DNA–chitosan complexation: A dynamic light scattering study

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Abstract

The interactions of DNA in mixed solutions with cationic macromolecular compounds have attracted great interest of biomedical sciences, in particular for application in gene therapy. One such compound is chitosan that in addition to being positive in charge has demonstrated to be non-toxic in animal and human trials.

In this work the formation of complexes between chitosan and calf thymus DNA was studied by dynamic light scattering. The study was done in an attempt to characterize the effect of pH of the media and of molecular weight of chitosan, Mw, on (i) the hydrodynamic radii, RH, of the complexes, (ii) the stability of the complexes and (iii) the critical ratio of chitosan to DNA mass concentrations, rc, at which the complexation is completely achieved. Three chitosans of different viscosities (low, middle and high) were used. The viscosity average molecular weight of each chitosan was determined by means of the Mark-Houwink equation. Before determinations, the quality of all autocorrelation functions was confirmed by the calculation of the coherence factor f of the instrument. We demonstrate that the DNA–chitosan complexes obtained are stable, and that pH and Mw influence rc and RH, respectively. We found that the higher was the pH, the larger was the quantity of chitosan needed to completely complex DNA, which was an especially pronounced effect for the highest molecular weight chitosans (middle and highly viscous). By contrast, with increasing Mw, the size of the complexes grew. In general, compacted DNA–chitosan complexes were detectable and reproducibly measured to have average hydrodynamic radii RH around 190–250 nm only at ratios of chitosan to DNA mass concentrations higher than rc  3.

Introduction

The interaction of DNA in mixed solutions with cationic cosolutes, such as multivalent ions, cationic amphiphiles and cationic macromolecules, has attracted great interest of biomedical sciences, not only because of its direct biological implications but also for its potential applications in separation, purification and transfection of DNA. This assembly process is promoted by electrostatic forces where an increase in entropy is present due to release of counterions [1], [2]. On the other hand, the transfection of DNA, which consists in introducing foreign DNA into cells, is presently widely used in biotechnology and has received, over recent years, considerable attention in medicine for gene therapy in treating genetic diseases [3]. The basic requirements that a substance has to meet to be an effective transfection vector is the ability to (i) compact DNA, (ii) protect it against degradation and (iii) deliver it across the membrane with efficiency and specificity. The most efficient transfection vectors currently used are based on viruses, but they suffer from severe limitations such as host immune responses [4].

Due to their potential to be administered repeatedly with minimal host immune response, target-ability, stability on storage and ease of production, non-viral delivery systems for gene therapy have been increasingly proposed as safer alternatives to viral vectors [5], [6]. Among those, DNA–polycation complexes are special in that they are more stable than other non-viral gene delivery systems [7]. In recent years, the potential of chitosan as a polycationic gene carrier has been explored by several research groups [7], [8], [9], [10], [11], [12]. Preparation of self-assembling polymeric DNA–chitosan complexes was first described in 1995. The complex sizes (100–600 nm) were found to depend on the molecular weight of the chitosan used [13]. Other authors report comparable complex sizes as well as their dependence, in addition to the molecular weight of chitosan, on the chitosan–DNA mass concentration ratio, r [14], [15]. Chitosan is a biodegradable polyaminosaccharide [16]. Chemically it is a fully or partially N-deacetylated derivative of chitin [17] extracted from crustacean shells, and has been shown to be non-toxic in both animal [18] and human trials [19]. Chitosan has a positive charge and hydrophilic character at acidic pHs [20]. It is a continuum of primary aliphatic amine that can be protonated by acids, the pKa of the chitosan amine groups being around 6.3–6.5 [17]. The presence of amino groups in its backbone provides chitosan cationicity, and consequently a capacity to form polyelectrolyte complexes and nitrogen derivatives [17]. In practice, the terms “chitin” and “chitosan” refer to a sample of polymers that vary in the degree of acetylation (DA), with “chitin” referring to high DA (ideally 100%) polymers and “chitosan” referring to low DA (ideally 0%) polymers. When DA is 50% or lower, the polymer becomes water-soluble due to the protonation of the –NH2 groups, which is the reason why the term “chitosan” is typically used to refer to polymers that are soluble in dilute acid solutions and “chitin” to those that are insoluble [21]. Therefore, the chemical and biochemical properties of chitosan depend heavily on the degree of acetylation (DA). DA along with pH determines the charge density, which together with the valence (the total charge of the molecule, proportional to the molecular weight) are believed not to only exert an influence over the quantity of chitosan needed toward DNA complexation, but also over the final structures of the complexes [22], [23].

Hybrid DNA–chitosan systems can be classified into two categories, complexes and nanospheres, which differ in their mechanism of formation and morphology [17]. DNA–chitosan complexes are made by simple mixing of solutions of DNA and of chitosan, as a result of which a broad range of particulate complexes are formed with mean sizes between 100 and 600 nm, depending on the molecular weight of chitosan [13]. Stable complexes are usually formed when chitosan is added in molar excess relative to the negatively charged DNA, with zeta potential values between 0 and 20 mV, depending on the degree of excess [14]. By contrast, DNA–chitosan nanospheres are obtained by increasing the speed of mixing and temperature upon blending of the two solutions, the DNA solution having a dilute salt (Na2SO4) and the polymer solution having a desolvating agent added [9]. Such modifications induce significant changes in particle morphology, creating a monodisperse, spherical suspension of nanospheres of sizes between 200 and 500 nm, which sharply differ from the loose rod like [10], [20], doughnut [20] or toroidal [10], [14] structures reported for chitosan–DNA complexes.

The objective of this work was to characterize the influence the pH of the media and the molecular weight of chitosan exert on (i) the hydrodynamic radii, RH, of the complexes, (ii) the stability of the complexes, and (iii) the critical ratio of chitosan to DNA mass concentrations, rc, at which the complexation is completely achieved. These are three parameters of paramount importance in the final stage of gene therapy, the transfection, where as mentioned earlier, the proper penetration of DNA toward the interior of the cell is strongly dependent not only on the size of the complexes (intended to be equivalent to the size of a virus) but also on their stability in order to avoid the destructive action of the enzymes in the proximity of the membrane.

Section snippets

Materials

Chitosans (from crab shells) of low viscosity (≤200 mPa s, 1% in 1% acetic acid, at 20 °C), of middle viscosity (200–400 mPa s, 1% in 1% acetic acid, at 20 °C) and of high viscosity (≥400 mPa s, 1% in 1% acetic acid, at 20 °C) were purchased from Fluka. Calf thymus DNA sodium salt with 13,000 base pairs and an average molecular weight of 12.5 × 106 g mol−1 was bought from Sigma–Aldrich. Bis–Tris and EDTA (both with a purity grade of 99%) from Sigma–Aldrich were used as a buffer and as a quelant,

Results and discussion

Prior to the analysis of the correlation functions and of the derived results, the quality of the correlation functions of each and every one of the light scattering runs was evaluated by calculating the coherence factor. The calculation is described below.

Conclusions

The compaction of DNA by polyelectrolytes such as chitosan can be conveniently followed by dynamic light scattering. It was observed that both pH and molecular weight can have an effect on the properties of complexes formed, including their stability, hydrodynamic radii and complexation mass concentration ratio.

From this study performed on DNA complexation with chitosan, it is demonstrated that the charge density of chitosan plays an important role in the complexation of DNA. That is, that as

Acknowledgements

Authors thank the financial supports of the Dirección Xeral de I+D+I of the Xunta de Galicia and the European Regional Development Fund (INCITE07PXI206076ES), of the Spanish Ministry of Education and Science and the European Regional Development Fund (FIS2007 66823-C02-02) and of the National Council of Science and Technology of Mexico (CONACYT grant 25463).

Manuel Alatorre-Meda is supported by the Programme Alban, the European Union Programme of High Level Scholarships for Latin America,

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