Exosomes and exosome-like nanoparticles: applications for biomedicine

With the rise of the average life expectancy in the last century, the prevalence of life-threatening diseases has greatly increased. Thus, the need for effective, cost efficient and easy to produce therapeutic systems, combined with recent technological advances, boosted nanotechnology research. Since their discovery more than 30 years ago, naturally occurring exosomes are an increasingly interesting vehicle for drug delivery, with the disadvantage that they are difficult and expensive to extract, characterize and their encapsulation efficiency is poor. Liposomes, known since the mid-60’s, can be used for various types of therapies for their capacity to encapsulate almost any molecule, with efficient production and encapsulation processes, but are less biocompatible. A new type of systems, exosome-like nanoparticles, that combine the advantages of these two systems, mitigating their limitations, show potential to be excellent therapeutic options with almost no side-effects.This work has been supported by the strategic program UID/BIA/04050/2020 funded by National funds through the Fundação para a Ciência e Tecnologia I.P. Mário Fernandes (SFRH/BD/147819/2019) holds a scholarship from Fundação para a Ciência e Tecnologia. We also acknowledge support from FCT within the FUN2CYT project with the grant POCI-01-0145- FEDER-030568

Nanotechnology solutions for controlled cytokine delivery: an applied perspective

Around 200 cytokines with roles in cell signaling have been identified and studied, with the vast majority belonging to the four-α-helix bundle family. These proteins exert their function by binding to specific receptors and are implicated in many diseases. The use of several cytokines as therapeutic targets has been approved by the FDA, however their rapid clearance in vivo still greatly limits their efficacy. Nano-based drug delivery systems have been widely applied in nanomedicine to develop safe, specific and controlled delivery techniques. Nevertheless, each nanomaterial has its own specifications and their suitability towards the biochemical and biophysical properties of the selected drug needs to be determined, weighing in the final choice of the ideal nano drug delivery system. Nanoparticles remain the most used vehicle for cytokine delivery, where polymeric carriers represent the vast majority of the studied systems. Liposomes and gold or silica nanoparticles are also explored and discussed in this review. Additionally, surface functionalization is of great importance to facilitate the attachment of a wide variety of molecules and modify features such as bioavailability. Since the monitoring of cytokine levels has an important role in early clinical diagnosis and for assessing therapeutic efficacy, nanotechnological advances are also valuable for nanosensor development.This work was supported by the “Contrato-Programa” UIDB/04050/2020 funded by national funds through the FCT I.P. and project FUN2CYT: Harnessing the potential for biomedical applications of pleiotropic cytokines LIF and oncostatin M (POCI-01-0145-FEDER-030568) supported by Programa Operacional Competitividade e Internacionalização (FEDER) and FCT, IP. Anabela Gonçalves acknowledges her PhD scholarship from FCT (SFRH/BD/146807/2019)

Absence of light exposure increases pathogenicity of Pseudomonas aeruginosa pneumonia-associated clinical isolates

Pseudomonas aeruginosa can alter its lifestyle in response to changes in environmental conditions. The switch to a pathogenic host-associated lifestyle can be triggered by the luminosity settings, resorting to at least one photoreceptor which senses light and regulates cellular processes. This study aimed to address how light exposure affects the dynamic and adaptability of two P. aeruginosa pneumonia-associated isolates, HB13 and HB15. A phenotypic characterization of two opposing growth conditions, constant illumination and intensity of full-spectrum light and total absence of light, was performed. Given the nature of P. aeruginosa pathogenicity, distinct fractions were characterized, and its inherent pathogenic potential screened by comparing induced morphological alterations and cytotoxicity against human pulmonary epithelial cells (A549 cell line). Growth in the dark promoted some virulence-associated traits (e.g., pigment production, LasA proteolytic activity), which, together with higher cytotoxicity of secreted fractions, supported an increased pathogenic potential in conditions that better mimic the lung microenvironment of P. aeruginosa. These preliminary findings evidenced that light exposure settings may influence the P. aeruginosa pathogenic potential, likely owing to differential production of virulence factors. Thus, this study raised awareness towards the importance in controlling light conditions during bacterial pathogenicity evaluation approaches, to more accurately interpret bacterial responses.FCT -Fundação para a Ciência e a Tecnologia(SFRH/BD/98558/2013)info:eu-repo/semantics/publishedVersio

Uncovering the promiscuous activity of IL-6 proteins: a multi-dimensional analysis of phylogeny, classification and residue conservation

The IL-6 family of cytokines, known for their pleiotropic behavior, share binding to the gp130 receptor for signal transduction with the necessity to bind other receptors. Leukemia inhibitory factor receptor is triggered by the IL-6 family proteins: leukemia inhibitory factor (LIF), oncostatin-M (OSM), cardiotrophin-1 (CT-1), ciliary neurotrophic factor (CNTF), and cardiotrophin-like cytokine factor 1 (CLCF1). Besides the conserved binding sites to the receptor, not much is known in terms of the diversity and characteristics of these proteins in different organisms. Herein, we describe the sequence analysis of LIF, OSM, and CT-1 from several organisms, and m17, a LIF ortholog found in fishes, regarding its phylogenetics, intrinsic properties, and the impact of conserved residues on structural features. Sequences were identified in seven classes of vertebrates, showing high conservation values in binding site III, but protein-dependent results on binding site II. GRAVY, isoelectric point, and molecular weight parameters were relevant to differentiate classes in each protein and to enable, for the first time and with high fidelity, the prediction of both organism class and protein type just using machine learning approaches. OSM sequences from primates showed an increased BC loop when compared to the remaining mammals, which could influence binding to OSM receptor and tune signaling pathways. Overall, this study highlights the potential of sequence diversity analysis to understand IL-6 cytokine family evolution, showing the conservation of function-related motifs and evolution of class and protein-dependent characteristics. Our results could impact future medical treatment of disorders associated with imbalances in these cytokines.This work was supported by the “Contrato-Programa” UIDB/04050/2020 funded by national funds through the FCT I.P. and project FUN2CYT: Harnessing the potential for biomedical applications of pleiotropic cytokines LIF and oncostatin M (POCI-01-0145-FEDER-030568) supported by Programa Operacional Competitividade e Internacionalizaçao (FEDER) and FCT, I.P. Raul ˜ Machado acknowledges FCT I.P. for funding within the Scientific Employment Stimulus instrument (CEECIND/00526/2018)

Folate-targeted nanoparticles for rheumatoid arthritis therapy

Rheumatoid arthritis (RA) is the most common inflammatory rheumatic disease, affecting almost 1% of the world population. Although the cause of RA remains unknown, the complex interaction between immune mediators (cytokines and effector cells) is responsible for the joint damage that begins at the synovial membrane. Activated macrophages are critical in the pathogenesis of RA and have been shown to specifically express a receptor for the vitamin folic acid (FA), folate receptor (FR). This particular receptor allows internalization of FA-coupled cargo. In this review we will address the potential of nanoparticles as an effective drug delivery system for therapies that will directly target activated macrophages. Special attention will be given to stealth degree of the nanoparticles as a strategy to avoid clearance by macrophages of the mononuclear phagocytic system (MPS). This review summarizes the application of FA-target nanoparticles as drug delivery systems for RA and proposes prospective future directions.Eugénia Nogueira (SFRH/BD/81269/2011) holds a scholarship from Fundação para a Ciência e a Tecnologia (FCT). This study was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit, the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the Project “BioHealth — Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2 — O Novo Norte), QREN, FEDER. This work was also supported by FCT I.P. through the strategic funding UID/BIA/04050/2013. We thank the Centro Hospitalar do Alto Ave (Guimarães, Portugal) for providing radiographic joint images

Protein microspheres as suitable devices for piroxicam release

Bovine serum albumin-piroxicam (BSA-piroxicam) and human serum albumin-piroxicam (HSA-piroxicam) microspheres were sonochemically prepared and characterized. The use of polyvinyl alcohol (PVA) lead to an improvement of formulation characteristics, including smaller size, lower polydispersity index (PDl), higher entrapment efficiency and higher stability. The release kinetics of these proteinaceous microspheres was determined in presence of protease, indicating an anomalous drug transport mechanism (diffusion and polymer degradation). In presence of higher protease concentration, BSA microspheres exhibit Case II transport, leading to zero order release (protein degradation). These proteinaceous devices did not show cytotoxicity against human skin fibroblasts in vitro, for range concentrations below to 300 mg L−1, greatly supporting their potential application in the treatment of inflammatory diseases.We would like to acknowledge the financial support of European project Lidwine (contract no. NMP2-CT-2006-026741), and to POPH/FSE for co-financing and FCT for fellowship SFRH/BPD/38939/2007

Low-Complexity Equalisers for Offset Constellations in Massive MIMO Schemes

This work was supported in part by the European Regional Development Fund (FEDER), through the Competitiveness and Internationalization Operational Program of the Portugal 2020 Framework, in part by the Regional OP Centro under Grant POCI-01-0145-FEDER-030588, in part by the Regional OP Lisboa under Grant Lisboa-01-0145-FEDER-03058, in part by the FCT/MEC through national funds of MASSIVE5G Project under Grant SAICT-45-2017-02 and PES3N Project under Grant 2018-SAICT-45-2017-POCI-01-0145-FEDER-030629, in part by the UID/EEE/50008/2019 Project, and in part by the FCT Ph.D. under Grant SFRH/BD/108522/2015.Massive multi-input-multi-output (m-MIMO) schemes require low-complexity implementations at both the transmitter and the receiver side, especially for systems operation at millimeter wave (mmWave) bands. In this paper, we consider the use of offset constellations in m-MIMO systems operating at mmWave frequencies. These signals are designed to have either an almost constant envelope or be decomposed as the sum of constant-envelope signals, making them compatible with strongly nonlinear power amplifiers, which can have low-implementation complexity and high amplification efficient, making them particularly interesting for mmWave communications. We design and evaluate low-complexity frequency-domain receivers for offset signals. It is shown that the proposed receivers can have excellent performance/complexity trade-offs in m-MIMO scenarios, making them particularly interesting for future wireless systems operating at mmWave bands.publishersversionpublishe

Enzymatic phosphorylation of silk fibroins : a platform for the production of biocompatible, cell-static, materials

Silks are natural protein polymers produced by insects. Silk heavy chain of B.mori is primarily composed of hydrophobic, –(–Ala–Gly–)n– -sheet crystalline domains. Based on silk biocompatibility, biodegradability and strength, different materials were developed. Silk offers a stabilizing environment for incorporated proteins and molecules. Silk properties can be controlled via structure manipulation, by coupling molecules of biological significance; its Tyr and Ser residues can be modified. Once incorporated into a protein, the phosphate group establishes hydrogen bonds that affect intra- and inter-molecular interactions16. Phosphorylation is stable under physiological conditions, thus directing the formation and reorganization of protein networks. Curiously, using phosphorylation for protein functionalization is largely unexplored. Significant research is devoted to bio-inspired materials with various cell-differentiating and cell-supporting features. However, little attention is paid to develop cell-static bio-materials. Such materials do not promote cell growth. That can be achieved by lowering the probability of cell attachment to the material, via creation of negatively charged material surface. The goal of this study was to produce bio-compatible materials with the cell-static properties by phosphorylation. Silk solutions were made to cast films of variable pH and phosphorylated content. Obtained materials were tested and a dependency between amount of phosphorylation and bio-chemical properties confirmed

Lipid-based Nanocarriers for siRNA Delivery: Challenges, Strategies and the Lessons Learned from the DODAX: MO Liposomal System

The possibility of using the RNA interference (RNAi) mechanisms in gene therapy was one of the scientific breakthroughs of the last century. Despite the extraordinary therapeutic potential of this approach, the need for an efficient gene carrier is hampering the translation of the RNAi technology to the clinical setting. Although a diversity of nanocarriers has been described, liposomes continue to be one of the most attractive siRNA vehicles due to their relatively low toxicity, facilitated siRNA complexation, high transfection efficiency and enhanced pharmacokinetic properties. This review focuses on RNAi as a therapeutic approach, the challenges to its application, namely the nucleic acids’ delivery process, and current strategies to improve therapeutic efficacy. Additionally, lipid-based nanocarriers are described, and lessons learned from the relation between biophysical properties and biological performance of the dioctadecyldimethylammonium:monoolein (DODAX: MO) system are explored. Liposomes show great potential as siRNA delivery systems, being safe nanocarriers to protect nucleic acids in circulation, extend their half-life time, target specific cells and reduce off-target effects. Nevertheless, several issues related to delivery must be overcome before RNAi therapies reach their full potential, namely target-cell specificity and endosomal escape. Understanding the relationship between biophysical properties and biological performance is an essential step in the gene therapy field.This work was further supported by FEDER through POFC-COMPETE and by national funds from Fundacao para a Ciencia e a Tecnologia (FCT), through the projects PEst-OE/BIA/UI4050/2014 (CBMA) and PEst-C/FIS/UI0607/2013 (CFUM). Ana Oliveira was the recipient of a FCT scholarship (SFRH/BD/68588/2010). The authors would also like to acknowledge Andre Seixas Pereira for all the assistance with figures and graphs.info:eu-repo/semantics/publishedVersio

Stimuli-sensitive self-assembled tubules based on lysine-derived surfactants as nanocarriers for proteins

Drug delivery vectors based on amphiphilic molecules present considerable advantages, namely versatility in physicochemical properties and sensitivity to stimuli. Amino acid-based surfactants, in particular, are rather promising amphiphiles for this purpose1 because of their enhanced biocompatibility compared to conventional surfactants. In addition to forming micelles and vesicles, they can self-organize into other complex supramolecular structures, such as fibers, twisted ribbons, helical tapes and nanotubes.2,3 Herein, we have studied a family of novel anionic double-chained lysine-based surfactants, with variable degree of chain length mismatch. Because of their peculiar structure, these compounds are able to form in water tubular structures with assorted morphologies, as evidenced by video-enhanced light microscopy (VELM), scanning electron microscopy (SEM and cryo-SEM), cryogenic transmission electron microscopy (cryo-TEM) and atomic force microscopy (AFM).3 The loading ability of the tubules towards lysozyme, under varying experimental conditions, has been investigated inter alia by differential scanning microcalorimetry, gel electrophoresis and UV/VIS spectroscopy, with the goal of assessing the efficiency of these aggregates as pH- and temperature-sensitive nanocarriers for a model biomolecule. Results on the stability of the native and loaded tubules when in contact with different fluids (serum, artificial saliva, artificial sweat, blood), and on their toxicity in human cells, are also presented and discussed.FCT is gratefully acknowledged for financial support through Ph.D. grant SFRH/BD/108629/2015. CIQUP acknowledges financial support from FEDER/COMPETE and FCT through grants UID/QUI/00081/2013, POCI-01-0145-FEDER- 006980 and NORTE-01-0145-FEDER-000028