Comparison of cytotoxicity of Miltefosine and its niosomal form on chick embryo model

Abstract

Various drugs have been used for the treatment of leishmaniasis, but they often have adverse effects on the body's organs. In this study, we aimed to explore the effects of one type of drug, Miltefosine (MIL), and its analogue or modifier, liposomal Miltefosine (NMIL), on several fetal organs using both in silico analysis and practical tests on chicken embryos. Our in silico approach involved predicting the affinities of MIL and NMIL to critical proteins involved in leishmaniasis, including Vascular Endothelial Growth Factor A (VEGF-A), the Kinase insert domain receptor (KDR1), and apoptotic-regulator proteins (Bcl-2-associate). We then validated and supported these predictions through in vivo investigations, analyzing gene expression and pathological changes in angiogenesis and apoptotic mediators in MIL- and NMIL-treated chicken embryos. The results showed that NMIL had a more effective action towards VEGF-A and KDR1 in leishmaniasis, making it a better candidate for potential operative treatment during pregnancy than MIL alone. In vivo, studies also showed that chicken embryos under MIL treatment displayed less vascular mass and more degenerative and apoptotic changes than those treated with NMIL. These results suggest that NMIL could be a better treatment option for leishmaniasis during pregnancy.

Figure 1

(I) Conformer of (a) MIL and (b) NMIL structures used in docking process. (II) FTIR spectra of (A) free Miltefosine, (B) niosomal Miltefosine (III) Gaussian size distribution for (A) unloaded niosome and (B) niosomal Miltefosine (IV) (A) SEM, and (B) TEM image of niosomal Miltefosine.

Figure 2

Diagram of UV–Vis absorption spectroscopy of (A) 2.5, 5, 10, 20, 40 ppm of MIL (MIL) (B) 30 ppm of NMIL. (C) Typical standardization curve of the MIL. (D) In vitro drug release profile of MIL.

Figure 3

Representation of the best score docking solution of the MIL ligands and (A) BAX and (C) Bcl-2 receptor and NMIL and (B) BAX and (D)Bcl-2 receptor with the designated crystal structure of 5W5X and 5JSN, respectively, along with the ligand map with various chemical bonds of Discovery Studio.

Figure 4

Representation of the best score docking solution of the MIL ligands and (A) Caspase-8 and (C) VEGF-A receptor and NMIL and (B) Caspase-8 and (D) VEGF-A receptor with the designated crystal structure of 1I4E and 5T89, respectively, along with the ligand map with various chemical bonds of Discovery Studio.

Figure 5

Effect of MIL and its NMIL within the blood vessel system. (A) Control embryo with characteristic blood vessel system. MIL (A) 1 × and (C) 2x, respectively, where the blood vessel system is interrupted. NMIL (D) 1X and (E) 2X, respectively, in contrast to MIL, which demonstrated a minor decrease in vascular mass. Vascular densities following treatments of MIL and its NMIL. A significant reduction in vascular density can be seen in both groups that received MIL and its NMIL. The embryos that received the MIL treatment revealed less vascular mass than NMIL.

Figure 6

MIL and NMIL induced apoptotic moderator and angiogenesis genes in the chick’s extra-embryonic membrane vasculature. The expression level of the apoptotic mediator (A) Bax, (B) Bcl2, (C) TP53, and (D) Apaf 1. The angiogenesis genes (E) VEGF and (F) KDR the MIL and its NMIL treated embryos compared to controls. The expression profiles were standardized to GAPDH and HPRT and calibrated to controls (error bars demonstration standard mean error; *p < 0.05).

Figure 7

Microscopy images of H&E staining of chick embryo (A) control group, MIL (B) 1X, (C) 2X, (D) NMIL 1X, and (E) 2X.

Figure 8

Microscopy images of IHC staining Bax (up) and Bcl 2 (down) of chick embryo (A) control group, MIL (B) 1X, (C) 2X, (D) NMIL 1X, and (E) 2X.

Figure 9

Microscopy images of IHC staining CD34 of chick embryo (A) control group, MIL (B) 1X, (C) 2X, (D) NMIL 1X, and (E) 2X.