The MRI-Visible Nanocomposite Facilitates the Delivery and Tracking of siRNA Loaded DC Vaccine in the Breast Cancer Model

Abstract

Dendritic cell (DC) vaccines have recently been developed for the treatment of various cancers but often do not function as well as expected, primarily due to the highly complex in vivo immune environment. This proof-of-principle study aimed to test the feasibility of modulating the in vivo behaviors of DC vaccines (DCVs) by introducing siRNA-laden magnetic resonance (MR) imaging nanovectors into cells, while providing visible information on their homing to lymph nodes. The N-alkyl-PEI2k-LAC/SPIO nanocomposites were prepared and characterized, showing favorable properties of siRNA transfection and MRI labeling efficiency in DCs. Cell viability assays revealed no observable effects on the survival and phenotype of DCs if the concentration of the complex was within 8 μg Fe/ml. An orthotopic mouse model of breast cancer was developed. The DCVs transfected with IDO siRNA contained nanocomposites were adoptively transferred to start the treatment. MR imaging clearly visualized the homing of DCVs into lymph nodes. At the end of the treatment, DCVs presented significantly better tumor suppression than DCs or PBS (P < 0.05). Generally, the N-alkyl-PEI2k-LAC/SPIO nanocomposites represent a highly efficient MR imaging platform for siRNA transfection that is potentially useful for in vivo tracking of vaccine cells.

Keywords: anticancer immunotherapy; dendritic cell; gene transfection; magnetic resonance imaging; nanocomposite.

Figure 1

Schematic of the experimental procedures.

Figure 2

¹H-NMR spectra of N-alkyl-PEI2k-LAC (DMSO) and N-alkyl-PEI2k (CDCl3).

Figure 3

DLS of SPIO nanocrystals in hexane and N-alkyl-PEI2k-LAC/SPIO nanocomposites.

Figure 4

SEM (A) and TEM (B) images of N-alkyl-PEI2k-LAC/SPIO nanocomposites.

Figure 5

T ₂ relaxation rate as a function of the Fe concentration for N-alkyl-PEI2k-LAC/SPIO nanocomposites at 1.5 T (A); T ₂-weighted MR images of N-alkyl-PEI2k-LAC/SPIO nanocomposites in water (B).

Figure 6

Agarose gel electrophoresis analysis of the N-alkyl-PEI2k-LAC/SPIO/siRNA complexes. (A) At an N:P ratio ≥ 5, the nanocomposites completely bound the siRNA. (B) At a heparin/siRNA mass ratio ≥ 5, a substantial amount of the siRNA was released. (C) Serum stability of the siRNA bound to or free from N-alkyl-PEI2k-LAC/SPIO. The free siRNA or N-alkyl-PEI2k-LAC/SPIO/siRNA complexes were incubated with 50% serum for the indicated times. Heparin was used to release the bound siRNA from the nanocomposites.

Figure 7

CLSM images of immature siRNA-laden DC showing clusters of green fluorescent particles around the DAPI blue-stained nuclei. DIC images display the pseudopodia of the cells.

Figure 8

The siRNA transfection efficiency was altered by the N-alkyl-PEI2k-LAC/SPIO/siRNA concentration (A) and transfection time period (B). Higher concentrations of the complexes or a longer transfection time period increases the Fe content within DCs (n = 5).

Figure 9

Effects of N-alkyl-PEI2k-LAC/SPIO/siRNA transfection on the maturation phenotype and marker expression of DCs. FACS assay showed that siRNA-laden mature DCs expressed similar levels in biomarkers MHC-II, CD80, CD86, and CCR7 to siRNA-free mature DCs, but significantly higher levels than immature DCs in CCR7, CD80 and CD86. A two-tailed unpaired Student’s t test was performed for two-group comparisons, and the results with significant difference were marked in * P < 0.05, **P < 0.01, or *** P < 0.001, respectively. ns, no significance.

Figure 10

In vitro experiments showing the effects of IFN-γ on IDO expression in DCs.

Figure 11

The left hind footpads were injected three times (1 injection/week) with the siRNA/DC vaccine (siRNA/DCVs), DC vaccine (DCVs), DC or PBS. At 24 h after each injection, MR images of all groups but the PBS group showed decreased signals within enlarged popliteal lymph nodes (yellow arrows).

Figure 12

Photos taken on the 6^th day after tumor cell inoculation. Tumors grew fastest in the PBS group, followed by the DC group and the two vaccine groups.

Figure 13

Tumor volumes were compared among the four groups. A significant difference was observed between the PBS group and the other groups (n = 5, P < 0.05). Moreover, measurements at the second to fifth time points showed that tumors in the siRNA/DCV group or the DCV group were both smaller than tumors in the DC group (n = 5, P < 0.05), suggesting the presence of a specific immune response.