The Development of Magnolol-Loaded Intravenous Emulsion with Low Hepatotoxic Potential

Abstract

Intestinal failure-associated liver disease (IFALD) is a severe liver injury occurring due to factors related to intestinal failure and parenteral nutrition administration. Different approaches are studied to reduce the risk or ameliorate the course of IFALD, including providing omega-3 fatty acids instead of soybean oil-based lipid emulsion or administering active compounds that exert a hepatoprotective effect. This study aimed to develop, optimize, and characterize magnolol-loaded intravenous lipid emulsion for parenteral nutrition. The preformulation studies allowed for chosen oils mixture of the highest capacity of magnolol solubilization. Then, magnolol-loaded SMOFlipid was developed using the passive incorporation method. The Box-Behnken design and response surface methodology were used to optimize the entrapment efficiency. The optimal formulation was subjected to short-term stress tests, and its effect on normal human liver cells and erythrocytes was determined using the MTT and hemolysis tests, respectively. The optimized magnolol-loaded SMOFlipid was characterized by the mean droplet diameter of 327.6 ± 2.9 nm with a polydispersity index of 0.12 ± 0.02 and zeta potential of -32.8 ± 1.2 mV. The entrapment efficiency of magnolol was above 98%, and pH and osmolality were sufficient for intravenous administration. The magnolol-loaded SMOFlipid samples showed a significantly lower toxic effect than bare SMOFlipid in the same concentration on THLE-2 cells, and revealed an acceptable hemolytic effect of 8.3%. The developed formulation was characterized by satisfactory stability. The in vitro studies showed the reduced cytotoxic effect of MAG-SMOF applied in high concentrations compared to bare SMOFlipid and the non-hemolytic effect on human blood cells. The magnolol-loaded SMOFlipid is promising for further development of hepatoprotective lipid emulsion for parenteral nutrition.

Keywords: Box–Behnken design; liver disease; magnolol; parenteral nutrition.

Figure 1

The mechanism of hepatoprotective action of magnolol. PI3K—phosphoinositide-3-kinase; AKT—AKT kinase; Nrf2—nuclear factor erythroid 2-related factor 2; Keap1—kelch-like ECH associated protein 1; PPARγ—peroxisome proliferator-activated receptor gamma; ARE—antioxidant response element; SOD2—superoxide dismutase 2; HO-1—heme oxygenase 1; GSH-Px—glutathione peroxidase; MAPK—mitogen-activated protein kinase; NF-κB—nuclear factor kappa B; TNF-α—tumor necrosis factor alpha; iNOS—inducible nitric oxide synthase; IL-6—interleukin-6; COX-2—cyclo-oxygenase-2; NLRP3—nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3; proIL-1β—prointerleukin-1β; proIL-18—prointerleukin-18; IL-1β—interleukin-1β; IL-18—interleukin-18; CYP2E1—cytochrome P450 2E1; Hsl—hormone-sensitive lipase; Mgl—monoacylglycerol lipase; Atgl—adipose triglyceride lipase; LXR—liver X receptor; LXRE—LXR response element; SREBP-1c—sterol-regulatory element binding protein-1c; ACC—acetyl-CoA carboxylase; FAS—fatty acid synthase; AMPK—adenosine monophosphate (AMP)-activated protein kinase; PPARα—peroxisome proliferator-activated receptor alpha (based on [9,10,12]).

Figure 2

Solubility of magnolol in oils mixtures.

Figure 3

Pareto chart for entrapment efficiency of magnolol in MAG-SMOF formulations.

Figure 4

Response surface plots present the interaction effect of (1) time of shaking (B) and shaking speed (A), (2) concentration of magnolol (C) and shaking speed (A), and (3) concentration of magnolol (C) and time of shaking (B) entrapment efficiency.

Figure 4

Response surface plots present the interaction effect of (1) time of shaking (B) and shaking speed (A), (2) concentration of magnolol (C) and shaking speed (A), and (3) concentration of magnolol (C) and time of shaking (B) entrapment efficiency.

Figure 5

Results of MDD and PDI of MAG-SMOF immediately after preparation (t = 0 h) and after 50 days of storage (t = 50 days) at 4 ± 1 °C without light access. The left axis corresponds to MDD, and the right one to PDI.

Figure 6

Results of ZP of MAG-SMOF immediately after preparation (t = 0 h) and after 50 days of storage (t = 50 days) at 4 ± 1 °C without light access.

Figure 7

Results of MAG-SMOF (A) and MAG_aq (B) recovery during storage for 7 days exposed to various stress factors.

Figure 8

Results of MDD of magnolol-loaded SMOFlipid (MAG-SMOF) and bare SMOFlipid (SMOF) during storage for 7 days exposed to various stress factors. The left axis corresponds to MDD and the right one to PDI.

Figure 9

The effect of the magnolol, SMOFlipid, and MAG-SMOF on the viability of THLE-2 cells after 24 h incubation. The cell viability was measured by MTT assay. A cell culture medium was used as a control. Data are expressed as the mean ± SEM from three separate experiments. Dunnett’s multiple comparison test assessed statistical significance between groups (* p < 0.05 as the comparison between SMOFlipid and MAG-SMOF; # p < 0.05 as the comparison between SMOFlipid and magnolol).