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Non-viral vectors represent versatile and immunologically safer alternatives for nucleic acid delivery. Nanoneedles and high-aspect ratio nanostructures are unconventional but interesting delivery systems, in which delivery is mediated by surface interactions. Herein, nanoneedles are synergistically combined with polysaccharide-polyplex nanofilms and enhanced transfection efficiency is observed, compared to polyplexes in suspension. Different polyplex-polyelectrolyte nanofilm combinations are assessed and it is found that transfection efficiency is enhanced when using polysaccharide-based polyanions, rather than being only specific for hyaluronic acid, as suggested in earlier studies. Moreover, results show that enhanced transfection is not mediated by interactions with the CD44 receptor, previously hypothesized as a major mechanism mediating enhancement via hyaluronate. In cardiac tissue, nanoneedles are shown to increase the transfection efficiency of nanofilms compared to flat substrates; while in vitro, high transfection efficiencies are observed in nanostructures where cells present large interfacing areas with the substrate. The results of this study demonstrate that surface-mediated transfection using this system is efficient and safe, requiring amounts of nucleic acid with an order of magnitude lower than standard culture transfection. These findings expand the spectrum of possible polyelectrolyte combinations that can be used for the development of suitable non-viral vectors for exploration in further clinical trials.
Assuntos
Técnicas de Transferência de Genes , Ácidos Nucleicos , Terapia Genética/métodos , Polieletrólitos , TransfecçãoRESUMO
Polymersomes are vesicular structures self-assembled from amphiphilic block copolymers and are considered an alternative to liposomes for applications in drug delivery, immunotherapy, biosensing, and as nanoreactors and artificial organelles. However, the limited availability of systematic stability, protein fouling (protein corona formation), and blood circulation studies hampers their clinical translation. Poly(2-oxazoline)s (POx) are valuable antifouling hydrophilic polymers that can replace the current gold-standard, poly(ethylene glycol) (PEG), yet investigations of POx functionality on nanoparticles are relatively sparse. Herein, a systematic study is reported of the structural, dynamic and antifouling properties of polymersomes made of poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b-PMOXA). The study relates in vitro antifouling performance of the polymersomes to atomistic molecular dynamics simulations of polymersome membrane hydration behavior. These observations support the experimentally demonstrated benefit of maximizing the length of PMOXA (degree of polymerization (DP) > 6) while keeping PDMS at a minimal length that still provides sufficient membrane stability (DP > 19). In vitro macrophage association and in vivo blood circulation evaluation of polymersomes in zebrafish embryos corroborate these findings. They further suggest that single copolymer presentation on polymersomes is outperformed by blends of varied copolymer lengths. This study helps to rationalize design rules for stable and low-fouling polymersomes for future medical applications.
Assuntos
Sistemas de Liberação de Medicamentos , Peixe-Zebra , Animais , Interações Hidrofóbicas e Hidrofílicas , Macrófagos , OxazóisRESUMO
The generation and sensing of membrane curvature by proteins has become of increasing interest to researchers with multiple mechanisms, from hydrophobic insertion to protein crowding, being identified. However, the role of charged lipids in the membrane curvature-sensing process is still far from understood. Many proteins involved in endocytosis bind phosphatidylinositol 4,5-bisphosphate (PIP2) lipids, allowing these proteins to accumulate at regions of local curvature. Here, using coarse-grained molecular dynamics simulations, we study the curvature-sensing behavior of the ANTH domain, a protein crucial for endocytosis. We selected three ANTH crystal structures containing either an intact, split, or truncated terminal amphipathic helix. On neutral membranes, the ANTH domain has innate curvature-sensing ability. In the presence of PIP2, however, only the domain with an intact helix senses curvature. Our work sheds light on the role of PIP2 and its modulation of membrane curvature sensing by proteins.
Assuntos
Endocitose , Fosfatidilinositóis , Membrana Celular/química , Simulação de Dinâmica Molecular , Fosfatidilinositóis/metabolismo , Proteínas/metabolismoRESUMO
The therapeutic and research potentials of oligonucleotides (ONs) have been hampered in part by their inability to effectively escape endosomal compartments to reach their cytosolic and nuclear targets. Splice-switching ONs (SSOs) can be used with endosomolytic small molecule compounds to increase functional delivery. So far, development of these compounds has been hindered by a lack of high-resolution methods that can correlate SSO trafficking with SSO activity. Here we present in-depth characterization of two novel endosomolytic compounds by using a combination of microscopic and functional assays with high spatiotemporal resolution. This system allows the visualization of SSO trafficking, evaluation of endosomal membrane rupture, and quantitates SSO functional activity on a protein level in the presence of endosomolytic compounds. We confirm that the leakage of SSO into the cytosol occurs in parallel with the physical engorgement of LAMP1-positive late endosomes and lysosomes. We conclude that the new compounds interfere with SSO trafficking to the LAMP1-positive endosomal compartments while inducing endosomal membrane rupture and concurrent ON escape into the cytosol. The efficacy of these compounds advocates their use as novel, potent, and quick-acting transfection reagents for antisense ONs.
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Oligonucleotídeos Antissenso , Oligonucleotídeos , Endossomos/metabolismo , Membranas Intracelulares , Lisossomos , Oligonucleotídeos/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologiaRESUMO
As research becomes predominantly digitalized, scientists have the option of using electronic laboratory notebooks to record and access entries. These systems can more readily meet volume, complexity, accessibility and preservation requirements than paper notebooks. Although the technology can yield many benefits, these can be realized only by choosing a system that properly fulfills the requirements of a given context. This review explores the factors that should be considered when introducing electronic laboratory notebooks to an academically focused research group. We cite pertinent studies and discuss our own experience implementing a system within a multidisciplinary research environment. We also consider how the required financial and time investment is shared between individuals and institutions. Finally, we discuss how electronic laboratory notebooks fit into the broader context of research data management. This article is not a product review; it provides a framework for both the initial consideration of an electronic laboratory notebook and the evaluation of specific software packages.
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LaboratóriosRESUMO
We investigated the biomaterial interface between the bacteria Escherichia coli DH5α and silicon nanowire patterned surfaces. We optimised the engineering of silicon nanowire coated surfaces using metal-assisted chemical etching. Using a combination of focussed ion beam scanning electron microscopy, and cell viability and transformation assays, we found that with increasing interfacing force, cell viability decreases, as a result of increasing cell rupture. However, despite this aggressive interfacing regime, a proportion of the bacterial cell population remains viable. We found that the silicon nanowires neither resulted in complete loss of cell viability nor partial membrane disruption and corresponding DNA plasmid transformation. Critically, assay choice was observed to be important, as a reduction-based metabolic reagent was found to yield false-positive results on the silicon nanowire substrate. We discuss the implications of these results for the future design and assessment of bacteria-nanostructure interfacing experiments.
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Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Viabilidade Microbiana/efeitos dos fármacos , Nanofios , Silício/química , Silício/farmacologia , Biotransformação/efeitos dos fármacos , Escherichia coli/metabolismo , Propriedades de SuperfícieRESUMO
The evolution of life on earth eventually leads to the emergence of species with increased complexity and diversity. Similarly, evolutionary chemical space exploration in the laboratory is a key step to pursue the structural and functional diversity of supramolecular systems. Here, we present a powerful tool that enables rapid peptide diversification and employ it to expand the chemical space for supramolecular functions. Central to this strategy is the exploitation of palladium-catalyzed Suzuki-Miyaura cross-coupling reactions to direct combinatorial synthesis of peptide arrays in microtiter plates under an open atmosphere. Taking advantage of this in situ library design, our results unambiguously deliver a fertile platform for creating a set of intriguing peptide functions including green fluorescent protein-like peptide emitters with chemically encoded emission colors, hierarchical self-assembly into nano-objects, and macroscopic hydrogels. This work also offers opportunities for quickly surveying the diversified peptide arrays and thereby identifying the structural factors that modulate peptide properties.
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Paládio , Peptídeos , HidrogéisRESUMO
Nanoscale membrane curvature is a common feature in cell biology required for functions such as endocytosis, exocytosis and cell migration. These processes require the cytoskeleton to exert forces on the membrane to deform it. Cytosolic proteins contain specific motifs which bind to the membrane, connecting it to the internal cytoskeletal machinery. These motifs often bind charged phosphatidylinositol phosphate lipids present in the cell membrane which play significant roles in signaling. These lipids are important for membrane deforming processes, such as endocytosis, but much remains unknown about their role in the sensing of membrane nanocurvature by protein domains. Using coarse-grained molecular dynamics simulations, we investigated the interaction of a model curvature active protein domain, the epsin N-terminal homology domain (ENTH), with curved lipid membranes. The combination of anionic lipids (phosphatidylinositol 4,5-bisphosphate and phosphatidylserine) within the membrane, protein backbone flexibility, and structural changes within the domain were found to affect the domain's ability to sense, bind, and localize with nanoscale precision at curved membrane regions. The findings suggest that the ENTH domain can sense membrane curvature without the presence of its terminal amphipathic α helix via another structural region we have denoted as H3, re-emphasizing the critical relationship between nanoscale membrane curvature and protein function.
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High-aspect-ratio nanostructures have emerged as versatile platforms for intracellular sensing and biomolecule delivery. Here, we present a microfabrication approach in which a combination of reactive ion etching protocols were used to produce high-aspect-ratio, nondegradable silicon nanoneedle arrays with tip diameters that could be finely tuned between 20 and 700 nm. We used these arrays to guide the long-term culture of human mesenchymal stem cells (hMSCs). Notably, we used changes in the nanoneedle tip diameter to control the morphology, nuclear size, and F-actin alignment of interfaced hMSCs and to regulate the expression of nuclear lamina genes, Yes-associated protein (YAP) target genes, and focal adhesion genes. These topography-driven changes were attributed to signaling by Rho-family GTPase pathways, differences in the effective stiffness of the nanoneedle arrays, and the degree of nuclear membrane impingement, with the latter clearly visualized using focused ion beam scanning electron microscopy (FIB-SEM). Our approach to design high-aspect-ratio nanostructures will be broadly applicable to design biomaterials and biomedical devices used for long-term cell stimulation and monitoring.
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Nanoestruturas , Membrana Nuclear , Expressão Gênica , Humanos , Silício , Células-TroncoRESUMO
Materials patterned with high-aspect-ratio nanostructures have features on similar length scales to cellular components. These surfaces are an extreme topography on the cellular level and have become useful tools for perturbing and sensing the cellular environment. Motivation comes from the ability of high-aspect-ratio nanostructures to deliver cargoes into cells and tissues, access the intracellular environment, and control cell behavior. These structures directly perturb cells' ability to sense and respond to external forces, influencing cell fate, and enabling new mechanistic studies. Through careful design of their nanoscale structure, these systems act as biological metamaterials, eliciting unusual biological responses. While predominantly used to interface eukaryotic cells, there is growing interest in nonanimal and prokaryotic cell interfacing. Both experimental and theoretical studies have attempted to develop a mechanistic understanding for the observed behaviors, predominantly focusing on the cell-nanostructure interface. This review considers how high-aspect-ratio nanostructured surfaces are used to both stimulate and sense biological systems.
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Materiais Biocompatíveis/química , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Células Eucarióticas/ultraestrutura , Nanoestruturas/química , Animais , Fenômenos Biomecânicos , Adesão Celular , Diferenciação Celular , Permeabilidade da Membrana Celular , Técnicas Eletroquímicas , Humanos , Metais/química , Processos Fotoquímicos , Polímeros/química , Porosidade , Silício/química , Propriedades de SuperfícieRESUMO
Nanoporous surfaces are used in many applications in intracellular sensing and drug delivery. However, the effects of such nanostructures on cell membrane properties are still far from understood. Here, we use coarse-grained molecular dynamics simulations to show that nanoporous substrates can stimulate membrane-curvature effects and that this curvature-sensing effect is much more sensitive than previously thought. We define a series of design parameters for inducing a nanoscale membrane curvature and show that nanopore taper plays a key role in membrane deformation, elucidating a previously unexplored fabrication parameter applicable to many nanostructured biomaterials. We report significant changes in the membrane area per lipid and thickness at regions of curvature. Finally, we demonstrate that regions of the nanopore-induced membrane curvature act as local hotspots for an increased bioactivity. We show spontaneous binding and localization of the epsin N-terminal homology (ENTH) domain to the regions of curvature. Understanding this interplay between the membrane curvature and nanoporosity at the biointerface helps both explain recent biological results and suggests a pathway for developing the next generation of cell-active substrates.
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Proteínas Adaptadoras de Transporte Vesicular/química , Membrana Celular/química , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Nanoporos , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismoRESUMO
Organic diodes manufactured on a plastic substrate capable of rectifying a high-frequency radio-frequency identification signal (13.56 MHz), with sufficient power to operate an interactive smart tag, are reported. A high-performance conjugated semiconductor (an indacenodithiophene-benzothiadiazole copolymer) is combined with a carefully optimized architecture to satisfy the electrical requirements for an organic-semiconductor-based logic chip.
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Here we demonstrate the optimization of gravure printed metal ink, dielectric, and semiconductor formulations. We present a technique for nondestructively imaging printed films using a commercially available flatbed scanner, combined with image analysis to quantify print behavior. Print speed, cliché screen density, nip pressure, the orientation of print structures, and doctor blade extension were found to have a significant impact on the quality of printed films, as characterized by the spreading of printed structures and variation in print homogeneity. Organic semiconductor prints were observed to exhibit multiple periodic modulations, which are correlated to the underlying cell structure.