Morin-VitaminE-β-CyclodextrinInclusionComplexLoadedChitosanNanoparticles (M-Vit.E-CD-CSNPs) Ameliorate Arsenic-Induced Hepatotoxicityina Murine Model

Abstract

The special features of cyclodextrins (CDs), hydrophilic outer surfaces and hydrophobic inner surfaces, allow for development of inclusion complexes. The two bioactive strong antioxidant hepatoprotective compounds, Morin and vitamin E, are water insoluble. The present study aimed to prepare Morin-vitamin E-β-cyclodextrin inclusion complex loaded chitosan nanoparticles (M-Vit.E-CD-CS NPs) and to examine their hepatoprotective efficacy against arsenic-induced toxicity in a murine model. The NPs were characterized by FTIR, DLS, NMR, DSC, XRD, AFM, and a TEM study. The NPs were spherical in shape, 178 ± 1.5 nm in size with a polydispersity index (PDI) value of 0.18 and a zeta potential value of −22.4 ± 0.31 mV, with >50% encapsulation and drug loading efficacy. Mice were exposed to arsenic via drinking water, followed by treatment without or with the NPs on every alternate day up to 30 days by oral gavaging. Administration of NPs inhibited the arsenic-induced elevation of liver function markers, inflammatory and proapoptotic factors, reactive oxygen species (ROS) production, alteration in the level of blood parameters and antioxidant factors, and liver damage, which was measured by different biochemical assays, ELISA, Western blot, and histological study. Organ distribution of nanoparticles was measured by HPLC. M-Vit.E-CD-CS NPs showing potent hepatoprotective activity may be therapeutically beneficial.

Keywords: Morin; apoptosis; chitosan; hepatoprotection; inflammation; nanoformulation; oxidative stress; vitamin E; β-cyclodextrin.

Figure 1

Characterization of Morin, vitamin E, and β-CD inclusion complex loaded chitosan nanoparticles. (A) Fourier transform infrared spectroscopy (FTIR) spectra of Morin, vitamin E, β-CD, chitosan, and M-Vit.E-CD-CS NPs. (B) Particle size distribution from differential light scattering (DLS) with M-Vit.E-CD-CS NPs. (C) TEM images of M-Vit.E-CD-CS NPs. (D) M-Vit.E-CD-CS NPs particle surface topology determination using atomic force microscopy (AFM). The acquired images were analyzed using scanning probe microscopy (SPM) tools for laboratory study. (E) The encapsulation efficiency percentage and the drug-loading percentage of M-Vit.E-CD-CS NPs. (F) The percentage of release of Morin from M-Vit.E-CD-CS NPs over a time period of 0–72 h. Result is the mean ± standard deviation (SD) from triplicate independent experiments.

Figure 2

(A) 1HNMR spectra of MOR, Vitamin E, β-CD, chitosan, and M-Vit.E-CD-CS NPs. (B,C) Thermogravimetric analysis (TGA) was performed using a TGA Q500 system from TA Instruments Inc. under a N2 atmosphere from 0–500 °C at a heating rate of 5 °C/min. (D) Differential Scanning Calorimetry (DSC) was performed using a DSC Q200 RCS system from TA Instruments Inc. (New Castle, DE, USA), with a refrigerated cooling system. The sample was heated with a constant ramp rate of 10 °C/min between −30 °C and 90 °C. (E) XRD: X-Ray Diffraction.

Figure 3

Effect of MOR, vitamin E, and M-Vit.E-CD-CS NPs on arsenic treated HepG2 cells. (A) % cell survivability was measured by MTT assay. (B) ROS level was detected fluorometrically. (C) Cell morphology was analyzed, using nuclear staining DAPI and bright field (Scale bar 10 μM).

Figure 4

Effect of MOR, vitamin E, and M-Vit.E-CD-CS NPs on arsenic-induced elevation of (A) ALT, (B) AST, (C) ROS generation, and (D) MDA level. MOR, vitamin E, and M-Vit.E-CD-CS NPs level with the duration of its exposure. Indicated doses of MOR, vitamin E, and M-Vit.E-CD-CS NPs were treated during arsenic exposure. Data is one of the three representative experiments ± SD (ns—nonsignificant,* p, 0.1, ** p, 0.01, *** p, 0.001, **** p, 0.0001).

Figure 5

Effect of MOR, vitamin E, and M-Vit.E-CD-CS NPs on antioxidant factors. M-Vit.E-CD-CS NPs (20mg/kg bwt), vitamin E (50 mg/kg bwt), and MOR (200 mg/kg bwt) were orally treated in mice during their exposure to arsenic. The level of (A) SOD (B) catalase (C) GSH in the liver tissue lysate of arsenic was measured by using assay kits (ns—nonsignificant, ** p, 0.01, *** p, 0.001, **** p, 0.0001). (D) the effect of MOR, vitamin E, and M-Vit.E-CD-CS NPs on protein expression of cytosolic Nrf2, nuclear Nrf2, GPx, HO-1, and NQO1 (western blot analysis) in the liver tissue lysate. (E) Relative change of protein expression of the respective protein with respect to β-actin (** p, 0.01, *** p, 0.001, **** p, 0.0001).

Figure 6

Effect of MOR, vitamin E, and M-Vit.E-CD-CS NPs on liver tissue apoptosis. (A) The level of active caspase-3, active caspase-9, and cytosolic cytochrome C obtained using respective colorimetric assay kits (*** p, 0.001, **** p, 0.0001). (B,C) Level of DNA fragmentation obtained using DNA fragmentation kit (ns—nonsignificant, * p, 0.1, ** p, 0.01, *** p, 0.001, **** p, 0.0001). (D) the effect of MOR, vitamin E, and M-Vit.E-CD-CS NPs on protein expression (western blot analysis) of Bcl-2, Bax, Bad, p53, Apaf-1, and PUMA in mice exposed to arsenic. (E) Relative change of protein expression of the respective protein with respect to β-actin (**** p, 0.0001).

Figure 7

Effect of MOR, vitamin E, and M-Vit.E-CD-CS NPs on the level of (A) TNF-α, IL-β, IL-6, and TGF-β in the liver tissue lysate of arsenic challenged mice as seen in ELISA analysis. Data are one of the three representative experiments ± SD (**** p, 0.0001). (B) the effect of MOR, vitamin E, and M-Vit.E-CD-CS NPs on protein expression of nuclear NF-kBp65, NLRP3, Caspase-1, and IL-18 (western blot analysis). (C) Relative change of protein expression of the respective protein with respect to β-actin (** p, 0.01, *** p, 0.001, **** p, 0.0001).

Figure 8

(A) Effect of MOR, vitamin E, and M-Vit.E-CD-CS NPs on liver tissue histology architecture of liver tissue section, following treatment without or with MCNPs and MOR in arsenic-exposed mice (Scale bar 100 microns). (B) Histopathological scoring of the liver tissue (*** p, 0.001, **** p, 0.0001). (C) Tissue distribution studies of M-Vit.E-CD-CS NPs in various organs. Mean concentration of Morin in liver, lungs, kidneys, spleen, and serum at 2, 6, 12, 24, 48, and 72 h after oral administration of single dose of M-Vit.E-CD-CS NPs (n = 3, mean ± SD) in mice.