Short communicationSurface charging and dimensions of chitosan coacervated nanoparticles
Graphical abstract
Highlights
► Chitosan nanoparticles obtained using the coacervation method are subjected to enthalpic and entropic influences. ► Enthalpic effect relates to interactions between electrically charged sites and enthalpic effect to spatial arrangement of counter-ions. ► Initially chitosan macromolecular coils shrink, then they collapse, forming clusters of nanometric dimensions. ► If acid concentration is high enough, nanoparticle formation is mainly driven by entropic effects.
Introduction
Since its popularization by Drexler [1] nanotechnology has itself turned from a promise into a reality in many areas, such as textile industry [2], agriculture [3], forensic science [4], and biomedicine [5]. As a matter of fact, this technology has found an increasingly important role in controlled release of therapeutic agents for diseases like cancer, AIDS, diabetes malaria, and tuberculosis [6].
Biopolymer-based systems have been studied as pharmaceutical vehicles for controlled release devices due to their stability in blood and low toxicity [7], [8]. Some of these polymers have been approved for administration in humans and have been widely studied, such as poly(glycolic acid) (PLGA), polycaprolactam (PCL), gelatin, and chitosan [9]. In specific case of chitosan, it is a copolymer formed from glucosamine and N-acetyl-glucosamine units, which is prepared by partial deacetylation of chitin, usually obtained from crustacean shells [10], [11], [12], [13]. Chitosan has been chosen for drug delivery systems based on its capability of being protonated, acquiring positive charges, which give to this polymer mucoadhesive characteristic, increasing drug residence time in the site of action [14], [15].
The preparation of chitosan-based nanoparticles can be carried out using different methods, which, on their basis, involve one (or both) of two kinds of association between chitosan macromolecules:
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Covalent crosslinking: needs a crosslinking agent, e.g., glutaraldehyde [16] and genipin [17];
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Physical interactions: chitosan particles are formed as a result of a pronounced decrease in free energy as a result chitosan/chitosan association. The techniques used to prepare such particles may involve methods such as spray-drying [18], ionic gelification [19], reverse microemulsion method [20], polyelectrolytic complexation [21], and coacervation/precipitation [22], [23].
The interaction between polyelectrolytes, not only natural but also synthetic, and multivalent counter-ions has been intensively investigated, both for academic research and practical reasons [24], [25]. Berthold et al. developed a method of coacervation of chitosan particles, consisting in the addition of a sulfate salt to an acid solution of chitosan containing surfactant under constant stirring [26]. Tiyaboonchai and Limpeanchob reported that the electrostatic interactions between the negative charges of dextran sulfate and zinc sulfate with positively charged chitosan moieties resulted in the formation of virtual crosslinks, leading to particle coacervation [27] and Mao et al. used this very principle to obtain chitosan nanoparticles to be used as gene carriers by the addition of negatively charged DNA to acidic chitosan solutions [28].
Our group has carried out some work on the preparation of chitosan nanoparticles using the coacervation method, employing sulfate as virtual crosslinker as well as poly(methacrylic acid) [29], [30], [31], [32]. The aim of the present work is to obtain chitosan nanoparticles with a different perspective of analysis: now we analyze the process of nanoparticle formation much before the development of turbidity within the solution using SAXS and relate it to surface charging development, as described in the next sections.
Section snippets
Experimental
Chitosan used in this work was purchased from Polymar LTD (Brazil) and was purified as reported in the literature [33]. It had a deacetylation degree of XD = 0.88, determined by CHN elemental analysis and conductometric titration as described elsewhere [34], and average viscometric molar mass, , determined using Mark–Houwink–Sakurada equation [35], [36]. Acetic acid (P.A., Cromato Produtos Químicos LTD, Brazil), HCl (P.A., Cromato Produtos Químicos LTD, Brazil), and sodium
Results and discussion
Before analyzing the results in terms of sulfate:amino molar ratio, rSA, we have carried out experiments to determine if the system reached its thermodynamic equilibrium before any measurement was made. This was done by measuring zeta potential, ζ, for dispersions with 4 different rSA's, as shown in Fig. 1. It is clearly verified that 4 h represents an adequate time for the dispersions to reach equilibrium, since ζ randomically oscillates within this time range, indicating it has reached its
Conclusions
The process of formation of chitosan nanoparticles by the coacervation method can be characterized as occurring in two steps: (i) as polyvalent ion is added, macromolecular chitosan chains contract, due to polyelectrolyte effect and (ii) contraction leads macromolecules to their minimum dimensions and the addition of polycharged ion results in the collapse of solubilized complexes. SAXS results were a function of counter-ion concentration, indicating that the higher sulfate:amino ratio needed
Acknowledgements
The authors thank Brazil's Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Pró-Reitoria de Pesquisa da Universidade Federal do Rio Grande do Norte (PROPESQ-UFRN) for financial support during the course of this work. The authors also thank Ms. Camila R.M. de Lima for her help in carrying out the experiments as her part in an undergraduate student scientific initiation program.
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