Long-Circulation and Brain Targeted Isoliquiritigenin Micelle Nanoparticles: Formation, Characterization, Tissue Distribution, Pharmacokinetics and Effects for Ischemic Stroke

Abstract

Purpose: We designed a novel isoliquiritigenin (ISL) loaded micelle prepared with DSPE-PEG₂₀₀₀ as the drug carrier modified with the brain-targeting polypeptide angiopep-2 to improve the poor water solubility and low bioavailability of ISL for the treatment of acute ischemic stroke.

Methods: Thin film evaporation was used to synthesize the ISL micelles (ISL-M) modified with angiopep-2 as the brain targeted ligands. The morphology of the micelles was observed by the TEM. The particle size and zeta potential were measured via the nanometer particle size analyzer. The drug loading, encapsulation and in vitro release rates of micelles were detected by the HPLC. The UPLC-ESI-MS/MS methods were used to measure the ISL concentrations of ISL in plasma and main tissues after intravenous administration, and compared the pharmacokinetics and tissue distributions between ISL and ISL-M. In the MCAO mice model, the protective effects of ISL and ISL-M were confirmed via the behavioral and molecular biology experiments.

Results: The results showed that the drug loading of ISL-M was 7.63 ± 2.62%, the encapsulation efficiency was 68.17 ± 6.23%, the particle size was 40.87 ± 4.82 nm, and the zeta potential was -34.23 ± 3.35 mV. The in vitro release experiments showed that ISL-M had good sustained-release effect and pH sensitivity. Compared with ISL monomers, the ISL-M could significantly prolong the in vivo circulation time of ISL and enhance the accumulation in the brain tissues. The ISL-M could ameliorate the brain injury induced by the MCAO mice via inhibition of cellular autophagy and neuronal apoptosis. There were no the cellular structural damages and other adverse effects for ISL-M on the main tissues and organs.

Conclusion: The ISL-M could serve as a promising and ideal drug candidate for the clinical application of ISL in the treatment of acute ischemic stroke.

Keywords: MCAO; brain distribution; ischemic stroke; isoliquiritigenin; micelle; pharmacokinetics.

Scheme 1

Preparation of ISL micelles and its mechanism of LRP-1 receptor-mediated penetration of the blood-brain barrier.

Figure 1

Characterization of the DSPE-PEG₂₀₀₀-Ang. The UV (A) and FT-IR spectra (B) of angiopep-2, DSPE-PEG₂₀₀₀-Mal and DSPE-PEG₂₀₀₀-Ang; ¹H-NMR spectra of angiopep-2 (C1), DSPE-PEG₂₀₀₀-Mal (C2) and DSPE-PEG₂₀₀₀-Ang (C3).

Figure 2

The physicochemical properties of ISL-M. (A) Particle size distribution of ISL-M; (B) TEM image of ISL-M; (C) zeta potential of ISL-M; (D) in vitro release profile of ISL and ISL-M in PBS at pH = 7.4 and 5.

Figure 3

The mean plasma concentration-time curve of ISL and ISL-M after a single intravenous injection (2 mg/kg) to rats.

Figure 4

The tissue distribution of ISL in (A) heart, (B) liver, (C) lung, (D) kidney and (E) brain after single i.v. administration of ISL and ISL-M (2.8 mg/kg) to mice.

Figure 5

Neuroprotection by the treatment of ISL-M in MCAO-induced ischemic and reperfusion injury. Longa scores of mice in different groups on (A) the first and (B) seventh days after the operation. (C) The weight of mice in different groups in seven days. Behavioral manifestation of MCAO in mice in the rotarod experiments (D) and tape removal test (E and F).

Figure 6

Inhibitory effect on apoptosis and autophagy of ISL-M in MCAO mice model was proved by Nissl staining (A) and immunofluorescence staining of LC3 (B) and TUNEL (C). Record the number of cells in the same fields of view stained with LC3 (D) and TUNEL (E) by image J.

Figure 7

Hematoxylin-eosin staining of heart, liver, spleen, lung and kidney from control, MCAO, MCAO + ISL, MCAO + M, MCAO + ISL-M group of mice (Scale bar: 50 μm).