Neuroprotective effect of chitosan nanoparticle gene delivery system grafted with acteoside (ACT) in Parkinson’s disease models

doi:10.1016/j.jmst.2019.10.013

摘要/Abstract

Abstract:

Developing new drugs to treat Parkinson's disease efficiently is challenging. Here we report that chitosan nanoparticles (APPDNs) could serve as novel candidates for the design of anti-PD drugs. In this study, we investigated the effects of chitosan poly ethyleneglycol-poly lactic acid (PEG-PLA) nanoparticles conjugated with nerve growth factor (NGF), acteoside (ACT) and plasmid DNA (pDNA) for PD therapy using in vitro and in vivo models. Using PD cell models, we demonstrated that APPDN had good neuroprotective effects. More significantly, experiments using mouse PD models demonstrated that APPDNs could ameliorate the behavioral disorders of sick mice. Immunohistochemical and western blot (WB) analyses demonstrated that APPDNs could significantly reverse dopaminergic (DA) neuron loss in the substantia nigra and striatum of sick mice. This study opens up a novel avenue to develop anti-PD drugs.

Key words: Parkinson's disease, Chitosan nanoparticles, Acteoside, Gene therapy, pDNA

. [J]. 材料科学与技术, 2020, 43(0): 197-207.

Yongyong Xue, Na Wang, Zhi Zeng, Jinpeng Huang, Zhiming Xiang, Yan-Qing Guan. Neuroprotective effect of chitosan nanoparticle gene delivery system grafted with acteoside (ACT) in Parkinson’s disease models[J]. J. Mater. Sci. Technol., 2020, 43(0): 197-207.

图/表 8

Fig. 1. Schematic illustration of the preparation of mPEG-PLA-ACT-CTS-pDNA-NGF (APPDN).

Fig. 2. (A) FITC spectra of mPEG-PLA, ACT-PLA-mPEG, ACT and ACT-PLA-mPEG-CTS-pDNA; (B) UV-vis spect of mPEG-PLA, ACT and ACT-PLA-mPEG; (C) The Grafting rate and Loading capacities of pDNA, NGF and ACT.

Fig. 3. (A) Size distribution histograms and TEM image of mPEG-PLA, ACT-PLA-mPEG, ACT-PLA-mPEG-CTS-pDNA and ACT-PLA-mPEG-CTS-pDNA-NGF; (B) AFM observation of ACT-PLA-mPEG-CTS-pDNA and ACT-PLA-mPEG-CTS-pDNA-NGF; (C) Zeta potential of mPEG-PLA, ACT-PLA-mPEG, ACT-PLA-mPEG-CTS-pDNA and ACT-PLA-mPEG-CTS-pDNA-NGF; (D) Variation of ACT-PLA-mPEG-CTS-pDNA-NGF particle size with time gradient in cell culture medium.

Fig. 4. (A) Viabilities of PC12 cells 24 h after MPP+ treatment in the absence or presence of pre-incubated nano-drug with different groups were measured with MTT assays; (B) Bright field and confocal fluorescence images of PC12 cells treated with different groups. The nuclei of cells werestained with DAPI (blue); (C) Apoptosis rate of different groups were assessed by flow cytometry methods; (D) The PC12 cells apoptosis quantified by the flow cytometry at 24 h after various treated with different groups (PBS + PBS; MPP++PBS; MPP++ mPEG-PLA; MPP++mPEG-PLA-ACT; MPP++mPEG-PLA-ACT-CTS-pDNA; MPP++mPEG-PLA-ACT-pDNA-NGF) (Q1: dead cells; Q2: late apoptotic cells; Q3: live cells; Q4: early apoptotic cells)).

Fig. 5. (A, B) Confocal images of PC12 cells at 24 h after various treatments. Immunofluorescence staining was used to detect the nucleus (blue fluorescence) of DAPI, alpha synuclein (SNCA) and nerve growth factor receptor (NGFR) (green fluorescence) staining; (C, D) Expression of NGFR and SNCA after various treatment with different groups. The relative levels are plotted at a significance of p＜0.05 indicated by *, 0.001＜p＜0.01 indicated by **, and p＜0.001 indicated by ***, in comparison to the MPP++PBS group.

Fig. 6. (A) Schematic representation of the open field system; (B) The photos of mice after various treatments (Saline + Saline; MPTP + Saline; MPTP + APPDN); (C) The body weight changes of mice during various treatments; (D-H) The behavior detection in mice after various treatments; (D, G) Open field test; (E) Pole-jump test; (H) Gait analysis Morris water maze (n = 5 biologically independent samples; two-way ANOVA with Tukey HSD test). The relative levels are plotted at a significance of p＜0.05 indicated by *, 0.001＜p＜0.01 indicated by **, and p＜0.001 indicated by ***, in comparison to the MPTP + saline group.

Fig. 7. (A1, A2) Representative images and head semi-quantitative analysis of APPDN/CY5 fluorescence imaging; (B) A representative picture of the retention of fluorescent labeled drugs in the substantia nigra at different times after intraperitoneal injection of APPDN/CY5 in mice. The nucleus is marked blue with DAPI and the APPDN is marked pink with CY5; (C, D) Immunofluorescence and alpha-syn immunohistochemical images of TH in substantia nigra of three groups (Saline + Saline; MPTP + Saline; MPTP + APPDN). The red circle indicates abnormal accumulation of alpha -syn cells, TH uses green fluorescent labeling; (E1, E2) Expression levels of TH and α-syn in the substantia nigra pars compacta of C57bl/6 mice and the quantitative results of ratio; (F) The images of H&E stained brain, lung, liver, spleen, kidney and heart after various treatments; (G-I) Serological and toxicology test of C57 mice, including the density of platelet, the density of white blood cell, and the density of red blood cell in different groups (Saline + Saline; MPTP + Saline; MPTP + APPDN). The relative levels are plotted at a significance of p＜0.05 indicated by *, 0.001＜p＜0.01 indicated by **, and p＜0.001 indicated by ***, in comparison to the MPTP + saline group.

Fig. 8. Schematic illustration of the APPDN treatment process. The APPDNs nano- micelle complex is internalized by PC12 cells, and PDNA degrades mRNA through a series of actions, thus inhibiting SNCA expression. ACT inhibits cell senescence by increasing the activity of telomerase and reducing the production of malondialdehyde in the cytoplasm.

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