Hybrid block and graft copolymers made from macrolactones and α-amino acids for applications as drug delivery nanosystems

Premi Extraordinari de Doctorat, promoció 2018-2019. Àmbit d’Enginyeria IndustrialNaturally produced peptides or proteins can be regarded as highly refined polymers. When synthetic polymers are married to proteins or peptides, the resulting bioconjugates can synergistically combine the properties of the individual components and overcome their separate limitations. This Thesis is focused on the study of hybrid copolymers based on polypeptides and polymacrolactones. Block and graft copolymers have been synthesized by making use of the ring opening polymerization method (ROP) mainly and extensively characterized including both their chemical structure and their structure in the solid state. The self-assembly properties of the new copolymers have been preliminary examined regarding their potential application as nanocarriers for pharmaceutical compounds. This Thesis initially reports the ROP of w-pentadecalactone (PDL) using different amino-ended initiators and assisted by either organic or enzymatic catalysts. This method was then extended for the ROP of PDL using bisamino-ended poly(ethylene glycol) (PEG) for the preparation of poly(w-pentadecalactone)-b-poly(ethylene glycol)-b-poly(wpentadecalactone) [PPDLx-PEG-PPDLx] triblock copolymers. These amphiphilic ABA-type copolymers were able to selfassemble in water to form nanoparticles with diameters between 100 and 200 nm. Hybrid copolymers of poly(ester-peptide) or poly(ether-ester-peptide) type exhibiting different architectures (e.g. diblock, triblock, graft or triblock/grafted) respectively, were then synthesized using as building blocks: poly(w-pentadecalactone), poly(globalide) (PGl), PEG as well as polypeptides derived from the L-glutamic acid (Glu), L-lysine (Lys), L-alanine (Ala) and L-phenylalanine (Phe) a-amino acids. The hybrid copolymers were synthesized through several stages depending on the desired architecture. The first stage in the preparation of these copolymers was the synthesis of macroinitiators from PDL or PGl containing either an amino group at the end of the chain or multiple amine groups along their polymeric chain. In the second stage, such macroinitiators were used to trigger the polymerization of the a-amino acid N-carboxyanhyrides (NCA) with the COOH group of L-glutamic acid and NH2 of L-lysine duly protected as g-benzyl-L-glutamate (BLG) and eNcarbobenzoxy-L-lysine (ZLL) respectively. Some copolymers containing BLG or ZLL units were treated with acids to render copolymers bearing the amino acids residues with their COOH or NH2 functionalities in the free form. All of the synthesized copolymers were fully characterized through GPC and NMR spectroscopy. The thermal properties were studied by TGA and DSC techniques. The conformation adopted by the peptide-based copolymers in the solid-state was assessed by FTIR, and their crystalline structure was examined by X-ray diffraction using synchrotron radiation in most cases. The conformation in aqueous solution of water-soluble copolymers containing Glu or Lys residues in the free form was explored by circular dichroism. The self-assembly behavior in aqueous medium of all the amphiphilic copolymers was investigated with the purpose of obtaining nanoparticles with the appropriated diameters required for their application as biomedical nanocarriers. The nanoparticles were duly characterized by light scattering and SEM and TEM microscopies. Block and graft copolymers were able to load doxorubicin and release it under pH control. Copolymers containing L-lysine were shown to be able of condensing DNA. The potential of these copolymers as DDS of anticancer drugs and vectors for transfection have been evidenced.Los polipéptidos o proteínas obtenidos de manera natural son considerados como polímeros altamente refinados. Cuando los polímeros sintéticos se unen a proteínas o polipéptidos, los sistemas bioconjugados que se obtienen pueden sinérgicamente combinar las propiedades de sus componentes individuales y mejorar las propias limitaciones que tienen por separado. La proteína o el elemento polipeptídico puede impartir propiedades bifuncionales al bioconjugado, mientras que el polímero sintético puede mejorar la estabilidad proteica, la solubilidad y la biocompatibilidad. Esta tesis está enfocada en el estudio de copolímeros híbridos basados en polipeptidos y polimacrolactonas. Copolímeros tipo bloque e injerto fueron sintetizados utilizando principalmente la polimerización por apertura de anillo y extensamente caracterizados tanto su estructura química, como su estructura en estado sólido. Las propiedades de auto-agregación de los nuevos copolímeros han sido anteriomente examinadas respecto a su potencial aplicación como nanotransportadores de compuestos farmacéuticos. Esta Tesis inicialmente reporta la homopolimerización de w-pentadecalactona (PDL) usando diferentes iniciadores aminoterminados mediante el uso de catalizadores tanto orgánicos como enzimáticos. Este se extiende a la ROP de PDL usando poli(etilén glicol) bisamino-terminado (PEG) para la preparar copolímeros tribloque poli(w-pentadecalactona)-b-poli(etilén glicol)-b-poli(w-pentadecalactona) [PPDLx-PEG-PPDLx]. Estos copolímeros de tipo ABA fueron capaces de auto-agregarse en agua para formar nanopartículas con diámetros entre 100 y 200 nm. Por otra parte, sistemas híbridos de tipo poli(éster-péptido) o poli(éter-éster-péptido) que presentan distintas arquitecturas (por ejemplo dibloque, tribloque, injerto, o tribloque-injertado) respectivamente, se sintetizaron utilizando como bloques de construcción derivados de macrolactonas (w-pentadecalactona), globalida) y a-amino ácidos (ácido L-glutámico (Glu), Llisina (Lys), L-alanina (Ala) y L-fenilalanina (Phe) así como poli(etien glicol) telequélico. Los copolímeros híbridos fueron sintetizados en varias etapas dependiendo de cuál fuese la arquitectura deseada. La primera etapa fue la preparación de los macroiniciadores a partir de PDL o PGl conteniendo en su estructura ya sea un grupo amino en el extremo de la cadena, o múltiples grupos aminos a lo largo de la cadena polimérica. En la segunda etapa, los macroiniciadores fueron utilizados en la polimerización de a-amino ácidos N-carboxianhídridos (NCA), con los grupos COOH del ácido L-glutámico y el grupo NH2 de la L-lisina apropiadamente protegidos como g-bencil-L-glutamato (BLG) y eN-carbobenzoxi-L-lisina (ZLL) respectivamente. Para los copolímeros que contienen bloques peptídicos de BLG o ZLL, las funcionalidades COOH o NH2 fueron regeneradas bajo condiciones ácidas, para producir así los copolímeros conteniendo el amino ácido en su forma libre. Todos los copolímeros sintetizados fueron completamente caracterizados mediante GPC y espectroscopia de RMN. Las propiedades térmicas fueron estudiadas por las técnicas de TGA y DSC. La conformación adoptada por los copolímeros en el estado sólido fue estudiada por FTIR, y su estructura cristalina fue analizada mediante difracción de rayos X usando radiación sincrotrón en la mayoría de los casos. La conformación en solución acuosa de los copolímeros solubles en agua, que contienen residuos de Glu o Lys, fue analizada por dicroísmo circular. Se estudió el comportamiento de todos los copolímeros para auto-agregarse en agua obteniéndose partículas con diámetros del orden nanométrico, como se demostró por DLS así como también por SEM y TEM, las cuales son apropiadas para ser aplicadas en biomedicina. Las nanopartículas de copolímeros dibloque y de injerto conteniendo ácido L-glutámico fueron capaces de incorporar doxorubicina y efectuar su liberación bajo control por medio del pH. Por otro lado, los copolímeros dibloque y de injerto con bloques conteniendo L-lisina mostraron la habilidad de condensar el ADN, demostrando así su potencial uso como vectores en transfección.Award-winningPostprint (published version

Hybrid block and graft copolymers made from macrolactones and α-amino acids for applications as drug delivery nanosystems

Naturally produced peptides or proteins can be regarded as highly refined polymers. When synthetic polymers are married to proteins or peptides, the resulting bioconjugates can synergistically combine the properties of the individual components and overcome their separate limitations. This Thesis is focused on the study of hybrid copolymers based on polypeptides and polymacrolactones. Block and graft copolymers have been synthesized by making use of the ring opening polymerization method (ROP) mainly and extensively characterized including both their chemical structure and their structure in the solid state. The self-assembly properties of the new copolymers have been preliminary examined regarding their potential application as nanocarriers for pharmaceutical compounds. This Thesis initially reports the ROP of w-pentadecalactone (PDL) using different amino-ended initiators and assisted by either organic or enzymatic catalysts. This method was then extended for the ROP of PDL using bisamino-ended poly(ethylene glycol) (PEG) for the preparation of poly(w-pentadecalactone)-b-poly(ethylene glycol)-b-poly(wpentadecalactone) [PPDLx-PEG-PPDLx] triblock copolymers. These amphiphilic ABA-type copolymers were able to selfassemble in water to form nanoparticles with diameters between 100 and 200 nm. Hybrid copolymers of poly(ester-peptide) or poly(ether-ester-peptide) type exhibiting different architectures (e.g. diblock, triblock, graft or triblock/grafted) respectively, were then synthesized using as building blocks: poly(w-pentadecalactone), poly(globalide) (PGl), PEG as well as polypeptides derived from the L-glutamic acid (Glu), L-lysine (Lys), L-alanine (Ala) and L-phenylalanine (Phe) a-amino acids. The hybrid copolymers were synthesized through several stages depending on the desired architecture. The first stage in the preparation of these copolymers was the synthesis of macroinitiators from PDL or PGl containing either an amino group at the end of the chain or multiple amine groups along their polymeric chain. In the second stage, such macroinitiators were used to trigger the polymerization of the a-amino acid N-carboxyanhyrides (NCA) with the COOH group of L-glutamic acid and NH2 of L-lysine duly protected as g-benzyl-L-glutamate (BLG) and eNcarbobenzoxy-L-lysine (ZLL) respectively. Some copolymers containing BLG or ZLL units were treated with acids to render copolymers bearing the amino acids residues with their COOH or NH2 functionalities in the free form. All of the synthesized copolymers were fully characterized through GPC and NMR spectroscopy. The thermal properties were studied by TGA and DSC techniques. The conformation adopted by the peptide-based copolymers in the solid-state was assessed by FTIR, and their crystalline structure was examined by X-ray diffraction using synchrotron radiation in most cases. The conformation in aqueous solution of water-soluble copolymers containing Glu or Lys residues in the free form was explored by circular dichroism. The self-assembly behavior in aqueous medium of all the amphiphilic copolymers was investigated with the purpose of obtaining nanoparticles with the appropriated diameters required for their application as biomedical nanocarriers. The nanoparticles were duly characterized by light scattering and SEM and TEM microscopies. Block and graft copolymers were able to load doxorubicin and release it under pH control. Copolymers containing L-lysine were shown to be able of condensing DNA. The potential of these copolymers as DDS of anticancer drugs and vectors for transfection have been evidenced.Los polipéptidos o proteínas obtenidos de manera natural son considerados como polímeros altamente refinados. Cuando los polímeros sintéticos se unen a proteínas o polipéptidos, los sistemas bioconjugados que se obtienen pueden sinérgicamente combinar las propiedades de sus componentes individuales y mejorar las propias limitaciones que tienen por separado. La proteína o el elemento polipeptídico puede impartir propiedades bifuncionales al bioconjugado, mientras que el polímero sintético puede mejorar la estabilidad proteica, la solubilidad y la biocompatibilidad. Esta tesis está enfocada en el estudio de copolímeros híbridos basados en polipeptidos y polimacrolactonas. Copolímeros tipo bloque e injerto fueron sintetizados utilizando principalmente la polimerización por apertura de anillo y extensamente caracterizados tanto su estructura química, como su estructura en estado sólido. Las propiedades de auto-agregación de los nuevos copolímeros han sido anteriomente examinadas respecto a su potencial aplicación como nanotransportadores de compuestos farmacéuticos. Esta Tesis inicialmente reporta la homopolimerización de w-pentadecalactona (PDL) usando diferentes iniciadores aminoterminados mediante el uso de catalizadores tanto orgánicos como enzimáticos. Este se extiende a la ROP de PDL usando poli(etilén glicol) bisamino-terminado (PEG) para la preparar copolímeros tribloque poli(w-pentadecalactona)-b-poli(etilén glicol)-b-poli(w-pentadecalactona) [PPDLx-PEG-PPDLx]. Estos copolímeros de tipo ABA fueron capaces de auto-agregarse en agua para formar nanopartículas con diámetros entre 100 y 200 nm. Por otra parte, sistemas híbridos de tipo poli(éster-péptido) o poli(éter-éster-péptido) que presentan distintas arquitecturas (por ejemplo dibloque, tribloque, injerto, o tribloque-injertado) respectivamente, se sintetizaron utilizando como bloques de construcción derivados de macrolactonas (w-pentadecalactona), globalida) y a-amino ácidos (ácido L-glutámico (Glu), Llisina (Lys), L-alanina (Ala) y L-fenilalanina (Phe) así como poli(etien glicol) telequélico. Los copolímeros híbridos fueron sintetizados en varias etapas dependiendo de cuál fuese la arquitectura deseada. La primera etapa fue la preparación de los macroiniciadores a partir de PDL o PGl conteniendo en su estructura ya sea un grupo amino en el extremo de la cadena, o múltiples grupos aminos a lo largo de la cadena polimérica. En la segunda etapa, los macroiniciadores fueron utilizados en la polimerización de a-amino ácidos N-carboxianhídridos (NCA), con los grupos COOH del ácido L-glutámico y el grupo NH2 de la L-lisina apropiadamente protegidos como g-bencil-L-glutamato (BLG) y eN-carbobenzoxi-L-lisina (ZLL) respectivamente. Para los copolímeros que contienen bloques peptídicos de BLG o ZLL, las funcionalidades COOH o NH2 fueron regeneradas bajo condiciones ácidas, para producir así los copolímeros conteniendo el amino ácido en su forma libre. Todos los copolímeros sintetizados fueron completamente caracterizados mediante GPC y espectroscopia de RMN. Las propiedades térmicas fueron estudiadas por las técnicas de TGA y DSC. La conformación adoptada por los copolímeros en el estado sólido fue estudiada por FTIR, y su estructura cristalina fue analizada mediante difracción de rayos X usando radiación sincrotrón en la mayoría de los casos. La conformación en solución acuosa de los copolímeros solubles en agua, que contienen residuos de Glu o Lys, fue analizada por dicroísmo circular. Se estudió el comportamiento de todos los copolímeros para auto-agregarse en agua obteniéndose partículas con diámetros del orden nanométrico, como se demostró por DLS así como también por SEM y TEM, las cuales son apropiadas para ser aplicadas en biomedicina. Las nanopartículas de copolímeros dibloque y de injerto conteniendo ácido L-glutámico fueron capaces de incorporar doxorubicina y efectuar su liberación bajo control por medio del pH. Por otro lado, los copolímeros dibloque y de injerto con bloques conteniendo L-lisina mostraron la habilidad de condensar el ADN, demostrando así su potencial uso como vectores en transfección

Validation of smart nanoparticles as controlled drug delivery systems: loading and pH-dependent release of pilocarpine

Micelles are good devices for use as controlled drug delivery systems because they exhibit the ability to protect the encapsulated substance from the routes of degradation until they reach the site of action. The present work assesses loading kinetics of a hydrophobic drug, pilocarpine, in polymeric micellar nanoparticles (NPs) and its pH-dependent release in hydrophilic environments. The trigger pH stimulus, pH 5.5, was the value encountered in damaged tissues in solid tumors. The new nanoparticles were prepared from an amphiphilic block copolymer, [(HEMA19%-DMA31%)-(FMA5%-DEA45%)]. For the present research, three systems were validated, two of them with cross-linked cores and the other without chemical stabilization. A comparison of their loading kinetics and release profiles is discussed, with the support of additional data obtained by scanning electron microscopy and dynamic light scattering. The drug was loaded into the NPs within the first minutes; the load was dependent on the degree of cross-linking. All of the systems experienced a boost in drug release at acidic pH, ranging from 50 to 80% within the first 48 h. NPs with the highest degree (20%) of core cross-linking delivered the highest percentage of drug at fixed times. The studied systems exhibited fine-tuned sustained release features, which may provide a continuous delivery of the drug at specific acidic locations, thereby diminishing side effects and increasing therapeutic rates. Hence, the studied NPs proved to behave as smart controlled drug delivery systems capable of responding to changes in pH.Peer ReviewedPostprint (published version

Recontextualização da política de gestão em escolas indígenas em tempo integral na Baja California

This article reports a study on the model of school management followed by indigenous public elementary schools participating in the Full-Time Schools program (PETC) in Baja California, Mexico. It is based on Stephen Ball´s theoretical proposal on policy enactments. The objective was to infer the processes involved in policy interpretation and translation at work when endorsing the PETC policy by supervisor, principals and teachers. From a qualitative perspective, the data consisted of school observations and semi-structured interviews; for their study, we applied James Paul Gee´s analysis of I-statements in order to identify the participants´ stances regarding their everyday schoolwork. The results highlight the restrictions of the contexts under which the program is implemented, as well as the participants’ actions taken to fulfill the demands of the PETC, on top of their everyday tasks. In this way, the program and its generic elements are recontextualized in a context marked by historical inequality and marginalization.El presente artículo reporta un estudio sobre el modelo de gestión escolar en cinco escuelas primarias públicas indígenas adscritas al Programa Escuelas de Tiempo Completo (PETC) en el estado de Baja California, México desde la propuesta teórica de los procesos de recontextualización empleada por Stephen Ball. El propósito fue inferir los procesos de interpretación y traducción en la puesta en práctica del modelo de gestión del PETC por parte de diferentes actores educativos (supervisora, directores y docentes). Desde una perspectiva cualitativa, se realizaron observaciones en las escuelas y se recabaron entrevistas semiestructuradas. Para el análisis de éstas, utilizamos el análisis de enunciados en primera persona propuesto por James Paul Gee. La finalidad fue identificar las posturas ante los bienes sociales en el discurso de los interlocutores en cuanto al trabajo escolar cotidiano. En los resultados se destacan las restricciones del contexto bajo las cuales el PETC es puesto en práctica, así como las acciones que los agentes educativos llevan a cabo para cumplir con las demandas del programa, además de las funciones que realizan de forma cotidiana. De este modo, el programa y sus elementos genéricos se recontextualizan en un contexto marcado por una desigualdad y marginación históricas.Este artigo relata um estudo sobre o modelo de gestão escolar em cinco escolas públicas indígenas vinculadas ao Programa Escolas de Tempo Integral (PETC) no estado de Baja California, México, a partir da proposta teórica dos processos de recontextualização utilizada por Stephen Ball. O objetivo foi inferir os processos de interpretação e tradução na implementação do modelo de gestão do PETC por diferentes atores educacionais (supervisores, diretores e professores). Do ponto de vista qualitativo, foram realizadas observações nas escolas e entrevistas semiestruturadas foram coletadas. Para a análise destes, usamos a análise de primeira pessoa proposta por James Paul Gee. O objetivo foi identificar as posições diante dos bens sociais no discurso dos interlocutores sobre o cotidiano escolar. Os resultados destacam as restrições do contexto em que o PETC é implementado, bem como as ações que os agentes educacionais realizam para atender às demandas do programa, além das funções que desempenham no dia a dia. Desta forma, o programa e seus elementos genéricos são recontextualizados em um contexto marcado pela desigualdade histórica e pela marginalização

Copolymacrolactones grafted with L-glutamic acid: Synthesis, structure, and nanocarrier properties

The enzymatic ring-opening copolymerization (eROP) of globalide (Gl) and pentadecalactone (PDL) was performed in solution from mixtures of the two macrolactones at ratios covering the whole range of comonomeric compositions. The resulting P(Glx-r-PDLy) random copolyesters were aminofunctionalized by thiol-ene reaction with aminoethanethiol. ROP of ¿-benzyl-l-glutamate N-carboxyanhydride initiated by P(Glx-r-PDLy)-NH2 provided neutral poly(¿-benzyl-L-glutamate)-grafted copolyesters, which were converted by hydrolysis into negatively charged hybrid copolymers. Both water-soluble and nonsoluble copolymers were produced depending on copolymer charge and their grafting degree, and their capacity for self-assembling in nano-objects were comparatively examined. The emulsion solvent-evaporation technique applied to the chloroform-soluble copolymers grafted with benzyl glutamate rendered well-delineated spherical nanoparticles with an average diameter of 200–300 nm. Conversely, micellar solutions in water were produced from copolyesters bearing grafted chains composed of at least 10 units of glutamic acid in the free form. The copolymer micelles were shown to be able to load doxorubicin (DOX) efficiently through electrostatic interactions and also to release the drug at a rate that was markedly pH dependent.Peer ReviewedPostprint (published version

Poly(amino acid)-grafted polymacrolactones. Synthesis, self-assembling and ionic coupling properties

Polyglobalide (PGl) with number average polymerization degree of ~20 was prepared by enzymatic ROP and then polyfunctionalized at 60% with aminothioethylene groups. The PGl20-(NH2)12 copolymer was used as macroinitiator for the ROP of NCAs of BLG (¿-benzyl L-glutamate) and ZLL (eN-carbobenzoxy-l-lysine) protected amino acids to produce neutral polypeptide-grafted polyglobalides poly[Gl20-graft-(AA)z] with z¿=¿5 and 12, which upon deprotection, afforded anionic and cationic copolymers, respectively. Both protected and deprotected graft copolymers were characterized in full detail by NMR, and their thermal properties were evaluated by TGA and DSC. The structure of these copolymers in the solid-state was examined by FTIR and XRD using synchrotron radiation. All grafted polyglobalides were amorphous but the polypeptide side chains were arranged in either alpha-helix or beta-sheet conformation, and reliable indications on the occurrence of supramolecular structures were frequently found. The capacity of poly[Gl20-graft-(AA)z] copolymers to self-assemble in aqueous medium was evidenced by the preparation of well-shaped spheroidal nanoparticles with a diversity of sizes depending on copolymer composition and charge. Loading and release of doxorubicin (DOX) from nanoparticles made of negatively charged poly[Gl20-graft-(LGA)12] as well as DNA complexation with cationic poly[Gl20-graft-(LL)5] were explored to appraise the potential of these copolymers for building drug delivery systems.Peer ReviewedPostprint (author's final draft

Metal-free catalyzed ring-opening polymerization and block copolymerization of ¿-pentadecalactone using amino-ended initiators

Metal-free catalysis was successfully applied to polymerize ¿-pentadecalactone (PDL) by ring-opening polymerization (ROP) using several amino-ended initiators, namely hexylamine, allylamine and O,O'-bis(3-aminopropyl)diethylene glycol. This polymerization method was suitable to prepare telechelic polyesters carrying functional-end groups. The technique was then extended to the synthesis of block copolymers by ROP of PDL using bisamino-ended poly(ethylene glycol) (Mn¿=¿2600) as macroinitiator. PPDLx-PEG56-PPDLx triblock copolymers with Mn ranging between ~4000 and ~90,000¿g¿·mol-1 were synthesized and extensively characterized by NMR, DSC, TGA and XRD. The amphiphilic copolymers thus produced were demonstrated to be able to self-assemble in nanoparticles with average diameters of ~100–200¿nm and morphologies highly depending on blocks lengths. The described synthetic route distinguishes in providing “clean” amphiphilic copolymers, which are attractive candidates for biomedical applications.Peer ReviewedPostprint (author's final draft

Ring opening polymerization of macrolactones using amines as initiators

The ring opening polymerization (ROP) of these macrolactones mediated by alcohols as initiators and organic and metal-based catalysts has been reported before with satisfactory results [4,5]. In the present work, the polymerization of these macrolactones has been studied using amines as initiators. The ROP of both PDL and 6HDL was carried out in bulk at 100 oC using 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) as organocatalyst. Distinct types of amines were tested as initiators for the ROP of these macrolactones. The copolymerization reaction was also studied by ROP of PDL and 6HDL with an oligo-polyethylene glycol bis(3-aminopropyl) terminated and an amino-ended homopolypeptide (poly(¿-benzyl a,Lglutamate) as initiator.Postprint (published version

Green metallic nanoparticles for cancer therapy: evaluation models and cancer applications

Metal-based nanoparticles are widely used to deliver bioactive molecules and drugs to improve cancer therapy. Several research works have highlighted the synthesis of gold and silver nanoparticles by green chemistry, using biological entities to minimize the use of solvents and control their physicochemical and biological properties. Recent advances in evaluating the anticancer effect of green biogenic Au and Ag nanoparticles are mainly focused on the use of conventional 2D cell culture and in vivo murine models that allow determination of the half-maximal inhibitory concentration, a critical parameter to move forward clinical trials. However, the interaction between nanoparticles and the tumor microenvironment is not yet fully understood. Therefore, it is necessary to develop more human-like evaluation models or to improve the existing ones for a better understanding of the molecular bases of cancer. This review provides recent advances in biosynthesized Au and Ag nanoparticles for seven of the most common and relevant cancers and their biological assessment. In addition, it provides a general idea of the in silico, in vitro, ex vivo, and in vivo models used for the anticancer evaluation of green biogenic metal-based nanoparticles.Peer ReviewedObjectius de Desenvolupament Sostenible::8 - Treball Decent i Creixement EconòmicObjectius de Desenvolupament Sostenible::16 - Pau, Justícia i Institucions SòlidesObjectius de Desenvolupament Sostenible::4 - Educació de QualitatPostprint (published version

Hybrid block and graft copolymers made from macrolactones and α-amino acids for applications as drug delivery nanosystems

Naturally produced peptides or proteins can be regarded as highly refined polymers. When synthetic polymers are married to proteins or peptides, the resulting bioconjugates can synergistically combine the properties of the individual components and overcome their separate limitations. This Thesis is focused on the study of hybrid copolymers based on polypeptides and polymacrolactones. Block and graft copolymers have been synthesized by making use of the ring opening polymerization method (ROP) mainly and extensively characterized including both their chemical structure and their structure in the solid state. The self-assembly properties of the new copolymers have been preliminary examined regarding their potential application as nanocarriers for pharmaceutical compounds. This Thesis initially reports the ROP of w-pentadecalactone (PDL) using different amino-ended initiators and assisted by either organic or enzymatic catalysts. This method was then extended for the ROP of PDL using bisamino-ended poly(ethylene glycol) (PEG) for the preparation of poly(w-pentadecalactone)-b-poly(ethylene glycol)-b-poly(wpentadecalactone) [PPDLx-PEG-PPDLx] triblock copolymers. These amphiphilic ABA-type copolymers were able to selfassemble in water to form nanoparticles with diameters between 100 and 200 nm. Hybrid copolymers of poly(ester-peptide) or poly(ether-ester-peptide) type exhibiting different architectures (e.g. diblock, triblock, graft or triblock/grafted) respectively, were then synthesized using as building blocks: poly(w-pentadecalactone), poly(globalide) (PGl), PEG as well as polypeptides derived from the L-glutamic acid (Glu), L-lysine (Lys), L-alanine (Ala) and L-phenylalanine (Phe) a-amino acids. The hybrid copolymers were synthesized through several stages depending on the desired architecture. The first stage in the preparation of these copolymers was the synthesis of macroinitiators from PDL or PGl containing either an amino group at the end of the chain or multiple amine groups along their polymeric chain. In the second stage, such macroinitiators were used to trigger the polymerization of the a-amino acid N-carboxyanhyrides (NCA) with the COOH group of L-glutamic acid and NH2 of L-lysine duly protected as g-benzyl-L-glutamate (BLG) and eNcarbobenzoxy-L-lysine (ZLL) respectively. Some copolymers containing BLG or ZLL units were treated with acids to render copolymers bearing the amino acids residues with their COOH or NH2 functionalities in the free form. All of the synthesized copolymers were fully characterized through GPC and NMR spectroscopy. The thermal properties were studied by TGA and DSC techniques. The conformation adopted by the peptide-based copolymers in the solid-state was assessed by FTIR, and their crystalline structure was examined by X-ray diffraction using synchrotron radiation in most cases. The conformation in aqueous solution of water-soluble copolymers containing Glu or Lys residues in the free form was explored by circular dichroism. The self-assembly behavior in aqueous medium of all the amphiphilic copolymers was investigated with the purpose of obtaining nanoparticles with the appropriated diameters required for their application as biomedical nanocarriers. The nanoparticles were duly characterized by light scattering and SEM and TEM microscopies. Block and graft copolymers were able to load doxorubicin and release it under pH control. Copolymers containing L-lysine were shown to be able of condensing DNA. The potential of these copolymers as DDS of anticancer drugs and vectors for transfection have been evidenced.Los polipéptidos o proteínas obtenidos de manera natural son considerados como polímeros altamente refinados. Cuando los polímeros sintéticos se unen a proteínas o polipéptidos, los sistemas bioconjugados que se obtienen pueden sinérgicamente combinar las propiedades de sus componentes individuales y mejorar las propias limitaciones que tienen por separado. La proteína o el elemento polipeptídico puede impartir propiedades bifuncionales al bioconjugado, mientras que el polímero sintético puede mejorar la estabilidad proteica, la solubilidad y la biocompatibilidad. Esta tesis está enfocada en el estudio de copolímeros híbridos basados en polipeptidos y polimacrolactonas. Copolímeros tipo bloque e injerto fueron sintetizados utilizando principalmente la polimerización por apertura de anillo y extensamente caracterizados tanto su estructura química, como su estructura en estado sólido. Las propiedades de auto-agregación de los nuevos copolímeros han sido anteriomente examinadas respecto a su potencial aplicación como nanotransportadores de compuestos farmacéuticos. Esta Tesis inicialmente reporta la homopolimerización de w-pentadecalactona (PDL) usando diferentes iniciadores aminoterminados mediante el uso de catalizadores tanto orgánicos como enzimáticos. Este se extiende a la ROP de PDL usando poli(etilén glicol) bisamino-terminado (PEG) para la preparar copolímeros tribloque poli(w-pentadecalactona)-b-poli(etilén glicol)-b-poli(w-pentadecalactona) [PPDLx-PEG-PPDLx]. Estos copolímeros de tipo ABA fueron capaces de auto-agregarse en agua para formar nanopartículas con diámetros entre 100 y 200 nm. Por otra parte, sistemas híbridos de tipo poli(éster-péptido) o poli(éter-éster-péptido) que presentan distintas arquitecturas (por ejemplo dibloque, tribloque, injerto, o tribloque-injertado) respectivamente, se sintetizaron utilizando como bloques de construcción derivados de macrolactonas (w-pentadecalactona), globalida) y a-amino ácidos (ácido L-glutámico (Glu), Llisina (Lys), L-alanina (Ala) y L-fenilalanina (Phe) así como poli(etien glicol) telequélico. Los copolímeros híbridos fueron sintetizados en varias etapas dependiendo de cuál fuese la arquitectura deseada. La primera etapa fue la preparación de los macroiniciadores a partir de PDL o PGl conteniendo en su estructura ya sea un grupo amino en el extremo de la cadena, o múltiples grupos aminos a lo largo de la cadena polimérica. En la segunda etapa, los macroiniciadores fueron utilizados en la polimerización de a-amino ácidos N-carboxianhídridos (NCA), con los grupos COOH del ácido L-glutámico y el grupo NH2 de la L-lisina apropiadamente protegidos como g-bencil-L-glutamato (BLG) y eN-carbobenzoxi-L-lisina (ZLL) respectivamente. Para los copolímeros que contienen bloques peptídicos de BLG o ZLL, las funcionalidades COOH o NH2 fueron regeneradas bajo condiciones ácidas, para producir así los copolímeros conteniendo el amino ácido en su forma libre. Todos los copolímeros sintetizados fueron completamente caracterizados mediante GPC y espectroscopia de RMN. Las propiedades térmicas fueron estudiadas por las técnicas de TGA y DSC. La conformación adoptada por los copolímeros en el estado sólido fue estudiada por FTIR, y su estructura cristalina fue analizada mediante difracción de rayos X usando radiación sincrotrón en la mayoría de los casos. La conformación en solución acuosa de los copolímeros solubles en agua, que contienen residuos de Glu o Lys, fue analizada por dicroísmo circular. Se estudió el comportamiento de todos los copolímeros para auto-agregarse en agua obteniéndose partículas con diámetros del orden nanométrico, como se demostró por DLS así como también por SEM y TEM, las cuales son apropiadas para ser aplicadas en biomedicina. Las nanopartículas de copolímeros dibloque y de injerto conteniendo ácido L-glutámico fueron capaces de incorporar doxorubicina y efectuar su liberación bajo control por medio del pH. Por otro lado, los copolímeros dibloque y de injerto con bloques conteniendo L-lisina mostraron la habilidad de condensar el ADN, demostrando así su potencial uso como vectores en transfección