Immunostimulatory nanoparticles delivering cytokines as a novel cancer nanoadjuvant to empower glioblastoma immunotherapy

Abstract

Glioblastoma (GBM) stands as a highly aggressive and deadly malignant primary brain tumor with a median survival time of under 15 months upon disease diagnosis. While immunotherapies have shown promising results in solid cancers, brain cancers are still unresponsive to immunotherapy due to immunological dysfunction and the presence of a blood-brain barrier. Interleukin-12 (IL-12) emerges as a potent cytokine in fostering anti-tumor immunity by triggering interferon-gamma production in T and natural killer cells and changing macrophages to a tumoricidal phenotype. However, systemic administration of IL-12 toxicity in clinical trials often leads to significant toxicity, posing a critical hurdle. To overcome this major drawback, we have formulated a novel nanoadjuvant composed of immunostimulatory nanoparticles (ISN) loaded with IL-12 to decrease IL-12 toxicity and enhance the immune response by macrophages and GBM cancer cells. Our in vitro results reveal that ISN substantially increase the production of pro-inflammatory cytokines in GBM cancer cells (e.g. 2.6 × increase in IL-8 expression compared to free IL-12) and macrophages (e.g. 2 × increase in TNF-α expression and 6 × increase in IL-6 expression compared to the free IL-12). These findings suggest a potential modulation of the tumor microenvironment. Additionally, our study demonstrates the effective intracellular delivery of IL-12 by ISN, triggering alterations in the levels of pro-inflammatory cytokines at both transcriptional and protein expression levels. These results highlight the promise of the nanoadjuvant as a prospective platform for resharing the GBM microenvironment and empowering immunotherapy.

Keywords: Cancer nanoadjuvant; Glioblastoma; IL-12; Immunostimulatory cytokines; Immunotherapy; Nanoparticles.

Fig. 1

Morphology of immunostimulatory nanoparticles (ISN). The morphology of the nanoparticles was analyzed by A, C transmission electron microscopy (TEM) and B, D scanning electron microscopy (SEM). Scale bar of TEM microphotographs: 2 µm (B, C) and 500nm (D, E). Scale bar of SEM microphotographs: 2 µm

Fig. 2

Development of empty cationic polymeric nanoparticles. Empty CPN were analyzed by A particle size, B polydispersity index (PdI), and C zeta potential. Different concentrations of DOTAP were incorporated into the PLGA matrix: 0, 5, and 15% (w/w). All data are presented as means ± SD. Graphs represent pooled data from at least three independent experiments. D Representative SEM microphotographs of CPN with 0, 5, and 15% DOTAP. Scale bar: 2 µm. Representative TEM microphotographs of empty CPN with 0, 5, and 15% of DOTAP. Scale bar: 500nm

Fig. 3

Immunostimulatory nanoparticles are less cytotoxic than cationic polymeric nanoparticles. A Percentage of metabolic activity measured by reduction of MTT after 24 h of exposure of free IL-12, ISN, and PLGA at 20 ng/mL in U-87 MG and J774A.1 cells. The metabolic activity of empty and loaded CPN was performed at 5 ng/mL. B Percentage of cell viability measured by LDH assay after 24 h of exposure of the previous treatment groups. The same concentrations were used in the LDH assay. In all graphs, bars represent mean values ± SEM (n = 8 values per condition with 3 biological experiments). Statistically significant differences among the groups (one-way ANOVA post-test for multiple comparisons). ns, non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, determined as described in the “Materials and methods” section

Fig. 4

Immunostimulatory nanoparticles induced a higher release of pro-inflammatory cytokines. A Release of inflammatory cytokines in the cell culture media, interleukin-6 (IL-6) and interleukin-8 (IL-8), by U-87 MG cancer cells exposed to free IL-12, ISN, PLGA NPs at 20 ng/mL for 24h. Cells treated with lipopolysaccharide (LPS) were used as a positive control for inflammation. A supernatant was collected, and an enzyme-linked immunosorbent assay (ELISA) was performed as described in the "Materials and methods" section. B Release of inflammatory cytokines in the cell culture media, interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNFα), by J77A.1 cells exposed to free IL-12, ISN, PLGA NPs for 24h. In all graphs, bars represent mean values ± SEM (n = 3 biological replicates). ns, non-significant, *p < 0.05, **p < 0.01, ****p < 0.0001, determined as described in the "Materials and methods"

Fig. 5

ISN induced an increase in gene expression for IL6 and IL8 genes upon exposure to U-87 MG cancer cells. A Real-time qRT-PCR results representing the expression of IL-6 and B IL-8 upon 24h of exposure to free IL-12, ISN, PLGA NPs, and LPS in human glioblastoma cancer cells (U-87 MG cell line). In all graphs, bars represent mean values ± SEM (n = 3 biological replicates). Statistically significant differences among the groups (one-way ANOVA post-test for multiple comparisons). *p < 0.05

Fig. 6

Immunostimulatory nanoparticles led to a decrease in intracellular IL-12. U-87 MG cancer cells were treated with medium (control), free IL-12, empty PLGA NPs, and ISN at a concentration of 20 ng/mL, and a quantitative immunophenotyping analysis by flow cytometry was performed after (A) 24h and (B) 48h of incubation. Bar graphs represent the median fluorescence intensity (MFI) of IL12 normalized to the control. The data are expressed as mean ± SD of three independent biological experiments. Statistically significant differences among the groups (one-way ANOVA post-test for multiple comparisons). ns, non-significant, *p < 0.05