Intranasal Delivery of Genistein-Loaded Nanoparticles as a Potential Preventive System against Neurodegenerative Disorders

Abstract

Genistein has been reported to have antioxidant and neuroprotective activity. Despite encouraging in vitro and in vivo results, several disadvantages such as poor water solubility, rapid metabolism, and low oral bioavailability limit the clinical application of genistein. The aim of this study was to design and characterize genistein-loaded chitosan nanoparticles for intranasal drug delivery, prepared by the ionic gelation technique by using sodium hexametaphosphate. Nanoparticles were characterized in vitro and their cytotoxicity was tested on PC12 cells. Genistein-loaded nanoparticles were prepared, and sodium hexametaphosphate was used as a valid alternative to well-known cross-linkers. Nanoparticle characteristics as well as their physical stability were affected by formulation composition and manufacturing. Small (mean diameters of 200⁻300 nm) and homogeneous nanoparticles were obtained and were able to improve genistein penetration through the nasal mucosa as compared to pure genistein. Nanoparticle dispersions showed a pH consistent with the nasal fluid and preserved PC12 cell vitality.

Keywords: chitosan; genistein; intranasal delivery; nanoparticles; neurodegenerative disease; nose-to-brain; sodium hexametaphosphate.

Figure 1

Influence of chitosan (CS) concentration on the particle size. CS:SHMP mass ratio of 4:1 is used. The error bar indicates the standard deviation averaged from three measurements. * p < 0.05: 0.5 vs. 0.8, 1, and 2; ^# p < 0.05: 0.8 vs. 1 and 2. SHMP: sodium hexametaphosphate.

Figure 2

Influence of storage (at 25 °C and 4 °C) on the mean particle size of B-NPAc2 and NPAc2 nanoparticles. p < 0.05 NPAc2 at 25 °C: day 0 vs. day 1; day 0 vs. day 7; p < 0.05 NPAc2 at 4 °C: day 0 vs. day 1; day 0 vs. day 7.

Figure 3

Influence of storage (at 25 °C and 4 °C) on the PDI of B-NPAc2 and NPAc2 nanoparticles. p < 0.05 B-NPAc2 at 25 °C: day 1 vs. day 14; p < 0.05 B-NPAc2 at 4 °C: day 1 vs. day 14.

Figure 4

Influence of storage (at 25 °C and 4 °C) on the mean particle size of B-NPEt1 and NPEt1 nanoparticles. p < 0.05 B-NPEt1 at 25 °C: day 0 vs. day 7 and day 14, day 1 vs. day 7 and day 14. p < 0.05 B-NPEt1 at 4 °C: day 0 vs. day 1, day 7, and day 14, day 1 vs. day 14. p < 0.05 NPEt1 at 25 °C: day 0 vs. day 14. p < 0.05 B-NPEt1 at 4 °C: day 0 vs. day 7 and day 14, day 1 vs. day 14, day 7 vs. day 14.

Figure 5

Influence of storage (at 25 °C and 4 °C) on the particle size distribution (PDI) of B-NPEt1 and NPEt1 nanoparticles. p < 0.05 B-NPEt1 at 25 °C: day 0 vs. day 7 and day 14, day 1 vs. day 7 and day 14; p < 0.05 B-NPEt1 at 4 °C: day 0 vs. day 14, day 1 vs. day 14; p < 0.05 NPEt1 at 25 °C: day 0 vs. day 7 and day 14, day 1 vs. day 7 and day 14; p < 0.05 NPEt1 at 4 °C: day 0 vs. day 14.

Figure 6

SEM pictures of pure drug (a), purified chitosan (b), and air-dried nanoparticles (c). Magnifications are included in each picture.

Figure 7

TEM images of loaded (a,c) and unloaded (b,d) nanoparticles.

Figure 8

Topographic images of loaded (a,c) and unloaded (b,d) nanoparticles. Three-dimensional views of 5 × 5 μm scan of loaded (e) and unloaded (f) nanoparticles.

Figure 9

XRD patterns of chitosan nanoparticles prepared with SHMP.

Figure 10

Ex vivo distribution of GEN after the permeation test (120 min) from nanoparticle (NPEt1) and from dispersion in phosphate buffer through the porcine nasal mucosa (n = 3). *§# = p < 0.05.

Figure 11

Effects of two different chitosan unloaded (B-NPAc2 and B-NPEt1) and loaded (NPEt1 and NPAc2) nanoparticles prepared in acetone (pink bars) and ethanol (green bars) on PC12 cells viability after 24 h of exposure, evaluated by MTT assay (a) and trypan blue assay (b).

Figure 12

Apoptosis analysis by flow cytometry performed on PC12 cells exposed for 24 h to B-NPEt1 and NPEt1; no significant differences were found between samples (p > 0.05 CTRL vs. B-NPEt1 and CTRL vs. NPEt1).