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. 2019 Apr 20;17(2):e2008.
doi: 10.21859/ijb.2008. eCollection 2019 Apr.

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

Hassan Bardania  1 , Seyed Abbas Shojaosadati  2 , Farzad Kobarfard  3 , Dina Morshedi  4 , Farhang Aliakbari  4 , Mohammad Taher Tahoori  5 , Elahe Roshani  6
Affiliations

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

Hassan Bardania et al. Iran J Biotechnol. .

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: Cytotoxicity; Eptifibatide; Liposomes; Materials Testing.

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Figures

Figure 1.
Figure 1.
TEM images of phospholipid-based bilayer vesicles containing eptifibatide prepared by freeze/thawing method.
Figure 2.
Figure 2.
Cytotoxicity of nano-liposomes determined by MTT assay on HeLa cells after 24 h exposures. Data are expressed as percent of control mean ± SD of three independent experiments. *, *** denotes a statistically significant (P<0.05, P<0.001) difference from the untreated control.
Figure 3.
Figure 3.
Effects of liposomal samples on LDH release from (A) HUVEC and (B) HeLa cell lines during treatment for 24 h. Data are shown as mean ± SD (n=3) significantly (*P<0.05, ****P<0.0001) different relative to untreated control cells. (Lip=Liposome, Epf = eptifibatide, RGD=GRGDSPA ligand).
Figure 4.
Figure 4.
Quantification of oxidative stress in (A) HUVEC and (B) HeLa cells treated with different concentrations liposomal samples during a 24 h period. H2O2 was used as a positive control of oxidative stress and was correlated to 100 %. Data are shown as mean ± SD (n = 4) significantly (*P < 0.05, **P < 0.01) different relative to untreated control cells. (Lip = Liposome, Epf = Eptifibatide, RGD = GRGDSPA ligand).
Figure 5.
Figure 5.
Membrane integrity assessed by release of hemoglobin in whole blood samples treated with liposomal samples. Data are shown as mean ± SD (n=3) significantly (**** P<0.0001) different relative to untreated control (Lip=Liposome, Epf = Eptifibatide, RGD=GRGDSPA ligand, PBS= phosphate-buffered saline).
Figure 6.
Figure 6.
LDH release from red blood cells after liposomal treatment. Triton X-100 (2 % v/v) was considered as 100 % of cell damage. Data are shown as mean ± SD (n=3) significantly (*P<0.05, ****P<0.0001) (Lip=Liposome, Epf=Eptifibatide, RGD=GRGDSPA ligand).
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