Polymer nanoparticles for cross-presentation of exogenous antigens and enhanced cytotoxic T-lymphocyte immune response

Abstract

Effective induction of an antigen-specific cytotoxic T lymphocyte (CTL) immune response is one of the key goals of cancer immunotherapy. We report the design and fabrication of polyethylenimine (PEI)-coated polymer nanoparticles (NPs) as efficient antigen-delivery carriers that can induce antigen cross-presentation and a strong CTL response. After synthesis of poly(d,l-lactide-co-glycolide) (PLGA) NPs containing ovalbumin (OVA) by the double-emulsion solvent-evaporation method, cationic-charged PLGA NPs were generated by coating them with PEI. In a methyl tetrazolium salt assay, no discernible cytotoxic effect of PEI-coated PLGA (OVA) NPs was observed. The capacity and mechanism of PEI-coated PLGA (OVA) NPs for antigen delivery and cross-presentation on dendritic cells (DCs) were determined by fluorescence microscopy and flow cytometry. PEI-coated PLGA (OVA) NPs were internalized efficiently via phagocytosis or macropinocytosis in DCs and induced efficient cross-presentation of the antigen on MHC class I molecules via both endosome escape and a lysosomal processing mechanism. The DCs treated with PEI-coated PLGA (OVA) NPs induced a release of IL-2 cytokine from OVA-specific CD8-OVA1.3 T cells more efficiently than DCs treated with PLGA (OVA) NPs. Therefore, the PEI-coated PLGA (OVA) NPs can induce antigen cross-presentation and are expected to be used for induction of a strong CTL immune response and for efficient anticancer immunotherapy.

Keywords:

• antigen delivery
• dendritic cells
• polymer NPs
• vaccine
• cross-presentation

Supplementary materials

Figure S1 Effects of PEI-coated PLGA (OVA) NPs on the maturation of DCs.

Notes: (A, B) DCs were stimulated with PEI-coated PLGA (OVA) NPs (20 μg/mL OVA) or LPS (1 μg/mL) for 6 hours. Expression patterns of DC-maturation markers (MHC I and CD86) were assessed by flow cytometry.

Abbreviations: PEI, polyethylenimine; PLGA, poly(d,l-lactide-co-glycolide); OVA, ovalbumin; NPs, nanoparticles; DCs, dendritic cells; LPS, lipopolysaccharide; CD, cluster of differentiation; NS, not significant.

Figure S2 Effect of cytochalasin D on cell viability.

Notes: DCs were incubated with cytochalasin D for 30 minutes, then pulsed with PEI-coated PLGA (OVA) NPs (20 μg/mL OVA) for 3 hours. Cell viability analyzed by MTS assay. Results expressed as means ± SD of three samples.

Abbreviations: DCs, dendritic cells; PEI, polyethylenimine; PLGA, poly(d,l-lactide-co-glycolide); OVA, ovalbumin; NPs, nanoparticles; MTS, methyl tetrazolium salt; NS, not significant; SD, standard deviation.

Figure S3 Intracellular localization of PEI-coated PLGA (OVA) NPs.

Notes: DCs were stained with 50 nM LysoTracker red and incubated with PLGA (OVA) or PEI-coated PLGA (OVA) NPs (20 μg/mL OVA) for 1 hour. Cells were washed twice and incubated without NPs for an additional 12 hours. Intracellular localization of OVA was determined by fluorescence microscopy. Scale bar 10 μm.

Abbreviations: PEI, polyethylenimine; PLGA, poly(d,l-lactide-co-glycolide); OVA, ovalbumin; NPs, nanoparticles; DCs, dendritic cells; DIC, differential interference contrast; FITC, fluorescein isothiocyanate.

Figure S4 Effects of cross-presentation of PEI-coated PLGA (OVA) NPs.

Notes: For the cross-presentation assay, DCs were incubated with soluble OVA (20 μg/mL), PLGA (OVA), or PEI-coated PLGA (OVA) NPs (20 μg/mL OVA) for 6 hours. Cells were stained with antimouse H-2K^b bound to the OVA_257–264 peptide (clone 25-D1.16). Expression patterns of the H-2K^b–OVA_257–264 complex on DCs were assessed by flow cytometry. The value of each sample represent average percentage of H-2K^b-SIINFEKL-positive cells. Graphs show means ± SD of duplicates. **P<0.01.

Abbreviations: DCs, dendritic cells; PEI, polyethylenimine; PLGA, poly(d,l-lactide-co-glycolide); OVA, ovalbumin; NPs, nanoparticles; SD, standard deviation.

Acknowledgments

This work was supported by a grant from the National Research Foundation of Korea (NRF), funded by the Korean government (MEST; grant 2014R1A2A1A10049960 and 2015R1A2A1A15051980), and a grant from the Korea Health Technology R&D Project, provided through the Korea Health Industry Development Institute, funded by the Ministry of Health and Welfare (grant HI14C2680).

Disclosure

The authors report no conflicts of interest in this work.