Tailored PGE2 Immunomodulation of moDCs by Nano-Encapsulated EP2/EP4 Antagonists

Abstract

Prostaglandin E2 (PGE2) is an important maturation mediator for dendritic cells (DCs). However, increased PGE2 levels in the tumor exert immunosuppressive effects on DCs by signaling through two E-Prostanoid (EP) receptors: EP2 and EP4. Blocking EP-receptor signaling of PGE2 with antagonists is currently being investigated for clinical applications to enhance anti-tumor immunity. In this study, we investigated a new delivery approach by encapsulating EP2/EP4 antagonists in polymeric nanoparticles. The nanoparticles were characterized for size, antagonist loading, and release. The efficacy of the encapsulated antagonists to block PGE2 signaling was analyzed using monocyte-derived DCs (moDCs). The obtained nanoparticles were sized between 210 and 260 nm. The encapsulation efficacy of the EP2/EP4 antagonists was 20% and 17%, respectively, and was further increased with the co-encapsulation of both antagonists. The treatment of moDCs with co-encapsulation EP2/EP4 antagonists prevented PGE2-induced co-stimulatory marker expression. Even though both antagonists showed a burst release within 15 min at 37 °C, the nanoparticles executed the immunomodulatory effects on moDCs. In summary, we demonstrate the functionality of EP2/EP4 antagonist-loaded nanoparticles to overcome PGE2 modulation of moDCs.

Keywords: EP2/EP4 antagonist; delivery; dendritic cells; nanoparticles; prostaglandin E2.

Figure 1

Normalized expression of CD83, CD40, HLA-DR, and CD86 on moDCs after 24 h in the presence or absence of NP formulations. Control moDCs were left untreated (NT, white bar), stimulated with a maturation cocktail (MC, gray bar), or MC with 0.2 µM PGE2 (MC + PGE2, black bar). Test condition moDCs were stimulated with MC + PGE2 and NPs with either aEP2 or aEP4 alone or in combination (green, pink, orange, and yellow bars, respectively). The NPs were added in a final concentration of 60 µM aEP2 and the aEP4 concentration was adjusted to the according concentration calculated in the combination NPs. Empty NPs were used as control (blue bar). Expression was normalized to the expression of the marker on moDCs stimulated with MC + PGE2. Each dot represents one donor and the bar is the mean ± SD for n = 5. p values were calculated on non-normalized data with one-way ANOVA with Tukey multiple comparison correction.

Figure 2

Normalized expression of CD83, CD40, HLA-DR, and CD86 on moDCs after 24 h of stimulation. Control moDCs were left NT or stimulated with MC either in the presence or absence of PGE2. MoDCs were stimulated with MC + PGE2 with either NPs PBS-incubated or non-incubated NPs at a 60 µM aEP2 concentration. PBS-incubated NPs (empty) were used as control matching the highest NP concentration. The expression was normalized to the MC + PGE2 control moDCs. Each dot represents one donor and the bar is the mean ± SD for n = 5. p values were calculated on non-normalized data with one-way ANOVA with Tukey multiple comparison correction.

Figure 3

Normalized expression of CD83, CD40, HLA-DR, and CD86 on moDCs 24 h after the addition of NPs, either directly or washed cells. Controls included NT, stimulated with MC and MC with PGE2. MoDCs were stimulated with NPs directly or the cells were washed after one hour of stimulation. Two batches of NPs were used with a matched 60 µM aEP2 concentration. The expression was normalized to the MC + PGE2 control. Each dot represents one donor and the bar is the mean ±SD for n = 5 for direct and n = 4 for washed condition. p values were calculated on non-normalized data with one-way ANOVA with Tukey multiple comparison correction.

Figure 4

Normalized expression of CD83, CD40, HLA-DR, and CD86 on moDCs after 24 h of stimulation with NPs for varied hours. The control with MC + PGE2 was repeated for each timepoint. The expression was normalized to the MC + PGE2 expression after 1 h of stimulation. The NT and MC control are shown in Supplementary Figure S5. Each dot represents the mean ± SD for n = 4.

Figure 5

Percentage of CD83 and CCR7 expressing moDCs after 24 h of stimulation with encapsulated or soluble aEP2 + aEP4 and increasing PGE2 concentration. Control moDCs were left NT, stimulated with MC and MC with three different concentrations of PGE2: 0.2, 1, or 28 µM. MoDCs were stimulated with encapsulated or soluble aEP2 + aEP4 and MC + PGE2 with the three different PGE2 concentrations. The encapsulated and soluble antagonist concentration matched a final concentration of 60 µM aEP2 and 1.25 µM aEP4. Each dot represents one donor and the bar is the mean ± SD for n = 4. p values were calculated on non-normalized data with one-way ANOVA with Tukey multiple comparison correction.