E14 - Microgels with Cell-Like Tunability for Drug Delivery Systems

Introduction: : Intravenous administration of hydrogel-based drug delivery particles holds immense promise for the targeted treatment of a wide range of diseases, from cancer to autoimmune disorders. However, a major challenge lies in overcoming rapid sequestration by biological barriers, such as the liver and spleen, which can significantly reduce circulation time and therapeutic efficacy. The ability of these particles to evade clearance is governed by key physical and chemical properties, including size, elastic modulus, and surface chemistry, which influence interactions with immune cells and vascular structures. The objective of this study is to optimize the properties of hydrogel microcarriers so they can be engineered to enhance vascular persistence, improve biodistribution, and ultimately enable more effective and sustained delivery of drugs gradually released from encapsulated nanoparticles into the systemic circulation. Based on prior theoretical and experimental findings summarized by Baranov et al., we hypothesized that soft, large, and uncoated microparticles would be most effective at evading sequestration during systemic circulation.

Materials and

Methods: : In recent years, there have been important strides made to engineer particles with stealth properties to extend circulation time within the body. Here we engineered monodisperse, tunable, cell-like microparticle (MP) carriers that could be loaded with drug release nanotechnologies. These MPs were fabricated from polyacrylamide hydrogel using a microfluidic droplet generator. The elastic modulus was governed by the concentration of bis-acrylamide crosslinker in the polymer precursor solution. Importantly, our fabricated MPs were softer than typical hydrogel MPs, potentially extending their circulation time in the body. To simulate the adsorption effects of prolonged circulation in the bloodstream, where MPs are expected to acquire specific and non-specific proteins on their surface, the MPs were incubated for 24 hours with a coating of interest (FBS or BSA) prior to their use in an cell engulfment assay. To further assess the particle interaction with cells, MPs were coated with IgG via Sulfo-SANPAH crosslinking, to mimic cell-cell adhesion. We then utilized in vitro methods with J774a.1 murine macrophages and primary human neutrophils to characterize the potential of these MPs as protective microcarriers, plating MPs to cells in a 1:1 ratio and incubating for 2-hours before imaging (Figure 1). Engulfment scores of MPs of varying stiffness, protein coating, and size were then assessed by dividing the number of MPs engulfed by the total MPs for multiple fields of view. Statistical comparisons were made using ANOVA followed by Tukey’s post hoc test, with p < 0.05 regarded as statistically significant.

Results, Conclusions, and Discussions:: This study systematically examined how microparticle (MP) properties—stiffness, size, and surface coating—influenced phagocytic engulfment by immune cells, using J774a.1 macrophages and primary human neutrophils. MPs were fabricated with tunable elastic moduli: soft (~0.25 kPa), stiff (~10 kPa), as well as a rigid polystyrene control (~3 MPa). Engulfment scores revealed that stiffness significantly enhanced phagocytic uptake: polystyrene MPs were engulfed most efficiently, followed by stiff MPs, with soft MPs showing minimal internalization. This was confirmed both quantitatively (Figure 2A) and qualitatively via epifluorescent microscopy (Figure 2B). These findings align with prior mechanistic models suggesting that stiffer particles facilitate membrane wrapping by enhancing cytoskeletal recruitment and ligand-receptor engagement.

Surface chemistry further modulated uptake. Coating MPs in fetal bovine serum (FBS), which mimics protein corona formation and enhances opsonin-mediated recognition, increased engulfment by 90.7 % across all particle types, particularly for stiffer MPs. In contrast, bovine serum albumin (BSA) coating suppressed phagocytosis by 4.3%, supporting its non-opsonic “stealth” behavior (Figure 3A). Notably, IgG functionalization led to increased engulfment across all stiffnesses, reinforcing the role of targeted receptor-mediated recognition (Figure 3B). However, even with IgG, soft MPs were still less readily internalized, indicating that surface cues alone cannot overcome mechanical impediments to phagocytosis.

To further investigate the role of phagocytic cells in microparticle engulfment, experiments were repeated using primary neutrophils from human donors. Results revealed trends consistent with those observed in macrophages; however, neutrophil engulfment scores were consistently 2–3 times lower, hypothesized to be due to their smaller average diameter (12–15 µm) compared to cell line macrophages (~20 µm), which may limit their engulfment capacity. To investigate the effect of MP size relative to phagocyte size, engulfment assays were performed using polystyrene and stiff MPs of increasing diameters. Results demonstrated that increasing MP diameter from 15 to 25 µm significantly decreased engulfment (Figure 3C). This size-dependent effect was more pronounced in softer MPs, where increased size mimicked the evasion typically associated with deformable materials.

These findings underscore the potential of tuning MP stiffness and size to evade immune detection, with implications for drug delivery applications.

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