Size- and charge-dependent non-specific uptake of PEGylated nanoparticles by macrophages

Abstract

The assessment of macrophage response to nanoparticles is a central component in the evaluation of new nanoparticle designs for future in vivo application. This work investigates which feature, nanoparticle size or charge, is more predictive of non-specific uptake of nanoparticles by macrophages. This was investigated by synthesizing a library of polymer-coated iron oxide micelles, spanning a range of 30-100 nm in diameter and -23 mV to +9 mV, and measuring internalization into macrophages in vitro. Nanoparticle size and charge both contributed towards non-specific uptake, but within the ranges investigated, size appears to be a more dominant predictor of uptake. Based on these results, a protease-responsive nanoparticle was synthesized, displaying a matrix metalloproteinase-9 (MMP-9)-cleavable polymeric corona. These nanoparticles are able to respond to MMP-9 activity through the shedding of 10-20 nm of hydrodynamic diameter. This MMP-9-triggered decrease in nanoparticle size also led to up to a six-fold decrease in nanoparticle internalization by macrophages and is observable by T(2)-weighted magnetic resonance imaging. These findings guide the design of imaging or therapeutic nanoparticles for in vivo targeting of macrophage activity in pathologic states.

Keywords: iron oxides; macrophage targeting; opsonization; poly(ethylene glycol) (PEG); poly(propylene sulfide) (PPS).

Figure 1

Characterization of USPIOs and PEG-PPS-USPIO micelles. HRTEM images of (A) 3 nm and (B) 12 nm hydrophobic, oleic acid-stabilized USPIO cores (γ-Fe₂O₃), which were synthesized via thermal decomposition. To render particles water-soluble, they were coated with PEG-PPS block copolymers via thin-film hydration to yield, respectively, (C) 30 nm and (D) 40 nm PEG-PPS-USPIO micelles. (E) 100 nm PEG-PPS-USPIO micelles can also be synthesized via direct hydration using the same feed materials used to create micelles in (D); this TEM image has been counterstained with 3% uranyl acetate. (F) Size-number distributions of these PEG-PPS-USPIO micelles were obtained by dynamic light scattering. (G) As shown in this representative photograph, 40 nm PEG-PPS-USPIOs remain stable in water and do not flocculate even after storage at room temperature over 4 months. Note: Scale bars: (A and C) 20 nm; (B and D) 100 nm; (E) 500 nm. Abbreviations: HRTEM, high resolution transmission electron microscope; PEG, poly(ethylene glycol); PPS, poly(propylene sulfide); TEM, transmission electron spectroscopy; USPIO, ultrasmall superparamagnetic iron oxides.

Figure 2

Dose- and size-dependent internalization of PEG-PPS-USPIOs by THP-1 macrophages. THP-1 cells were treated for up to 24 hours with standard growth serum medium supplemented with varying doses of PEG-PPS-USPIOs in PBS. As a negative control, PBS was used in place of the PEG-PPS-USPIO colloidal suspension. Iron internalization and initial doses were quantified using a colorimetric phenanthroline assay, and internalized iron content was normalized to cell number indirectly via a protein assay. (A) Internalization of nanoparticles over the time period of interest is described by first-order rate kinetics, indicating that initial dose of nanoparticles is the primary determinant of internalization rate and total internalization amount. Relative to the initial doses of USPIOs, macrophages receiving 30 μM, 60 μM, and 120 μM of iron endocytosed 8.4% ± 3.7%, 7.7% ± 3.2%, and 6.2% ± 0.9% of the maximum possible USPIOs, respectively. Error bars indicate standard deviations from six independent experiments. (B) Derivatives of the best-fit kinetic equations plotted in (A) demonstrate further the dependence of uptake rate on initial dose of PEG-PPS-USPIOs. (C) Of the three sizes investigated, 100 nm nanoparticles were most effectively internalized by the macrophages. Smaller nanoparticles were internalized less effectively, and 30 nm nanoparticles experienced almost negligible uptake levels over the 24 hour experimental period. Normalization of the 24 hour uptake amounts to the initially administered doses shows that macrophages internalized 6.2% ± 0.9%, 1.4% ± 2.3%, and 1.1% ± 0.3% of the 100 nm, 40 nm, and 30 nm PEG-PPS-USPIOs, respectively. Error bars represent standard deviation from three to six independent experiments. (D) Fluorescent imaging of the delivery of 40 nm and 100 nm fluorescent PEG-PPS-USPIO micelles. The uptake of 100 nm nanoparticles was easily visualized at 20× magnification with a 0.7 second exposure time, but even with a lower magnification and roughly a three-fold higher exposure time, the microscope was insufficiently sensitive to visualize the internalization of the 40 nm nanoparticles. (E) 40 nm PA-USPIOs (at 200 μM Fe) or 100 nm PEG-PPS-USPIOs (at 120 μM Fe) were co-administered to THP-1 macrophages with varying amounts of fucoidan for 24 hours, and allowed to incubate overnight prior to cell lysis and measurement of internalized iron. Increasing concentrations of fucoidan correlated with decreased uptake of the nanoparticles, suggesting that the mechanism of PEG-PPS-USPIO uptake is via receptor-mediated endocytosis, and facilitated by the scavenger receptor CD204. Error bars represent standard deviation of three independent experiments. Abbreviations: PA-USPIOs, proximity-activated ultrasmall superparamagnetic iron oxides; PBS, phosphate buffered saline; PEG, poly(ethylene glycol); PPS, poly(propylene sulfide); THP, human acute monocytic leukemia cell line.

Figure 3

Effects of nanoparticle surface charge and chemistry on macrophage uptake. Representative TEM images of (A) hydrophobic, unloaded 3 nm OA-USPIOs and (B) water-soluble Pluronic-PPS nanoparticles after loading with the OA-USPIOs. (C) The loading process does not significantly affect the hydrodynamic diameters or the ζ potentials inherent to the Pluronic-PPS nanoparticles. (D) ζ-potential of all nanoparticle formulations (color-coded by surface chemistry) was originally measured in PBS following synthesis, and again following incubation in 10% serum media. While modulation of surface chemistry allows for a wide range of ζ-potentials, this range is compressed due to interactions between nanoparticles and media components. Uptake of nanoparticles correlated with their surface charge as measured in media (inset; red dotted boxes indicate source of data for x-axis), according to a parabolic distribution. To account for differences in USPIO loading efficiency across the different Pluronic- PPS nanoparticle formulations, nanoparticle uptake was normalized to the initial dose administered as well as cell content indirectly, via a protein assay. Error bars indicate standard deviation for three independent experiments. (E) Cell internalization data is plotted versus nanoparticle ζ-potentials measured in 10% serum media (solid squares). In order to determine which nanoparticle feature may be more determinant of non-specific interactions with macrophages, the effects of nanoparticle diameter have also been plotted for comparison (open squares). Abbreviations: OA-USPIOs, oleic acid-stabilized ultrasmall superparamagnetic iron oxides; PBS, phosphate buffered saline; PPS, poly(propylene sulfide); TEM, transmission electron microscopy.

Figure 4

Behavior of MMP-9-responsive PA-USPIOs. (A) Synthesis of MMP-9-cleavable PEG-PPS chains (mPEG-[M9C]-PEG-PPS; PA) and (B) encapsulation of USPIOs to form PA-USPIOs. MMP-9 is able to recognize and cleave the (M9C) peptide sequence, resulting in release of a layer of PEG from the nanoparticle surface, accompanied by a decrease in nanoparticle hydrodynamic diameter. (C) DLS characterization of hydrodynamic diameters of as-synthesized 60 nm PA-USPIOs (C; green) and 30 nm PA-USPIOs (D; blue) demonstrates a loss in hydrodynamic diameter following treatment with MMP-9. (E) Buffer-treated or MMP-9-pretreated nanoparticles were delivered to THP-1 macrophages for 24 hours in standard growth medium. As a control, PBS was used in place of the nanoparticles. For both PA-USPIO formulations tested, the decrease in nanoparticle size following MMP-9 treatment results in less effective nanoparticle internalization by the macrophages. Error bars represent standard deviations from three to six independent experiments. (F) T₂-weighted MRI of THP-1 cells treated with MMP-9-cleaved PA-USPIOs appeared brighter than cells incubated with untreated PA-USPIOs, indicating that less cleaved nanoparticles were internalized by the macrophages versus the untreated PA-USPIOs. Note: *P < 0.05 by Student’s t-test. Abbreviations: DLS, dynamic light scattering; MMP-9, matrix metalloproteinase-9; PA-USPIOs, proximity-activated ultrasmall superparamagnetic iron oxides; MRI, magnetic resonance imagery; PBS, phosphate buffered saline; PEG, poly(ethylene glycol); PPS, poly(propylene sulfide).