Nanomaterial Interactions with Human Neutrophils

Abstract

Neutrophils are the most abundant circulating leukocyte and the first point of contact between many drug delivery formulations and human cells. Despite their prevalence and implication in a range of immune functions, little is known about how human neutrophils respond to synthetic particulates. Here, we describe how ex vivo human neutrophils respond to particles which vary in both size (5 nm to 2 μm) and chemistry (lipids, poly(styrene), poly(lactic-co-glycolic acid), and gold). In particular, we show that (i) particle uptake is rapid, typically plateauing within 15 min; (ii) for a given particle chemistry, neutrophils preferentially take up larger particles at the nanoscale, up to 200 nm in size; (iii) uptake of nanoscale poly(styrene) and liposomal particles at concentrations of up to 5 μg/mL does not enhance apoptosis, activation, or cell death; (iv) particle-laden neutrophils retain the ability to degranulate normally in response to chemical stimulation; and (v) ingested particles reside in intracellular compartments that are retained during activation and degranulation. Aside from the implications for design of intravenously delivered particulate formulations in general, we expect these observations to be of particular use for targeting nanoparticles to circulating neutrophils, their clearance site (bone marrow), or distal sites of active inflammation.

Keywords: drug delivery; leukocytes; nanomaterials; nanoparticles; neutrophils.

Figure 1.

Schematic illustrating (a) the interaction of neutrophils with small (nano- to microscale), synthetic particles, (b) the impact of physiologically relevant quantities of serum on such interactions, and (c) how particle-loaded neutrophils respond to degranulation stimuli.

Figure 2.

Nanoparticles are rapidly internalized by ex vivo human neutrophils in the absence of serum proteins. (a–c) Flow cytometry and confocal microscopy z-stacks indicate internalization of NPs by neutrophils incubated with NPs (b and c) for 15 min (FC) or 3 h (confocal) compared to untreated controls (a). (d) Mean fluorescence intensity and (e) histogram distributions for the NP fluorescence channel for neutrophils incubated with NPs for varying lengths of time. Uptake occurs rapidly and plateaus for all particle types showing uptake, with the majority of uptake occurring within 15 min. Note that the results here should not be interpreted as a comparison of uptake efficiencies between cell types due to differing fluorophores and fluorophore concentrations between certain formulations. All NP fluorescence data from flow cytometry was first gated on live, nonapoptotic, nonactivated neutrophils as described in Figure S1. Cells were incubated with P) nanoparticles at 1 μg/mL and liposomes at 10 μM.

Figure 3.

Neutrophil internalization of nanomaterials is size-dependent. All particle incubation times are 3 h unless otherwise noted. (a) Theoretical expected relative fluorescence intensities for different particle formulations as they would be observed by a flow cytometer, calculated for fixed particle concentrations with known detector gains, fluorophore properties, and cytometer excitation/emission wavelengths (top). Comparison of actual fluorescence emission intensities observed at 1.0 μg/mL for gold (middle) and poly(styrene) (bottom) particles on a fluorescence plate reader. (b) NP fluorescence intensity distribution histograms for poly(styrene) and liposomes taken up by nonapoptotic, nonactivated neutrophils as a function of particle size. PS particles incubated at 1 μg/mL. Liposomes incubated at 0.5 μg/mL. (c) Mean NP fluorescence intensities as observed by the flow cytometer for nonapoptotic, nonactivated neutrophils. Particle concentrations were 1.0 μg/mL. Fluorescence intensities as observed by the cytometer were adjusted by subtracting the untreated control fluorescence and applying the appropriate correction factor calculated in Figure 3a. (d) Dose response of NP fluorescence intensities for liposomes in nonapoptotic, nonactivated neutrophils. (e) Dose response for gold, PS, and liposomal formulations in nonapoptotic, nonactivated neutrophils. Percent cells with fluorescence exceeding the gate (maximum value of the control) calculated according to the gating shown in Figures 2a–c. Particle incubation time was 12 h.

Figure 4.

Serum dramatically reduces particle uptake for PS and liposomal nanoformulations but enhances uptake of PLGA microparticles and nanoparticles. (a) NP fluorescence after a 3 h incubation with PS particles of various sizes (at 1 μg/mL) is completely abrogated. Uptake of liposomes (at 5 μg/mL) is reduced by approximately an order of magnitude. Gating is for nonapoptotic, nonactivated neutrophils. (b) The presence of serum improves the uptake of PVA-stabilized PLGA microparticles. Particle preadsorption with human serum albumin even further improves uptake as assessed by NP fluorescence on flow cytometry, gated for nonapoptotic, nonactivated neutrophils. Incubation time was 2 h. (c) Confocal microscopy images of untreated neutrophils and neutrophils incubated with HSA-preadsorbed PLGA microparticles at 500 μg/mL for 2 h. (d) Uptake of PLGA particles is size-dependent. In formulations both with and without HSA, uptake of PLGA microparticles exceeds uptake of nanoparticles at a dose of 500 μg/mL. Incubation time was 2 h.

Figure 5.

Critical aspects of neutrophil phenotype are not perturbed by particle uptake; particles remain inside the cell following degranulation. Incubation times were 3 h unless otherwise noted. (a) Cell viability is not affected by particle uptake. At least 40 000 cells from 2 separate experiments were used to generate the chart. (b) Neutrophil apoptosis as measured by CD16 shedding was not impacted by particle uptake. (c) Neutrophil activation as measured by CD62L shedding was not impacted by particle uptake. (d and e) Neutrophils incubated with 1 μg/mL PS or 5 μg/mL liposomes for 3 h and then treated with 50 nM PMA, 1 μM ionomycin, or 50 nM PMA + 1 μM ionomycin for 30 min at 37 °C do not exocytose particles previously internalized.

Figure 6.

Release of NGAL by neutrophils during degranulation is unchanged by prior uptake of NPs. Neutrophils previously incubated with nanoparticles for 2 h at 1 μg/mL (PS) or 5 μg/mL (liposomes) exhibit no change in their ability to release NGAL, a critical degranulation marker, upon stimulation with 1 μM fMLP, 50 nM PMA, or 1 μM ionomycin for 30 min at 37 °C. n = 2. * = p < 0.05.