RGD-Modified Nano-Liposomes Encapsulated Eptifibatide with Proper Hemocompatibility and Cytotoxicity Effect

Document Type: Research Paper

Authors

1 Cellular and Molecular Research Center, Yasuj University of Medical Sciences, Yasuj, Iran

2 Biotechnology Group, Department of Chemical Engineering, Tarbiat Modares University, Tehran, Iran

3 Department of Medicinal Chemistry, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran

4 Industrial and Environmental Biotechnology, National Inst. of Genetic Engineering and Biotechnology, Tehran, Iran

5 Department of Immunology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

6 Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

Abstract

Background: Eptifibatide (Integrilin®) is a hepta-peptide drug which specifically prevents the aggregation of activated platelets. The peptide drugs are encapsulated into nanolipisomes in order to decreasing their side effects and improving their half-life and bioavailability.
Objectives: In this study, the in vitro cytotoxicity and hemocompatibility of RGD-modified nano-liposomes (RGD-MNL) encapsulated a highly potent antiplatelet drug (eptifibatide) was investigated.
Material and Methods: RGD-MNL encapsulated eptifibatide was prepared using lipid film hydration and freeze/thawing method. The morphology and size distribution (about 90 nm) of RGD-MNL were characterized using transmission electron microscopy (TEM). The in-vitro cytotoxicity of nano-liposomes was examined using the MTT, LDH release and reactive oxygen species (ROS) generation assays. The effect of RGD-MNL on red blood cells (RBC) was investigated using hemolysis and LDH release assays.
Results: The results revealed that RGD-MNL had no significant cytotoxic effect on HeLa and HUVEC cell lines, and also no ROS generation increase in the cells. In addition, the adverse effect of RGD-MNL on LDH release and membrane integrity of RBC was not observed.
Conclusions: In conclusion, the recommended RGD-MNL formulations have not any significant cytotoxicity on normal cells or RBC and have potential for protecting and enhancing the activity of antiplatelet drugs.

Keywords

Main Subjects


1.           Chang K, Chiu J-J. Clinical applications of nanotechnology in atherosclerotic diseases. Curr Nanosci. 2005;1(2):107-115.

2.           Tcheng JE, O'Shea JC. Eptifibatide: a potent inhibitor of the platelet receptor integrin, glycoprotein IIb/IIIa. Expert Opin Investig Drugs. 1999;8(11):1893-1905. pmid: 11139832

3.           Addeo R, Faiola V, Guarrasi R, Montella L, Vincenzi B, Capasso E, et al. Liposomal pegylated doxorubicin plus vinorelbine combination as first-line chemotherapy for metastatic breast cancer in elderly women > or = 65 years of age. Cancer Chemother Pharmacol. 2008;62(2):285-292. doi: 10.1007/s00280-007-0605-6 pmid: 17922275

4.           Torchilin VP. Targeting of drugs and drug carriers within the cardiovascular system Adv Drug Delivery Rev. 1995;17 75-101.

5.           Mohammadian M, Farzampanah L, Behtash-oskouie A, Majdi S, Mohseni G, Imandar M, et al. A biosensor for detect nitrite (NO2-) and hydroxylamine (nh2oh) by using of hydroxylamine oxidase and modified electrode with ZnO nanoparticles. Int J Electrochem Sci. 2013;8(9):11215-11227.

6.           Schiener M., Hossann M., Viola JR., Ortega-Gomez A., Weber C., Lauber K., et al. Nanomedicine-based strategies for treatment of atherosclerosis. Trends Mol Med. 2014;20(5):271-281. doi: 10.1016/j.molmed.2 013.12.001 pmid: 24594264

7.           Haller C. A., Cui W, Wen J, Robson SC, Chaikof E. L. Reconstitution of CD39 in liposomes amplifies nucleoside triphosphate diphosphohydrolase activity and restores thromboregulatory properties. J Vasc Surg. 2006;43(4):816-823. doi: 10.1016/j.jvs.2005.11.057 pmid: 16616242

8.           Elbayoumi TA, Torchilin VP. Liposomes for targeted delivery of antithrombotic drugs. Expert Opin Drug Deliv. 2008;5(11):1185-1198. doi: 10.1517/17425240802497457 pmid: 18976130

9.           Bardania H, Tarvirdipour S, Dorkoosh F. Liposome-targeted delivery for highly potent drugs. Artif Cells Nanomed Biotechnol. 2017;45(8):1478-1489. doi: 10.1080/21691401.2017.1290647 pmid: 28278584

10.        Marques-Gallego P, de Kroon AI. Ligation strategies for targeting liposomal nanocarriers. Biomed Res Int. 2014;2014:129458. doi: 10.1155/2014/129458 pmid: 25126543

11.        Noble GT, Stefanick JF, Ashley JD, Kiziltepe T, Bilgicer B. Ligand-targeted liposome design: challenges and fundamental considerations. Trends Biotechnol. 2014;32(1):32-45. doi: 10.1016/j.tibtech.2013.09.007 pmid: 24210498

12.        Vyas SP, Vaidya B. Targeted delivery of thrombolytic agents: role of integrin receptors. Expert Opin Drug Deliv. 2009;6(5):499-508. doi: 10.1517/17425240902878002 pmid: 19413457

13.        Srinivasan R, Marchant RE, Gupta AS. In vitro and in vivo platelet targeting by cyclic RGD-modified liposomes. J Biomed Mater Res A. 2010;93(3):1004-1015. doi: 10.1002/jbm.a.32549 pmid: 19743511

14.        Huang G, Zhou Z, Srinivasan R, Penn MS, Kottke-Marchant K, Marchant RE, et al. Affinity manipulation of surface-conjugated RGD peptide to modulate binding of liposomes to activated platelets. Biomaterials. 2008;29(11):1676-1685. doi: 10.1016/j.biomaterial s.2007.12.015 pmid: 18192005

15.        Mousavizadeh A, Jabbari A, Akrami M, Bardania HJC, Biointerfaces SB. Cell targeting peptides as smart ligands for targeting of therapeutic or diagnostic agents: A systematic review. Biointerfaces. 2017;158:507-517.

16.        Vaidya B, Nayak MK, Dash D, Agrawal GP, Vyas SP. Development and characterization of site specific target sensitive liposomes for the delivery of thrombolytic agents. Int J Pharm. 2011;403(1-2):254-261. doi: 10.1016/j.ijpharm.2010.10.028 pmid: 20971175

17.        Lestini BJ, Sagnella SM, Xu Z, Shive MS, Richter NJ, Jayaseharan J, et al. Surface modification of liposomes for selective cell targeting in cardiovascular drug delivery. J Control Release. 2002;78(1-3):235-247. pmid: 11772464

18.        Goodman SL, Cooper SL, Albrecht RM. Integrin receptors and platelet adhesion to synthetic surfaces. J Biomed Mater Res. 1993;27(5):683-695. doi: 10.1002/jbm.820270516 pmid: 8390998

19.        Bardania H, Shojaosadati SA, Kobarfard F, Dorkoosh F. Optimization of RGD-modified Nano-liposomes Encapsulating Eptifibatide. Iran J Biotechnol. 2016;14(2):33-40. doi: 10.15171/ijb.1399 pmid: 28959324

20.        Bardania H, Shojaosadati SA, Kobarfard F, Dorkoosh F, Zadeh ME, Naraki M, et al. Encapsulation of eptifibatide in RGD-modified nanoliposomes improves platelet aggregation inhibitory activity. J Thromb Thrombolysis. 2017;43(2):184-193. doi: 10.1007/s11239-016-1440-6 pmid: 27778144

21.        Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1-2):55-63. pmid: 6606682

22.        Krejsa CM, Schieven GL. Detection of oxidative stress in lymphocytes using dichlorodihydrofluorescein diacetate.  Stress Response: Springer; 2000. p. 35-47.

23.        Mohammad-Beigi H, Shojaosadati SA, Morshedi D, Arpanaei A, Marvian AT. Preparation and in vitro characterization of gallic acid-loaded human serum albumin nanoparticles. J Nanopart Res. 2015;17(4):1-16.

24.        Kuznetsova NR, Sevrin C, Lespineux D, Bovin NV, Vodovozova EL, Meszaros T, et al. Hemocompatibility of liposomes loaded with lipophilic prodrugs of methotrexate and melphalan in the lipid bilayer. J Control Release. 2012;160(2):394-400. doi: 10.1016/j.jconrel.2011.12.010 pmid: 22210161

25.        Bender EA, Adorne MD, Colome LM, Abdalla DSP, Guterres SS, Pohlmann AR. Hemocompatibility of poly(varepsilon-caprolactone) lipid-core nanocapsules stabilized with polysorbate 80-lecithin and uncoated or coated with chitosan. Int J Pharm. 2012;426(1-2):271-279. doi: 10.1016/j.ijpharm.2012.01.051 pmid: 22322210

26.        Smistad G, Jacobsen J, Sande SA. Multivariate toxicity screening of liposomal formulations on a human buccal cell line. Int J Pharm. 2007;330(1-2):14-22. doi: 10.1016/j.ijpharm.2006.08.044 pmid: 16997516

27.        Dokka S, Toledo D, Shi X, Castranova V, Rojanasakul Y. Oxygen radical-mediated pulmonary toxicity induced by some cationic liposomes. Pharm Res. 2000;17(5):521-525. pmid: 10888302

28.        Filion MC, Phillips NC. Toxicity and immunomodulatory activity of liposomal vectors formulated with cationic lipids toward immune effector cells. Biochim Biophys Acta. 1997;1329(2):345-356. pmid: 9371426

29.        Maurer-Jones MA, Bantz KC, Love SA, Marquis BJ, Haynes CL. Toxicity of therapeutic nanoparticles. Nanomedicine (Lond). 2009;4(2):219-241. doi: 10.2217/17435889.4.2.219 pmid: 19193187

30.        Cadenas E, Davies KJ. Mitochondrial free radical generation, oxidative stress, and aging. Free Radic Biol Med. 2000;29(3-4):222-230. pmid: 11035250

31.          Mayer A, Vadon M, Rinner B, Novak A, Wintersteiger R, Frohlich E. The role of nanoparticle size in hemocompatibility. Toxicology. 2009;258(2-3):139-147. doi: 10.1016/j.tox.2009.01.015 pmid: 19428933