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

Nanoperforations in poly(lactic acid) free-standing nanomembranes to promote interactions with cell filopodia

Nanoperforated poly(lactic acid) (PLA) free-standing nanomembranes (FsNMs) have been prepared using a two-step process: (1) spin-coating a mixture of immiscible polymers to provoke phase segregation and formation of appropriated nanofeatures (i.e. phase separation domains with dimensions similar to the entire film thickness); and (2) selective solvent etching to transform such nanofeatures into nanoperforations. For this purpose, PLA has been mixed with polyethylene glycol (PEG) and poly(vinyl alcohol) (PVA). Unfortunately, the characteristics of PLA:PEG mixtures were not appropriated to prepare nanoperforated FsNMs. In contrast, perforated PLA FsNMs with pores crossing the entire film thickness, which have been characterized by scanning electron microscopy and atomic force microscopy, were obtained using PLA:PVA mixtures. The diameter (¿) of such pores has been controlled through both the PLA:PVA ratio and the processing conditions of the mixtures, FsNMs with pores of ¿ ˜ 0.8 µm, 170 nm and 65 nm being achieved. Investigations on nanoperforated FsNMs (i.e. those with ¿ ˜ 170 and 65 nm), which are the more regular, reveal that pores crossing the entire membrane thickness do not affect the surface wettability of PLA but drastically enhances the cellular response of this biomaterial. Thus, cell proliferation assays indicate that cell viability in PLA with perforations of ¿ ˜ 170 nm is ~2.6 and ~2.2 higher than in non-perforated PLA and PLA with perforations of ¿ ˜ 65 nm, respectively. This excellent response has been attributed to the similarity between the nanoperforations with ¿ ˜ 170 nm and the filopodia filaments in cells (¿ ˜ 100–200 nm), which play a crucial role in cell migration processes. The favorable interaction between the perforated membrane nanofeatures and cell filopodia has been corroborated by optical and scanning electron microscopies.Peer ReviewedPostprint (author's final draft

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 ReviewedPostprint (author's final draft

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

Encapsulation and storage of therapeutic fibrin-homing peptides using conducting polymer nanoparticles for programmed release by electrical stimulation

Cys-Arg-Glu-Lys-Ala (CREKA) is an important fibrin-homing pentapeptide that has been extensively demonstrated for diagnoses and therapies (e.g., image diagnosis of tumors and to inhibit tumor cell migration and invasion). Although CREKA-loaded nanoparticles (NPs) have received major interest as efficient biomedical systems for cancer diagnosis and treatment, almost no control on the peptide release has been achieved yet. Herein, we report the development of conductive polymer NPs as therapeutic CREKA carriers for controlled dose administration through electric stimuli. Furthermore, the study was extended to CR(NMe)EKA, a previously engineered CREKA analogue in which Glu was replaced by N-methyl-Glu for improvement of the peptide resistance against proteolysis, which is one of the major weaknesses of therapeutic peptide delivery, and for enhancement of the tumor homing capacity by overstabilizing the bioactive conformation. Particularly, the present work is focused on understanding the interactions between the newly designed nanoengineered materials and biological fluids and the achievement of a modulated peptide release by fine-tuning the electrical stimuli. Two different types of stimuli were compared, chronoamperometry versus cyclic voltammetry, the latter being more effectivePeer ReviewedPostprint (author's final draft

Thermomechanical Response of a Representative Porin for Biomimetics

The thermomechanical response of Omp2a, a representative porin used for the fabrication of smart biomimetic nanomembranes, has been characterized using microcantilever technology and compared with standard proteins. For this purpose, thermally induced transitions involving the conversion of stable trimers to bigger aggregates, local reorganizations based on the strengthening or weakening of intermolecular interactions, and protein denaturation have been detected by the microcantilever resonance frequency and deflection as a function of the temperature. Measurements have been carried out on arrays of 8-microcantilevers functionalized with proteins (Omp2a, lysozyme and bovine serum albumin). To interpret the measured nanofeatures, the response of proteins to temperature has been also examined using other characterization techniques, including real time wide angle X-ray diffraction. Results not only demonstrate the complex behavior of porins, which exhibit multiple local thermal transitions before undergoing denaturation at temperatures higher than 105 °C, but also suggest a posttreatment to control the orientation of immobilized Omp2a molecules in functionalized biomimetic nanomembranes and, thus, increase their efficacy in ion transport.Peer Reviewe

Perforated polyester nanomebranes as templates of electroactive and robust free-standing films

Robust and flexible free-standing films made of spin-coated poly(lactic acid) (PLA) and poly(3,4-ethylenedioxythiophene) (PEDOT) nanolayers have been prepared. A steel sheet coated with a sacrificial layer of PEDOT:poly(styrenesulfonate) (PSS) and a spin-coated nanolayer of PLA was used as working electrode for the anodic polymerization of 3,4-ethylenedioxythiophene monomer. The latter was only successfully accomplished when rounded-shape nanoperforations of average diameter 49¿±¿14¿nm were introduced into PLA layers, which was achieved by combining the phase segregation processes undergone by immiscible PLA:poly(vinyl alcohol) (PVA) mixtures with selective solvent etching to remove PVA domains. Nanoperforations allowed the utilization of the semiconducting PEDOT:PSS sacrificial layer to immobilize the electropolymerized PEDOT chains. Morphological and topographical studies show the templating effect of PEDOT layers. In addition of flexibility and mechanical strength, free-standing 5-layered films present good electrochemical activity, evidencing their potential ability to reversibly exchange ions with the medium. These properties offer important advantages with respect to those of neat PLA and supported PEDOT films, as has been illustrated by cell culture and protein adsorption assays. Cell cultures evidenced the superior behavior of 5-layered films as bioactive platforms for fibroblast and epithelial cells proliferation, while adsorption assays reflected their potential as selective bioadhesive surfaces for protein separationPeer 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

Remote spatiotemporal control of a magnetic and electroconductive hydrogel network via magnetic fields for soft electronic applications

Multifunctional hydrogels are a class of materials offering new opportunities for interfacing living organisms with machines due to their mechanical compliance, biocompatibility, and capacity to be triggered by external stimuli. Here, we report a dual magnetic- and electric-stimuli-responsive hydrogel with the capacity to be disassembled and reassembled up to three times through reversible cross-links. This allows its use as an electronic device (e.g., temperature sensor) in the cross-linked state and spatiotemporal control through narrow channels in the disassembled state via the application of magnetic fields, followed by reassembly. The hydrogel consists of an interpenetrated polymer network of alginate (Alg) and poly(3,4-ethylenedioxythiophene) (PEDOT), which imparts mechanical and electrical properties, respectively. In addition, the incorporation of magnetite nanoparticles (Fe3O4 NPs) endows the hydrogel with magnetic properties. After structural, (electro)chemical, and physical characterization, we successfully performed dynamic and continuous transport of the hydrogel through disassembly, transporting the polymer–Fe3O4 NP aggregates toward a target using magnetic fields and its final reassembly to recover the multifunctional hydrogel in the cross-linked state. We also successfully tested the PEDOT/Alg/Fe3O4 NP hydrogel for temperature sensing and magnetic hyperthermia after various disassembly/re-cross-linking cycles. The present methodology can pave the way to a new generation of soft electronic devices with the capacity to be remotely transported.Peer ReviewedPostprint (author's final draft