Uptake of Cyclodextrin Nanoparticles by Macrophages is Dependent on Particle Size and Receptor-Mediated Interactions

Abstract

Physiochemical properties of nanoparticles, such as their size and chemical composition, dictate their interaction with professional phagocytes of the innate immune system. Macrophages, in particular, are key regulators of the immune microenvironment that heavily influence particle biodistribution as a result of their uptake. This attribute enables macrophage-targeted delivery, including for phenotypic modulation. Saccharide-based materials, including polyglucose polymers and nanoparticles, are efficient vehicles for macrophage-targeted delivery. Here, we investigate the influence of particle size on cyclodextrin nanoparticle (CDNP) uptake by macrophages and further examine the receptor-mediated interactions that drive macrophage-targeted delivery. We designed and synthesized CDNPs ranging in size from 25 nm to >100 nm in diameter. Increasing particle size was correlated with greater uptake by macrophages in vitro. Both scavenger receptor A1 and mannose receptor were critical mediators of macrophage-targeted delivery, inhibition of which reduced the extent of uptake. Finally, we investigated the cellular bioavailability of drug-loaded CDNPs using a model anti-inflammatory drug, celastrol, which demonstrated that drug bioactivity is improved by CDNP loading relative to free drug alone. This study thus elucidates the interactions between the polyglucose nanoparticles and macrophages, thereby facilitating their application in macrophage-targeted drug delivery that has applications in the context of tissue injury and repair.

Keywords: Nanomedicine; cyclodextrin; macrophage; nanoparticle; receptor-mediated uptake.

Figure 1

CDNP synthesis and characterization. (a) Schematic of cyclodextrin nanoparticle (CDNP) preparation via EDC/NHS-catalyzed cross-linking of succinyl-β-cyclodextrin by l-lysine and subsequent fluorescence derivatization by reaction with the succinimidyl ester of Alexa Fluor 555. (b) The dependence of CDNP on the molar feed ratio of EDC to succinyl groups (3.3% w/v cyclodextrin, 0.5:1 l-lysine), n = 3. (c) Absorbance (solid line) and emission (dashed line) spectra of CDNP-AF555 in PBS.

Figure 2

Time-course imaging of CDNP uptake in RAW264.7 macrophages. (a) Representative fluorescence images of CDNP-AF555 (12.5:1 equiv of EDC) uptake at 0, 6, and 24 h. Staining: DAPI (nuclei, blue), WGA-AF488 (cell membrane, green), and CDNP-AF555 (nanoparticle, red). Scale bars: 50 μm, primary; 10 μm, inset. (b) Quantification of CDNP-AF555 uptake over time and dependence on varying CDNP diameter over 24 h normalized to untreated controls; mean ± SEM, n > 100 cells. (c) Integrated density at 24 h normalized to untreated cell controls; mean ± SD, n > 100 cells; ****P < 0.0001; ANOVA, Tukey’s HSD.

Figure 3

Flow cytometry of CDNP uptake by RAW264.7 macrophages. (a) Representative histograms and (b) corresponding quantification of CDNP-AF555 mean fluorescence intensity (MFI) for M0, M1, and M2 polarized cells with and without CDNP treatment. (c) Quantified frequency of CDNP⁺ cells. Data represents the mean ± SD, n = 3; ****P < 0.0001; ANOVA, Tukey’s HSD.

Figure 4

Flow cytometry of receptor expression and CDNP uptake in RAW264.7 macrophages. (a) Representative flow cytometry plots for RAW264.7 macrophages with or without CDNP-AF555 treatment. Gating represents regions of CDNP⁺ uptake coincident with CD204^hi (left) or CD206^hi (right) expression. Inset values represent percentage of the live population that is (b) CDNP⁺CD204^hi or (c) CDNP⁺CD206^hi. Data represents the mean ± SD, n = 3; ****P < 0.0001; ANOVA, Tukey’s HSD.

Figure 5

Cell imaging for CDNP uptake inhibition. (a) Representative fluorescence images of CDNP-AF555 uptake in RAW264.7 macrophages at 24 h after indicated antibody blocking (MRC-1, SR-A1) or CytoD treatment. Staining: DAPI (nuclei, blue), WGA-AF488 (cell membrane, green), and CDNP-AF555 (nanoparticle, red). Scale bar: 50 μm. (b) Corresponding quantification of CDNP-AF555 uptake normalized to CDNP-AF555 treated controls; mean ± SEM, n > 100; **P < 0.01, ****P < 0.0001; ANOVA, Tukey’s HSD.

Figure 6

Bioavailability of celastrol-loaded CDNP (Cel-CDNP). (a) Anti-inflammatory activity of Cel-CDNP treatment on RAW-Blue cells assessed 24 h after concurrent drug treatment and zymosan stimulation; mean ± SD, n = 3. (b) Anti-inflammatory activity of unloaded CDNP vehicle, free celastrol (1 μM), and Cel-CDNP of varying sizes; mean ± SD, n = 3; −P < 0.05 relative to zymosan (positive) control; *P < 0.05 relative to free celastrol treatment; ANOVA, Tukey’s HSD.