In Vitro and In Vivo Evaluation of Liposomes Mediated ApoE2 Gene Therapy for the Treatment of Alzheimer’s Disease
Purpose: Alzheimer’s disease (AD) is a debilitating neurodegenerative disorder which currently has no cure. Approximately 6 million people are suffering from this disorder in US alone. One of the major risk factor for AD is Apolipoprotein E4 (ApoE4) isoform whereas ApolipoproteinE2 (ApoE2) isoform was found to have a protective role in brain. Therefore, gene delivery of ApoE2 to brain has enormous potential as a therapeutic strategy against AD. However, delivering gene inside brain is majorly limited owing to presence of various physiological barriers like cellular barriers and nucleases. Additionally, the presence of blood brain barrier is another major obstacle in the path of gene delivery. Blood brain barrier (BBB) is a highly selective semi permeable membrane which prevents the entry of unwanted substances while facilitating transportation of selective molecules necessary for proper functioning of brain. Glucose, which is the major energy source to brain, is one such molecule. Therefore, to meet brain energy demand, Glucose transporter-1 (GLUT-1) are present at BBB in high density. Hence, in this study lipid bilayered vesicles called liposomes were prepared entrapping the pDNA for ApoE2. These nanoparticles were surface modified with mannose which can target GLUT-1 transporters present on the apical side of the blood brain barrier. Also, cell penetrating peptides, Penetratin or Rabies virus glycoprotein (RVG), were also attached to the surface to enhance cell penetration. These dual functionalized liposomes entrapping pDNA for ApoE2 were tested in-vitro and in-vivo for their transport and transfection efficiency in the brain cells. Methods: Liposomes modified with 4-aminophenyl α-D-Mannopyranoside (MAN) were prepared by cross-linking amino group of lipid (DSPE-PEG-NH2, 2000 Da) to the amino group of MAN with the help of homobifunctional crosslinker glutaraldehyde. CPPs was coupled to ester group of lipid (DSPE-PEG-NHS) via formation of an amide bond. Conjugation efficiency of MAN and Pen was determined as per manufacturer’s instructions using 2,4,6-trinitrobenzene sulfonic acid reagent and BCA protein assay kit, respectively. Liposomes were prepared using film hydration technique using specific ratios of selected lipids (DOTAP/DOPE/Cholesterol/DSPE-PEG-MAN/DSPE-PEG-PEN). Size and zeta potential of the liposomes was determined using dynamic light scattering technique. pDNA was complexed with chitosan prior to encapsulation into the liposomes to facilitate pDNA condensation while providing efficient protection of the genetic material from lysosomal nuclease degradation. In-vitro triple co-culture blood brain barrier model, was developed by culturing bEnd.3 cell and primary rat glial cells on the inner and outer membrane of transwell culture insert respectively, placed in 24 well plate with primary neuronal cells seeded at the bottom. Transportation ability of dual modified liposomes across BBB model was analyzed at different time points. Transfection ability of these nanoparticles encapsulating ApoE2 pDNA was also analyzed in triple co-culture model using ELISA. Dual modified liposomes were also assessed for their transfection ability in brain cells of the C57BL/6 mice post intravenous administration. Results: Particle size of liposomes was found to be < 200nm with net positive zeta potential that is helpful in promoting binding to negatively charged cell membrane. Approximately 16% of dual modified liposomes were transported across the in-vitro BBB over a period of 8 hours. These liposomes also showed higher transfection efficiency compared to unmodified, MAN-only, PEN-only, and RVG only modified liposomes in primary neuronal cells seeded across the in vitro barrier. In-vivo, liposomes modified using PenMAN and RVGMAN showed approximately 2 times higher transfection efficiency than baseline ApoE level in the brain of the mice. Dual modified liposomes also showed significantly higher transfection efficiency than other formulation controls. Conclusion: Dual modified liposomes conjugated to mannose and cell penetrating peptides were successfully prepared. pDNA was entrapped inside the liposomes with high efficiency. Modification with mannose and CPPs significantly enhanced transportation of these dual modified nanoparticles across the in vitro BBB model without disrupting the integrity of the membrane. Finally, these nanoparticles were able to transport across the BBB in-vivo and transfect the brain cells. Overall, these lipid nanoparticles can potentially deliver therapeutic genes across the blood brain barrier in a safe and effective manner for the treatment of Alzheimer’s disease. This research was supported by the National Institutes of Health (NIH) grant # RO1AG051574.