(T1530-06-35) Brain-Targeted ApoE2 Gene Delivery Across the Blood-Brain Barrier using Dual-functionalized Liposomal Nanoparticles for Alzheimer's Disease Therapy

Purpose: Alzheimer's disease (AD) remains an incurable neurodegenerative disorder, with the apolipoprotein E (ApoE) isoform playing a central role in disease onset and progression. While ApoE4 accelerates AD pathology, ApoE2 offers neuroprotection by reducing amyloid beta burden and tau tangles, enhancing synaptic plasticity, and improving cognitive function¹. Building on this therapeutic potential, our study aims to develop a dual-functionalized liposomal nanocarrier for efficient delivery of plasmid ApoE2 (pApoE2) across the blood-brain barrier (BBB). The liposomes are functionalized with transferrin and cell-penetrating peptides (RDP and Cgn) to enhance brain-specific targeting and improve gene transfection efficiency. We will evaluate its therapeutic efficacy in two transgenic AD models: APP/PS1 to assess amyloid pathology and PS19 to study tauopathy. This strategy represents a novel gene-based intervention to combat AD at the molecular level and aims to unlock new avenues for disease-modifying therapy.

Methods: Liposomal formulations were prepared using thin-film hydration with 45mol% DOTAP, 45mol% DOPE, 8mol% DSPE-PEG, and 2mol% cholesterol. Cell-penetrating peptides and transferrin were pegylated using DSPE-PEG(2000)-NHS. The liposomes' physicochemical properties, including size, zeta potential, and polydispersity index were characterized via dynamic light scattering (DLS) technique². Encapsulation efficiency of pApoE2 complexed with chitosan (N:P ratio, 5:1) within the liposome was further determined using a spectrophotometer³. Biocompatibility and biodistribution of the liposomal formulations encapsulating the pApoE2-chitosan polyplex (15.2umoles of total lipids containing 1mg pDNA/kg b.w) were assessed in 3-month-old C57BL/6 (WT) mice. For biocompatibility, a) alanine aminotransferase (ALT/GPT), b) aspartate aminotransferase (AST/GOT), c) creatinine, and d) cardiac troponin I Type 3 levels were analyzed 7-days post-treatment. Biodistribution levels of lissamine rhodamine-labeled liposomes in C57BL6/J mice brains were determined using HPLC 24 h post-treatment via single tail vein injection. ApoE2 expression levels in the brain of 3-months old WT and ApoE-knockout (KO) mice treated with Apoe2 loaded-liposomes were sacrificed 7 days after single dose treatment. The ApoE2 protein levels were quantified in brain lysate using ApoE2 ELISA. For neuron-specific targeting of dual-functionalized liposomes, ApoE immunostaining was performed in WT mice following intravenous administration of RDP-Tf liposomes encapsulating ApoE2 on days 1, 3, and 5. Mice were sacrificed one week after the final dose. Brain sections were stained with an anti-ApoE2 antibody (FITC, green) to detect ApoE2 localization, NeuN (Alexa Fluor 594, red) to label neurons, and DAPI (blue) to visualize nuclei. Confocal imaging was performed to confirm neuron-specific localization of ApoE2.

Results: The liposomal formulations exhibited a cationic charge, uniform size distribution ( < 200 nm), low polydispersity index ( < 0.3), and an encapsulation efficiency of over 85% (Figure 1). Biocompatibility evaluations revealed no signs of systemic toxicity, with all physiological parameters remaining within normal range. No statistically significant differences were observed between liposome-treated and saline control groups (p > 0.05), a finding corroborated by hematoxylin and eosin (H&E) staining, which confirmed preserved tissue morphology (Figure 2). Dual-functionalized liposomes (RDP-Tf and Cgn-Tf) exhibited significantly higher brain accumulation (p < 0.0001) compared to single-functionalized or plain liposomes, indicating enhanced BBB permeability and targeting efficacy. In both C57Bl/6 and ApoE-knockout (ApoE-KO) mice, RDP-Tf and Cgn-Tf liposomes showed a markedly elevated expression of ApoE2 (p < 0.0001), compared to single-functionalized liposomes, plain liposomes, or naked pApoE2 treatment. Immunofluorescent staining confirmed ApoE2 localization within the hippocampus and dentate gyrus, with clear colocalization of ApoE2 and the neuronal marker NeuN, indicative of neuron-specific delivery (Figure 3). Saline-treated mice served as negative controls for all experimental comparisons.

Conclusion: The liposomal formulations demonstrated excellent biocompatibility, and the dual-functionalized liposomes (RDP-Tf and Cgn-Tf) significantly enhanced brain biodistribution and efficiently facilitated the delivery of the pApoE2 gene across the BBB. This was evidenced by significant increases in ApoE2 expression in the brains of both WT and ApoE-KO mice. Immunostaining confirmed that ApoE2 was predominantly expressed in neuronal cells, validating targeted gene delivery. Further studies, including neurobehavioral assessments, amyloid and tau pathology analysis, and synaptic function evaluation in AD transgenic mouse models, will further substantiate the therapeutic efficacy of this gene delivery system and its potential to reverse neurodegeneration.

References: (1) Michaelson, D. M. APOE ε4: the most prevalent yet understudied risk factor for Alzheimer's disease. Alzheimers Dement 2014, 10 (6), 861-868. DOI: 10.1016/j.jalz.2014.06.015.
(2) Dos Santos Rodrigues, B.; Oue, H.; Banerjee, A.; Kanekiyo, T.; Singh, J. Dual functionalized liposome-mediated gene delivery across triple co-culture blood brain barrier model and specific in vivo neuronal transfection. J Control Release 2018, 286, 264-278. DOI: 10.1016/j.jconrel.2018.07.043.
(3) Lakkadwala, S.; Singh, J. Co-delivery of doxorubicin and erlotinib through liposomal nanoparticles for glioblastoma tumor regression using an in vitro brain tumor model. Colloids Surf B Biointerfaces 2019, 173, 27-35. DOI: 10.1016/j.colsurfb.2018.09.047.

Acknowledgements: This research was supported by the National Institutes of Health grants RF1 AG068034, RF1 AG068034-01S1, R01 AG068034-02 and R01 AG083981.


Figure 1: Liposome characterization A) Transmission electron microscopy (TEM) image displaying morphological characteristics of liposomes, Scale 50nm. B) Characterization of Different Liposomal Formulations. Data are expressed as mean ± SD (n =4).


Figure 2a: Plasma biochemical assessments of I) alanine aminotransferase (ALT/GPT), II) aspartate aminotransferase (AST/GOT), III) creatinine, and IV) cardiac troponin I Type 3 levels 7-days post treatment with liposomes (15.2umoles of total lipids containing 1mg pDNA/kg b.w) in 3-months old WT mice. Data are expressed as mean ± SD (n =6) (p > 0.05).
Figure 2b: Hematoxylin and eosin staining of the brain, heart, lungs, kidney, spleen and liver of 3-month-old mice 7 days post-treatment with a single dose of ApoE2-loaded liposomes.



Figure 3a: ApoE2 expression levels in the brain of 3-months old mice treated with liposomal formulations containing chitosan-plasmid ApoE2 polyplex after 7days post treatment (15.2umoles of total lipids containing 1mg pDNA/kg b.w). I) C57BL/6 (WT) and II) ApoE Knock-out Mice. Data are expressed as mean ± SD (n =6) (p < 0.05).
Figure 3b: Immunofluorescence staining to assess neuronal targeting of dual-functionalized (RDP-Tf) liposomes in wild-type (WT) mouse brain. WT mice received intravenous administration of RDP-Tf liposomes encapsulating ApoE2 on days 1, 3, and 5, and were sacrificed one week after the final dose. Brain sections were stained with an anti-ApoE2 antibody (FITC, green) to detect ApoE2 localization, NeuN (Alexa Fluor 594, red) to label neurons, and DAPI (blue) to visualize nuclei. Representative images from the hippocampus and dentate gyrus (DG) regions show colocalization of ApoE2 and NeuN (orange in merged images), indicating neuron-specific uptake of the liposomes. Saline-treated WT mice served as control.