Fatty Acid-Mimetic Micelles for Dual Delivery of Antigens and Imidazoquinoline Adjuvants

Abstract

Vaccine design has undergone a shift towards the use of purified protein subunit vaccines, which offer increased safety and greater control over antigen specificity, but at the expense of immunogenicity. Here we report the development of a new polymer-based vaccine delivery platform engineered to enhance immunity through the co-delivery of protein antigens and the Toll-like receptor 7 (TLR7) agonist imiquimod (IMQ). Owing to the preferential solubility of IMQ in fatty acids, a series of block copolymer micelles with a fatty acid-mimetic core comprising lauryl methacrylate (LMA) and methacrylic acid (MAA), and a poly(ethylene glycol) methyl ether methacrylate (PEGMA) corona decorated with pyridyl disulfide ethyl methacrylate (PDSM) moieties for antigen conjugation were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Carriers composed of 50 mole% LMA (LMA50) demonstrated the highest IMQ loading (2.2 w/w%) and significantly enhanced the immunostimulatory capacity of IMQ to induce dendritic cell maturation and proinflammatory cytokine production. Conjugation of a model antigen, ovalbumin (OVA), to the corona of IMQ-loaded LMA50 micelles enhanced in vitro antigen uptake and cross-presentation on MHC class I (MHC-I). A single intranasal (IN) immunization of mice with carriers co-loaded with IMQ and OVA elicited significantly higher pulmonary and systemic CD8⁺ T cell responses and increased serum IgG titer relative to a soluble formulation of antigen and adjuvant. Collectively, these data demonstrate that rationally designed fatty acid-mimetic micelles enhance intracellular antigen and IMQ delivery and have potential as synthetic vectors for enhancing the immunogenicity of subunit vaccines.

Keywords: Imiquimod; RAFT polymerization; Toll-like receptor 7; block copolymer micelles; mucosal immunity; subunit vaccine.

Figure 1

Fatty acid-mimetic micelles for dual-delivery of antigen and adjuvant. (A) P(PEGMA-co-PDSM)-b-(LMA-co-MAA) polymer with varying LMA composition were synthesized via RAFT polymerization. (B) The hydrophilic shell was composed of PEGMA and a small percentage of thiol-reactive PDSM for the conjugation of antigens. The hydrophobic and fatty acid-mimetic core comprising LMA and MAA drives micelle assembly, creating an optimal environment for IMQ loading. (C) Representative size distribution (number average) measured via DLS of LMA75, LMA50 and LMA25. (D) Representative cell viability of BMDCs after incubation with P(PEGMA-co-PDSM)-b-(LMA-co-MAA) micelles for 24 hours as measured with Alamar Blue assay. The assay was repeated three times in 4 replicates and the viability results were normalized according to the control treatment (untreated cells). Error bars represents standard deviation.

Figure 2

The core composition of fatty acid-mimetic micelles influences the release kinetics of IMQ at physiological, early, and late endosomal pH values. IMQ was encapsulated in (A) LMA75, (B) LMA50, and (C) LMA25 polymer micelles, dialyzed against buffers at pH 7.4, 6.2, and 5.0 and cumulative release was quantified over 48 h. 50% IMQ release values were determined by using exponential one-phase decay nonlinear fit analysis with R² values greater than 0.98. The 50% release time points for LMA75, LMA50, and LMA25 polymer micelles were revealed to be 12.1 ± 0.6, 25.7 ± 0.3, 32.7 ± 0.4 h (at pH 7.4) and 2.6 ± 0.7, 5.1 ± 0.6, 8.4 ± 0.4 h (at pH 5.0), respectively. Graphs represent the means and standard deviation from four individual experiments.

Figure 3

Micellar IMQ delivery enhances NF-κB activity resulting in increased cytokine production. (A) In vitro evaluation of the NF-κB activation in RAW-Blue cells stimulated with 0.1 – 5 μg/mL free and encapsulated IMQ for 24 h. Statistical differences were observed between the loaded polymer micelles and soluble IMQ at higher doses and are represented as (#) LMA75-IMQ vs IMQ; (*) LMA50-IMQ vs IMQ; and (ˆ) LMA25-IMQ vs IMQ. BMDCs were stimulated for 24 h with free and encapsulated IMQ at varying doses (0.2, 1, and 2 μg/mL). A higher IMQ dose, 10 μg/mL, was included in the studies with LMA50-IMQ and LMA25-IMQ being the only two formulations able to achieve this dose. Levels of proinflammatory cytokines (B) TNFα, (C) IL-6, and (D) IL-1β were measured by ELISA. The lower detection limit of each cytokine is represented by a dotted line. Statistical analysis revealed no significant difference between treatment groups at 0.2 and 10 μg/mL for all cytokines. Statistical analysis illustrated by (*) represents significance for each loaded micelle vs free IMQ, while significance amongst polymer micelles are indicated with tracing lines.

Figure 4

Micellar IMQ delivery enhances TLR7-driven BMDC activation and maturation. Flow cytometry was used to measure surface expression of BMDC maturation markers (A) MHC-II, (B) CD86, (C) CD40, and (D) CD80 induced by free and encapsulated IMQ. BMDCs were treated with 2 μg/mL IMQ for 24 h and were evaluated for their relative marker surface expressions (top) and percentage of marker positive cells (bottom). Representative histograms of untreated (control), IMQ, and LMA50-IMQ are shown for each marker. (*) represents the significant difference between micellar-IMQ vs free IMQ, while connected lines illustrate significance amongst different polymer micelles.

Figure 5

Conjugation of OVA to PDSM groups on micelle coronas enables co-loading of antigen and IMQ, resulting in enhanced intracellular uptake and antigen cross-presentation. (A) FITC-labeled OVA was thiolated (4-5 thiols/OVA) (represented as I in the top gel and V in the bottom gel) and reacted with empty (III: OVA-LMA50) or IMQ-loaded micelles (VI: OVA-LMA50-IMQ) at 1:20 (OVA:polymer) ratio and SDS-PAGE was used to assess antigen conjugation and release of OVA upon incubation with TCEP (VII: OVA-LMA50-IMQ + TCEP). A physical mixture of non-thiolated OVA and LMA50 (II: OVA+LMA50) as well as formulations lacking OVA (IV: LMA50 and VIII: LMA50-IMQ) were run as controls. Flow cytometry was used to assess the effect of micellar delivery on the uptake of OVA488 and a model hydrophobic, fluorescent drug, NR by DC2.4 cells after 24 h incubation at 37 °C. (B) Uptake of OVA488: Representative histograms (top) and MFI values (bottom) of DC2.4 cells incubated with OVA488 (red), OVA488-LMA50 (orange), and dual-delivery carriers (OVA488-LMA50-IMQ; green) treatment groups for 24 h. (C) Uptake of NR: DC2.4s treated with NR (red), OVA488-LMA50 (orange), a physical mixture of OVA488 and NR (OVA488+NR) (blue), and dual-delivery carriers (OVA488-LMA50--NR; green) are illustrated in representative histograms (top) and MFI graphs (bottom). (D) The effect of IMQ and/or micellar delivery on MHC-I presentation by DC2.4 cells was assessed via flow cytometry using an antibody that recognizes MHC-I (H-2K^b)-bound SIINFEKL. Antigen cross presentation was illustrated by representative histograms (top) and MFI graphs (bottom) of DC2.4 cells incubated with OVA (red), OVA-LMA50 (orange), OVA+IMQ (blue), and dual-delivery carriers (OVA-LMA50-IMQ; green) for 24 h. Statistical analysis illustrated by (*) represents significance for each ova conjugated-loaded micelle vs control (free antigen or dye) and significance between different groups are shown with connected lines.

Figure 6

IN vaccination with OVA and IMQ-loaded micelles enhances antigen-specific CD8⁺ T cell and IgG antibody responses. Mice were vaccinated IN with either OVA-LMA50-IMQ carriers or a soluble OVA+IMQ formulation. Mice were sacrificed on d 12 after treatment and frequencies of OVA-specific CD8⁺ T cells in the (A) airway fluid (BAL: Tetramer⁺ anti-CD45^-), (B) lung vasculature (lung parenchyma: Tetramer⁺ i.v. anti-CD45⁺), (C) lung interstitium (lung parenchyma: Tetramer⁺ i.v. anti-CD45^-), and (D) spleen were assessed using intravascular (in vivo) CD45⁺ staining coupled ex vivo SIINFEKL-H-2K^b tetramer staining. (E) IgG antibody titer was measured by ELISA from serum collected on d 10 post vaccination. ND: not detectable.