Mannosylated Polyrotaxanes for Increasing Cellular Uptake Efficiency in Macrophages through Receptor-Mediated Endocytosis

Abstract

Macrophages play an important role in the regulation of inflammation and immune response as well as the pathogenesis of chronic inflammatory diseases and cancer. Therefore, targeted delivery of therapeutic reagents to macrophages is an effective method for treatment and diagnosis. We previously examined the therapeutic applications of polyrotaxanes (PRXs) comprised of multiple cyclodextrins (CDs) threaded on a polymer chain and capped with bulky stopper molecules. In the present study, we designed an α-d-mannose-modified α-CD/poly(ethylene glycol)-based PRX (Man-PRX). The intracellular uptake of Man-PRX through the interaction with macrophage mannose receptor (MMR) in macrophage-like RAW264.7 cells was examined. Intracellular Man-PRX uptake was observed in MMR-positive RAW264.7 cells but was negligible in MMR-negative NIH/3T3 cells. In addition, the intracellular Man-PRX uptake in RAW264.7 cells was significantly inhibited in the presence of free α-d-mannose and an anti-MMR antibody, which suggests that MMR is involved in the intracellular uptake of Man-PRX. Moreover, the polarization of RAW264.7 cells affected the Man-PRX internalization efficiency. These results indicate that Man-PRX is an effective candidate for selective targeting of macrophages through a specific interaction with the MMR.

Keywords: cyclodextrin; macrophage; macrophage mannose receptor; mannose; polyrotaxane.

Figure 1

(A) Scheme for synthesizing α-d-mannose-modified α-CD/PEG-based PRX (Man-PRX) and (B) chemical structure of 2-(2-hydroxyethoxy)ethyl carbamate-modified PRX (HEE-PRX), where n and m denote the polymerization degree of the PEG axle and the number of threaded α-CDs, respectively.

Figure 2

(A) SEC charts of Man-NH₂, α-CD, axle PEG (biscarboxy-PEG, M_n: 9800), unmodified PRX, and Man-PRX in DMSO containing 10 mM LiBr at 60 °C. (B) FT-IR spectra of Man-NH₂, α-CD, axle PEG, unmodified PRX, and Man-PRX.

Figure 3

(A) ¹H NMR and (B) ¹³C NMR spectra of Man-NH₂, unmodified PRX, and Man-PRX in DMSO-d₆ at 25 °C.

Figure 4

(A) SPR sensor grams of Man-PRX (5 mg/mL) and HEE-PRX (5 mg/mL) on the mouse MMR-immobilized sensor chip surfaces. (B) Relationship between PRX concentration and the SPR response. The circles and squares in the plot indicate Man-PRX and HEE-PRX, respectively.

Figure 5

Fluorescence intensities of (A) NIH/3T3 and (B) RAW264.7 cells treated with the APC-labeled IgG2a,κ isotype control and APC-anti-MMR antibody. The time course of fluorescence intensity of (C) NIH/3T3 and (D) RAW264.7 cells treated with BODIPY-HEE-PRX (10 μM, open circles) and BODIPY-Man-PRX (10 μM, closed squares). Data are expressed as mean ± standard deviation (n = 3, **** p < 0.001).

Figure 6

CLSM images of RAW264.7 cells treated with BODIPY-HEE-PRX (10 μM, green, first row) and BODIPY-Man-PRX (10 μM, green, first row) for 3 h and 24 h (scale bars: 20 μm). The cells were stained with Hoechst 33,342 (blue) and LysoTracker Red (red, second row) to visualize the cell nuclei and lysosomes, respectively. The third row depicts overlay images.

Figure 7

Fluorescence intensity of RAW 264.7 cells pretreated with various concentrations of (A) α-d-mannose and (B) anti-MMR antibody for 3 h, followed by treatment with BODIPY-Man-PRX for 3 h. The concentration of BODIPY-Man-PRX was 10 μM, which corresponded to 565 μM α-d-mannose molecules modified on PRX. Data are expressed as mean ± standard deviation (n = 3, *** p < 0.005 and **** p < 0.001).

Figure 8

(A) mRNA expression levels of TNF-α, IL-6, and Arg-1 in RAW 264.7 cells polarized with LPS (100 ng/mL) + IFN-γ (100 ng/mL) (M1 polarization), or IL-4 (400 ng/mL) (M2 polarization), for 24 h. The phenotype of untreated RAW264.7 cells was denoted as M0. The expression levels of each gene were normalized by the expression level of the β-actin gene (n = 4). (B,C) The fluorescence intensity of RAW264.7 cells polarized with LPS + IFN-γ or IL-4 for 24 h, followed by treatment with the APC-labeled IgG2a,κ isotype control and APC-anti-MMR antibody (n = 3). (D) The fluorescence intensity of RAW264.7 cells polarized with LPS + IFN-γ or IL-4 for 24 h was followed by treatment with BODIPY-HEE-PRX (10 μM) and BODIPY-Man-PRX (10 μM) for 3 h. Data are expressed as mean ± standard deviation (n = 3, * p < 0.05, *** p < 0.005, and **** p < 0.001. N.S. indicates no significance).