Delivery of rapamycin to dendritic cells using degradable microparticles

Abstract

Degradable microparticles have the potential to protect and release drugs over extended periods and, if sized appropriately, can be passively targeted to phagocytic cells in vivo. Dendritic cells (DC) are a class of phagocytic cells known to play important roles in transplant rejection. Previously, we have demonstrated that DC treated with an immunosuppressive drug, rapamycin, have the ability to suppress transplant rejection. Herein, we describe a strategy to deliver an intracellular depot of rapamycin to DC. To achieve this, rapamycin was encapsulated into ~3.4 microm sized poly(lactic-co-glycolic)acid (PLGA) microparticles (rapaMPs), and release behavior was examined under intra-phagosomal (pH=5) and extracellular (pH=7.4) conditions. It was observed that 4 days following phagocytosis of rapaMP, DC have significantly reduced ability to activate T cells, in comparison to DC treated with soluble rapamycin. Hence, we conclude that DC-specific intracellular delivery of rapamycin results in better efficacy of the drug, with respect to its ability to modulate DC function, when compared to treating DC with extracellular rapamycin.

Figure 1

SEM images of rapamycin containing PLGA particles. These images confirm that the particles exhibit surface integrity. Also, sizes are consistent with measurements via volume impedance (Avg. Vol. Dia – 3.4 μm).

Figure 2

In vitro controlled release of rapamycin from small-rapaMPs in pH 5 and pH 7.4 buffer. Percentages are based on amount encapsulated. 100% indicates ∼3.7 μg of rapamycin/mg of rapaMPs – the maximum amount that would be released if the particles degraded completely. Standard deviations are based on n = 6 samples for release at pH 7.4, and n=3 samples for release at pH 5.

Figure 3

Representative images of live DC cultured for 1 day with rapaMPs. NT (no treatment) implies that no particles were added to these DC cultures. The ratios indicate the number of particles added to the number of DC. Scale = 50 μm.

Figure 4

Annexin-V and 7-AAD staining of DC to examine potential toxicity of rapaMPs. Apoptosis of DC incubated for (A) - 1 day, or (B) − 4 days with different number of rapaMPs was compared to untreated DC. Flow diagrams were generated by gating on CD11c⁺ (DC restricted marker) cells.

Figure 5

A mixed leukocyte reaction indicating the amount of radioactive thymidine incorporated into responding T lymphocytes. DC were loaded with rapaMPs using the indicated MP:cell ratio and then used to stimulate allo-reactive CD4⁺ T cells. NT (no treatment) group was assigned the value of 100% and all other percentages are normalized based on this value. The ratio of MPs to DC in the blank particles group is 5:1. Standard deviations are based on n = 6 experiments per group. * indicates comparison between 5:1 and NT groups - p <0.005; ** indicates comparison between 5:1 and soluble Rapa treatment groups - p < 0.01.

Figure 6

Flow Cytometric Analysis of DC activation markers on cells that have taken up fluorescently labeled rapaMPs (A647⁺). Flow diagrams were generated by gating on CD11c⁺ and Alexa-fluor 647^high or Alexa-fluor 647^low, as described in the top-center plot. Numbers on flow diagrams indicate mean fluorescence intensity values. DC were cultured with rapaMPs at a ratio of 5:1 (rapaMPs to DC) for 24 hours, prior to flow analysis.