High-throughput platforms for the screening of new therapeutic targets for neurodegenerative diseases

Despite the recent progress in the understanding of neurodegenerative disorders, a lack of solid fundamental knowledge on the etiology of many of the major neurodegenerative diseases has made it difficult to obtain effective therapies to treat these conditions. Scientists have been looking to carry out more-human-relevant studies, with strong statistical power, to overcome the limitations of preclinical animal models that have contributed to the failure of numerous therapeutics in clinical trials. Here, we identify currently existing platforms to mimic central nervous system tissues, healthy and diseased, mainly focusing on cell-based platforms and discussing their strengths and limitations in the context of the high-throughput screening of new therapeutic targets and drugs.This work had the financial support of Fundação para a Ciência e Tecnologia ( FCT ) through National Funds and, when applicable, co-financed by the FEDER through the PT2020 Partnership Agreement under the 4293 Unit I&D. D.N. Rocha acknowledges FCT for her PhD grant

Biocompatibility of Platinum Nanoparticles in Brain ex vivo Models in Physiological and Pathological Conditions

Platinum nanoparticles (PtNPs) have unique physico-chemical properties that led to their use in many branches of medicine. Recently, PtNPs gathered growing interest as delivery vectors for drugs, biosensors and as surface coating on chronically implanted biomedical devices for improving electrochemical properties. However, there are contradictory statements about their biocompatibility and impact on target organs such as the brain tissue, where these NPs are finding many applications. Furthermore, many of the reported studies are conducted in homeostasis conditions and, consequently, neglect the impact of the pathologic conditions on the tissue response. To expand our knowledge on the effects of PtNPs on neuronal and glial cells, we investigated the acute effects of monodisperse sodium citrate-coated PtNPs on rat organotypic hippocampal cultures in physiological or neuronal excitotoxic conditions induced by kainic acid (KA). The cellular responses of the PtNPs were evaluated through cytotoxic assays and confocal microscopy analysis. To mimic a pathologic scenario, 7-day organotypic hippocampal cultures were exposed to KA for 24 h. Subsequently, PtNPs were added to each slice. We show that incubation of the slices with PtNPs for 24 h, does not severely impact cell viability in normal conditions, with no significant differences when comparing the dentate gyrus (DG), as well as CA3 and CA1 pyramidal cell layers. Such effects are not exacerbated in KA-treated slices, where the presence of PtNPs does not cause additional neuronal propidium iodide (PI) uptake in CA3 and CA1 pyramidal cell layers. However, PtNPs cause microglial cell activation and morphological alterations in CA3 and DG regions indicating the establishment of an inflammatory reaction. Morphological analysis revealed that microglia acquire activated ameboid morphology with loss of ramifications, as a result of their response to PtNPs contact. Surprisingly, this effect is not increased in pathological conditions. Taken together, these results show that PtNPs cause microglia alterations in short-term studies. Additionally, there is no worsening of the tissue response in a neuropathological induced scenario. This work highlights the need of further research to allow for the safe use of PtNPs. Also, it supports the demand of the development of novel and more biocompatible NPs to be applied in the brain.The authors acknowledge the funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie grant agreement No. 764977, in the framework of the mCBEEs project (Advanced integrative solutions to corrosion problems beyond micro-scale: toward long-term durability of miniaturized biomedical, electronic, and energy systems). This work was also financed by Portuguese funds through FCT/MCTES in the framework of the project “Institute for Research and Innovation in Health Sciences”– UIDB/04293/2020. The authors thank the support of the i3S Scientific Platforms Bioimaging and Advanced Light Microscopy (ALM), members of the national infrastructure PPBI–Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122). SS acknowledges FCT for her contract under the Norma Transitória–DL57/2016/CP/CP1360/CT0013

Scavenging activity of Magnéli phases as a function of Ti(4+)/Ti(3+) ratios

TiO2 is able to scavenge reactive oxygen and nitrogen species (ROS and RNS) in the absence of light. The scavenging mechanism has been related to the chemistry of defects (oxygen vacancy reduced oxidation states of Ti) but it is still unknown. This study describes the ROS scavenging activity of different titanium oxide phases and relates their scavenging activities with the Ti4+/Ti3+ molar ratio as well as the band gap value. The Ti5O9 phase, with a mixture of both oxidation states, presented a substantially higher percentage of 2,2-diphenyl-1-picrylhydracyl radicals (DPPH) eliminated per m2 of specific surface area in comparison to phases with predominant oxidation states Ti4+ or Ti3+ such as TiO2 and Ti2O3, respectively. The obtained results indicate that the DPPH scavenging mechanism corresponds to a catalytic process on the Ti5O9 surface which is facilitated by the presence of charges that can easily move through the material. The mobility of charges and electrons in the semiconductor surface, related to the presence of oxidation states Ti4+ and Ti3+ and a small band gap, could create an attractive surface for radical species such as DPPH. This puts forward Ti5O9 as a promising candidate coating for implantable biomedical devices, as an electrode, since it can cushion inflammatory processes which could lead to device encapsulation and, consequently, failure.The authors would like to thank the Fundación General CSIC and Obra Social “La Caixa” (Project BIOAMD) for providing financial support to this work. Dr Canillas acknowledges the financial support received from the CSIC for her PhD JAEpre grant and Dr Moreno is grateful to the Fondo Social Europeo and CSIC for her JAEDoc contract

Therapeutic antisense oligonucleotides against cancer: Hurdling to the clinic

Under clinical development since the early 90's and with two successfully approved drugs (Fomivirsen and Mipomersen), oligonucleotide-based therapeutics has not yet delivered a clinical drug to the market in the cancer field. Whilst many pre-clinical data has been generated, a lack of understanding still exists on how to efficiently tackle all the different challenges presented for cancer targeting in a clinical setting. Namely, effective drug vectorization, careful choice of target gene or synergistic multi-gene targeting are surely decisive, while caution must be exerted to avoid potential toxic, often misleading off-target-effects. Here a brief overview will be given on the nucleic acid chemistry advances that established oligonucleotide technologies as a promising therapeutic alternative and ongoing cancer related clinical trials. Special attention will be given toward a perspective on the hurdles encountered specifically in the cancer field by this class of therapeutic oligonucleotides and a view on possible avenues for success is presented, with particular focus on the contribution from nanotechnology to the field.The authors would like to acknowledge the FEDER funds through the Programa Operacional Factores de Competitividade - COMPETE and the Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia (PTDC/CTM-NAN/115124/2009, HMSP-ICT/0020/2010 and PEst-C/SAU/LA0002/2013) that supported this work. Pedro M. D. Moreno is supported by a Marie Curie Action of the European Community’s Seventh Framework Program (PIEF-GA2 011300485)

Three-dimensional culture of single embryonic stem-derived neural/stem progenitor cells in fibrin hydrogels: neuronal network formation and matrix remodelling

In an attempt to improve the efficacy of neural stem/progenitor cell (NSPC) based therapies, fibrin hydrogels are being explored to provide a favourable microenvironment for cell survival and differentiation following transplantation. In the present work, the ability of fibrin to support the survival, proliferation, and neuronal differentiation of NSPCs derived from embryonic stem (ES) cells under monolayer culture was explored. Single mouse ES-NSPCs were cultured within fibrin (fibrinogen concentration: 6 mg/ml) under neuronal differentiation conditions up to 14 days. The ES-NSPCs retained high cell viability and proliferated within small-sized spheroids. Neuronal differentiation was confirmed by an increase in the levels of ßIII-tubulin and NF200 over time. At day 14, cell-matrix constructs mainly comprised NSPCs and neurons (46.5% ßIII-tubulin + cells). Gamma-aminobutyric acid (GABA)ergic and dopaminergic/noradrenergic neurons were also observed, along with a network of synaptic proteins. The ES-NSPCs expressed matriptase and secreted MMP-2/9, with MMP-2 activity increasing along time. Fibronectin, laminin and collagen type IV deposition was also detected. Fibrin gels prepared with higher fibrinogen concentrations (8/10 mg/ml) were less permissive to neurite extension and neuronal differentiation, possibly owing to their smaller pore area and higher rigidity. Overall, it is shown that ES-NSPCs within fibrin are able to establish neuronal networks and to remodel fibrin through MMP secretion and extracellular matrix (ECM) deposition. This three-dimensional (3D) culture system was also shown to support cell viability, neuronal differentiation and ECM deposition of human ES-NSPCs. The settled 3D platform is expected to constitute a valuable tool to develop fibrin-based hydrogels for ES-NSPC delivery into the injured central nervous system.The authors would like to acknowledge Prof. Domingos Henrique (Instituto de Medicina Molecular, Lisbon) for providing the ES 46C cell line. This work was supported by FEDER funds through the Programa Operacional Factores de Competitividade – COMPETE (FCOMP‐01–0124‐FEDER‐021125) and by National funds FCT – Fundação para a Ciência e a Tecnologia (PTDC/SAU‐BMA/118869/2010). A.R.B. and M.J. Oliveira are supported by FCT (SFRH/BD/86200/2012; Investigator FCT)

Breaking Barriers: Bioinspired Strategies for Targeted Neuronal Delivery to the Central Nervous System

Central nervous system (CNS) disorders encompass a vast spectrum of pathological conditions and represent a growing concern worldwide. Despite the high social and clinical interest in trying to solve these pathologies, there are many challenges to bridge in order to achieve an effective therapy. One of the main obstacles to advancements in this field that has hampered many of the therapeutic strategies proposed to date is the presence of the CNS barriers that restrict the access to the brain. However, adequate brain biodistribution and neuronal cells specific accumulation in the targeted site also represent major hurdles to the attainment of a successful CNS treatment. Over the last few years, nanotechnology has taken a step forward towards the development of therapeutics in neurologic diseases and different approaches have been developed to surpass these obstacles. The versatility of the designed nanocarriers in terms of physical and chemical properties, and the possibility to functionalize them with specific moieties, have resulted in improved neurotargeted delivery profiles. With the concomitant progress in biology research, many of these strategies have been inspired by nature and have taken advantage of physiological processes to achieve brain delivery. Here, the different nanosystems and targeting moieties used to achieve a neuronal delivery reported in the open literature are comprehensively reviewed and critically discussed, with emphasis on the most recent bioinspired advances in the field. Finally, we express our view on the paramount challenges in targeted neuronal delivery that need to be overcome for these promising therapeutics to move from the bench to the bedside.This work was financially supported by the project PTDC/CTM-NAN/3547/2014 (POCI-01-0145-FEDER-016639) funded by FEDER funds through the Programa Operacional Competitividade e Internacionalização-COMPETE 2020 and Portuguese funds through FCT–Fundação para a Ciência e a Tecnologia. A.P.S., B.C. and S.D.S. acknowledge FCT for the Ph.D. scholarships (SFRH/BD/137073/2018 and SFRH/BD/145652/2019) and the contract under the Norma Transitória–DL57/2016/CP/CP1360/CT0013, respectively. V.L. acknowledges her contract in the framework of the project NORTE-01-0247-FEDER-033399, funded by FEDER funds through the Sistema de Incentivos à Investigação e Desenvolvimento Tecnológico (SI I&DT), Aviso nº 03/SI/2017, Projetos em Co-promoção do Programa Interfac

Biomimetic synthetic self-assembled hydrogels for cell transplantation

The development of three-dimensional matrices capable of recapitulating the main features of native extracellular matrix and contribute for the establishment of a favorable microenvironment for cell behavior and fate is expected to circumvent some of the main limitations of cell-based therapies. In this context, self-assembly has emerged as a promising strategy to engineer cell-compatible hydrogels. A wide number of synthetically-derived biopolymers, such as proteins, peptides and DNA/RNA, with intrinsic ability to self-assemble into well-defined nanofibrous structures, are being explored. The resulting hydrogels, in addition to closely resembling the architecture of native cellular microenvironments, present a versatile and dynamic behavior that allows them to be designed to undergo sol-to-gel transition in response to exogenous stimulus. This review presents an overview on the state-of-the-art of the different strategies being explored for the development of injectable synthetic self-assembled hydrogels for cell transplantation and/or recruitment of endogenous cells, with an emphasis on their biological performance, both in vitro and in vivo. Systems based on peptides are the most widely explored and have already generated promising results in pre-clinical in vivo studies involving different repair/regenerative scenarios, including cartilage, bone, nerve and heart. On the other hand, systems based on DNA and hybrid hydrogels are now emerging for application in the biomedical field with high potential. Finally, the main challenges hampering the translation of these systems to the clinic and the issues that need to be addressed for these to progress from bench-to-bedside are discussed.The authors would like to acknowledge the FEDER funds through the Programa Operacional Factores de Competitividade – COMPETE and the Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia (HMSPICT/0020/2010, PTDC/SAU-BMA/118869/2010 and PEst/SAU/LA0002/2013) that supported this work. D Barros is supported by FCT (PD/BD/105953/2014) and I.F. Amaral by QREN through program ON.2, in the framework of "Project on Biomedical Engineering for Regenerative Therapies and Cancer” (NORTE-07-0124-FEDER-000005)

Delivering siRNA with dendrimers: In vivo applications

Over the last decades, gene therapy has emerged as a pioneering therapeutic approach to treat or prevent several diseases. Among the explored strategies, the short-term silencing of protein coding genes mediated by siRNAs has a good therapeutic potential in a clinical setting. However, the widespread use of siRNA will require the development of clinically suitable, safe and effective vehicles with the ability to complex and deliver siRNA into target cells with minimal toxicity. Lately, dendrimers have gained considerable attention as non-viral vectors in nucleic acid delivery due to their unique structural characteristics (globular, well defined and highly branched structure, multivalency, low polydispersity and tunable nanosize), along with their relevant capacity to complex and protect nucleic acids in compact nanostructures, which can be functionalized with targeting moieties in order to get cell specificity. Here, we present an overview of the state-of-the-art of the most significant and recent advances on the use of dendrimers as siRNA delivery vectors, with particular focus on the in vivo applications. We will cover the use of different dendrimers, distinct administration routes, toxicity issues, as well as the target tissue or disease, highlighting the potential of dendrimers as nanocarriers for therapeutic and biomedical applications.The authors acknowledge the financial support of the Fundo para a Investigacao em Saude (INFARMED, project reference FIS-2015-01_CCV_20150630-88), as well as the FEDER funds through the Programa Operacional Competitividade e Internacionalizacao - COMPETE 2020 and the Portuguese funds through FCT - Fundacao para a Ciencia e a Tecnologia in the frame of the project POCI-01-0145-FEDER-016639 (reference PTDC/CTM-NAN/3547/2014). V. Leiro is supported by the project NORTE-01-0145-FEDER-000012, financed by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). S.D. Santos is supported by FCT (SFRH/BPD/109297/2015). We thank Joao Pedro Garcia for his contribution to the dendriplex picture

Seeing is believing but quantifying is deciding

The work was financed by Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia in the framework of project UID/BIM/04293/2013. CDF Lopes acknowledges FCT for her PhD scholarship (SFRH/BD/77933/2011). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript

The role of the surface on microglia function: Implications for central nervous system tissue engineering

In tissue engineering, it is well accepted that a scaffold surface has a decisive impact on cell behaviour. Here we focused on microglia-the resident immune cells of the central nervous system (CNS)-and on their response to poly(trimethylene carbonate-co-1-caprolactone) (P(TMC-CL)) fibrous and flat surfaces obtained by electrospinning and solvent cast, respectively. This study aims to provide cues for the design of instructive surfaces that can contribute to the challenging process of CNS regeneration. Cell morphology was evidently affected by the substrate, mirroring the surface main features. Cells cultured on flat substrates presented a round shape, while cells with elongated processes were observed on the electrospun fibres. A higher concentration of the pro-inflammatory cytokine tumour necrosis factor-a was detected in culture media from microglia on fibres. Still, astrogliosis is not exacerbated when astrocytes are cultured in the presence of microgliaconditioned media obtained from cultures in contact with either substrate. Furthermore, a significant percentage of microglia was found to participate in the process of myelin phagocytosis, with the formation of multinucleated giant cells being observed only on films. Altogether, the results presented suggest that microglia in contact with the tested substrates may contribute to the regeneration process, putting forward P(TMC-CL) substrates as supporting matrices for nerve regeneration.This work was financed by FEDER funds through the Programa Operacional Factores de Competitividade – COMPETE and by Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia in the framework of the project PEst-C/SAU/LA0002/2011. L.P. and D.R. thank FCT for their PhD grants (SFRH/BD/46015/2008 and SFRH/BD/64079/2009)