Donepezil-Loaded Nanocarriers for the Treatment of Alzheimer’s Disease: Superior Efficacy of Extracellular Vesicles Over Polymeric Nanoparticles

Abstract

Introduction

Drug delivery across the blood-brain barrier (BBB) is challenging and therefore severely restricts neurodegenerative diseases therapy such as Alzheimer’s disease (AD). Donepezil (DNZ) is an acetylcholinesterase (AChE) inhibitor largely prescribed to AD patients, but its use is limited due to peripheral adverse events. Nanodelivery strategies with the polymer Poly (lactic acid)-poly(ethylene glycol)-based nanoparticles (NPs-PLA-PEG) and the extracellular vesicles (EVs) were developed with the aim to improve the ability of DNZ to cross the BBB, its brain targeting and efficacy.

Methods

EVs were isolated from human plasma and PLA-PEG NPs were synthesized by nanoprecipitation. The toxicity, brain targeting capacity and cholinergic activities of the formulations were evaluated both in vitro and in vivo.

Results

EVs and NPs-PLA-PEG were designed to be similar in size and charge, efficiently encapsulated DNZ and allowed sustained drug release. In vitro study showed that both formulations EVs-DNZ and NPs-PLA-PEG-DNZ were highly internalized by the endothelial cells bEnd.3. These cells cultured on the Transwell^® model were used to analyze the transcytosis of both formulations after validation of the presence of tight junctions, the transendothelial electrical resistance (TEER) values and the permeability of the Dextran-FITC. In vivo study showed that both formulations were not toxic to zebrafish larvae (Danio rerio). However, hyperactivity was evidenced in the NPs-PLA-PEG-DNZ and free DNZ groups but not the EVs-DNZ formulations. Biodistribution analysis in zebrafish larvae showed that EVs were present in the brain parenchyma, while NPs-PLA-PEG remained mainly in the bloodstream.

Conclusion

The EVs-DNZ formulation was more efficient to inhibit the AChE enzyme activity in the zebrafish larvae head. Thus, the bioinspired delivery system (EVs) is a promising alternative strategy for brain-targeted delivery by substantially improving the activity of DNZ for the treatment of AD.

Graphical Abstract

Keywords:

• Alzheimer’s disease
• donepezil
• extracellular vesicular
• PLA-PEG
• bEnd.3 cells
• zebrafish

Abbreviations

AChE, Acetylcholinesterase; AD, Alzheimer’s disease; BCA, Bicinchoninic acid; BBB, blood-brain barrier; CNS, Central Nervous System; DNZ, Donepezil; DMEM, Dulbecco’s Modified Eagle’s Media; DL%, Drug loading; EMEM, Eagle’s minimal essential medium; EDTA, Ethylenediaminetetraacetic acid; EE %, Encapsulation efficiency; FDA, United States Food and Drug Administration; hpf, Hours Post-Fertilization; INRS-CER, Ethical Committee of the Institut National de la Recherche Scientifique; EVs, Extracellular Vesicles; FBS, Fetal Bovine Serum; FACS, Fluorescence-activated cell sorting; nFCM, nanoscale flow cytometry; NPs nanoparticles; NTA, Nanoparticle tracking analysis; PET, Polyester; PTU, phenylthiourea; PDI, Polydispersity index; PLA-PEG, Poly (lactic acid)-poly(ethylene glycol); SEM, Standard Error of the Mean; TEER, Transendothelial Electrical Resistance; TJ, tight junctions; TEM, Transmission electron microscopy.

Acknowledgments

This study was supported by Research Chair Louise & André Charron on Alzheimer’s disease, the Armand-Frappier Foundation, NSERC (RGPIN-2018-06182) Government of Canada), Canadian Institute of Health Research, NSERC/CRSNG, and Canada Foundation for Innovation. The authors are grateful to Pr D. Girard for the use of the DLS Malvern Zetasizer; Pr Maritza Jaramillo for the use of the Microplate Spectrophotometer; Jessy Tremblay (INRS, Center Armand-Frappier) for help with experiments involving flow cytometry and confocal microscopy; Arnaldo Nakamura (INRS, Center Armand-Frappier) for help with the transmission electron microscope. Tg (flk1:EGFP) was a generous gift from Dr. Xiao-Yan Wen (Univ. of Toronto, Toronto, ON, Canada). Graphical abstract is created by the main author using BioRender.com.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.