In vivo targeting of alveolar macrophages via RAFT-based glycopolymers

Abstract

Targeting cell populations via endogenous carbohydrate receptors is an appealing approach for drug delivery. However, to be effective, this strategy requires the production of high affinity carbohydrate ligands capable of engaging with specific cell-surface lectins. To develop materials that exhibit high affinity towards these receptors, we synthesized glycopolymers displaying pendent carbohydrate moieties from carbohydrate-functionalized monomer precursors via reversible addition-fragmentation chain transfer (RAFT) polymerization. These glycopolymers were fluorescently labeled and used to determine macrophage-specific targeting both in vitro and in vivo. Mannose- and N-acetylglucosamine-containing glycopolymers were shown to specifically target mouse bone marrow-derived macrophages (BMDMs) in vitro in a dose-dependent manner as compared to a galactose-containing glycopolymer (30- and 19-fold higher uptake, respectively). In addition, upon macrophage differentiation, the mannose glycopolymer exhibited enhanced uptake in M2-polarized macrophages, an anti-inflammatory macrophage phenotype prevalent in injured tissue. This carbohydrate-specific uptake was retained in vivo, as alveolar macrophages demonstrated 6-fold higher internalization of mannose glycopolymer, as compared to galactose, following intratracheal administration in mice. We have shown the successful synthesis of a class of functional RAFT glycopolymers capable of macrophage-type specific uptake both in vitro and in vivo, with significant implications for the design of future targeted drug delivery systems.

Fig. 1. In vitro BMDM glycopolymer uptake

Representative histograms (left) and fluorescence microscopy images (right) of BMDMs incubated in vitro with indicated AF488-glycopolymer (1.5 μM) for 4 hr. Black line in histograms represents no polymer control. Inset shows a higher magnification image of the AF488-pManEMA treated cells.

Fig. 2. Dose dependent internalization

BMDMs were incubated with AF488-glycopolymers for 15 min at 37°C. Data are reported as mean fluorescence intensity ± standard deviation from three independent experiments.

Fig. 3. Competitive glycopolymer uptake

BMDMs were preincubated with 15 μM unlabeled glycopolymer for 15 min followed by 1 μM AF488-glycopolymer for 30 min at 37°C. NC represents no competition. Data are rep orted as mean fluorescence intensity ± standard deviation from three independent experiments.

Fig. 4. Time course of mannose uptake in stimulated macrophages

AF488-pManEMA (1 μM) uptake by unstimulated (M0), LPS-stimulated (M1), or IL-4/IL-13 stimulated (M2) BMDM’s (n=3).

Fig. 5. Cell specificity of glycopolymer internalization

Internalization of AF488-glycopolymers (1.5 μM) by BMDMs, mouse lung epithelial cells (MLE-12), or primary mouse lung fibroblasts (MLF) after 2h (A) or 24h (B) incubation.

Fig. 6. Internalization of glycopolymers by alveolar macrophages in vivo

Mice were given 10 μM AF488-glycopolymers intratracheally. Bronchoalveolar lavage (BAL) was performed after 30 min and BAL cells were analyzed by flow cytometry. Data are reported as mean fluorescence intensity ± standard deviation from three independent experiments. *p<0.01 compared to no polymer.

Scheme 1

RAFT-mediated glycopolymer synthesis and subsequent fluorophore conjugation and proposed mechanism of MRC-1-mediated macrophage uptake of the glycopolymers.