PEGylation of model drug carriers enhances phagocytosis by primary human neutrophils

Abstract

Targeted drug carriers are attractive for the delivery of therapeutics directly to the site of a disease, reducing systemic side effects and enhancing the efficacy of therapeutic molecules. However, the use of particulate carriers for drug delivery comes with its own set of challenges and barriers. Among these, a great deal of research effort has focused on protecting carriers from clearance by phagocytes via altering carrier surface chemistry, mostly with the use of polyethylene glycol (PEG) chain coatings. However, few papers have explored the effects of PEGylation on uptake by freshly-obtained primary human phagocytes in physiological conditions. In this work, we investigate the effect of PEGylation on particle uptake by primary human neutrophils in vitro and compare these effects to several cell lines and other model phagocytic cells systems. We find that human neutrophils in whole blood preferentially phagocytose PEGylated particles (e.g., ∼40% particle positive neutrophils for PEGylated versus ∼20% for carboxylated polystyrene microspheres) and that this effect is linked to factors present in human plasma. Model phagocytes internalized PEGylated particles less efficiently or equivalently to carboxylated particles in culture medium but preferentially phagocytosed PEGylated particles in the human plasma (e.g., ∼86% versus ∼63% PEGylated versus carboxylated particle positive cells, respectively). These findings have significant implications for the efficacy of PEGylation in designing long-circulating drug carriers, as well as the need for thorough characterization of drug carrier platforms in a wide array of in vitro and in vivo assays.

Statement of significance: The work in this manuscript is highly significant to the field of drug delivery, as it explores in-depth the effects of polyethylene glycol (PEG) coatings, which are frequently used to prevent phagocytic clearance of particulate drug carriers, on the phagocytosis of such carriers by neutrophils, the most abundant leukocyte in blood circulation. Surprisingly, we find that PEGylation enhances uptake by primary human neutrophils, specifically in the presence of human plasma. This result suggests that PEGylation may not confer the benefits in humans once thought, and may help to explain why PEG has not become the "magic bullet" it was once thought to be in the field of particulate drug delivery.

Keywords: Microparticles; Nanoparticles; Neutrophils; Opsonization; PEGylation; Particle drug carriers; Phagocytosis.

Figure 1—

Uptake of carboxylated and PEGylated 2 μm polystyrene particles by primary human neutrophils, as determined by flow cytometry. (a) Uptake by neutrophils from individual human donors. (b) Neutrophil uptake of 2 μm carboxylated and PEGylated polystyrene particles. (c) Aggregated neutrophil particle uptake for 2 μm, 500 nm, and 200 nm carboxylated and PEGylated polystyrene particles. (d) Neutrophil uptake of 2 μm carboxylated and PEGylated polystyrene particles at varying concentrations.

Figure 2—

Characterization of the Impact of PEG Density and Chain Length on PEGylated Particle Uptake in Whole Blood. Uptake of (a) 2 μm, (b) 500 nm, and (c) 200 nm carboxylated and PEGylated polystyrene particles of varying PEG densities. (d) Uptake of 2 μm PEGylated polystyrene particles with varying PEG chain endgroups. (e) Uptake of 2 μm PEGylated polystyrene particles with varying PEG chain lengths.

Figure 3—

Uptake of 2 μm, 500 nm, and 200 nm carboxylated and PEGylated polystyrene particles by differentiated HL-60 derived neutrophils in the presence of human plasma.

Figure 4—

Uptake of carboxylated and PEGylated polystyrene particles by various cell types in presence of human plasma. Uptake by (a) bone marrow-derived macrophages from BALB/c mice of particles soaked in human plasma; (b) bone marrow-derived macrophages from C57BL/6 mice of particles soaked in human plasma; and (c) THP1 monocytes cultured in human plasma; particles soaked in human plasma.

Figure 5—

SDS PAGE analysis of proteins adsorbed to carboxylated vs PEGylated 2 μm polystyrene particles and proteins of interest found in bands exclusive to each particle.

Figure 6—

Uptake of (a) carboxylated and (b) PEGylated polystyrene particles by primary human neutrophils in plasma, RPMI + 10% FBS, RPMI + human complement sera, RPMI + human complement C3, RPMI + human serum albumin, and RPMI + human serum album with particles soaked in human serum albumin.