Fibrous braided stents with antibacterial properties

Tese de Doutoramento (Programa Doutoral em Engenharia Biomédica)Nowadays, cardiovascular diseases are reported as a major cause of death and disability in developed countries. Therefore, stents have emerged as a solution to overcome this problem. However, common commercialize stents, are metallic, which present several disadvantages as corrosion, higher risk of restenosis and infection. In order to minimize the disadvantages associated with these stents, new materials, like fibrous materials, have begun to be used as well as surface modifications of biomaterials begun to be applied, in order to avoid those drawbacks, especially infection. Therefore, the main objective of this work was the development of a fibrous stent, able to compete with the mechanical properties of the commercial ones, with the advantaged of being coated with an antibacterial agent, able to avoid infections. For this purpose, braided stents were produced by varying materials, structural and process parameters, such as monofilament type and diameter, braiding angle and mandrel diameter. The influence of these design parameters on mechanical behavior, as well as stent's porosity, was thoroughly investigated, and suitable parameters were selected for developing a stent with mechanical characteristics and porosity matching with the commercial stents. According to the experimental results, suitable performance was achieved with a polyester stent designed with: monofilament diameter of 270 µm, braiding angle of 35°; and mandrel diameter of 6 mm, providing similar properties to commercial Nitinol stents (porosity above 70%, unchanged diameter during bending tests above 75%, force for longitudinal compression between [0.16-5.28] N and force for radial compression between [1.13-2.90] N). After the stent’s development, silver and silver oxide thin films were deposited by non-reactive and reactive pulsed dc magnetron sputtering. The coatings were characterized chemical, physical and structurally. This first approach in stent’s functionalization revealed that silver thin film formed a continuous layer, while silver oxide layer was composed of islands with hundreds of nanometers surrounded by small nanoparticles with tens of nanometers. In order to verify the antibacterial behavior of the coatings, halo inhibition zone tests were realized for Staphylococcus epidermidis and Staphylococcus aureus. Silver coatings did not show antibacterial behavior, contrarily to silver oxide coatings, which presented antibacterial properties against the studied bacteria. The presence of silver oxide phase along with the development of different morphology were pointed as the main factors in the origin of the antibacterial effect found in silver oxide thin film. With the purpose of understand the influence of oxygen species in the physical, chemical and structural properties of thin films new silver and silver oxide thin films were deposited, by nonreactive and reactive pulsed dc magnetron sputtering, with the variation of oxygen content. The resulted coatings revealed that silver coating forms a continuous layer. The incorporation of oxygen leads to the formation of a mixture of Ag2O + AgO phases. However, with the increase of the oxygen fraction, the mixture of oxides disappear and the resultant thin film became only AgO. Cytotoxicity tests were performed, demonstrating that in the silver oxide coating no cytotoxicity was found, making this coating able to be used in stents applications. The antibacterial behavior of coatings was, quantitatively, tested against Staphylococcus epidermis, showing that AgxO unlike Ag coating presented antibacterial behavior. The presence of silver oxide is the main reason for the antibacterial effect, probably due to the increased production of reactive oxygen species (ROS). Finally, and in order to evaluate the coating’s behavior when subjected to mechanical forces, coated silver and silver oxide stents were, again, mechanically tested, and no loss of adhesion or delamination were observed, indicating that coatings will support, adequately, the mechanical forces and the dangerous of delivering parts of coating in blood current will not occur. In conclusion, fibrous stents coated with silver oxide, present a good mechanical behavior, when compared with nitinol commercial stents, possess antibacterial properties and are no cytotoxic, which made this stent a promising candidate as a biomaterial and a viable substitute to metallic stents.Atualmente, as doenças cardiovasculares são consideradas a principal causa de morte e incapacidade nos países desenvolvidos. Os stents surgiram como uma solução para este problema, contudo, os que se encontram no mercado são metálicos, pelo que apresentam várias desvantagens como: corrosão, maior risco de reestenose e infeção. A fim de minimizar as desvantagens associadas a estes stents, novos materiais, como materiais fibrosos, começaram a ser utilizados, assim como, a modificação da superfície dos biomateriais, de forma a evitar a infeção. Consequentemente, o principal objetivo deste trabalho foi o desenvolvimento de um stent fibroso, capaz de competir com as propriedades mecânicas dos stents comerciais, com a vantagem de ser revestido com um agente antibacteriano, evitando, deste modo, a possibilidade de infeção. Para este efeito, foram produzidos diferentes stents entrançados, através da variação dos materiais, parâmetros estruturais e de processo, tais como: o tipo de monofilamento e o seu diâmetro, ângulo de entrançamento e diâmetro do mandril. A influência destes parâmetros no comportamento mecânico, bem como a porosidade dos stents foram estudadas detalhadamente. Para o desenvolvimento do stent foram selecionados os parâmetros adequados com o objetivo de obter caraterísticas mecânicas e porosidade compatíveis com os stents comerciais. De acordo com os resultados experimentais, a melhor performance foi obtida com um stent de poliéster com diâmetro de monofilamento de 270 µm, ângulo de entrançamento de 35° e o diâmetro do mandril de 6 mm, proporcionando propriedades semelhantes ao stents comerciais de nitinol (porosidade acima de70%, diâmetro inalterado durante os testes de dobragem acima de 75%, força de compressão longitudinal compreendida entre [0.16-5.28] N e força de compressão radial compreendida entre [1.13-2.90] N). Após a produção do stent, foram depositados filmes finos de prata e óxidos de prata, através da pulverização catódica pulsada em magnetrão, em regime não-reativo e reativo. Os revestimentos foram caracterizados química, física e estruturalmente. Esta primeira abordagem na funcionalização do stent revelou que o revestimento de prata formou um filme contínuo, enquanto o filme de óxido de prata era composto de ilhas, com centenas de nanómetros rodeadas por nanopartículas com dezenas de nanómetros. A fim de verificar o comportamento antibacteriano dos revestimentos, foram realizados testes de halo para Staphylococcus epidermidis e Staphylococcus aureus. Como resultado, o revestimento de prata não apresentou comportamento antibacteriano, ao contrário do revestimento de óxido de prata, que apresentou propriedades antibacterianas contra as bactérias estudadas. A presença da fase de óxido de prata juntamente com o desenvolvimento de morfologias diferentes foram apontados como os principais fatores na origem do efeito antibacteriano encontrado no revestimento de óxido de prata. Com o propósito de compreender a influência das espécies de oxigênio nas propriedades físicas, químicas e estruturais dos filmes finos, foram depositados novos revestimentos de prata e óxido de prata, por pulverização catódica em magnetrão, em regime não-reativo e reativo, com variação do teor de oxigénio. Os revestimentos resultantes revelaram que os revestimentos de prata formaram uma camada contínua. A incorporação do oxigénio levou à formação de uma mistura de fases de Ag2O + AgO. Contudo, com o aumento da fração de oxigénio, a mistura de óxidos desapareceu e os filmes finos resultantes apresentaram apenas a fase de AgO. Foram realizados ensaios de citotoxicidade, demonstrando que o revestimento de óxido de prata escolhido não é tóxico, tornando-o adequado para ser aplicado em stents. O comportamento antibacteriano dos revestimentos foi, quantitativamente, testado contra Staphylococcus epidermis, revelando que os revestimentos de AgxO, ao contrário dos revestimentos de Ag, apresentam um comportamento antibacteriano. A presença do óxido de prata é um dos principais fatores para o efeito antibacteriano, provavelmente, devido ao aumento da produção de espécies reativas de oxigénio (ROS). Finalmente, e de modo a avaliar o comportamento do revestimento quando submetido a forças mecânicas, os stents revestidos com prata e óxido de prata foram, novamente, testados mecanicamente, e não se observou perda de adesão ou a delaminação do filme. Estes resultados indicam que os revestimentos conseguem suportar, de forma adequada, as forças mecânicas e que não haverá risco de perda de revestimento para a corrente sanguínea. Em conclusão, os stents fibrosos revestidos com óxido de prata, apresentam um bom comportamento mecânico, quando comparado com os stents comerciais de nitinol, além de apresentarem propriedades antibacterianas e não serem citotóxicos, o que torna este stent um candidato promissor como substituto para os stents metálicos atuais

Current nanotechnology advances in diagnostic biosensors

Current diagnostics present challenges that are imposed by increased life expectancy in the worldwide population. These challenges are related, not only to satisfy the need for higher performance of diagnostic tests, but also to the capacity of creating pointâ ofâ care, wearable, multiplexing and implantable diagnostic platforms that will allow early detection, continuous monitoring and treatment of health conditions in a personalized manner. These health challenges are translated into technological issues that need to be solved with multidisciplinary knowledge. Nanoscience and technology play a fundamental role in the development of miniaturized sensors that are cheap, accurate, sensitive and consume less power. At nanometre scale, these materials possess higher volumeâ toâ surface ratio and display novel properties (composition, charge, reactive sites, physical structure and potential) that are exploited for sensing purposes. These nanomaterials can therefore be integrated into diagnostic sensing platforms allowing the creation of novel technologies that tackle current health challenges. These nanomaterialâ enhanced sensors are extremely diverse, since they use numerous types of materials, nanostructures and detection modes for a multitude of biomarkers. The purpose of this review is to summarize the current stateâ ofâ theâ art of nanomaterialâ enhanced sensors, emphasizing and discussing the diagnostic challenges that are addressed by the different engineering and nanotechnology approaches. This review also aims to identify the drawbacks of nanomaterialâ enhanced sensors, as well as point out future developmental directions.This research was funded by FCT- FUNDAÇÃO PARA A CIÊNCIA E TECNOLOGIA, grant numbers: PTDC/EMD-EMD/31590/2017 and PTDC/BTM-ORG/28168/2017

Aging effect on functionalized silver-based nanocoating braided coronary stents

A previously developed fiber-based polyester (PES) stent, with mechanical properties comparable to commercial nitinol stents, was coated with metallic silver (Ag0) and silver oxides (AgxO) thin films through direct current (DC) magnetron sputtering. Ag0 and AgxO coatings provide antimicrobial properties to the stents to minimize the occurrence of coronary stent infections. Nevertheless, the stent interacts with the atmosphere and then with the biological fluids and may lead to the generation of silver species with diminished antimicrobial efficiency and/or prone to induce cytotoxicity. Therefore, stent coating nanostructures aged 3 months were thoroughly analyzed by X-ray photoelectron spectroscopy (XPS) and their antimicrobial and cytotoxicity properties were assessed. Aging led to the presence of silver carbonate and bicarbonate as well as chemisorbed oxygen species in Ag0 and AgxO coatings. Bactericidal efficacy was tested against an important nosocomial bacterium, particularly associated to indwelling devices: Staphylococcus epidermidis. Aged Ag0 and AgxO coating presented a Log reduction of 1 and 2 at their surface; respectively. However, aged stents were able to induce a Log reduction of 2 (Ag0) and 4 (AgxO) on the surrounding medium. Only aged AgxO stent was able to provide a mild reduction of the bacterium at its surface and a clear antimicrobial effect (Log reduction >3) within its vicinity. More importantly, both aged Ag0 and AgxO stents were shown to be compatible with fibroblasts cells indicating that they can be safely used as indwelling devices, despite the aging effectThis work was funded by FEDER funds through the Operational Competitiveness ProgramCOMPETE and by National Funds through Fundacao para a Ciencia e Tecnologia (FCT)-under the projects PTDC/CTM-TEX/28295/2017 and UID/CTM/00264/2019

3D biosensors in advanced medical diagnostics of high mortality diseases

Cardiovascular diseases, cancer, and diabetes are high mortality diseases, which account for almost two thirds of all deaths worldwide. Their early detection and continuous evaluation is fundamental for an improved patient prognosis and reduced socioeconomic impact. Current biosensor technologies are typically based on the analysis of whole blood samples from patients for the detection of disease-specific biomarkers. However, these technologies display serious shortcomings, such as reduced sensitivity and dynamic range, limited in vivo applicability, and lack of continuous monitoring. There is the urgent need for new diagnostic and treatment follow-up tools, which allow for the early detection of the pathology as well as for the continuous monitoring of the physiological response to specific therapies. During the last years, a new generation of biosensor technologies with improved performance has emerged in the biomedical sector. The combination of advanced biomaterial methods, biochemical tools, and micro/nanotechnology approaches has resulted in the development of innovative three-dimensional (3D) biosensor platforms for advanced medical diagnosis. In this review, we report the most recent advances in the field of 3D biosensors for clinical applications, focusing on the diagnosis and monitoring of cardiovascular diseases, cancer, and diabetes. We discuss about their clinical performance compared to standard biosensor technologies, their implantable capability, and their integration into microfluidic devices to develop clinically-relevant models. Overall, we anticipate that 3D biosensors will drive us toward a new paradigm in medical diagnosis, resulting in real-time in vivo biosensors capable to significantly improve patient prognosis.V.M.C., S.C.K, and D.C. acknowledge thefinancial support from theEuropean Union Framework Programme for Research and InnovationHorizon 2020 on Forefront Research in 3D Disease Cancer Models asinvitroScreening Technologies (FoReCaST) under Grant agreement no.668983. V.M.C also thanks the Portuguese Foundation for Science andTechnology (FCT) for his distinction attributed under the FCTInvestigator program (IF/01214/2014). D.C. and S.C.K also acknowl-edge the support from the FCT under the scope of the project ModellingCancer Metastasis into the Human Microcirculation System using aMulti-organ-on-a-Chip Approach (2MATCH) (PTDC/BTM-ORG/28070/2017) funded by the Programa Operacional Regional do Norte sup-ported by Fundo Europeu de Desenvolvimento Regional (FEDER). A.I.B.acknowledges thefinancial support of project FROnTHERA (NORTE-01-0145-FEDER-000023

Chitosan micro-membranes with integrated gold nanoparticles as an LSPR-based sensing platform

Currently, there is an increasing need to develop highly sensitive plasmonic sensors able to provide good biocompatibility, flexibility, and optical stability to detect low levels of analytes in biological media. In this study, gold nanoparticles (Au NPs) were dispersed into chitosan membranes by spin coating. It has been demonstrated that these membranes are particularly stable and can be successfully employed as versatile plasmonic platforms for molecular sensing. The optical response of the chitosan/Au NPs interfaces and their capability to sense the medium’s refractive index (RI) changes, either in a liquid or gas media, were investigated by high-resolution localized surface plasmon resonance (HR-LSPR) spectroscopy, as a proof of concept for biosensing applications. The results revealed that the lowest polymer concentration (chitosan (0.5%)/Au-NPs membrane) presented the most suitable plasmonic response. An LSPR band redshift was observed as the RI of the surrounding media was incremented, resulting in a sensitivity value of 28 ± 1 nm/RIU. Furthermore, the plasmonic membrane showed an outstanding performance when tested in gaseous atmospheres, being capable of distinguishing inert gases with only a 10−5 RI unit difference. The potential of chitosan/Au-NPs membranes was confirmed for application in LSPR-based sensing applications, despite the fact that further materials optimization should be performed to enhance sensitivity.This research was sponsored by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding UIDB/04650/2020, by the project CO2Plasmon with reference EXPL/CTM-REF/0750/2021 and by the project with reference PTDC/CTM-CTM/2846/2020. Diana I. Meira acknowledges her Ph.D. Scholarship from FCT, with reference SFRH/BD/143262/2019. Manuela Proença acknowledges her Ph.D. Scholarship from FCT, with reference SFRH/BD/137076/2018

Development of label-free plasmonic Au-TiO2 thin film immunosensor devices

"Available online 09 March 2019"This work reports on the development of a label-free immunosensor technology, based on nanoplasmonic Au-TiO2 thin films. The Au-TiO2 thin films were prepared by cost-effective reactive DC magnetron sputtering, followed by a thermal annealing procedure. The latter promoted the growth of the Au nanoparticles throughout the TiO2 matrix and induced some morphological changes, which are the base for the immunosensor device functionality. A posterior plasma etching treatment was required to partially expose the nanoparticles to the biological environment. It gave rise to a 6-fold increase of the total area of gold exposed, allowing further possibilities for the sensor sensitivity enhancement. Experimental results demonstrated the successful functionalization of the films' surface with antibodies, with the immobilization occurring preferentially in the exposed nanoparticles and negligibly on the TiO2 matrix. Antibody adsorption surface coverage studies revealed antibody low affinity to the film's surface. Nevertheless, immunoassay development experiments showed a strong and active immobilized antibody monolayer at an optimized antibody concentration. This allowed a 236 signal-to-noise-ratio in a confocal microscope, using mouse IgG and 100 ng/ml of Fab-specific anti-mouse IgG-FITC conjugated. Label-free detection of the optimized antibody monolayer on Au-TiO2 thin films was also tested, revealing an expected redshift in the LSPR band, which demonstrates the suitability for the development of cost-effective, label-free LSPR based immunosensor devices.Authors would like to acknowledge FROnTHERA (NORTE-01-0145-FEDER-0000232), VM Correlo acknowledges Investigator FCT program(IF/01214/2014). Coauthors from CFUM acknowledge FCT in the fra-mework of the Strategic Funding UID/FIS/04650/2013; project POCI-01-0145-FEDER-016902, with FCT reference PTDC/FIS-NAN/1154/2014; and project POCI-01-0145-FEDER-032299, with FCT referencePTDC/FIS-MAC/32299/2017. Joel Borges also acknowledges FCT forhis Post-Doc Grant SFRH/BPD/117010/2016 and Marco S. Rodriguesacknowledges his PhD-Grant from FCT, with reference SFRH/BD/118684/2016.info:eu-repo/semantics/publishedVersio