Characterization of insulin-loaded alginate nanoparticles produced by ionotropic pre-gelation through DSC and FTIR studies
Introduction
Nanoparticles made of polyelectrolytes complexation have shown potential for use as drug delivery systems. Polyelectrolyte complexes (PEC) are formed when oppositely charged polyelectrolytes are mixed and interact via electrostatic interactions. Chitosan and alginate are polycation and polyanion polyelectrolytes, respectively, that can be used to form a polyelectrolyte complex and to deliver proteins (Sarmento et al., in press), peptidic drugs (Coppi, Iannuccelli, Leo, Bernabei, & Cameroni, 2001) and DNA (Douglas & Tabrizian, 2005).
The interaction between alginate in dilute solution with Ca2+ occurs at a certain ion concentration (Rajaonarivony, Vauthier, Couarraze, Puisieux, & Couvreur, 1993). A pre-gel state results with stirring, avoiding the gel point and forming a continuous system. Subsequent addition of an aqueous polycationic solution (chitosan) results in a polyelectrolyte complex, stabilizing the alginate pre-gel nucleus into individual sponge-like nanoparticles (Sarmento, Ribeiro, Veiga, Neufeld, & Ferreira, 2005).
Many studies have been published on individual chitosan–alginate systems (De and Robinson, 2003, Lee et al., 1997). Although parameters such as the molecular weight, temperature, and pH have been studied to some extent, most of these studies are performed under specific and limited pH and stoichiometric conditions. Few attempts have been made to compare the properties of complexes formed between chitosan and alginate with and without protein and to rationalize the results in terms of the molecular properties of the protein-containing complexes.
The use of polyelectrolyte complexes as protein delivery systems may result in an overall higher level of electrostatic interactions due to the presence of protein. The control of particles size remains a main challenge but the retention of biological activity of encapsulant has been reported (Silva, Ribeiro, Figueiredo, Goncalves, & Veiga, 2006).
Differential scanning calorimetry (DSC) can be used to characterize the thermal behaviour of polyelectrolytes and biomolecules which is correlated to their structure, hydrophilic properties and association state. It is based on the heat capacity of the sample as a function of the temperature. It is possible in one experiment to obtain the complete temperature profile of the Gibbs energy change associated with the loss of water in polymers, with the denaturation process in proteins and with depolymerization at high temperatures. Shifts of exothermic and endothermic peaks are usually associated with interactions between drugs and polymer (Borges et al., 2005, Ribeiro et al., 2005, Wong et al., 2002).
Fourier transform infrared (FTIR) analysis has also been proposed to examine interactions between polyelectrolyte complexes (Dupuy et al., 1994, Ribeiro et al., 2005).
With these series of experiments it was intended to monitorize the complexation of contrary charged polyelectrolytes as insulin nanoparticulate carriers. Systems resulting from complexation of polyelectrolytes at varying pH and stoichiometric relationship between polyelectrolytes were lyophilized, and analyzed by DSC and FTIR to verify interactions between polyelectrolytes and between polymers and insulin.
Section snippets
Materials and methods
Low viscosity sodium alginate with a low guluronic content (FG = 0.39), low molecular weight (MW) chitosan (≈50 kDa) and calcium chloride were purchased from Sigma (Oakville, Canada). Alginate solution was prepared in deionized water (Milli-Q®) overnight under magnetic stirring and chitosan sample was dissolved in 1% acetic acid solution in deionized water followed by filtration by using a Millipore #2 paper filter and stored at 4 °C. Human zinc–insulin crystal was a gift from Lilly Farma, Portugal.
Results and discussion
Particle size is determinant in mucosal and epithelial tissue uptake of particles and in the intracellular traffic of the particles (Panyam & Labhasetwar, 2003). Drug carriers sizing few microns have shown higher potential as oral delivery systems of proteins and peptides (Chen & Langer, 1998). Granulometric distribution of chitosan–alginate nanoparticles depend on concentration and molecular weight of both polyelectrolytes, and conditions of mixing (Sarmento et al., in press). Besides, protein
Conclusion
DSC and FTIR were successfully used to characterize the nanoparticulate systems made up by polyelectrolyte complexes. Shifts on endothermic and exothermic peaks and shifts on maximum infrared peaks observed between individual polyelectrolytes and final nanoparticle carriers were understand as ionic interactions which led to the formation of new chemical entities with different thermal and absorption properties. Additionally, insulin entrapment also originated small shifts on same nanoparticle
Acknowledgements
This work was supported by Fundação para a Ciência e Tecnologia, Portugal and the Natural Sciences and Engineering Research Council of Canada. The authors thank Lilly Portugal for insulin supply and Marco van de Weert and Lene Jorgensen from Danish University of Pharmaceutical Sciences for FTIR equipment facilities.
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