nucleus (Figure 31). Figure 31. Schematic illustration of the methodology. A) Mechanical disruption of the cell membrane when the cell passes trough the constriction and B) the subsequent electric pulses driving DNA into the cytoplasm and nucleus through the disrupted membrane. (Doted arrows represent electric field). 215 Ding, X.; Stewart, M. P.; Sharei, A.; Weaver, J. C.; Langer, R. S.; Jensen, K. F. Nat. Biomed. Eng., 2017, 1, 1–7. Targeting ligand PGA HO H NN H H NH O OO O OH NAb O O O xy NH NOONOOH NN N O O OH O O N n PBAE polymer Polyglutamic acid Plasmid DNA Nanoparticle MTAS-NLS peptide GRYLTQETNKVETYKEQPLKTPGKKKKGKPGKRKRQEKKKRRTR MTAS NLS CAR-programming of cultured T cells via DNA nanocarriers We first assessed the ability of the engineered nanoparticles to program specificities against leukaemia by incubating mouse splenocytes with the particles at various ratios. We found that CD3-targeted nanoparticles selectively bind T lymphocytes, as their interactions with off-target cells were low (Fig. 2a). Confocal imaging established that the particles are rapidly (120 min) internalized into the cytoplasm, presumably as a result of receptor-induced endocytosis (Fig. 2b). As early as 30 h post-transfection, 194-1BBz receptors were detected on the surfaces of the treated cells (mean 3.8% CAR + Tcells±0.3%, Polymer Plasmid DNA co-delivery PGA Targeting ligand Targeting ligand PGA CD19-specific scFv m194-1BBz CAR 4-1BB CD3ζ a b Anti-CD3e f(ab’)2 PGA Nanoparticle Lyophilization Plasmid DNA PBAE polymer CAR MTAS-NLS peptide EF1A 3’PB 5’PB iPB7 WPRE BGH PA AMP ORI WPRE BGH PA AMP ORI EF1A CAR 2A GFP/Luc PBAE polymer 447 Polyglutamic acid (PGA) MTAS-NLS peptide NLS MTAS GR YLTQE TNKVE TYKE QPLKTP GKKKKGKPGKR KE QE KKKR RTR N N H NOO O O N O O O OH H NN N OHO OH N O N H OH NH OHOOHO H N OAb n xy Figure 1 | Design and manufacture of lymphocyte-programming nanoparticles. a, Schematic of the T-cell-targeted DNA nanocarrier used in our experiments. The inset shows a transmission electron micrograph of a representative nanoparticle. Scale bar, 100 nm. Also depicted are the two plasmids that were encapsulated into the nanoparticles; these encode an all-murine 194-1BBz CAR and the hyperactive iPB7 transposase. EF1A, eukaryotic translation elongation factor 1 alpha 1; BGH PA, bovine growth hormone polyadenylation signal; ampicillin resistance gene; ORI, origin of replication. b,Diagram describing the fabrication of the poly(β-amino ester) nanoparticles. Also shown are the chemical structures of the PBAE 447 polymer and polyglutamic acid, as well as the amino acid sequence of the microtubule-associated-nuclear localization (MTAS-NLS) peptide. ARTICLES NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO.2017.57 NATURE NANOTECHNOLOGY | VOL 12 | AUGUST 2017 | www.nature.com/naturenanotechnology814 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. =CAR: chimeric antigen receptor Plasmid DNA PBAE polymer Diffusion-based molecules DNA A) B) 2 MESSENGER RNA DELIVERY BY HYDRAZONEACTIVATED POLYMERS Chapter 3: Messenger RNA delivery by hydrazone-activated polymers 121 2.1 Precedents and Objectives As commented above, polymers are very promising materials for cell delivery and release of macromolecules of interest. Our research group has previously reported the synthesis of polyhydrazides functionalized with hydrophobic and cationic aldehydes for the delivery of siRNA and pDNA.7,8 The general objective of this chapter is to further expand the delivery properties of polyhydrazone polymers by tackling the challenging delivery of mRNA. A high molecular weight polyhidrazide polymer will be prepared using free-radical polymerization, which will be condensed with six different hydrophobic aldehydes and a cationic aldehyde (t-guanidinium), to obtain the corresponding amphiphilic polyhydrazones. This polyhydrazones will be complexed with mRNA cargo, encoded for the synthesis of the enhanced green fluorescent protein (EGFP), for studying its delivery in Hek293 cells. The formation of these polyhydrazones will be carried out in biocompatible conditions, allowing their combination with mRNA without needing any isolation or purification steps (Figure 32). Quantification of transfection will be performed by flow cytometry. Transfection efficiency will be optimized by trying different hydrophobic aldehyde tails changing the molar fraction of cationic/hydrophobic aldehydes present in the polyhydrazone scaffold for determining which is the most promising ratio for mRNA delivery. Control experiments will be carried out using the octaarginine penetrating peptide, the pore forming peptide GALA, the cationic lipid DOTAP and PEI polymer. The MTT colorimetric assay will be used to evaluate the toxicity of the polyhydrazones at the transfecting concentrations. The interaction between mRNA and the six different polyhydrazones will be evaluated by gel electrophoresis, DLS and Zeta potential measurements. Figure 32. A) Polyhydrazone formation and B) delivery of mRNA for translation. 7 Priegue, J. M.; Crisan, D. N.; Martínez-Costas, J.; Granja, J. R.; Fernandez-Trillo, F.; Montenegro, J. Angew. Chemie Int. Ed., 2016, 55, 7492–7495. 8 Priegue, J. M.; Lostalé-Seijo, I.; Crisan, D.; Granja, J. R.; Fernández-Trillo, F.; Montenegro, J. Biomacromolecules, 2018, 19, 2638–2649. + + ++ n O HN NH2 Polyhydrazide = H O H O H O + Hydrophobic aldehydes H2NN HN HH NH2O O Cationic aldehydes + mRNA complexation Transport Release polyplex formation + + + + + + + + + + ++ A) B) Polyhydrazone 60 ºC, 2h DMSO:100 mM AcOH https://pubs.rsc.org/en/content/articlelanding/2019/md/c9md00231f#!divAbstract Supporting Information 137 1.1 Supporting Figures Figure S1.General synthetic scheme for the Solid Phase Peptide Synthesis (SPPS). KL LRK L RR LL RR Fmoc N H N H Fmoc 1) 20% piperidine/DMF 2) 4 equiv Fmoc-Aa-OH, 4 equiv HBTU 0.195 M DIEA/DMF 13 x Mtt Mtt RL K RK L RR LL R RN H Mtt Mtt O Coupling cycle 1) 20% piperidine/DMF 2) Acetic anhydride/2,6 -lutidine (1:1) Mtt cleavage and Alkoxyamine coupling 1) DCM/HFIP/TFE/TIS (6.5:2:1:0.5) (2x2h) 2) 2,5 equiv [(tert-Butoxycarbonyl)aminooxy]acetic acid, 2,5 equiv HATU,4 equiv DIEA/DMF RL K RK L RR LL R RN H O O HN ONH O O RL K RK L RR L L RR N H O O HN ONH O O Deprotection 20% piperidine/DMF RL K RK L RR LL R RN H O O HN ONH O O H2N RL K RK L RR LL R RN H O O HN ONH O O RL K RK L RR LL R RN H O O HN ONH O O RL K RK L RR LL R RNH2 O O H2N ONH2 O O Linker coupling 1) 20% piperidine/DMF 2) 4 equiv Fmoc-6Ahx-OH, 4 equiv HBTU 0,195 M DIEA/DMF RL K RK L RR LL RR N H Mtt Mtt O Acetylation TAMRA DIEA/DMF OxDex HATU, DIEA Resin cleavage AcP(Ox)2 TmP(Ox)2 DexP(Ox)2 Resin cleavage Resin cleavage FmocHN 5 FmocHN 5 5 H N O O F HO HO 5RL K RK L RR LL R RNH2 O O H2N ONH2 O O H N O O F HO HO 5 H N O O N N OO RL K RK L RR LL R RNH2 O O H2N ONH2 O O H N O O N N OO TFA/DCM/TIS/H2O (9:0.5:0.25:0.25) 55 TFA/DCM/TIS/H2O (9:0.5:0.25:0.25) TFA/DCM/TIS/H2O (9:0.5:0.25:0.25) Boc Boc Boc Boc Boc Boc Boc Boc Boc Boc RL L L LL L L L L L MARISA JUANES CARRASCO 138 Figure S2. General synthetic scheme for the mannose aldehyde coupling. Peptides were obtained with an overall yield of: 28 % for AcP(Man)2, 5.6 % for TmP(Man)2 and 5 % for DexP(Man)2. Figure S3. SPR. Sensorgrams of the interaction of ConA (9668 µRiU) in the concentration range [AcP(Alloc)(Man)] = 6-2000 nM. R LK RKLRRLL R R NH 2 R1 O O H 2 N ONH 2 O R1P(Man)2 H N O O N N O O R1 = H N O O F HO HO O O O Acetyl (Ac) TAMRA (Tm) Dexamethasone (Dex) , , R LK RKLRRLL R R NH 2 R1 O O N ON O O O HO HO OH OH O O OH OH HO HO O O OH HO HO OH O 2 equiv H 2 O, r.t. 5 min 5 5 LL RKRKRR R R NH 2 R1 Man Man A A AA A RKRKRRRR NH 2 R1 Man Man RKRKRR R R NH 2 R1 Alloc Man L L LL L RKRKRR R R NH 2 R1 Acetone Acetone L L LL L R RRRR NH 2 R1RRR Time / seconds SPR Response / µRiu Supporting Information 139 Figure S4. A) TmArg2(Man)2 peptide structure. B) Fluorescence anisotropy titration and best fitting to a simple 1:1 binding model of TmArg2(Man)2 with increasing amounts of ConA in HKR buffer (pH 7.4) at 22 ºC. Calculated Kd = 9 ± 1 µM. C) Epifluorescence and D) CLSM images of TmArg2(Man)2 (3 µM, red) treated with ConAFITC (30 nM, green) and incubated with HeLa cells. Nuclei were counterstained with Hoechst (blue). 0,04 0,06 0,08 0,1 0,12 0,14 0,16 0,18 0 50 100 150 200 250 300 350 Fluorescence Anisotropy [ConA]/µM A) B) C) D) H NH 2 N O NH H 2 NNH 2 N HO H N O HN N HO NH 2 H 2 N NH O O N O HO HO HO N H O O N N O O O O O OOH HO OH OH O N O NH HO [ConA] / µM MARISA JUANES CARRASCO 140 Figure S5. A) TmArg6Gly5(Man)2 peptide structure. B) Circular dichroism of TmArg6Gly5(Man)2 measured in HKR buffer at different temperatures. C) Fluorescence anisotropy titration and best fitting to a simple 1:1 binding model of TmArg6Gly5(Man)2 with increasing amounts of ConA in HKR buffer (pH 7.4) at 22 ºC. Calculated Kd = 19 ± 2 µM. D) Epifluorescence image of TmArg6Gly5(Man)2 (3 µM, red) treated with ConAFITC (30 nM, green) and incubated with HeLa cells. Nuclei were counterstained with Hoechst (blue). -12000 -10000 -8000 -6000 -4000 -2000 0 2000 200 220 240 260 280 300 10 ºC 20 ºC 30 ºC 40 ºC 50 ºC 60 ºC Wavelength/nm [θ] MRt Wavelength / nm 0,06 0,08 0,1 0,12 0,14 0,16 0,18 0,2 0 100 200 300 400 500 600 Fluorescence Anisotropy [Con A]/µM [ConA] / µM A) B) C) D) H NN H NH 2 O N H O H N O N H O H N O N H O H N O N H O HN H N NH NH 2 H 2 N H NN H O O OO O HN N H O NH NH 2 H 2 N NH NH 2 H 2 N NH H 2 NNH 2 NH NH 2 H 2 N NH NH 2 H 2 N H N O O N N O O O N O O N O O O O O HO OH OH OH OH OH HO HO Supporting Information 141 Figure S6. A) TmArg6(Man)2 peptide structure. B) Circular dichroism of TmArg6(Man)2 measured in HKR buffer at different temperatures. C) Fluorescence anisotropy titration and best fitting to a simple 1:1 binding model of TmArg6(Man)2 with increasing amounts of ConA in HKR buffer (pH 7.4) at 22 ºC. Calculated Kd = 16 ± 1 µM. D) Epifluorescence image of TmArg6(Man)2 (3 µM, red) treated with ConAFITC (30 nM, green) and incubated with HeLa cells. Nuclei were counterstained with Hoechst (blue). 0,06 0,08 0,1 0,12 0,14 0,16 0,18 0,2 0,22 0 100 200 300 400 500 600 Fluorescence Anisotropy [ConA]/µM A) B) C) D) -12000 -10000 -8000 -6000 -4000 -2000 0 2000 200 220 240 260 280 300 10 ºC 20 ºC 30 ºC 40 ºC 50 ºC 60 ºC Wavelength/nm [θ] MRt O O N N O O NH 2 H N O NH N HO NH 2 H 2 N NH H N O NH 2 H 2 N NH N HO NH 2 H 2 N NH H N O HN N HO NH 2 H 2 N NH H N O NH 2 H 2 N NH N HO NH 2 H 2 N NH O N H O O N O N O O O OO OH OH OH OH OH OH OH OH Wavelength / nm [ConA] / µM MARISA JUANES CARRASCO 142 Figure S7. Cell viability in HeLa Cells. A) MTT assay at different concentrations of AcP(Man)2 (red) and AcP(Acetone)2 (blue); B) MTT assay at different concentrations of ConA using 5 µM of AcP(Man)2, the viability for AcP(Man)2 alone is shown in red at 0 nM of ConA; C) Propidium iodide staining of dead cells for flow cytometry at different concentrations of AcP(Man)2. The staining of untreated cells (control) is represented in green; D) Propidium iodide staining of dead cells for flow cytometry at different concentrations of ConA using 5 µM of AcP(Man)2 (red). Blue bars represent the cytotoxicity of the ConA alone. 0 10 20 30 40 50 0 0.1 0.5 1 2.5 5 10 20 % PI positive [peptide] / µM 0 20 40 60 80 100 0 1 25 100 % Viability [ConA] / nM 0 20 40 60 80 100 0.1 0.5 1 2.5 5 10 20 % Viability [peptide] / µM A) B) C) D) 0 20 40 60 80 100 0 1 5 10 25 50 75 100 % PI positive [ConA] / nM Supporting Information 143 Figure S8. Cell viability in HepG2 Cells. A) MTT assay at different concentrations of AcP(Man)2 (red); B) MTT assay at different concentrations of ConA using 5 µM of AcP(Man)2, the viability for AcP(Man)2 alone is shown in red at 0 nM of ConA; C) Propidium iodide staining of dead cells for flow cytometry at different concentrations of AcP(Man)2. The staining of untreated cells (control) is represented in green; D) Propidium iodide staining of dead cells for flow cytometry at different concentrations of ConA using 5 µM of AcP(Man)2 (red). Blue bars represent the cytotoxicity of the ConA alone. 0 20 40 60 80 100 0 1 5 10 25 50 75 100 %PI positive [ConA] / nM 0 20 40 60 80 100 0 1 25 100 % Viability [ConA] / nM 0 20 40 60 80 100 120 140 0,1 0,5 1 2,5 5 10 20 % Viability [peptide] / µM A) B) C) D) 0 10 20 30 40 50 0 0.1 0.5 1 2.5 5 10 20 % PI positive [peptide] / µM MARISA JUANES CARRASCO 144 Figure S9. Glucocorticoid induced GFP translocation assay (GIGT). A) Cells transfected with the plasmid pK7-GR-GFP show green fluorescence mostly on the cytoplasm, and so, the ratio between the median fluorescence in the nucleus and in the surrounding cytosolic region (red circle) will be low. Upon incubation with dexamethasone-labelled peptides, if the peptide remains trapped in the endosome (B) or it does not enter the cell, the glucocorticoid receptor will not be able to bind the dexamethasone and the translocation ratio will remain low. However, if the peptide is able to reach the cytosol, by direct translocation or endosomal escape (C), the binding to the glucocorticoid receptor triggers a conformational change that exposes NLSs. This causes the accumulation of GR-GFP in the nucleus and the increase in the translocation ratio. Figure S10. General synthetic scheme for the Solid Phase Peptide Synthesis (SPPS) for synthesizing the CFArg8 peptide. Fmoc N H 1) 20% piperidine/DMF 2) 4 equiv Fmoc-Aa-OH, 4 equiv HBTU 0.195 M DIEA/DMF 9 x K R RR R RN H Fmoc Coupling cycle 1) 20% piperidine/DMF 2) Acetic anhydride/2,6 -lutidine (1:1) Mtt cleavage and CF coupling 1) DCM/HFIP/TFE/TIS (6.5:2:1:0.5) (2x2h) 2) 2 equiv5(6)-Carboxyfluorescein, 2 equiv HBTU,4 equiv DIEA/DMF TFA/DCM/TIS/H2O (9:0.5:0.25:0.25) Acetylation Resin cleavage R R R K R RR R RN H O R R R Mtt Mtt K R RR R RN H R R R O O O HO O OH O H NN H O O NH2 O N H NH NH2 H2N O H N NH O N H NH O H N NH O NH2 H2N NH2 H2N NH2 H2N N H NH NH2 H2N O H N NH NH2 H2N O N H O HN O O HO OO OH NH NH2 H2N NH NH2 H2N Supporting Information 145 Figure S11. General synthetic scheme for the Solid Phase Peptide Synthesis (SPPS) for synthesizing the AcP(Biot)2 peptide. Figure S12. Lipofectamine is not a selective carrier. CLSM images of HeLa cells incubated with a mixture of 30 nM ConAFITC, 30 nM Streptavidin594 and 2 ng/µL of Lipofectamine 2000 (protein/Lipofectamine complex formation was done by incubation in a tenth of the final volume for 20 min) and incubated 30 min at 37 ºC. Both proteins co-localize in punctate structures, located at the perifery of the cell. Nuclei were counterstained with Hoechst (blue). R LK RKLRRLL R R Fmoc N H N H Fmoc 1) 20% piperidine/DMF 2) 4 equiv Fmoc-Aa-OH, 4 equiv HBTU 0.195 M DIEA/DMF 13 x Mtt Mtt R LK RKLRRLL R RN H Mtt Mtt O Coupling cycle 1) 20% piperidine/DMF 2) Acetic anhydride/2,6 -lutidine (1:1) 1) DCM/HFIP/TFE/TIS (6.5:2:1:0.5) (2x2h) 2) 2,5 equiv D-(+)-Biotin, 2,5 equiv HATU, 4 equiv DIEA/DMF R LK RKLRRLL R RN H O O O TFA/DCM/TIS/H2O (9:0.5:0.25:0.25) Acetylation Resin cleavage S H NH H O HN Biotin coupling HN O H N HH S H NN H NH2 O N H O H N O N H O H N O N H O H N O N H O HN H N NH NH2 H2N H NN H O O OO O NH N H NH NH2 H2N NH NH2 H2N NH H2NNH2 NH NH2 H2N NH NH2 H2N O HN O H NHH S O NH O H HN H S O L L L MARISA JUANES CARRASCO 152 solution of α-amino acids (4 equiv), N-HBTU (3.95 equiv) in DMF (2 mL), which was mixed with DIEA (0.195 M solution in DMF, 1.2 equiv) 1 min before the addition and the resulting mixture was shaken by bubbling Ar for 15 min. Finally, the resin was washed with DMF (3 x 2 mL, 1 min). The efficiency of each amino acid coupling and deprotection was monitored employing the TNBS testS4. Once the linear peptide was finished, two different ending protocols were used: A) Acetylation: the acetylation capping of N-terminal group was performed by standard Fmoc removal conditions (20% piperidine in DMF) followed by treatment with a solution of acetic anhydride and 2,6-lutidine (1:1, 1 mL) for 30 min. B) Linker coupling: after Fmoc cleavage with piperidine/DMF (20%, 2 mL), the linear peptide was treated with a solution of N-Fmoc-6-aminohexanoic acid (4 equiv), N-HBTU (3.95 equiv) and DIEA (0.195 M solution in DMF, 1.2 equiv) in DMF. The resin was washed with DCM (2 x 2 mL, 5 min), and the Mtt protecting group was selectively removed by mechanical shaking of the resin with a mixture of DCM/HFIP/TFE/TIS (6.5:2:1:0.5, 2 x 2 mL, 2 h). Finally, the mixture was filtered and the resin was washed with DCM (2 x 2 mL, 2 min) and DMF (2 mL, 20 min). A solution of [(tert-butoxycarbonyl)aminooxy]acetic acid (2.5 equiv per free amine) and N-HATU (2.5 equiv) in DMF (1 mL) was added to the resin followed by the dropwise addition of a solution of DIEA (4 equiv) in DMF (0.5 mL). The resin was shaken by bubbling Ar for 30 min and finally washed with DMF (3 x 2 mL, 2 min) and DCM (3 x 2 mL, 2 min). 1.4.2 General protocol for N-terminal functionalization Fluorescently labelled peptides, the Fmoc-protecting group of the previously attached linker was removed by using a solution of piperidine in DMF (20%, 4 mL) for 15 min and the resin was washed with DMF (3 x 3 mL). The coupling was carried out by the addition of a solution of 5(6)-Carboxytetramethylrhodamine succinimidyl ester (1 equiv) and DIEA (0.195 M, 1 equiv) in DMF (2 mL) and the mixture was stirred by bubbling Ar for 30 min. Finally, the resin was washed with DMF (3 x 3 mL) and DCM (3 x 3 mL). Ox-Dex terminating peptide, the Fmoc-protecting group of the linker was removed by using a solution of piperidine in DMF (20%, 4 mL) during 15 min and the resin was washed with DMF (3 x 3 mL). A solution of Ox-Dex (3 equiv), N-HATU (2.9 equiv) and DIEA (0.195 M, 3 equiv) in DMF (2 mL) was added and the mixture was shaken by bubbling Ar for 30 min. Finally, the resin was washed with DMF (3 x 3 mL) and DCM (3 x 3 mL). 1.4.3 General protocol for peptide cleavage Finally, peptides were deprotected and cleaved from the resin by standard TFA cleavage procedure at rt by using TFA/DCM/H2O/TIS (90:5:2.5:2.5, 1 mL per 70 mg of resin) for 2 h. Then, the mixture was filtered, washed with TFA (1 mL) and the peptide was precipitated with ice-cold Et2O (25 mL). The precipitate was centrifuged and dissolved in H2O (5 mL). Supporting Information 153 Peptides were obtained following the previously described procedure, and were treated with the different ligands without purification. 1.4.4 General protocol for ligand coupling A solution of peptide (R1P(Ox)2) in H2O (5 mM) was reacted with a solution of corresponding ligands (2 equiv per alkoxyamine) [1-α-formylmethyl-mannopyranoside or acetone] in H2O (120 mM) for 5 min. Then, peptides were purified by RP-HPLC for removing the ligand excess. The purification was carried by a C18 RP-HPLC [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] with a binary gradient of Solvent A and Solvent B, the collected fractions were lyophilized and stored at -20 °C. Purity and identity were confirmed by analytical HPLC, 1HNMR and low and high resolution mass spectrometry. 1.5 Synthesis of peptides 1.5.1 Synthesis of AcP(Man)2 Following the general protocol of the SPPS for synthesizing an acetylated peptide with two mannoses, AcP(Man)2 was obtained after RP-HPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] with an overall yield of 28% and 99.3% purity. Rt 3.8 min (Fig. S16) [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min)]. 1H NMR (300 MHz, D2O, δ): 7.77 (t, J = 5.3 Hz, 1H), 7.17 (t, J = 3.9 Hz, 1H), 4.93 (s, 2H), 4.59 (s, 4H), 4.45-4.22 (m, 17H), 4.07-3.64 (m, 12H), 3.33-3.16 (m, 16H), 2.07 (s, 3H), 1.98-1.32 (m, 51H), 1.03-0.82 (m, 30H). MS (ESI, H2O): 1300 (19, [M+2H+2TFA]2+), 1243 (21, [M+2H+TFA]2+), 1187 (23, [M+2H]2+), 867 (42, [M+3H+2TFA]3+), 827 (67, [M+3H+TFA]3+), 791 (100, [M+3H]3+), 593 (38, [M+4H]4+). HRMS (ESI): Calcd for C100H188N36O30: 1186.7139; found: 1186.7141 ([M+2H]2+). 1.5.2 Synthesis of TmP(Man)2 Following the general protocol of the SPPS for synthesizing a TAMRA labelled peptide with two mannoses, TmP(Man)2 was obtained after RP-HPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] with an overall yield of 6% and 100% purity. Rt 4.1 min (Fig. S17) [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min)]. 1H NMR (500 MHz, D2O, δ): 8.21 (s, 1H), 7.92 (d, J = 8.1 Hz, 1H), 7.58 (m, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.11 (t, J = 9.9 Hz, 2H), 6.97 (m, 1H), 6.83 (d, J = 9.6 Hz, 2H), 6.75 (s, 2H), 4.80-4.73 (m, 2H), 4.68 (s, 4H), 4.40 (d, J = 24.1 Hz, 4H), 4.28-3.96 (m, 13H), 3.87-3.24 (m, 12H), 3.13 (s, 12H), 3.10-2.95 (m, 16H), 2.91-2.80 (m, 2H), 2.25 (t, J = 7.1 Hz, 2H), 1.79-1.11 (m, 57H), 0.87-0.67 (m, 30H). MS (ESI, H2O): 1657 (7, [M+2H+4TFA]2+), 1599 (17, [M+2H+3TFA]2+), 1542 (15, [M+2H+TFA]2+), 1027 (100, [M+3H+2TFA]3+), 989 MARISA JUANES CARRASCO 154 (99, [M+3H+TFA]3+), 743 (62, [M+4H+TFA]4+), 716 (53, [M+4H]4+). HRMS (ESI): Calcd for C129H217N39O34: 1428.3209; found: 1428.3220 ([M+2H]2+). 1.5.3 Synthesis of AcP(Alloc)(Man) Following the general protocol of the SPPS for synthesizing an acetylated peptide with one mannose, and using Alloc protected Lysine, AcP(Alloc)(Man) was obtained after RPHPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] with an overall yield of 15% and 100% purity. Rt 4.1 min (Fig. S18) [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min)]. 1H NMR (300 MHz, D2O, δ): 7.62 (t, J = 5.3 Hz, 1H), 7.02 (t, J = 3.7 Hz, 1H), 5.90-5.73 (m, 1H), 5.23-5.06 (m, 2H), 4.78 (s, 1H), 4.48-4.39 (m, 5H), 4.31-4.04 (m, 15H), 3.89-3.46 (m, 6H), 3.15-2.95 (m, 16H), 1.92 (s, 3H), 1.78-1.12 (m, 51H), 0.87-0.70 (m, 30H). MS (ESI, H2O): 1260 (18, [M+2H+3TFA]2+), 1203 (27, [M+2H+2TFA]2+), 1147 (20, [M+2H+TFA]2+), 803 (38, [M+3H+2TFA]3+), 763 (100, [M+3H+TFA]3+), 727 (42, [M+3H]3+), 574 (10, [M+4H+TFA]4+), 547 (27, [M+4H]4+). HRMS (ESI): Calcd for C94H175N35O24: 1090.6868; found: 1090.6871 ([M+2H]2+). 1.5.4 Synthesis of TmP(Alloc)(Man) Following the general protocol of the SPPS for synthesizing a TAMRA labelled peptide with one mannose, and using Alloc protected Lysine, TmP(Alloc)(Man) was obtained after RP-HPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] with an overall yield of 10% and 98.4% purity. Rt 4.3 min (Fig. S19) [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min)]. 1H NMR (300 MHz, D2O, ): 8.24 (s, 1H), 8.08-7.87 (m, 2H), 7.55 (m, 1H), 7.03 (s, 2H), 6.88-6.61 (m, 3H), 5.71 (m, 2H), 5.07 (m, 3H), 4.52-4.30 (m, 4H), 4.29 (s, 2H), 4.26-3.92 (m, 13H), 3.86-3.44 (m, 6H), 3.30 (m, 2H), 3.09 (s, 12H), 3.08 (s, 16H), 2.22 (m, 2H), 1.96-1.12 (m, 57H), 0.95-0.59 (m, 30H). MS (ESI, H2O): 965 (43, [M+3H+2TFA]3+), 927 (100, [M+3H+TFA]3+), 668 (40, [M+4H]4+), 533 (10, [M+5H]5+). HRMS (ESI): Calcd for C123H204N38O28: 888.5326; found: 888.5330 ([M+3H]3+). 1.5.5 Synthesis of AcP(Acetone)2 Following the general protocol of the SPPS for synthesizing an acetylated peptide capped with acetone, AcP(Acetone)2 was obtained after RP-HPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] with an overall yield of 4% and 100% purity. Rt 4.3 min (Fig. S20) [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min)]. 1H NMR (300 MHz, D2O, ): 4.36 (s, 4H), 4.27-4.05 (m, 13H), 3.20-3.00 (m, 16H), 1.92 (s, 3H), 1.83 (s, 6H), 1.76 (s, 6H), 1.71-1.17 (m, 51H), 0.85-0.69 (m, 30H); MS (ESI, H2O): 1193 (30, [M+2H+3TFA]2+), 1133 (39, [M+2H+2TFA]2+), 1077 (23, [M+2H+TFA]2+), 757 (30, [M+3H+3TFA]3+), 720 (100, [M+3H+TFA]3+), 680 (61, [M+3H]3+), 513 (30, [M+4H]4+). HRMS (ESI): Calcd for C90H172N36O18: 1022.6820; found: 1022.6820 ([M+2H]2+). Supporting Information 155 1.5.6 Synthesis of TmP(Acetone)2 Following the general protocol of the SPPS for synthesizing a TAMRA labelled peptide capped with acetone, TmP(Acetone)2 was obtained after RP-HPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] with an overall yield of 12% and 99.9% purity. Rt 4.4 min (Fig. S21) [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min)]. 1H NMR (300 MHz, D2O, ): 8.07-7.89 (m, 2H), 7.55 (s, 1H), 7.03 (d, J = 9.2 Hz, 2H), 6.89-6.69 (m, 4H), 4.34 (d, J = 7.9 Hz, 4H), 4.27-3.93 (m, 13H), 3.40-3.22 (m, 2H), 3.10 (s, 12H), 3.07-2.98 (m, 16H), 2.31-2.11 (m, 2H), 1.89-1.63 (m, 12H), 1.60-1.10 (m, 57H), 0.88-0.64 (m, 30H). MS (ESI, H2O): 1492 (8, [M+2H+4TFA]2+), 1434 (10, [M+2H+3TFA]2+), 1376 (8, [M+2H+2TFA]2+), 918 (100, [M+3H+2TFA]3+), 881 (100, [M+3H+TFA]3+), 690 (18, [M+4H+2TFA]4+), 662 (40, [M+4H+TFA]4+), 632 (18, [M+4H]4+). HRMS (ESI): Calcd for C119H201N39O22: 1264.2902; found: 1264.2899 ([M+2H]2+). 1.5.7 Synthesis of AcArg2(Man)2 Following the general protocol of the SPPS for synthesizing an acetylated tetrapeptide with two mannoses, AcArg2(Man)2 was obtained after RPHPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] with an overall yield of 15% and 100% purity. Rt 3.1 min (Fig. S22) [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min)]. 1H NMR (300 MHz, D2O, δ): 7.62 (t, J = 5.3 Hz, 1H), 7.02 (t, J = 3.9 Hz, 1H), 4.78 (s, 2H), 4.44 (s, 4H), 4.25-4.07 (m, 8H), 3.90-3.47 (m, 12H), 3.20-2.98 (m, 8H), 1.91 (s, 3H), 1.83-1.13 (m, 20H). MS (ESI, H2O): 1183 (17, [M+H]+), 590 (100, [M+2H]2+). HRMS (ESI): Calcd for C46H84N15O21: 1182.5963; found: 1182.5961 ([M+H]+). 1.5.8 Synthesis of TmArg2(Man)2 Following the general protocol of the SPPS for synthesizing a TAMRA labelled tetrapeptide with two mannoses, TmArg2(Man)2 was obtained after RP-HPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] with an overall yield of 26% and 100% purity. Rt 3.8 min (Fig. S23) [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min)]. 1H NMR (300 MHz, D2O, δ): 8.35 (d, J = 14.1 Hz, 1H), 7.99 (t, J = 5.5 Hz, 1H), 7.56 (t, J = 10.4 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.11-6.98 (m, 2H), 6.95 (s, 1H), 6.80 (d, J = 9.5 Hz, 2H), 6.64 (d, J = 7.0 Hz, 2H), 4.73 (s, 2H), 4.38 (s, 4H), 4.22-3.99 (m, 4H), 3.85-3.33 (m, 12H), 3.10 (s, 12H), 3.08-2.98 (m, 12H), 2.86 (t, J = 7.5 Hz, 2H), 2.24 (t, J = 7.0 Hz, 2H), 1.81-1.12 (m, 26H). MS (ESI, H2O): 834 (100, [M+2H]2+), 556 (95, [M+3H]3+). HRMS (ESI): Calcd for C75H114N18O25: 833.4088; found: 833.4096 ([M+2H]2+). MARISA JUANES CARRASCO 156 1.5.9 Synthesis of TmArg6Gly5(Man)2 Following the general protocol of the SPPS for synthesizing a TAMRA labelled peptide with two mannoses, TmArg6Gly5(Man)2 was obtained after RP-HPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] with an overall yield of 9% and 100% purity. Rt 3.4 min (Fig. S24) [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min)]. 1H NMR (500 MHz, D2O, δ): 8.42 (s, 1H), 8.02 (s, 1H), 7.66-7.49 (m, 1H), 7.41 (s, 1H), 7.09-6.88 (m, 3H), 6.71 (s, 2H), 6.29 (s, 2H), 4.77 (s, 2H), 4.74 (s, 4H), 4.46-4.30 (m, 4H), 4.27-4.04 (m, 10H), 3.98-3.73 (m, 16H), 3.75-3.45 (m, 12H), 3.37 (d, J = 5.3 Hz, 2H), 3.17-3.01 (m, 12H), 2.98 (s, 8H), 2.24 (s, 2H), 1.85-1.10 (m, 42H). MS (ESI, H2O): 1402 (7, [M+2H+2TFA]2+), 973 (42, [M+3H+3TFA]3+), 936 (100, [M+3H+2TFA]3+), 898 (65, [M+3H+TFA]3+), 702 (60, [M+4H+2TFA]4+), 674 (95, [M+4H+TFA]4+), 645 (35, [M+4H]4+), 516 (45, [M+5H]5+). HRMS (ESI): Calcd for C109H177N39O34: 1288.1651; found: 1288.1655 ([M+2H]2+). 1.5.10 Synthesis of TmArg6(Man)2 Following the general protocol of the SPPS for synthesizing a TAMRA labelled peptide with two mannoses, TmArg6(Man)2 was obtained after RP-HPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] with an overall yield of 9% and 100% purity. Rt 3.4 min (Fig. S25) [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min)]. 1H NMR (300 MHz, D2O, δ): 8.41 (s, 1H), 7.99 (d, J = 8.5 Hz, 1H), 7.57-7.29 (m, 2H), 7.00-6.85 (m, 3H), 6.68 (d, J = 9.3 Hz, 2H), 6.33 (s, 2H), 4.84-4.66 (m, 2H), 4.704.65 (m, 4H), 4.32 (d, J = 15.0 Hz, 4H), 4.17-3.91 (m, 8H), 3.88-3.37 (m, 12H), 3.34-3.24 (m, 2H), 2.98 (s, 12H), 2.96 (s, 16H), 2.23-2.08 (m, 2H), 1.77-1.03 (m, 42H). MS (ESI, H2O): 840 (90, [M+3H+2TFA]3+), 803 (100, [M+3H+TFA]3+), 765 (30, [M+3H]3+), 629 (30, [M+4H+2TFA]4+), 602 (25, [M+4H+TFA]4+). HRMS (ESI): Calcd for C99H162N34O29 : 1145.6119; found: 1145.6118 ([M+2H]2+). 1.5.11 Synthesis of DexP(Man)2 Following the general protocol of the SPPS for synthesizing an Dex labelled peptide with two mannoses, DexP(Man)2 was obtained after RP-HPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] with an overall yield of 5% and 99.6% purity. Rt 4.2 min (Fig. S26) [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min)]. 1H NMR (300 MHz, D2O, δ): 7.62 (s, 1H), 7.38 (d, J = 10.4 Hz, 1H), 7.00 (s, 1H), 6.27 (d, J = 9.9 Hz, 1H), 6.07 (s, 1H), 4.77 (s, 2H), 4.43 (s, 8H), 4.13 (m, 13H), 3.873.44 (m, 12H), 3.23 (s, 2H), 3.15-2.90 (m, 16H), 2.84 (m, 2H), 2.26 (m, 2H), 2.03-1.11 (m, 70H), 0.93-0.62 (m, 36H). MS (ESI, H2O): 1573 (18, [M+2H+3TFA]2+), 1517 (20, [M+2H+2TFA]2+), 1049 (22, [M+3H+3TFA]3+), 1011 (90 [M+3H+2TFA]3+), 974 (100, [M+3H+TFA]3+), 938 (50, [M+3H]3+). HRMS (ESI): Calcd for C125H222FN37O34: 1402.3371; found: 1402.3376 ([M+2H] 2+). Supporting Information 157 1.5.12 Synthesis of CFArg8 For the preparation of the control peptide CFArg8, the carboxyfluorescein was coupled in the amino group of a lysine Mtt localized at the end of the peptide sequence. Therefore, following the general protocol of the SPPS for growing the peptide, the Mtt protecting group was selectively removed by mechanically stirring the resin with a mixture of DCM/HFIP/TFE/TIS (6.5:2:1:0.5, 2 x 1 mL per 70 mg of resin) for 2 h. Finally, the mixture was filtered and the resin was washed with DCM (2 x 2 mL, 2 min) and DMF (2 mL, 20 min). Then, a solution of 5(6)-carboxyfluorescein (2 equiv) and N-HBTU (2 equiv) in DMF (1 mL) was added to the vessel followed by the drop wise addition of DIEA (4 equiv). The resulting mixture was shaken by bubbling Ar for 30 min and finally the filtered resin was washed with DMF (3 x 2 ml, 2 min) and DCM (3 x 2 ml, 2 min) (Fig. S10). CFArg8 was obtained after RP-HPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] with an overall yield of 11% and 100% purity. Rt 3.2 min (Fig. S27) [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min)]. 1H NMR (500 MHz, D2O, δ): 8.16 (d, J = 1.8 Hz, 1H), 7.93 (t, J = 7.9 Hz, 1H), 7.58 (s, 1H), 7.36 (d, J = 7.9 Hz, 1H), 7.17-7.06 (m, 2H), 6.86-6.77 (m, 1H), 6.72 (td, J = 9.3 and 1.8 Hz, 1H), 6.60 (dd, J = 9.2 and 2.4 Hz, 1H), 4.24-3.86 (m, 9H), 3.32 (m, 2H), 3.12-2.87 (m, 16H), 1.88 (d, J = 10.2 Hz, 3H), 1.76-1.16 (m, 38H). MS (ESI, H2O): 675 (50, [M+3H+2TFA]3+), 638 (100, [M+3H+TFA]3+), 599 (45, [M+3H]3+), 477 (50, [M+4H+TFA]4+), 450 (58, [M+4H]4+), 360 (17, [M+5H]5+). HRMS (ESI): Calcd for C77H125N35O16: 898.0008; found: 898.0016 ([M+2H]2+). 1.5.13 Synthesis of AcP(Biot)2 For the preparation of the biotinylated peptide, D-(+)-Biotin was coupled in the amino group of the lysines Mtt localized at the peptidic sequence. Therefore, following the general protocol of the SPPS for growing the peptide, the Mtt protecting group was selectively removed by mechanically stirring the resin with a mixture of DCM/HFIP/TFE/TIS (6.5:2:1:0.5, 2 x 1 mL per 70 mg of resin) for 2 h. Finally, the mixture was filtered and the resin was washed with DCM (2 x 2 mL, 2 min) and DMF (2 mL, 20 min). Then, a solution of D-(+)-Biotin (2 .5 equiv) and N-HATU (2.5 equiv) in DMF (1 mL) was added to the vessel followed by the drop wise addition of DIEA (4 equiv). The resulting mixture was shaken by bubbling Ar for 30 min and finally the filtered resin was washed with DMF (3 x 2 ml, 2 min) and DCM (3 x 2 ml, 2 min) (Fig. S11). AcP(Biot)2 was obtained after RP-HPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min), 95:5→5:95 (5→35 min)] with an overall yield of 11% and 100% purity. Rt 4.1 min (Fig. S28) [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min)]. 1H NMR (500 MHz, D2O, δ): 4.52-4.39 (m, 2H), 4.34-4.22 (m, 2H), 4.22-3.83 (m, 13H), 3.22-2.95 (m, 20H), 2.90-2.74 (m, 2H), 2.69-2.53 (m, 2H), 2.10 (s, 3H), 1.97-1.88 (m, 2H), 1.88-1.14 (m, 63H), 0.84-0.65 (m, 30H). MS (ESI, H2O): 1362 (10, [M+2H+4TFA]2+), 1306 (32, MARISA JUANES CARRASCO 158 [M+2H+3TFA]2+), 1249 (30, [M+2H+2TFA]2+), 1191 (7, [M+2H+TFA]2+), 870 (18, [M+3H+3TFA]3+), 832 (80, [M+3H+2TFA]3+), 795 (100, [M+3H+TFA]3+), 756 (35, [M+3H]3+), 624 (20, [M+4H+2TFA]4+), 596 (25, [M+4H+TFA]4+), 567 (40, [M+4H]4+). HRMS (ESI): Calcd for C100H186N38O18S2: 1135.7120; found: 1135.7119 ([M+2H] 2+). 1.6 Preparation of Dexamethasone labelled Concanavalin A 1.6.1 Synthesis of Dexamethasone-NHS Dexamethasone-NHS was prepared by the reaction of Ox-DexS2 (0.106 mmol) with NHydroxysuccinimide (1.5 equiv) in DMF, using EDC (1 equiv) and DMAP (cat.) for 4 h. The DMF was removed by rotary evaporation and the oil obtained was dried. The compound was washed with 1 M solution of HCl and after that with a saturated solution of NaHCO3. Purification by silica gel column chromatography (DCM/MeOH, 99:1) provided final product with an overall yield of 35%. 1H NMR (500 MHz, CDCl3, δ): 7.22 (d, J = 10.1 Hz, 1H), 6.32 (dd, J = 10.1, 1.9 Hz, 1H), 6.12 (s, 1H), 4.37 (d, J = 8.1 Hz, 1H), 3.22 (s, 1H), 3.12-2.97 (m, 1H), 2.84 (s, 4H), 2.63 (td, J = 13.8, 13.0, 6.1 Hz, 1H), 2.49-2.17 (m, 4H), 1.87-1.72 (m, 3H), 1.63-1.56 (m, 1H), 1.55 (s, 3H), 1.35-1.28 (m, 2H), 1.22 (s, 3H), 1.07 (d, J = 7.1 Hz, 3H). 13C NMR (500 MHz, CDCl3, δ): 186.56 (q), 168.40 (q), 166.04 (q), 152.12 (CH), 129.81 (CH), 125.13 (CH), 101.02 (q), 99.62 (q), 86.46 (q), 71.99 (CH), 48.62 (q), 48.17 (q), 42.96 (CH), 37.01 (CH), 35.88 (CH2), 34.32 (CH), 32.35 (CH2), 30.99 (CH2), 29.70 (CH2), 25.67 (CH2), 22.90 (CH3), 16.70 (CH3), 14.57 (CH3). (Fig. S29). HRMS (ESI): Calcd for C25H31FNO7: 476.2082; found: 476.2079. 1.6.2 Synthesis of ConA-Dex ConA-Dex was prepared by the reaction of Concanavalin A (3 mg/mL) with Dexamethasone-NHS (50 equiv) in NaHCO3 buffer (pH 9) at room temperature for 2 h. The resulting solution was dialysed overnight in HKR buffer. To quantify the number of dexamethasones per Concanavalin A the absorbance of the mixture was measured using a NanoDrop UV-Vis Spectrophotometer. Absorbance values at 242 nm (maximum absorbance for dexamethasone) and 280 nm (maximum absorbance for the protein) were measured for various dilutions of the dialysed ConA-Dex, and several known concentrations of ConA or dexamethasone. Data in the linear range were analysed using the following system of equations: A!"! !"#$!!"# =x·A!"! !"#$ +y·A!"! !"# A!"# !"#$!!"# =x·A!"# !"#$ +y·A!"# !"# O O OH HO F HO 1.5 equiv. NHS 1 equiv. EDC, DMAPcat., DMF, 35% O O O HO F HO N O O Ox-Dex DexNHS Supporting Information 159 Where the values x and y were used to estimate the concentration of each component in ConA-Dex by multiplying the concentration of pure ConA or dexamethasone by x or y respectively, and the extent of labelling was calculated as the ratio between dexamethasone and ConA concentrations. A value of around 16 Dex per ConA tetramer was obtained. 1.7 General Procedure for Circular Dichroism Circular dichroism measurements were carried out using the following settings: acquisition range: 300-190 nm; band width: 1.0 nm; accumulation: 3 scans; data pitch: 1 nm; CD scale 200 mdeg/1.0 dOD; D.I.T. (Data Integration Time): 1 s; scanning mode: continuous; scanning speed: 200 nm/min. Measurements were done from 10 ºC to 60 ºC (data interval: 10 ºC; temp. gradient 5 ºC/min) in a quartz cell of 0.2 cm path length at a final volume of 0.5 mL (HKR buffer or TFE) with a final peptide concentration of 200 µM. For the measurements in liposomes, samples were prepared by drying under reduced pressure L--phosphatidylcholine (8.5 µL, 100 mg/mL solution in CHCl3) and peptide (325 µL, 200 µM) in TFE to obtain a ratio lipid/peptide of 18:1. Lipids were suspended in HKR buffer (650 µL) and sonicated for 45 min until a clear solution was obtained. Spectra were recorded in a 0.2 cm path length quartz cell. The results are expressed as the mean residue molar ellipticity [θ]MRt with units of degrees·cm2·dmol-1 and calculated using the equation S1, [!]!"# = 100 ·! !·!·!".!" !"#$%&"# (S1) where θ is the ellipticity (mdeg), C is the peptide concentration (M) and l is the cell path length (cm). 1.8 Cells Lines and Culture HeLa cells were incubated at 37 ºC/ 5% CO2/ 95% humidity in an INCO 108 incubator (Memmert) with Dulbecco’s Modified Eagle’s Medium (4500 mg/L glucose, L-glutamine, sodium pyruvate and sodium bicarbonate; Sigma-Aldrich), supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% of Penicillin-Streptomycin-Glutamine Mix (Fisher). 1.9 Cell transport experiments in HepG2 cells Internalization experiments in HepG2 cells were done as described for HeLa cells (see Methods in the manuscript). In the case of streptavidin, incubation time was increased to 5 h. 1.10 Cell viability: MTT Assay Cell viability was established by a standard MTT assay (Fig. S7 and S8). One day before the assay, a suspension of HeLa or HepG2 cells was plated in 96-well tissue culture plates (Costar 96 Flat Bottom Transparent Polystyrol) by adding 100 µL (150.000 cells/mL) per well. The next day, the medium was aspirated and cells were incubated with different concentrations of peptide and peptide/protein complexes diluted in HKR (50 µL/well). After MARISA JUANES CARRASCO 160 30 min of incubation at 37 ºC, the medium was aspirated and replaced by fresh medium (DMEM) containing 10% FBS (100 µL). Control cells were given only cell culture medium (100 µL final medium). The viability was measured by quantifying the cellular ability to reduce the water-soluble tetrazolium dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) to its insoluble formazan salt as follows. MTT (5 mg/mL in PBS, 10 µL/well) was added to the wells and the cells were further incubated for 6 h. The supernatant was carefully removed and the water-insoluble formazan salt was dissolved in DMSO (100 µL/well). The absorbance was measured at 570 nm using a microplate reader (Infinite F200pro, Tecan). Data points were collected in triplicate and expressed as normalized values for untreated control cells (100%). 1.11 Propidium Iodide Assay Cell viability was established by a standard PI assay (Fig. S7 and S8). One day before the assay, a suspension of HeLa or HepG2 cells was plated in 96-well tissue culture plates (Costar 96 Flat Bottom Transparent Polystyrol) by adding 100 µL (150.000 cells/mL) per well. The next day, the medium was aspirated and cells were incubated with different concentrations of peptide and peptide/protein complexes diluted in DMEM (50 µL/well). After 30 min of incubation at 37 ºC, the medium was aspirated; cells were washed with fresh medium and then 100 µL of trypsin were added in each well and cells were incubated for 15 min at 37 ºC. After this, 100 µL of a solution of 2% FBS and 5 mM EDTA in PBS containing PI (0.25 µg/mL) were added and the plate was incubated for 5 min in the dark. Samples were analysed on a Guava EasyCyteTM cytometer. Propidium iodide was measured by excitation at 532 nm and collecting emission at 695/50 nm. Data points were collected in triplicate and analysis was performed with InCyte software included in GuavaSoft 3.2 (Millipore). 1.12 Quantification of the uptake by fluorometry For internalization assays, a modification of the quantification of CPP uptake by fluorometry protocol described in Holm et al.S5 was used. Briefly, semiconfluent monolayers of HeLa cells seeded the day before were washed twice with HKR before incubation with 4 µM of TAMRA labelled peptides, with TmP(Man)2/ConA or with AcP(Man)2/ConAFITC complexes (previously prepared by co-incubation for 7 min rt) diluted in the same buffer for 30 min at 37 ºC (unless otherwise stated). Then, cells were washed twice with HKR and trypsinized, and after the addition of 1 mL of HKR, centrifuged for 5 min at 1000 g. The resultant pellets were lysed by incubation in aqueous solution of NaOH (0.1 M) at 4 ºC for 60 min and frozen until analysis. Where indicated, cells were pretreated for 30 min before the incubation of the peptide and during the incubation with the following inhibitors: Wortmannin (200 nM), chlorpromazine (30 µM), methyl-ß-cyclodextrin (5 mM), EIPA (50 µM), ammonium chloride (50 mM), chloroquine (100 µM) or heparin (5 µg/mL). For the incubation at low temperature, cells were placed on ice 15 min before the incubation with the peptides, and ice-cold solutions were used for the washes and incubation. Supporting Information 161 Fluorescence levels of the lysates were measured using a microplate reader (Infinite F200Pro, Tecan), both for TAMRA (ex 560 nm, em 610 nm) and fluorescein (ex 485 nm, em 535 nm) labelled compounds, and the concentration of the peptides or ConA was calculated by comparison to a standard curve. After neutralization with a volume of an aqueous solution of HCl (0.1 M), protein concentration in the lysates was determined using a Coomassie (Bradford) protein assay kit following the manufacturer instructions, and measuring the absorbance at 570 nm (Infinite F200Pro, Tecan). The uptake was calculated as pmol of peptide/mg of total protein, and normalized to the uptake in untreated cells in the case of the inhibition studies. 1.13 Glucocorticoid assay The ability of the peptide to reach the cytosol was determined with a glucocorticoid induced GFP translocation assay (GIGT)S6. In this assay, a GFP protein fused to the steroid binding domain of the glucocorticoid receptor (GR) accumulates in the nucleus of the cell in response to dexamethasone binding, so the translocation ratio (the ratio between the nuclear and cytoplasmic fluorescence) can be used as an indicator of the presence of dexamethasonelabelled peptides in the cytosol (Fig. S9). HeLa cells grown in four chamber glass bottom dishes were transfected with the plasmid pK7-GR-GFP (a gift from Ian MacaraS7, Addgene plasmid #15534) using Lipofectamine 2000 and, 24 h post-transfection, cells were washed with HKR and incubated for 30 min with Hoechst 33342 (1 µM), and endocytosis inhibitors where indicated, at the following concentrations: chlorpromazine (50 µM), methyl-ß-cyclodextrin (5 mM), Wortmannin (200 nM), chloroquine (100 µM), NH4Cl (50 mM), heparin (5 µg/mL) or EIPA (50 µM). Cells were then incubated for 1 h with DexP(Man)2 (4 µM), in the presence or absence of unlabelled ConA (30 nM), with dexamethasone (1 µM) (as positive control) or just with HKR (as negative control) and immediately imaged. To study the cytosolic release of ConA-Dex, cells were incubated with 7 nM of Dex-labelled ConA (equivalent to around 100 nM Dexamethasone) previously incubated with 4 µM of AcP(Man)2. As controls, cells were incubated with the same amount of ConA-Dex in the absence of peptide or after incubation for 20 min with 1 µg of Lipofectamine 2000. Twenty to thirty images of each sample were acquired with an Andor Zyla 4.2 digital camera mounted on a Nikon Eclipse Ti-E microscope at 60x magnification and the translocation ratio (the ratio of the median intensities of GFP in the nucleus and in the 2 µm wide surrounding region) was calculated with CellProfilerS8 as follows. Nuclei were identified as Hoechst stained objects using the three-class thresholding Otsu method and the cytoplasmic region was defined as the 2 µm surrounding area. To ensure a better separation of the cytoplasmic and nuclear region, nuclei were shrunk 0.5 µm before measuring object intensity. Cells falling below the 20% of the maximum intensity of the image were considered untransfected and discarded for the analysis. A total of 40 to 80 cells were analyzed for each sample. Statistical analysis of the data was performed with R softwareS9. Results were subjected to pairwise two-tailed Student's t-test and p-values were adjusted using Bonferroni's correction. MARISA JUANES CARRASCO 168 Figure S21. A) 1H-NMR spectra in D2O of TmP(Acetone)2. B) RP-HPLC [Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min)] (Rt 4.4 min) and ESI-MS for TmP(Acetone)2. H NN H NH2 O N H O H N O N H O H N O N H O H N O N H O HN H N NH NH2 H2N H NN H O O OO O HN N H O NH NH2 H2N NH NH2 H2N NH H2NNH2 NH NH2 H2N NH NH2 H2N H N O O N N O O O N O O N O -100 0 100 200 300 400 500 600 0 1 2 3 4 5 6 7 Abs 222 nm (a.u.) time/min 0 20 40 60 80 100 120 400 600 800 1000 1200 1400 1600 m/z 1376,1 1433,9 1491,7 881,4 917,5 632,2 661,5 690,0 A) B) Supporting Information 169 Figure S22. A) 1H-NMR spectra in D2O of AcArg2(Man)2. B) RP-HPLC [Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min)] (Rt 3.1 min) and ESI-MS for AcArg2(Man)2. H NH 2 N O NH H 2 NNH 2 N HO HN H N O HN N HO NH 2 H 2 N NH O O N O O N O O O O O OH HO OH OH HO HO HO HO -50 0 50 100 150 200 250 300 350 0 1 2 3 4 5 6 7 Abs 222 nm (a.u.) time/min 0 20 40 60 80 100 120 400 600 800 1000 1200 1400 1183,3 m/z 590,0 A) B) MARISA JUANES CARRASCO 170 Figure S23. A) 1H-NMR spectra in D2O of TmArg2(Man)2. B) RP-HPLC [Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min)] (Rt 3.8 min) and ESI-MS for TmArg2(Man)2. H NH 2 N O NH H 2 NNH 2 N HO H N O HN N HO NH 2 H 2 N NH O O N O HO HO OH N H O O N N O O O O O OOH HO OH OH O N O NH OH 0 20 40 60 80 100 120 300 400 500 600 700 800 900 1000 833,5 m/z 556,3 0 50 100 150 200 0 1 2 3 4 5 6 7 Abs 222 nm (a.u.) time/min A) B) Supporting Information 171 Figure S24. A) 1H-NMR spectra in D2O of TmArg6Gly5(Man)2. B) RP-HPLC [Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min)] (Rt 3.4 min) and ESI-MS for TmArg6Gly5(Man)2. H NN H NH2 O N H O H N O N H O H N O N H O H N O N H O HN H N NH NH2 H2N H NN H O O OO O HN N H O NH NH2 H2N NH NH2 H2N NH H2NNH2 NH NH2 H2N NH NH2 H2N H N O O N N O O O N O O N O O O O O OH OH OH OH OH OH HO HO 0 50 100 150 200 250 300 350 0 1 2 3 4 5 6 7 time/min Abs 222 nm (a. u.) 0 20 40 60 80 100 120 400 600 800 1000 1200 1400 516.2 645.0 673.5 701.8 897.7 935.5 973.3 m/z 1402.1 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5 ppm 42.3 2.4 8.2 12.5 2.2 12.2 16.1 12.3 4.1 3.8 2.2 1.5 2.1 2.5 0.5 0.6 0.6 0.7 A) B) MARISA JUANES CARRASCO 172 Figure S25. A) 1H-NMR spectra in D2O of TmArg6(Man)2. B) RP-HPLC [Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min)] (Rt 3.4 min) and ESI-MS for TmArg6(Man)2. O O N N O O NH 2 H N O NH N HO NH 2 H 2 N NH H N O NH 2 H 2 N NH N HO NH 2 H 2 N NH H N O HN N HO NH 2 H 2 N NH H N O NH 2 H 2 N NH N HO NH 2 H 2 N NH O N H O O N O N O O O OO OH OH OH OH OH OH OH OH 0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0 ppm 43.3 2.0 15.7 12.6 2.2 12.9 8.5 4.4 4.2 2.1 2.4 2.2 3.1 1.8 1.0 1.0 0 20 40 60 80 100 120 300 400 500 600 700 800 900 1000 602.2 629.3 764.5 802.5 840.3 % Abundance m/z -50 0 50 100 150 200 250 0 1 2 3 4 5 6 7 Abs 222 nm (a. u.) time/min A) B) Supporting Information 173 Figure S26. A) 1H-NMR spectra in D2O of DexP(Man)2. B) RP-HPLC [Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min)] (Rt 4.2 min) and ESI-MS for DexP(Man)2. H NN H NH2 O N H O H N O N H O H N O N H O H N O N H O HN H N NH NH2 H2N H NN H O O OO O HN N H NH NH2 H2N NH NH2 H2N NH H2NNH2 NH NH2 H2N NH NH2 H2N O H N O N O O N O O O F HO HO O O O O HO HO OH OH OH OH OH HO 0 100 200 300 400 0 1 2 3 4 5 6 7 time/min Abs 222 nm (a.u.) 0 20 40 60 80 100 120 400 600 800 1000 1200 1400 1600 m/z 973,7 1011,0 1049,4 1517,2 1573,0 937,7 A) B) 8. 0 MARISA JUANES CARRASCO 174 Figure S27. A) 1H-NMR spectra in D2O of CFArg8. B) RP-HPLC [Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min)] (Rt 3.2 min) and ESI-MS for CFArg8. H NN H O O NH2 O N H NH NH2 H2N O H N NH O N H NH O H N NH O NH2 H2N NH2 H2N NH2 H2N N H NH NH2 H2N O H N NH NH2 H2N O N H O HN O O HO OO OH NH NH2 H2N NH NH2 H2N 0 50 100 150 200 250 300 0 1 2 3 4 5 6 7 time/min Abs 222 nm (a.u.) 0 20 40 60 80 100 120 320 400 480 560 640 720 800 880 960 m/z 449,7 477,3 360,1 598,6 637,9 675,3 A) B) Supporting Information 175 Figure S28. A) 1H-NMR spectra in D2O of AcP(Biot)2 B) RP-HPLC [Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→5 min)] (Rt 4.1 min) and ESI-MS for AcP(Biot)2. 0.00.51.01.52.02.53.03.54.04.55.05.5 ppm 32.4 64.3 2.2 3.0 1.8 1.7 19.6 13.2 1.6 1.5 0 20 40 60 80 100 120 400 600 800 1000 1200 1400 m/z 1191.2 1248.5 1305.5 1362.3 756.3 794.6 832.4 870.2 567.4 595.8 624.3 0 200 400 600 800 1000 1200 012345678 Abs 222 nm (a. u.) time/min H NN H NH 2 O N H O H N O N H O H N O N H O H N O N H O HN H N NH NH 2 H 2 N H NN H O O OO O NH N H NH NH 2 H 2 N NH NH 2 H 2 N NH H 2 NNH 2 NH NH 2 H 2 N NH NH 2 H 2 N O HN O H NHH S O NH O H HN H S O A) B) MARISA JUANES CARRASCO 176 Figure S29. 1H-NMR, DEPT and 13C-NMR spectra in CDCl3 of DexNHS. 0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.5 ppm 3.0 3.1 1.7 3.2 1.1 2.9 4.1 1.0 4.1 1.0 0.8 1.0 1.0 1.0 1.0 0102030405060708090100110120130140150160170180190200 ppm O O O HO F HO N O O Supporting Information 177 1.18 Supporting References [S1] W. Yao, Y. Jiao, J. Luo, M. Du, L. Zong, Int. J. Biol. Macromol. 2012, 50, 821–825. [S2] M. V Govindan, B. Manz, 1980, 53, 47–53. [S3] R. Behrendt, P. White, J. Offer, J. Pept. Sci. 2016, 22, 4–27. [S4] C. Kay, O. E. Lorthioir, N. J. Parr, M. Congreve, S. C. McKeown, J. J. Scicinski, S. V Ley, Biotechnol. Bioeng. 2000, 71, 110–8. [S5] T. Holm, H. Johansson, P. Lundberg, M. Pooga, M. Lindgren, U. Langel, Nat. Protoc. 2006, 1, 1001–5. [S6] J. M. Holub, J. R. Larochelle, J. S. Appelbaum, A. Schepartz, Biochemistry 2013, 52, 9036–46. [S7] K. L. Carey, S. A. Richards, K. M. Lounsbury, I. G. Macara, J. Cell Biol. 1996, 133, 985–96. [S8] A. E. Carpenter, T. R. Jones, M. R. Lamprecht, C. Clarke, I. H. Kang, O. Friman, D. A. Guertin, J. H. Chang, R. A. Lindquist, J. Moffat, et al., Genome Biol. 2006, 7, R100. [S9] R Core Team, R: A Language and Environment for Statistical Computing, 2015.