High-density sub-100-nm peptide-gold nanoparticle complexes improve vaccine presentation by dendritic cells in vitro

Abstract

Nanocarriers have been explored to improve the delivery of tumor antigens to dendritic cells (DCs). Gold nanoparticles are attractive nanocarriers because they are inert, non-toxic, and can be readily endocytosed by DCs. Here, we designed novel gold-based nanovaccines (AuNVs) using a simple self-assembling bottom-up conjugation method to generate high-peptide density delivery and effective immune responses with limited toxicity. AuNVs were synthesized using a self-assembling conjugation method and optimized using DC-to-splenocyte interferon-γ enzyme-linked immunosorbent spot assays. The AuNV design has shown successful peptide conjugation with approximately 90% yield while remaining smaller than 80 nm in diameter. DCs uptake AuNVs with minimal toxicity and are able to process the vaccine peptides on the particles to stimulate cytotoxic T lymphocytes (CTLs). These high-peptide density AuNVs can stimulate CTLs better than free peptides and have great potential as carriers for various vaccine types.

Figure 1

Schematic of gold-based nanovaccine design synthesis. The AuNPs were coated with self-assembled monolayers of 5000-MW PEG-SH. The AuNPs were subsequently conjugated with the desired peptides using EDC and sulfo-NHS as linkers.

Figure 2

Characterization of AuNV conjugation process. (A) The absorbance spectra of the initial peptide AuNP conjugates. The full view shows the 400- to 800-nm range, and the zoom insert shows the peaks between 510 and 545 nm. Preconjugate refers to the carboxyl-PEG-AuNPs. The NH₂OH control refers to capping the active carboxyl groups on the particles with hydroxylamine. The preconjugates and NH₂OH control particles had the same peak, verifying that the conjugation protocol does not alter the absorbance peak. The particles conjugated with peptides show a 2-nm red shift. (B) TEM images of a 30-nm AuNP coated with PEG and a 30-nm gp100 AuNV. The surface of the peptide-coated AuNV appears rougher and thicker (red arrow) than the PEG-coated AuNP, indicating successful conjugation (scale bar = 10 nm).

Figure 3

Image and hyperspectral analysis of BMDC loaded AuNVs. (A) Dark-field and hyperspectral images of DCs loaded with AuNVs or DCs only. Only DCs loaded with AuNVs appeared in the dark-field images with the same exposure time. The hyperspectral images show a spectral shift from purple blue to yellow green when the DCs were loaded with AuNVs (scale bars = 10 um). (B) The average spectral data for BMDCs with or without AuNVs, using each cell as regions of interest. The intensities were calibrated to the lamp spectra baseline.

Figure 4

IFN-γ ELISPOT results from gp100 AuNV induction of pmel-1 splenocytes. At 10¹¹ particles/ml or 25 μg/ml, AuNVs stimulated threefold more IFN-γ secreting cells compared to the free-peptide control. At 10¹⁰ particles/ml or 2.5 μg/ml maximum dose, the gp100 AuNVs exhibited similar effects as the free-peptide control (10 μg/ml) with no significant difference (p = 0.4).

Figure 5

gp100 AuNVs ELISPOT results for conjugation time optimization and comparison of the two-step and one-step methods. (A) The DC-to-pmel-1 splenocyte ELISPOT results for the gp100 AuNVs at different conjugation times. The 1-h method AuNVs gave the most optimal stimulation results between the various incubation times (single asterisk denotes p < 0.05). (B) The DC-to-pmel-1 splenocyte ELISPOT results for a comparison of the two-step and one-step method AuNV (double asterisk denotes p < 0.01).

Figure 6

ELISPOT results comparing efficacies of AuNVs using the modified DC-to-splenocyte assays. (A) The DC-to-OT-I splenocyte IFN-γ ELISPOT data comparing the efficacies of the OVA AuNVs and the free peptides. The AuNVs (max dose 10 μg/ml) were able to induce a significantly stronger response than the free peptides (10 μg/ml) (double asterisk denotes p < 0.01). (B) The DC-to-OT-I and pmel-1 splenocyte IFN-γ ELISPOT data comparing the PEG linker AuNVs and the DNA spacer AuNVs for OVA and gp100 (double asterisk denotes p < 0.01; n.s, not significant). (C) The DC-to-OT-I and pmel-1 splenocyte IFN-γ ELISPOT with the OVA and gp100 AuNVs. Each particle responded to its corresponding splenocyte significantly more than the unmatched AuNV (double asterisk denotes p < 0.01).