Design and development of bioinspired PEG-ylated nanoliposomes loaded with rosemary extract for treatment of Alzheimer's disease

Abstract

Literature data suggest that the pathogenesis of several neurodegenerative diseases, including Alzheimer's disease (AD), is associated with the state of oxidative stress. Hence, the use of antioxidant and anti-inflammatory molecules, such as polyphenols present in the rosemary extract (RE), is recommended as a useful strategy for preventing AD progression. Taking into consideration the limitations arising from the poor systemic bioavailability of these compounds, and additionally, the complexity of the blood-brain barrier (BBB), the development of nanoliposomes is considered to be suitable and effective solution for transport of the extract across BBB, with the possibility of targeted brain delivery, and thus, successful AD treatment. In this direction, non-PEG-ylated empty nanoliposome formulations (NLs) were firstly formulated, while the further steps involved preparation and characterization of NLs with a polymer for steric stabilization (PEG) and subsequently, PEG-ylated NLs with encapsulated RE. In order to obtain the optimal formulations, central composite design was implemented. In this phase of the research, 20 different NLs formulations were prepared with a modified method of dry lipid film hydration and were completely characterized in terms of physico-chemical and biopharmaceutical properties. The optimal nanoliposomal formulations were characterized with mean size ~ 120 nm, narrow unimodal distribution (SPAN ~ 1) and a negative zeta potential (from -18.50 to -48.3 mV). The encapsulation of the rosmarinic acid (RA) in the liposomal vesicles was ~ 90%, while the in vitro release within 24 hours ranged from 24.83 - 48.39%. These results indicated that the prepared nanoliposomes have high encapsulation efficacy and the rosmarinic acid follows prolonged and controlled release in a period of 24 hours. All optimal nanoliposomal samples showed a statistically significant higher antioxidant capacity (> 94.15%) compared to the extract (RE) (90.04%). The lowest percentage of adsorbed proteins in the plasma of healthy volunteers and the plasma from patients with AD, was obtained for the formulation with the highest amount of polyethilene glycol (PEG) on its surface, while IR-ATR spectroscopic analysis of the liposome-protein complex indicated that this same formulation provided a stronger hydrogen bonding between PEG and Bovine Serum Albumin. The results obtained from the in vitro viability and cytotoxicity assays on BBB cells (hCMEC/D3) and neuroblastoma cells (SH-SY5Y) in a period of 24 hours, after their treatment with 3 different concentrations of blank nanoliposome samples (5, 10 and 100 μg/mL ) did not show a significant decrease in the cell viability, nor a violation of the integrity of their cell membrane. In the comparative studies of quantitative cellular uptake of fluorescently labeled blank NLs formulations by these two cell lines it was found that the concentration of the nano-vesicles and the incubation time play an essential role on the percentage of their internalization. In addition, all tested fluorescently labeled blank NLs formulations followed energy-dependent transport of endocytosis and passive diffusion, while the transport studies on the combined hCMEC/D3/SH-SY5Y cell lines confirmed the successful transport of the nanoformulations across the BBB cells and their subsequent uptake by neurons (ranging from 25.17 to 27.54%). Micro-Raman spectras confirmed the successful internalization of the nano-vesicles, while the fluorescence and confocal microscopy micrographs revealed that the internalized nanoliposomes co-localize in the perinuclear cell regions.