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

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

Dendrimers as Powerful Building Blocks in Central Nervous System Disease: Headed for Successful Nanomedicine

Dendrimers have emerged as a powerful class of nanomaterials in the nanomedicine field due to their unique structural features: globular, well-defined, highly branched and controllable structure, nanosize-scale, low polydispersity, and the presence of several terminal groups that can be functionalized with different ligands simulating the multivalency present in different biological systems. Although in its infancy, the application of dendrimers as therapeutics or theranostic tools in central nervous system (CNS) disorders is already significant and has opened promising avenues in the treatment of many conditions where the inherent "smartness" of the dendritic structures is being explored to effectively target the CNS. Here we present an overview of the past and future challenges of the use of dendrimers to respond to one of the ultimate challenges in the (nano)medicine field: to attain CNS repair and regeneration.V Leiro, SD Santos, and CDF Lopes contributed equally to this work. The authors would like to acknowledge the financial support of the Fundo para a Investigação em Saúde (INFARMED, project reference FIS-2015-01_CCV_20150630-88), as well as the FEDER funds through the Programa Operacional Competitividade e Internacionalização – COMPETE 2020 and the Portuguese funds through FCT − Fundação para a Ciência e a Tecnologia in the frame of the project POCI-01-0145-FEDER-016639 (reference PTDC/CTM-NAN/3547/2014). SD Santos is supported by FCT funds (SFRH/BPD/109297/2015). 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)

The Present and the Future of Degradable Dendrimers and Derivatives in Theranostics

Interest in dendrimer-based nanomedicines has been growing recently, as it is possible to precisely manipulate the molecular weight, chemical composition, and surface functionality of dendrimers, tuning their properties according to the desired biomedical application. However, one important concern about dendrimer-based therapeutics remains-the nondegradability under physiological conditions of the most commonly used dendrimers. Therefore, biodegradable dendrimers represent an attractive class of nanomaterials, since they present advantages over conventional nondegradable dendrimers regarding the release of the loaded molecules and the prevention of bioaccumulation of synthetic materials and subsequent cytotoxicity. Here, we present an overview of the state-of-the-art of the design of biodegradable dendritic structures, with particular focus on the hurdles regarding the use of these as vectors of drugs and nucleic acids, as well as macromolecular contrast agents.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/112428/2009, PEst/SAU/LA0002/2013 and PEst-OE/QUI/UI0674/2013-2014) that supported this work. V. Leiro is supported by FCT (SFRH/BPD/69110/2010)

Disentangling the determinants of symbiotic species richness in native and invasive gammarids (Crustacea, Amphipoda) of the Baltic region

Dispersal of alien species is a global problem threatening native biodiversity. Co-introduction of nonnative parasites and pathogens adds to the severity of this threat, but this indirect impact has received less attention. To shed light on the key factors determining the richness of microorganisms in native and invasive host species, we compared symbiotic (parasitic and epibiotic) communities of gammarids across different habitats and localities along the Baltic coast of Poland. Seven gammarid species, two native and five invasive, were sampled from 16 freshwater and brackish localities. Sixty symbiotic species of microorganisms of nine phyla were identified. This taxonomically diverse species assemblage of symbionts allowed us to assess the effect of host translocation and regional ecological determinants driving assembly richness in the gammarid hosts. Our results revealed that (i) the current assemblages of symbionts of gammarid hosts in the Baltic region are formed by native and co-introduced species; (ii) species richness of the symbiotic community was higher in the native Gammarus pulex than in the invasive hosts, probably reflecting a process of species loss by invasive gammarids in the new area and the distinct habitat conditions occupied by G. pulex and invasive hosts; (iii) both host species and locality were key drivers shaping assembly composition of symbionts, whereas habitat condition (freshwater versus brackish) was a stronger determinant of communities than geographic distance; (iv) the dispersion patterns of the individual species richness of symbiotic communities were best described by Poisson distributions; in the case of an invasive host, the dispersion of the rich species diversity may switch to a right-skewed negative binomial distribution, suggesting a host-mediated regulation process. We believe this is the first analysis of the symbiotic species richness in native and invasive gammarid hosts in European waters based on original field data and a broad range of taxonomic groups including Microsporidia, Choanozoa, Ciliophora, Apicomplexa, Platyhelminthes, Nematoda, Nematomorha, Acanthocephala and Rotifera, to document the patterns of species composition and distributio

Resonant inelastic x-ray scattering (RIXS) spectra of magnesium diboride

Using the tight-binding linear muffin-tin orbitals method, the soft x-ray fluorescence K-emission spectra of boron in MgB_2, excited close to the absorption edge, are estimated. In the calculations the angle of incidence between the direction of the incoming photon and the hexagonal axis of the specimen is 60 degrees and 75 degrees. Comparison with experiment is possible in the former case where good agreement is found. Furthermore, another resonant feature below the Fermi energy is predicted for the larger angle. This feature can be related to the excitations to the antibonding B pi-band in the neighbourhood of the L-H line in the Brillouin zone.Comment: 4 pages with 4 figure

Evaluation of potential of high Si high C steel nanostructured bainite for wear and fatigue applications

The present study is concerned with the potential of high carbon, high silicon steel grades isothermally transformed to bainite at low temperature (<300 C). The first part gives an overview of the design principles, allowing very high strength and ductility to be achieved while minimising transformation duration. Wear and fatigue properties are then investigated for over 10 variants of such materials, manufactured in the laboratory or industrially. The results are discussed against published data. Tensile strengths above 2 GPa are routinely achieved, with, in one case, an exceptional and unprecedented total elongation of over 20%. Bainite plate thickness and retained austenite content are shown to be important factors in controlling the yield strength, though additional, non-negligible parameters remain to be quantified. Rolling-sliding wear performances are found to be exceptional, with as little as 1% of the specific wear rate of conventional 100Cr6 isothermally transformed to bainite. It is suggested that this results from the decomposition of retained austenite in the worn layer, which considerably increases hardness and presumably introduces compressive residual stresses. Fatigue performance was slightly improved over 100Cr6 for one of the two industrially produced materials but significantly lower otherwise. Factors controlling fatigue resistance require further investigations. © 2013 Institute of Materials, Minerals and Mining Published by Maney on behalf of the Institute.Peer Reviewe

Functionalized chitosan derivatives as nonviral vectors: Physicochemical properties of acylated N,N,N-trimethyl chitosan/oligonucleotide nanopolyplexes

Cationic polymers have recently attracted attention due to their proven potential for nonviral gene delivery. In this study, we report novel biocompatible nanocomplexes produced using chemically functionalized N,N,N-trimethyl chitosan (TMC) with different N-acyl chain lengths (C 5 -C 18 ) associated with single-stranded oligonucleotides. The TMC derivatives were synthesized by covalent coupling reactions of quaternized chitosan with n-pentanoic (C 5 ), n-decanoic (C 10 ), and n-octadecanoic (C 18 ) fatty acids, which were extensively characterized by Fourier transform-infrared spectroscopy (FT-IR) and proton nuclear magnetic resonance ( 1 H NMR). These N-acylated TMC derivatives (TMC n ) were used as cationic polymeric matrices for encapsulating anionic 18-base single-stranded thiophosphorylated oligonucleotides (ssONs), leading to the formation of polyplexes further characterized by zeta potential (ZP), dynamic light scattering (DLS), binding affinity, transfection efficiency and in vitro cytotoxicity assays. The results demonstrated that the length of the grafted hydrophobic N-acyl chain and the relative amino:phosphate groups ratio (N/P ratio) between the TMC derivatives and ssON played crucial roles in determining the physicochemical properties of the obtained nanocomplexes. While none of the tested derivatives showed appreciable cytotoxicity, the type of acyl chain had a remarkable influence on the cell transfection capacity of TMC-ssON nanocomplexes with the derivatives based on stearic acid showing the best performance based on the results of in vitro assays using a model cell line expressing luciferase (HeLa/Luc705).We acknowledge the financial support from the following Brazilian agencies: CAPES, FAPEMIG, CNPq, and FINEP. This work was co-financed by Fundação para a Ciência e a Tecnologia (FCT, Portugal) within the projects PTDC/CTM-NAN/NAN/115124/2009 and HMSP-ICT/0020/2010. Additionally, PMDM thanks the European Commission – Marie Curie Actions (PIEF-GA-2011-300485) for the postdoctoral fellowship. VL thanks the FCT fo the fellowship (SFRH/BPD/69110/2010). We are grateful to Dr Sandhra Carvalho (UFMG, Brazil) for the bioimaging analyses. The authors acknowledge the Centro de Materiais daUniversidade do Porto (CEMUP) for SEM and1H NMR analysis

A high-throughput bioimaging study to assess the impact of chitosan-based nanoparticle degradation on DNA delivery performance

By using imaging flow cytometry as a powerful statistical high-throughput technique we investigated the impact of degradation on the biological performance of trimethyl chitosan (TMC)-based nanoparticles (NPs). In order to achieve high transfection efficiencies, a precise balance between NP stability and degradation must occur. We altered the biodegradation rate of the TMC NPs by varying the degree of acetylation (DA) of the polymer (DA ranged from 4 to 21%), giving rise to NPs with different enzymatic degradation profiles. While this parameter did not affect NP size, charge or ability to protect plasmid DNA, NPs based on TMC with an intermediate DA (16%) showed the highest transfection efficiency. Subsequently, by means of a single quantitative technique, we were able to follow, for each tested formulation, major steps of the NP-mediated gene delivery process – NP cell membrane association, internalization and intracellular trafficking, including plasmid DNA transport towards the nucleus. NP cytotoxicity was also possible to determine by quantification of cell apoptosis. Overall, the obtained data revealed that the biodegradation rate of these NPs affects their intracellular trafficking and, consequently, their efficiency to transfect cells. Thus, one can use the polymer DA to modulate the NPs towards attaining different degradation rates and tune their bioactivity according to the desired application. Furthermore, this novel technical approach revealed to be a valuable tool for the initial steps of nucleic acid vector design. Statement of Significance By changing the biodegradation rate of trimethyl chitosan-based nanoparticles (NPs) one was able to alter the NP ability to protect or efficiently release DNA and consequently, to modulate their intracellular dynamics. To address the influence of NP degradation rate in their transfection efficiency we took advantage of imaging flow cytometry, a high-throughput bioimaging technique, to unravel some critical aspects about NP formulation such as the distinction between internalized versus cell-associated/adsorbed NP, and even explore NP intracellular localization. Overall, our work provides novel information about the importance of vector degradation rate for gene delivery into cells, as a way to tune gene expression as a function of the desired application, and advances novel approaches to optimize nanoparticle formulation.The work was financed by Portuguese funds through FCT (Fundação para a Ciência e a Tecnologia) in the framework of the projects UID/BIM/04293/2013, PTDC/CTM-NAN/115124/2009 and PTDC/CTM-NAN/3547/2014. CP Gomes, CDF Lopes and PMD Moreno acknowledge FCT for their scholarships (SFRH/BD/79930/2011, SFRH/BD/77933/2011 and SFRH/BPD/108738/2015, respectively). Authors acknowledge the Bioimaging Center for Biomaterials and Regenerative Therapies of INEB (b.IMAGE) for the support with Imaging Flow Cytometry and the Biointerfaces and Nanotechnology Service (INEB-i3S) for size and zeta potential analysis