SUMMARY: Neuro-degenerative diseases have become the most common cause of dementia among the elderly and have been reported to affect about 5% of Americans over age of 65, and 20% over the age of 80 years. There were 36 million people living with dementia worldwide in 2010, increasing to 66 million by 2030 and 115 million by 2050. In 2010, the global cost of dementia was $604 billion. This is 1% of global GDP and it is likely that these costs will increase in proportion to the number of people with dementia. Gene therapy has been identified to possess a broad potential for the treatment of numerous neurological diseases, including Alzheimer’s disease (AD). AD is a progressive neurodegenerative disease and the most common form of dementia caused by accumulation of toxic amyloid-β (Aβ) peptides in the brain, in which the development of effective therapies have been desired. However, the major challenge in the field of gene therapy is the design of safe non-viral vectors that can cross the blood brain barrier (BBB). It has been found that the transferrin receptors are present on the surface of brain endothelial cells. The liposomes, lipid based nanoparticles, can be surface modified with transferrin (Tf) protein for targeting the brain endothelial receptors and conjugated to cell penetrating peptide (CPP) for improving their internalization into brain by overcoming receptor saturation. Therefore, we propose to conjugate the liposomes with two ligands (1) a receptor targeting protein (Tf) and (2) a CPP. Thus we will design near-neutral, PEGylated liposomes by modifying the surface with Tf and CPP. Furthermore, the transfection properties of low molecular weight chitosan will be utilized for improving the transfection of gene by facilitating endosomal escape inside the cells. The long term goal of the proposed research is to design a gene delivery carrier for efficient delivery of Nerve Growth Factor (NGF) to brain for prevention and treatment of AD. We propose the following three specific aims: (1). To synthesize and characterize Tf and CPP coupled liposomes loaded with chitosan-pDNA polyplexes: The CPP-liposomes will be synthesized using thin film hydration technique followed by insertion of Tf coupled micelles using post- insertion technique. We propose to use three types of CPPs based on the physico-chemical properties [cationic hydrophilic (HIV-Tat, PasR8 and R9F2), cationic amphiphilic (pVec, penetratin and Mellittin) and hydrophobic (PFVYLI, pentapeptide QLPVM and Kaposi Fibroblast Growth Factor derived peptide). The gene of interest (pGFP or pNGF) will be complexed with chitosan to improve transfection properties of liposomes. The liposomes will be evaluated for particle size, zeta potential, encapsulation efficiency, cell uptake and uptake mechanism(s), transfection efficiency, cell cytotoxicity, and hemolysis assay. (2). To evaluate the transport efficacy of liposomes across the barrier layer using 2-Dimensional (2D) BBB model: The transport...