Enhancement of MHC-I antigen presentation via architectural control of pH-responsive, endosomolytic polymer nanoparticles

Abstract

Protein-based vaccines offer a number of important advantages over organism-based vaccines but generally elicit poor CD8(+) T cell responses. We have previously demonstrated that pH-responsive, endosomolytic polymers can enhance protein antigen delivery to major histocompatibility complex class I (MHC-I) antigen presentation pathways thereby augmenting CD8(+) T cell responses following immunization. Here, we describe a new family of nanocarriers for protein antigen delivery assembled using architecturally distinct pH-responsive polymers. Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize linear, hyperbranched, and core-crosslinked copolymers of 2-(N,N-diethylamino)ethyl methacrylate (DEAEMA) and butyl methacrylate (BMA) that were subsequently chain extended with a hydrophilic N,N-dimethylacrylamide (DMA) segment copolymerized with thiol-reactive pyridyl disulfide (PDS) groups. In aqueous solution, polymer chains assembled into 25 nm micellar nanoparticles and enabled efficient and reducible conjugation of a thiolated protein antigen, ovalbumin. Polymers demonstrated pH-dependent membrane-destabilizing activity in an erythrocyte lysis assay, with the hyperbranched and cross-linked polymer architectures exhibiting significantly higher hemolysis at pH ≤ 7.0 than the linear diblock. Antigen delivery with the hyperbranched and cross-linked polymer architecture enhanced in vitro MHC-I antigen presentation relative to free antigen, whereas the linear construct did not have a discernible effect. The hyperbranched system elicited a four- to fivefold increase in MHC-I presentation relative to the cross-linked architecture, demonstrating the superior capacity of the hyperbranched architecture in enhancing MHC-I presentation. This work demonstrates that the architecture of pH-responsive, endosomolytic polymers can have dramatic effects on intracellular antigen delivery, and offers a promising strategy for enhancing CD8(+) T cell responses to protein-based vaccines.

Fig. 1

pH-responsive nanoparticles assembled using polymer chains of different architectures were utilized as carriers for delivery of protein antigen into the MHC-I antigen processing pathway. a pH-responsive diblock copolymers were synthesized with linear, core-crosslinked, or hyperbranched architectures. The pH-responsive, endosomolytic component (red) is a copolymer of butyl methacrylate (BMA) and 2-(N,N-diethylamino)ethyl methacrylate (DEAEMA) which was chain extended with a copolymer of dimethylacrylamide (DMA) doped with a pyridyl disulfide functionalized monomer (PDSMA) for antigen conjugation. b Polymer chains of all architectures assembled into nanoparticles under aqueous conditions at neutral pH, PDS groups were used for conjugation of protein antigen via disulfide exchange. c Nanocarriers composed of architecturally distinct polymer chains were evaluated for their ability to enhance MHC-I antigen presentation by dendritic cells

Scheme 1

Synthesis of [BMA-co-DEAEMA]-b-[DMA-co-PDSMA] polymers of linear (a), hyperbranched (b), or core-crosslinked (c) architecture. Block extension of these architectures by DMA and PDSMA (d)

Fig. 2

Evolution plot of polydispersity (Ð) and molar mass progression (M _n (GPC)) with DEAEMA conversion vs. calculated molar mass (M _n (calc)) for DEAEMA-co-BMA polymers (top) of linear (a), cross-linked (b), and hyperbranched (c) architecture. Representative molar mass distributions (bottom) of DEAEMA-co-BMA mCTA (dotted line) and [DEAEMA-co-BMA]-b-[DMA-co-PDSMA] diblock copolymer (solid line) of linear (a), hyperbranched (b), and cross-linked (c) architectures

Fig. 3

Dynamic light scattering (DLS) characterization of pH-responsive diblock copolymers (high pH-responsive polymer content; see Table I) in aqueous solution. a Representative size distribution (number average diameter) of particles at pH 7.4 in HEPES-buffered glucose. b Particle size measurements of copolymers as a function of pH (0.5 mg/mL in 10 mM sodium phosphate buffer with 150 mM NaCl)

Fig. 4

Erythrocyte lysis assay demonstrating pH-dependent membrane-destabilizing activity of copolymers with linear, cross-linked, and hyperbranched architectures. a Total polymer concentration fixed at 1.25 μg/mL. b Polymer concentration normalized to 1.25 μg/mL of the pH-responsive element (DEAEMA-co-BMA). c Hemolytic activity of polymers with higher mass fraction of hydrophilic DMA-co-PDSMA block (lower pH-responsive content), normalized to 1.25 μg/mL of the pH-responsive element. Data represent mean ± SD, n = 4

Fig. 5

Antigen conjugation to pH-responsive polymer nanoparticles via disulfide exchange reaction. a Size of nanoparticles (number average diameter) by dynamic light scattering after reaction with thiolated ovalbumin (ova-SH) at 0, 0.35, 0.175, and 0.0875 mg ova/mg polymer. b SDS-PAGE of fluorescently labeled ovalbumin (ova) and ova-nanoparticle conjugates (0.35 mg ova/mg polymer) prepared using linear (L), hyperbranched (HB), or cross-linked (CL) polymers. Incubation of conjugates with TCEP liberates ova from the carrier. c SDS-PAGE of fluorescently labeled ova and ova-nanoparticle conjugates (c) and a physical mixture (m) of polymers and non-thiolated ova

Fig. 6

a Effect of polymer architecture on MHC class I presentation in an in vitro co-culture model. Murine dendritic cells (DC2.4) were incubated with free antigen (ova) or conjugates prepared using polymers of different architecture and subsequently co-cultured with B3Z T cells which produce β-galactosidase in response to antigen presentation on MHC-I. Data are from a single representative experiment conducted in quadruplicate (mean ± standard deviation). b Uptake of Alexa Fluor 488-labeled ova measured by DC2.4 cells after 5 h at 37°C as measured by flow cytometry (MFI median fluorescent intensity). Data are from a single experiment conducted in triplicate (mean ± standard deviation)