Crosslinked Chitosan Nanoparticles with Muco-Adhesive Potential for Intranasal Delivery Applications

Abstract

Intranasal drug delivery is convenient and provides a high bioavailability but requires the use of mucoadhesive nanocarriers. Chitosan is a well-established polymer for mucoadhesive applications but can suffer from poor cytocompatibility and stability upon administration. In this work, we present a method to obtain stable and cytocompatible crosslinked chitosan nanoparticles. We used 2,6-pyridinedicarboxylic acid as a biocompatible crosslinker and compared the obtained particles with those prepared by ionotropic gelation using sodium tripolyphosphate. Nanoparticles were tested to evaluate the size and the surface charge, as well as their stability in storage conditions (4 °C), at the nasal cavity temperature (32 °C), and at the body temperature (37 °C). The crosslinked chitosan nanoparticles showed a size around 150 nm and a surface charge of 10.3 mV ± 0.9 mV, both compatible with the intranasal drug administration. Size and surface charge parameters did not significantly vary over time, indicating the good stability of these nanoparticles. We finally tested their cytocompatibility in vitro using SHSY5Y human neuroblastoma and RPMI 2650 human nasal epithelial cells, with positive results. In conclusion, the proposed synthetic system shows an interesting potential as a drug carrier for intranasal delivery.

Keywords: chitosan nanoparticles; drug delivery; intranasal administration; mucoadhesion.

Figure 1

Size and surface charge of produced nanoparticles: (a) mean diameter (nm) and (b) z-potential (mV) of CHI-TPP-NPs with varying concentrations of TPP solution used for ionotropic gelation (0.1 and 1.0 mg/mL); (c) mean size (nm) and (d) z-potential (mV) of CHI-DA-NPs synthesized with different DA concentrations. Values are reported as mean ± SEM, replicate experiments correspond to different syntheses, n = 6 for CHI-TPP-NPs-0.1, n = 7 for CHI-TPP-NPs-1, n = 7 for CHI-DA-NPs-1.25.

Figure 2

Evaluation of size and z-potential trends measured during stability tests at different temperatures (4 °C, 32 °C, and 37 °C) up to 16 days: (a1–a3) size and (b1–b3) z-potential measured for CHI-TPP-NPs-0.1; (c1–c3) size and (d1–d3) z-potential measured for CHI-TPP-NPs-1. Values are reported as mean ± SEM.

Figure 3

Evaluation of size and z-potential trends measured during stability tests at different temperatures (4 °C, 32 °C, and 37 °C) up to 16 days: (a1–a3) size and (b1–b3) z-potential measured for CHI-DA-NPs-1.25. Values are reported as mean ± SEM.

Figure 4

Biocompatibility tests on neuronal and nasal epithelial cells. (a,b) The proliferation rate of SH cells (a) or RPMI-2650 cells (b) treated for 24 h with different concentrations (0.25–2.5 mg/mL) of CHI-TPP-NPs formulated with 0.1 mg/mL TPP (left) or 1 mg/mL TPP (right), reported in % over their untreated conditions (Ctrl). */**/*** p < 0.05/0.01/0.001, one-way ANOVA, Dunnett’s test. Inset: legend of NP concentrations. (c,d) The proliferation rate of SH (c) or RPMI-2650 (d) cells treated with CHI-DA-NPs-1.25 at different concentrations (0.25–2.5 mg/mL) for 24 and 48 h, reported in % over untreated conditions (Ctrl). Data = mean ± SEM, n ≥ 3; only Ctrl is reported as mean ± SD, to show the intra-assay variability. (e–h) Vitality test: adherent vital cells are visible in green (calcein-positive), all cell nuclei in blue, and the level of necrotic/dying cells in red (PI-positive). (e,f) Fluorescence microscopy images of SHs (e) or RPMI-2650 (f) at t = 24 h after selected CHI-TPP-NP treatments. (g,h) Fluorescence microscopy images of SHs (g) or RPMI-2650 (h) at t = 24 h after all CHI-DA-NP treatments. Scale bar = 100 µm.