A Novel Brain-Targeting Nanoparticle Loaded with Biatractylolide and Its Protective Effect on Alzheimer's Disease

Abstract

Background: To enhance the bioavailability and neuroprotective efficacy of biatractylolide against Alzheimer's disease by developing a novel Tween-80-modified pullulan-chenodeoxycholic acid nanoparticle as a delivery vehicle. Methods: Chenodeoxycholic acid (CDCA) was chemically conjugated to pullulan to yield hydrophobically modified pullulan (PUC), onto which polysorbate 80 (Tween-80) was subsequently adsorbed. The PUC polymers with CDCA substitution levels were analyzed by ¹H NMR spectroscopy. Nanoparticles were fabricated via the dialysis method and characterized by transmission electron microscopy and dynamic light scattering for morphology, size, and surface charge. In vitro neuroprotection was assessed by exposing SH-SY5Y and PC12 cells to 20 µM Aβ_25-35 to induce cytotoxicity, followed by pretreatment with biatractylolide-loaded PUC (BD-PUC) nanoparticle solutions at various biatractylolide concentrations. The in vivo brain-targeting capability of both empty PUC and BD-PUC particles was evaluated using a live imaging system. Results: The ¹H NMR analysis confirmed three distinct CDCA substitution degrees (8.97%, 10.66%, 13.92%). Transmission electron microscopy revealed uniformly dispersed, spherical nanoparticles. Dynamic light scattering measurements showed a hydrodynamic diameter of ~200 nm and a negative zeta potential. Exposure to 20 µM Aβ_25-35 significantly reduced SH-SY5Y and PC12 cell viability; pretreatment with BD-PUC nanoparticles markedly enhanced cell survival rates and preserved cellular morphology compared to cells treated with free biatractylolide. Notably, the cytoprotective effect of BD-PUC exceeded that of the free drug. In vivo imaging demonstrated that both empty PUC and Tween-80-adsorbed BD-PUC nanoparticles effectively accumulated in the brain. Conclusions: The protective effect of BD-PUC on SH-SY5Y and PC12 cells induced by Aβ_25-35 was higher than free biatractylolide solution, and the BD-PUC nanosolution modified with Tween-80 showed a brain-targeting effect.

Keywords: Alzheimer’s disease; biatractylolide; brain targeting; nanoparticle; pullulan polysaccharide.

Figure 1

(A). PUC polymer synthesis using infrared spectrophotometry, (a) pullulan, (b) 0.13 PUC polymer, (c) 0.15 PUC polymer, (d) 0.18 PUC polymer. (B). NMR detection of PUC polymer synthesis, (a) 0.13 PUC polymer, (b) 0.15 PUC polymer, (c) 0.18 PUC polymer, (d) pullulan.

Figure 2

(A). Characterization of PUC nanoparticles at different feeding ratios by DLS; (a,b) shows the particle size and zeta potential of 0.13-scale nanoparticles; (c,d) shows the particle size and zeta potential of 0.15-scale nanoparticles; (e,f) shows the particle size and zeta potential of 0.18-scale nanoparticles. (B). (a,b) shows the particle size and potential values of adsorbed Tween-80-PUC nanoparticles; (c,d) are the particle size and potential values of BD-PUC. (C) Transmission electron microscopy observation of the morphology (a) for PUC nanoparticles; (b) for BD-PUC nanoparticles. (D). The standard curve for BD is y = 0.002924 × X − 0.004724 (R² = 0.9991). (E). MST detection of Tween-80 and FITC-PUC nanoparticles by MST assay to verify the binding ability of both.

Figure 3

(A). After staining with DAPI and FITC-PUC nanoparticles: (a) inverted fluorescence images of SH-SY5Y cells, SH-SY5Y cells were able to take up PUC nanoparticles; (b) inverted fluorescence images of PC12 cells showed green fluorescence in PC12 cells, PC12 cells were able to take up PUC nanoparticles. (Scale length: 200 µm) (B). After treating cells with different concentrations of blank PUC nanoparticles: (a) PUC nanoparticles were not toxic to SH-SY5Y cells, (b) PUC nanoparticles were not toxic to PC12 cells. (C). After treatment of cells with different concentrations of Aβ_25-35: (a) the effect of Aβ_25-35 on the survival rate of SH-SY5Y cells, (b) the effect of Aβ_25-35 on the survival rate of PC12 cells (*** p < 0.001 vs. control group; * p < 0.05 vs. control group). (D). Pretreatment of cells with different concentrations of BD-, BD-PUC-, Aβ_25-35-simulated cell injury: (a) effect of BD-PUC on the activity of Aβ_25-35-injured SH-SY5Y cells, (b) effect of BD-PUC on the activity of Aβ_25-35-injured PC12 cell activity (*** p < 0.001 vs. control group; ^# p < 0.05 vs. Aβ_25-35 group; ^## p < 0.01 vs. Aβ_25-35 group; ^### p < 0.001 vs. Aβ_25-35 group). (E). Pretreatment of cells with different concentrations of BD, BD-PUC, Aβ_25-35 mock cell injury: (a) BD-PUC on apoptosis of Aβ_25-35-injured SH-SY5Y cells, (b) BD-PUC on apoptosis of Aβ_25-35-injured PC12 cells (green fluorescence: living cells; yellow fluorescence: early apoptosis; red fluorescence: late-stage apoptosis).

Figure 4

(A). In vivo imaging system for (a) free FITC injection, (b) FITC-PUC nanoparticle injection, (c) mice injected with Tween-80-modified FITC-PUC nanoparticles. (B). (a) Brain of mice injected with free FITC. (b) Brain of mice injected with FITC-PUC nanoparticles. (c) Brain of mice injected with FITC-PUC nanoparticles modified with Tween-80. (C). Imaging of organs of mice injected with Tween-80-modified FITC-PUC nanoparticles (1, 2, 3, 4, 5, 6 are heart, liver, spleen, lung, kidney, and brain, respectively).

Figure 5

(A). In vivo imaging system assay: (a) mouse injected with free FITC; (b) mouse injected with FITC-BD-PUC nanoparticles; (c) mouse injected with Tween-80-modified FITC-BD-PUC nanoparticles. (B). In vivo imaging: (a) brain of mouse injected with free FITC; (b) brain of mouse injected with FITC-BD-PUC nanoparticles; (c) brain of mouse injected with Tween-80-modified FITC-BD-PUC nanoparticle mice. (C). Imaging of organs in mice injected with Tween-80-modified FITC-BD-PUC nanoparticles (1, 2, 3, 4, 5, 6 in the figure are heart, liver, spleen, lung, kidney, and brain, respectively).

Figure 6

PUC polymer synthesis chemical reaction formula.