Fluorescent nanodiamonds as innovative delivery systems for MiR-34a replacement in breast cancer

Abstract

Nanodiamonds are innovative nanocrystalline carbon particles able to deliver chemically conjugated miRNAs. In oncology, the use of miRNA-based therapies may represent an advantage, based on their ability to simultaneously target multiple intracellular oncogenic targets. Here, nanodiamonds were tested and optimized to deliver miR-34a, a miRNA playing a key role in inhibiting tumor development and progression in many cancers. The physical-chemical properties of nanodiamonds were investigated suggesting electrical stability and uniformity of structure and size. Moreover, we evaluated nanodiamond cytotoxicity on two breast cancer cell models and confirmed their excellent biocompatibility. Subsequently, nanodiamonds were conjugated with miR-34a, using the chemical crosslinker polyethyleneimine; real-time PCR analysis revealed a higher level of miR-34a in cancer cells treated with the different formulations of nanodiamonds than with commercial transfectant. A significant and early nanodiamond-miR-34a uptake was recorded by FACS and fluorescence microscopy analysis in MCF7 and MDA-MB-231 cells. Moreover, nanodiamond-miR-34a significantly inhibited both cell proliferation and migration. Finally, a remarkable anti-tumor effect of miR-34a-conjugated nanodiamonds was observed in both heterotopic and orthotopic murine xenograft models. In conclusion, this study provides a rationale for the development of new therapeutic strategies based on use of miR-34a delivered by nanodiamonds to improve the clinical treatment of neoplasms.

Keywords: MT: Delivery Strategies; MiR-34a; MicroRNA; breast cancer; gene delivery; gene therapy; miRNA replacement therapy; nanodiamonds; nanomedicine; nanotechnology.

Graphical abstract

Figure 1

Analysis of cytotoxicity by evaluating LDH release induced by plain ND and Expression of miR-34a in breast cancer cell lines assessed by real-time PCR Bars, cytotoxicity of nanoparticles in the MDA-MB-231 (A) and MCF7 (B) cell lines was assessed after treatment for 7, 10, 14, and 17 days, with ND treatment every 3 days. (C and D) Delivery to MDA-MB-231 and MCF7 mediated by both the transfectant agent Lipofectamine 2000 and ND conjugated with different concentrations of miR-34a, 500, 250, 100, and 50 nM, respectively, was reported. Each experiment was repeated at least three times and data are shown as mean ± SD. The samples were statistically analyzed against positive controls in LDH assays and against untreated controls for real-time PCR.∗p ≤ 0.05, ∗∗p ≤ 0.01.

Figure 2

Cell uptake of ND in breast cancer cell lines assessed by FACS Graphical representation of peaks obtained from cell uptake of ND by flow cytometry in MCF7 and MDA-MB-231 cells after 24 h incubation with ND-PEI-miR-34a evaluated at the absorption peak (BL3 channel-633nm wave-length) (A). Bar graph of the fluorescence emission of ND-PEI-miRNA in solution and in MCF7 (B) and MDA-MB-231 (C) breast cancer cell lines. Each experiment was repeated at least three times and data are shown as mean ± SD. ∗p ≤ 0.05, ∗∗p ≤ 0.01 (ND-PEI-miR solution vs. cells treated with ND-PEI-miR).

Figure 3

Cell internalization of ND-PEI and ND-PEI-miR-34a in breast cancer cell lines Intracellular localization of nanoparticles is evident after 2, 6, and 24 h incubation through fluorescence microscopy images (original magnification ×100). Overlay of blue (DAPI, nuclei), red (ND), and green (antibody anti-actin, cytoskeleton) channels in MCF7 (A) and MDA-MB-231 (C) cells. Merge image of brightfield, blue (DAPI, nuclei), and red (ND) channels in MCF7 (B) and MDA-MB-231 (D) cells. Bar graph of the fluorescence emission of ND-PEI and ND-PEI-miRNA in MCF7 (E) and MDA-MB-231 (F) breast cancer cell lines.

Figure 4

Effects of ND-PEI-miR-34a on tumorigenic potential and apoptosis of cancer cell lines Different levels of colony formation after treatment with ND-PEI-miR-34a100nM in MCF7 and MDA-MB-231 breast cancer cell lines (A). In detail, the reported samples are untreated control, ND-PEI, ND-PEI-NC, and ND-PEI-miR-34a 100-nM samples. The bars represent the percentage of colony area after treatment for 15 days (B). Identification of the wells as untreated controls, ND-PEI, ND-PEI-NC, and ND-PEI-miR-34a 500nM samples for MCF7 and MDA-MB-231 breast cancer cell lines (C), with corresponding bars for the evaluation of the percentage of colony area (D). MCF7 and MDA-MB-231 were treated with ND-PEI and ND-PEI-miR-34a for 72 h at concentrations of 100 nM and 500 nM. Early apoptosis was assessed by FACS analysis after labeling with FITC-conjugated annexin V, evidenced by fluorescence peaks with emission in the green channel (BL1 channel) (E). (F) Bars of cells undergoing early apoptosis after treatment with the conjugated nanoparticles in MCF7 and MDA-MB-231. The experiments were performed at least three times and the results were always similar. Data are shown as mean ± SD. Statistical analysis was performed on the samples by comparing CTR with the treated samples and ND-PEI with the treated samples, respectively. ∗p ≤ 0.05, ∗∗p ≤ 0.01.

Figure 5

Effects of miR-34 on Migration of MCF7 and MDA-MB-231 Cells Confluent monolayer of breast cancer cells, transduced with miR-34 by conventional transfectant or treated with ND-PEI-miR-34a, were scratched to create an artificial wound viewed by microscopy (original magnification ×20). (A) miR-34a mimic effects on cell migratory ability was recorded by microscopy at T0 and after 24 and 48 h and analyzed in comparison with untreated controls (CTR), quantitated with ImageJ. (B) MDA-MB-231 cells transduced with miR-34a were assayed in comparison to CTR. Error bars show mean ± SD. ∗p ≤ 0.05, ∗∗p ≤ 0.01.

Figure 6

Effects of ND-PEI-miR-34a on orthotopic and heterotopic xenograft mouse breast cancer models and evaluation of miR-34a levels in brain, lungs, kidneys, spleen, and liver (A) Mice were randomized into three groups (each group n = 8 mouse) and treated intravenously (iv) with vehicle, ND-PEI, and ND-PEI-miR-34a at 20μg/mice twice a week for 4 weeks. MCF7 was inoculated into the CD-1 nude mice and, when the tumors reached a mass of approximately 200 mm³, the treatments were started. Tumor growth was followed by caliber measurements (A). (B) Tumor weight in untreated mice or mice treated with ND-PEI plain and ND-PEI-miRNA was reported at the nadir of the effect. (C) Anti-tumor efficacy of the ND formulations was also evaluated in an orthotopic xenograft model of mammary carcinoma by inoculation of MDA-MB-231 cell line in female NOD SCID mice. Real-time tumor growth was monitored with the IVIS Lumina II CCD camera system (PerkinElmer). Representative pictures (mouse #1 and mouse #2) (C) of in vivo bioluminescence imaging analyzed before administration of compounds (day 0), during the treatments at days 23 and at the end at day 35. Quantitative analysis (D) of in vivo luciferase activity at different time points. Data were acquired and analyzed using the Living Image Software version 4.3 (PerkinElmer). The luminescent signals are expressed as mean ± SD of the total photon flux/s/cm²/steradian (p/s/cm²/sr). Data were analyzed by t test (∗p = 0.04 [day 23]; ∗p = 0.01 [day 35]; n = 8). (E) Subcutaneous tumor xenotransplantation model for breast cancer in mice CD-1 nude with relative expression of miR-34a levels accumulated in brain, lungs, kidneys, spleen, and liver represented by bars. (F) Expression of miR-34 in organs selected for the orthotopic NOD SCID model of tumor xenotransplantation. Statistical analysis was performed on the samples compared with untreated controls.∗p ≤ 0.05, ∗∗p ≤ 0.01.