Design and Validation of Liposomal ApoE2 Gene Delivery System to Evade Blood-Brain Barrier for Effective Treatment of Alzheimer's Disease

Abstract

Targeting gene-based therapeutics to the brain is a strategy actively sought to treat Alzheimer's disease (AD). Recent findings discovered the role of apolipoprotein E (ApoE) isoforms in the clearance of toxic amyloid beta proteins from the brain. ApoE2 isoform is beneficial for preventing AD development, whereas ApoE4 is a major contributing factor to the disease. In this paper, we demonstrated efficient brain-targeted delivery of ApoE2 encoding plasmid DNA (pApoE2) using glucose transporter-1 (glut-1) targeted liposomes. Liposomes were surface-functionalized with a glut-1 targeting ligand mannose (MAN) and a cell-penetrating peptide (CPP) to enhance brain-targeting and cellular internalization, respectively. Among various CPPs, rabies virus glycoprotein peptide (RVG) or penetratin (Pen) was selected as a cell-penetration enhancer. Dual (RVGMAN and PenMAN)-functionalized liposomes were cytocompatible at 100 nM phospholipid concentration and demonstrated significantly higher expression of ApoE2 in bEnd.3 cells, primary neurons, and astrocytes compared to monofunctionalized and unmodified (plain) liposomes. Dual-modified liposomes also showed ∼2 times higher protein expression than other formulation controls in neurons cultured below the in vitro BBB model. These results translated well to in vivo efficacy study with significantly higher transfection of pApoE2 in the C57BL/6 mice brain following single tail vein administration of RVGMAN and PenMAN functionalized liposomes without any noticeable signs of toxicity. These results illustrate the potential of surface-modified liposomes for safe and brain-targeted delivery of the pApoE2 gene for effective AD therapy.

Keywords: Alzheimer’s disease; Apolipoprotein; cell penetrating peptides; glucose transporter; liposomes.

Figure 1.

(A) Distribution of particle size for different liposome formulations using DLS method. AFM images (normal and phase contrast) of (B) PenMAN and (C) RVGMAN liposomes.

Figure 2.

DNase I protection assay. Liposomes containing chitosan/pApoE2 complex was incubated with DNase I enzyme. Lane A, naked pApoE2; lane B, pApoE2 + DNase I; lanes C–H, pApoE2/chitosan complexes loaded in plain, MAN, Pen, PenMAN, RVG, or RVGMAN liposomes, respectively, plus DNase I.

Figure 3.

In vitro cytocompatibility of liposomes at various phospholipid concentrations on (A) bEnd.3, (B) primary astrocytes, and (C) primary neurons. Data represent mean ± SD of four replicates.

Figure 4.

Quantitative analysis of (A) cellular uptake mechanism and (B) competition assay in bEnd.3 cells. Data represent mean ± SD of four replicates. # indicates statistically (p < 0.05) different from their respective PBS control.

Figure 5.

Transfection of ApoE2 in (A) bEnd.3 cells, (B) primary astrocytes, and (C) primary neurons using various chitosan/pApoE2 (1 μg) loaded liposomes. Data represent mean ± SD of four replicates. *, #, +, &, !, and ~ indicate statistically (p < 0.05) different from plain, MAN, Pen, RVG liposomes, naked DNA, and lipofectamine, respectively.

Figure 6.

In vitro BBB model. (A) TEER value and (B) Na–F permeability coefficient. Data represent mean ± SD of four replicates (*p < 0.05). (C) ApoE2 protein expression in primary neurons. Data represent mean ± SD of four replicates. *, #, +, and & indicate statistically (p < 0.05) different from plain, MAN, Pen, and RVG liposomes, respectively.

Figure 7.

ApoE concentration in various mouse tissues ((A) brain; (B) heart; (C) liver; (D) spleen; (E) lungs; (F) kidney; (G) plasma) following tail vein administration of pApoE2/chitosan loaded liposomes (15.2 nM of total lipids containing 1 μg pApoE2/g body weight). Data represent mean ± SD of four replicates. *, #, +, &, !, and ~ indicate statistically (p < 0.05) different from plain, MAN, Pen, RVG liposomes, naked DNA, and control, respectively.

Figure 8.

Biocompatibility assessment of different liposomal formulations following H&E staining post-ApoE2 transfection in different organs.