Effects of cholesterol incorporation on the physicochemical, colloidal, and biological characteristics of pH-sensitive AB₂ miktoarm polymer-based polymersomes

Abstract

In our previous study, a histidine-based AB2 miktoarm polymer, methoxy poly(ethylene glycol)-b-poly(l-histidine)2 (mPEG-b-(PolyHis)2), was designed to construct pH-sensitive polymersomes that transform in acidic pH; the polymer self-assembles into a structure that mimics phospholipids. In this study, the polymersomes further imitated liposomes due to the incorporation of cholesterol (CL). The hydrodynamic radii of the polymersomes increased with increasing CLwt% (e.g., 70 nm for 0 wt% vs. 91 nm for 1 wt%), resulting in an increased capacity for encapsulating hydrophilic drugs (e.g., 0.92 μL/mg for 0 wt% vs. 1.42 μL/mg for 1 wt%). The CL incorporation enhanced the colloidal stability of the polymersomes in the presence of serum protein and retarded their payload release. However, CL-incorporating polymersomes still demonstrated accelerated release of a hydrophilic dye (e.g., 5(6)-carboxyfluorescein (CF)) below pH 6.8 without losing their desirable pH sensitivity. CF-loaded CL-incorporating polymersomes showed better cellular internalization than the hydrophilic CF, whereas doxorubicin (DOX)-loaded CL-incorporating polymersomes presented similar or somewhat lower anti-tumor effects than free hydrophobic DOX. The findings suggest that CL-incorporating mPEG-b-(PolyHis)2-based polymersomes may have potential for intracellular drug delivery of chemical drugs due to their improved colloidal stability, lower drug loss during circulation, acidic pH-induced drug release, and endosomal disruption.

Keywords: Cholesterol; Colloidal stability; Miktoarm polymer; Poly(histidine); Polymersome; pH-sensitive.

Fig. 1

Schematic illustration of a unilamellar vesicle (CL-Polymersome) formed by the AB₂ 3-miktoarm star polymer (one mPEG block (2 kDa) and two polyHis (2.9 kDa) blocks) and cholesterol (CL) and its TEM image. The degree of polymerization (DP) for ethylene glycol (k) and histidine (l) in each arm and the weight fraction (f) of PEG are indicated.

Fig. 2

Angle-dependent relaxation times of CL-Polymersome solutions (0.3 mg/mL, pH 9.0) measured by DLS. D₀ is the intercept of the regression line (dash line) against the D_app-axis, and R_h is calculated based on D₀. The CL_0wt%-Polymersome is the CL-free polymersome, and its plot used as a control was taken from Yin et al., J. Mater. Chem. 22 (2012) 19168–19178.

Fig. 3

Partial Zimm plots of CL-Polymersome solutions (0.3 mg/mL, pH 9.0) measured by SLS. R_g is calculated from the slope and 1/I_ex-intercept of the regression line (dash line). The CL_0wt%-Polymersome is the CL-free polymersome, and its plot, used as a control was taken from Yin et al., J. Mater. Chem. 22 (2012) 19168–19178.

Fig. 4

(a) Time-dependent R_h,app distribution and (b) time-dependent colloidal stability of the CL_0wt%-Polymersome and the CL_1wt%-Polymersome in the presence of BSA (1 mg/mL). The colloidal stability of CL-Polymersome (0.1 mg/mL) was determined by subtracting the portion of BSA from the total scattered light intensity of the mixtures (CL-Polymersome and BSA) at 90°.

Fig. 5

In vitro pH-dependent 5(6)-carboxyfluorescein (CF) release profiles of the CF-loaded CL_1wt%-Polymersome: (a) 72 hr release profile and (b) the comparison of CF release between the CF-loaded CL_1wt%-Polymersome and the CF-loaded CL_0wt%-Polymersome at pH 6.8 and pH 6.0. The CF release data of the CF-loaded CL_0wt%-Polymersome, used as the control, was taken from Yin et al., J. Mater. Chem. 22 (2012) 19168–19178. (Mean ± standard deviation; n=3)

Fig. 6

Cellular uptake of CF-loaded CL-Polymersome in A2780 cells cultured at pH 7.4: (a) FACS histograms and (b) confocal microscopy images. The data were obtained at 4 hr post-incubation.

Fig. 7

In vitro anti-tumor effect of the DOX-loaded CL-Polymersome in (a) MCF7/ADR-RES and (b) A2780/ADR cells. Cells were incubated for 5 days following treatment with the DOX-loaded CL-Polymersome or DOX. (Mean ± standard error; n=6)