Magnetic Enrichment of Dendritic Cell Vaccine in Lymph Node with Fluorescent-Magnetic Nanoparticles Enhanced Cancer Immunotherapy

Abstract

Dendritic cell (DC) migration to the lymph node is a key component of DC-based immunotherapy. However, the DC homing rate to the lymphoid tissues is poor, thus hindering the DC-mediated activation of antigen-specific T cells. Here, we developed a system using fluorescent magnetic nanoparticles (α-AP-fmNPs; loaded with antigen peptide, iron oxide nanoparticles, and indocyanine green) in combination with magnetic pull force (MPF) to successfully manipulate DC migration in vitro and in vivo. α-AP-fmNPs endowed DCs with MPF-responsiveness, antigen presentation, and simultaneous optical and magnetic resonance imaging detectability. We showed for the first time that α-AP-fmNP-loaded DCs were sensitive to MPF, and their migration efficiency could be dramatically improved both in vitro and in vivo through MPF treatment. Due to the enhanced migration of DCs, MPF treatment significantly augmented antitumor efficacy of the nanoparticle-loaded DCs. Therefore, we have developed a biocompatible approach with which to improve the homing efficiency of DCs and subsequent anti-tumor efficacy, and track their migration by multi-modality imaging, with great potential applications for DC-based cancer immunotherapy.

Keywords: DC migration tracking; DC vaccine; antigen delivery; immunotherapy; magnetic targeting..

Figure 1

Synthesis and characterization of α-AP-fmNPs. a) Schematic representation of α-AP-fmNPs. The synthesis of α-AP-fmNPs involved coating oleic acid-stabilized iron oxide nanocrystals with phospholipids according to a solvent exchange method. To load AP, a fusion peptide comprising AP and α-peptide sequences was designed (α-AP). α-AP-fmNPs were formed by self-association between ICG, α-AP, and the iron oxide@phospholipid complexes. b) TEM image of α-AP_gp100-fmNPs. Scale bar represents 100 nm. c) Size distribution of α-AP_gp100-fmNPs determined with DLS. d) Photograph of the α-AP_gp100-fmNPs solution in PBS. e) Absorption measurement of α-AP_gp100-fmNPs and ICG in PBS. f) MR images of PBS solutions of α-AP_gp100-fmNPs with various concentrations. g) Analysis of MR signals versus α-AP_gp100-fmNPs concentration.

Figure 2

Analysis of α-AP-fmNPs uptake by BMDCs and DC2.4 cells and in vitro evaluation of MPF-promoted DC migration. a) Flow cytometric assessment of cellular α-AP-fmNPs uptake. The mean fluorescence intensity (MFI) was recorded and presented as the mean ± SEM (n = 3). b) Effect of α-AP-fmNPs labeling on the viability of BMDCs and DC2.4 cells. c) Prussian blue staining of α-AP-fmNP-internalized DC2.4 cells. d-e) Comparison of intracellular uptake of FAM-α-AP-fmNPs and FAM-AP in DC2.4 cells (d) and BMDCs (e). All experiments were carried out in triplicate. f) Determination of the subcellular localization of α-AP-fmNPs. Confocal imaging of DC2.4 cells incubated with FAM-α-AP-fmNPs for 6 h and stained with LysoTracker and Hoechst 33258. g) Representative images of migrated cells in the absence (-MPF) or presence (+MPF) of MPF. α-AP-fmNP-loaded DC2.4 cells were resuspended in a cell culture flask with a magnet attached to one side of the flask for 24 h, and the photograph of the migrated cells was acquired. h) Calculations of the migrated cells from bright images. Data were acquired from nine fields of view of three independent images. i) Analysis of the migration ability of α-AP-fmNP-loaded BMDCs. The number of the migrated BMDCs in the lower chamber was counted via flow cytometry. All experiments were carried out in triplicate. Data are presented as mean ± SEM (n = 3).

Figure 3

Bimodal imaging of MPF-promoted DC migration to the LN. a) Schematic representation of the α-AP-fmNP/MPF-based strategy for improving BMDCs migration in mice. The α-AP-fmNP-loaded BMDCs were injected into the hind-leg footpad and subjected to MPF treatment for promotion of migration to PLN. b-c) Optical imaging of BMDCs migration at 0 h (b) and 24 h (c) post-injection in the presence (+MPF, bottom panel) or absence (-MPF, top panel) of magnetic field exposure. d) Average fluorescence measurements from c). Data are presented as the mean ± SEM (3-4 mice per group). The experiment was repeated at least three times with similar results. e) Representative MRI images of α-AP-fmNP-treated mice. MRI images reveal the accumulation of iron oxide particles in the PLN (blue arrow: BMDCs alone; red arrow: α-AP-fmNP-loaded BMDCs).

Figure 4

Validation of MPF-assisted in vivo DC migration. a) Representative immunofluorescence images of PLNs from either MPF-treated mice (+MPF, top panels) or control mice (-MPF, bottom panels). The green, blue, and red signals indicate EGFP-expressing BMDCs, B cell zone (Alexa fluor 647 anti-B220), and T cell zone (Alexa fluor 594 anti-CD3), respectively. b) Calculation of the average percentage of migrated cells to PLN by EGFP fluorescence. The EGFP-BMDCs were obtained from EGFP-transgenic mice. The PLNs were dissected and mechanically disrupted to obtain single cell suspensions, and the EGFP-positive cells in these solutions were analyzed by flow cytometry. Each group contained 2-3 mice and the indicated results represent the means ± SEM of three independent experiments.

Figure 5

Immune function assessment. a-b) In vitro OVA-specific CD8⁺ T cell activation by α-AP-fmNP-loaded BMDCs. T cells collected from the OT-I mice were stained with CFSE and co-cultured with mature BMDCs loaded with PBS, AP_OVA, α-AP_OVA, or α-AP_OVA-fmNPs at a T cell: DC ratio of 50: 1. T cell proliferation was measured via CFSE fluorescence dilution using flow cytometry. c) Measurement of IFN-γ concentrations. d-e) Measurement of the killing activity of CTL. Data are presented as the mean ± SEM (n = 3).

Figure 6

Evaluation of in vivo anti-tumor efficiency. a) Flow chart of the treatment schedule for α-AP-fmNPs/MPF-assisted, DC-based tumor immunotherapy. b) Tumor growth curve of immunized mice. c) Photograph of the dissected tumor tissues. Data are presented as the mean ± SEM (p1 = 0.0016; p2 = 0.0018; p3 = 0.0349; p4 = 0.0029; n = 6).