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

ABSTRACT 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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    • "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.
    Colloids and surfaces B: Biointerfaces 10/2013; 114C:1-10. DOI:10.1016/j.colsurfb.2013.09.029 · 4.15 Impact Factor
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    • "Therefore, novel composites of HA and organic polymers that can compensate for the weak mechanical properties of HA have become of great interest [4] [5]. Chitosan, (C 6 H 11 O 4 N) n is an N-deacetylation product of chitin and is a unique polysaccharide based biopolymer that shares a number of chemical and structural similarities with collagen and has been used as a skin grafting template, hemostatic agent, DNA and drug delivery vehicle, and as a wound healing material [6] [7] [8] [9] [10] [11]. Also, chitosan (CTS) films support the growth, function, and cellular activity of osteoblasts and chondrocytes [12]. "
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    ABSTRACT: Incorporation of hydroxyapatite (HA) with organic polymer in favor of composites would be used in biomaterial engineering. According to prior researches, because of its chemical similarity to natural bone and dental, this product could improve bioactivity and bone bonding ability. In this research, nano-hydroxyapatite/chitosan composite material was prepared via in situ Hybridization route. The surface chemical characterization on the nanocomposite was evaluated by Fourier transformed infrared (FTIR) and X-ray diffraction (XRD). Surface topography, roughness and morphology of the samples were observed by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The characterization results confirmed homogeneity, interaction and integration between the HA and chitosan matrix. It was indicated that composite samples consist of homogeneous aggregations around 40–100 nm, in which many HA nanocrystals align along the chitosan molecules. HA grain gradually decreased in size when amount of chitosan increased from 0 to 6 g into 100 cc solution. It can be seen that by increasing chitosan, the aggregation of nanoparticles enhance and subsequently, improve the expected compatibility among HA filler and chitosan matrix. Furthermore, the mechanical compressive testing indicated that the synthesized composites have acceptable mechanical behavior for tissue substitution. The mechanistic of the biodegradable nanocomposite systems, their preparation and characterization for medical usage are strongly discussed.
    Composites Part B Engineering 06/2012; 43(4):1881–1886. DOI:10.1016/j.compositesb.2012.01.056 · 2.98 Impact Factor
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    • "For instance the binding affinities for pDNA/cationic polymer systems measured by ITC were also heavily exothermic [18], however they were reported two to three logarithmic units lower for liposomal or cationic peptide/nucleic acid interactions [26] [27]. This increased complex stability may explain the inferior transfection effectiveness of these siRNA/chitosan polyplexes compared to liposomal systems reflecting an impact on the siRNA release from the carrier system [28]. ITC also allowed for direct quantification of N:P ratios between chitosan and siRNA at which full saturation of the polymer with siRNA, or vice versa, is obtained. "
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    ABSTRACT: Chitosans are naturally occurring polymers widely used in life science to mediate intracellular uptake of nucleic acids such as siRNA. Four chitosans of fungal origin (Agaricus bisporus; molecular weights MW=44, 63, 93 and 143 kDa) were used in this study and profiled for size, viscosity and hydrodynamic radius using gel permeation chromatography (GPC). Polyplexes made of these chitosans and siRNA were developed and optimized for transfection efficacy in vitro. The characteristics of these polyplexes were low chitosan:siRNA ratios (4-8; N:P) similar positive zeta potential (20-30 mV) and comparable particle sizes (about 150 nm). Endogenous luciferase reporter gene down-regulation in human epithelial H1299 cells at nanomolar concentrations (37.5-150 nM) was significantly stronger for the lower molecular weight chitosans. The impact of these low N:P polyplexes on the cellular viability was minimal also at 150 nM. To help develop an understanding of these differences, an energetic profile of the molecular interactions and polyplex formation was established by isothermal titration calorimetry (ITC). The four polyplexes exhibited strong binding enthalpies delta H(bind)(-84 to -102 kcal/mol) resulting in nanomolar dissociation constants. Intracellular trafficking studies using rhodamine labeled siRNA revealed that polyplexes made from smaller MW chitosans exhibited faster cellular uptake kinetics than their higher MW counterpart. Transmission electron microscopy and small angle X-ray scattering studies (SAXS) revealed that the 44 kDa derived polyplexes exhibited regular spherical structure, whereas the 143 kDa chitosan polyplex was rather irregularly shaped. With regards to adverse effects these low N:P chitosan/siRNA formulations represent an interesting alternative to so far reported chitosan polyplexes that used vast N:P excess to achieve similar bioactivity.
    Journal of Controlled Release 08/2011; 157(2):297-304. DOI:10.1016/j.jconrel.2011.08.023 · 7.26 Impact Factor
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