The influence of chitosan valence on the complexation and transfection of DNA The weaker the DNA-chitosan binding the higher the transfection efficiency

Grupo de Nanomateriales y Materia Blanda, Departamento de Física de Materia Condensada, Facultad de Física, Universidad de Santiago de Compostela, E-15782 Santiago de Compostela, Spain.
Colloids and surfaces B: Biointerfaces (Impact Factor: 4.15). 01/2011; 82(1):54-62. DOI: 10.1016/j.colsurfb.2010.08.013
Source: PubMed


The DNA-chitosan polyplexes have attracted for some years now the attention of physical-chemists and biologists for their potential use in gene therapy, however, the correlation between the physicochemical properties of these polyplexes with their transfection efficiency remains still unclear. In a recent paper we demonstrated by means of DLS that the DNA-chitosan complexation is favored at acidic conditions considering that fewer amounts of chitosan were required to compact the DNA. As a second study, in the present work we analyze the influence of chitosan valence on the complexation and transfection of DNA. Three chitosans of different molecular weights (three different valences) are characterized as gene carriers at 25°C and pH 5 over a wide range of chitosan-Nitrogen to DNA-Phosphate molar ratios, N/P, by means of conductometry, electrophoretic mobility, isothermal titration calorimetry (ITC), transmission electron microscopy (TEM), atomic force microscopy (AFM), and β-galactosidase and luciferase expression assays.

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Available from: Manuel Alatorre-Meda, Feb 14, 2014
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    • "−7 M). Our CS-PLGA binding constant reached similar value as the one of Ma et al [63] (10 9 ) and Alatorre-Meda et al [62] (10 7 ) for the CS-DNA interaction. We obtained for the average number of binding sites a value of 3.4, which can be considered as the number of CS chains per NP in our experiment, as was confirmed with the zeta potential results (next section). "
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    ABSTRACT: In this work, we report the synthesis and characterization of a new hybrid nanoparticles system performed by magnetite nanoparticles, loaded in a PLGA matrix, and stabilized by different concentrations of chitosan. Magnetite nanoparticles were hydrophobized with oleic acid and entrapped in a PLGA matrix by the emulsion solvent evaporation method, after that, magnetite/PLGA/chitosan nanoparticles were obtained by adding dropwise magnetite/PLGA nanoparticles in chitosan solutions. Magnetite/PLGA nanoparticles produced with different molar ratios did not show significant differences in size and the 3:1 molar ratio showed best spherical shapes as well as uniform particle size. Isothermal titration calorimetry studies demonstrated that the first stage of PLGAchitosan interaction is mostly regulated by electrostatic forces. Based on a single set of identical sites model, we obtained for the average number of binding sites a value of 3.4, which can be considered as the number of chitosan chains per nanoparticle. This value was confirmed by using a model based on theDLVO theory and fitting zeta potential measurements of magnetite/PLGA/chitosan nanoparticles. From the adjusted parameters, we found that an average number of chitosan molecules of 3.6 per nanoparticle are attached onto the surface of the PLGA matrix. Finally, we evaluated the effect of surface charge of nanoparticles on a membrane model of endothelial cells performed by a mixture of three phospholipids at the air–water interface. Different isotherms and adsorption curves show that cationic surface of charged nanoparticles strongly interact with the phospholipids mixture and these results can be the basis of future experiments to understand the nanoparticles- cell membrane interaction.
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    • "Moreover, its properties can be tuned to a considerable extent by simply controlling the degree of deacetylation and polymerization, or the pH-dependent degree of ionization [34]. Chitosan properties controlled by these parameters, affect its interaction with DNA [35] [36] [5] and, as a consequence, the properties and performance of the resulting polyplexes [6] as vectors for gene delivery. However, the effects of the different parameters are interconnected in a non trivial way. "
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    ABSTRACT: The design of biocompatible polyelectrolyte complexes is a promising strategy for in vivo delivery of biologically active macromolecules. Particularly, the condensation of DNA by polycations received considerable attention for its potential in gene delivery applications, where the development of safe and effective non-viral vectors remains a central challenge. Among polymeric polycations, Chitosan has recently emerged as a very interesting material for these applications. In this study, we compare the observed aggregation behavior of Chitosan-DNA complexes with the predictions of existing models for the complexation of oppositely charged polyelectrolytes. By using different and complementary microscopy approaches (AFM, FESEM and TEM), light scattering and electrophoretic mobility techniques, we characterized the structures of the complexes formed at different charge ratios and Chitosan molecular weight. In good agreement with theoretical predictions, a reentrant condensation, accompanied by charge inversion, is clearly observed as the polycation/DNA charge ratio is increased. In fact, the aggregates reach their maximum size in correspondence of a value of the charge ratio where their measured net charge inverts its sign. This value does not correspond to the stoichiometric 1:1 charge ratio, but is inversely correlated with the polycation length. Distinctive "tadpole-like" aggregates are observed in excess polycation, while only globular aggregates are found in excess DNA. Close to the isoelectric point, elongated fiber-like structures appear. Within the framework of the models discussed, different apparently uncorrelated observations reported in the literature find a systematic interpretation. These results suggest that these models are useful tools to guide the design of new and more efficient polycation-based vectors for a more effective delivery of genetic material.
    Full-text · Article · Oct 2013 · Colloids and surfaces B: Biointerfaces
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    • "With a view to overcoming these disadvantages, a number of materials such as chitosan, poly(vinyl pyrrolidone) (PVP), collagen, chitin, poly(vinyl alcohol), and alginate polymers have been applied as curative materials to improve biological applications. Chitosan (CS) is a biodegradable polymer, a unique polysaccharidebased biopolymer that shares a number of chemical and structural similarities with collagen; it has been used as a haemostatic agent, a skin grafting template, a DNA and drug-delivery vehicle and a wound-healing material (Park et al., 2010; Alatorre-Meda et al., 2011; S ¸entürk et al., 2011; Khor & Lim, 2003; Ragetly et al., 2010). The fact that chitosan films support the growth, function, and cellular activity of osteoblasts, and chondrocytes renders this material an appropriate candidate for tissue engineering, particularly bone, and cartilage scaffolds (Nettles et al., 2002; Francis Suh & Matthew, 2000). "
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    ABSTRACT: The purpose of this work was to compare hydroxyapatite (HAP) and composites of HAP, HAP with chitosan (CS), and HAP with poly(vinyl pyrrolidone) (PVP), in terms of their particle size and morphology, using different methods, such as Coulter counter analysis, X-ray diffraction (XRD), and transmission electron microscopy (TEM). Although many researchers have studied HAP and CS/HAP and PVP/HAP composites extensively, there is no evidence of a comparative study of their particle sizes. For this reason, different complementary methods have been used so as to provide a more complete image of final product properties — particle size — from the perspective of possible applications. The syntheses of HAP and HAP with polymer nanoparticles were carried out employing a precipitation method. Variation in particle size with synthesis time and influence of the reactants’ concentration on the materials’ preparation were systematically explored. Crystallite size calculated from XRD data revealed nanosized particles of HAP, CS/HAP, and PVP/HAP materials in the range of 2.5–9.2 nm. Coulter counter analysis revealed mean particle sizes of one thousand orders of magnitude larger, confirming that this technique measures agglomerates, not individual particles. In addition, the particles’ morphology and an assessment of their binding mode were completed by TEM measurements.
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