(T1230-02-09) Development and In Vitro Evaluation of Donepezil, Memantine and siRNA Encapsulated Liposomes

Purpose: Alzheimer’s Disease (AD) is a chronic neurological disorder and accounts for the major cause of dementia. Current pharmacotherapies are frequently used to improve symptoms in patients with AD. However, poor pharmacokinetics and biodistribution of drugs after systemic administration decreases their accumulation in the brain, and clinical efficacy and increases the risk of adverse side effects. siRNA can have a significant role in regulating gene expression in AD, but it can be easily degraded once administered systemically. Delivery of the medications directly to the brain via the nasal route potentially could enhance the efficacy of the treatment and limit its adverse effects. The present study is aimed at developing and testing liposomal formulation of donepezil, memantine, and Bace-1 siRNA for intranasal delivery to target the brain.

Methods: Donepezil, memantine, and Bace-1 siRNA were individually loaded in liposomes. Particle size, zeta-potential, and drug encapsulation efficiency of liposomes were measured. Calu-3 cells were used for the evaluation of the nasal penetration of the liposomal drugs. HBEC-5i and human astrocytes were used for the evaluation of cellular internalization. SH-SY5Y cells were differentiated with retinoic acid(RA)/Brain-Derived Neurotrophic Factor (BDNF) and a beta-amyloid plaque was used as a model of AD. The cells were treated with drug-loaded liposomes. Beta-amyloid plaque levels, caspase 3/7 activities and BACE-1 activities were studied using these liposomes.

Results: Drug-loaded liposomes (125-140 nm) demonstrated a sufficient drug encapsulation efficiency (81.6% donepezil; 39% memantine; 36.5% Bace-1 siRNA). Confocal microscope studies showed excellent cellular penetration and internalization of liposomes. Caspase activity and BACE-1 activity were decreased in the treated cells.

Conclusion: The present study demonstrated the possibility of encapsulating donepezil, memantine, and Bace-1 siRNA into liposomes for nose-to-brain delivery. These experiments formed the basis for the treatment of Alzheimer’s Disease via the nasal route of administration.

Acknowledgements: Rutgers, The State University of New Jersey, Ernest Mario School of Pharmacy, Dept. of Pharmaceutics, Piscataway, NJ
The research was supported in part by R01 CA238871 grant

HBEC-5i endothelial cells (left) and astrocytes (right) were incubated for 24 h with liposomes (green fluorescence) containing siRNA (red fluorescence). Nuclei were labeled with DAPI (blue fluorescence). The superimposition of images allows for the detection of cytoplasmic co-localization of liposomes with siRNA (yellow color). Liposomes internalized into the cells and localized in the cytoplasm; siRNA released from liposomes and localized in the cytoplasm.

Western blot of BACE-1 activity in differentiated SH-SY5Y cells. Aβ42 5 µM was incubated for 24 hours and sequentially treated for 48 hours with Bace-1 siRNA 25 and 100nM. Used GAPDH Alexa Fluor 647, rabbit monoclonal anti-BACE1, and secondary goat anti-rabbit IGG (IRDYE® 800CW)

RA/BDNF differentiated SH-SY5Y cells were incubated with Aβ42 5µM for 24 hours and sequentially treated for 24 hours with stausporine 20 nM, memantine liposome (1,5,10 µM). Cells treated with 10µM memantine liposome had lower caspase-3/7 activity compared to cells treated with Aβ 5µM (* p < 0.01)