Role of particle size in phagocytosis of polymeric microspheres

Abstract

Purpose: Polymeric microspheres are extensively researched for applications in drug and vaccine delivery. However, upon administration into the body, microspheres are primarily cleared via phagocytosis by macrophages. Although numerous studies have reported on the biochemical pathways of phagocytosis, relatively little is known about the dependence of phagocytosis on particle size. Here, we investigate the previously unexplained dependence of phagocytosis on particle size.

Methods: Rat alveolar macrophages and IgG-opsonized and non-opsonized polystyrene microspheres were used as model macrophages and drug delivery particles. Phagocytosis, attachment and internalization were measured by flow cytometry and time-lapse video microscopy.

Results: Particles possessing diameters of 2-3 microm exhibited maximal phagocytosis and attachment. Rate of internalization, however, was not affected significantly by particle size. Maximal attachment of 2-3 microm microspheres is hypothesized to originate from the characteristic features of membrane ruffles in macrophages. Elimination of ruffles via osmotic swelling nearly eliminated the peculiar size-dependence of phagocytosis. A simple mathematical model is presented to describe the dependence of phagocytosis on particle size.

Conclusions: The dependence of phagocytosis on particle size originated primarily from the attachment step. These results reveal the importance of controlling drug delivery particle size distribution and selecting the size appropriate for avoiding or encouraging phagocytosis.

Fig. 1

Overall phagocytosis uptake, combined attachment and internalization, for IgG-opsonized (open circles) and non-opsonized (closed circles) microspheres by rat alveolar macrophages (n ≥ 3, error bars show one standard deviation).

Fig. 2

(a) K values for attachment of IgG-opsonized (open circles) and non-opsonized (closed circles) particles by rat alveolar macrophages. K exhibits a peak for particles with diameters of 2–3 μm, p<0.00007 (n≥ 3, error bars show one standard deviation). (b) Internalization velocities for internalization of IgG-opsonized (open circles) and non-opsonized (closed circles) particles by rat alveolar macrophages. Internalization velocity is not significantly affected by particle size, p>0.15 comparing largest and smallest particles.

Fig. 3

Comparison of K values for attachment of IgG-opsonized particles by rat alveolar (closed diamonds), murine peritoneal (closed circles) and human spleen (open circles) macrophages. Each macrophage type exhibits a peak in K values around 3 μm diameters, p< 0.02 for each type (n≥3, error bars show one standard deviation).

Fig. 4

(a) Scanning electron micrograph of alveolar macrophage displaying a ruffled membrane. Scale bar=5 μm. (b) Scanning electron micrograph of osmotically swollen alveolar macrophage with smooth membrane surface. Scale bar=5 μm. (c) K values for attachment of IgG-opsonized particles by normal (open circles) and swollen (closed circles) rat alveolar macrophages. K values for swollen cells decreased significantly for particles with diameters of 2–3 μm, p<0.0006 (n≥3, error bars show one standard deviation). (d) K values for attachment of non-opsonized particles by normal (open circles) and swollen (closed circles) rat alveolar macrophages. K values for swollen cells decreased significantly for particles with diameters of ~2 μm, p<0.000002 (n≥3, error bars show one standard deviation).

Fig. 5

(a) Schematic of membrane ruffles (black) and attached particles (white). Small (D ≪ L, left) and large particles (D ≫ L, right) can make 1 or 2 contacts with the membrane. Mid-sized particles (D~L, center) can make up to 3 contacts. (b) Schematic of ruffle and particle length scales used to calculate the particle diameter capable of making the most membrane contact (h = height of ruffles, L=distance between ruffles and D=particle diameter).