Development of pomegranate seed oil nanoliposomes for the treatment of neurodegenerative diseases

Abstract

Neurodegenerative diseases such as dementia, Alzheimer's Disease (AD), Parkinson's Disease (PD), and multiple sclerosis are leading causes of disability, loss of life quality, and death worldwide. The loss of neuronal function and structure as well as mental impairments are the consequence of chronic neuroinflammation and oxidative stress in brain tissue. No definite treatment has been found, but numerous natural compounds have been proposed for disease delay, showing low to mild results. Punicic Acid (PuA), obtained from Pomegranate Seed Oil (PSO), is one of the most potent antioxidants in the world, with high antioxidative and anti-inflammatory properties. The biggest problem for studying the effect of PuA and other polyunsaturated fatty acids in the brain is its low bioavailability because of its lack of permeability across the blood-brain barrier. In this Master's in biotechnology program project, we deviate from the classical approach of high content PuA PSO consumption for treating neurodegenerative diseases to a liposome-controlled release delivery system from brain tissue. The project was divided into three experimental phases. The first one was PSO extraction from pomegranate seeds using two methods: traditional solvent extraction with n-hexane and a more sustainable, green technology known as supercritical fluid extraction (SFE), using carbon dioxide as the solvent. Supercritical carbon dioxide (ScCO2) extraction of PSO provided the highest lipid peroxidation inhibition capacity at 16.43% compared to n-hexane PSO extraction. Even though n-hexane obtained a higher yield and higher PuA content, longer extraction hours at high temperatures deteriorate PuA. The second stage was preparing a transferrin-conjugated PSO liposome delivery system (TF-PSO-Lip). The best size, polydispersity index, and zeta potential were obtained at phosphatidylcholine: cholesterol of ratio of 2:1, the amplitude of 70%, pulses of 5 ON 5 OFF sec for 10 min, and a PSO% of 1%. The best encapsulation % at 60% was achieved at 1% PSO. The final size of the TF-PSO-Lip was 198.88 ± 6.72, and the z-potential of -36.98 ± 2.83 mV. The third and final stage of the experimentation process consisted of in-vitro assessments of the ScCO2 and n-hexane PSO, the liposome treatments with (TF-PSO-Lip), without transferrin (PSO-Lip), and without the oil (Lip), and PuA standard for cell viability, nitric oxide inhibition, cellular antioxidative activity, and cellular uptake on murine astrocyte (C8-D1A) and macrophage (RAW 264.7) cell culture, respectively. No treatment showed cytotoxicity. Liposomes with encapsulated PSO maintain the oil's antioxidative and anti-inflammatory properties and provide a controlled release delivery system that delays PuA metabolism inside the cell.