Sterically stabilized recombined HDL composed of modified apolipoprotein A-I for efficient targeting toward glioma cells

Abstract

Reconstituted high density lipoprotein (rHDL) has been regarded as a promising brain-targeting vehicle for anti-glioma drugs under the mediation of apolipoprotein A-I (apoA-I). However, some stability issues relating to drug leakage and consequent reduced targeting efficiency in the course of discoidal rHDL (d-rHDL) circulating in blood hinder its broad application. The objective of the study was to develop a novel stabilized d-rHDL by replacing cholesterol and apoA-I with mono-cholesterol glutarate (MCG) modified apoA-I (termed as mA) and to evaluate its allosteric behavior and glioma targeting. MCG was synthesized through esterifying the hydroxyl of cholesterol with glutaric anhydride and characterized by FI-IR and ¹H NMR. d-rHDL assembled with mA (termed as m-d-rHDL) presented similar properties such as minute particle size and disk-like appearance resembling nascent HDL. Morphological transformation observation and in vitro release plots convinced that the modification of cholesterol could effectively inhibit the remolding of d-rHDL. The uptake of m-d-rHDL by LCAT-pretreated bEND.3 cells was significantly higher than that of d-rHDL, thereby serving as another proof for the capability of m-d-rHDL in enhancing targeting property. Besides, apoA-I anchoring into m-d-rHDL played a critical role in the endocytosis process into bEND.3 cells and C6 cells, which implied the possibility of traversing blood brain barrier and accumulating in the brain and glioma. These results suggested that the modification toward cholesterol to improve the stability of d-rHDL is advantageous, and that this obtained m-d-rHDL revealed great potential for realization of suppressing the remolding of d-rHDL in the brain-targeted treatment of glioma for drug delivery.

Keywords: BBB; LCAT; allosteric; cholesterol modification; glioma; rHDL.

Figure 1.

Graphical synthetic route of mono-cholesteryl glutarate (MCG).

Figure 2.

SDS-PAGE (left) and Western blot (right) patterns of samples. (A) Marker; (B) FIV precipitate; (C, D) extracted products; (E) standardized apoA-I.

Figure 3.

(A) Infrared spectra of cholesterol and MCG, (B) ¹H NMR of cholesterol (a: the characteristic chemical shift of 3′-methine of cholesterol), (C) ¹H NMR of MCG (b: the characteristic chemical shifts of methylene of MCG; c: the characteristic chemical shift of 3′-methine of MCG).

Figure 4.

SDS-PAGE diagram of marker (A), unmodified apoA-I (B), and mA (C).

Figure 5.

The surface tension of natural apoA-I and mA with concentrations ranging from 0.02 to 0.5 mg/mL (n = 3).

Figure 6.

Transmission electron microscope of lipid core (A) and m-d-rHDL (B). The bar of A was 100 nm. The bar of B was 20 nm.

Figure 7.

The particle size and PDI changes of d-rHDL and m-d-rHDL in PBS 7.4 containing 10% FBS.

Figure 8.

Microphotographs of different preparations using transmission electron microscope. (A) d-rHDL without LCAT; (B) d-rHDL incubation with LCAT; (C) m-d-rHDL without LCAT; (D) m-d-rHDL incubation with LCAT. The bar of A was 50 nm. The bar of B was 100 nm. The bar of C and D was 20 nm.

Figure 9.

In vitro release profiles of coumarin 6 from d-rHDL and m-d-rHDL with or without LCAT under PBS 7.4 containing 10% FBS at 37 °C (n = 3).

Figure 10.

In vitro cytotoxicity of lipid core, d-rHDL and m-d-rHDL on bEND.3 cells (A) and C6 cells (B) at 10, 500, and 1000 μg/mL concentrations.

Figure 11.

Fluorescence images of bEND.3 cells after incubation with lipid core (A–C), d-rHDL (D–F), and m-d-rHDL (G–I) at 37 °C for 1 h, 2 h, and 4 h, respectively.

Figure 12.

Fluorescence images of C6 cells after incubation with lipid core (A–C) and m-d-rHDL (D–F) at 37 °C for 1 h, 2 h, and 4 h, respectively.

Figure 13.

Uptake of lipid core, d-rHDL and m-d-rHDL in bEND.3 cells (A) and C6 cells (B) observed qualitatively by a multi-mode microplate reader (n = 5), *p<.05, **p<.01.