Effects of microparticle size and Fc density on macrophage phagocytosis

Abstract

Controlled induction of phagocytosis in macrophages offers the ability to therapeutically regulate the immune system as well as improve delivery of chemicals or biologicals for immune processing. Maximizing particle uptake by macrophages through Fc receptor-mediated phagocytosis could lead to new delivery mechanisms in drug or vaccine development. Fc ligand density and particle size were examined independently and in combination in order to optimize and tune the phagocytosis of opsonized microparticles. We show the internalization efficiency of small polystyrene particles (0.5 µm to 2 µm) is significantly affected by changes in Fc ligand density, while particles greater than 2 µm show little correlation between internalization and Fc density. We found that while macrophages can efficiently phagocytose a large number of smaller particles, the total volume of phagocytosed particles is maximized through the non-specific uptake of larger microparticles. Therefore, larger microparticles may be more efficient at delivering a greater therapeutic payload to macrophages, but smaller opsonized microparticles can deliver bio-active substances to a greater percentage of the macrophage population. This study is the first to treat as independent variables the physical and biological properties of Fc density and microparticle size that initiate macrophage phagocytosis. Defining the physical and biological parameters that affect phagocytosis efficiency will lead to improved methods of microparticle delivery to macrophages.

Figure 1. Microparticle opsonization.

(A) Carboxylated polystyrene microparticles are first incubated with a BSA solution to ensure the BSA antigen is adsorbed onto the microparticle surface. (B) Varying amounts of sheep anti-BSA IgG was then added corresponding to a specific ratio of IgG to antigen. This process ensures external presentation of the Fc portion of the antibody molecule and was repeated for each microparticle size.

Figure 2. Normalized mean fluorescence intensity of microparticles labeled with fluorescently conjugated secondary antibodies.

Decreasing normalized MFI corresponds to decreased Fc density for fluorescent particles.

Figure 3. Average number of attached microparticles for each particle size and Fc density condition.

As particle size decreases, so does the attachment efficiency. Overall, Fc density was not a significant variable for opsonized and BSA-only particles. †- Significantly different (SD) from 1∶1, ‡- SD from 1∶2, with p<0.05, (N = 2).

Figure 4. Average number of microparticles internalized per cell for each particle size and Fc density condition.

(A) Average number of microparticles per cell for each particle size for the various Fc density conditions. (†- Significantly different (SD) from 1∶1, ‡- SD from 1∶2, ♦- SD from BSA Only, with p<0.05). These values were also illustrated through fluorescent microscopy by comparing images taken of RAW264.7 macrophages incubated with Fc functionalized particles. (B) Representative images for each size with the maximum Fc density ratio of 1∶1. (C) Representative images for each size with the minimum Fc density ratio of 1∶50. Scale bar represents 20 µm. (N = 4).

Figure 5. Average total volume and surface area of internalized microparticles.

Average calculated volume and surface area of microparticles internalized for the maximum Fc condition of 1∶1 for each particle size. (N = 4) The results for each particle size are significantly different from each other (p<0.01).

Figure 6. Effects of Fc density and particle size on the macrophage population.

(A) Percentage of phagocytic cells at each Fc density condition for each particle size with a dashed line to illustrate trends. (N = 4) (B) Representative histograms of the FITC intensity for macrophages incubated with particles functionalized with the maximum Fc density of 1∶1 and the minimum Fc density of 1∶50. These histograms illustrate two groups: macrophages that have internalized at least one particle (phagocytic cells) and macrophages that did not internalize a particle (non-phagocytic cells).

Figure 7. Summary of the average number of attached and internalized microparticles per cell for each particle size and Fc density condition.

(A) The attachment of particles to macrophages does not have a strong dependence on microparticles size and Fc density. (B) The internalization of particles by macrophages is highly dependent on microparticle size and Fc density with an overall trend of the average number of internalized particles decreasing as microparticle size increases and Fc density decreases.