Functionalized CoCr surfaces with adhesive molecules to improve endothelialization

Versió amb diverses seccions encriptades, per drets a'autorCobalt-chromium (CoCr) alloys are widely used as biomaterials for coronary stents due to their excellent mechanical properties, biocompatibility and corrosion resistance. However, these materials are bioinert, retarding the complete endothelialization and resulting in a higher risk of restenosis, narrowing of the artery, and late-stent thrombosis. Therefore, the improvement of implants surface endothelialization has acquired importance in the last years. Immobilization of cell adhesive biomolecules onto biomaterials surface is a well-known strategy to control cell response. However, the strategy of immobilization, the optimal combination or the appropriate spatial presentation of the bioactive sequences to enhance endothelialization for cardiovascular applications, remains to be elucidated. The present PhD thesis focused on the development of a new biofunctionalized CoCr alloy surfaces in order to improve the endothelialization. To that end, elastin-like recombinamers (ELR) genetically modified with an REDV (Arg-Glu-Asp-Val) sequence and short synthetized peptides RGDS (Arg-Gly-Asp-Ser), REDV, YIGSR (Tyr-Ile-Gly-Ser-Arg) and their equimolar combination, were attached by physisorption and covalent bonding onto CoCr alloy surfaces and thoroughly characterized physico-chemically and evaluated in vitro with human umbilical vein endothelial cells (HUVECs), coronary artery smooth muscle cells (CASMCs) and platelets from blood donors. First, biofunctionalized surfaces with ELR were developed and optimized by evaluating different surface activation treatments, oxygen plasma and sodium hydroxide etching, and different binding strategies, physisorption and covalent bonding. The functionalized surfaces demonstrated a higher cell adhesion and spreading of HUVEC cells, this effect is emphasized as increases the amount of immobilized biomolecules and directly related to the immobilization technique: covalent bonding. Nevertheless, the silanization process was not completely effective since a mixture of covalent and physisorption behavior was observed probably due to the use of big molecules that decreased the control of the bonding between the biomolecule and the surface. Secondly, it was synthetized immobilized RGDS, REDV, YIGSR and their equimolar combination peptides onto the different surfaces. Cell studies demonstrated that the covalent functionalization of CoCr surfaces with an equimolar combination of RGDS/YIGSR represented the most powerful strategy to enhance the early stages of HUVECs adhesion, proliferation and migration, indicating a positive synergistic effect between the two peptide motifs. Besides, gene expression and platelet adhesion studies showed that surfaces silanized with the combination RGDS/YIGSR improved anti-thrombogenicity compared to non-modified surfaces. Finally, cell co-cultures of HUVECs/CASMCs found that functionalization increased the amount of adhered HUVECs onto modified surfaces compared to plain CoCr, independently of the used peptide and the strategy of immobilization. Overall, the present thesis offer a comprehensive view of the effectiveness of immobilizing cell adhesive molecules onto CoCr alloy surfaces to enhance endothelialization while preventing restenosis and thrombosis for cardiovascular applications.Las aleaciones de cobalto-cromo (CoCr) son ampliamente utilizadas como biomateriales para stents coronarios debido a sus excelentes propiedades mecánicas, biocompatibilidad y resistencia a la corrosión. Sin embargo, estos materiales son bio-inertales, retardando la completa endotelialización y resultando en un mayor riesgo de reestenosis, estrechamiento de la arteria y trombosis tardía. Por lo tanto, la mejora de la endotelización de superficie de los implantes ha adquirido importancia en los últimos años. La inmovilización de biomoléculas adhesivas celulares sobre la superficie de los biomateriales es una estrategia bien conocida para controlar la respuesta celular. Sin embargo, queda por aclararse la estrategia de inmovilización, la combinación óptima o la presentación espacial apropiada de las secuencias bioactivas para potenciar la endotelización en aplicaciones cardiovasculares. La presente tesis doctoral se centró en el desarrollo de una nueva superficie biofuncionalizada de la aleación CoCr con el fin de mejorar la endotelialización. Para ello, los recombinameros tipo elastina (ELR) modificados genéticamente con una secuencia REDV (Arg-Glu-Asp-Val) y péptidos cortos sintetizados RGDS (Arg-Gly-Asp-Ser), REDV, YIGSR (Tyr-Ile-Gly -Ser-Arg) y su combinación equimolar, fueron anclados por fisisorción y unión covalente a las superficies de la aleación, se caracterizaron físicamente-químicamente y evaluó in vitro con células endoteliales de vena umbilical humana (HUVECs), células de músculo liso de arteria coronaria (CASMCs) y plaquetas de donantes de sangre. En primer lugar, se desarrollaron y optimizaron las superficies biofuncionalizadas con ELR mediante la evaluación de diferentes tratamientos de activación superficial: plasma de oxígeno y ataque con hidróxido de sodio, y diferentes estrategias de unión: fisisorción y unión covalente. Las superficies funcionalizadas demostraron una mayor adhesión celular y propagación de células HUVEC, este efecto se enfatiza a medida que aumenta la cantidad de biomoléculas inmovilizadas y se relaciona directamente con la técnica de inmovilización: la unión covalente. Sin embargo, el proceso de silanización no fue completamente efectivo, ya que se observó una mezcla de uniones covalentes y fisisorbidas, probablemente debido al uso de grandes moléculas que disminuyeron el control de la unión entre la biomolécula y la superficie. En segundo lugar, se inmovilizado los péptidos sintetizados RGDS, REDV, YIGSR y sus combinaciones equimolares sobre las diferentes superficies. Los estudios celulares demostraron que la funcionalización covalente de las superficies de CoCr con una combinación equimolar de RGDS/YIGSR representó la estrategia más potente para potenciar las etapas tempranas de la adhesión, proliferación y migración de HUVECs, indicando un efecto sinérgico positivo entre los dos motivos peptídicos. Además, la expresión génica y los estudios de adhesión plaquetaria mostraron que las superficies silanizadas con la combinación RGDS/YIGSR mejoraron la antitrombogenicidad en comparación con las superficies no modificadas. Finalmente, con los co-cultivos celulares de HUVECs/CASMCs se encontró que la funcionalización aumentaba la cantidad de HUVEC adheridas sobre las superficies modificadas en comparación con CoCr simple, independientemente del péptido usado y la estrategia de inmovilización. En general, la presente tesis ofrece una visión completa de la efectividad de la inmovilización de moléculas adhesivas celulares en la superficie de la aleación CoCr para mejorar la endotelialización, mientras que previene la reestenosis y trombosis en aplicaciones cardiovasculares.Postprint (published version

Safety and efficacy of ApTOLL in patients with ischemic stroke undergoing endovascular treatment: a phase 1/2 randomized clinical trial

Importance ApTOLL is a TLR4 antagonist with proven preclinical neuroprotective effect and a safe profile in healthy volunteers. Objective To assess the safety and efficacy of ApTOLL in combination with endovascular treatment (EVT) for patients with ischemic stroke. Design, Setting, and Participants This phase 1b/2a, double-blind, randomized, placebo-controlled study was conducted at 15 sites in Spain and France from 2020 to 2022. Participants included patients aged 18 to 90 years who had ischemic stroke due to large vessel occlusion and were seen within 6 hours after stroke onset; other criteria were an Alberta Stroke Program Early CT Score of 6 to 10, estimated infarct core volume on baseline computed tomography perfusion of 5 to 70 mL, and the intention to undergo EVT. During the study period, 4174 patients underwent EVT. Interventions In phase 1b, 0.025, 0.05, 0.1, or 0.2 mg/kg of ApTOLL or placebo; in phase 2a, 0.05 or 0.2 mg/kg of ApTOLL or placebo; and in both phases, treatment with EVT and intravenous thrombolysis if indicated. Main Outcomes and Measures The primary end point was the safety of ApTOLL based on death, symptomatic intracranial hemorrhage (sICH), malignant stroke, and recurrent stroke. Secondary efficacy end points included final infarct volume (via MRI at 72 hours), NIHSS score at 72 hours, and disability at 90 days (modified Rankin Scale [mRS] score). Results In phase Ib, 32 patients were allocated evenly to the 4 dose groups. After phase 1b was completed with no safety concerns, 2 doses were selected for phase 2a; these 119 patients were randomized to receive ApTOLL, 0.05 mg/kg (n¿=¿36); ApTOLL, 0.2 mg/kg (n¿=¿36), or placebo (n¿=¿47) in a 1:1:v2 ratio. The pooled population of 139 patients had a mean (SD) age of 70 (12) years, 81 patients (58%) were male, and 58 (42%) were female. The primary end point occurred in 16 of 55 patients (29%) receiving placebo (10 deaths [18.2%], 4 sICH [7.3%], 4 malignant strokes [7.3%], and 2 recurrent strokes [3.6%]); in 15 of 42 patients (36%) receiving ApTOLL, 0.05 mg/kg (11 deaths [26.2%], 3 sICH [7.2%], 2 malignant strokes [4.8%], and 2 recurrent strokes [4.8%]); and in 6 of 42 patients (14%) receiving ApTOLL, 0.2 mg/kg (2 deaths [4.8%], 2 sICH [4.8%], and 3 recurrent strokes [7.1%]). ApTOLL, 0.2 mg/kg, was associated with lower NIHSS score at 72 hours (mean difference log-transformed vs placebo, -45%; 95% CI, -67% to -10%), smaller final infarct volume (mean difference log-transformed vs placebo, -42%; 95% CI, -66% to 1%), and lower degrees of disability at 90 days (common odds ratio for a better outcome vs placebo, 2.44; 95% CI, 1.76 to 5.00). Conclusions and Relevance In acute ischemic stroke, 0.2 mg/kg of ApTOLL administered within 6 hours of onset in combination with EVT was safe and associated with a potential meaningful clinical effect, reducing mortality and disability at 90 days compared with placebo. These preliminary findings await confirmation from larger pivotal trials.Peer ReviewedPostprint (author's final draft