Supramolecular strategies for intracellular delivery and design of new thermoresponsive materials

TESIS DOCTORAL SUPRAMOLECULAR STRATEGIES FOR INTRACELLULAR DELIVERY AND DESIGN OF NEW THERMORESPONSIVE MATERIALS Héctor Fernández Caro ESCUELA DE DOCTORADO INTERNACIONAL PROGRAMA DE DOCTORADO EN CIENCIA Y TECNOLOGÍA QUÍMICA SANTIAGO DE COMPOSTELA AÑO 2020 DECLARACIÓN DEL AUTOR DE LA TESIS SUPRAMOLECULAR STRATEGIES FOR INTRACELLULAR DELIVERY AND DESIGN OF NEW THERMORESPONSIVE MATERIALS Para defensas telemáticas D. Héctor Fernández Caro Presento mi Tesis, siguiendo el procedimiento adecuado al Reglamento, y declaro que: 1) La Tesis abarca los resultados de la elaboración de mi trabajo. 2) De ser el caso, en la Tesis se hará 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 mí para la obtención de otros títulos. Y me comprometo a presentar el ejemplar impreso de la Tesis en el plazo de un mes desde que la EDIUS me lo requiera, así como del Compromiso Documental de Supervisión en el caso que el original no esté depositado en la Escuela. En Santiago de Compostela, ... de ..... de 2020 Fdo. Héctor Fernández Caro AUTORIZACIÓN DEL DIRECTOR / TUTOR DE LA TESIS SUPRAMOLECULAR STRATEGIES FOR INTRACELLULAR DELIVERY AND DESIGN OF NEW THERMORESPONSIVE MATERIALS D. Juan Ramón Granja Guillán D. Javier Montenegro García INFORMAN: Que la presente Tesis se corresponde con el trabajo realizado por D. Héctor Fernández Caro, bajo nuestra dirección, y autorizamos su presentación, considerando que reúne los requisitos exigidos en el Reglamento de Estudios de Doctorado de la USC, y que como directores de esta no incurre en las causas de abstención establecidas en la Ley 40/2015. En Santiago de Compostela, ... de ..... de 2020 Fdo. Juan Ramón Granja Guillán Fdo. Javier Montenegro García Agradecimientos En primer lugar, me gustaría expresar mi enorme agradecimiento a los directores de esta Tesis doctoral, Javier Montenegro y Juan R. Granja. Gracias por darme esta oportunidad y permitir que todo esto haya sido posible. A Javier Montenegro, por darme la oportunidad de formar parte de este proyecto. Gracias por trasmitirnos día tras día tu pasión por la investigación, por tus multiples enseñanzas para poder desarrollar investigación de alta calidad y por tu apoyo a nivel personal en los momentos difíciles. Todavía recuerdo mis inicios cuando me enseñabas a hacer mis primeras columnas cromatográficas. Sin duda, todo lo conseguido ha sido fruto de innumerables horas de trabajo, esfuerzo y aprendizaje. Gracias por confiar en mí. A Juan R. Granja, por tus innumerables enseñanzas, ideas para mejorar el desarrollo de los proyectos y discusiones científicas en los seminarios. Por transmitirnos tu enorme rigor científico y por enseñarnos y demostrarnos los valores que debe cumplir un excelente investigador y profesor. Por enseñarnos que de los errores también se aprende. Muchas gracias. Agradecer a la Xunta de Galicia la concesión de mi beca predoctoral (ED481A-2017/047) que me ha permitido desarrollar esta Tesis doctoral y por haberme dado la oportunidad de realizar una estancia predoctoral en Northwestern University. Además, también me gustaría dar las gracias a todas las instituciones que han financiado la labor investigadora del grupo durante estos años. A Alejandro Méndez, por todo lo que me has enseñado. Por poseer esa capacidad para reconducir los proyectos y para hacer que lo difícil parezca fácil. A Irene Lostalé, por enseñarme a hacer los primeros experimentos en células y explicarme todo lo necesario para poder entender la parte biológica de los proyectos. A Julián Bergueiro, por confiar en mí y permitirme aprender de tí, día tras día, en los distintos proyectos que compartimos. Muchas gracias. A los doctores Manuel Amorín, Rebeca García-Fandiño y al prof. Luis Castedo. Por las interesantes discusiones científicas durante los seminarios de grupo. A Patricia Lago, por tu por tu ayuda y amabilidad a la hora de resolver cualquier tipo de gestión. Al prof. Nathan Gianneschi por haberme brindado la oportunidad de realizar una estancia en su grupo de investigación en Northwestern University, Evaston, Illinois. A Andrea S. Carlini, Mollie A. Touve, Matthew P. Thompson, Wei Cao, Mary F. Cassidy, Spencer Burton, Claudia Battistella, Hao Sun, Chris Forman, Wonmin Choi y al resto del grupo por haberme ayudado a crecer como investigador y acompañarme en esta gran experiencia. Al prof. Jose Luis Mascareñas por sus ideas, consejos y ayuda en el desarrollo del proyecto. A Miguel Martínez-Calvo por su ayuda en la preparación de los transportadores y, también, a Jesús Mosquera. Al prof. Jonathan R. Nitschke y su grupo de investigación por la preparación de la caja supramolecular. A Rebeca Menaya-Vargas por su ayuda con los cultivos celulares y su enorme interés y dedicación para que todo salga siempre a la perfección. A la RIAIDT de la Universidad de Santiago de Compostela por poner a mi disposición sus medios técnicos para el correcto desarrollo de esta Tesis doctoral. En especial me gustaría destacar a Esteban Guitián por todos los análisis de espectrometría de masas realizados. Además, me gustaría agradecer a todo el personal técnico del CIQUS, en especial a Arcadio, Laura, Pablo y Noela. Me gustaría agradecerle enormemente a todas/os y cada una/o de mis compañeras/os de laboratorio, que después de todo este tiempo, tengo el placer de, a muchas/os, poder llamarlas/os amigas/os. A Ale, creo que ninguna palabra puede agradecer todo lo que has hecho por mí durante estos años, ya no solo todo lo que me has enseñado en el laboratorio, sino como amigo, me has hecho reir, has sabido comprenderme y animarme en todo momento. Eres una persona y un amigo impresionante y te deseo todo lo mejor porque te lo mereces. A Alfonso, gracias por recorrer este duro camino conmigo. Pienso en el pasado y recuerdo millones de buenos momentos, chistes, risas, viajes, fiestas, festivales, conversaciones en el desierto. Pero, sin duda, lo que nos ha hecho ser grandes amigos es el mantenernos unidos y animarnos en los malos momentos. Muchas gracias por todo, mago. A Marisa, por enseñarme y cuidarme desde que llegué al laboratorio y ayudarme a confiar en el péptido. Marisa, confía en el péptido. A Juanillo, por enseñarme en los inicios y querer siempre lo mejor para mí. A Iván, por todos esos abrazos y animos reconfortantes, por las gominolas a últimas horas de la tarde. A Jose Juan ¨Josete¨, por ayudarme cuando las cosas no salían, por hacerme reir día a día, por aconsejarme como a un hijo, por tu forma de aplicar matemáticas. A Marta, por acompañarme en este camino y permitirme observar en primera persona la gran investigadora en la que te has convertido. A Irene, por ayudarnos siempre en todo lo necesario, por tus bromas. A Alberto, por tus millones de consejos, por ayudarme con el proyecto inicial y con muchas otras cosas, por contarnos miles de veces la misma historia, haciéndonos creer, a veces, que tu eras el protagonista, por los partidos de padel mañaneros. A Álvaro, por ser parte del grupo, por nuestras comiditas y cafecitos, por proporcionarnos siempre cualquier reactivo. A Julián, por crear en menos de un segundo millones de ideas para millones de nuevos proyectos, por confiar en mí para los nuevos proyectos, por tener siempre un chiste malo para compartir. A Geert, por llegar al laboratorio todos los días con una sonrisa. También a Mariette, gracias por tu enorme generosidad. A Rebe, por tus ganas de aprender y por suministrarme chocolate en tiempos de escritura. También, a Jose María. A Lamas, por todos los grandes momentos compartidos, por demostrarnos como se hace un buen revés en tenis. A Nacho, por las clases prácticas, por las risas y bromas compartidas, por tus inesperados lanzamientos de pipas. A Giulia, por todas las conversaciones, por enseñarnos italiano, por organizar las mejores cenas y fiestas. A Martín Calvelo, por todas las anécdotas graciosas que vivimos, por formar parte del equipo trámite. A Evita, por todas las conversaciones de camino al trabajo, aunque, a veces, te quedases dormida, por tu apoyo y tus enseñanzas. A Ángel, por tu bondaz, tu esfuerzo y dedicación que me han enseñado que todo esfuerzo, al final, tiene su recompensa. A Vicky, por irradiar esa alegría característica. A Fede, por tu generosidad, espero que todo vaya increíble en tu nueva etapa. A Alicia, por ser una magnifica vecina de vitrina. A Iria, por ayudarme en los inicios. A los más jóvenes, Sandra, Marcos, Ezequiel, Antía, Charlene etc. estoy seguro que os irá muy bien. A Richard, Ghibom y a Sahnawaz, por todos los momentos compartidos. Además, también, a todos los compañeros de otros grupos de investigación, en especial a Jaime, por animarme día tras día, por esperarme mientras acababa los experimentos antes de ir a entrenar, por ser el gran capitán. También a Felipe, por recibirme siempre con una sonrisa y algo nuevo que contar, por compartir tu sabiduría. A Tomás Pose, por animarme siempre a superar mis limites, por los maratones nocturnos a la ciudad de la cultura y vuelta. A Jacobo, a David y a Marc, por todas las charlas siempre divertidas. A Bea, por tratarme siempre tan bien. A todas las demás personas que han contribuído en mayor o menor medida al desarrollo de este período de mi vida y no los haya citado anteriormente. Todas estas personas han aportado su granito de arena, tanto dentro como fuera del laboratorio, en esta Tesis. Muchísimas gracias a todos. Entrando en un ámbito más personal, me gustaría agradecerle todos los animos recibidos y experiencias vividas a todos mis amigos/as durante estos años. A mis amigos que conocí hace ya muchos años en la universidad y todavía forman parte de mi vida. En especial voy a destacar a los pilares básicos en mi vida: A Andrea, por acompañarme en mi desarrollo como investigador y, a la vez, personal, por hacer evaporarse todos los problemas con un par de palabras, por tu empuje y motivación durante todos estos años. A Bustelo, por ser el claro ejemplo de optimismo y transmitírselo a toda la gente que te rodea. Tengo suerte de tenerte cerca. A Martín, por acudir al rescate siempre que lo necesito y hacerme reir hasta más no poder. A Reims, por comprenderme y aconsejarme, a Rocío, a Nerea y a Andrea López por su apoyo continuado. A mis amigos de toda la vida, en especial a Rica. Siempre dispuestos a aconsejarme, ayudarme y vivir nuevas experiencias. Por muchos años que pasen conservamos nuestra amistad y eso es un auténtico tesoro. A mis compañeros/amigos del equipo de fútbol, que me permitisteis liberar todas las tensiones del doctorado y llegar a formar parte de vuestra ¨familia¨. A Carliños, por tratarme tan bien y enseñarme tanto inglés. A Jorge, por convertirte en una pieza muy importante en mi vida, por recorrer el mundo conmigo, por querer siempre lo mejor para mí, por regarlarme siempre los mejores consejos. A Sara, gracias por transmitirme todos los días tus ganas de vivir y tu alegría, por convertirte en la perfecta compañera de aventuras, experiencias y viajes, por tirar de mi cuando las cosas no salen bien, por hacer que este baile valga la pena. Gracias por todo lo vivido, ha sido apasionante. Me alegra pensar todo lo que nos queda por vivir. Agradecer, también, a Ana, César y Anita todo el apoyo que me habéis brindado y por tratarme siempre tan bien. Por último, me gustaría dedicarle unas líneas a mi familia por apoyarme y animarme en todo momento. A mis abuelos, en especial a mi abuela Lola, que me demuestra día a día que la edad no importa sino las ganas de vivir. A mi sobrino Álvaro, que desde que llegó a la familia, nos ha proporcionado un enorme soplo de aire fresco. A Luis. A mi hermana, Jessica, siempre dispuesta a ayudarme y, sin duda, uno de mis grandes apoyos en la vida. A mis padres, por avanzar en tiempos en los que sólo estudiaban algunos privilegiados, por prometerse, hasta conseguirlo, que sus hijos, al revés que ellos, podrían ir a la universidad y por formar esta familia de la cual estoy tan orgulloso. Gracias por tanto. Abbreviations 17 Abbreviations δ ζ λem λex Aa Abs Ac Ad ACPPs ACN AFM Ahx Ala anti-EGFR Arg ATRP AuNPs BCECF BCECF-AM Boc br s 13C-NMR CA calcd CB β-CD CD CDs β-CD@AuNPs CD@AuNPs chemical shift (ppm) zeta emission wavelength excitation wavelength amino acid absorbance acetyl adamantane activable cell-penetrating peptides acetonitrile atomic force microscope 6-aminohexanoic acid alanine anti-epidermal growth factor receptor arginine atom transfer radical polymerization gold nanoparticles 2′,7′-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein 2′,7′-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester tert-Butoxycarbonyl broad singlet 13C nuclear magnetic resonance calixarene calculated cucurbituril β-cyclodextrin circular dichroism cyclodextrins β-cyclodextrin-modified AuNPs cyclodextrin-modified AuNPs Héctor Fernández Caro 18 CDP CF CP CPPs CPT CRISPR CTC d DCM DLS DNA DMEM DMSO DIEA DMF DP DYN E9 EDC EDTA EBPs EBPPA ELPs EPR EYPG Fab′ FBS FDA FITC Fmoc FRET FSC Gln cyclodextrin-containing polymer carboxyfluorescein cyclic peptide cell-penetrating peptides camptothecin clustered regularly interspace short palindromic repeats 2- chlorotrityl chloride doublet dichloromethane dynamic light scattering deoxyribonucleic acid Dulbecco's Modified Eagle Medium dimethylsulfoxide N, N-Diisopropylethylamine N, N-Dimethylformamide degree of polymerization dynasore nona-L-glutamic acid 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride ethylenediaminetetraacetic acid elastin-based polymers elastin-based side-chain helical poly(phenylacetylene) elastin-like polypeptides enhanced permeability and retention egg yolk phosphatidylglycerol antigen-binding fragment fetal bovine serum food and drug administration fluorescein isothiocyanate 9- Fluorenylmethoxycarbonyl fluorescence resonance energy transfer forward scatter channel glutamine Abbreviations 19 Gly Glu GSH 1H-NMR HEPES HFIP His HKR HPLC-MS HPTS HR-MS ICP-MS J Ka Kd LCST Leu Lys m MeOH MES mRNA MS MTT Mtt N-HATU N-HBTU NHS glycine glutamic acid glutathione proton nuclear magnetic resonance 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 1,1,1,3,3,3-hexafluoro-2-propanol histidine HEPES-Krebs-Ringer buffer high-performance liquid chromatography coupled with mass spectrometry 8-hydroxypyrene-1,3,6-trisulfonic acid (pyranine) high-resolution mass spectrometry inductively coupled plasma mass spectrometry coupling constant affinity constant dissociation constant lower critical solution temperature leucine lysine multiplet methanol 2-(N-morpholino)ethanesulfonic acid messenger RNA mass spectrometry (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium; 4-methyltrityl N-[(Dimethylamino)-1H1,2,3-triazolo[4,5-b]pyridine 1ylmethylene]-N-methylmethanaminium-hexafluorophosphate N-oxide; N-[(1HBenzotriazol-1-yl)-(dimethylamino)methylene]-N- methylmethanaminium hexafluorophosphate N-oxide N-hydroxysuccinimide Héctor Fernández Caro 20 NMR NPs O2Oc PAMAM Pbf PBS Phe pDNA PEG PEI PNAs PNA-AuNPs PNIPAM POEG Ppm Pro PyAOP PVCL RAFT R4 R8 RGD RP-HPLC Rt s SD Ser SCID SCPNs siRNA sgRNA shRNA nuclear magnetic resonance nanoparticles 8-amino-3,6-dioxaoctanoic acid polyamidoamine dendrimers 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl phosphate-buffered saline phenylalanine plasmid DNA polyethylene glycol polyethyleneimine peptide nucleic acids polyvalent nucleic acid capped AuNPs poly(N-isopropylacrylamide) poly(oligoethyleneglycol) parts per million proline (7-Azabenzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate; poly(N-vinlycaprolactam) reversible addition−fragmentation chain-transfer tetraarginine octaarginine arginine-glycine-aspartic acid peptide reversed-phase high-performance liquid chromatography retention time singlet standard deviation serine severe combined immunodeficiency self-assembling cyclic peptide nanotubes small interfering RNA single guide RNA small hairpin RNA Abbreviations 21 SPNs SPPS t TAMRA TCP TEM TF TFA TFE TFRC TGA TIS TNF TRAIL UCST uHPLC UV-vis Val supramolecular nanoparticles solid-phase peptide synthesis triplet 5-carboxytetramethyl rhodamine cloud point temperature transmission electron microscopy transcription factor trifluoroacetic acid trifluoroethanol transferrin receptor thermogravimetric analysis triisopropylsilane tumor necrosis factor tumor necrosis factor-related apoptosis-inducing ligand upper critical solution temperature ultra-high performance liquid chromatography ultraviolet-visible valine Summary 23 Summary Synthetic organic chemistry has played a key role in the development of novel strategies for obtaining chemically functional compounds. Despite the great advance that organic synthesis has undergone in the last decades, covalent strategies present intrinsic limits for achieving structures with larger dimensions such as architectures commonly found in Nature, such as those formed by the assembling of proteins. The preparation of these complex molecules by only using strategies based on the formation of covalent bonds is almost unapproachable from the point of view of time and human effort required together with the lack of appropriated synthetic strategies. Additionally, certain collective phenomena such as selforganization require the coordinated interaction between different chemical entities, which is outside the scope of traditional covalent chemistry. Accordingly, a novel discipline coined supramolecular chemistry emerged in the 70-80’s. This field was first defined by Jean-Marie Lehn as ¨the chemistry beyond the molecule¨. This discipline focuses on the study of interactions between different chemical entities through non-covalent interactions, which include Van de Waals forces, hydrogen or halogen bonding, ion-ion/dipole, dipole-dipole, cation or anion π interactions, or π-π interactions. This strategy allows the construction of extremely complex three-dimensional structures from synthetically simple precursors. Furthermore, the use of supramolecular chemistry confers to these structures many different advantages such as reversibility, self-healing, or adaptive response to different stimuli. For the preparation of novel supramolecular systems mainly two processes have been explored the molecular recognition and self-assembly. Molecular recognition has been defined as the specific binding of a guest entity to a complementary host molecule to generate a host-guest complex by using non-covalent interactions. Importantly, in some examples, host-guest binding constants have reached values almost comparable to covalent bonds. However, these host-guest complexes still maintain the advantages that offer the use of supramolecular chemistry. A relevant example in this sense is the molecular recognition of biotin by the protein, streptavidin. On the other hand, self-assembly has been defined as a process by which a non-organized system of molecular components, or a part of a molecule, as a consequence of non-covalent interactions, spontaneously generates an organized structure. Nature has also used molecular self-assembly to achieve complex and functional structures such as the double helix structure of DNA, the assembly of the tobacco mosaic virus, or the construction of lipid membranes. Since the day of its inception to nowadays, supramolecular systems have gained momentum in the development of functional materials, such as diverse delivery and thermoresponsive systems. In this Ph.D. dissertation, we have applied supramolecular strategies to promote intracellular delivery of diverse molecules and design potential thermoresponsive systems. Host-guest chemistry has been exploited to encapsulate and deliver different anionic probes such as pyranine, as well as anionic peptides with low self-delivery efficiency. Finally, we have designed potential thermoresponsive supramolecular materials based on self-assembling cyclic peptide nanotubes (SCPNs). During the last decades, new promising therapeutic and diagnostic molecules have been synthesized. However, some of these molecules presented limitations to cross the cellular membrane. To overcome this problem, a wide range of cell delivery systems have been described to promote the intracellular internalization of different entities such as therapeutic nucleic acids, proteins, or small molecules such as drugs or fluorescent probes. Different examples of delivery systems commonly used to internalize biologically important Héctor Fernández Caro 24 macromolecules and small molecules are liposomes, polymers, or metal nanoparticles. A relevant example of promising delivery vehicles is cell-penetrating peptides (CPPs). These peptides are small-sized peptides that showed impressive capabilities for internalizing a wide variety of membrane-impermeable therapeutic and diagnostic macromolecules and also small molecules into the cell cytosol. CPPs with different physicochemical properties have been described such as cationic, hydrophobic, or amphipathic. Some relevant examples of widely studied CPPs are penetratin, TAT, octaarginine, MPG, or PEP-1. During the last decades, different designs and structural modifications have been explored to improve the translocation capabilities of these molecules. An innovative strategy used to prepare new CPPs has been the formation of dynamic bonds such as oxime, hydrazone, or disulfide bonds. This strategy allowed to prepare a library of CPPs in a short period of time, larger quantities, and high yields. For instance, in our group, we have used the alcoxyamine group for attaching different electrophilic aldehydes and to achieve the easy modulation of the properties of the model CPP. Moreover, our group has also explored the hydrazide group for the incorporation of different hydrophobic aldehydes such as oleic acid into the CPP scaffold. It was found that this modification importantly improved the translocation of a pseudohelical model peptide. The incorporation of the cargo into the delivery system has been achieved mainly by applying covalent or non-covalent supramolecular strategies. Covalent attachment of the cargo has been traditionally used for the preparation of the drug-delivery system entity. On the other hand, supramolecular methodologies have shown important advantages such as improvement of cargo release and reduction of the synthetic effort. For instance, cell-penetrating peptides have been non-covalently attached to therapeutic proteins such as Cas9 ribonucleoprotein, nucleic acids such as siRNA or plasmid DNA, and small molecules such as drugs or probes. In the first chapter of this thesis, we have developed a novel generation of highly efficient and biocompatible supramolecular vehicles for the transport of molecular probes inside living cells. Negatively charged molecular probes such as pyranine or Alexa dyes are of interest for probing the intracellular space. However, the inefficient transport of these promising molecules across the plasma membrane, due to the repulsive interactions between molecular probes and the cellular membrane, currently remains a fundamental limitation. To overcome this problem, we have demonstrated that a hybrid system composed of a membrane-impermeable supramolecular cage and a covalently anchored tetraarginine peptide can be exploited for the cytosolic delivery of negatively charged fluorophores to different cell lines (HeLa and Vero cells). The supramolecular cage C used for the preparation of these carriers shows a trisbipyridyl structure and, in a previous report by prof. Nitschke group, demonstrated to encapsulate with high-affinity different anions of large sizes, such as pyranine, in an aqueous environment. Based on these previous results, we prepared acetylated and fluorescently labeled (TAMRA) R4 peptides by using solid-phase peptide synthesis, and then the cationic supramolecular cage C was coupled by standard conditions for amide bond formation to generate delivery agents AcR4C and TmR4C. Fluorescence experiments showed supramolecular complexation between these hybrid vectors and pyranine probe, with calculated dissociation constants of Kd = 189 nM and Kd = 12.6 µM, respectively. In previous reports by the group of prof. Matile, in vitro U-tube experiments showed the ability of different molecules to act as carriers. In this experiment, chloroform was placed at the bottom of a U-tube, and two different aqueous buffers were added at both sides of the organic phase (cis and trans buffers). Then, the transference from cis to trans buffer was monitored. To assess transport across the model apolar organic solvent -chloroform-, we carried out in vitro U-tube experiments in the presence of naturally occurring phospholipids. Time-dependent fluorescence measurements indicated that the peptide-cage carrier AcR4C favored the transport of pyranine probe from cis to trans aqueous Summary 25 phase. After a period of 12 h, the trans aqueous phase contained 4 times more pyranine dye when the peptide carrier was used in comparison with control experiments. This indicated that the peptide-cage carrier AcR4C acts as a carrier of the pyranine probe across the bulk apolar layer. Delivery experiments with pyranine-complexed to AcR4C and TmR4C showed cytosolic release in HeLa and Vero cells. Competition assays in the presence of a competing molecular probe (TAMRA) showed that TmR4C was able to selectively internalize pyranine into the cytosol by endocytosis. This mechanism of entry was confirmed by experiments in the presence of endocytosis inhibitors and by observing pyranine-carrier colocalization by confocal microscopy experiments. We confirmed the integrity of the cellular membrane in the pyranine transport by nuclear staining. In these experiments, after the transport experiments with the probe, we incubated the cells with DAPI and also with propidium iodide, probes that would cause nuclear staining when the cellular membrane is damaged. Moreover, competition experiments also ensured non-membrane permeabilization. Finally, we extrapolated this strategy to the delivery of multiple anionic probes, namely carboxyfluorescein and Alexa Fluors 488, 546, and 568, and we also confirmed their release in the cytosolic medium by confocal microscopy experiments. On the other hand, the modification of the physiological intracellular pH has been described to be related to the emergence of different pathologies such as cancer. For instance, cancer cells present a slightly acidic pH compared to normal cells. Therefore, the meticulous study of the intracellular pH would allow a better understanding of important cellular processes and diseases. A wide range of molecules can be used to carry out intracellular pH measurements. For instance, pyranine presents wonderful properties and can be used as a pH indicator. However, aggressive strategies, such as electroporation, have to be used for the intracellular delivery of this molecule. Therefore, in this chapter, we finally decided to use the previously synthesized carriers to efficiently internalize pyranine and to track the intracellular pH. Using confocal microscopy experiments, we confirmed the efficiency of the developed methodology to carry out the ratiometric intracellular pH tracking using the pyranine probe. Interestingly, we were able to discriminate the pH of different cellular compartments such as the neutral pH of cytosol (pH: 7.5), the moderately acidic pH of the early endosomes (pH: 7.0), and the more acidic pH of late endosomes (pH: 6.5). In the second chapter, we have explored supramolecular nanoparticles for the delivery of hydrophilic peptides. The term supramolecular nanoparticles (SNPs) has normally been used to define nanoparticles constructed mainly by non-covalent interactions. Supramolecular interactions have been used for the functionalization with different moieties which provide different functionalities to the system. The scaffolds used to construct these materials can be soft or hard, which have relevant consequences not only in their properties but also in their therapeutic effect. Supramolecular nanoparticles based on soft materials have been used for the intracellular delivery of not only therapeutic nucleic acids but also small molecules such as drugs. Interestingly, examples of SNPs that have already reached clinical trials have been reported. Moreover, co-delivery using SPNs of different cargos, such as small drugs and nucleic acids, has been also explored. On the other hand, metallic nanoparticles have been widely used and studied in Nanotechnology. For instance, these particles have been used in the development of novel drug delivery vehicles. During the last decades, gold nanoparticles (AuNPs) have shown applications in catalysis, as sensors, in biomedicine, etc. For the development of efficient and non-toxic therapeutics, multicomponent functionalization of the nanoparticle surface is fundamental. Furthermore, the incorporation of reversibility by supramolecular interactions has emerged as an innovative approach for the design of novel multicomponent nanoparticles. A relevant example is the combination of AuNPs and supramolecular macrocycles. The use of supramolecular interactions allowed the preparation of complex systems by easy mixing of the Héctor Fernández Caro 32 funcionalización con diferentes entidades que proporcionan diferentes funcionalidades al sistema. Los materiales utilizados para construir estos sistemas pueden ser blandos o duros, lo que tiene consecuencias relevantes no solo en sus propiedades sino también en su efecto terapéutico. Las nanopartículas supramoleculares basadas en materiales blandos se han utilizado para el transporte intracelular no solo de ácidos nucleicos terapéuticos sino también de moléculas pequeñas como los fármacos. Curiosamente, se han publicado ejemplos de SNPs que han alcanzado ensayos clínicos. Además, también se han preparado SPNs que permiten el transporte intracelular simultáneo de diferentes cargos, como fármacos y ácidos nucleicos. Por otro lado, las nanopartículas metálicas han sido ampliamente utilizadas y estudiadas en nanotecnología. Por ejemplo, estas partículas se han utilizado en el desarrollo de nuevos vehículos de transporte de fármacos. Durante las últimas décadas, las nanopartículas de oro (AuNPs) han mostrado aplicaciones en catálisis, como sensores, en biomedicina, etc. Para el desarrollo de terapias altamente eficientes y con baja toxicidad, la incorporación de varios componentes en la superficie de las nanopartículas es fundamental. Además, el empleo de interacciones supramoleculares se ha convertido en un enfoque innovador para el diseño de nuevas nanopartículas multicomponente. Un ejemplo relevante es la combinación de AuNPs y macrociclos supramoleculares. El uso de interacciones supramoleculares permite la preparación de sistemas complejos simplemente mezclando los diferentes componentes. Además, esta metodología evita pasos sintéticos difíciles y purificaciones complicadas de los materiales finales. Durante los últimos años, se han preparado AuNPs funcionalizadas con anfitriones macrocíclicos como cucurbiturilos o ciclodextrinas (CDs). Por ejemplo, las CDs contienen una cavidad interna altamente hidrofóbica, lo que hace que estas entidades sean excelentes anfitriones para un amplio rango de moléculas hidrofóbicas, como los fármacos hidrofóbicos. Durante las últimas décadas, el uso de nanopartículas metálicas, basadas en química anfitriónhuésped, con aplicaciones terapéuticas ha suscitado un gran interés en la comunidad científica. La formación de complejos anfitrión-huésped se ha utilizado tradicionalmente en la solubilización e integración en nanopartículas de diferentes fármacos hidrofóbicos como los profármacos de cisplatino, doxorrubicina, paclitaxel o metotrexato. Sin embargo, la mayoría de estas estrategias han explorado la liberación e intercambio de cargos hidrofóbicos. Hasta donde sabemos, aún no se ha investigado la liberación y el intercambio dinámico entre péptidos huésped hidrofílicos y nanopartículas decoradas con anfitriones. En este proyecto, uno de los principales desafíos es la complejidad química de los péptidos, que puede conducir a interacciones inespecíficas como, por ejemplo, interacciones electrostáticas o de van der Waals. Por otro lado, tradicionalmente para la funcionalización de la superficie de las nanopartículas con péptidos hidrofílicos se han empleado enlaces covalente e interacciones electrostáticas. Sin embargo, ejemplos que utilizan el reconocimiento supramolecular anfitrión-huésped siguen siendo limitados. El segundo capítulo de esta Tesis, se desarrolló una estrategia supramolecular para la incorporación de péptidos y polímeros hidrofílicos en β-CD@AuNPs mediante el empleo de procesos supramoleculares anfitrión-huésped. Preparamos nanopartículas de oro ancladas a βciclodextrinas (β-CD@AuNPs) y las caracterizamos mediante experimentos de TEM, DLS, UV-Visible y TGA. Los experimentos de TGA indicaron que estas poseen un promedio de 20 β-CD por nanopartícula, mientras que experimentos de TEM y DLS demostraron la preparación de nanopartículas de un tamaño de 2-3 nm y un diámetro hidrodinámico de aproximadamente 8 nm. Se sintetizó una quimioteca de péptidos penetrantes de células hidrófilicos (tetra y octaarginina) modificados con adamantanos, mono (AdR4 y AdR8) y divalentes (Ad2R4 y Ad2R8) empleando la condensación alcoxiamina-aldehído entre el grupo alcoxiamina del péptido y el grupo aldehído del huésped. En este trabajo, elegimos el adamantano (Ad) como huésped Resumen 33 debido a su elevada constante de afinidad por el anfitrión, β-CD. Por otro lado, los huéspedes mono y divalentes se utilizaron para explorar el intercambio dinámico de péptidos en la superficie de las β-CD@AuNPs mediante efectos estadísticos o multivalentes. Experimentos de potencial ζ confirmaron la incorporación de estos péptidos mono y divalentes en las β- CD@AuNPs mediante interacciones específicas anfitrión-huésped. Además, los péptidos control sin huésped no recubrieron de forma relevante las β-CD@AuNPs. Los resultados de los experimentos de potencial ζ se utilizaron para proporcionar una idea estimada de las constantes de asociación anfitrión-huésped, que se encontraron en un rango adecuado para la interacción individual Ad-CD. Además, la interpretación de las constantes de asociación aparentes calculadas indica que el proceso predominante es una unión estadística o interacciones entre partículas. Además, los experimentos de competición en presencia de β-CD libre mostraron un descenso en el potencial ζ de las nanopartículas que indica reversibilidad termodinámica. Por otro lado, los polímeros biocompatibles como el polietilenglicol (PEG) se han utilizado para prevenir el recubrimiento de la superficie de nanopartículas con diferentes proteínas del medio biológico. Aquí, decidimos sintetizar y caracterizar cadenas de PEG hidrofílicos mono (AdPEG) y divalentes (Ad2PEG), utilizando la metodología sintética descrita anteriormente, para incorporar otra funcionalidad en el nanosistema. Construimos nanopartículas multicomponente compuestas por péptidos de poliarginina y cadenas de PEG funcionalizados con el huésped adamantano. Los experimentos de tamaño y potencial ζ en presencia de una mezcla de poliarginina y PEG monovalentes/divalentes sugirieron que la estabilización electrostática es la causante de la estabilización del sistema, mientras que el PEG podría tener un efecto negativo al obstaculizar el alcance de valores elevados de potencial. Exclusivamente en zonas de estabilidad electrostática, la adición de PEG dio como resultado una modulación favorable del tamaño. Por último, planteamos la hipótesis de que el intercambio de péptidos sobre las β-CD@AuNPs, mediante interacciones anfitrión-huésped, podría llevarse a cabo empleando péptidos que presentasen una valencia superior. Demostramos que la formación de complejos entre péptidos poliglutámicos impermeables a la membrana (AdE9) y β-CD@AuNPs promueve el transporte intracelular de estos péptidos. Además, los experimentos de microscopía confocal y los ensayos de citometría celular confirmaron el intercambio de estos péptidos aniónicos AdE9 por péptidos divalentes como Ad2R8 en el interior de células vivas. El último capítulo de esta Tesis se ha centrado en el desarrollo de nuevos sistemas nanotubulares supramoleculares termorresponsivos. Durante las últimas décadas, los sistemas que responden a los estímulos, también llamados "materiales inteligentes", han atraído un gran interés debido a su capacidad para responder a una amplia gama de estímulos, como el pH, la fuerza iónica, la luz, el campo magnético o eléctrico o la temperatura. Entre ellos, los materiales termorresponsivos, que poseen la capacidad de adaptarse a los cambios de la temperatura ambiental, han demostrado numerosas aplicaciones biomédicas, como en terapia génica o en la administración de fármacos. Se ha demostrado que la elastina, que es una proteína estructural, presenta propiedades termorresponsivas. La tropoelastina se definió como el precursor de la elastina y su estructura primaria está compuesta por secuencias repetidas de pentapéptidos. La secuencia peptídica más comúnmente encontrada es Val1Pro2Gly3Val4Gly5 o VPGVG. Durante las últimas décadas, se han sintetizado polipéptidos tipo elastina (ELP) y polímeros basados en elastina (EBP). Al investigar este tipo de materiales, se ha encontradon comportamientos LCST (temperatura de solución crítica inferior) o comportamientos UCST (temperatura de solución crítica superior) y múltiples aplicaciones, como por ejemplo como vehículos termorresponsivos para la internalización celular de fármacos. Por otro lado, los nanotubos constituidos por péptidos cíclicos (SCPNs) representan uno de los ejemplos más relevantes de entidades Héctor Fernández Caro 34 supramoleculares. Por ejemplo, SCPNs se han preparado utilizando D,L-α-ciclopéptidos (D,L- α-CP) que consisten en un número par de aminoácidos con quiralidad alternante. El autoensamblaje de péptidos cíclicos se basa en el apilamiento de múltiples subunidades mediante la formación de una red de enlaces de hidrógeno. Desde el descubrimiento de los SCPNs, se han llevado a cabo diferentes modificaciones estructurales y también se han descrito una amplia variedad de aplicaciones. Los SCPNs han demostrado capacidades como análogos de canales de iones de membrana o como sensores de iones. Además, estas entidades supramoleculares se han utilizado no solo como vehículos de administración de fármacos sino también como agentes antibacterianos o antivirales. Durante las últimas décadas, nuestro grupo de investigación ha diseñado y sintetizado un amplio rango de estructuras de péptidos cíclicos de diferentes tamaños capaces de autoensamblarse en nanotubos adquiriendo una alta experiencia en este campo. Recientemente, en nuestro grupo, se ha logrado demostrar el autoensamblaje de péptidos cíclicos dentro de vesículas. Más recientemente, la formación de nanohojas se ha demostrado en condiciones fisiológicas y se caracterizaron como bicapas de SCPNs. Para proporcionarle a estos sistemas supramoleculares la capacidad para responder a distintos estímulos, péptidos cíclicos se han funcionalizado con polímeros que conducen a la construcción de nanotubos basados en híbridos de péptidos cíclicos y polímeros. Estos materiales híbridos han demostrado responder a estímulos ambientales como la temperatura o el pH. Sin embargo, la unión de los polímeros artificiales termorresponsivos a péptidos cíclicos carece de una estrategia sintética fácil que permita la modificación controlada de las temperaturas de transición y un estudio detallado del autoensamblaje de los SCPNs con la variación de la temperatura. Además, cuando se usan en aplicaciones biomédicas, estos híbridos cíclicos péptido-polímero normalmente producen toxicidad celular. Por lo tanto, la unión de cadenas peptídicas termorresponsivas, como la elastina, a péptidos cíclicos permitiría construir no solo materiales supramoleculares termorresponsivos de una forma más sencilla sino también sistemas plenamente biocompatibles. Por lo tanto, en el tercer capítulo de esta Tesis doctoral, hemos estudiado las diferentes posibilidades para diseñar péptidos cíclicos modificados con elastina con potenciales capacidades para autoensamblarse en nanotubos de péptidos cíclicos mediante variaciones en el pH del medio. Estos péptidos cíclicos están constituidos por ocho aminoácidos con quiralidad alternante (D/L-α-CP) decorados con una cadena de elastina, que confiere capacidades termorresponsivas al sistema. Diseñamos tres péptidos cíclicos base diferentes que poseen diferentes aminoácidos sensibles al pH para promover el autoensamblaje a pH básico (CP1.1 y CP1.2), a pH neutro (CP2.1 y CP2.2) y a pH ácido (CP3.1 y CP3.2). En este diseño, decidimos incorporar una cadena de elastina a los péptidos cíclicos en dos orientaciones diferentes: el segmento de elastina natural [H-(V-P-G-V-G-)OH] y la estructura inversa, también termorresponsiva (H-(G-V-G-P-V-)OH). Además, también diseñamos los péptidos de control sin la cadena de elastina CP1, CP2 y CP3. En esta Tesis, sintetizamos dos péptidos cíclicos diferentes modificados con elastina CP1.1 y CP3.1, en ambos péptidos incorporamos el segmento natural de elastina [H-(V-P-G-V-G-)OH]. Aquí planteamos la hipótesis de que CP1.1 podría autoensamblarse en estructuras nanotubulares a pH básico, mientras que CP3.1 a pH ácido. En CP1.1, incorporamos a la secuencia de péptido cíclico base (CP1) tres residuos sensibles al pH (2 His y 1 Lys) para lograr el autoensamblaje en nanotubos de péptidos cíclicos a pH básico. En este peptido cíclico, la basificación del medio acuoso promovería la desprotonación de las cadenas laterales de histidina (pKa ≈ 6.0) que conduciría a la aparición de enlaces de hidrógeno histidina-histidina. Además, las cadenas laterales de Lys (pKa ≈ 10.5) a pH básico estarían desprotonadas, por lo tanto, las repulsiones electrostáticas catiónicas se minimizarían. En CP3.1, para lograr la formación de nanotubos a pH ácido, incorporamos tres Resumen 35 residuos de Glu a la secuencia base del péptido cíclico (CP3). En este péptido, la acidificación del medio acuoso produciría la protonación de las cadenas laterales de Glu (pKa ≈ 4.2) que conduciría a la formación de enlaces de hidrógeno. Para la preparación de estos dos péptidos cíclicos diferentes modificados con elastina (CP1.1 y CP3.1), utilizamos exclusivamente la síntesis de péptidos en fase sólida. Usando esta estrategia sintética, el crecimiento del fragmento de elastina y la síntesis y ciclación del fragmento D, L se llevaron a cabo en la resina clorotritilo. Además, hemos estudiado el autoensamblaje y la capacidad de respuesta térmica de estos péptidos cíclicos modificados con elastina. Estudiamos el autoensamblaje de CP1.1 a pH básico mediante experimentos de dicroísmo circular (CD). Sin embargo, no pudimos correlacionar la señal de CD obtenida con el autoensamblaje de péptidos cíclicos a pH básico. También investigamos, mediante experimentos de turbidimetría, la capacidad de respuesta térmica de CP1.1 y, desafortunadamente, no encontramos comportamiento termorresponsivo a diferentes temperaturas y/o pH. Por otro lado, también llevamos a cabo la exploración de las capacidades de autoensamblaje de CP3.1 en condiciones de purificación previamente descritas. General Introduction General Introduction 39 1. Supramolecular chemistry Jean-Marie Lehn defined supramolecular chemistry as the “chemistry beyond the molecule”. 1 Due to their important discoveries in this area, Lehn1, Cram 2 , and Pedersen 3 received the Nobel prize in 1987. Supramolecular chemistry focuses on the design and preparation of complex systems that held together chemical components by non-covalent interactions. 4 , 5 , 6 Instead of networks of covalent bonds, the driving force for the assembly of supramolecular systems relies on a gamut of non-covalent forces, such as ion-ion/dipole, dipole-dipole, hydrogen and halogen bonding, cation or anion-π interactions, van der Waals forces or π-π interactions.6, 7 The emergence of supramolecular chemistry allowed to obtain complex structures that present functional groups oriented in a precise direction of the three-dimensional space. The preparation of these complex ensembles by the formation of covalent bonds results extremely challenging from the point of view of time and human resources. 8 Moreover, supramolecular chemistry provided a possibility of manipulating supramolecular building blocks at a molecular level, allowing the “bottom-up” synthesis of complex molecular architectures with controlled size and shape. 9 Due to the use of non-covalent interactions, supramolecular materials have shown reversibility, allowing convenient complex dissociation and association at a low energy cost. 10 Interestingly, this type of systems has also shown adaptive behavior in response to external stimuli that can trigger the structure variation of supramolecular materials. 11 2. Molecular recognition and self-assembly Supramolecular systems are mainly assembled by molecular recognition or self-assembly. In both cases, the interaction between the molecules occurs based on their structural information. This is constituted mainly of two elements: chemical complementarity, which implies the correct distribution of the matching in shape and chemical groups in the molecule, and the preorganization, which refers to the adequate conformational disposition of the interacting units. The main difference between both processes is related to the complexity of the obtained supramolecular systems. Molecular recognition is focused on simple processes in which only a few molecules are interacting, while self-assembly deals with more complex systems, generally involving an infinite number of molecules. Molecular recognition is defined as the specific binding of a guest molecule to a complementary host entity to generate a host- 1 J.-M. Lehn, Angew. Chem. Int. Ed. 1988, 27, 89-112. 2 D. J. Cram, Angew. Chem. Int. Ed. 1988, 27, 1009-1020. 3 C. J. Pedersen, Angew. Chem. Int. Ed. 1988, 27, 1021-1027. 4 J.-M. Lehn, Chem. Soc. Rev. 2017, 46, 2378-2379. 5 J.-M. Lehn, Eur. Rev. 2009, 17, 263-280. 6 J. W. Steed, D. R. Turner, K. Wallace, Core Concepts in Supramolecular Chemistry and Nanotechnology, Jon Wiley & Sons, Chichester, 2007. 7 J. D. Badjić, A. Nelson, S. J. Cantrill, W. B. Turnbull, J. F. Stoddart, Acc. Chem. Res. 2005, 38, 723-732. 8 D. N. Reinhoudt, Science 2002, 295, 2403-2407. 9 V. Balzani, A. Credi, M. Venturi, Chem. Eur. J. 2002, 8, 5524-5532. 10 K. Liu, Y. Kang, Z. Wang, X. Zhang, Adv. Mater. 2013, 25, 5530-5548. 11 B. Rybtchinski, ACS Nano 2011, 5, 6791-6818. Héctor Fernández Caro 40 guest complex by the use of non-covalent interactions. On the other hand, molecular selfassembly is defined as the construction of systems without guidance from an outside source.6, 12 3. Molecular self-assembly Self-assembly is a process by which a non-organized system of molecular components, or a part of a molecule, spontaneously generates an organized structure as a consequence of noncovalent interactions. 13 The molecules involved in this type of process are programmed chemical entities. Therefore, these molecules include functional complementary groups that direct the organization of matter towards the generation of architectures with a pre-defined topology and properties. 14 Molecular self-assembly has been also the strategy selected by nature to construct complex and functional architectures. 15 In fact, the most sophisticated examples of self-assembly can be found in natural systems, such as the double helix structure of DNA, the assembly of the tobacco mosaic virus, the folding of proteins to obtain their three-dimensional structure that determines its biological function or the formation of lipid membranes. 16 The implementation of self-assembly in synthetic chemistry allowed to construct a wide range of complex structures with different topologies such as spheres, 17 helicates, 18 barrels, 19 nanowires, 20 cubes 21 , or nanotubes. 22 4. Host-guest chemistry 4.1. General principles An important phenomenon studied by supramolecular chemistry is the formation of inclusion complexes. Here, a host molecule can interact with a guest entity in order to form a host-guest complex, as shown in Figure 1. 23 The driving force is the formation of non-covalent supramolecular interactions such as van der Waals forces. 24 The geometrical fitting of the guest to the host pocket is therefore an essential factor in maximizing these stabilizing interactions while minimizing sterical repulsion. The stability of the host-guest complex at a given temperature and solvent is typically measured by the thermodynamic equilibrium formation constant (Ka), or the dissociation constant (Kd, being Kd = Ka−1). Ka is expressed as the concentration of formed host-guest complex divided by concentrations of free host and guest (Figure 1, right). In some cases, host-guest binding constants can reach magnitudes almost 6 J. W. Steed, D. R. Turner, K. Wallace, Core Concepts in Supramolecular Chemistry and Nanotechnology, Jon Wiley & Sons, Chichester, 2007. 12 D. Rasale, A. Das, Int. J. Mol. Sci. 2015, 16, 10797-10820. 13 Whitesides, G. M.; Grzybowski, B. Science 2002, 295, 2418-2421. 14 Hosseini, M. W. Chem. Commun. 2005, 5825-5829. 15 Lehn, J.-M. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 4763-4768. 16 Whitesides, G. M.; Mathias, J. P.; Seto, C. T. Science 1991, 254, 1312-1319. 17 A. R. Stefankiewicz, J. K. M. Sanders, Science 2010, 328, 1115-1116. 18 M. Boiocchi, L. Fabbrizzi, Chem. Soc. Rev. 2014, 43, 1835-1847. 19 Y. Lim, M. Lee, J. Mater. Chem. 2011, 21, 11680-11685. 20 C. Qian, F. Kim, L. Ma, F. Tsui, P. Yang, J. Liu, J. Am. Chem. Soc. 2004, 126, 1195-1198. 21 A. Stephenson, M. D. Ward, Dalton Trans. 2011, 40, 10360-10369. 22 Chapman, R.; Danial, M.; Koh, M. L.; Jolliffe, K. A.; Perrier, S. Chem. Soc. Rev. 2012, 41, 6023-6041. 23 S. M. Mantooth, B. G. Munoz-Robles, M. J. Webber, Macromol. Biosci. 2019, 19, 1800281. 24 C. B. Rodell, J. E. Mealy, J. A. Burdick, Bioconjug. Chem. 2015, 26, 2279-2289. General Introduction 41 comparable to covalent bonds. A clear example in this sense is the molecular recognition of biotin by the binding pocket of streptavidin protein, 25 with a Kd ~ 4 × 10-14 M-1. Figure 1. Interaction between a cavitand host (red) and a fitting guest (blue) to give a host-guest complex, where kon and koff are the formation and dissociation rate constants respectively. The binding constant (Ka) determines the speciation of free host, free guest, and host-guest complex.23 4.2. Multivalency In supramolecular chemistry, multivalency typically describes multiple non-covalent interactions that occur between a multivalent host and a multivalent guest. This results, generally, in enhanced binding affinities and specificities, 26 which plays a pivotal role in intercellular adhesion 27 or protein-carbohydrate recognition. 28 Depending on the spacing and flexibility of the multivalent motives, a multivalent guest can bind to a multivalent host in an intramolecular or intermolecular fashion. Intramolecular binding typically leads to relatively high association constants with respect to monovalent binding and the formation of welldefined complexes. In contrast, intermolecular binding might lead to the formation of large aggregates that often precipitate from solution. Binding shows association constants comparable to those of the corresponding monovalent interactions, while irreversible precipitation and aggregation lead to decreased dissociation rates and consequently to an apparent binding enhancement (Figure 2A). 29 In addition to its importance in relevant biological processes, multivalent interactions are the foundation of several technologies in nanomedicine and material sciences. For example, viral infections typically proceed by an initial binding to membrane receptors which subsequently triggers cell endocytosis and starts the infection. Viral inhibitors can be designed by including multiple copies of the antigen into a multivalent platform, which shows higher affinities towards the host cell and therefore prevents virus binding by multivalent competition. As example, Nazario Martin and col. demonstrated that giant multivalent glycofullerenes are potent inhibitors of Ebola virus infection showing subnanomolar half-maximum inhibitory concentrations. These multivalent compounds demonstrated inhibition of lectin-mediated viral infections in cellular experiments. Interestingly, the half-maximum inhibitory concentrations overpassed by three orders of magnitude (two if the number of mannoses is considered) those exhibited by hexakis adducts showing 12 mannoses (Figure 2B). 30 On the other hand, materials with enhanced adhesion can be designed by covering large surfaces with host-guest pairs, yielding underwater “velcros”. For example, multivalent interactions arising between a host 25 N. M. Green, Methods Enzymol. 1990, 184, 51-67. 26 M. J. W. Ludden, D. N. Reinhoudt, J. Huskens, Chem. Soc. Rev. 2006, 35, 1122-1134. 27 Y. Zhang, S. Sivasankar, W. J. Nelson, S. Chu, Proc. Natl. Acad. Sci. 2009, 106, 109-114. 28 T. K. Dam, R. Roy, S. K. Das, S. Oscarson, C. F. Brewer, J. Biol. Chem. 2000, 275, 14223-14230. 29 A. Mulder, J. Huskens, D. N. Reinhoudt, Org. Biomol. Chem. 2004, 2, 3409-3424. 30 A. Muñoz, D. Sigwalt, B. M. Illescas, J. Luczkowiak, L. Rodríguez-Pérez, I. Nierengarten, M. Holler, J. Remy, K. Buffet, S. P. Vincent, et al., Nat. Chem. 2016, 8, 50-57. Introduction Chapter I: Introduction 51 1. The cellular membrane as a selective natural barrier The cellular membrane is primarily responsible for the protection and the strict control of the exchange of molecules between the cellular interior and the extracellular environment. 73 This selective natural barrier is mainly formed by two components: lipids and proteins. Membrane lipids have a hydrophobic tail and a hydrophilic head that are oriented to form a protective barrier, the lipid bilayer. In addition to lipids, the cellular membrane contains different types of proteins. These membrane proteins can act, for example, as transporters (controlling the exchange of different molecules between the cellular interior and its environment) or as receptors (being responsible for cell signaling). 74 The transport of different substances inside of the cells can be performed by using a variety of mechanisms that can be mainly classified in passive diffusion, facilitated diffusion, and active transport. Transport by passive diffusion allows the cells to transport different substances across the cell membrane in favor of an electrochemical or a concentration gradient. Therefore, this mechanism does not require energy from the cell. An important point is that this mechanism only allows the transport of hydrophobic small molecules across the cell membrane. To improve the transport of biologically important hydrophilic molecules for the cell such as ions or sugars, cells use facilitated diffusion. This mechanism occurs helped by different membrane proteins and it does not require energy from the cell. 75 Figure 5. Schematic representation of the different endocytic pathways. Reprinted with permission from ref. 77. Copyright 2007 Springer Nature. Active transport allows cells to transport extracellular molecules against an electrochemical or a concentration gradient, a mechanism that demands energy consumption by the cell. An important type of active transport is endocytosis, which allows the cell to transport fluids and solutes from its surroundings to the cell interior. Endocytosis is a phenomenon where there is a conformational change of the cell membrane to introduce different extracellular substances 73 J. Lombard, Biol. Direct 2014, 9, 32. 74 A. Uzman, B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, P. Walter, Molecular Biology of the Cell, 4th edition, Garland Science, New York, 2003. 75 N. J. Yang, M. J. Hinner, Methods Mol. Biol. 2015, 1266, 29-53. Héctor Fernández Caro 52 through the formation of small transport vesicles. Endocytosis can be classified into two major classes: phagocytosis and pinocytosis. Phagocytosis is an important process that allows the cell to internalize large particles and occurs exclusively in specialized cells such as neutrophils and macrophages. However, pinocytosis is a mechanism that allows the non-specialized cells to transport a variety of extracellular molecules and fluids through the cell membrane. Furthermore, pinocytosis can be also classified in different internalization pathways such as macropinocytosis, caveolin-dependent endocytosis, clathrin-dependent endocytosis, and clathrin and caveolin-independent endocytosis (Figure 5). 76 , 77 This meticulous control of the transport through the cell membrane importantly influences the delivery of therapeutics as the effectiveness of drugs and other bioactive compounds depends on their effective delivery. The main reason is that the site of action of many molecules is placed in the cell interior. Thus, the drug needs to cross the plasma membrane to accomplish its pharmaceutical effect. For this reason, in the last years, many innovative cell delivery systems have emerged to improve the transport across the cell membrane of large hydrophilic molecules. Over the last twenty years, cell-penetrating peptides have emerged as an innovative approach to improve the intracellular delivery of different therapeutic and diagnostic cargoes such as drugs, imaging agents, nucleic acids, proteins, etc. 78 2. Cell-penetrating peptides In 1988, it was discovered that the TAT protein of the human immunodeficiency virus (HIV-1), consisting of 86 amino acids, had the ability to cross the cell membrane. 79 , 80 After studying this phenomenon, it was discovered that the sequence that provided these transport capabilities was a small peptide (RKKRRQRRR) called TAT. 81 In 1994, translocation capabilities were also found in the homeodomain of Antennapedia. 82 It was discovered that these transport properties were provided also by a small peptide which was called Penetratin (RQIKIWFQNRRMKWKK). 83 Following these preliminary findings, the scientific community was fascinated by the transport capabilities of these peptide sequences and began to develop modifications of these sequences designing new structures with penetrating capabilities, these peptides were named as cell-penetrating peptides (CPPs). 84 Cell-penetrating peptides (CPPs) are small-sized peptides (usually less than 30 amino acids) capable of transporting a wide range of membrane-impermeable therapeutic agents into the cell interior. An important characteristic of these peptides is that they have the ability to transport molecules with low cellular toxicity at low micromolar concentrations. Different classifications have emerged for the wide variety of CPPs, for example, CPPs can be easily classified according to their physical-chemical properties in different types: 76 G. J. Doherty, H. T. McMahon, Annu. Rev. Biochem. 2009, 78, 857-902. 77 S. Mayor, R. E. Pagano, Nat. Rev. Mol. Cell Biol. 2007, 8, 603-612. 78 F. Heitz, M. C. Morris, G. Divita, Br. J. Pharmacol. 2009, 157, 195-206. 79 A. D. Frankel, C. O. Pabo, Cell 1988, 55, 1189-1193. 80 M. Green, P. M. Loewenstein, Cell 1988, 55, 1179-1188. 81 E. Vivès, P. Brodin, B. Lebleu, J. Biol. Chem. 1997, 272, 16010-16017. 82 D. Derossi, A. H. Joliot, G. Chassaing, A. Prochiantz, J. Biol. Chem. 1994, 269, 10444-10450. 83 D. Derossi, G. Chassaing, A. Prochiantz, Trends Cell Biol. 1998, 8, 84-87. 84 J. D. Ramsey, N. H. Flynn, Pharmacol. Ther. 2015, 154, 78-86. Chapter I: Introduction 53 Cationic CPPs: This type of CPPs is characterized by their content of mainly cationic residues such as arginine and lysine, which confer a high net positive charge to the CPP. Some examples in this category are TAT, penetratin, or polyarginines. Hydrophobic CPPs: These peptides mainly consist of hydrophobic amino acids. An example of a hydrophobic peptide is K-FGF (AAVLLPVVLLAAP). Amphipathic CPPs: these peptides contain two different regions, a hydrophobic domain, and hydrophilic and cationic domain. 85 This class of CPPs normally can form stable noncovalent complexes with different cargos inducing the internalization in the cell. An amphipathic peptide, Pep-1 (KETWWETWWTEWSQPKKKRKV) has been applied for delivering a wide number of cargos. For instance, this peptide can form non-covalent interactions with other peptides and protein cargos to allow their membrane translocation. Another example of this category is the MPG (GALFLGFLGAAGSTMGAWSQPKKKRKV), a peptide that has been discovered able to form stable complexes with different nucleic acids. 86 Moreover, amphipathic proline-rich peptides (VXLPPP)n have been prepared and also showed good membrane translocation capabilities. 87 , 88 Many studies have been published regarding the mechanism of uptake of the CPPs. This topic has generated great controversy. Normally, these peptides are translocated inside of the cell following different endocytic pathways. It is known that there is a high influence of the concentration of the peptide and the characteristics of the cargo delivered and the type of CPP. 89 A powerful strategy for the synthesis of new cell-penetrating peptides is the incorporation of dynamic bonds, such as oximes, hydrazones, or disulfides. Dynamic bonds are responsive to different external stimuli and, during their internalization pathway, these bonds are sensitive to the environment. From the synthetic point of view, dynamic bonds are also interesting as they lead to the final products with high yields, with the generation of a water molecule as the only side product, and in short periods of time. 90 Recently, in our research group, we have designed new penetrating peptides that contain dynamic bonds.91 First, we synthesized cationic peptides containing amino acids modified with an alcoxyamine group. These peptides were then reacted with different aldehydes resulting in new oxime amphiphilic peptides. These amphiphilic cationic peptides were able to transport a variety of anionic DNA molecules in vesicle transport experiments. 91 Subsequently, we applied the same strategy to synthesize an amphiphilic pseudo-helical parent peptide, which was easily reacted with different hydrophobic and hydrophilic electrophiles (Figure 6).92 In this work, we explored the capacity of DNA transport in vesicles of this type of peptides and the difference that exists depending on the hydrophilicity of the electrophiles used. Moreover, we studied the modification of the membrane translocation capabilities of the peptides in cells with the modulation of their amphiphilicity. 92 In our research group, dynamic linkages, such as hydrazone bonds, have also been applied to synthesize 85 D. Raucher, J. S. Ryu, Trends Mol. Med. 2015, 21, 560-570. 86 M. C. Morris, S. Deshayes, F. Heitz, G. Divita, Biol. Cell 2008, 100, 201-217. 87 A. D. Frankel, C. O. Pabo, Cell 1988, 55, 1189-1193. 88 Y. A. Fillon, J. P. Anderson, J. Chmielewski, J. Am. Chem. Soc. 2005, 127, 11798-11803. 89 A. Rioboo, I. Gallego, J. Montenegro, An. Química 2019, 115, 9-21. 90 C. Gehin, J. Montenegro, E. K. Bang, A. Cajaraville, S. Takayama, H. Hirose, S. Futaki, S. Matile, H. Riezman, J. Am. Chem. Soc. 2013, 135, 9295-9298. 91 J. M. Priegue, J. Montenegro, J. R. Granja, Small 2014, 10, 3613-3618. 92 M. Pazo, H. Fernández-Caro, J. Priegue, I. Lostalé-Seijo, J. Montenegro, Synlett 2017, 28, 924-928. Héctor Fernández Caro 54 dynamic penetrating peptides with hydrophobic tails which formed complexes by non-covalent interactions and, then, triggered the transport of plasmid DNA 93 and Cas9 94 in cells. Figure 6. A) Chemical structure of the amphiphilic peptide showing two alcoxyamine reactive groups where the different electrophilic aldehydes were attached. B) Top-view helical diagram and circular dichroism spectra of the parent peptide. C) Schematic representation of peptide functionalization with the different aldehydes to afford oxime-modulated peptides for membrane transport applications. Reprinted from ref. 92 under the terms of the Creative Commons CC-BY-NC-ND license. 3. Cell delivery of biologically active macromolecules Proteins and nucleic acids have emerged as excellent therapeutics for the treatment of a variety of diseases. 95 , 96 Macromolecular therapeutics, such as nucleic acids or recombinant proteins, have to efficiently cross the cellular membrane due to their site of action is located in the cytosol or the cell nucleus. Unfortunately, the cytosolic delivery of nucleic acids and proteins is inefficient mainly because of endosomal entrapment and enzymatic digestion. 97 , 98 Therefore, the delivery of macromolecules into cells and tissues remains a major challenge, and new delivery systems to overcome these limitations are needed. During the last years, different methods can be differentiated to deliver therapeutic macromolecules into the cell interior including physical and mechanical methods and chemical covalent and non-covalent methods. 93 I. Louzao, R. García-Fandiño, J. Montenegro, J. Mater. Chem. B 2017, 5, 4426-4434. 94 I. Lostalé-Seijo, I. Louzao, M. Juanes, J. Montenegro, Chem. Sci. 2017, 8, 7923-7931. 95 J. Nguyen, F. C. Szoka, Acc. Chem. Res. 2012, 45, 1153-1162. 96 Y. Zhang, J. J. Røise, K. Lee, J. Li, N. Murthy, Curr. Opin. Biotechnol. 2018, 52, 25-31. 97 V. Torchilin, Drug Discov. Today Technol. 2008, 5, 95-103. 98 M. L. Read, A. Logan, L. W. Seymour, Adv. Genet. 2005, 53, 19-46. A) B) C) Chapter I: Introduction 55 3.1. Physical and mechanical methods Several physical and mechanical strategies have been developed to achieve direct cytosolic delivery of macromolecules such as DNA or proteins. 99 , 100 Mechanical methods, such as microinjection, are a typical powerful strategy. Microinjection is one of the most direct and simplest methods to incorporate DNA into the nucleus or the cytosol of cells. In this technique, the release of the biomolecule is carried out using a needle on a single cell. This strategy is carried out when a precise and accurate delivery of the macromolecule is necessary. However, this methodology does not show good results when a high number of cell transfection is required. 101 Physical methods, such as electroporation have been also widely applied. This technique induces short high voltage electric cycles to the cell suspensions. This voltage produces transient pores in the cell membrane and the soluble desired proteins can cross the cell membrane by simple diffusion. 102 The main advantage of this technique is that it can be applied to different cell lines and a large number of cells can be transfected. However, the main limitation of electroporation methods is the important cytotoxicity induced by the high voltage cycles and the damage produced to the cell membrane. Moreover, this technique is limited to in vitro usage. 103 3.2. Chemical covalent methods During the last years, macromolecules, such as DNA or proteins, have been covalently linked to different delivery systems including cell-penetrating peptides, 104 gold nanoparticles, 105 polymers, 106 and strain-promoted thiols. 107 3.2.1. Cell-penetrating peptides The covalent attachment of macromolecules to cell-penetrating peptides has been applied to deliver different therapeutic macromolecules such as proteins or nucleic acids.104 Delivery of proteins. Different CPPs such as TAT peptide, Penetratin, Transportan, etc. have demonstrated, in different in vitro examples, their abilities to efficiently deliver proteins. 108 , 109 For instance, it has been demonstrated that TAT peptide has the ability to deliver large proteins such as β-galactosidase. The in vivo administration of the covalent conjugate 99 M. S. Al-Dosari, X. Gao, AAPS J. 2009, 11, 671. 100 S. Du, S. S. Liew, L. Li, S. Q. Yao, J. Am. Chem. Soc. 2018, 140, 15986-15996. 101 S. Mehier-Humbert, R. H. Guy, Adv. Drug Deliv. Rev. 2005, 57, 733-753. 102 M. Chiper, K. Niederreither, G. Zuber, Adv. Healthc. Mater. 2018, 7, e1701040. 103 A. Fu, R. Tang, J. Hardie, M. E. Farkas, V. M. Rotello, Bioconjug. Chem. 2014, 25, 1602-1608. 104 D. Kalafatovic, E. Giralt, Molecules 2017, 22, 1929. 105 D. A. Giljohann, D. S. Seferos, W. L. Daniel, M. D. Massich, P. C. Patel, C. A. Mirkin, Angew. Chem. Int. Ed. 2010, 49, 3280-3294. 106 R. Duncan, Nat. Rev. Drug Discov. 2003, 2, 347-360. 107 N. Chuard, G. Gasparini, D. Moreau, S. Lörcher, C. Palivan, W. Meier, N. Sakai, S. Matile, Angew. Chem. Int. Ed. 2017, 56, 2947-2950. 108 S. G. Patel, E. J. Sayers, L. He, R. Narayan, T. L. Williams, E. M. Mills, R. K. Allemann, L. Y. P. Luk, A. T. Jones, Y. H. Tsai, Sci. Rep. 2019, 9, 6298. 109 M. Mäe, Ü. Langel, Curr. Opin. Pharmacol. 2006, 6, 509-514. Héctor Fernández Caro 56 between β-galactosidase and TAT has shown an efficient delivery not only in the different tissues of the mice but also in the brain. 110 To successfully reach its intracellular target and produce its therapeutic effect the protein should not remain trapped inside of the endosome. 111 To promote protein delivery, the use of covalently attached endosomolytic peptides has also been reported. 112 The covalent conjugation between proteins and CPPs can be achieved by expressing CPP-fusion proteins. 113 In addition, the CPP can be covalently linked to the side chain of a lysine of the protein or to a thiol of a cysteine to achieve specific modifications. 114 , 115 Delivery of nucleic acids. CPPs have also demonstrated their abilities to efficiently deliver bioactive nucleic acids. 5´-thiol functionalized siRNA has been covalently attached to Transportan and Penetratin peptides modified with terminal cysteines. This complex CCP- siRNA showed an efficient knockdown of mammalian cells. 116 Moreover, peptide nucleic acids (PNAs) have been also covalently attached to different CPPs to improve their translocation capabilities. 117 , 118 The use of covalent bonds with CPPs has been successfully applied for the cellular delivery of a variety of therapeutic macromolecules. However, this strategy has also drawbacks such as modification of the chemical properties of the cargo, which can lead to changes in its biological activity. 119 In addition, this strategy is not as compatible with the functional delivery of large biomolecules such as pDNA and the synthesis and purification of the corresponding conjugates results tedious as it normally requires multiple steps. 120 3.2.2. Metal nanoparticle conjugates Gold nanoparticles (AuNPs) have also been applied to deliver macromolecules by covalent strategies. The group of prof. Chad Mirkin prepared a type of polyvalent nucleic acid capped AuNPs (pNA-AuNPs) by covalently functionalizing AuNPs with thiol-modified oligonucleotides for siRNA-based gene silencing. 121 The incorporation of the oligonucleotides on the surface of these AuNPs showed protection of the cargo and inhibition of its degradation by nucleases. Interestingly, these pNA-AuNPs were efficiently internalized in more than fifty different cell lines.105, 122 More recently, to target specific cells, this group modified the pNA- 110 S. R. Schwarze, A. Ho, A. Vocero-Akbani, S. F. Dowdy, Science 1999, 285, 1569-1572. 111 P. Lönn, A. D. Kacsinta, X. S. Cui, A. S. Hamil, M. Kaulich, K. Gogoi, S. F. Dowdy, Sci. Rep. 2016, 6, 32301. 112 M. Li, Y. Tao, Y. Shu, J. R. LaRochelle, A. Steinauer, D. Thompson, A. Schepartz, Z. Y. Chen, D. R. Liu, J. Am. Chem. Soc. 2015, 137, 14084-14093. 113 J. Jo, S. Hong, W. Y. Choi, D. R. Lee, Sci. Rep. 2014, 4, 4378. 114 S. B. Gunnoo, A. Iyer, W. Vannecke, K. W. Decoene, T. Hebbrecht, J. Gettemans, M. Laga, S. Loverix, I. Lasters, A. Madder, Chem. Commun. 2018, 54, 11929-11932. 115 D. A. Shannon, E. Weerapana, Curr. Opin. Chem. Biol. 2015, 24, 18-26. 116 A. Muratovska, M. R. Eccles, FEBS Lett. 2004, 558, 63-68. 117 Y. Wolf, S. Pritz, S. Abes, M. Bienert, B. Lebleu, J. Oehlke, Biochemistry 2006, 45, 14944-14954. 118 S. Abes, J. J. Turner, G. D. Ivanova, D. Owen, D. Williams, A. Arzumanov, P. Clair, M. J. Gait, B. Lebleu, Nucleic Acids Res. 2007, 35, 4495-4502. 119 G. Guidotti, L. Brambilla, D. Rossi, Trends Pharmacol. Sci. 2017, 38, 406-424. 120 T. Lehto, K. Kurrikoff, Ü. Langel, Expert Opin. Drug Deliv. 2012, 9, 823-836. 121 D. S. Seferos, A. E. Prigodich, D. A. Giljohann, P. C. Patel, C. A. Mirkin, Nano Lett. 2009, 9, 308-311. 105 D. A. Giljohann, D. S. Seferos, W. L. Daniel, M. D. Massich, P. C. Patel, C. A. Mirkin, Angew. Chem. Int. Ed. 2010, 49, 3280-3294. 122 P. C. Patel, D. A. Giljohann, W. L. Daniel, D. Zheng, A. E. Prigodich, C. A. Mirkin, Bioconjug. Chem. 2010, 21, 2250-2256. Chapter I: Introduction 57 AuNPs with monoclonal antibody-DNA conjugates (Figure 7, left). Importantly, higher cell selectivity and gene knockdown were achieved in cells that overexpress the target antigen. 123 Figure 7. Left: Hybridization of pNA-AuNPs using monoclonal antibody-DNA conjugates. Reprinted with permission from ref. 123. Copyright 2012 American Chemical Society. Right: Strain-promoted thiols strategy applied to deliver liposomes and polymersomes inside living cells. Reprinted from ref. 107 with permission from John Wiley and Sons. Copyright 2017 Wiley‐VCH Verlag GmbH & Co. KGaA, Weinheim. 3.2.3. Strain-promoted thiols Strain-promoted thiols are a recently developed strategy from the group of the prof. Matile.107 The application of this strategy allowed the delivery of polymersomes and liposomes into HeLa Kyoto cells.107 Based on previous work from this group on strained cyclic disulfides, the transferrin receptor (TFRC) was found to be related to the cellular internalization of the corresponding penetrating peptides. 124 Recently, cationic amphiphiles were modified with cyclic disulfides to obtain liposomes and polymersomes and, after disulfide exchange with cellular exofacial thiols, the highly strained disulfides lead to an efficient cytosolic release of these giant substrates (Figure 7, right).107 3.2.4. Polymer-drug conjugates Polymer-drug conjugates are attractive molecules to deliver drugs inside cells. The incorporation of different macromolecules, such as proteins, into polymeric scaffolds, has emerged as an innovative approach to overcome endosomal entrapment. For instance, the limitations of protein therapeutics such as fast clearance, low stability, and high immunogenicity can be solved by PEGylation. 125 Conjugation of PEG with protein therapeutics reduce immunogenicity, by shielding its antigenic epitopes, and improving its therapeutic safety. Moreover, protein PEGylation demonstrated to avoid opsonization and digestion by proteolytic enzymes. Furthermore, molecular mass increase helps to reduce plasma clearance and therefore the dosage frequency. 126 During the last years, several PEGylated proteins such 123 K. Zhang, L. Hao, S. J. Hurst, C. A. Mirkin, J. Am. Chem. Soc. 2012, 134, 16488-16491. 107 N. Chuard, G. Gasparini, D. Moreau, S. Lörcher, C. Palivan, W. Meier, N. Sakai, S. Matile, Angew. Chem. Int. Ed. 2017, 56, 2947-2950. 124 D. Abegg, G. Gasparini, D. G. Hoch, A. Shuster, E. Bartolami, S. Matile, A. Adibekian, J. Am. Chem. Soc. 2017, 139, 231-238. 125 P. Caliceti, F. M. Veronese, Adv. Drug Deliv. Rev. 2003, 55, 1261-1277. 126 I. Ekladious, Y. L. Colson, M. W. Grinstaff, Nat. Rev. Drug Discov. 2019, 18, 273-294. Héctor Fernández Caro 64 4. Cell delivery of small molecules Small molecules have a strong potential as intracellular probes and drugs for the treatment of a wide range of diseases. 157 , 158 , 159 However, many small molecules, such as drugs, have their site of action located in the cytosol, in the cell nucleus, or intracellular organelles. As examples, reverse transcriptase inhibitors or statins can be highlighted. 160 , 161 Moreover, some drugs suffer from cell efflux mediated by P-glycoproteins (P-gp) or multidrug resistance proteins (MRP). 162 Therefore, to achieve the desired effect, small molecules have to be able to cross the cell membrane, to be efficiently delivered into the cell cytosol, and to avoid the cellular efflux. Hydrophilic small drugs with intracellular targets, such as doxorubicin, have emerged as valuable anticancer drugs. Doxorubicin is a widely administer anticancer drug whose mechanism of cell damage is related to inhibition of the DNA-topoisomerase II complex. 163 Unfortunately, its hydrophilic character makes its cytosolic delivery very inefficient and the efflux pumping, by multidrug resistance proteins (MRPs) and P-glycoproteins (P-gp), makes mandatory to administrate a high dose of doxorubicin to reach the proper anticancer effect. 164 Therefore, the efficient delivery of small hydrophilic drugs into cells and tissues remains a major challenge, and new delivery methods are needed to overcome these problems. Hydrophilic anionic small fluorescent molecules are relevant intracellular probes such as sulfonated Alexas, pyranine, or carboxyfluorescein (Figure 13). Moreover, pyranine has shown excellent properties as a pH indicator. Unfortunately, the negative net charge at physiological pH of these hydrophilic anionic small molecules makes their cytosolic delivery very inefficient. 165 Therefore, the delivery of small hydrophilic fluorescent molecules into cells and tissues remains a major challenge, and new delivery systems to overcome these limitations are needed. Figure 13. Examples of hydrophilic and anionic fluorescent probes which do not pass through the cellular membrane at low micromolar concentration. 157 S. Weggen, M. Rogers, J. Eriksen, Trends Pharmacol. Sci. 2007, 28, 536-543. 158 M. Vogler, D. Dinsdale, M. J. S. Dyer, G. M. Cohen, Cell Death Differ. 2009, 16, 360-367. 159 N. K. Devaraj, S. Hilderbrand, R. Upadhyay, R. Mazitschek, R. Weissleder, Angew. Chem. Int. Ed. 2010, 49, 2869-2872. 160 W. Lewis, B. J. Day, W. C. Copeland, Nat. Rev. Drug Discov. 2003, 2, 812-822. 161 C. Stancu, A. Sima, J. Cell. Mol. Med. 2001, 5, 378-387. 162 M. M. Gottesman, Annu. Rev. Med. 2002, 53, 615-627. 163 S. Darwish, S. Mozaffari, K. Parang, R. Tiwari, Tetrahedron Lett. 2017, 58, 4617-4622. 164 A. Nasrolahi Shirazi, R. Tiwari, B. S. Chhikara, D. Mandal, K. Parang, Mol. Pharm. 2013, 10, 488-499. 165 G. Y. Wiederschain, Biochem. 2011, 76, 1276-1276. Chapter I: Introduction 65 During the last years, different methods have been explored for the delivery of small hydrophilic molecules to the cell interior including physical and mechanical methods, cellpenetrating peptides (CPPs), supramolecular methods, or by transitory reduction of charge. 4.1. Physical and mechanical methods Physical and mechanical methods have been applied to cytosolic deliver small molecules such as anionic probes. For example, microinjection has been used when the number of cells to transfect is small. Moreover, the use of strong hypotonic stress to the cells has been also applied. 166 Furthermore, small molecules, such as anticancer drugs or fluorophores, have been incorporated inside the cell by electropermeabilization. This technique uses short high-voltage cycles to produce transitory permeabilization of the cell membrane to allow small molecules to cross the cell membrane by simple diffusion. 167 , 168 4.2. Covalent methods using cell-penetrating peptides The covalent attachment to a CPP constitutes an alternative strategy for the cytosolic delivery of small molecules. Different approaches have been developed where the CPP is covalently attached to small anticancer drugs such as doxorubicin or methotrexate 169 and also to fluorophores. 170 Traditionally, the covalent bond formation was one of the most used methods. To incorporate the cargo into the peptide by covalent bonding, one of the possible strategies is based on the anchoring of the cargo into the cysteine or the lysine side chain of one of the peptide residues. 171 Among the different types of covalent bonding between the cargo and the CPP, it is necessary to highlight the disulfide bond, as this bond has the advantage of being sensitive to the cytosolic glutathione, which allows the dynamic disconnection of the cargo inside the cells. An exceptional example was described by the group of prof. Paul Wender, in which a CPP was connected to luciferin by a disulfide bond. The cargo-luciferin complex can cross the plasma membrane and once inside the cell, glutathione reduces the disulfide bond causing the release of the luciferin that triggers luciferase activity and light emission (Figure 14). 172 166 B. S. Gan, E. Krump, L. D. Shrode, S. Grinstein, Am. J. Physiol. 1998, 275, C1158-C1166. 167 M.-P. Rols, Biochim. Biophys. Acta 2006, 1758, 423-428. 168 A. Pena, J. Ramirez, G. Rosas, M. Calahorra, J. Bacteriol. 1995, 177, 1017-1022. 169 K. M. Stewart, K. L. Horton, S. O. Kelley, Org. Biomol. Chem. 2008, 6, 2242-2255. 170 Z. Qian, P. G. Dougherty, D. Pei, Chem. Commun. 2015, 51, 2162-2165. 171 M. Zorko, U. Langel, Adv. Drug Deliv. Rev. 2005, 57, 529-545. 172 L. R. Jones, E. A. Goun, R. Shinde, J. B. Rothbard, C. H. Contag, P. A. Wender, J. Am. Chem. Soc. 2006, 128, 6526-6527. Héctor Fernández Caro 66 Figure 14. Schematic illustration showing intracellular transport of luciferin covalently attached to a cell-penetrating peptide through a releasable linker and, the subsequent, intracellular release of the cargo mediated by glutathione. Reprinted with permission from 172. Copyright 2006 American Chemical Society. CPPs have been used to improve the internalization of a wide range of drugs (Figure 15). For example, the prodrug 5-aminolevulinic acid was attached to penetratin for cell delivery. 173 Furthermore, cyclosporine A-CPP conjugates were also synthesized and it was found that this conjugate is able to pass through the stratum corneum and to carry out its therapeutic effect. 174 Some of these covalent conjugates were also applied in vivo, showing good properties to cross the brain membrane. For example, the attachment of benzylpenicillin to a CPP importantly increased the drug delivered into the brain in comparison with the drug alone. 175 Interestingly, the covalent attachment of CPPs to anticancer drugs also showed to overcome anticancer-drug resistance in cells. 176 These conjugates showed the ability to avoid the efflux pump proteins, such as in the case of the covalent conjugation of doxorubicin to TAT which has shown high cytotoxicity against doxorubicin-resistant cell lines. 177 A methotrexate-CPP conjugate has also shown internalization and toxicity against cell lines resistant to this anticancer drug. 178 A prominent strategy based on activable cell-penetrating peptides (ACPPs) was developed by the group of prof. Roger Tsien (Figure 15, right).179 A fusion peptide was prepared by a combination of a cationic CPP and an anionic amino acid sequence that acts as an inhibitor of the penetrating activity. A consensus peptide sequence recognized by proteases that are overexpressed in tumors cells was incorporated between the cationic and the anionic sequences. Therefore, proteases cleavage of the inhibitor sequence triggers selective penetration of the cargo such as a fluorophore to selectively label tumor cells. 179 Imaging agents have been 173 L. Bourré, F. Giuntini, I. M. Eggleston, M. Wilson, A. J. MacRobert, Br. J. Cancer 2009, 100, 723-731. 174 J. B. Rothbard, S. Garlington, Q. Lin, T. Kirschberg, E. Kreider, P. L. McGrane, P. A. Wender, P. A. Khavari, Nat. Med. 2000, 6, 1253-1257. 175 C. Rousselle, P. Clair, J. Temsamani, J. M. Scherrmann, J. Drug Target. 2002, 10, 309-315. 176 S. B. Fonseca, M. P. Pereira, S. O. Kelley, Adv. Drug Deliv. Rev. 2009, 61, 953-964. 177 J. F. Liang, V. C. Yang, Bioorganic Med. Chem. Lett. 2005, 15, 5071-5075. 178 M. Lindgren, K. Rosenthal-Aizman, K. Saar, E. Eiríksdóttir, Y. Jiang, M. Sassian, P. Östlund, M. Hällbrink, Ü. Langel, Biochem. Pharmacol. 2006, 71, 416-425. 179 T. Jiang, E. S. Olson, Q. T. Nguyen, M. Roy, P. A. Jennings, R. Y. Tsien, Proc. Natl. Acad. Sci. 2004, 101, 17867-17872. Chapter I: Introduction 67 incorporated into these peptides for their use as tumor tissues labelling probes in vivo 180 , 181 and for guided surgery. 182 , 183 Figure 15. Left: Examples of drugs delivered into cells by applying covalent attachment to CPPs. Reproduced from Ref. 169 with permission from The Royal Society of Chemistry. Right: Schematic illustration of the mechanism of internalization of activable cell-penetrating peptides (ACPPs). Reprinted from ref. 179 Copyright 2004 National Academy of Sciences. Moreover, this strategy has been extended to the field of chemotherapy by the incorporation of an anticancer drug such as doxorubicin. This idea allowed to increase the specificity in tumors that overexpress enzymes such as MMPs (matrix metalloproteinases). 184 Although there are many examples of CPP-molecular conjugates by covalent bonds, this strategy has also some limitations such as alterations of the biological effect of the cargo and sometimes tedious synthetic steps. For this reason, during the last decades, different supramolecular strategies have been developed to overcome some of the limitations of the covalent attachment. 4.3. Non-covalent supramolecular methods Supramolecular strategies can use different non-covalent interactions, such as host-guest encapsulation, electrostatic interactions, or hydrophobic forces, to incorporate small molecules, such as drugs or probes, into efficient delivery vehicles. During the last years, multiple supramolecular strategies have emerged for the cytosolic delivery of a wide range of small molecules including peptides, 185 ionophores, 186 liposomes, 187 macrocyclic capsules,188 supramolecular nanoparticles 188 or stimuli-responsive vesicles. 189 180 T. A. Aguilera, E. S. Olson, M. M. Timmers, T. Jiang, R. Y. Tsien, Integr. Biol. 2009, 1, 371-381. 181 E. S. Olson, T. A. Aguilera, T. Jiang, L. G. Ellies, Q. T. Nguyen, E. H. Wong, L. A. Gross, R. Y. Tsien, Integr. Biol. 2009, 1, 382-393. 182 Q. T. Nguyen, R. Y. Tsien, Nat. Rev. Cancer 2013, 13, 653-662. 183 Q. T. Nguyen, E. S. Olson, T. A. Aguilera, T. Jiang, M. Scadeng, L. G. Ellies, R. Y. Tsien, Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 4317-4322. 184 N. Q. Shi, W. Gao, B. Xiang, X. R. Qi, Int. J. Nanomedicine 2012, 7, 1613-1621. 185 S. J. Song, S. Lee, K. S. Ryu, J. S. Choi, Bioconjug. Chem. 2017, 28, 2266-2276. 186 P. R. Brotherhood, A. P. Davis, Chem. Soc. Rev. 2010, 39, 3633-3647. 187 U. Bulbake, S. Doppalapudi, N. Kommineni, W. Khan, Pharmaceutics 2017, 9, 12. 188 M. J. Webber, R. Langer, Chem. Soc. Rev. 2017, 46, 6600-6620. 189 W. C. de Vries, D. Grill, M. Tesch, A. Ricker, H. Nüsse, J. Klingauf, A. Studer, V. Gerke, B. J. Ravoo, Angew. Chem. Int. Ed. 2017, 56, 9603-9607. Héctor Fernández Caro 68 4.3.1. Cell-penetrating peptides Non-covalent conjugates between cell-penetrating peptides and small molecules constitute an excellent method to accomplish the cytosolic delivery of small molecules. Despite covalent conjugation has been extensively used, the preparation of the corresponding peptide conjugates sometimes requires multiple steps. In contrast, the procedure to cellular delivery by noncovalent interactions using penetrating peptides represents a much more simple and straightforward idea. An elegant example of non-covalent delivery of fluorophores has been developed in the group of prof. Tampé (Figure 16, left).190 A peptide consisting of six N-terminal histidines was connected by a linker (GGGS) to the typical penetrating peptide TAT (RKKRRQRRR). On the other hand, a fluorescent multivalent chelator made of a multivalent derivative of the N- nitrilotriacetic acid (trisNTA) was modified with different fluorophores, such as Alexa Fluor 647. The trisNTA was able to coordinate with the histidine amino acids and when mixed together the trisNTA and the peptide His6-TAT in the presence of Ni (II), a complex was formed. This complex was able to cross the cell membrane and the fluorescent multivalent chelator was released inside of the cell. 190 Another example of non-covalent delivery of fluorophores with amphiphilic cell-penetrating peptides was shown with a non-covalent complex including thermally activated delayed fluorescence (TADF) moieties.191 In this work, the authors designed an amphiphilic cell-penetrating peptide, F6G6(rR)3R2, that was able to form supramolecular complexes with TADF compounds such as 4CzIPN, NAI-DPAC, and BTZDMAC (Figure 16, right). These fluorescent molecules and the peptide self-assembled into fluorescent nanoparticles, these NPs were able to easily cross the cell membrane and to allow the acquisition of time-resolved luminescence images. 191 Furthermore, amphipathic peptides forming nanoribbons coated with a second layer of cell-penetrating peptides were also able to deliver fluorophores inside of the cell. 192 Figure 16. Left: Chemical structure of trisNTA fluorophore and His6-tagged TAT cell-penetrating peptide. Reprinted with permission from ref. 190. Copyright 2018 American Chemical Society. Right: Chemical structure of amphiphilic CPP and the different TADF fluorophores. Reprinted and adapted with permission from ref. 191. Copyright 2018 American Chemical Society. 190 R. Wieneke, N. Labòria, M. Rajan, A. Kollmannsperger, F. Natale, M. C. Cardoso, R. Tampé, J. Am. Chem. Soc. 2014, 136, 13975-13978. 191 Z. Zhu, D. Tian, P. Gao, K. Wang, Y. Li, X. Shu, J. Zhu, Q. Zhao, J. Am. Chem. Soc. 2018, 140, 17484-17491. 192 Y. Lim, E. Lee, M. Lee, Angew. Chem. Int. Ed. 2007, 46, 3475-3478. Chapter I: Introduction 69 On the other hand, amphiphilic peptides, using non-covalent interactions, were shown to transport drugs such as curcumin or doxorubicin.185,193 For example, amphiphilic peptide nanorods were recently used to encapsulate curcumin and subsequently to deliver this molecule in cancer cell lines and zebrafish models.185 Moreover, a new amphiphilic peptide was recently shown to self-assemble to form pH-responsive micelles that were able to entrap and deliver doxorubicin in cells. 193 4.3.2. Ionophores Another supramolecular method for the transport of small molecules is the ionophores. This family of synthetic molecules gets inspiration from a variety of naturally occurring products that are able to complex ions in a very tight and efficient manner and promote their translocation across lipid bilayers. 194 To carry out such a challenging task, natural ionophores present a structure that is generally cyclic or bent-shaped, in which the external surface of the molecules presents a strong hydrophobic character, which grants a good partitioning into the fatty-acid rich membrane lumen. The hydrophilic region of ionophores is rich in coordinating heteroatoms responsible for the interaction with the ions, normally being able to mimic the hydration sphere of the ion that is translocated. 195 , 196 Various research groups have modified natural molecules to design new transporters. In this group, prodigiosins are a class of molecules based on 4-methoxy-bipyrrole with different substituents (Figure 17). 197 In recent years, it has been found that this type of molecules can function as H+/Cl- 198 and Cl-/HCO3- 199 exchanger antiporters. To achieve segregation between an external lipophilic surface and a coordination-rich inner cavity, researchers have employed multipodal scaffolds to obtain a prearranged tridimensional disposition of key functional groups. 200 In this regard, the cholapods, which are a family of steroids derived from cholic acid, have been designed (Figure 17B). In these molecules, one of the hydrophobic faces is preserved and groups capable of complexing anions such as squaramides, thioureas, or ureas are incorporated into various positions of the polycyclic ring.186 Interestingly, cholapods with high efficiency for the transport of Clin model vesicles have been discovered. 201 Also applying this strategy, the functionalization with three ureas or 185 S. J. Song, S. Lee, K. S. Ryu, J. S. Choi, Bioconjug. Chem. 2017, 28, 2266-2276. 193 J. Liang, W. Wu, X. Xu, R. Zhuo, X. Zhang, Colloids Surfaces B Biointerfaces 2014, 114, 398-403. 194 A. Roy, D. Saha, A. Mukherjee, P. Talukdar, Org. Lett. 2016, 18, 5864-5867. 195 J. Rutkowski, B. Brzezinski, Biomed Res. Int. 2013, 2013, 162513. 196 P. A. Gale, Acc. Chem. Res. 2011, 44, 216-226. 197 J. L. Sessler, L. R. Eller, W. S. Cho, S. Nicolaou, A. Aguilar, J. T. Lee, V. M. Lynch, D. J. Magda, Angew. Chem. Int. Ed. 2005, 44, 5989-5992. 198 K. Tanigaki, T. Sato, Y. Tanaka, T. Ochi, A. Nishikawa, K. Nagai, H. Kawashima, S. Ohkuma, FEBS Lett. 2002, 524, 37-42. 199 J. T. Davis, P. A. Gale, O. A. Okunola, P. Prados, J. C. Iglesias-Sánchez, T. Torroba, R. Quesada, Nat. Chem. 2009, 1, 138-144. 200 N. Busschaert, M. Wenzel, M. E. Light, P. Iglesias-Hernndez, R. Prez-Toms, P. A. Gale, J. Am. Chem. Soc. 2011, 133, 14136-14148. 186 P. R. Brotherhood, A. P. Davis, Chem. Soc. Rev. 2010, 39, 3633-3647. 201 S. J. Edwards, H. Valkenier, N. Busschaert, P. A. Gale, A. P. Davis, Angew. Chem. Int. Ed. 2015, 54, 4592- 4596. Héctor Fernández Caro 70 thioureas has been carried out using tris(2-aminoethyl)amine, and the resulting compounds showed good efficiency as bicarbonate transporters. 202 Figure 17. Chemical structures of different examples of ionophores: A) Prodigiosin.197 B) Example of cholapod.186 C) Representative example of trans-decalin derivative.204 Other transporters of high interest are difunctionalized trans-decalins in the 4,8-diaxial positions. In this type of molecules, the hydrophobic moiety of the core molecule is maintained while in the other part is modified with two thiourea or urea groups with aromatic substituents. 203 Interestingly, one of the trans-decalin derivatives was the most effective transporter for the Clanion in model vesicles (Figure 17).204 This transporter consists of a modified trans-decalin with two aryl-thiourea groups in which the aromatic rings are functionalized with two CF3 groups in positions 3 and 5. 204 More recently, we should highlight that these transport systems are starting to be considered in vitro for the treatment of cystic fibrosis, in which these artificial molecules could have the potential to mimic Cltransporting channels (CFTR proteins) absent or malfunctioning in the patients that suffer from this disease. 205 , 206 4.3.3. Liposomes Liposomes are one of the most used drug delivery systems. Classical liposomes consist of spherical vesicles with an internal aqueous core that is surrounded by one or more lipid bilayers in the external layer. From the pharmaceutical point of view, these entities are capable of incorporating both hydrophobic (in the bilayer) and hydrophilic molecules (in the aqueous space). 207 When classical liposomes were administered following the intravenous pathway, it was found that they suffer from a fast clearance, having short circulation times. However, clearance was reduced carrying out the PEGylation of the liposome. A few years ago, the PEGylated liposomal doxorubicin (Doxil®) was FDA approved and showed an increase in the drug levels and reduction of cardiomyocyte damage in comparison with doxorubicin alone. Another example of using a PEGylated liposomal system, in this case, for irinotecan delivery is OnivydeTM.187 Recently, multifunctional liposomes (Figure 18, left) have emerged to improve 202 N. Busschaert, P. A. Gale, C. J. E. Haynes, M. E. Light, S. J. Moore, C. C. Tong, J. T. Davis, W. A. Harrell, Chem. Commun. 2010, 46, 6252-6254. 203 S. Hussain, P. R. Brotherhood, L. W. Judd, A. P. Davis, J. Am. Chem. Soc. 2011, 133, 1614-1617. 204 H. Valkenier, L. W. Judd, H. Li, S. Hussain, D. N. Sheppard, A. P. Davis, J. Am. Chem. Soc. 2014, 136, 12507- 12512. 205 K. A. Muraglia, R. S. Chorghade, B. R. Kim, X. X. Tang, V. S. Shah, A. S. Grillo, P. N. Daniels, A. G. Cioffi, P. H. Karp, L. Zhu, et al., Nature 2019, 567, 405-408. 206 P. A. Gale, J. T. Davis, R. Quesada, Chem. Soc. Rev. 2017, 46, 2497-2519. 207 T. O. B. Olusanya, R. R. H. Ahmad, D. M. Ibegbu, J. R. Smith, A. A. Elkordy, Molecules 2018, 23, 907. 187 U. Bulbake, S. Doppalapudi, N. Kommineni, W. Khan, Pharmaceutics 2017, 9, 12. A) B) C) Chapter I: Introduction 71 the pharmaceutical properties of liposomes. This kind of liposomes can incorporate different functionalities in the same entity such as targeting ligands (antibody or carbohydrates), imaging agents, hydrophilic or hydrophobic drugs, therapeutic nucleic acids, etc. 208 For instance, an innovative approach has been recently developed in the group of prof. Alexander Kros.209 In this work, a pair of complementary coiled-coil lipopeptides were designed and incorporated into liposomes. The fusion of the coiled-coil peptides allowed the release of the anticancer drug, doxorubicin, encapsulated inside the liposomes in cells, and zebrafish models. 209 4.3.4. Host-guest delivery systems Host-guest processes are a powerful tool to develop new carriers for the encapsulation and the delivery of small molecules. As we have previously commented, classical host molecules such as cyclodextrins, cucurbiturils, or calixarenes have been applied to deliver a variety of therapeutic molecules.188 Cyclodextrins (CDs) have been shown to be suitable delivery carriers for different cargos due to their high bioavailability and low cytotoxicity. For instance, β-CDs have been demonstrated not only to recognize a variety of substrates but also to promote the delivery of different anticancer drugs such as doxorubicin 210 or paclitaxel. 211 For example, hostguest molecular recognition between β-CDs and paclitaxel has been recently used to deliver this antitumoral drug in living cells and also in vivo (Figure 18, right).212 Kim and co-workers have designed a system constituted by poly-paclitaxel and poly-CDs conjugated by ester bonds to a polymer chain. Moreover, a peptidic targeting ligand was also incorporated into the system. Once inside the cell, intracellular enzymes cleave the ester bonds and the cyclodextrin/paclitaxel complexes are released from the polymers. Finally, these complexes dissociate and the free paclitaxel produces its tumor cytotoxic effect. 212 208 M. Riaz, M. Riaz, X. Zhang, C. Lin, K. Wong, X. Chen, G. Zhang, A. Lu, Z. Yang, Int. J. Mol. Sci. 2018, 19, 195. 209 J. Yang, A. Bahreman, G. Daudey, J. Bussmann, R. C. L. Olsthoorn, A. Kros, ACS Cent. Sci. 2016, 2, 621-630. 188 M. J. Webber, R. Langer, Chem. Soc. Rev. 2017, 46, 6600-6620. 210 H. Hyun, S. Lee, W. Lim, D. Jo, J. S. Jung, G. Jo, S. Y. Kim, D. Lee, S. Um, D. H. Yang, et al., J. Ind. Eng. Chem. 2019, 70, 145-151. 211 J. Jing, A. Szarpak-Jankowska, R. Guillot, I. Pignot-Paintrand, C. Picart, R. Auzély-Velty, Chem. Mater. 2013, 25, 3867-3873. 212 R. Namgung, Y. Mi Lee, J. Kim, Y. Jang, B. H. Lee, I. S. Kim, P. Sokkar, Y. M. Rhee, A. S. Hoffman, W. J. Kim, Nat. Commun. 2014, 5, 3702. Héctor Fernández Caro 72 Figure 18. Left. Schematic representation showing the different types of liposomes: A) Conventional liposomes, B) PEGylated liposomes, C) Ligand targeted liposomes and D) Multifunctional liposomes such as theranostic liposomes. Reprinted from ref. 208 under the terms of the Creative Commons CC BY license. Right. Schematic illustration of the mechanism of internalization and paclitaxel release from the supramolecular host-guest nano-assembly. Reprinted with permission from ref. 212. Copyright 2014 Springer Nature. 4.3.5. Redox and pH-responsive encapsulating vesicles Redox-responsive nanocontainers have been shown to deliver hydrophilic cargos such as pyranine or phalloidin. These nanocontainers consist of a shell formed by a redox-responsive polymer attached by host-guest processes to a cyclodextrin core through an adamantane moiety (Figure 19, left). The encapsulation of different cargos was achieved by disulfide bonds that were incorporated into the polymeric shell as redox cleavable functions. In this work, the authors showed that these nanocontainers were able to release different molecular payloads in the cell interior and driven by the redox trigger.189 On the other hand, pH-responsive core-shell nanoparticles have also been described to deliver fluorescent probes and small molecular drugs simultaneously (Figure 19, right).213 These nanoparticles consist of a shell formed by the negatively charged pyranine with a core of positively charged carboxymethylhexanoyl chitosan. The electrostatic interactions between the shell and core polymers produce the assembly into pH-responsive nanoparticles. Interestingly, the encapsulation of the antitumoral drug camptothecin into these NPs for pH-controlled drug release was achieved. The authors were able to develop a theranostic system with potential therapeutic and diagnosis capabilities. 213 189 W. C. de Vries, D. Grill, M. Tesch, A. Ricker, H. Nüsse, J. Klingauf, A. Studer, V. Gerke, B. J. Ravoo, Angew. Chem. Int. Ed. 2017, 56, 9603-9607. 213 H. S. Chou, M. H. Hsiao, W. Y. Hung, T. Y. Yen, H. Y. Lin, D. M. Liu, J. Mater. Chem. B 2014, 20, 6580- 6589. Chapter I: Introduction 73 Figure 19. Left. Preparation of redox-responsive nanocontainer and redox-triggered payload release. Reprinted ref. 189 with permission from John Wiley and Sons. Copyright 2017 Wiley‐VCH Verlag GmbH & Co. KGaA, Weinheim. Right: Schematic representation of pH-responsive core-shell nanoparticles. Reproduced from Ref. 213 with permission from The Royal Society of Chemistry. 4.4. Transitory reduction of charge Transitory reduction of charge is one of the most widely applied methods to trigger the intracellular release of small hydrophilic molecules. This strategy is based on increasing the hydrophobicity of the molecule by forming hydrophobic esters at phosphoric or carboxylic acids, among others. The resulting molecule, with higher hydrophobic properties, can cross the cell membrane and, after the action of intracellular enzymes, the hydrophilic molecule is released. 214 , 215 The main limitations of this strategy are the required synthetic modification of the active molecule and the need for efficient intracellular hydrolysis of the corresponding ester. A few decades ago, the prodrug strategy has been developed. 216 This strategy is based on the modification of active drugs that after suffering an intracellular cleavage, by enzymes or chemicals, will lead to the release of the active drug. This methodology has been applied to increase the efficiency of a wide range of drugs. 217 For instance, cidofovir is a highly hydrophilic drug, used as therapy for herpesviruses, which have strong limitations to cross the plasma membrane. However, when the phosphate group of this molecule is modified by esterification with a hydrophobic molecule, the resulting molecule can be internalized by simple diffusion, and subsequently, the ester enzymatic hydrolysis of the ester inside the cell allows the recovery of the active principle. Importantly, the administration of the prodrug of cidofovir causes a four-fold activity enhancement compared to the unmodified drug (Figure 20). 218 In addition to bioactive molecules and drugs, this strategy has also been applied for different fluorescent probes. 219 In recent years, fluorescein derivatives have been used to carry out intracellular pH measurements by fluorescence methods. One of the most used compounds is 2´,7´-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) (Figure 20). 220 This small highly 214 P. Ozkan, R. Mutharasan, Biochim. Biophys. Acta 2002, 1572, 143-148. 215 J. Rautio, H. Kumpulainen, T. Heimbach, R. Oliyai, D. Oh, T. Järvinen, J. Savolainen, Nat. Rev. Drug Discov. 2008, 7, 255-270. 216 J. Rautio, N. A. Meanwell, L. Di, M. J. Hageman, Nat. Rev. Drug Discov. 2018, 17, 559-587. 217 K. M. Huttunen, H. Raunio, J. Rautio, Pharmacol. Rev. 2011, 63, 750-771. 218 E. De Clercq, H. J. Field, Br. J. Pharmacol. 2006, 147, 1-11. 219 L. D. Lavis, T.-Y. Chao, R. T. Raines, Chem. Sci. 2011, 2, 521-530. 220 N. Boens, W. Qin, N. Basarić, A. Orte, E. M. Talavera, J. M. Alvarez-Pez, J. Phys. Chem. A 2006, 110, 9334- 9343. Chapter I: Objectives 81 Based on previous results on cage complexation of planar anionic molecules,69 we decided to explore the potential caging strategy for the transitory masking of the hydrophilicity of planar probes and to achieve their delivery in the cytosol. The supramolecular nature of the approach will allow us to deliver these probes into the cell cytosol driven by the different concentrations of the probe between the extracellular and the intracellular environment and the high concentration of different anions present in the cell interior (Figure 24). The specific objectives of this first chapter would be:  Conjugation of a membrane-impermeable supramolecular cationic cage with different cell-penetrating peptides of different lengths, which would donate cell membrane transport capabilities to the supramolecular cage and evaluation of their cytotoxicity.  Study of the interaction between the supramolecular cage and the pyranine probe by titration experiments, to understand the transport mechanism of the probe by U-tube experiments.  Intracellular cytosolic delivery of pyranine in different cell lines using the previously synthesized peptide-cage carriers.  Study of the mechanism of transport of these supramolecular complexes inside living cells by ensuring the non-permeabilization of the cell membrane and by using endocytosis inhibitors.  Evaluation of the specificity of the approach by competition assays with different cargos.  Extension of the approach to study interaction with the cage and cellular delivery efficiency of different anionic probes (CF and Alexa Fluor´s).  Cell pH tracking by using peptide-cage carrier and pyranine as a pH-sensitive probe. Figure 24. A) Objective: development of a peptide-cage carrier that consists of a peptide (blue spheres) and a positively charged supramolecular cage that is capable of delivering membrane-impermeable planar anionic probes into living cells and release them from endosomes. B) Examples of highly valuable fluorescent probes which are not able to cross cell membranes at low micromolar concentrations. 69 J. Mosquera, S. Zarra, J. R. Nitschke, Angew. Chem. Int. Ed. 2014, 53, 1556-1559. A) B) Results and discussions The results of this chapter have been published in: H. Fernández-Caro, I. Lostalé-Seijo, M. Martínez-Calvo, J. Mosquera, J. L. Mascareñas, J. Montenegro, Chem. Sci. 2019, 10, 8930-8938. Chapter I: Results and discussions 85 1. Antecedents The group of prof. Nitschke has recently developed a cationic supramolecular cage (Figure 25) that showed the ability to interact by host-guest molecular recognition with the pyranine probe in aqueous environment with a Kd of 1.2 nM.69 Later, in collaboration with Prof. Mascareñas group, a pyranine-peptide guest was designed by connecting a typical cellpenetrating peptide (octaarginine) to a polyanionic oligoglutamic terminated with a pyranine moiety. This peptide was not able to cross the cell membrane due to the anionic character of the pendant. However, when the supramolecular cage was incorporated, the cationic cage formed a host-guest complex with the anionic pyranine, masking its negative charge and promoting the cellular internalization of the membrane-impermeable pyranine-peptide (Figure 25). 241 More recently, in a fruitful collaboration between the group of prof. Mascareñas and the group of prof. Liz-Marzán, the supramolecular interaction pyranine-cage was further explored to switch the cellular uptake of gold nanoparticles (AuNPs) (Figure 25).242 In this work, AuNPs were functionalized with pyranine moieties. Due to their high negative charge, these AuNPs did not cross through the cell membrane. However, after the formation of the host-guest complex between the cage and the pyranine, their negative charge was masked and the cellular internalization of the pyranine-modified AuNPs was achieved. Interestingly, it was found that the cellular uptake of the pyranine-modified AuNPs could be reversibly turned on and off with the addition of the supramolecular cage. 242 Figure 25. Schematic representations showing the intracellular delivery of membrane-impermeable A) pyranine-functionalized peptide and B) pyranine-modified AuNPs triggered by the addition of the cationic supramolecular cage. A) Reprinted with permission from ref. 241. Copyright 2017 American Chemical Society. B) Reprinted with permission from ref. 242. Copyright 2018 American Chemical Society. 69 J. Mosquera, S. Zarra, J. R. Nitschke, Angew. Chem. Int. Ed. 2014, 53, 1556-1559. 241 J. Rodríguez, J. Mosquera, J. R. Couceiro, J. R. Nitschke, M. E. Vázquez, J. L. Mascareñas, J. Am. Chem. Soc. 2017, 139, 55-58. 242 J. Mosquera, M. Henriksen-Lacey, I. García, M. Martínez-Calvo, J. Rodríguez, J. L. Mascareñas, L. M. Liz- Marzán, J. Am. Chem. Soc. 2018, 140, 4469-4472. A) B) Héctor Fernández Caro 86 2. Design and synthesis 2.1. Synthesis of the cage C and initial cellular internalization experiments First, in collaboration with the group of prof. Nitschke, we followed the procedure reported in the literature for the synthesis and the purification of the supramolecular cage C (Figure 4A).69 Following the described procedure, the 3,3’-bipyridine-6,6’-dicarboxaldehyde was reacted with tris(2-aminoethyl)amine and cadmium(II) trifluoromethanesulfonate (Cd(OTf)2) in acetonitrile solution. The resulting CdII-templated cage was then reduced using sodium borohydride (NaBH4), leading to the demetallated cage C. Once, we have prepared the cationic cage C, we wondered if the cage C alone would have the ability to promote the cellular internalization of pyranine and, also, if pyranine alone would be able to cross the cell membrane at different concentrations (Figure 26). To study this phenomenon, we decided to carry out preliminary control experiments using confocal microscopy. Figure 26. A) Schematic representation of the control experiments showing neither internalization of the complex pyranine and cage C nor pyranine alone inside cells. B and C) Control transport experiments in HeLa cells. Confocal micrographs of HeLa cells incubated with: B) Pyranine (5 µM) C) Pyranine (5 µM) and only cage C (5µM). Nuclei stained with Hoechst (blue). DIC on the left and merge of fluorescence channels [Pyranine emission (green) + Hoechst 33342 emission (blue)] on the right. First, we decided to evaluate the potential of the cage C to internalize pyranine. For this, we co-incubated different concentrations of the cage C and pyranine with HeLa cells. To carry out this internalization experiment, we treated HeLa cells with Hoechst 33342 for 30 min (for nucleus staining), we washed (HKR buffer, 1x), and then cells were incubated with the different concentrations of cage and pyranine, for instance, 5 μM of pyranine and 5 μM of cage C (Figure 26), in HKR buffer for 30 min at 37 ⁰C. Cells were finally washed (HKR buffer, 3x) and 69 J. Mosquera, S. Zarra, J. R. Nitschke, Angew. Chem. Int. Ed. 2014, 53, 1556-1559. A) B) C) Chapter I: Results and discussions 87 observed under the confocal microscope. In the confocal micrographs (Figure 26), we did not observe any pyranine (green) fluorescence inside the cells. This result demonstrated that the supramolecular cage C does not promote the intracellular delivery of pyranine. On the other hand, we also evaluated the ability of pyranine alone to cross the cell membrane at different concentrations. Following the same procedure, HeLa cells incubated with 5 μM of pyranine alone did not show any green fluorescence inside the cells (Figure 26). 2.2. Initial design and synthesis of the peptide-cage hybrids TmAC and TmR8C After verifying by confocal experiments that the supramolecular cage C alone is not able to internalize pyranine at different concentrations, we then decided to covalently attach to one of the vertices of the cage different cationic peptides with excellent internalization abilities and high aqueous solubility. We hypothesized that these peptide pendants would promote the cellular internalization and also would increase the solubility of the cage in the aqueous environment. With this purpose, first, we decided to covalently attach to the cage a typical cellpenetrating peptide octaarginine (R8). This peptide (Figure 27) has been intensively studied and demonstrated to be able to improve the solubility and to promote the cellular translocation of different therapeutically active molecules.169 We also decided to incorporate the amphiphilic peptide A (Figure 27) to the cage.109 Furthermore, we incorporated the TAMRA fluorophore at the N-terminus of both peptides (Figure 27) to follow the distribution of the peptide in cellular experiments by confocal microscopy without causing any interference with the pyranine emission. Figure 27. Chemical structure of the peptide-cage hybrids TmR8C and TmAC. To promote the intracellular delivery of pyranine, we synthesized two different peptidecage hybrids TmR8C and TmAC in which the Tm superindex denotes the incorporation of TAMRA fluorophore at the N-terminus of the peptides (Figure 27). The synthetic strategy was divided into two steps, first, we synthesized TmA and TmR8 with a free carboxylic acid group at their terminal carboxylic ends and, in the second step, in collaboration with the group of the prof. Mascareñas, we attached the supramolecular cage C (Figure 28). To carry out the first step, SPPS by manual Fmoc solid-phase was applied by using a 2-chlorotrityl chloride resin. 243 This resin was employed to obtain the corresponding free C-terminal group for the subsequent 169 K. M. Stewart, K. L. Horton, S. O. Kelley, Org. Biomol. Chem. 2008, 6, 2242-2255. 109 M. Mäe, Ü. Langel, Curr. Opin. Pharmacol. 2006, 6, 509-514. 243 M. Alhassan, O. Al Musaimi, J. M. Collins, F. Albericio, B. G. de la Torre, Green Chem. 2020, 22, 2840-2845. Héctor Fernández Caro 88 attachment of the cage C. Importantly, we incorporated two different linkers, first, 8-amino- 3,6-dioxaoctanoic acid (O2Oc) and then, 6-aminohexanoic acid (Ahx), before both peptide sequences. We hypothesized that the incorporation of both linkers would decrease the steric hindrance and the electrostatic interactions and would increase the yield of the reaction with the cationic cage in the last step. The first coupling was carried out in CH2Cl2 using DIEA as the base. For the following couplings, we used N-HBTU as activator, DIEA as base, and DMF as solvent. The deprotection of the temporal Fmoc protecting group was performed by treating the resin with 20% piperidine in DMF. To couple the fluorophore at the N-terminus we used three equivalents of 5(6)-Carboxytetramethylrhodamine (TAMRA) with 3 equivalents of N- HATU and 5 equivalents of DIEA in DMF (0.2 M) for 60 min. The cleavage/deprotection step was performed by treatment of the resin-bound peptide for 2 h with the following cleavage cocktail: 900 mL TFA, 50 mL CH2Cl2, 25 mL H2O, and 25 mL TIS. Peptides were precipitated with Et2O and purified by RP-HPLC. The peptides were characterized by HPLC-MS. Once we have synthesized and characterized TmA and TmR8 with the free terminal carboxylic groups, we proceed with the second synthetic step, the coupling of the supramolecular cage. To synthesize TmAC and TmR8C, we first dissolved TmA and TmR8 peptides in DMF (0.1 mM, 100 mL) and then N-HATU (1 equiv.) and DIEA (10 equiv.) were added to the solution. This mixture was added to a 0.1 mM solution of the cage C (1 equiv.) and the reaction was left stirring overnight. The resulting product was purified by RP-HPLC and the peptide-cage peptides were characterized by mass spectrometry. Figure 28. Scheme for the synthesis of TmAC and TmR8C. 2.3. Preliminary internalization experiments using TmAC and TmR8C Once we had synthesized and characterized the peptides TmAC and TmR8C, we addressed if these peptide-cage hybrids would be able to internalize pyranine inside cells. To study the internalization of this probe, we decided to carry out preliminary transport experiments using low micromolar concentrations of both pyranine and the different peptides. First, we carried out pyranine transport experiments using TmAC. We incubated HeLa cells with Hoechst 33342 for 30 min, washed (HKR buffer, 1x), and then we co-incubated the cells with 5 µM of TmAC and 5 µM Pyranine in HKR buffer for 30 min at 37 ⁰C. The cells were finally washed (HKR buffer, 3x) and then observed under the confocal microscope. Unexpectedly, we found high cellular toxicity of the TmAC peptide which can be appreciated in the confocal images (Figure 29) by the morphological cellular alterations such as nuclear profiling. Moreover, we also found that this peptide forms aggregates in the presence of pyranine in the cellular environment. Chapter I: Results and discussions 89 Then, we carried out pyranine transport experiments with TmR8C following the same procedure as before but, in this case, using 5 µM of TmR8C and 5 µM Pyranine. However, we found again high cytotoxicity of TmR8C which was evident by the cellular morphological alterations and the lower number of cells observed (Figure 29). Moreover, as the previous hybrid, this peptide is also forming aggregates in the presence of pyranine in the cellular medium. Figure 29. Transport experiments in HeLa cells with TmAC and TmR8C. Confocal micrographs of HeLa cells incubated with: A) 5 µM of TmAC and 5 µM Pyranine B) 5 µM of TmR8C and 5 µM Pyranine. DIC on the left and merge channels [Pyranine emission (green) + TAMRA emission (red) + Hoechst 33342 emission (blue)] on the right. 2.4. Re-design and synthesis of the peptide cage-hybrids AcR4C/TmR4C Taking into account the high cellular toxicity and high ability to form aggregates when incubated with pyranine of previously designed peptides TmAC and TmR8C. We re-designed our peptide-cage hybrid and we decided to covalently attach a shorter tetraarginine peptide to one of the vertices of the cage instead of the octapeptide (Figure 30). We hypothesized that this shorter cationic peptide would avoid cytotoxicity and aggregation problems. Therefore, we synthesized two R4-cage hybrids, AcR4C and TmR4C (super indexes: Ac for the acetylated hybrid and Tm for the TAMRA labeled derivative). Again, we prepared the labeled version with the aim of following the distribution of the peptide in cellular experiments using confocal microscopy. On the other hand, the acetylated version AcR4C (Figure 30) was prepared to evaluate the influence of the fluorophore in the internalization. The synthesis of both AcR4C and TmR4C was carried out following the strategy previously described for TmAC and TmR8C. For the preparation of AcR4, the acetylation of the N-terminal group was performed under 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. Moreover, these peptide-cage vectors were purified and characterized by HPLC-MS showing that only one peptide was attached to a single cage. A) B) Héctor Fernández Caro 96 To achieve an efficient pyranine delivery in cells, first, we performed dose-response transport experiments with increasing concentrations of pyranine (Figure 37) in order to find the best concentration of carrier/pyranine for the best transport efficiency. To carry out this experiment, we decided to use the same concentration of TmR4C (5 μM) than in the previous experiment because it was shown to have good translocation efficiency at this concentration. Then, we used increasing concentrations of the pyranine (2,5 μM, 5 μM, and 10 μM). In this experiment, HeLa cells were incubated with the different concentrations in HKR buffer for 30 min at 37 ⁰C. The cells were finally washed (HKR buffer, 3x) and then observed under the confocal microscope. In this dose-response experiment, we found that TmR4C (5 µM) was able to transport pyranine to the cell cytosol at 2.5 µM and 5 µM concentration. The best transport efficiency was showed at TmR4C (5 µM) and pyranine (5 µM). Moreover, we found that when we increased the pyranine concentration to 10 μM, the transport of pyranine was inhibited and extracellular aggregation was observed (Figure 37). Figure 37. Dose-response transport experiments of pyranine in HeLa cells with TmR4C. Confocal micrographs of HeLa cells incubated with: A) TmR4C (5 µM), B) TmR4C (5 µM) and Pyranine (2.5 µM), C) TmR4C (5 µM) and Pyranine (5 µM), D) TmR4C (5 µM) and Pyranine (10 µM). DIC on the left and merge channels [Pyranine emission (green) + Tm emission (red)] on the right. Since the results of the dose-response experiment suggested that the best transport efficiency was reached at equimolar concentrations of TmR4C (5 µM) and pyranine (5 µM) in HeLa cells (Figure 37C), we decided to carry out further cell internalization experiments in a different cell line such as Vero cells, achieving also efficient cytosolic delivery of pyranine with TmR4C (Figure 38). Chapter I: Results and discussions 97 Figure 38. Pyranine transport experiments in HeLa (A) and Vero (B) cells. A) Top: confocal micrographs of HeLa cells co-incubated with pyranine (5 μM, green) and TmR4C (5 μM, red) for 30 min in HKR buffer. Nuclei were stained with Hoechst (blue). The white arrow shows the nucleolar accumulation of the peptide-cage carrier. Bottom: confocal micrographs and the orthogonal projection of the same HeLa cells showing pyranine (green) and TmR4C (red) cytosolic distribution and partial endosomal co-localization. B) Internalization of pyranine (15 μM, green) in the presence of TmR4C (15 μM) in Vero cells, after incubation for 30 min in HKR. Moreover, we carried out control transport experiments in HeLa cells using the tetraarginine peptide without the supramolecular cage C (TmR4). These experiments would allow us to confirm the key role of the supramolecular cage in the intracellular transport of pyranine. In this experiment, we followed the same procedure as before but using TmR4 instead of TmR4C. Interestingly, we found that TmR4 does not promote any pyranine internalization (Figure 39A). Therefore, we showed that the supramolecular cage has a key role to promote the pyranine transport inside of the cell. In addition, as we have previously mentioned, we also demonstrated that neither the cage C alone co-incubated with pyranine nor pyranine alone is able to enter the cellular cytosol (Figure 26B and C). Once we showed the ability of TmR4C to deliver pyranine, we studied the intracellular transport of pyranine using the acetylated version of the peptide-cage hybrid (AcR4C). Interestingly, we found that AcR4C is also able to deliver pyranine probe into the cytosol of both HeLa (Figure 39B) and Vero cells (Figure 39D). Moreover, as expected, we also showed that the acetylated peptide without the cage C (AcR4) does not promote intracellular delivery of pyranine inside cells (Figure 39C). A) B) Héctor Fernández Caro 98 After discovering the ability of both TmR4C and AcR4C to promote intracellular delivery of pyranine. We hypothesized if AcR4C would be able to capture free pyranine previously dissolved in the cellular medium and internalize this probe inside of the cells. With this purpose, we decided to carry out the sequential addition of both components, the pyranine, and then the peptide. In this experiment, we first incubated Vero cells for ten minutes only with pyranine, and then we added dropwise the peptide-cage hybrid AcR4C to Vero cells in a final concentration of 10 µM followed by an incubation for 30 minutes at 37 ⁰C. Interestingly, we found that AcR4C is able to capture and to internalize pyranine dissolved in the extracellular medium (Figure 39D). Figure 39. Experiments in HeLa (A and B) and Vero (C and D) cells. A) Confocal micrographs of HeLa cells incubated with TmR4 (5 µM) and Pyranine (5 µM). Nuclei were stained with Hoechst. DIC on the left and merge of fluorescence channels [Pyranine emission (green) + TAMRA emission (red) + Hoechst 33342 emission (blue)] on the right. B) Confocal micrographs of HeLa cells incubated with AcR4C (5 µM) and Pyranine (5 µM; green) for 30 min in HKR. C) Confocal micrographs of Vero cells incubated with Pyranine (10 µM, green) and AcR4 (10 µM) in HKR for 30 min. D) Sequential addition of pyranine and AcR4C to Vero cells. B, C, and D) DIC on the left and Pyranine emission (green) on the right. 6. Demonstration of cellular membrane integrity cytosolic delivery of pyranine Once we showed that both AcR4C and TmR4C can promote the intracellular transport of pyranine with high efficiency and reproducibility, we decided to study the mechanism of transport of the supramolecular complex inside living cells. To this aim, we carried out two different experiments using nuclear staining agents to demonstrate membrane integrity during pyranine internalization and also competition experiments. A) B) C) D) Chapter I: Results and discussions 99 6.1. Membrane integrity by nuclear staining First, we decided to demonstrate that the internalization of pyranine is not mediated by membrane permeabilization or by any cellular membrane damage. With this purpose, we carried out pyranine transport experiments but subsequently, we incubated the cells with two different dyes, DAPI or propidium iodide. Both dyes would produce nuclear staining in the case of membrane permeabilization. In the first experiment, we used DAPI (4',6-diamidino-2- phenylindole) which is a fluorescent probe with the ability to bind DNA. This dye will produce a blue (λex = 405 nm and λem = 450/50 nm) nuclear fluorescence in the case of membrane permeabilization (Figure 40A). First, we carried out an initial experiment to ensure the ability of DAPI to detect cell membrane damage. In this experiment, we treated the cells with a membrane detergent (Triton X) to cause membrane damage. Then, we incubated these cells for 30 min with 2 μM DAPI to check the dye intensity in the cellular nucleus. As expected, intense blue nuclear fluorescence was detected in all the damaged cells (Figure 40B). Figure 40. A) Chemical structure of DAPI, this dye produces nuclear staining in case of membrane damage. B) Confocal micrographs of membrane-damaged HeLa cells showing blue nuclear fluorescence of DAPI (2μM). Once we demonstrated the ability of DAPI to detect cell membrane damage. We carried out the pyranine transport experiment where we incubated pyranine (5 μM) with TmR4C (5 μM) for 30 min with HeLa cells. Cells were washed with HKR buffer several times, and subsequently, we incubated the cells for 30 min with 2 μM DAPI to check membrane integrity. We evaluated the cells under confocal microscopy and no DAPI nuclear fluorescence in the cells loaded with pyranine was found (Figure 41). Therefore, this experiment indicated that there is not permeabilization of the cell membrane during pyranine transport. Figure 41. Demonstration of membrane integrity by DAPI in pyranine-loaded HeLa cells. Confocal micrographs of HeLa cells incubated with TmR4 (5 µM) and Pyranine (5 µM), followed by washes (3x) with HKR and, then, incubated with 2 μM DAPI. Left panel: BF; second panel: pyranine (green); third panel: DAPI (blue); right panel: merge of fluorescence channels [Pyranine emission (green) + TAMRA emission (red) + DAPI emission (blue)]. A) B) Héctor Fernández Caro 100 We then confirmed by propidium iodide the membrane integrity. This probe produces red (λex = 493 nm and λem = 636 nm) nuclear staining in the case of membrane damage. In this experiment, we carried out a sequential addition of pyranine and AcR4C to Vero cells. First, cells were incubated for ten minutes with pyranine before the dropwise addition of AcR4C to a final concentration of 10 µM for 30 minutes at 37 ⁰C. Then, cells were washed with HKR several times and were incubated with propidium iodide for 10 minutes. This experiment also showed non-membrane permeabilization of pyranine-loaded cells. Interestingly, in Figure 42, we can compare a death cell with membrane damage which shows a red nucleus, with many healthy cells loaded with pyranine without fluorescence detected in the nucleus of cells. Figure 42. Demonstration of membrane integrity by propidium iodide in pyranine-loaded Vero cells. Confocal micrographs of Vero cells incubated with pyranine before the addition of AcR4C to a final concentration of 10 µM. Then, cells were washed with HKR and incubated with propidium iodide. Left panel: BF; second panel: pyranine (green); third panel: propidium iodide (red); right panel: merge of fluorescence channels [Pyranine emission (green) + Propidium iodide emission (red)]. 6.2. Membrane integrity by competition assays Subsequently, we wondered if the peptide-carrier system would be capable of selectively internalizing pyranine when co-incubated with other structurally-similar probes while maintaining the membrane integrity. With this purpose, we carried out competition experiments between pyranine and TAMRA mixed in the extracellular medium. First, we carried out two different internalization experiments with TAMRA alone and with AcR4C and TAMRA at low concentration (Figure 43). In these experiments, we demonstrated that TAMRA alone and in the presence of AcR4C is not able to cross the cell membrane (Figure 43). Moreover, we carried out a titration experiment by fluorescence to study the interaction between AcR4C and TAMRA and we found that this probe does not interact with AcR4C (Figure 43). Once we demonstrated that TAMRA alone and in the presence of AcR4C is not able to cross the plasma membrane and neither to interact with AcR4C, we decided to carry out competition experiments using TAMRA and pyranine. Therefore, if the carrier would have a high preference for pyranine, it would selectively capture and would introduce only this probe inside cells (Figure 43). Chapter I: Results and discussions 101 Figure 43. A and B) Confocal micrographs of Vero cells incubated in HKR buffer for 30 min at 37 ⁰C with: A) only TAMRA (20 µM, red channel). B) AcR4C (10 µM) in the presence of TAMRA (20 µM, red channel). Left panels correspond to DIC images. C) Fluorescence spectra of the titration of 30 nM TAMRA fluorophore with increasing equivalents (50, 100, and 200) of AcR4C. No evidence of interaction could be found. D) Schematic representation of the competition experiment showing pyranine, TAMRA, and the peptide-cage carrier (AcR4C). To carry out this experiment, we co-incubated Vero cells with AcR4C (10 μM) and with pyranine and TAMRA (20 μM each) for 30 min (Figure 44). Importantly, we found the internalization of pyranine (green fluorescence) but not TAMRA uptake (red fluorescence). This experiment demonstrated the carrier selectivity for pyranine when co-incubated with other structurally-similar probe and the absence of membrane permeabilization. Figure 44. TAMRA competition experiments in Vero cells. Confocal micrographs of Vero cells incubated in HKR for 30 min at 37 ⁰C with AcR4C (10 µM) mixed with pyranine (20 µM, green) in the presence of TAMRA (20 µM, red). Left panel: BF; second panel: TAMRA (red); right panel: Pyranine emission (green). A) B) D) C) Héctor Fernández Caro 102 7. Study of the mechanism of internalization using endocytosis inhibitors Previous confocal microscopy experiments (Figure 38) showed that beyond the general cytosolic distribution of both pyranine and carrier, a fraction of the peptide-cage carrier was able to colocalize with the probe in the endosomes. Therefore, we hypothesized that the peptidecage hybrid/pyranine complex was being internalized inside the cells following the endocytic pathway. Figure 45. Quantification of the uptake of TmR4C (15 µM) and pyranine (15 µM) in Vero cells under different conditions: 37 ⁰C (control), DYN (dynasore, 80 µM), and 4 ⁰C. A) and C) median fluorescence intensity (MFI) corresponding to pyranine fluorescence (A) or TmR4C (C). B) and D) uptake normalized to the control of TmR4C with pyranine incubated at 37 ⁰C, corresponding to pyranine (B) or TmR4C (D). The last two bars in all charts indicate the uptake of pyranine (A, B) or TmR4C (C, D) when incubated alone. Error bars indicate the SD of three replicates. To investigate the mechanism of entry of the complex, we decided to perform cell transport experiments using endocytosis inhibitors. For this, we quantified the internalization of TmR4C and pyranine by cell cytometry experiments in the presence of dynasore (DYN) endocytic inhibitor and at low temperature (Figure 45). The low-temperature experiments completely inhibit the energy-dependent uptake and dynasore is an inhibitor of macropinocytosis and all internalization pathways which depend on dynamin. Therefore, if the complex is entering inside the cells following the endocytic pathway, at low temperature and in the presence of dynasore, a reduction of the uptake of TmR4C would be shown. To carry out this experiment, we incubated Vero cells with TmR4C (15 µM) and pyranine (15 µM) and TmR4C (15 µM) alone at low temperatures, with the dynasore inhibitor, and at 37⁰C. Interestingly, we found a reduction of the uptake of TmR4C, with or without pyranine, at low temperature (4⁰C) and in the presence of dynasore (Figure 45). These results suggest that the internalization of the TmR4C and the TmR4C/pyranine complex is mediated by an energy-dependent endocytosis mechanism. Based on all previous results, we propose that the complex is entering inside the cell by an endocytosis mechanism, followed by an escape from the endosomes and, then, reach the cell A) B) C) D) Chapter I: Results and discussions 103 cytosol. The nucleolar distribution of the peptide also confirmed that the peptide is capable to escape from the endosome (Figure 38). A similar internalization mechanism for the acetylated peptide can also be proposed based on the analogous results showed by this carrier. 8. Interaction and intracellular delivery of different anionic fluorophores Once we delivered pyranine inside different cell lines using the designed peptide-cage carriers. We wondered if we would be able to internalize new fluorophores with similar structures to pyranine. We first studied the interaction between peptide-cage carriers AcR4C/TmR4C and various anionic fluorophores with potential biological interest. First, we decided to study the ability of the peptide-cage carrier (AcR4C) to interact with carboxyfluorescein (CF). With this purpose, we carried out titration experiments of this fluorophore with increasing concentrations of AcR4C in phosphate buffer at pH 7 (Figure 46). These experiments allowed the calculation of the dissociation constant Kd = 16.2 µM for AcR4C. Interestingly, when labeled, TmR4C also reduced its affinity with pyranine Kd = 51.5 µM (Figure 54). Figure 46. A) Chemical structure of carboxyfluorescein (CF). B) Fluorescence spectra of the titration of CF with AcR4C in phosphate buffer (pH 7). Arrow indicates the decrease of CF fluorescence with increasing amounts of the peptide. C) 516 nm fluorescence emission and curve fit for the titration of CF with AcR4C in phosphate buffer (pH 7). The best fit to the data using nonlinear analysis with Origin 8.5 to the Hill 1 equation. Encouraged by the interaction between CF and AcR4C, we decided to study the potential recognition of the peptide cage hybrid with different Alexa fluorophores. We carried out different titration studies with AcR4C and the different Alexa probes (Figure 47) and we found the interaction of AcR4C with all the probes with the following binding constants: Kd = 6 µM for Alexa Fluor 488, Kd = 1.37 µM for Alexa Fluor 546, and Kd = 9.5 µM for Alexa Fluor 568. A) B) C) Héctor Fernández Caro 104 Figure 47. A), D) and G): Chemical structure of Alexa Fluor 488, 546 and 568, respectively. B, E, and H: Fluorescence spectra of the titration of Alexa Fluor dyes with AcR4C. Arrows indicate the decrease of fluorophore emission with increasing amounts of AcR4C. C), F) and I) Fluorescence emission at the maximum of each fluorophore (513 nm for Alexa Fluor 488, 570 nm for Alexa Fluor 546 and 603 nm for Alexa Fluor 568), and curve fit for the titration with AcR4C in phosphate buffer (pH 7). B), C): Alexa Fluor 488; E), F): Alexa Fluor 546; H), I): Alexa Fluor 568. The best fit to the data using nonlinear analysis with Origin 8.5 to the Hill 1 equation. Once we discovered that our peptide-cage carrier could encapsulate different anionic/planar fluorescent probes, we decided to use our innovative system to transport these membrane-impermeable fluorophores to the cell interior. First, we demonstrated that AcR4C can transport carboxyfluorescein in two different cell lines such as Vero cells (Figure 48) and HeLa cells (Figure 54). To confirm the potential delivery of the Alexa probes, we incubated Vero cells with the different conditions showed in Figure 48. In these experiments, we have shown that the cage-peptide carrier was able to efficiently deliver into the cytosol of the Vero cells the different Alexa Fluor dyes (Figure 48). E) F) G) H) I) B) C) A) D) Chapter I: Results and discussions 105 Figure 48. Transport experiments of different anionic fluorophores into Vero cells. A) Emission wavelengths of the different fluorophores. B) to F) Chemical structures and pseudocolored micrographs of Vero cells incubated with the different compounds showed for 30 min and diluted in HKR buffer. B) Pyranine (10 µM) and TmR4C (10 µM). C) CF (10 µM) and TmR4C (10 µM). D) Alexa Fluor 488 (20 µM) and TmR4C (10 µM). E) Alexa Fluor 546 (35 µM) and AcR4C (15 µM). F) Alexa Fluor 568 (25 µM) and AcR4C (15 µM). In all cases, control panels indicate the incubation of the cells with the same amount of dye in the absence of the different peptide-cage hybrid. Insets show DIC images. Excitation and emission wavelengths: pyranine, CF, Alexa Fluor 488: ex = 488 nm, em = 525/50 nm; Alexa Fluor 546, Alexa Fluor 568: ex = 561 nm, em = 620/20 nm. 9. pH tracking in living cells After confirming the ability of peptide/cage hybrids to transport various anionic probes, we investigated the potential of pyranine as a pH-sensitive probe for intracellular ratiometric pH tracking. Pyranine has two ratiometric pH-dependent excitation maxima at 405 nm (protonated) and 450 nm (deprotonated).240 Interestingly, this property will allow to distinguish the pH of the different cellular organelles. As previously mentioned in the introduction, the current strategies to efficiently deliver pyranine inside cells such as physical methods have some limitations including cellular toxicity. Therefore, we decided to apply our methodology to carry out cellular pH tracking. For this, we incubated TmR4C (10 μM) and pyranine (10 μM) for 30 min with Vero cells. Interestingly, by just checking the acidic (exc = 405 nm) and the basic (exc = 488 nm) excitation wavelengths, 240 C. C. Overly, K. D. Lee, E. Berthiaume, P. J. Hollenbeck, Proc. Natl. Acad. Sci. 1995, 92, 3156-3160. A) B) E) D) C) F) Chapter I: Experimental section 113 1. Chemical structures A) TmA B) TmR8 C) TmR4 D) AcR4 Figure 50. Chemical structures of the peptides A) TmA, B) TmR8, C) TmR4, D) AcR4. O O H N O N H O H N NH H2NNH2 O N H O H N NH H2NNH2 O N H O H N NH H2NNH2 O N H O H N NH H2NNH2 O N H O O N N O O O H NOH O NH NH2 H2N NH NH2 H2N NH NH2 H2N NH NH2 H2N O O H N O N H O N H O H N NH H2NNH2 O N H O H N NH H2NNH2 O O N N O O O H NOH O NH NH2 H2N NH NH2 H2N O O H N O N H O N H O H N NH H2NNH2 O N H O H N NH H2NNH2 O OH O NH NHHN NH NHH2N Héctor Fernández Caro 114 A) TmAC B) TmR8C C) TmR4C D) AcR4C O O H N O N H O N H O H N NH H2NNH2 O N H O H N NH H2NNH2 O O N N O O O H N N N N HN N N O NH NH2 H2N NH NH2 H2N Figure 51. Chemical structures of the peptide-cage hybrids A) TmAC, B) TmR8C, C) TmR4C, D) AcR4C. Chapter I: Experimental section 115 2. Figures Figure 52. Viability assay in Vero cells with pyranine co-incubated in HKR with TmR4C (A, D) or AcR4C (B, E) and only pyranine (C, F) at different concentrations after 1 hour incubation time (A, B, C) and after 24 hours incubation time (D, E, F) at 37 ºC. Error bars represent the standard deviation of four replicates. Figure 53. A) Fluorescence titration of pyranine with the peptide/cage AcR4C in phosphate buffer (pH 7). Arrow indicates the decrease of pyranine fluorescence with increasing amounts of the peptide. B) 510 nm fluorescence emission and curve fit for the titration of pyranine with AcR4C in phosphate buffer pH 7. The best fit to the data using nonlinear analysis with Origin 8.5 to the Hill 1 equation. In B, fitting was done with a fixed value of n = 1, and Kd for AcR4C under these conditions was 149 nM. The small peak observed in A) at about 480 nm at the higher concentrations of peptide corresponds to the Raman scattering of water for an excitation wavelength of 415 nm. A) B) A) D) B) C) E) F) Héctor Fernández Caro 116 Figure 54. Transport experiments of Carboxyfluorescein (CF) in HeLa cells. Confocal micrographs of HeLa cells incubated with: A) TmR4C (5 µM) and Carboxyfluorescein (5 µM). B) TmR4 (5 µM) and Carboxyfluorescein (5 µM). DIC on the left and merge channels [Carboxyfluorescein emission (green) + Tm emission (red)] on the right. HeLa cells were incubated with the different components in the HKR buffer for 30 min at 37 ºC. The cells were finally washed (HKR buffer, 3x) and then observed under the confocal microscope. Figure 55. A) Fluorescence spectra of the titration of CF with TmR4C in phosphate buffer (pH 7). Arrow indicates the decrease of CF fluorescence with increasing amounts of the peptide. The right peak in C corresponds to the TAMRA emission from TmR4C. B) 516 nm fluorescence emission and curve fit for the titration of CF with TmR4C in phosphate buffer (pH 7). The best fit to the data using nonlinear analysis with Origin 8.5 to the Hill 1 equation. B) A) A) B) Chapter I: Experimental section 117 Figure 56. A) Absorption spectra of pyranine in buffers of different pH. B) The ratio of the absorbance at 450 and 405 nm plotted as a function of pH. C) Ratiometric method for determining the pKa. pKa = 7.01. A) B) C) Héctor Fernández Caro 118 Figure 57. pH studies in Vero cells. A) Ratiometric imaging after pH clamping with nigericin of Vero cells incubated with 10 µM pyranine and 10 µM TmR4C. The top row (BF) shows bright-field images. The second (PyrH) and third (Pyr-) rows show the emission of pyranine after excitation at 405 nm or 488 nm, respectively. Finally, the bottom row shows the processed images. B) Mean ratio values were obtained for each field and plotted against pH. Box and whisker plot of each dataset, green points represent individual observations. C) Logarithmic conversion of the previous data and linear fit. Error bars indicate SD. A) B) C) Chapter I: Experimental section 119 3. Materials and methods Chemicals were purchased from Carbosynth, Iris Biotech, Sigma Aldrich, Alfa Aesar, Stream Chemicals, and Novabiochem and used without further purification. Commercially available 2-Chlorotrityl chloride resin, Fmoc-Ahx-OH, Fmoc-O2Oc-OH, Fmoc-L-Ala-OH, Fmoc-L-Leu-OH, Fmoc-L-Arg(pbf)-OH, triisopropylsilane (TIS), Disopropylethyl amine (DIEA), Nigericin and 8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (pyranine) were obtained from Sigma-Aldrich. MTT and MES (2-(N- morpholino)ethanesulfonic acid) were purchased from Alfa Aesar; 5(6)-Carboxyfluorescein (CF) and 5-Carboxytetramethyl rhodamine (TAMRA) were available from Carbosynth. L-α- phosphatidylglycerol (Egg, Chicken) (sodium salt) was purchased from Avanti Polar Lipids and N-HATU from Glentham life sciences. N-HBTU was obtained from Iris. Hoechst 33342 Trihydrochloride Trihydrate, Alexa Fluor dyes (488, 546 and 594), and Dulbecco’s Modified Eagle’s Medium (4500 mg/L glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate) were purchased in ThermoFisher. HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)] was purchased from TCI Chemicals. The solvents for organic synthesis were of reagent grade. Dry solvents were bought from Sigma-Aldrich. N, N-dimethylformamide, and trifluoroacetic acid were purchased from Scharlau, dichloromethane from Panreac, and acetonitrile from Merck. Water was deionized and purified on a Millipore Milli-Q Integral system. The removal of solvents under reduced pressure was carried out on a rotary evaporator Büchi R-210 equipped with a thermostated bath B-491, a vacuum regulator V-850, and a vacuum pump V-700. Purification of products was accomplished using reversed-phase high-performance liquid chromatography (RP-HPLC) using an Agilent Technologies 1160 Infinity using H2O (+ 0.1% TFA) and CH3CN (+ 0.1% TFA) as eluents and a Luna (C18)-Phenomenex column and on Jasco LC-4000 with an Agilent Eclipse XDB-C18 column. High-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) analyses were carried out on Agilent Technologies 1260 Infinity II associated with a 6120 Quadrupole LC-MS using an Agilent SB-C18 column or on DIONEX Ultimate 3000 U-HPLC+ (Thermo Scientific) with an Acclaim RSLC 120-C18 column with Solvent A: Solvent B gradients between 5:95 (Solvent A: H2O with 0.1% TFA; Solvent B: CH3CN with 0.1% TFA). Fluorescence measurements were performed using a Varian Cary Eclipse fluorometer and UV-Vis spectra were measured in an Agilent 8453 UV-Vis diode-array spectrophotometer or a Biochrom Libra S60 UV-vis spectrophotometer. For taking confocal microscopy images a Dragonfly confocal spinning-disk system mounted on a Nikon Eclipse Ti-E equipped with an Andor Zyla 4.2 PLUS sCMOS digital camera was used. The 3D reconstructions were obtained from the different individual confocal planes with Imaris bitplane 9.0.0 software. A Tecan Infinite F200Pro microplate reader was used to measure directly in Costar cell culture 96-well plates UV-Vis absorbance for the MTT viability assays. Flow cytometry was performed on a Guava easyCyteTM cytometer. Data analysis was performed with InCyte software included in GuavaSoft 3.2 (Millipore). The “U-tubes” were house-made. Héctor Fernández Caro 120 4. General protocol for synthetic procedures Peptides TmA, TmR8, TmR4, and AcR4 were synthesized according to classic SPPS of peptides and further details are given below. The cage C was prepared and purified following the procedure reported in literature.69,241 4.1. Synthesis and characterization of the peptides TmA, TmR8, TmR4 and AcR4 All peptides were synthesized by manual Fmoc solid-phase peptide synthesis on a 2- Chlorotrityl chloride resin (1.14 mmol/g). The first coupling was performed in CH2Cl2 using DIEA as base whereas for the following couplings N-HBTU as the activator, DIEA as the base, and DMF as solvent were used. The deprotection of the temporal Fmoc protecting group was performed by treating the resin with 20% piperidine in DMF. 5(6)- carboxytetramethylrhodamine (TAMRA) was coupled using 3 equivalents (0.15 mmol, 64.5 mg), 3 equivalents of N-HATU, and 5 equivalents of DIEA 0.2 M in DMF for 60 min. Acetylation of the N-terminal group of AcR4 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, 2 mL) for 30 min. The cleavage/deprotection step was performed by treatment of the resin-bound peptide for 2 h with the following cleavage cocktail: 900 μL TFA, 50 μL CH2Cl2, 25 μL H2O and 25 μL TIS (1 mL of cocktail/40 mg resin) and peptides were precipitated in Et2O. 4.1.1. Synthesis and characterization of peptide TmA The synthesis of the peptide TmA (TAMRA-Ahx-R-R-L-R-R-L-L-R-R-L-L-R-A-Ahx- O2Oc-OH) was performed following the above methodology. TmA was obtained after RPHPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (5→35 min)] with an overall yield of 12%. Rt 8.0 min [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (0→12 min)] (Figure 58). MS (ESI, H2O) m/z: calcd for C118H204N39O23 [M+5H]5+: 507.3, found: 507.5; calcd for C118H203N39O23 [M+4H]4+: 633.9, found 634.0; calcd for C120H203N39O25F3 [M+3H+TFA]3+: 882.9, found: 883.0; calcd for C122H204N39O27F6 [M+3H+2TFA]3+: 920.9, found: 920.9; calcd for C122H203N39O27F6 [M+2H+2TFA]2+ : 1380.8, found: 1380.7. 69 J. Mosquera, S. Zarra, J. R. Nitschke, Angew. Chem. Int. Ed. 2014, 53, 1556-1559. 241 J. Rodríguez, J. Mosquera, J. R. Couceiro, J. R. Nitschke, M. E. Vázquez, J. L. Mascareñas, J. Am. Chem. Soc. 2017, 139, 55-58. Chapter I: Experimental section 121 Figure 58. Representation of the HPLC-MS for TmA. A) HPLC chromatogram (λabs = 550 nm) of TmA and B) ESI-MS recorded at 8.0 min of TmA. 4.1.2. Synthesis and characterization of peptide TmR8 The synthesis of the peptide TmR8 (TAMRA-Ahx-R-R-R-R-R-R-R-R-Ahx-O2Oc-OH) was performed following the above methodology. TmR8 was obtained after RP-HPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (5→35 min)] with an overall yield of 12%. Rt 10.9 min [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 75:5→5:75 (0→21 min)] (Figure 59). MS (ESI, H2O) m/z: calcd for C91H156N37O18 [M+5H]5+: 411.0, found: 411.3; calcd for C93H156N37O20F3 [M+4H+TFA]4+: 542.1, found: 542.3; calcd for C93H155N37O20F3 [M+3H+TFA]3+: 722.4, found: 722.9; calcd for C95H156N37O22F6 [M+3H+2TFA]3+: 760.4, found: 760.6; calcd for C97H156N37O24F9 [M+2H+3TFA]2+: 1197.1, found: 1197.6. Figure 59. Representation of the HPLC-MS for TmR8. A) HPLC chromatogram (λabs = 550 nm) of TmR8 and B) ESI-MS recorded at 10.9 min of TmR8. 4.1.3. Synthesis and characterization of peptide TmR4 The synthesis of the peptide TmR4 (TAMRA-Ahx-R-R-R-R-Ahx-O2Oc-OH) was performed following the above methodology. TmR4 was obtained after RP-HPLC purification [Phenomenex Luna C18(2) 100A column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 95:5→5:95 (5→35 min)] with an overall yield of 12%. Rt 11.1 min [RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/ CH3CN (0.1% TFA) 75:5→5:75 (0→21 min)] (Figure 60). MS (ESI, H2O) A) B) A) B) Héctor Fernández Caro 128 8. Cell transport experiments HeLa or Vero cells seeded the day before on glass-bottom dishes were washed with HEPES-Krebs-Ringer (HKR) buffer (5 mM HEPES, 137 mM NaCl, 2.68 mM KCl, 2.05 mM MgCl2, 1.8 mM CaCl2, pH 7.4) and incubated for 30 min with 1 µM Hoechst 33342 (ThermoFisher) in HKR to stain the nucleus. This solution was removed and cells were incubated for another 30 min with pyranine (or other fluorescent probes such as TAMRA, CF, or Alexa Fluor dyes) in combination with the peptide, cage, or peptide-cage hybrids at the concentrations indicated in the legend of the figures. After this incubation time, cells were washed twice with HKR and examined on the confocal microscope. In some cases, the step of nuclear staining with Hoechst was omitted. To check membrane integrity, after incubation with the complexes, cells were washed with HKR and further incubated with 2 µM of DAPI diluted in HKR buffer for 30 min or 0.5 µg/mL of propidium iodide for 10 min, before washing with HKR and imaging. To investigate the possibility of the in situ capture of the pyranine into the cage of the carrier, Vero cells were incubated with 12.5 µM pyranine in HKR for 10 min before adding, dropwise, a solution of AcR4C in HKR to a final concentration of both peptide and pyranine of 10 µM and incubated for 30 min at 37 °C before imaging. 9. In vitro pH measurements To confirm pH sensitivity of the dye in the calibration buffers used for in situ calibrations (10 mM MES, 10 mM HEPES, 20 mM glucose, 1 mM CaCl2, 1 mM MgCl2, 135 mM KCl, 20 mM NaCl; pH adjusted with KOH), absorption spectra of pyranine in the different buffers were obtained. Pyranine was diluted at 70 µM in each buffer and absorbance between 330-510 nm was measured in a Libra S60 spectrophotometer. The ratio between absorbances at 450 and 405 nm were calculated, and then transformed using the following equation: 𝑙𝑜𝑔10((𝑅𝑚𝑎𝑥−𝑅 𝑅−𝑅𝑚𝑖𝑛)·(𝐴405 𝑛𝑚 𝑏𝑎𝑠𝑖𝑐 𝐴405 𝑛𝑚 𝑎𝑐𝑖𝑑𝑖𝑐 )) in which R represents the ratio A450/A405, Rmax and Rmin the maximal and minimal ratio values, and Abasic405 and Aacidic405 the absorbance of the pyranine at the highest and lowest pH tested. Data were fitted to a linear model. Intercept (7.01) corresponds to the pKa under these conditions. 10. pH studies in cells For pH ratiometric measurements, Vero cells were incubated with 10 µM pyranine and 10 µM TmR4C peptide for 30 min in HKR buffer. Cells were then washed twice with HKR and imaged. A control with no pyranine was also prepared to quantify the background. To prepare a calibration curve, cells were first incubated with pyranine and TmR4C under standard conditions (30 min, pH = 7.4). Subsequently, the cells were washed twice with high-potassium pH calibration buffers (10 mM MES, 10 mM HEPES, 20 mM glucose, 1 mM CaCl2, 1 mM MgCl2, 135 mM KCl, 20 mM NaCl; pH adjusted with KOH) at each indicated pH. The cells were then incubated at the corresponding pH for 20 min with the calibration buffers containing 10 µg/mL of the ionophore nigericin for pH clamping before imaging. Images were acquired Chapter I: Experimental section 129 with a Zyla 4.2 PLUS camera mounted on a Dragonfly spinning disk confocal microscope (Andor), by excitation with 405 nm (protonated form, PyrH) and 488 nm (deprotonated form, Pyr-) lasers, and detecting the fluorescence at 500-550 nm. Images from the pyranine channels were processed with FIJI, 248 as follows: the background was subtracted from both channels and then image 488 was divided by image 405. Mean values per field were obtained (Figure 57B) and data was linearized by logarithmic conversion and adjusted to a linear model (Figure 57C). The linear fit was then used to assign estimated values of pH to the images. 11. Flow cytometry To further investigate the uptake mechanisms of the supramolecular complex formed by pyranine and TmR4C, Vero cells seeded the day before at 10.000 cells/well of a 96-well plate, were treated for 30 min with dynasore (80 µM) diluted in DMEM without serum or antibiotics. Cells were then washed with HKR and incubated with 15 µM TmR4C and 15 µM pyranine for 30 min at 37 °C with the same concentration of the inhibitor in HKR buffer. For the incubation at low temperature (4 °C), cells were placed on ice before the incubation, and ice-cold solutions were used for the washes and incubations. Controls with 15 µM TmR4C alone and 15 µM pyranine alone were also performed. After 30 min of incubation, cells were washed with HKR and trypsinized. Trypsin was neutralized with 2 % FBS in PBS with 5 mM EDTA and cell fluorescence was measured on a Guava EasyCyteTM cytometer using two lasers: a blue laser (488 nm) with emission collected at 512/18 nm (pyranine) and a green laser (532 nm) collecting the emission at 575/25 nm (TAMRA). Cells with typical FSC and SSC parameters were selected and the median fluorescence intensity (MFI) was calculated for each sample. Each condition was done in triplicate. Fluorescence values were normalized to the uptake of each untreated control (100%) after blank subtraction. In all cases, data analysis was performed with InCyte software included in GuavaSoft 3.2 (Millipore). 12. Cell viability Cell viability was measured by MTT assay. To evaluate the toxicity of the treatment of cells with TmR4C, AcR4C, and pyranine, Vero and HeLa cells were submitted to an MTT assay 1 hour and 24 h after the incubation. One day before the assay, a suspension of HeLa and Vero cells were plated in 96-well tissue culture plates by adding 100 µL (∼10.000 cells) per well. The next day, the medium was removed and cells were incubated in HKR in the presence of TmR4C, AcR4C, and pyranine at different concentrations (50 µL/well) during 1 hour of incubation at 37 °C. After the incubation, HKR with the compounds was removed and prewarmed DMEM containing 10 % FBS was added to the wells. To evaluate the toxicity at 1 hour, MTT (5 mg/mL in PBS, 10 µL/well) was added and the cells were further incubated for 4 h. To evaluate the toxicity at 24 h, cells were incubated for 24 h before adding the MTT. The cells were further incubated for 4 h in the presence of MTT. The supernatant was removed and the water-insoluble formazan salt was dissolved in DMSO (100 µL/well). The absorbance at 570 nm was measured. Data points were collected in triplicate and expressed as normalized values for untreated as control cells (100%) after blank subtraction. 248 J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, et al., Nat. Methods 2012, 9, 676-682. Chapter II: Peptide integration and exchange into AuNPs mediated by host-guest molecular recognition Introduction Chapter II: Introduction 135 1. Nanoparticles as a tool for nanomedicine Since the advent of Nanotechnology, nanosystems with diagnostic and/or therapeutic capabilities have been regarded as a new promising paradigm that could improve in a significant manner therapeutical problems such as cancer. 249 This has led to a vast research effort in order to understand how fundamental physicochemical properties, such as size, charge, and chemical composition, can be addressed in order to improve the pharmacokinetic capabilities and biodistribution. 250 In fact, many limitations, such as limited delivery to target tissues are still major hurdles for the development of safe and efficient therapies. 251 Regardless of these difficulties, several nanomedicines are currently on different stages of clinical trials. Up to 2016, the U.S. Food and Drug Administration had approved 51 nanomedicines, with 77 products in clinical trials. 252 An analysis of the nanocarrier category revealed that soft nanoparticles (e.g. micelles, polymers) had more importance in comparison with hard carriers (e.g. metallic nanoparticles). At first sight, this data suggest some inertia towards the successful use of nanoparticle systems in real therapeutic applications, but also it might reflect a pluralistic approach towards the delivery problem, i.e. a single vehicle might not be well suited for different delivery therapeutic problems. Therefore, it seems plausible that different strategies towards the preparation of nanosystems could expand the possible applications in nanomedicine. In this context, the diversity of structure and function accessible through supramolecular assembly holds promise as a versatile tool for the preparation of nanomedicine tools, including micellar, nanotubular, or nanoparticle-like systems.188 Here we will focus on the use of hostguest recognition and its integration on nanosystems intended for therapeutic applications. 2. Nanoparticles built up by using host-guest molecular recognition The use of host-guest molecular recognition offers the possibility to functionalize or even fully assemble nanoparticles by stable, yet reversible interactions. In the latter case, the term supramolecular nanoparticles (SNPs) has often been coined to indicate the formation of particles of nanometric size built fundamentally by non-covalent interactions. One of the simplest examples of the preparation of SNPs is the condensation, driven by electrostatic interactions, between polyanions (e.g. DNA) and positively charged lipids or polymers, which have been traditionally used for DNA delivery. 253 On the other hand, supramolecular interactions can be used for the reversible functionalization with motifs that impart certain functionality. The scaffolds used to build these systems can be either soft or hard, which have important consequences in their physicochemical properties and the therapeutic function. Here we will discuss examples of nanoparticles that integrate host-guest elements according to this classification. 249 R. van der Meel, E. Sulheim, Y. Shi, F. Kiessling, W. J. M. Mulder, T. Lammers, Nat. Nanotechnol. 2019, 14, 1007-1017. 250 B. Li, L. A. Lane, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2019, 11, e1542. 251 S. Wilhelm, A. J. Tavares, Q. Dai, S. Ohta, J. Audet, H. F. Dvorak, W. C. W. Chan, Nat. Rev. Mater. 2016, 1, 16014. 252 D. Bobo, K. J. Robinson, J. Islam, K. J. Thurecht, S. R. Corrie, Pharm. Res. 2016, 33, 2373-2387. 188 M. J. Webber, R. Langer, Chem. Soc. Rev. 2017, 46, 6600-6620. 253 O. Boussif, F. Lezoualc’h, M. A. Zanta, M. D. Mergny, D. Scherman, B. Demeneix, J. P. Behr, Proc. Natl. Acad. Sci. 1995, 92, 7297-7301. Héctor Fernández Caro 136 2.1. Soft supramolecular nanoparticles for delivery of therapeutics based on host-guest molecular recognition 2.1.1. Delivery of therapeutic nucleic acids A particularly successful example of SNPs that has moved from fundamental research to clinical trials is the work of Mark E. Davis group. Since the end of the 90´s, this group has developed targeted SNPs that can deliver small interfering RNA (siRNA) to inhibit tumor growth.254 These SNPs consist of three different building blocks (Figure 66A): a linear cyclodextrin-containing polymer (CDP), an adamantane-polyethylene glycol conjugate (Ad- PEG), and the targeting moiety transferrin linked to an adamantane moiety (Ad-PEG-Tf). The self-assembly of these three components into a hybrid polymer is driven by host-guest processes. The resulting conjugate is mixed with siRNA, which is subsequently captured by electrostatic interactions between the positively charged polymer and the negatively charged nucleic acid (Figure 66B). This four-component cocktail finally yields soft nanoparticles of around 70 nm size. The vehicle remained stable into the bloodstream and was able to enter into tumor tissues due to the EPR effect. Enhanced delivery efficiency in tumor cells was achieved due to the recognition between anchored transferrin and its receptor (TfR) which is overexpressed in certain types of tumor cells (Figure 66C). 254 , 255 This therapeutic product named CALAA-01 was evaluated in phase I clinical trials in human patients with solid tumors, administrated by intravenous systemic dosing. Although some adverse effects were observed (fatigue, chill), these studies indicated that this nanosystem could be safely used in humans. 256 This illustrative example was the inspiration for intensive research in the area of gene delivery by using host-guest systems. While Davis group work relied on the incorporation of the host into cationic polymers, many groups have exploited the chemical functionalization of the host as a way for designing homogeneous gene delivery vectors, which are in consequence “preorganized” towards interactions with nucleic acids. 257 In this context, remarkable examples include calixarenes 258 and cyclodextrins 259 hosts. In the examples above, the size of the soft nanoparticles was a consequence of the interaction between the nucleic acid and the host polymer/molecules, and no size-tuning was reported. However, control over nanoparticle size is desirable, since many cell-nanoparticle interactions are strongly influenced by their contact surface, (e.g. determining the number of receptor-antigen interactions). 260 Remarkably, the group of prof. Tseng developed sizecontrollable SNPs based on the self-assembly of three different molecular building blocks (Figure 66D): i) multivalent host poly(ethylene imine) functionalized with β-CD (CD-PEI), ii) multivalent guest, Ad-poly(amidoamine) dendrimer (Ad-PAMAM) and iii) monovalent guest linked to a steric stabilizer, Ad-PEG. The beauty of this work is that tuning of the nanoparticle size is achieved simply by changing the mixing ratio of the different building blocks. Importantly, it was shown that this strategy can be a promising strategy for the preparation of 254 M. E. Davis, Mol. Pharm. 2009, 6, 659-668. 255 M. E. Davis, J. E. Zuckerman, C. H. J. Choi, D. Seligson, A. Tolcher, C. A. Alabi, Y. Yen, J. D. Heidel, A. Ribas, Nature 2010, 464, 1067-1070. 256 J. E. Zuckerman, M. E. Davis, Nat. Rev. Drug Discov. 2015, 14, 843-856. 257 C. Ortiz Mellet, J. M. Benito, J. M. García Fernández, Chem. Eur. J. 2010, 16, 6728-6742. 258 F. Sansone, M. Dudi, G. Donofrio, C. Rivetti, L. Baldini, A. Casnati, S. Cellai, R. Ungaro, J. Am. Chem. Soc. 2006, 128, 14528-14536. 259 L. Gallego-Yerga, J. M. Benito, L. Blanco-Fernández, M. Martínez-Negro, I. Vélaz, E. Aicart, E. Junquera, C. Ortiz Mellet, C. Tros de Ilarduya, J. M. García Fernández, Chem. Eur. J. 2018, 24, 3825-3835. 260 N. Hoshyar, S. Gray, H. Han, G. Bao, Nanomedicine 2016, 11, 673-692. Chapter II: Introduction 137 NPs with sizes between 30 nm and 100 nm and with a good polydispersity. Interestingly, the size of the SNPs was easily tuned by increasing the quantity of multivalent guest, while keeping constant the concentration of the host and the stabilizer. 261 The control over the assembly and subsequent size distribution were further tuned by modulation of the mixing regimes in microfluidic chips. 262 , 263 These soft SPNs were used for the intracellular delivery of DNA. SPNs combined with DNA and the targeting ligand RGD (Figure 66E) were used to efficiently deliver DNA inside living cells. 264 Moreover, using this strategy, an intact artificial transcription factor (TF) (GAL4-VP16) was encapsulated into SPNs leading to the targeted delivery of an intact TF. In this work, a DNA plasmid (pG5E4T-Fluc) equipped with five tandem copies of GAL4- VP16 matching recognition sequences (Kd ≈ 10 nM) was used to improve the incorporation of the TF into the SPN by the formation of an anionic TF/DNA complex. 265 Further examples of the use of these platforms in delivery include the controlled release of the anticancer drug camptothecin 266 or intradermal release of ketoconazole -an antifungal drugin mice models. 267 261 H. Wang, S. Wang, H. Su, K.-J. Chen, A. L. Armijo, W.-Y. Lin, Y. Wang, J. Sun, K. Kamei, J. Czernin, et al., Angew. Chem. Int. Ed. 2009, 48, 4344-4348. 262 K. Liu, H. Wang, K.-J. Chen, F. Guo, W.-Y. Lin, Y.-C. Chen, D. L. Phung, H.-R. Tseng, C. K. F. Shen, Nanotechnology 2010, 21, 445603. 263 K. Liu, Y.-C. Chen, H.-R. Tseng, C. K.-F. Shen, R. M. van Dam, Microfluid. Nanofluidics 2010, 9, 933-943. 264 H. Wang, K.-J. Chen, S. Wang, M. Ohashi, K. Kamei, J. Sun, J. H. Ha, K. Liu, H.-R. Tseng, Chem. Commun. 2010, 46, 1851-1853. 265 Y. Liu, H. Wang, K. Kamei, M. Yan, K.-J. Chen, Q. Yuan, L. Shi, Y. Lu, H.-R. Tseng, Angew. Chem. Int. Ed. 2011, 50, 3058-3062. 266 K.-J. Chen, L. Tang, M. A. Garcia, H. Wang, H. Lu, W.-Y. Lin, S. Hou, Q. Yin, C. K. F. Shen, J. Cheng, et al., Biomaterials 2012, 33, 1162-1169. 267 F. Wang, P. Yang, J. Choi, P. Antovski, Y. Zhu, X. Xu, T.-H. Kuo, L.-E. Lin, D. N. H. Kim, P.-C. Huang, et al., ACS Nano 2018, 12, 6851-6859. Héctor Fernández Caro 144 work (Figure 68C), multifunctional CD@AuNPs for targeted cancer therapy were constructed by the incorporation of both adamantane-hydrazone-doxorubicin (Ad-Hyd-DOX) and adamantane-modified-PEG8-GRGDS (Ad-PEG8-GRGDS), playing the roles of anticancer drug and targeting moiety respectively. 302 The GRGDS peptide sequence has shown the ability to target cancer cells that overexpress αvβ3 integrin.264 On the other hand, the inclusion of the hydrazone bond enabled the proton-mediate hydrolysis of the linker, resulting in the release of the drug at acidic pH values. The authors showed that these multifunctional nanoparticles were able to be selectively internalized by cancer cells via receptor-mediated endocytosis. Moreover, this nanosystem showed cancer cell apoptosis due to the release of doxorubicin at lysosomal pH values. Overall, one of the most remarkable features of this work is the exclusive use of host-guest molecular recognition and the use of a pH-responsive therapeutic agent.302 302 W.-H. Chen, Q. Lei, G.-F. Luo, H.-Z. Jia, S. Hong, Y.-X. Liu, Y.-J. Cheng, X.-Z. Zhang, ACS Appl. Mater. Interfaces 2015, 7, 17171-17180. 264 H. Wang, K.-J. Chen, S. Wang, M. Ohashi, K. Kamei, J. Sun, J. H. Ha, K. Liu, H.-R. Tseng, Chem. Commun. 2010, 46, 1851-1853. Chapter II: Introduction 145 Figure 68. A) Left. Schematic and structural representation of the ferrocene dimer and the perthiolated cyclodextrin attached to the surface of the AuNPs. Right. Formation of the supramolecular network between ferrocene dimer and cyclodextrin-functionalized AuNPs.290 B) Schematic representation of the preparation of non-targeted and targeted CD@AuNPs for β-Lapachone intracellular delivery. Incorporation of building blocks into AuNPs i) SH-CD and PEG-SH ii) β-Lapachone, iii) SH-CD, PEGSH, and NHS-PEG-SH, iv) anti-EGFR and v) β-Lapachone.298 C) Schematic representation of building blocks and self-assembly of multifunctional host-guest CD@AuNPs for targeted anticancer therapy, internalization mechanism, and pH-mediated intracellular release of doxorubicin.302 A) B) C) Héctor Fernández Caro 146 2.2.2. Achieving therapeutic response by changes in physical/catalytic properties In addition to the incorporation of bioactive drugs, the use of hard nanoparticles decorated with host cavities can additionally exploit supramolecular processes in order to regulate a therapeutic response based on the physical or catalytic properties of the nanoparticles. A pioneering report of Rotello’s group showed how dynamic exchange of guests in living cells could be used for triggering a cytotoxic response.303 In particular, diaminohexanefunctionalized gold nanoparticles were capped with CB[7] guests. These particles were readily internalized in MFC-7 cells. Addition of 1-adamantylamine -a guest with a higher binding affinity towards CB[7] in comparison to diaminohexane- to the cell media triggered adamantane uptake and guest exchange in cells. Uncapped gold nanoparticles were able to promote endosomal escape and induce cell death. 303 Sketching a similar concept, the use of redoxresponsive guests have been exploited to trigger intracellular nanoparticle aggregation.304 In this report, CD-coated gold nanoparticles and oxidized divalent ferrocene crosslinkers were codelivered to HepG2 cells. Flow cell cytometry revealed increased cell toxicity when nanoparticles and crosslinkers were delivered at the same time in comparison with the individual components. As a mechanistic proposal, the authors claimed that intracellular Fc+ was reduced in the presence of intracellular glutathione, initiating nanoparticle aggregation by crosslinking. Quantitative ICP-MS revealed that the presence of the crosslinker resulted in higher Au retention in cells. This accumulation could induce apoptosis in cells. 304 On the other hand, Rotello’s group has shown the regulation of the enzymatic activity of nanoparticle based-nanoreactors.305 The design is composed of AuNPs coated with N-Benzyl- N,N-dimethyloligoethylenglycol ligands. These can encapsulate transition metal catalysts and hold them by supramolecular capping between CB[7] hosts and the ligands. Catalysts can be exposed to the media by releasing the host cap via competition with endocytosed adamantylamine. Once liberated, the catalytic activity is activated, which was demonstrated by the N-deacylation of an N-alkylated 5-fluorouracil prodrug. This triggered the toxic response of the drug. 305 303 C. Kim, S. S. Agasti, Z. Zhu, L. Isaacs, V. M. Rotello, Nat. Chem. 2010, 2, 962-966. 304 Y. Wang, H. Li, Q. Jin, J. Ji, Chem. Commun. 2016, 52, 582-585. 305 G. Y. Tonga, Y. Jeong, B. Duncan, T. Mizuhara, R. Mout, R. Das, S. T. Kim, Y.-C. Yeh, B. Yan, S. Hou, et al., Nat. Chem. 2015, 7, 597-603. Objectives Chapter II: Objectives 149 The use of metallic host-guest based-nanoparticles as motifs for nanotherapeutic applications has raised a great deal of interest. In this sense, the reversibility of the host-guest complexes has been traditionally used in the solubilization and integration of different hydrophobic drugs into nanoparticle systems. However, most of these strategies have been focused on the release and exchange of hydrophobic hosts cargos and, to the best of our knowledge, the release and dynamic exchange between host decorated nanoparticles and hydrophilic peptide guests has not been explored. Here, a potential challenge is the chemical complexity of peptides, which might promote unspecific interactions by electrostatic or van der Waals forces with NPs or any other component. On the other hand, the coating of nanoparticle surfaces with hydrophilic (cationic or anionic) peptides has been mostly approached by using covalent and electrostatic coating protocols, while examples using supramolecular recognition are scarce, with little information reporting the effect of progressive coating to control nanoparticle size and surface potential. This is essential to understand the critical parameters that determine nanoparticle stability. Therefore, in this second chapter, we will explore the incorporation of different guest-bearing hydrophilic peptides, such as polyarginine or oligoglutamic peptides, into β-cyclodextrin-functionalized gold nanoparticles (β-CD@AuNPs) driven by host-guest molecular recognition (Figure 69). Moreover, we will construct multicomponent nanoparticles with the incorporation of hydrophilic stabilizers such as polyethylene glycol chains. Finally, we will show the facilitated uptake of membrane-impermeable hydrophilic peptides driven by the formation of supramolecular peptide/β-CD@AuNP hybrids. We hypothesized that the functional dynamic guest exchange of hydrophilic biomolecules would be possible inside living cells based on the higher affinity of a multivalent guest, exploiting either statistical rebinding or multivalent binding processes. The specific objectives of this chapter would be:  The synthesis of β-CD@AuNPs by incorporation of per-thiolated-β-CD at the AuNP surface and their characterization by TEM, DLS, UV-Visible, and TGA experiments.  The synthesis of a small library of oligoarginine peptides bearing mono- (AdR4 and AdR8) or divalent (Ad2R4 and Ad2R8) adamantane guests attached. We will exploit the alcoxyamine-aldehyde condensation between peptide and guest respectively. Furthermore, mono- (AdPEG) and divalent (Ad2PEG) PEG moieties will be also prepared following a similar strategy.  The integration of these elements into multicomponent nanoparticles and their characterization by size and surface potential measurements. The later will be used to quantify the binding constants between functionalized peptides and β-CD@AuNPs.  The exploration of the in cellulo guest-bearing peptide exchange due to affinity differences based on guest valency. This will be carried out by first assessing the peptide exchange in vitro using fluorescence spectroscopy and later inside living cells by imaging (confocal microscopy experiments) and cell cytometry assays. Héctor Fernández Caro 150 Figure 69. A) Retrosynthetic strategy for the preparation of AdPEG exploiting the alcoxyaminealdehyde condensation between PEG and guest respectively. B) Guest pool composed of monovalent/divalent CPPs (tetra and octarginine based), monovalent/divalent PEG moieties, and monovalent model cargo oligoglutamic peptide (AdE9). C) Schematic representation of the incorporation of a monovalent peptide cargo AdE9 into β-CD@AuNP leading to the formation of the peptide/β-CD@AuNP supramolecular complex and dynamic exchange between guests according to their valence. B) C) A) Results and discussions Chapter II: Results and discussions 153 1. Synthesis and characterization of β-cyclodextrin-functionalized gold nanoparticles We started this project by optimizing the conditions for the preparation of the NPs. The β- CD@AuNPs were prepared following a slightly modified methodology described in the literature. 306 In our procedure, we added a solution of NaBH4 (reducing agent) and perthiolated-β-cyclodextrin 307 in dry DMSO to a solution of HAuCl4 in dry DMSO (Figure 70). During this period, the Au(III) cations were reduced to elementary, coalescing Au(0). Then, nanoparticles were precipitated by adding acetonitrile and collected after centrifugation. Finally, the particles were suspended in water and freeze-dried. Figure 70. Synthetic pathway for the preparation of β-CD@AuNPs. To characterize the β-CD@AuNPs, we carried out a range of measurements intended to provide information about the physical properties of the particles, as well as the degree of functionalization of the host molecule on the surface. First, we studied the size of the β- CD@AuNPs by TEM and DLS. TEM micrographs showed the spherical shape and low polydispersity of β-CD@AuNPs (Figure 71). Image analysis of the particle size distribution evidenced an average nanoparticle size of 2-3 nm (inset Figure 71). DLS experiments of β- CD@AuNPs suspended in Milli-Q water showed a hydrodynamic diameter of around 8 nm (Figure 71). This discrepancy is based on the fact that DLS measurements are influenced by diffusion factors, such as solvation layers. The UV spectra of β-CD@AuNPs dispersed in Milli- Q water showed the typical plasmon absorption band for well-dispersed β[email protected] Finally, we used thermogravimetric analysis (TGA) to quantify the amount of organic material at the surface of the prepared β-CD@AuNPs. The mass loss up to 100°C was attributed to water evaporation, while the weight loss attributed to organic components were observed between 280-320°C. This indicates about 13 % organic material, which would correspond to approximately 20 β-cyclodextrins covering each gold nanoparticle (Figure 71). This is in agreement with previous reports.306 306 R. Mejia-Ariza, J. Huskens, J. Mater. Chem. B 2014, 2, 210-216. 307 M. T. Rojas, R. Koeniger, J. F. Stoddart, A. E. Kaifer, J. Am. Chem. Soc. 1995, 117, 336-343.