An endosome-escaping microparticle facilitates delivery of RIG-I vaccine adjuvants

摘要

Introduction: DNA and RNA adjuvants can trigger innate immune responses by stimulating the Toll-like receptors (TLRs) and Retinoic-Acid-Inducible-Gene (RIG-I)-like receptors (RLRs). Foreign nucleic acid detection by RLRs induces type Ⅰinterferon production and co-stimulatory molecule expression on dendritic cells. Therefore, RIG-I adjuvants are an attractive means to boost vaccination efficacy. Their efficacy, however, is hindered by low level of transit to RLRs in the cytosol. In response, we have developed an endosomal-escaping RIG-I carrying microparticle formulation, which has the potential to amplify humoral and cell-mediated immune responses. The ability to control the particle's size, ligand-type, and ligand-density offers unique opportunities for cell-specific delivery, regulation of signaling kinetics, and combinatorial therapies; all of which could facilitate precise control of the innate immune response in vaccine and antiviral applications. Materials and Methods: Branched polyethylenimine (bPEI) was conjugated to PLGA microparticles formed by a double emulsion / solvent-evaporation method. PUUC (RIG-I ligand) was loaded by electrostatic interactions. Soluble PUUC or MP PUUC was co-incubated with dendritic cells for 24 hours and 48 hours. Flow cytometry was used to measure CD86 and MHC Ⅱ expression and type Ⅰinterferon in the supernatant was measured by ELISA. Cells were fixed and stained for endolysosomal markers (CD63/EEA1/LAMP1) and particle co-localization was evaluated by confocal microscopy. Soluble PUUC or MP PUUC was then injected subcutaneously into wild-type mice. Lymph nodes and tissue from the injection site were harvested after 24 hours. type Ⅰinterferon gene expression was measured by qPCR. Further studies evaluating (a) the effect of ligand density on particle-interaction with RLRs (using proximity ligation assay), (b) how the kinetics and magnitude of innate immune response differs between RLR and TLR ligands (e.g. CpG DNA adjuvants), and (c) how RLR-based signaling can synergize with TLR-based signaling in modulating and enhancing the innate and adoptive responses; are currently underway. Results and Discussion: The positive surface-charge (zeta potential of 40 mV) of the particles facilitates loading of the negatively charged PUUC by electrostatic interactions. In addition, the presence of secondary and tertiary amines facilitates particle endosomal escape by the 'proton sponge' mechanism. Analysis of microscopy images showed that fewer than 50% of particles co-localized with endolysosomal markers after 24 hours, indicating endosomal escape (Figure 1 A). We have previously reported that this type of particles carrying siRNA or CpG are non-toxic and efficacious in in vivo applications. After co-incubation with dendritic cells, the level of interferon-beta in the cell media was 40-fold higher in MP PUUC over soluble PUUC (Figure 1B). 24 hours post-injection, microparticle with ovalbumin antigen (MP-PUUC OVA) induced a 300-fold increase in interferon-alpha and 1000-fold increase in interferon beta gene expression over soluble PUUC with OVA (1C). Conclusion: Cationic polymeric particles have been developed that facilitate the cytosolic delivery of RNA. The RIG-I microparticle can induce a significant innate immune response, which enables its use as an adjuvant for vaccination.