Blood-brain barrier-penetrating siRNA nanomedicine for Alzheimer's disease therapy

Abstract

Toxic aggregated amyloid-β accumulation is a key pathogenic event in Alzheimer's disease (AD), which derives from amyloid precursor protein (APP) through sequential cleavage by BACE1 (β-site APP cleavage enzyme 1) and γ-secretase. Small interfering RNAs (siRNAs) show great promise for AD therapy by specific silencing of BACE1. However, lack of effective siRNA brain delivery approaches limits this strategy. Here, we developed a glycosylated "triple-interaction" stabilized polymeric siRNA nanomedicine (Gal-NP@siRNA) to target BACE1 in APP/PS1 transgenic AD mouse model. Gal-NP@siRNA exhibits superior blood stability and can efficiently penetrate the blood-brain barrier (BBB) via glycemia-controlled glucose transporter-1 (Glut1)-mediated transport, thereby ensuring that siRNAs decrease BACE1 expression and modify relative pathways. Noticeably, Gal-NP@siBACE1 administration restored the deterioration of cognitive capacity in AD mice without notable side effects. This "Trojan horse" strategy supports the utility of RNA interference therapy in neurodegenerative diseases.

Fig. 1. Illustration of the formation of the glycosylated “triple-interaction” stabilized siRNA nanomedicine (Gal-NP@siRNA) and the mechanism and approach to treat AD pathology in APP/PS1 transgenic mice.

(A) Schematic illustration of the fabrication of Gal-NP@siRNA. (B and C) Mechanism by which Gal-NP@siRNA penetrates the BBB and accumulates in the brain. Glut1 is overexpressed on the luminal membrane of the BBB after 24-hour fasting. After treatment with Gal-NP@siRNA, glucose replenishment in fasting mice results in Glut1 recycling from the luminal to the abluminal membrane of the BBB, which leads to the transport of Gal-NP@siRNA across the BBB. (D) Gal-NP@siRNA–mediated knockdown of BACE1 mRNA expression, which leads to reduced levels of amyloid plaques.

Fig. 2. Biophysical characterization and in vitro studies of Gal-NP@siRNA.

(A) Gel retardation assay of Gal-NP@siRNA at polymer/siRNA weight ratios of 1, 2.5, 5, 10, 15, and 20. (B) Size distribution and (C) transmission electron micrographs of Gal-NP@siRNA. (D) Confocal laser scanning microscopy images for NP cellular uptake. Images were collected for Neuro-2a cells after 4-hour NP incubation. Cell nuclei were stained with DAPI (blue), siRNA was labeled by FAM dye (green), and cell cytoskeleton was stained with TRITC-phalloidin (red) to indicate cytoplasm area. Scale bars, 10 μm. (E) Flow cytometry analysis of Neuro-2a cells following 4-hour incubation with free Cy5-siRNA, NP@Cy5-siRNA, and Gal-NP@Cy5-siRNA. (F and G) In vitro gene silencing effects of Gal-NP@siBACE1 and controls at day 3 post transfection. BACE1 mRNA (F) and protein (G) expression levels was quantified by qRT-PCR and western blot assay, respectively. Data are presented as mean ± SEM (n = 3, ***P < 0.001).

Fig. 3. Biodistribution and in vivo BACE1 targeting efficacy of Gal-NP@siRNA.

(A) In vivo pharmacokinetics as shown by Cy5-siRNA concentration/time curves in plasma after a single-dose injection. (B) (Left) Quantification of Cy5-siRNA accumulation in different organs. Cy5-siRNA levels were determined by fluorescence spectroscopy 1 hour after tail vein injection of siRNA nanomedicine after a single-dose injection. Data are presented as mean ± SEM (n = 3, *P < 0.05). (Right) Representative image for Cy5 signal in the brain of NP@siRNA and Gal-NP@siRNA groups 1 hour after injection. (C) Time course in vivo imaging of Gal-NP@Cy5-siRNA evaluated by fluorescence imaging after a single-dose injection. (D and E) BACE1 mRNA and protein expression level in cortex was quantified by (D) qRT-PCR and (E) Western blot assay from WT mice samples, and samples were collected at day 3 after two nanomedicine treatments. Data are presented as mean ± SEM (n = 3, *P < 0.05).

Fig. 4. Behavioral evaluation of Gal-NP@siBACE1 nanomedicine therapy in APP/PS1 mice.

(A) Schematic of the experimental timeline. APP/PS1 and WT mice were treated with siRNA nanomedicine or PBS via tail vein injection every 3 days (10 cycles). Mice were then subjected to nesting, NOR, and MWM tests for memory evaluation, and samples for molecular pathological assessments were collected. (B) Representative images and scoring criteria from the nest-building experiment in APP/PS1 and control WT mice. Photos were taken 24 hours after the introduction of nesting material to the home cage. Photo credits: Yutong Zhou, Nankai University. (C) Nest-building scores for each group. (D) Setup for NOR test. (E and F) Results for NOR test. (E) DI and (F) PI of each group after nanomedicine treatment. (G to J) Data for probe test in the MWM. (G) Representative swimming track, (H) swimming speed, (I) ratio of time spent in target quadrant, and (J) number of crossing the platform location of each group on the probe test day. All behavioral test bar or plot charts are presented as mean ± SEM (n = 6 to 8, *P < 0.05, **P < 0.01).

Fig. 5. Therapeutic evaluation of the ability of Gal-NP@siBACE1 treatment to modulate AD hallmarks in APP/PS1 mice.

(A) Mechanistic explanation for the effects of siBACE1 therapy. (B) Representative Western blot data for BACE1 protein expression in hippocampus and cortex from nanocarrier-treated APP/PS1 mice, control APP/PS1 groups, and WT mice. Quantification of Western blotting analysis of BACE1 expression was relative to β-actin (n = 3, mean with SEM, *P < 0.05, **P < 0.01). (C) Representative confocal laser scanning microscopy imaging data assessing amyloid plaque burden. Immunofluorescence of Aβ plaques (green) in hippocampus and cortex from APP/PS1 transgenic and WT mice. Nuclei were stained by DAPI (blue). Scale bars, 100 μm. (D) Percent surface area of amyloid plaques in hippocampus (left) and cortex (right) regions was quantified. Data are presented as mean ± SEM; n = 4, **P < 0.01. (E) p-tau and (F) MBP expression in the hippocampus and cortex for nanocarrier-treated APP/PS1 mice, control APP/PS1 groups, and WT mice (top). Quantification of Western blotting analysis was relative to β-actin (bottom) (n = 3, mean with SEM, *P < 0.05, **P < 0.01). All samples were collected after 10 administrations of nanomedicine.

Fig. 6. Cytotoxicity and in vivo biocompatibility assessment of the Gal-NP@siRNA nanomedicine.

(A and B) Blood chemistry examinations. Assessment of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), plasma urea (BUN), creatinine (CR), and uric acid (UA) levels after a single-dose nanomedicine treatment. n = 4, mean with SEM. (C and D) Core proinflammatory cytokines such as Il-1β, Il-6, and Tnf-α in liver (C) and kidney (D) were assessed after a single-dose PBS or Gal-NP@siRNA nanomedicine treatment at days 2 and 14. n = 3, mean with SEM. (E) Representative data for hematoxylin and eosin staining in major organs from APP/PS1 and control WT mice treated with Gal-NP@siBACE1 or PBS in the 10-time injection therapeutic experiments. Scale bars, 50 μm.