Efficient targeting of HIF-1α mediated by YC-1 and PX-12 encapsulated niosomes: potential application in colon cancer therapy

Abstract

A number of molecular biofactors have been documented in pathogenesis and poor prognosis of colorectal cancer (CRC). Among them, the Hypoxia-Inducible Factor (HIF-1a) is frequently reported to become over-expressed, and its targeting could restrict and control a variety of essential hallmarks of CRC. Niosomes are innovative drug delivery vehicles with the encapsulating capacity for co-loading both hydrophilic and hydrophobic drugs at the same time. Also, they can enhance the local accumulation while minimizing the dose and side effects of drugs. YC-1 and PX-12 are two inhibitors of HIF-1a. The purpose of this work was to synthesize dual-loaded YC-1 and PX-12 niosomes to efficiently target HIF-1α in CRC, HT-29 cells. The niosomes were prepared by the thin-film hydration method, then the niosomal formulation of YC-1 and PX-12 (NIO/PX-YC) was developed and optimized by the central composition method (CCD) using the Box-Behnken design in terms of size, polydispersity index (PDI), entrapment efficiency (EE). Also, they are characterized by DLS, FESEM, and TEM microscopy, as well as FTIR spectroscopy. Additionally, entrapment efficiency, in vitro drug release kinetics, and stability were assessed. Cytotoxicity, apoptosis, and cell cycle studies were performed after the treatment of HT-29 cells with NIO/PX-YC. The expression of HIF-1αat both mRNA and protein levels were studied after NIO/PX-YC treatment. The prepared NIO/PX-YC showed a mean particle size of 185 nm with a zeta potential of about-7.10 mv and a spherical morphology. Also, PX-12 and YC-1 represented the entrapment efficiency of about %78 and %91, respectively, with a sustainable and controllable release. The greater effect of NIO/PX-YC than the free state of PX-YC on the cell survival rate, cell apoptosis, and HIF-1α gene/protein expression were detected (p < 0.05). In conclusion, dual loading of niosomes with YC-1 and PX-12 enhanced the effect of drugs on HIF-1α inhibition, thus boosting their anticancer effects.

Keywords: Colon cancer; Drug delivery; HIF-1α; Niosome; PX-12; Targeting; YC-1.

Fig. 1

CCD method for average diameter as a function of lipid content and surfactant to cholesterol molar ratio

Fig. 2

CCD method for PDI as a function of lipid content and surfactant to cholesterol molar ratio

Fig. 3

CCD method for A) EE_PX-12, B) EE_YC-1 as a function of lipid content and surfactant to cholesterol molar ratio

Fig. 4

A Schematic illustration for the preparation of optimized niosomes by thin-layer hydration method. B-C Hydrodynamic size of blank noisomeandNIO/PX-YC, respectively. D-E Zeta potential for blank noisome and NIO/PX-YC, respectively

Fig. 5

A FTIR spectra of NIO/PX-YC, blank noisome, YC-1, and PX-12. B FESEM micrograph of the niosomes. C-E TEM images representing the structure of niosomes with a corresponding size distribution histogram

Fig. 6

A In vitro release profile of PX-12 and YC-1 from the NIO/PX-YC nanocarriers along with their comparison with free drugs in pH 7.4 and 5.4 at 37 °C. B-E Stability of optimal NIO/PX-YC formulation stored during 1 and 2 months at 4 ± 2 °C and 25 ± 2 °C. The data are shown based on (mean ± SD). *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 7

A-C Cell viability of HT-29 cells being exposed to different concentrations of PX-12, YC-1, PX/YC, NIO/YC-PX, and blank niosome after 24 h, 48 h, and 72 h, respectively. D-E Cell viability of HFF cells being exposed to different concentrations of PX-12, YC-1, YC/PX, NIO/YC-PX, and blank noisome after 24 h, 48 h, and 72 h, respectively. All tests were repeated three times, and the data are shown based on (mean ± SD). *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 8

A Flowcytometric analysis of HT-29 cells after treatment with IC₅₀ concentration of PX-12, YC-1, YC/PX, NIO/YC-PX, and free noisome. Lower left panel (Q4): live cells, upper left panel (Q1): necrosis, lower right panel (Q3): early apoptosis, upper right panel (Q2): late apoptosis. B-C Early and late apoptosis and necrosis of HT-29 cells treated with pX-12, YC-1, YC/PX, NIO/YC-PX, and Blank niosome after 24 and 48 h, respectively. All tests were repeated three times and the data are shown based on (mean ± SD). *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 9

A The effect of free PX-12, free YC-1, YC/PX, NIO/YC-PX, and blank niosome on the cell cycle of the HT-29 cell line. B HIF-1α gene expression in HT-29 cells after treatment with PX-12, YC-1, YC/PX, NIO/YC-PX, and blank niosome at their IC₅₀ values. C The cellular protein levels of HIF-1α were checked after treatments of HT-29 cells with YC-1, PX-12, YC/PX, and NIO/YC-PX using western blotting (up). The bands were quantified by Image J (down). All tests were repeated three times and the data are shown based on (mean ± SD). *p < 0.05, **p < 0.01, and ***p < 0.001