Biointerfaces based on the combination of synthetic polymers and biomolecules

Alicat embargament des de la defensa de la tesi fins al 31 de desembre de 2019Premi Extraordinari de Doctorat, promoció 2018-2019. Àmbit d’Enginyeria IndustrialDuring the last decades, research focused on the preparation of highly selective and smart materials has increased considerably. For instances, it has been possible to achieve intelligent drug nano-carriers, biomolecular sensors, platforms to promote cell growth and differentiation among many other striking applications. Two mentionable factors that helped such development are the incorporation of biological moieties onto this interfaces to gain specificity and the combination of more than one material in order to get a synergistic effect between the different components (i.e. conducting polymers suffer from poor mechanical strength, therefore its combination with polyesters can reduce their fragility). This thesis has been devoted to the design and development of high performance polymeric materials for multiple functions related to the biomedical field, such as passive ion transport membranes, drug delivery systems and the addition of selectivity in different surfaces. The work gives special emphasis to the characterization of these platforms, like its surface chemistry, topology, biocompatibility or its mechanical strength. Besides, these systems have been synthetized in a large variety of shapes, from free-standing nanomembranes to polymeric nanoparticles. The Thesis is divided in three blocs: Bloc A encloses all the studies realized for the generation of hybrid nanoperforated membranes in order to achieve controlled ion diffusion. Specifically, an outer membrane protein, Omp2a, was considered for these studies. Primarily, the protein was purified, folded and characterized in an ambient resembling to the one encountered in nature, its mechanical forces and conductivity were analysed. The project was followed by the immobilization of Omp2a into silicon microcantillevers to acquire greater knowledge of its folding and unfolding processes upon thermal stress. Next, artificial polymeric membranes containing nanofeatures were developed with the final purpose to immobilize Omp2a via protein confinement. Then, the conductivity of the membrane with different electrolyte media solutions was tested. Bloc B describes the state-of-the art of drug delivery systems prepared with intrinsically conducting polymers to achieve controlled drug release upon electrical stimuli. Furthermore, two systems based on poly(3,4-ethylenedioxythiophene) (PEDOT) nanoparticles are described. Particularly, curcumin was employed as a model neutral drug and incorporated within the PEDOT nanoparticles. The oxidation state of the PEDOT chains regulated the drug release. Later on, a similar system was generated with polyester microfibers loaded with curcumin and nanoparticles. The driving force for the later drug release was the actuation of the PEDOT nanoparticles. Lastly, Bloc C reports the immobilization of a pentapeptide called CREKA and its analog CR(NMe)EKA onto PEDOT and silicon surfaces. The addition of CREKA favoured the selectivity of those interfaces towards clotted plasma proteins such as fibrin and fibrinogen. PEDOT-peptide material allowed the electrochemical detection of the proteins by an increase in membrane resistance and these interactions were evaluated with microcantilevers by measuring the difference on weight when they were incubated with different protein concentrations. Overall, the compilation of the studies presented in this Thesis offer a comprehensive view on how modifying and generating hybrid materials is possible to optimize and exploit their capabilities for a wide range of applications.Durant les últimes dècades, la recerca centrada en la preparació de materials altament selectius i intel·ligents ha augmentat considerablement. Ha estat possible aconseguir nano-contenidors de fàrmacs, sensors de biomolècules, plataformes per promoure el creixement i la diferenciació cel·lular, entre moltes altres aplicacions interessants. Dos factors destacables que han ajudat aquest desenvolupament són la incorporació de cues biològiques en aquets materials per obtenir especificitat i la combinació de més d'un element per obtenir un efecte sinèrgic entre els diferents components (per exemple els polímers conductors pateixen d’una baixa resistència mecànica, per tant, la seva combinació amb polièsters pot reduir la seva fragilitat però seguir mantenint les seves propietats elèctriques). En resum, aquesta tesi s'ha centrat en el disseny i desenvolupament de materials polimèrics d'alt rendiment per a múltiples funcions relacionades amb el camp biomèdic, com ara membranes passives de transport iònic, sistemes de lliurament de fàrmacs i l'addició de selectivitat envers proteïnes del plasma en diferents superfícies. El treball fa especial èmfasi en la caracterització d'aquestes plataformes, com la seva química superficial, topologia, biocompatibilitat o resistència mecànica. A més, aquests sistemes s'han sintetitzat en una gran varietat de formes, des de films fins a nanopartícules polimèriques. La tesi es divideix en tres blocs: El bloc A inclou tots els estudis realitzats per a la generació de membranes híbrides nanoperforades amb la finalitat d’aconseguir una difusió controlada de ions. Concretament en aquests estudis es va emprar una proteïna transmembrana anomenada Omp2a. La primera etapa del treball es centra en la purificació, plegament i caracterització de la proteïna en un ambient similar al que es troba originàriament. A més a més, es van analitzar les seves forces mecàniques i de conductivitat. Seguidament, es va procedir a la immobilització d'Omp2a en microcantillevers de silici per adquirir un major coneixement sobre els seus processos de plegament i desplegament depenent de l'estrès tèrmic. Finalment, es van desenvolupar membranes polimèriques artificials amb nanoperforacions amb l'objectiu d'immobilitzar Omp2a a través del confinament de la proteïna en aquests porus. El Bloc B descriu l'estat de l’art dels sistemes d’alliberament controlat de fàrmacs, preparats amb polímers intrínsecament conductors, depenent d’estímuls elèctrics. En aquest apartat, es descriuen dos sistemes basats en nanopartícules de poli(3,4-etilendioxitiofé) (PEDOT). En el primer cas, l'estat d'oxidació de les cadenes PEDOT és el responsable de regular l'alliberament del medicament. En canvi, en el segon, on es va generar un sistema similar amb microfibres de polièster carregades de droga i nanopartícules per separat, la força motriu de l'alliberament del fàrmac és el moviment d’expansió i contracció de les nanopartícules PEDOT. Finalment, el Bloc C informa de la immobilització d'un pentapèptid anomenat CREKA i el seu anàleg CR(NMe)EKA en films de PEDOT i superfícies de silici. La incorporació de CREKA afavoreix la selectivitat d'aquestes interfícies cap a les proteïnes de coagulació del plasma com la fibrina i el fibrinogen. El material pèptid-PEDOT va permetre la detecció electroquímica de les proteïnes mitjançant un augment de la resistència a la membrana i aquestes interaccions van ser avaluades amb microcantilevers, concretament, mesurant la diferència de pes quan es van incubar amb diferents concentracions de proteïnes. En general, la recopilació d’aquets estudis ofereix una visió completa sobre com modificant i generant materials híbrids és possible optimitzar i explotar les seves capacitats particulars per a una àmplia gamma d'aplicacions.Award-winningPostprint (published version

Biomimetic hybrid membranes: incorporation of transport proteins/peptides into polymer supports

Molecular sensing, water purification and desalination, drug delivery, and DNA sequencing are some striking applications of biomimetic hybrid membranes. These devices take advantage of biomolecules, which have gained excellence in their specificity and efficiency during billions of years, and of artificial materials that load the purified biological molecules and provide technological properties, such as robustness, scalability, and suitable nanofeatures to confine the biomolecules. Recent methodological advances allow more precise control of polymer membranes that support the biomacromolecules, and are expected to improve the design of the next generation of membranes as well as their applicability. In the first section of this review we explain the biological relevance of membranes, membrane proteins, and the classification used for the latter. After this, we critically analyse the different approaches employed for the production of highly selective hybrid membranes, focusing on novel materials made of self-assembled block copolymers and nanostructured polymers. Finally, a summary of the advantages and disadvantages of the different methodologies is presented and the main characteristics of biomimetic hybrid membranes are highlightedPeer ReviewedPostprint (author's final draft

Properties of Omp2a-based supported lipid bilayers: comparison with polymeric bioinspired membranes

Omp2a ß-barrel outer membrane protein has been reconstituted into supported lipid bilayers (SLBs) to compare the nanomechanical properties (elastic modulus, adhesion forces, and deformation) and functionality of the resulting bioinspired system with those of Omp2a-based polymeric nanomembranes (NMs). Protein reconstitution into lipid bilayers has been performed using different strategies, the most successful one consisting of a detergent-mediated process into preformed liposomes. The elastic modulus obtained for the lipid bilayer and Omp2a are ~19 and 10.5 ± 1.7 MPa, respectively. Accordingly, the protein is softer than the lipid bilayer, whereas the latter exhibits less mechanical strength than polymeric NMs. Besides, the function of Omp2a in the SLB is similar to that observed for Omp2a-based polymeric NMs. Results open the door to hybrid bioinspired substrates based on the integration of Omp2a-proteoliposomes and nanoperforated polymeric freestanding NMs.Peer ReviewedPostprint (author's final draft

Biointerfaces based on the combination of synthetic polymers and biomolecules

During the last decades, research focused on the preparation of highly selective and smart materials has increased considerably. For instances, it has been possible to achieve intelligent drug nano-carriers, biomolecular sensors, platforms to promote cell growth and differentiation among many other striking applications. Two mentionable factors that helped such development are the incorporation of biological moieties onto this interfaces to gain specificity and the combination of more than one material in order to get a synergistic effect between the different components (i.e. conducting polymers suffer from poor mechanical strength, therefore its combination with polyesters can reduce their fragility). This thesis has been devoted to the design and development of high performance polymeric materials for multiple functions related to the biomedical field, such as passive ion transport membranes, drug delivery systems and the addition of selectivity in different surfaces. The work gives special emphasis to the characterization of these platforms, like its surface chemistry, topology, biocompatibility or its mechanical strength. Besides, these systems have been synthetized in a large variety of shapes, from free-standing nanomembranes to polymeric nanoparticles. The Thesis is divided in three blocs: Bloc A encloses all the studies realized for the generation of hybrid nanoperforated membranes in order to achieve controlled ion diffusion. Specifically, an outer membrane protein, Omp2a, was considered for these studies. Primarily, the protein was purified, folded and characterized in an ambient resembling to the one encountered in nature, its mechanical forces and conductivity were analysed. The project was followed by the immobilization of Omp2a into silicon microcantillevers to acquire greater knowledge of its folding and unfolding processes upon thermal stress. Next, artificial polymeric membranes containing nanofeatures were developed with the final purpose to immobilize Omp2a via protein confinement. Then, the conductivity of the membrane with different electrolyte media solutions was tested. Bloc B describes the state-of-the art of drug delivery systems prepared with intrinsically conducting polymers to achieve controlled drug release upon electrical stimuli. Furthermore, two systems based on poly(3,4-ethylenedioxythiophene) (PEDOT) nanoparticles are described. Particularly, curcumin was employed as a model neutral drug and incorporated within the PEDOT nanoparticles. The oxidation state of the PEDOT chains regulated the drug release. Later on, a similar system was generated with polyester microfibers loaded with curcumin and nanoparticles. The driving force for the later drug release was the actuation of the PEDOT nanoparticles. Lastly, Bloc C reports the immobilization of a pentapeptide called CREKA and its analog CR(NMe)EKA onto PEDOT and silicon surfaces. The addition of CREKA favoured the selectivity of those interfaces towards clotted plasma proteins such as fibrin and fibrinogen. PEDOT-peptide material allowed the electrochemical detection of the proteins by an increase in membrane resistance and these interactions were evaluated with microcantilevers by measuring the difference on weight when they were incubated with different protein concentrations. Overall, the compilation of the studies presented in this Thesis offer a comprehensive view on how modifying and generating hybrid materials is possible to optimize and exploit their capabilities for a wide range of applications.Durant les últimes dècades, la recerca centrada en la preparació de materials altament selectius i intel·ligents ha augmentat considerablement. Ha estat possible aconseguir nano-contenidors de fàrmacs, sensors de biomolècules, plataformes per promoure el creixement i la diferenciació cel·lular, entre moltes altres aplicacions interessants. Dos factors destacables que han ajudat aquest desenvolupament són la incorporació de cues biològiques en aquets materials per obtenir especificitat i la combinació de més d'un element per obtenir un efecte sinèrgic entre els diferents components (per exemple els polímers conductors pateixen d’una baixa resistència mecànica, per tant, la seva combinació amb polièsters pot reduir la seva fragilitat però seguir mantenint les seves propietats elèctriques). En resum, aquesta tesi s'ha centrat en el disseny i desenvolupament de materials polimèrics d'alt rendiment per a múltiples funcions relacionades amb el camp biomèdic, com ara membranes passives de transport iònic, sistemes de lliurament de fàrmacs i l'addició de selectivitat envers proteïnes del plasma en diferents superfícies. El treball fa especial èmfasi en la caracterització d'aquestes plataformes, com la seva química superficial, topologia, biocompatibilitat o resistència mecànica. A més, aquests sistemes s'han sintetitzat en una gran varietat de formes, des de films fins a nanopartícules polimèriques. La tesi es divideix en tres blocs: El bloc A inclou tots els estudis realitzats per a la generació de membranes híbrides nanoperforades amb la finalitat d’aconseguir una difusió controlada de ions. Concretament en aquests estudis es va emprar una proteïna transmembrana anomenada Omp2a. La primera etapa del treball es centra en la purificació, plegament i caracterització de la proteïna en un ambient similar al que es troba originàriament. A més a més, es van analitzar les seves forces mecàniques i de conductivitat. Seguidament, es va procedir a la immobilització d'Omp2a en microcantillevers de silici per adquirir un major coneixement sobre els seus processos de plegament i desplegament depenent de l'estrès tèrmic. Finalment, es van desenvolupar membranes polimèriques artificials amb nanoperforacions amb l'objectiu d'immobilitzar Omp2a a través del confinament de la proteïna en aquests porus. El Bloc B descriu l'estat de l’art dels sistemes d’alliberament controlat de fàrmacs, preparats amb polímers intrínsecament conductors, depenent d’estímuls elèctrics. En aquest apartat, es descriuen dos sistemes basats en nanopartícules de poli(3,4-etilendioxitiofé) (PEDOT). En el primer cas, l'estat d'oxidació de les cadenes PEDOT és el responsable de regular l'alliberament del medicament. En canvi, en el segon, on es va generar un sistema similar amb microfibres de polièster carregades de droga i nanopartícules per separat, la força motriu de l'alliberament del fàrmac és el moviment d’expansió i contracció de les nanopartícules PEDOT. Finalment, el Bloc C informa de la immobilització d'un pentapèptid anomenat CREKA i el seu anàleg CR(NMe)EKA en films de PEDOT i superfícies de silici. La incorporació de CREKA afavoreix la selectivitat d'aquestes interfícies cap a les proteïnes de coagulació del plasma com la fibrina i el fibrinogen. El material pèptid-PEDOT va permetre la detecció electroquímica de les proteïnes mitjançant un augment de la resistència a la membrana i aquestes interaccions van ser avaluades amb microcantilevers, concretament, mesurant la diferència de pes quan es van incubar amb diferents concentracions de proteïnes. En general, la recopilació d’aquets estudis ofereix una visió completa sobre com modificant i generant materials híbrids és possible optimitzar i explotar les seves capacitats particulars per a una àmplia gamma d'aplicacions

Biomimetic hybrid membranes: incorporation of transport proteins/peptides into polymer supports

Molecular sensing, water purification and desalination, drug delivery, and DNA sequencing are some striking applications of biomimetic hybrid membranes. These devices take advantage of biomolecules, which have gained excellence in their specificity and efficiency during billions of years, and of artificial materials that load the purified biological molecules and provide technological properties, such as robustness, scalability, and suitable nanofeatures to confine the biomolecules. Recent methodological advances allow more precise control of polymer membranes that support the biomacromolecules, and are expected to improve the design of the next generation of membranes as well as their applicability. In the first section of this review we explain the biological relevance of membranes, membrane proteins, and the classification used for the latter. After this, we critically analyse the different approaches employed for the production of highly selective hybrid membranes, focusing on novel materials made of self-assembled block copolymers and nanostructured polymers. Finally, a summary of the advantages and disadvantages of the different methodologies is presented and the main characteristics of biomimetic hybrid membranes are highlightedPeer Reviewe

Cell responses to electrical pulse stimulation for anticancer drug release

Electrical stimulation is an attractive approach to tune on-demand drug release in the body as it relies on simple setups and requires typically 1 V or less. Although many studies have been focused on the development of potential smart materials for electrically controlled drug release, as well as on the exploration of different delivery mechanisms, progress in the field is slow because the response of cells exposed to external electrical stimulus is frequently omitted from such investigations. In this work, we monitor the behavior of prostate and breast cancer cells (PC-3 and MCF7, respectively) exposed to electroactive platforms loaded with curcumin, a hydrophobic anticancer drug. These consist in conducting polymer nanoparticles, which release drug molecules by altering their interactions with polymer, and electrospun polyester microfibres that contain electroactive nanoparticles able to alter the porosity of the matrix through an electro-mechanical actuation mechanism. The response of the cells against different operating conditions has been examined considering their viability, metabolism, spreading and shape. Results have allowed us to differentiate the damage induced in the cell by the electrical stimulation from other effects, as for example, the anticancer activity of curcumin and/or the presence of curcumin-loaded nanoparticles or fibres, demonstrating that these kinds of platforms can be effective when the dosage of the drug occurs under restricted conditionsPeer Reviewe