RESUMO
UNLABELLED: AIM & METHODS: We have produced two chimerical peptides of 10.2 kDa, each contain four biologically active domains, which act as building blocks of protein-based nonviral vehicles for gene therapy. In solution, these peptides tend to aggregate as amorphous clusters of more than 1000 nm, while the presence of DNA promotes their architectonic reorganization as mechanically stable nanometric spherical entities of approximately 80 nm that penetrate mammalian cells through arginine-glycine-aspartic acid cell-binding domains and promote significant transgene expression levels. RESULTS & CONCLUSION: The structural analysis of the protein in these hybrid nanoparticles indicates a molecular conformation with predominance of α-helix and the absence of cross-molecular, ß-sheet-supported protein interactions. The nanoscale organizing forces generated by DNA-protein interactions can then be observed as a potentially tunable, critical factor in the design of protein-only based artificial viruses for gene therapy.
Assuntos
DNA/química , DNA/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Proteínas/química , Proteínas/metabolismo , Animais , Células Cultivadas , Dicroísmo Circular , DNA/genética , DNA/ultraestrutura , Terapia Genética/métodos , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Peptídeos/genética , Ligação Proteica , Proteínas/genética , Proteínas/ultraestrutura , Ratos , Ratos Sprague-DawleyRESUMO
AIMS: Arginine(R)-rich cationic peptides are powerful tools in drug delivery since, alone or when associated with polyplexes, proteins or chemicals, they confer DNA condensation, membrane translocation and blood-brain barrier crossing abilities. The unusual stability and high in vivo performance of their associated drugs suggest a particulate organization or R(n) complexes, which this study aimed to explore. MATERIALS & METHODS: We have analyzed the particulate organization and biological performance in DNA delivery of a model, R9-containing green fluorescent protein by dynamic light scattering, transmission electron microscopy, atomic force microscopy, single cell confocal microscopy and flow cytometry. RESULTS: A deep nanoscale examination of R9-powered constructs reveals a novel and promising feature of R9, that when fused to a scaffold green fluorescent protein, promote its efficient self-assembling as highly stable, regular disk-shaped nanoparticles of 20 x 3 nm. These constructs are efficiently internalized in mammalian cells and rapidly migrate through the cytoplasm towards the nucleus in a fully bioactive form. Besides, such particulate platforms accommodate, condense and deliver plasmid DNA to the nucleus and promote plasmid-driven transgene expression. CONCLUSION: The architectonic properties of arginine-rich peptides at the nanoscale reveal a new category of protein nanoparticles, namely nanodisks, and provide novel strategic concepts and architectonic tools for the tailored construction of new-generation artificial viruses for gene therapy and drug delivery.
Assuntos
Arginina/química , Nanopartículas/química , Peptídeos/química , Barreira Hematoencefálica , Cátions , DNA/química , DNA/genética , Citometria de Fluxo/métodos , Corantes Fluorescentes/farmacologia , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/química , Células HeLa , Humanos , Microscopia de Força Atômica/métodos , Microscopia Eletrônica de Transmissão/métodos , Nanocompostos/químicaRESUMO
MOTIVATION: This application aims at assisting researchers with the extraction of significant medical and biological knowledge from data sets with complex relationships among their variables. RESULTS: Non-hypothesis-driven approaches like Principal Curves of Oriented Points (PCOP) are a very suitable method for this objective. PCOP allows for obtaining of a representative pattern from a huge quantity of data of independent variables in a very flexible and direct way. A web server has been designed to automatically realize 'non-linear pattern' analysis, 'hidden-variable-dependent' clustering, and new samples 'local-dispersion-dependent' classification from the data involving new statistical techniques using the PCOP calculus. The tools facilitate the managing, comparison and visualization of results in a user-friendly graphical interface. AVAILABILITY: http://ibb.uab.es/revresearch.
