Abstract
Objective: The objective of the present study was to develop and optimize the methods for preparation and characterization of the liposomal delivery system of natural heme protein.
 Methods: Cytochrome C containing liposomes (Cyt-LS) were prepared by high-pressure homogenization technique using phosphatidylcholine (PC) and dipalmitoyl phosphatidylglycerol (DPPG). Nanoparticles were characterized by using: dynamic light scattering, zeta potential measurements, scanning electron microscopy and HPLC. The specific activity was studied in vitro.
 Results: The study of homogenization regimes for obtaining unilamellar Cyt-LS was carried out. The selected temperature regime of homogenization was kept within 38–44 °С with optimal homogenization pressure of 800 bar. The obtained Cyt-LS were characterized by the main physicochemical parameters showed: Encapsulation efficiency 95.8±2.0%, Zeta potential-57±1.0 mV, pH-6.95±0.05. Phospholipid impurities had the following content: lysophosphatidylcholine-0.60±0.05% and free fatty acids-0.4±0.05%. The average particle diameter was 156±2 nm. Also, the size of Cyt-LS particles was confirmed by the ability of emulsion subjected to the sterilizing filtration with the preservation of its main physicochemical properties. Cyt-LS exhibit specific activity, similar to non-liposomal Cyt-C solution.
 Conclusion: The formulation of the liposomal delivery system of heme protein was successfully prepared using natural components and evaluated for different parameters.
Highlights
One of the known natural heme protein involved in the respiratory electron transport chain and cell apoptosis is cytochrome C (Cyt-C)
The ability to capture and neutralize reactive oxygen species (ROS) defines some properties of Cyt-C as a medical preparation for the prevention and treatment of the oxidant-induced injury caused by ROS formation under oxidative stress which often accompanies the processes of tissue hypoxia
It is well known that Cyt-C can form complexes with anionic phospholipids in biological membranes [27,28,29], which is determinant in Cyt-C protein-lipid interaction
Summary
One of the known natural heme protein involved in the respiratory electron transport chain and cell apoptosis is cytochrome C (Cyt-C). The water-soluble Cyt C form has a number of significant drawbacks: Cyt C insignificantly penetrates cells through biological membranes and is rapidly removed from parts of the body damaged by hypoxia, which causes its extremely low bioavailability after administration. Under hypoxia, the structural damage leads to the simultaneous loss of Cyt-C and phospholipids in the mitochondrial respiratory chain [4]. LS derived from phospholipids of natural origin have a number of undoubted advantages: they are nonimmunogenic, low-toxic and under certain conditions can connect with the phospholipids of cell membranes and deliver their contents inside the cells [5, 6]
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