Introduction: Vascular targeted carriers (VTCs) are a promising drug delivery approach for treating various conditions, including cancer and coronary artery disease. However, a major issue that limits VTCs’ in vivo efficacy is their rapid removal from circulation through filtration via RES organs and clearance by phagocytic leukocytes. Over the years, researchers have explored ways to design VTCs to avoid phagocytic uptake and achieve longer circulation times. Most of these efforts have focused on non-fouling surface coatings such as polyethylene glycol (PEG), cell membrane-derived, and zwitterionic coatings. Particle elasticity emerged as an important factor in designing polymeric particles for intravenous drug delivery applications in recent years due to its favorable effects on reducing phagocytosis. Softer particles have been shown to have a lower tendency to be phagocytosed by macrophages and monocytes, suggesting they would be more effective for drug delivery. However, most work evaluating particle deformability as a viable approach to minimize or avoid drug carrier phagocytosis has been in vitro with macrophages and monocytic cell lines. Neutrophils are highly efficient phagocytes that comprise 50 – 70% of all immune cells circulating in human blood, with monocytes and lymphocytes at 25 – 33% and < 10%, respectively. However, little is known about how neutrophils interact with soft particles and whether the relationship between particle elasticity and phagocytosis holds in human circulation. In this work, we investigate the effects of particle elasticity on neutrophil phagocytosis.
Materials and
Methods: Our experiments were designed to investigate the effect of particle elasticity on the association of hydrogel particles with different types of phagocytes. PEG and HA particles of varying elastic moduli were fabricated by adjusting the polymer concentration, and their association with neutrophils and macrophages was quantified. Particles were modified with surface conjugation or altered zeta potential to assess interplay between these parameters and particle deformability. All the human donors for the experiments were healthy. The animal blood was collected from healthy mice. In vivo experiments were performed on healthy mice. All the experiments were replicated at least three times.
Human blood from healthy donors was obtained via venipuncture per a protocol approved by the University of Michigan Internal Review Board. A written consent was obtained from the individuals before the blood draw. Animal studies were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the University of Michigan. BALB/c mice were obtained from The Jackson Laboratory. All mice were housed under specific pathogen-free conditions and maintained at the University of Michigan in compliance with the University Committee on Use and Care of Animal regulations.
Results, Conclusions, and Discussions: Based on the vast amount of literature on interactions between immune cells and particles of varying moduli, we expected to see a reduction in uptake as we moved from a rigid polystyrene control to softer PEG-derived particles. Instead, neutrophils were as likely or more likely to phagocytose softer particles compared to the non-deformable polystyrene, suggesting that modulus may not be as effective of a parameter to leverage when designing particles to avoid clearance from the bloodstream in humans where neutrophils are likely the first phagocytes encountered by intravenously delivered particles. The data furthermore suggests that the mechanism by which deformable particles are more likely to be phagocytosed is exaggerated when neutrophils encounter nanoparticles instead of microparticles. Given the relevance of nanoparticles compared to microparticles in current vascular delivery technologies, these results indicate there may, in fact, be a loss of efficacy by using more deformable particles, given the higher propensity for clearance by neutrophils. We hypothesize that these differences in uptake may arise from the deformation of the PEG particles as they undergo membrane wrapping by neutrophils, resulting in an enhanced phagocytosis effect due to an apparent elongated particle shape as the particle is engulfed. Similarly, we see a reduction in deformable particle uptake by J774 macrophages because of their reported inability to effectively phagocytose rod-like particles, which has been linked to a higher energy barrier of membrane wrapping. The results with HA microparticles, with different hydrogel chemistry from PEG, support the hypothesis that the difference in uptake is due to neutrophils sensing the particle stiffness. Notably, the neutrophil uptake levels of HA particles were comparable to that of PEG particles, providing strong evidence that this enhanced phagocytosis effect is indeed linked to particle modulus. Overall, our results here suggest that the reported benefits of deformable particles, including longer circulation times and more effective avoidance of clearance by tissue-resident macrophages and monocytes, may not hold with respect to neutrophils. We find that a deformable particle instead exhibits an enhanced phagocytic effect, which might undermine efforts to improve vascular-targeted drug delivery.
