PREPARATION AND CHARACTERIZATION OF LIPOSOMAL DELIVERY SYSTEM OF NATURAL HEME PROTEIN

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.

Keywords: Liposome, Heme protein, Cytochrome C, Phospholipids, Oxidative stress, Antioxidant, Homogenization

References

1. Korshunov SS, Krasnikov BF, Pereverzev MO, Skulachev VP. The antioxidant functions of cytochrome c. FEBS Lett 1999;462:192-8.
2. Bowman SE, Bren KL. The chemistry and biochemistry of heme c: functional bases for covalent attachment. Nat Prod Rep 2008;25:18-30.
3. Pereverzev MO, Vygodina TV, Konstantinov AA, Skulachev VP. Cytochrome c, an ideal antioxidant. Biochem Soc Trans 2003;31:1312-5.
4. Krivtsova IM, Alekseeva NN. Cytochrome C–phospholipid complex (preparation and study in the experiment). In: Selivanov EA. editor. Cytochrome C and its clinical application. Leningrad: Leningrad Res Instit of Hematology and Blood Transfusion; 1990. p. 74-6.
5. Li J, Wang X, Zhang T, Wang C, Huang Z, Luo X, et al. A review on phospholipids and their main applications in drug delivery systems. Asian J Pharm Sci 2015;10:81-98.
6. Bonde S, Nair S. Advances in liposomal drug delivery system: fascinating types and potential applications. Int J Appl Pharm 2017;9:1-7.
7. Tatode AA, Patil AT, Umekar MJ, Telange DR. Investigation of the effect of phospholipids on physical and functional characterization of paclitaxel liposomes. Int J Pharm Pharm Sci 2017;9:141-6.
8. Awad R, W Abdelwahed, Y Bitar. Evaluating the impact of preparation conditions and formulation on the accelerated stability of tretinoin loaded liposomes prepared by the heating method. Int J Pharm Pharm Sci 2015;7:171-8.
9. Sakai H. Overview of potential clinical applications of hemoglobin vesicles (HbV) as artificial red cells, evidenced by preclinical studies of the academic research consortium. J Funct Biomater 2017;8:10.
10. Yuan L, Geng L, Ge L. Effect of iron liposomes on anemia of inflammation, Int J Pharm 2013;454:82-9.
11. Yadav VR, Nag O, Awasthi V. Biological evaluation of liposome-encapsulated hemoglobin surface-modified with a novel PEGylated nonphospholipid amphiphile. Artif Organs 2014;38:625-33.
12. Zhang J, Guan P, Wang T, Chang D, Jiang T, Wang S. Freeze-dried liposomes as potential carriers for ocular administration of cytochrome c against selenite cataract formation. J Pharm Pharmacol 2009;61:1171–8.
13. Katsai OG, Ruban OA, Krasnopolskyi YM. Preparation and in vivo evaluation of cytochrome-c-containing liposomes. Pharmazie 2017;72:736-40.
14. Dobrelya NV, Karazuba ?A, Gula NS, Dunyak YO, Boytsova LV, Danova IV, et al. The correction of hemostatic disorders with liposomal drugs in acute massive blood loss. Pharm Drug Tox 2016;47:66–71.
15. Pylypenko D, Katsai A, Prokhorov V, Konakhovich N, Grigoreva A, Krasnopolsky Y. Investigation of antiarrhythmic activity of liposomal cytochrome C. Sci Rise: Pharm Sci 2017;3:51-7.
16. Liposome Drug Products Chemistry, Manufacturing, and Controls; Human Pharmacokinetics and Bioavailability; and Labeling Documentation. Guidance for Industry. U. S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER); 2018. p. 1-18.
17. State Pharmacopoeia of Ukraine. 2.0. Liposomal praeperationes. Kharkov. Derzhavne pidpriemstvo “Naukovo-ekspertnii farmakopeinii tsentr; 2015. p. 1036–8.
18. Reflection paper on the data requirements for intravenous liposomal products developed with reference to an innovator liposomal product. EMA. Committee for Human Medicinal Products (CHMP); 2013. p. 1-13.
19. Indian Pharmacopoeia. Liposomal preparations. Ghaziabad. Indian Pharmacopoeia Commission; 2010. p. 742–3.
20. Katsai O, Ruban O, Krasnopolskyi Y. The influence of the composition of liposomes on the encapsulation efficiency and the particle size when creating the liposomal form of cytochrome C. Sci Rise: Pharm Sci 2017;4:32-6.
21. Krasnopolskii YM, Grigoreva AS, Katsai AG, Konakhovich NF, Prokhorov VV, Stadnichenko AV, et al. Technologies and perspectives of liposomal drug application in clinical practice. Nanotechnol Russia 2017;12:461–70.
22. Laouini A, Jaafar Maalej C, Limayem Blouza I, Sfar S, Charcosset C, Fessi H. Preparation, characterization and applications of liposomes: state of the art. J Colloid Sci Biotechnol 2012;1:147–68.
23. Perrie Y, Mohammed AUR, Vangala A, Mcneil SE. Environmental scanning electron microscopy offers a real-time morphological analysis of liposomes and niosomes. J Liposome Res 2007;17:27-37.
24. United States Pharmacopeia (USP NF 30). Egg phospholipids. The United States Pharmacopeial Convention; 2012.
25. European Pharmacopoeia (EP). Sterility. The European Pharmacopoeia Commission; 2016.
26. Chinese Pharmacopoeia (ChP). Cytochrome C for Injection. Pharmacopoeia Commission of the Ministry of Health of the People's Republic of China; 1997.
27. Mohn ES, Lee JM, Beaver C, Tobbe G, McCarthy SM, O’Neil E, et al. Interactions of cytochrome c with n-acylated phosphatidyl-ethanolamine lipids. J Phys Chem A 2014;118:8287-92.
28. Rytömaa M, Kinnunen PKJ. Reversibility of the Binding of Cytochrome c to Liposomes: implications for lipid-protein interactions J Biol Chem 1995;270:3197.
29. Gorbenko GP, Molotkovsky JG, Kinnunen PK. Cytochrome c interaction with cardiolipin/phosphatidylcholine model membranes: effect of cardiolipin protonation. Biophys J 2006;90:4093-103.
30. Torchilin VP, Weissig V. Liposomes: a practical approach. Oxford: Oxford University Press; 2003. p. 16-23.
31. Vemuri S, Yu Ch, Wangsatorntanakun V, Roosdorp N. Large-scale production of liposomes by a microfluidizer. Drug Dev Ind Pharm 1990;16:2243-56.
32. Jafari MS, He Y, Bhandari B. Nano-emulsion production by sonication and microfluidization-a comparison. Int J Food Prop 2006;9:3,475-85.
33. Mustafa Grit, Daan JA. Crommelin, chemical stability of liposomes: implications for their physical stability. Chem Phys Lipids 1993;64:3-18.
34. Heurtault B. Physico-chemical stability of colloidal lipid particles. Biomaterials 2003;24:4283-300.
35. Berger N, Sachse A, Bender J, Schubert R, Brandl M. Filter extrusion of liposomes using different devices: comparison of liposome size, encapsulation efficiency, and process characteristics. Int J Pharm 2001;223:55-68.
36. Hinna A, Steiniger F, Hupfeld S, Stein P, Kuntsche J, Brandl M. Filter-extruded liposomes revisited: a study into size distributions and morphologies in relation to lipid-composition and process parameters. J Liposome Res 2015;26:11-20.
37. Vemuri S, Rhodes C. Separation of liposomes by a gel filtration chromatographic technique: a preliminary evaluation. Pharm Acta Helv 1994;69:107-13.
38. Yamamoto E, Miyazaki S, Aoyama C, Kato M. A simple and rapid measurement method of encapsulation efficiency of doxorubicin-loaded liposomes by direct injection of the liposomal suspension to liquid chromatography. Int J Pharm 2018;536:21-8.
39. Toh MR, Chiu GN. Liposomes as sterile preparations and limitations of sterilization techniques in liposomal manufacturing. Asian J Pharm Sci 2013;8:88-95.
40. Wagner A, Vorauer Uhl K. Liposome technology for industrial purposes. J Drug Delivery 2011;2011:1-9.
41. Zuidam NJ, Lee SSL, Crommelin DJA. Sterilization of liposomes by heat treatment. Pharm Res 1993;10:1591-6.
42. Brandl M. Vesicular phospholipid gels. In: Weissig V. editor. Liposomes. methods in molecular biology (Methods and Protocols). Vol. 605. Humana Press; 2009. p. 205-12.
43. Driscoll DF. Lipid injectable emulsions. Nutr Clin Pract 2006;21:381-6.
44. Goldbach P, Brochart H, Wehrle P, Stamm A. Sterile filtration of liposomes: Retention of encapsulated carboxyfluorescein. Int J Pharm 1995;117:225-30.
45. Folmsbee M, Moussourakis M. Sterilizing filtration of liposome and related lipid-containing solutions: enhancing successful filter qualification. J Pharm Sci Technol 2012;66:161-7.
46. Creutz C, Sutin N. Reduction of ferricytochrome c by dithionite ion: electron transfer by parallel adjacent and remote pathways. Proc Natl Acad Sci USA 1973;70:1701-3.
47. Craig DB, Nichols ER. Spectroscopic measurement of the redox potential of cytochrome c for the undergraduate biochemistry laboratory. J Chem Educ 2006;83:1325.
48. Schweitzer Stenner R, Soffer JB. Optical spectroscopy. In: Dyson HJ. editor. Biophysical techniques for structural characterization of macromolecules, Oxford Academic Press; 2012. p. 533–91.
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How to Cite
Katsai, O., & Ruban, O. (2019). PREPARATION AND CHARACTERIZATION OF LIPOSOMAL DELIVERY SYSTEM OF NATURAL HEME PROTEIN. International Journal of Applied Pharmaceutics, 11(4), 418-425. https://doi.org/10.22159/ijap.2019v11i4.32080
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