Nanostructured functional multilayer coatings incorporating biomimetic macromolecules for biomedical applications

Tese de doutoramento do Programa Doutoral em Engenharia BiomédicaThe modification of surfaces has been a key aspect in biology and biotechnology, for applications including cell expansion, biomaterials development and preparation of substrates for regenerative medicine. In this thesis, the layer-by-layer (LbL) technique was employed in the modification of surfaces for multiple purposes, namely for films with improved adhesiveness, enhanced cell adhesion, drug delivery capsules, and magnetic spatial positioning. The working hypothesis was that LbL could be used in the modification of surfaces not only planar but also three-dimensional, using solely polymeric materials from a variety of natural origins or obtained by recombinant routes. Herein, chitosan (CHI), a well-known polycation with marine origins, was used recurrently as an ingredient in various multilayer formulations, driven by intermolecular forces such as electrostatic and hydrophobic interactions. First, multilayer coatings of CHI and an adhesive bacterial exopolysaccharide, levan, were fabricated. As a control, CHI and alginate films – two marine polysaccharides often regarded as good natural adhesives – were assembled in parallel. The adhesive strength of 50 CHI/levan bilayers was determined to be about 3 times higher than the control. The adhesion of L929 cells was also higher in levan-based films. Next, CHI was used along polyanionic elastin-like recombinamers (ELRs), a recombinant class of elastin-like polymers, exhibiting the cell adhesion motif arginine-glycine-aspartic acid (RGD). Although electrostatic interactions were relied upon to construct LbL coatings, they were not the sole mechanism of buildup between CHI and ELRs. The film construction of CHI and one of nine ELRs differing in amino acid content, length and biofunctionality was followed in situ at pH 4.0 and 5.5 using a quartz-crystal microbalance. Their thicknesses were estimated using the Voigt-based model for viscoelastic films, revealing that thicker films were obtained in the presence of hydrophobic interactions between ELRs and partially neutralized chitosan, i.e., when the pH was adjusted to 5.5, near its pKa. ELR/alginate coatings were also monitored, completing a total of 36 combinations studied. The results agreed with the ones from the CHI-based films: thicker films were obtained for partially neutralized alginate, at a pH value of 4.0. Bidimensional coatings of CHI and CHI/ELR-RGD were demonstrated to be stimuli-responsive by means of wettability measurements: they exhibited a moderate hydrophobic surface but switched to an extremely hydrophilic one when temperature, pH or ionic strength were raised above 50ºC, 11, or 1.25 M, respectively. The presence of RGD enhanced the adhesion of SaOs-2 cells, in comparison to films ending either in CHI or a nonbioactive RDG analogue. CHI/ELR-RGD coatings were extrapolated to the third dimension as spherical microcapsules assembled around calcium carbonate particles. Aiming at drug administration, two mechanisms were studied: (i) simple diffusion out of the microcapsules, and (ii) cellular uptake. In the first case, release studies at 25 and 37°C with “bovine serum albumin”-loaded microcapsules demonstrated that they provided a sustained release over 14 days, but with reduced capsule permeability at physiological temperature, due to the temperature-induced molecular transition of ELRs. Furthermore, a higher numbers of layers provided an added barrier to the diffusion of the encapsulated protein. The microcapsules were also noncytotoxic towards L929 cells. In the second case, the internalization efficacy and intracellular traffic of RGD- or nonbioactive RDG-functionalized microcapsules by human mesenchymal stem cells (hMSCs) was assessed. The data suggests that microcapsules were internalized mainly through macropinocytosis and that surface functionalization did not play a significant role on this phenomenon, although intracellular processing was faster for RGD-functionalized microcapsules. Both microcapsule types were noncytotoxic toward hMSCs for microcapsule/cell ratios between 5:1 and 100:1. Finally, inspired by the complex hierarchical and compartmentalized structure of cells, liquefied alginate macroscopic spheres coated with a chitosan/alginate shell were conceived. Within this liquefied environment, rhodamine and multilayer microcapsules were confined, with the latter encapsulating further either rhodamine or magnetic nanoparticles (MNPs). At 25 and 37ºC, rhodamine when encapsulated within the inner microcapsules showed a sustained release profile, with the diffusion kinetics being even more reduced at 25ºC. The release of rhodamine encapsulated in the outer liquefied alginate compartment did not show significant differences as a function of temperature. Encapsulating MNPs within the microcapsules provided magnetic responsiveness to the whole compartmentalized device and guided mobility capability. The developed work shows that LbL is a versatile technique that provides the means to develop a wide array of systems useful in biomedical applications, from adhesive and cell seeding planar supports to three dimensional structures for smart drug delivery, guided therapies and customized microreactors as disease models and microtissue production in laboratory.A modificação de superfícies tem sido um aspeto fundamental em biologia e biotecnologia, em aplicações como a expansão de células, desenvolvimento de biomateriais e preparação de substratos para medicina regenerativa. Neste trabalho, a técnica de camada-a-camada foi utilizada na modificação de superfícies para vários fins, como filmes com adesividade e adesão celular melhoradas, cápsulas para administração de drogas, e o posicionamento espacial magnético. A hipótese foi a de que esta técnica poderia ser utilizada na modificação de superfícies planares ou tridimensionais, usando exclusivamente polímeros obtidos de várias origens naturais ou a partir de vias recombinantes. O quitosano (CHI), um reconhecido policatião com origens marinhas, foi recorrentemente utilizado como ingrediente em várias formulações de multicamadas, impulsionado por forças intermoleculares, tais como interações eletrostáticas e hidrofóbicas. Primeiro, revestimentos de CHI e um exopolissacarídeo bacteriano adesivo, levano, foram construídos. Como controlo, foram comparados com filmes de CHI e alginato – polissacarídeos marinhos considerados bons adesivos naturais. A força adesiva de 50 bicamadas de CHI/levano foi determinada como sendo cerca de 3 vezes mais elevada do que o controlo. A adesão de células L929 foi também maior em filmes contendo levano. Em seguida, CHI foi utilizado juntamente com recombinâmeros tipo-elastina (ELRs), uma classe de polímeros tipo-elastina, exibindo a sequência de adesão celular “arginina-glicina-ácido aspártico” (RGD). Embora interações eletrostáticas tenham sido invocadas para a construção de multicamadas auto-organizadas, estas não foram o único mecanismo de interação entre CHI e ELRs. A construção de filmes de CHI e um de nove ELRs diferentes em conteúdo aminoacídico, tamanho e biofuncionalidade foi monitorizado in situ a pH 4.0 e 5.5 utilizando uma microbalança de cristais de quartzo. As suas espessuras foram estimadas usando o modelo de Voigt para filmes viscoelásticos, revelando que os filmes mais espessos foram obtidos na presença de interações hidrofóbicas entre ELRs e CHI parcialmente neutralizado, isto é, quando o pH foi ajustado para 5.5, próximo do seu pKa. Filmes de ELR/alginato também foram monitorados, completando um total de 36 combinações estudadas. Os resultados obtidos estiveram em concordância com os dados dos filmes baseados em CHI: filmes mais espessos foram obtidos para alginato parcialmente neutralizado, a um valor de pH de 4.0. Através de medições de ângulos de contato, demonstrou-se que os revestimentos bidimensionais de CHI e ELR-RGD eram responsivos a estímulos: exibiram uma superfície moderadamente hidrofóbica mas converteram-se em extremamente hidrofílicas quando se aumentou a temperatura, o pH ou força iónica acima de 50ºC, 11, ou 1,25 M, respetivamente. A presença de RGD melhorou a adesão de células SaOs-2, em comparação com filmes terminados ou em CHI ou num análogo não bioativo, RDG. Os revestimentos de CHI/ELR-RGD foram extrapolados para a terceira dimensão sob a forma de microcápsulas esféricas construídas em torno de partículas de carbonato de cálcio. Com a finalidade de administração de drogas, foram estudados dois mecanismos: (i) a difusão simples para o exterior das microcápsulas e (ii) a incorporação celular. No primeiro caso, estudos de libertação a 25 e 37°C com microcápsulas contendo albumina do soro bovino demonstraram uma libertação sustentada ao longo de 14 dias, mas sendo as cápsulas menos permeáveis a uma temperatura fisiológica, devido à transição molecular dos ELRs induzida pela temperatura. Além disso, um número mais elevado de camadas proporcionou uma barreira adicional à difusão da proteína encapsulada. As microcápsulas foram também não citotóxicas para células L929. No segundo caso, a eficácia de internalização e tráfego intracelular de microcápsulas funcionalizadas com RGD ou a sequência não bioativa RDG por células estaminais mesenquimais humanas (hMSCs) foi avaliada. Os dados sugerem que as microcápsulas foram internalizadas principalmente através de macropinocitose, e que a funcionalização da superfície não desempenhou um papel significativo neste fenómeno, embora o processamento intracelular tenha sido mais rápido para microcápsulas funcionalizadas com RGD. Ambos os tipos de microcápsulas foram não citotóxicas para hMSCs para rácios de microcápsula/célula entre 5:1 e 100:1. Finalmente, com inspiração na hierarquia complexa e estrutura compartimentada das células, esferas macroscópicas de alginato liquefeito revestidas com camadas de CHI/alginato foram concebidas. Dentro deste ambiente liquefeito, rodamina e microcápsulas foram confinadas, com estas últimas podendo conter ou mais rodamina ou nanopartículas magnéticas (MNPs). A 25 e 37ºC, rodamina quando encapsulada no interior de microcápsulas mostrou um perfil de libertação sustentada, sendo a cinética de difusão ainda mais reduzida a 25°C. A libertação de rodamina encapsulada no compartimento externo de alginato não exibiu diferenças significativas em função da temperatura. MNPs encapsuladas dentro das microcápsulas providenciaram resposta magnética a todo o dispositivo compartimentado e capacidade de mobilidade dirigida. O trabalho aqui desenvolvido mostra que a técnica de modificação camada-a-camada é uma técnica versátil capaz de fornecer meios para desenvolver uma ampla gama de sistemas úteis em aplicações biomédicas, desde suportes planos adesivos e para adesão celular até estruturas tridimensionais para a administração “inteligente” de drogas, terapias guiadas e microreatores personalizados para o desenvolvimento de modelos de doenças e produção de microtecidos em laboratório

Layer-by-layer coated calcium carbonate nanoparticles for targeting breast cancer cells

Breast cancer is resistant to conventional treatments due to the specific tumour microenvironment, the associated acidic pH and the overexpression of receptors that enhance cells tumorigenicity. Herein, we optimized the synthesis of acidic resorbable calcium carbonate (CaCO3) nanoparticles and the encapsulation of a low molecular weight model molecule (Rhodamine). The addition of ethylene glycol during the synthetic process resulted in a particle size decrease: we obtained homogeneous CaCO3 particles with an average size of 564 nm. Their negative charge enabled the assembly of layer-by-layer (LbL) coatings with surface-exposed hyaluronic acid (HA), a ligand of tumour-associated receptor CD44. The coating decreased Rhodamine release by two-fold compared to uncoated nanoparticles. We demonstrated the effect of nanoparticles on two breast cancer cell lines with different aggressiveness â SK-BR-3 and the more aggressive MDA-MB-231 â and compared them with the normal breast cell line MCF10A. CaCO3 nanoparticles (coated and uncoated) significantly decreased the metabolic activity of the breast cancer cells. The interactions between LbL-coated nanoparticles and cells depended on HA expression on the cell surface: more particles were observed on the surface of MDA-MB-231 cells, which had the thickest endogenous HA coating. We concluded that CaCO3 nanoparticles are potential candidates to carry low molecular weight chemotherapeutics and deliver them to aggressive breast cancer sites with an HA-abundant pericellular matrix. This work was supported by the Fundação para a Ciência e Tecnologia (project OncoNeoTreat, grant number PTDC/CTM-REF/0022/2020, co-financed by FCT – OE component); and the European program FEDER/FEEI. R.R.C. acknowledges FCT for support through grants 2022.00764.CEECIND and CEECIND/02842/2017. D.S.C. thanks FCT for the grant SFRH/BPD/85790/2012. Parts of Fig. A.1 were drawn by using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/)

Mineralization of layer-by-layer ultrathin films containing microfluidic-produced hydroxyapatite nanorods

We describe the assembly of layer-by-layer (LbL) ultrathin films containing bioactive hydroxyapatite (HAp) rod-shaped nanoparticles with mineralizing capacity. Monodisperse 96 nm long and 9 nm wide HAp nanorods with a surface charge of â 14 mV were produced with a microfluidic system. The negatively charged HAp nanorods were assembled with the polycation poly-L-lysine (PLL) in LbL fashion. The successful deposition of alternating layers was confirmed by quartz-crystal microbalance with dissipation monitoring. The Voigt-based viscoelastic model demonstrated steady film growth where three PLL/HAp bilayers reached a thickness of 70 nm. The bioactivity of [PLL/HAp]3 was evaluated in vitro by following the formation of a mineralized hydroxyapatite layer in simulated body fluid (SBF). X-ray diffraction, energy-dispersive X-ray spectroscopy and scanning electron microscopy (SEM) demonstrated formation of a crystalline hydroxyapatite layer and complete surface coverage within 7 days. SaOs-2 osteoblasts-like cells attached to the mineralized surfaces and developed longer filopodia extensions when compared to non-mineralized samples. Our results showed that [PLL/HAp]3 films are feasible osteoconductive coatings applicable to orthopedic implants and fixation devices.The authors acknowledge Fundación Ramón Areces, Xunta de Galicia (ED431B 2017/21, ED41E2018/08), Programa Iacobus, financial support from “Fundação para a Ciência e Tecnologia” (grant CEECIND/02842/2017 to R. R. C. and project Norte-01-0145-FEDER-022190) and the EC H2020 programme through the projects ELASTISLET (NMP-2014-646075

MAMMALS IN PORTUGAL : A data set of terrestrial, volant, and marine mammal occurrences in P ortugal

Mammals are threatened worldwide, with 26% of all species being includedin the IUCN threatened categories. This overall pattern is primarily associatedwith habitat loss or degradation, and human persecution for terrestrial mam-mals, and pollution, open net fishing, climate change, and prey depletion formarine mammals. Mammals play a key role in maintaining ecosystems func-tionality and resilience, and therefore information on their distribution is cru-cial to delineate and support conservation actions. MAMMALS INPORTUGAL is a publicly available data set compiling unpublishedgeoreferenced occurrence records of 92 terrestrial, volant, and marine mam-mals in mainland Portugal and archipelagos of the Azores and Madeira thatincludes 105,026 data entries between 1873 and 2021 (72% of the data occur-ring in 2000 and 2021). The methods used to collect the data were: live obser-vations/captures (43%), sign surveys (35%), camera trapping (16%),bioacoustics surveys (4%) and radiotracking, and inquiries that represent lessthan 1% of the records. The data set includes 13 types of records: (1) burrowsjsoil moundsjtunnel, (2) capture, (3) colony, (4) dead animaljhairjskullsjjaws, (5) genetic confirmation, (6) inquiries, (7) observation of live animal (8),observation in shelters, (9) photo trappingjvideo, (10) predators dietjpelletsjpine cones/nuts, (11) scatjtrackjditch, (12) telemetry and (13) vocalizationjecholocation. The spatial uncertainty of most records ranges between 0 and100 m (76%). Rodentia (n=31,573) has the highest number of records followedby Chiroptera (n=18,857), Carnivora (n=18,594), Lagomorpha (n=17,496),Cetartiodactyla (n=11,568) and Eulipotyphla (n=7008). The data setincludes records of species classified by the IUCN as threatened(e.g.,Oryctolagus cuniculus[n=12,159],Monachus monachus[n=1,512],andLynx pardinus[n=197]). We believe that this data set may stimulate thepublication of other European countries data sets that would certainly contrib-ute to ecology and conservation-related research, and therefore assisting onthe development of more accurate and tailored conservation managementstrategies for each species. There are no copyright restrictions; please cite thisdata paper when the data are used in publications.info:eu-repo/semantics/publishedVersio

Nationwide access to endovascular treatment for acute ischemic stroke in portugal

Publisher Copyright: Copyright Ordem dos M dicos 2021.Introduction: Since the publication of endovascular treatment trials and European Stroke Guidelines, Portugal has re-organized stroke healthcare. The nine centers performing endovascular treatment are not equally distributed within the country, which may lead to differential access to endovascular treatment. Our main aim was to perform a descriptive analysis of the main treatment metrics regarding endovascular treatment in mainland Portugal and its administrative districts. Material and Methods: A retrospective national multicentric cohort study was conducted, including all ischemic stroke patients treated with endovascular treatment in mainland Portugal over two years (July 2015 to June 2017). All endovascular treatment centers contributed to an anonymized database. Demographic, stroke-related and procedure-related variables were collected. Crude endovascular treatment rates were calculated per 100 000 inhabitants for mainland Portugal, and each district and endovascular treatment standardized ratios (indirect age-sex standardization) were also calculated. Patient time metrics were computed as the median time between stroke onset, first-door, and puncture. Results: A total of 1625 endovascular treatment procedures were registered. The endovascular treatment rate was 8.27/100 000 inhabitants/year. We found regional heterogeneity in endovascular treatment rates (1.58 to 16.53/100 000/year), with higher rates in districts closer to endovascular treatment centers. When analyzed by district, the median time from stroke onset to puncture ranged from 212 to 432 minutes, reflecting regional heterogeneity. Discussion: Overall endovascular treatment rates and procedural times in Portugal are comparable to other international registries. We found geographic heterogeneity, with lower endovascular treatment rates and longer onset-to-puncture time in southern and inner regions. Conclusion: The overall national rate of EVT in the first two years after the organization of EVT-capable centers is one of the highest among European countries, however, significant regional disparities were documented. Moreover, stroke-onset-to-first-door times and in-hospital procedural times in the EVT centers were comparable to those reported in the randomized controlled trials performed in high-volume tertiary hospitalspublishersversionpublishe

Acesso a Tratamento Endovascular para Acidente Vascular Cerebral Isquémico em Portugal

Introduction: Since the publication of endovascular treatment trials and European Stroke Guidelines, Portugal has re-organized stroke healthcare. The nine centers performing endovascular treatment are not equally distributed within the country, which may lead to differential access to endovascular treatment. Our main aim was to perform a descriptive analysis of the main treatment metrics regarding endovascular treatment in mainland Portugal and its administrative districts. Material and Methods: A retrospective national multicentric cohort study was conducted, including all ischemic stroke patients treated with endovascular treatment in mainland Portugal over two years (July 2015 to June 2017). All endovascular treatment centers contributed to an anonymized database. Demographic, stroke-related and procedure-related variables were collected. Crude endovascular treatment rates were calculated per 100 000 inhabitants for mainland Portugal, and each district and endovascular treatment standardized ratios (indirect age-sex standardization) were also calculated. Patient time metrics were computed as the median time between stroke onset, first-door, and puncture. Results: A total of 1625 endovascular treatment procedures were registered. The endovascular treatment rate was 8.27/100 000 inhabitants/year. We found regional heterogeneity in endovascular treatment rates (1.58 to 16.53/100 000/year), with higher rates in districts closer to endovascular treatment centers. When analyzed by district, the median time from stroke onset to puncture ranged from 212 to 432 minutes, reflecting regional heterogeneity. Conclusion: The overall national rate of EVT in the first two years after the organization of EVT-capable centers is one of the highest among European countries, however, significant regional disparities were documented. Moreover, stroke-onset-to-first-door times and in-hospital procedural times in the EVT centers were comparable to those reported in the randomized controlled trials performed in high-volume tertiary hospitals.Introdução: A aprovação do tratamento endovascular para o acidente vascular cerebral isquémico obrigou à reorganização dos cuidados de saúde em Portugal. Os nove centros que realizam tratamento endovascular não estão distribuídos equitativamente pelo território, o que poderá causar acesso diferencial a tratamento. O principal objetivo deste estudo é realizar uma análise descritiva da frequência e métricas temporais do tratamento endovascular em Portugal continental e seus distritos. Material e Métodos: Estudo de coorte nacional multicêntrico, incluindo todos os doentes com acidente vascular cerebral isquémico submetidos a tratamento endovascular em Portugal continental durante um período de dois anos (julho 2015 a junho 2017). Foram colhidos dados demográficos, relacionados com o acidente vascular cerebral e variáveis do procedimento. Taxas de tratamento endovascular brutas e ajustadas (ajuste indireto a idade e sexo) foram calculadas por 100 000 habitantes/ano para Portugal continental e cada distrito. Métricas de procedimento como tempo entre instalação, primeira porta e punção foram também analisadas. Resultados: Foram registados 1625 tratamentos endovasculares, indicando uma taxa bruta nacional de tratamento endovascular de 8,27/100 000 habitantes/ano. As taxas de tratamento endovascular entre distritos variaram entre 1,58 e 16,53/100 000/ano, com taxas mais elevadas nos distritos próximos a hospitais com tratamento endovascular. O tempo entre sintomas e punção femural entre distritos variou entre 212 e 432 minutos. Conclusão: Portugal continental apresenta uma taxa nacional de tratamento endovascular elevada, apresentando, contudo, assimetrias regionais no acesso. As métricas temporais foram comparáveis com as observadas nos ensaios clínicos piloto

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements

Mammals in Portugal: a data set of terrestrial, volant, and marine mammal occurrences in Portugal

Mammals are threatened worldwide, with ~26% of all species being included in the IUCN threatened categories. This overall pattern is primarily associated with habitat loss or degradation, and human persecution for terrestrial mammals, and pollution, open net fishing, climate change, and prey depletion for marine mammals. Mammals play a key role in maintaining ecosystems functionality and resilience, and therefore information on their distribution is crucial to delineate and support conservation actions. MAMMALS IN PORTUGAL is a publicly available data set compiling unpublished georeferenced occurrence records of 92 terrestrial, volant, and marine mammals in mainland Portugal and archipelagos of the Azores and Madeira that includes 105,026 data entries between 1873 and 2021 (72% of the data occurring in 2000 and 2021). The methods used to collect the data were: live observations/captures (43%), sign surveys (35%), camera trapping (16%), bioacoustics surveys (4%) and radiotracking, and inquiries that represent less than 1% of the records. The data set includes 13 types of records: (1) burrows | soil mounds | tunnel, (2) capture, (3) colony, (4) dead animal | hair | skulls | jaws, (5) genetic confirmation, (6) inquiries, (7) observation of live animal (8), observation in shelters, (9) photo trapping | video, (10) predators diet | pellets | pine cones/nuts, (11) scat | track | ditch, (12) telemetry and (13) vocalization | echolocation. The spatial uncertainty of most records ranges between 0 and 100 m (76%). Rodentia (n =31,573) has the highest number of records followed by Chiroptera (n = 18,857), Carnivora (n = 18,594), Lagomorpha (n = 17,496), Cetartiodactyla (n = 11,568) and Eulipotyphla (n = 7008). The data set includes records of species classified by the IUCN as threatened (e.g., Oryctolagus cuniculus [n = 12,159], Monachus monachus [n = 1,512], and Lynx pardinus [n = 197]). We believe that this data set may stimulate the publication of other European countries data sets that would certainly contribute to ecology and conservation-related research, and therefore assisting on the development of more accurate and tailored conservation management strategies for each species. There are no copyright restrictions; please cite this data paper when the data are used in publications