Characterization and stability of chitosan-based nanoparticles as efficient non-viral gene delivery systems

LIMERES MJ; PRIETO-SANCHEZ S; HERNANDEZ-MUNAIN C; SUÑE C; NUSBLAT A; CUESTAS M; SUÑÉ-NEGRE JM; SUÑÉ

Congreso; NanoBio&Med 2018.; 2018

Introduction: Chitosan (CS) is a linear cationic polysaccharide that is obtained commercially by the deacetylation of the chitin which is a biopolymer that is widely distributed in nature. Due to its characteristics of biocompatibility, biodegradability and non-immunogenicity, constitutes a suitable biomaterial for the design of nanoparticles, allowing in vivo applications [1]. It is also considered as a GRAS substance by the FDA. This polysaccharide has in its structure primary amino groups with a pKa of 6.5, therefore, in moderately acidic media these groups are protonated allowing the interaction with multivalent negatively charged molecules such as the polyelectrolyte sodium tripolyphosphate (TPP). Thus, the interaction between them in defined proportions and with the established parameters of a suitable technique leads to the self-assembly and the formation of chitosan-TPP nanoparticles. The aim of this work is to define and characterize the most suitable formulation of chitosan cationic polymeric nanoparticles with the addition of a diblock copolymer of ethylene oxide and propylene oxide (poloxamer P188), evaluating their potential application as non-viral gene delivery system [2, 3].Materials & Methods:The CS-based nanoparticles were produced using a modified ionotropic gelation method [4,5] in the presence of the TPP ionic cross-linker molecule. Briefly, a solution of chitosan (low molecular weight) at 0.2% (w/v) was prepared in acetic buffer solution at pH=5 and was stirred overnight to allow the complete dissolution of the polymer. In one part of the chitosan solution, 1% (w/v) and 0.5% (w/v) poloxamer P188 was added to evaluate the stability of nanoparticles in the presence of this surfactant. The solutions were filtered to discard not dissolved chitosan molecules. Then, a solution of 0.84 mg/mL TPP was added dropwise to the chitosan solution for 5 minutes under constant stirring at room temperature. The final suspension was centrifuged and filtered to obtain de CS-based nanoparticles. The stability of two different RATIO of CS-TPP nanoparticles, 6:1 and 3:1, was assessed at 4 °C and 25 °C. The particle size and the zeta potential were measured by the laser diffraction method (Mastersizer) and the laser Doppler microelectrophoresis method (Zetasizer) respectively. The morphology of the nanoparticles of CS-TPP-1%P188 was characterized by transmission electron microscopy (TEM), and finally the loading efficiency with different amounts of plasmidic DNA was performed by agarose gel electrophoresis.Results:Stability studies showed the presence of aggregates in the nanoparticles formulation of CS-TPP without P188 at day 7 at 4 °C and at day 3 at 25 °C, while formulations containing the surfactant maintained their size at that time. In addition, we observed that the nanoparticles with the highest amount of P188 were smaller in size, which may explain their higher stability. Regarding the zeta potential, all the formulations maintained the values over the time. However, the initial zeta potential of the RATIO 3: 1 nanoparticles was lower, therefore being discarded as non-viral gene delivery system. The TEM images confirm the results obtained with the Mastersizer, showing spherical nanoparticles with a homogenous distribution. Finally, the loading efficiency performed by agarose gel electrophoresis suggested that CS-TPP-1%P188 nanoparticles are capable to complex DNA at the studied proportions, and no free DNA was observed in the gel.Conclusions:The characterization of the different chitosan nanoparticles obtained and the stability studies determining particle size and zeta potential over time at different conditions allow the selection of the most stable and suitable formulation. In addition, polyplexes with DNA were obtained with 100% loading efficiency, demonstrating its potential application a non-viral gene delivery system.