Dextran and protamine-based solid lipid nanoparticles as potential vectors for the treatment of X-linked juvenile retinoschisis

This is a copy of an article published in the Human gene therapy © 2012 copyright Mary Ann Liebert, Inc.; Human gene therapy is available online at: http://online.liebertpub.com.

Design,optimization and in vivo evaluation of non-viral vectors for gene therapy. Applications in ocular disorders

227 págs.

Desarrollo de nanopartículas sólidas lipídicas como sistemas de administración de ADN para terapia génica

290 p. (Bibliogr. 257-290) Correo electrónico de la autora: ana.delpozo@ehu.es

Metal-Free Polymethyl Methacrylate (PMMA) Nanoparticles by Enamine "Click" Chemistry at Room Temperature

"Click" chemistry has become an efficient avenue to unimolecular polymeric nanoparticles through the self-crosslinking of individual polymer chains containing appropriate functional groups. Herein we report the synthesis of ultra-small (7 nm in size) polymethyl methacrylate (PMMA) nanoparticles (NPs) by the "metal-free" cross-linking of PMMA-precursor chains prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization containing beta-ketoester functional groups. Intramolecular collapse was performed by the one-pot reaction of beta-ketoester moieties with alkyl diamines in tetrahydrofurane at r.t. (i.e., by enamine formation). The collapsing process was followed by size exclusion chromatography and by nuclear magnetic resonance spectroscopy. The size of the resulting PMMA-NPs was determined by dynamic light scattering. Enamine "click" chemistry increases the synthetic toolbox for the efficient synthesis of metal-free, ultra-small polymeric NPs.

Design and synthesis of biofunctional magnetic/fluorescent glyco-nanoparticles and quantum dots and their application as specific molecular imaging probes

206 p.

Lipid Nanoparticles as Carriers for RNAi against Viral Infections: Current Status and Future Perspectives

The efforts made to develop RNAi-based therapies have led to productive research in the field of infections in humans, such as hepatitis C virus (HCV), hepatitis B virus (HBV), human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), herpetic keratitis, human papillomavirus, or influenza virus. Naked RNAi molecules are rapidly digested by nucleases in the serum, and due to their negative surface charge, entry into the cell cytoplasm is also hampered, which makes necessary the use of delivery systems to exploit the full potential of RNAi therapeutics. Lipid nanoparticles (LNP) represent one of the most widely used delivery systems for in vivo application of RNAi due to their relative safety and simplicity of production, joint with the enhanced payload and protection of encapsulated RNAs. Moreover, LNP may be functionalized to reach target cells, and they may be used to combine RNAi molecules with conventional drug substances to reduce resistance or improve efficiency. This review features the current application of LNP in RNAi mediated therapy against viral infections and aims to explore possible future lines of action in this field.

Development of nucleic acid vaccines: use of self-amplifying RNA in lipid nanoparticles

Self-amplifying RNA or RNA replicon is a form of nucleic acid-based vaccine derived from either positive-strand or negative-strand RNA viruses. The gene sequences encoding structural proteins in these RNA viruses are replaced by mRNA encoding antigens of interest as well as by RNA polymerase for replication and transcription. This kind of vaccine has been successfully assayed with many different antigens as vaccines candidates, and has been shown to be potent in several animal species, including mice, nonhuman primates, and humans. A key challenge to realizing the broad potential of self-amplifying vaccines is the need for safe and effective delivery methods. Ideally, an RNA nanocarrier should provide protection from blood nucleases and extended blood circulation, which ultimately would increase the possibility of reaching the target tissue. The delivery system must then be internalized by the target cell and, upon receptor-mediated endocytosis, must be able to escape from the endosomal compartment into the cell cytoplasm, where the RNA machinery is located, while avoiding degradation by lysosomal enzymes. Further, delivery systems for systemic administration ought to be well tolerated upon administration. They should be safe, enabling the multiadministration treatment modalities required for improved clinical outcomes and, from a developmental point of view, production of large batches with reproducible specifications is also desirable. In this review, the concept of self-amplifying RNA vaccines and the most promising lipid-based delivery systems are discussed.

Mechanisms of Toxicity of Ag Nanoparticles in Comparison to Bulk and Ionic Ag on Mussel Hemocytes and Gill Cells

Silver nanoparticles (Ag NPs) are increasingly used in many products and are expected to end up in the aquatic environment. Mussels have been proposed as marine model species to evaluate NP toxicity in vitro. The objective of this work was to assess the mechanisms of toxicity of Ag NPs on mussel hemocytes and gill cells, in comparison to ionic and bulk Ag. Firstly, cytotoxicity of commercial and maltose stabilized Ag NPs was screened in parallel with the ionic and bulk forms at a wide range of concentrations in isolated mussel cells using cell viability assays. Toxicity of maltose alone was also tested. LC50 values were calculated and the most toxic Ag NPs tested were selected for a second step where sublethal concentrations of each Ag form were tested using a wide array of mechanistic tests in both cell types. Maltose-stabilized Ag NPs showed size-dependent cytotoxicity, smaller (20 nm) NPs being more toxic than larger (40 and 100 nm) NPs. Maltose alone provoked minor effects on cell viability. Ionic Ag was the most cytotoxic Ag form tested whereas bulk Ag showed similar cytotoxicity to the commercial Ag NPs. Main mechanisms of action of Ag NPs involved oxidative stress and genotoxicity in the two cell types, activation of lysosomal AcP activity, disruption of actin cytoskeleton and stimulation of phagocytosis in hemocytes and increase of MXR transport activity and inhibition of Na-K-ATPase in gill cells. Similar effects were observed after exposure to ionic and bulk Ag in the two cell types, although generally effects were more marked for the ionic form. In conclusion, results suggest that most observed responses were due at least in part to dissolved Ag.

Two Glass Transitions Associated to Different Dynamic Disorders in the Nematic Glassy State of a Non-Symmetric Liquid Crystal Dimer Dopped with gamma-Alumina Nanoparticles

In the present work, the nematic glassy state of the non-symmetric LC dimer -(4-cyanobiphenyl-4-yloxy)--(1-pyrenimine-benzylidene-4-oxy) undecane is studied by means of calorimetric and dielectric measurements. The most striking result of the work is the presence of two different glass transition temperatures: one due to the freezing of the flip-flop motions of the bulkier unit of the dimer and the other, at a lower temperature, related to the freezing of the flip-flop and precessional motions of the cyanobiphenyl unit. This result shows the fact that glass transition is the consequence of the freezing of one or more coupled dynamic disorders and not of the disordered phase itself. In order to avoid crystallization when the bulk sample is cooled down, the LC dimer has been confined via the dispersion of -alumina nanoparticles, in several concentrations.

Early response to nanoparticles in the Arabidopsis transcriptome compromises plant defence and root-hair development through salicylic acid signalling

Background: The impact of nano-scaled materials on photosynthetic organisms needs to be evaluated. Plants represent the largest interface between the environment and biosphere, so understanding how nanoparticles affect them is especially relevant for environmental assessments. Nanotoxicology studies in plants allude to quantum size effects and other properties specific of the nano-stage to explain increased toxicity respect to bulk compounds. However, gene expression profiles after exposure to nanoparticles and other sources of environmental stress have not been compared and the impact on plant defence has not been analysed. Results: Arabidopsis plants were exposed to TiO2-nanoparticles, Ag-nanoparticles, and multi-walled carbon nanotubes as well as different sources of biotic (microbial pathogens) or abiotic (saline, drought, or wounding) stresses. Changes in gene expression profiles and plant phenotypic responses were evaluated. Transcriptome analysis shows similarity of expression patterns for all plants exposed to nanoparticles and a low impact on gene expression compared to other stress inducers. Nanoparticle exposure repressed transcriptional responses to microbial pathogens, resulting in increased bacterial colonization during an experimental infection. Inhibition of root hair development and transcriptional patterns characteristic of phosphate starvation response were also observed. The exogenous addition of salicylic acid prevented some nano-specific transcriptional and phenotypic effects, including the reduction in root hair formation and the colonization of distal leaves by bacteria. Conclusions: This study integrates the effect of nanoparticles on gene expression with plant responses to major sources of environmental stress and paves the way to remediate the impact of these potentially damaging compounds through hormonal priming.

Cellular and molecular responses of zebrafish to legacy and emerging pollutants: the specific cases of PAHs and metal oxide nanoparticles

270 p.

Surface plasmons and effects of losses in propagating modes in a chain of silver nanoparticles

In this work a chain of 4000 silver nanoparticles embedded in a glass medium is considered, and its leftmost particle is excited by an electric field pulse of Gaussian shape. Considering Drude’s model, losses of the system are taken into account by γ factor, which stands for the Ohmic losses, and different quantities, such as frequencies of excited modes and group velocities are calculated. Besides, these results are compared to those obtained from the dispersion relation of an infinite chain. The increase of losses affects the lifetime and propagation length of the plasmon; besides, although the response dispersion relation for an infinite chain seems to remain invariable, this is not the case for a finite chain. The mismatches are bigger for higher losses. Furthermore, plasmon propagation velocities are analysed, and an explanation for the mismatch of longitudinal modes close to the intersection point with the dispersion of light is suggested. Finally, some concepts to treat this problem from the energy transport point of view are introduced.

Efficient Synthesis of Single-Chain Polymer Nanoparticles via Amide Formation

Single-chain technology (SCT) allows the transformation of individual polymer chains to folded/collapsed unimolecular soft nanoparticles. In this work we contribute to the enlargement of the SCT toolbox by demonstrating the efficient synthesis of single-chain polymer nanoparticles (SCNPs) via intrachain amide formation. In particular, we exploit cross-linking between active methylene groups and isocyanate moieties as powerful "click" chemistry driving force for SCNP construction. By employing poly(methyl methacrylate)- (PMMA-) based copolymers bearing beta-ketoester units distributed randomly along the copolymer chains and bifunctional isocyanate cross-linkers, SCNPs were successfully synthesized at r.t. under appropriate reaction conditions. Characterization of the resulting SCNPs was carried out by means of a combination of techniques including size exclusion chromatography (SEC), infrared (IR) spectroscopy, proton nuclear magnetic resonance (H-1 NMR) spectroscopy, dynamic light scattering (DLS), and elemental analysis (EA).