Original Article
Retro-inverso peptide inhibitor nanoparticles as potent inhibitors of aggregation of the Alzheimer's Aβ peptide

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Abstract

Aggregation of amyloid-β peptide (Aβ) is a key event in the pathogenesis of Alzheimer's disease (AD). We investigated the effects of nanoliposomes decorated with the retro-inverso peptide RI-OR2-TAT (Ac-rGffvlkGrrrrqrrkkrGy-NH2) on the aggregation and toxicity of Aβ. Remarkably low concentrations of these peptide inhibitor nanoparticles (PINPs) were required to inhibit the formation of Aβ oligomers and fibrils in vitro, with 50% inhibition occurring at a molar ratio of ~1:2000 of liposome-bound RI-OR2-TAT to Aβ. PINPs also bound to Aβ with high affinity (Kd = 13.2-50 nM), rescued SHSY-5Y cells from the toxic effect of pre-aggregated Aβ, crossed an in vitro blood–brain barrier model (hCMEC/D3 cell monolayer), entered the brains of C57 BL/6 mice, and protected against memory loss in APPSWE transgenic mice in a novel object recognition test. As the most potent aggregation inhibitor that we have tested so far, we propose to develop PINPs as a potential disease-modifying treatment for AD.

Graphical Abstract

Retro-inverso peptides attached to the surface of nanoliposomes prevent the aggregation of β-amyloid monomers into oligomers and fibrils. The ‘ffvlk’ sequence of the inhibitory peptide is designed to interact with the ‘KLVFF’ sequence on the Aβ molecule. However, Aβ could also interact with the positive charge of the ‘TAT’ sequence and Aβ oligomers, once captured, could insert themselves into the lipid bilayer of the liposomes.

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Section snippets

Materials

Chemical reagents and Sepharose 4B-CL were from Sigma-Aldrich. Bovine brain sphingomyelin (Sm), cholesterol (Chol) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (mal-PEG-PE) were from Avanti Polar Lipids Inc., USA. [3H]-Sm, [3H]-propranolol, [14C]-sucrose, Ultima Gold scintillation cocktail and solvable tissue solubilizer were from PerkinElmer (Waltham, MA, USA). [14C]-Chol was provided by Quotient Bioresearch Ltd. Polycarbonate filters for liposome

Preparation and characterization of peptide inhibitor nanoparticles (PINPs)

To attach RI-OR2-TAT (Figure 1, A) covalently to the NL surface, we exploited a thiol-maleimide reaction employing an additional cysteine residue to provide the necessary thiol group.23 This thiol function at the C-terminus of RI-OR2-TAT reacted with a maleimide-functionalized phospholipid present in the NL formulation (mal-PEG-PE) (Figure 1, B). The yield of coupling was 80%-90% and, consequently, PINPs contained 2%-2.5 mol% of peptide. The total lipid recovery of NL, after the reaction with

Discussion

Here, we linked RI-OR2-TAT covalently to the surface of NL composed of sphingomyelin and cholesterol. This lipid formulation has been widely utilized in vivo for therapeutic purposes and displays good blood circulation times, good biocompatibility, and high resistance to hydrolysis.26 In addition, the 130-140 nm diameter is optimal for moving at an appreciable rate through the brain extracellular space.27 We found that the presence of the carrier appears to increase the potency of RI-OR2-TAT as

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  • Cited by (0)

    The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 212043, and from The Alzheimer's Society UK (grant number 210 (AS-PG-2013-032)). There were also contributions from Lancaster University's Defying Dementia campaign.

    Competing interests: The University of Lancaster has filed a patent (WO2013054110) on intellectual property related to this work, based on inventions of DA and MT. The patent has been licensed to MAC Clinical Research, UK.

    The authors thank Pierre-Olivier Couraud from Institut National de la Santé et de la Recherche Médicale (INSERM, Paris, France) for providing the hCMEC/D3 cells.

    1

    Maria Gregori and Mark Taylor contributed equally to this paper.

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