Co-delivery of Dual Toll-Like Receptor Agonists and Antigen in Poly(Lactic-Co-Glycolic) Acid/Polyethylenimine Cationic Hybrid Nanoparticles Promote Efficient In Vivo Immune Responses

Abstract

Strategies to design delivery vehicles are critical in modern vaccine-adjuvant development. Nanoparticles (NPs) encapsulating antigen(s) and adjuvant(s) are promising vehicles to deliver antigen(s) and adjuvant(s) to antigen-presenting cells (APCs), allowing optimal immune responses against a specific pathogen. In this study, we developed a novel adjuvant delivery approach for induction of efficient in vivo immune responses. Polyethylenimine (PEI) was physically conjugated to poly(lactic-co-glycolic) acid (PLGA) to form PLGA/PEI NPs. This complex was encapsulated with resiquimod (R848) as toll-like receptor (TLR) 7/8 agonist, or monophosphoryl lipid A (MPLA) as TLR4 agonist and co-assembled with cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG ODN) as TLR9 agonist to form a tripartite formulation [two TLR agonists (inside and outside NPs) and PLGA/PEI NPs as delivery system]. The physicochemical characteristics, cytotoxicity and cellular uptake of these synthesized delivery vehicles were investigated. Cellular viability test revealed no pronounced cytotoxicity as well as increased cellular uptake compared to control groups in murine macrophage cells (J774 cell line). In the next step, PLGA (MPLA or R848)/PEI (CpG ODN) were co-delivered with ovalbumin (OVA) encapsulated into PLGA NPs to enhance the induction of immune responses. The immunogenicity properties of these co-delivery formulations were examined in vivo by evaluating the cytokine (IFN-γ, IL-4, and IL-1β) secretion and antibody (IgG1, IgG2a) production. Robust and efficient immune responses were achieved after in vivo administration of PLGA (MPLA or R848)/PEI (CpG ODN) co-delivered with OVA encapsulated in PLGA NPs in BALB/c mice. Our results demonstrate a rational design of using dual TLR agonists in a context-dependent manner for efficient nanoparticulate adjuvant-vaccine development.

Keywords: CpG ODN; adjuvants; monophosphoryl lipid A; poly(lactic-co-glycolic) acid nanoparticles; polyethylenimine; resiquimod; toll-like receptor agonist; vaccine.

Graphical Abstract

PEI was physically conjugated to PLGA NPs to form PLGA/PEI NPs. This complex was encapsulated with R848 or MPLA and co-assembled with CpG ODN on the surface of NPs to form a tripartite complex [two TLR agonists (inside and outside NPs) and PLGA/PEI NPs as delivery system]. The immunogenicity properties of these co-delivery formulations were examined in vivo by evaluating the cytokines secretion and antibody production.

Figure 1

Scanning electron microscopy image of poly(lactic-co-glycolic) acid/polyethylenimine structure.

Figure 2

Transmission electron microscopy image of poly(lactic-co-glycolic) acid/polyethylenimine core–shell structure.

Figure 3

Agarose gel retardation assay of cationic poly(lactic-co-glycolic) acid (PLGA)/polyethylenimine (PEI) nanoparticles. A: Ladder, B–D: PLGA/PEI CP 2-4-6, E–G: PEI CP 2-4-6, H: cytosine–phosphorothioate–guanine oligodeoxynucleotide alone.

Figure 4

In vitro release profile of monophosphoryl lipid A, resiquimod, and ovalbumin from poly(lactic-co-glycolic) acid nanoparticles over the time. The experiment was performed in PBS in 37°C. Indicated values are mean (±SD) of at least three experiments.

Figure 5

Stability of vector–cytosine–phosphorothioate–guanine oligodeoxynucleotide conjugation by gel electrophoresis after 7 days. L: Ladder 100k, ODN: CpG ODN, D1–7: Days 1–7.

Figure 6

Comparison of cellular viability of polyethylenimine (PEI) 25 kDa (C/P 0.8) as control with PEI and cationic poly(lactic-co-glycolic) acid/PEI complexed with cytosine–phosphorothioate–guanine oligodeoxynucleotide up to C/P 6 in J774 cells. Cells were treated for 48 h under the condition used in uptake assay, and then cell viability was assessed using MTT. The results are reported as mean ± SD, n = 3.

Figure 7

Flow cytometry histogram showing the uptake of polyethylenimine (PEI) 10/cytosine–phosphorothioate–guanine oligodeoxynucleotide (CpG ODN, blue line) and PEI25/CpG ODN (green line) as positive control groups and poly(lactic-co-glycolic) acid (R or M)/PEI10/CpG ODN (black line) in J774 cells after 48 h incubation. J774 cells were used as negative control group. The R or M stands for either R848 or monophosphoryl lipid A.

Figure 8

The effect of different poly(lactic-co-glycolic) acid (PLGA)/polyethylenimine (PEI) formulations harboring dual toll-like receptor (TLR) agonists as adjuvant in inducing cellular immune responses evaluated by measuring the number of IFN-γ and IL-4 producing splenocytes using ELISpot assay. BALB/c mice (three mice per group) were immunized twice (at days 0 and 21) with different dual core–shell adjuvant and PLGA [ovalbumin (OVA)] NP formulations. On day 7 post booster, mice from each group were sacrificed, and their splenocytes were stimulated with OVA protein for 24 h. IFN-γ and IL-4 release from splenocytes induced by different PLGA/PEI adjuvant formulations were determined using ELISpot assay. The data indicate the mean ± SEM (n = 3) **P ≤ 0.01, ***P ≤ 0.001 (one-way analysis of variance). The asterisks located on top of the lines showed comparison of significance between the main (dual TLRs agonist) and control (OVA + aluminum hydroxide) group.

Figure 9

The effect of co-administration of poly(lactic-co-glycolic) acid (PLG)A [ovalbumin (OVA)] nanoparticles along with PLGA [monophosphoryl lipid A (MPLA) or resiquimod (R848)]/polyethylenimine core–shell complexed with cytosine–phosphorothioate–guanine oligodeoxynucleotide (CpG ODN) on IgG1 and IgG2a isotype responses in immunized BALB/c mice. The IgG1 and IgG2a antibody titers in the serum samples were measured by enzyme-linked immunosorbent assay. IgG1 (A) and IgG2a (B) isotype antibody titers 4 weeks after primary immunization (mean ± SD with three mice per treatment group). ***P < 0.001, **P < 0.01, and *P < 0.05 (one-way analysis of variance). The asterisks located on top of error bars showed comparison of significance between the main [dual toll-like receptor (TLR) agonist] and control (OVA + aluminum hydroxide) group and the asterisks located on top of the lines showed comparison between the main (dual TLR agonist) groups.

Figure 10

Detection of IL-1β level in the serum in BALB/c mice immunized with various toll-like receptor (TLR) agonists adjuvant formulations along with poly(lactic-co-glycolic) acid (PLGA) [ovalbumin (OVA)] NPs. Serum IL-1β level was determined using enzyme-linked immunosorbent assay. There was no significant difference in IL-1β secretion between different adjuvant formulations in comparison with positive control (OVA + aluminum hydroxide). The asterisks located on top of the lines showed comparison of significance between the main (dual TLRs agonist) and naive (untreated mice) as well as mice immunized with empty PLGA and PLGA (OVA). The data are indicated the mean ± SD (n = 3).