(592a) Pegylation of Model Drug Carriers Enhances Uptake By Primary Human Neutrophils

Introduction: The advent of targeted drug carriers has opened many new avenues for the delivery of therapeutics directly to the site of disease, reducing systemic side effects and enhancing the efficacy of therapeutic molecules. However, the packaging of therapeutics into particulate carriers for 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 by altering carrier surface chemistry. Many groups have explored the use of polyethylene glycol (PEG) chain coatings to mitigate unwanted phagocytosis, as PEG is highly hydrophilic and is well-known for its anti-fouling properties. However, this strategy has seen limited success in the clinic, as carriers are often still rapidly cleared with little improvement in therapeutic benefit. Notably, very few papers have explored the effects of PEG on uptake by freshly obtained primary human phagocytes in physiological conditions, creating a disconnect between the prevailing literature and ultimate applications. In this work, we investigate the effect of PEGylation on uptake by primary human neutrophils in vitro, and compare these effects to several cell lines and other model phagocytic cells systems in evaluating the effects of surface chemistry on phagocytosis.

Materials and Methods: Human blood was obtained from healthy donors via venipuncture per a protocol approved by the University of Michigan Internal Review Board. Mouse blood was obtained via cardiac puncture per a protocol approved by the University of Michigan Institutional Animal Care and Use Committee (IACUC) and in accordance with the National Institute of Health guidelines for the care and use of laboratory animals. Carboxylated polystyrene spheres (PS-COOH) were conjugated with methoxy-terminated polyethylene glycol (PEG, 5k molecular weight, Fisher) using N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, Sigma) chemistry. PEG chain density was quantified using rhodamine-labeled PEG and a standard curve created using a fluorescence plate reader. Particles of varying size and PEG densities were introduced to human blood, mouse blood, and other phagocytic cells (mouse bone marrow-derived macrophages, THP-1 monocytes, HL-60 derived neutrophils) for varying time periods (0-2 hours). Post-phagocytosis, cells were stained (for CD45, CD11b, Ly-6G, etc.) and fixed for flow cytometry.

Results and Discussion: Surprisingly, we found that primary human neutrophils preferentially phagocytose PEGylated carriers relative to their carboxylated counterparts (~40% particle positive neutrophils for PEGylated particles, compared to ~20% for carboxylated) (see Figure 1). This phenomenon was observed in nearly every donor used in the study (15/16), and was found to be consistent for individual donors. Additionally, this effect was consistently observed for 2m, 500 nm, and 200 nm particles with a wide variety of PEG conjugation densities. This phenomenon was not observed, however, in THP1 monocytes, mouse bone marrow-derived macrophages, or freshly-isolated mouse neutrophils; however, addition of human plasma to model phagocytic cells dramatically increased uptake of PEGylated particles, suggesting the involvement of factors in human plasma in elevated uptake. Further work to elucidate the precise factors responsible for this phenomenon is currently underway.

Conclusions: The addition of PEG chains to model drug carriers results in increased uptake by primary human neutrophils. This phenomenon is observed across a range of carrier sizes and PEG densities, but is not observed in typical model phagocytic cells. This result is seemingly linked to factors in human plasma resulting in preferential uptake of PEGylated carriers as compared to carboxylated counterparts. The surprising results have implications in the design of effective drug carriers, and possibly shed light as to why PEGylation has not proven to be the “magic bullet” in creating stealth drug carriers, and may lead to a more thorough understanding of the mechanisms of phagocytosis by neutrophils, which are thus far less studied than other phagocytes such as macrophages and monocytes.