PLGA nanoparticles modified with a BBB-penetrating peptide co-delivering Aβ generation inhibitor and curcumin attenuate memory deficits and neuropathology in Alzheimer's disease mice

Abstract

Alzheimer's disease (AD) is the most common form of dementia, characterized by the formation of extracellular senile plaques and neuronal loss caused by amyloid β (Aβ) aggregates in the brains of AD patients. Conventional strategies failed to treat AD in clinical trials, partly due to the poor solubility, low bioavailability and ineffectiveness of the tested drugs to cross the blood-brain barrier (BBB). Moreover, AD is a complex, multifactorial neurodegenerative disease; one-target strategies may be insufficient to prevent the processes of AD. Here, we designed novel kind of poly(lactide-co-glycolic acid) (PLGA) nanoparticles by loading with Aβ generation inhibitor S1 (PQVGHL peptide) and curcumin to target the detrimental factors in AD development and by conjugating with brain targeting peptide CRT (cyclic CRTIGPSVC peptide), an iron-mimic peptide that targets transferrin receptor (TfR), to improve BBB penetration. The average particle size of drug-loaded PLGA nanoparticles and CRT-conjugated PLGA nanoparticles were 128.6 nm and 139.8 nm, respectively. The results of Y-maze and new object recognition test demonstrated that our PLGA nanoparticles significantly improved the spatial memory and recognition in transgenic AD mice. Moreover, PLGA nanoparticles remarkably decreased the level of Aβ, reactive oxygen species (ROS), TNF-α and IL-6, and enhanced the activities of super oxide dismutase (SOD) and synapse numbers in the AD mouse brains. Compared with other PLGA nanoparticles, CRT peptide modified-PLGA nanoparticles co-delivering S1 and curcumin exhibited most beneficial effect on the treatment of AD mice, suggesting that conjugated CRT peptide, and encapsulated S1 and curcumin exerted their corresponding functions for the treatment.

Keywords: Alzheimer’s disease; curcumin; nanoparticles; peptide; β-amyloid.

Figure 1. Characterization of PLGA nanoparticles

(A) Schematic illustration of the PLGA NP fabrication. The PLGA NPs were prepared by self-assembly of PEG-PLGA with curcumin and S1 peptide, and then were conjugated with CRT peptide. The morphologies and diameters of NP-S1+Cur (B) and CRT-NP-S1+Cur (C) were determined by TEM and DLS, respectively (scale bar, 200 nm). The release profiles of curcumin and S1 from CRT-NP-S1+Cur in PBS (pH 7.4, containing 0.5% polysorbitol-80) at 37°C were detected by measuring absorbance at 430 nm and BCA, respectively (D). The cytotoxicity of NP-S1+Cur conjugated with or without CRT was determined via MTT assay by adding NPs to SH-SY5Y cells (E), BV2 cells (F) and bEnd.3 cells (G). The data shown are expressed as the percentage of control values from three independent experiments with each experimental value being the average of six replicate wells.

Figure 2. CRT peptide increased the uptake of PLGA NPs by bEnd

3 cells and improved the penetration of PLGA NP to brains. (A) Coumarin-6-labeled PLGA NP control, NP-S1+Cur and CRT-NP-S1+Cur were added to bEnd.3 cells. The fluorescence in cells was detected by a confocal microscope (scale bar, 20 μm). (B) ICG-labeled NP-S1+Cur and CRT-NP-S1+Cur were injected to nude mice via the tail vein. The fluorescence was detected by IVIS spectrum imaging system at different time points. (C) The mice were sacrificed and the fluorescence in various organs was detected.

Figure 3. PLGA NPs attenuated memory defects in AD model mice

The eight-month-old transgenic AD mice were treated with NP control, NP-S1, NP-Cur, NP-S1+Cur and CRT-NP-S1+Cur, respectively, and their memory was determined via Y maze (A, B) and NOR (C). The number of entries to the novel arm by the mice (A) and the time in the novel arm spent by the mice (B) were measured. (C) The investigation to novel target by mice was determined via NOR test. (*, P < 0.05, **, P < 0.01, compared with NP control-treated AD mice).

Figure 4. PLGA NPs reduced Aβ40 and Aβ42 levels in AD mouse brains

The levels of soluble Aβ40 (A) and Aβ42 (B), insoluble Aβ40 (C) and Aβ42 (D) in the brain lysates of AD mice treated with NP control, NP-S1, NP-Cur, NP-S1+Cur and CRT-NP-S1+Cur were detected by Aβ40 and Aβ42 sandwich ELISA kits, respectively. The senile plaques in the brains of AD transgenic mice treated with NP control, NP-S1, NP-Cur, NP-S1+Cur and CRT-NP-S1+Cur were detected by immunohistochemistry (E) and quantitatively analyzed by Image-Pro Plus software (F) (*, P < 0.05, **, P < 0.01, compared with NP control-treated AD mice).

Figure 5. PLGA NPs reduced microgliosis and astrogliosis in AD mice

(A) Microgliosis and astrogliosis in the brains of AD mice treated with NP control, NP-S1, NP-Cur, NP-S1+Cur and CRT-NP-S1+Cur were detected by IBA-1 and GFAP immunostaining, and qualified by Image-Pro Plus software (B), respectively. The protein levels of GFAP and Iba-1 in the brain lysates of AD transgenic mice were detected by western blot (C) and quantitatively analyzed by ImageQuant software (D). GAPDH was used as a loading control (*, P < 0.05, **, P < 0.01, ***, P < 0.001, compared with AD control mice).

Figure 6. PLGA NPs increased synapse number in AD mouse brains

The AD mice were treated with NP control, NP-S1, NP-Cur, NP-S1+Cur and CRT-NP-S1+Cur, and the synapses in the cerebral cortex and hippocampus were detected by anti-PSD95 and YE2681 antibodies, respectively (A). (B) The fluorescence intensity in (A) was quantitatively analyzed by Image-Pro Plus software (*, P < 0.05, **, P < 0.01, compared with NP control-treated AD mice).

Figure 7. PLGA NPs decreased inflammatory cytokine production and attenuated oxidative stress in AD mice

Levels of IL-6 (A) and TNF-α (B) in the brain lysates of AD mice treated with NP control, NP-S1, NP-Cur, NP-S1+Cur and CRT-NP-S1+Cur were determined using corresponding ELISA kits, respectively. The levels of SOD (C) and ROS (D) in the brain lysates of AD mice were determined using corresponding commercial kits, respectively (*, p < 0.05, **, p < 0.01, compared with NP control-treated AD mice).