Intelligent lesion blood-brain barrier targeting nano-missiles for Alzheimer's disease treatment by anti-neuroinflammation and neuroprotection

Abstract

The treatment of Alzheimer's disease (AD) is one of the most difficult challenges in neurodegenerative diseases due to the insufficient blood‒brain barrier (BBB) permeability and unsatisfactory intra-brain distribution of drugs. Therefore, we established an ibuprofen and FK506 encapsulated drug co-delivery system (Ibu&FK@RNPs), which can target the receptor of advanced glycation endproducts (RAGE) and response to the high level of reactive oxygen species (ROS) in AD. RAGE is highly and specifically expressed on the lesion neurovascular unit of AD, this property helps to improve targeting specificity of the system and reduce unselective distribution in normal brain. Meanwhile, these two drugs can be specifically released in astrocytes of AD lesion in response to high levels of ROS. As a result, the cognition of AD mice was significantly improved and the quantity of Aβ plaques was decreased. Neurotoxicity was also alleviated with structural regeneration and functional recovery of neurons. Besides, the neuroinflammation dominated by NF-κB pathway was significantly inhibited with decreased NF-κB and IL-1β in the brain. Overall, Ibu&FK@RNPs can efficiently and successively target diseased BBB and astrocytes in AD lesion. Thus it significantly enhances intracephalic accumulation of drugs and efficiently treats AD by anti-neuroinflammation and neuroprotection.

Keywords: Alzheimer's disease; Anti-neuroinflammation; Blood‒brain barrier transcytosis; Drug combination; Nano drug delivery system; Neuroprotection; ROS-responsive; Receptor for advanced glycation end products.

Graphical abstract

Figure 1

(A) Diagram depicting the preparation of ROS-responsive programmed RAGE-targeted delivery depot of Ibu&FK@RNPs. (B) Schematic illustration of Ibu&FK@RNPs target RAGE on lesion BBB and brain parenchymal cells. Amplification: schematic diagram of process of the ROS-responsive drug release from nanoparticles in astrocytes and neurons, and then treat AD by anti-neuroinflammation and neuroprotection.

Figure 2

Synthetic routes and characterization of nanoparticles. (A) Synthetic routes of PEP. Compound 1: EDT-MAA; compound 2: MAL-PEG-EDT-MAA; compound 3: MAL-PEG-MAL-EDT-MAH; compound 4: PEP. (B) TEM images of nanoparticles. Scale bars represent 100 nm. (C) Cumulative release efficiency of FK506-loaded nanoparticles incubated in different solutions. Data are presented as mean ± SD (n = 3).

Figure 3

Intracellular behavioral studies. (A) The Aβ pre-incubated bEnd.3 cell viability of ibuprofen and FK506 measured by MTT assay. Data are presented as mean ± SD (n = 3). (B) The Aβ pre-incubated bEnd.3 cell viability of nanoparticles measured by MTT assay. Data are presented as mean ± SD (n = 3). (C) Quantitative uptake of nanoparticles incubated in bEnd.3 cells for 2 h. Data are presented as mean ± SD (n = 3). (D) Confocal fluorescence images of nanoparticles internalized by bEnd.3 cells. Scale bar represents 20 μm. (E) 3D confocal images of bEnd.3 monolayers in the donor chamber of transwell model after the introduction of different nanoparticles for 4 and 12 h. Scale bar represents 10 μm.

Figure 4

In vivo distribution of nanoparticles in AD mice. (A) Living imaging depicting the in vivo distribution of different formulations at different time. (B) Ex vivo imaging of brains in different groups after 6 h. (C) The semiquantitative fluorescence intensity of (B). Data were presented as mean ± SD (n = 3). (D) Representative confocal images of brains showing the accumulation of different nanoparticles and immunofluorescence colocation with RAGE. Blue: nuclei stained by DAPI, green: FITC-PEG-PCL, red: the positive rate of RAGE expression, pink: Cy5.5-Ibu. Scale bars represent 100 and 10 μm, respectively. (E) The in vivo concentration‒time curves of FK506 measured by LC‒MS/MS analysis.

Figure 5

Learning acquisition and Aβ plaques reduction. (A) Average time to reach the platform in the training process of AD mice in Morris water maze test. Data are presented as mean ± SD (n = 5). (B) The representative swimming paths of AD mice in Morris water maze test, numbers in the lower right indicate the average time spent to reach the platform. (C) The time for mice to reach the platform of AD mice in the Morris water maze test. Data are presented as mean ± SD (n = 5). (D) The frequency for the AD mice passing through the platform in the spatial probe test. Data are presented as mean ± SD (n = 5). (E) Representative images of amyloid plaques stained by immunohistochemical in hippocampus from APP/PS1 transgenic mice. Scale bar represents 100 μm. (F) The semiquantitative integrated intensity of Aβ plaques by immunohistochemical staining in APP/PS1 transgenic mice. Data are presented as mean ± SD (n = 3).

Figure 6

Neuroprotective effects in APP/PS1 mice. (A) Nissl staining in the hippocampus. The black box shows the hippocampal CA1 area. Scale bars represent 100 and 10 μm, respectively. (B) Quantitative expression of Nissl's body in the CA1 area. (C) Immunofluorescence of MAP2 in the hippocampus. Green represents the positive rate of MAP2 expression, blue represents nuclei stained by DAPI and scale bar represents 20 μm. (D) Quantification of MAP2 in the hippocampus of mice in different treatment groups. Data are presented as mean ± SD (n = 3).

Figure 7

Anti-neuroinflammatory effects in APP/PS1 mice. (A) Immunofluorescence of GFAP in the cerebral cortex. Green represented the positive rate of GFAP expression, blue represented nuclei stained by DAPI and scale bar represents 20 μm. (B) Western Blot of NF-κB in the brain. (C) The semiquantitative integrated intensity of NF-κB by Western Blot. Data are presented as mean ± SD (n = 3). (D) The proportion of IL-1β in the brain proteins. Data are presented as mean ± SD (n = 3).