Comparing the Variants of Iron Oxide Nanoparticle-Mediated Delivery of miRNA34a for Efficiency in Silencing of PD-L1 Genes in Cancer Cells

Abstract

The blocking of programmed death-ligand 1 (PD-L1) in tumor cells represents a powerful strategy in cancer immunotherapy. Using viral vectors to deliver the cargo for inactivating the PD-L1 gene could be associated with host cell genotoxicity and concomitant immune attack. To develop an alternative safe gene delivery method, we designed a unique combination for miRNA34a delivery using a transgene carrier in the form of iron oxide magnetic nanoparticles (IONPs) via magnetofection to downregulate PD-L1 expression in cancer cells. We synthesized IONPs of multiple shapes (IONRs (iron oxide nanorods), IONSs (iron oxide nanospheres), and ITOHs (iron oxide truncated octahedrons)), surface-functionalized with polyethyleneimine (PEI) using the ligand exchange method, as gene delivery systems. Under the guidance of an external magnetic field, PEI@IONPs loaded with plasmid DNA (DNA/PEI@IONPs) encoding GFP showed high transfection efficiency at different weight ratios and time points in A549 and MDA-MB-231 cells. Additionally, the DNA/PEI@IONPs with miRNA34a inserts under a static magnetic field resulted in significant knockdown of the PD-L1 gene, as demonstrated via immunoblotting of the PD-L1 protein. Among the three shapes of IONPs, IONRs showed the highest PD-L1 knockdown efficiency. The genetic expression of miRNA34a was also studied using qPCR and it showed high expression of miRNA in cells treated with PEI@IONRs. Flow cytometry and a live/dead assay confirmed apoptosis after transfection with miRNA34a. To conclude, in this paper, a promising transgene carrier with low cost, negligible cytotoxicity, and high transfection efficiency has been successfully established for miRNA gene delivery in the context of cancer immunotherapy.

Keywords: PD-L1 gene; immunotherapy; iron oxide nanoparticles; miRNA34a; transfection efficiency.

Scheme 1

Illustration of the synthesis of iron oxide nanoparticles (IONPs) with three different morphologies, and surface functionalization with positively charged branched polyethylenimine (B-PEI). Plasmid DNA (pDNA) with miRNA34a inserts is encapsulated on the surface of B-PEI-coated IONPs (PEI@IONPs) with electrostatic interactions as DNA is negatively charged. The PEI@IONPs and pDNA magnetoplexes are transfected into cancer cells using a static external magnet to study gene delivery efficiency. Upon PD-L1 interaction with the programmed cell death protein 1 (PD-1) on T cell surfaces, the T cells become inactive and unable to help the immune system to fight against tumor growth. The PD-L1 present on the tumor cell surfaces is targeted by miRNA34a-encoded pDNA using PEI@IONPs. pDNA internalization and release inside the cell happen mostly via the enhanced permeability and retention (EPR)/proton sponge effects, and the desired PD-L1 gene silencing takes place gradually due to various factors inside the cell.

Figure 1

TEM images of iron oxide nanoparticles displaying monodispersed and diverse morphologies. (a) FeOOH nanorod precursors after hydrolysis of FeCl₃; (b) Fe₃O₄ nanorods after reduction of FeOOH using branched PEI (B-PEI) and tetraethylene glycol (TEG) at 230 °C; (c) Fe₃O₄ nanospheres formed via thermal decomposition of metal-oleate precursors at 320 °C; (d) Fe₃O₄ truncated octahedrons formed via thermal decomposition reaction of Fe(acac)₃, oleic acid, and trioctylamine (Scale bar: 100 nm); (e) the properties of nanoparticles before and after the surface functionalization using B-PEI 10K via a ligand exchange method; and (f) demonstration of the magnetic nature of the Fe₃O₄ nanoparticles using a strong static magnet.

Figure 2

Percentage of cell viability with and without miRNA34a transfection. Cell viability of (a) MDA-MB-231 and (b) A549 after 48 hours of incubation with PEI-functionalized iron oxide nanoparticles (PEI@IONPs) of different morphologies at different concentrations ranging from 0 to 200 𝜇g/mL. The percentage of cell viability was compared with the cells alone, i.e., 0 𝜇g/mL. The data are expressed as mean ± standard deviation (n = 8). (c) The percentage of cell viability after transfection with miRNA34a and without miRNA34a for 48 hours with PEI@IONPs of different morphologies at a weight ratio of 5 (w/w = 5). After miRNA34a transfection, the percentage of cell viability decreased drastically in the cells treated with PEI@IONPs compared to PEI 25K (_black) and PolyMag (_red). (d) Without miRNA34a transfection, the cell viability was higher with PEI@IONPs compared to PEI 25K _(black) and PolyMag (_red). The data are expressed as mean ± standard deviation (n = 3, * p < 0.05, ** p < 0.01)

Figure 3

Transfection efficiency study, involving the delivery of plasmid DNA, using PEI@IONPs. (a) Expression of green fluorescent protein (GFP), observed through fluorescence microscope in MDA-MB-231 and A549 cell lines transfected with GFP-tagged miRNA34a intact pDNA using PEI@IONPs and incubated with and without (w/o) a static magnet, for 45 min, at a DNA/IONP weight ratio of 5 (W/W = 5), compared with PolyMag as a positive control. (b) Corrected total cell fluorescence (CTCF) of MDA-MB-231 and A549 cells calculated using ImageJ software. The results are represented as mean value ± standard deviation (n = 3, ** p < 0.01).

Figure 4

Western blot analysis for PD-L1 protein and its signaling pathways. After transfection with miRNA34a in the (a) MDA-MB-231 and (b) A549 cell lines, the results show the success and higher percentages of knockdown in PD-L1 protein levels after treatment with PEI@IONPs compared to PEI 25K alone. PD-L1 silencing is also seen with PolyMag treatment, mostly due to the cell cytotoxicity after treatment leading to lower concentrations of protein lysate. The signaling proteins AKT and pAKT are downregulated as they are responsible for promoting and regulating PD-L1 in cancer cells. PTEN is seen to be upregulated as it acts as a tumor suppressor for the PD-L1 gene. The apoptosis markers caspase 3, cleaved caspase 3, and caspase 7 show upregulation after transfection with miRNA34a/PEI@IONPs, inducing apoptosis in cancer cells. Protein expression was analyzed via ImageJ using the fold change method.

Figure 5

Live/dead cell staining. (a) A549 and (b) MDA-MB-231 cells transfected with miRNA34a and incubated for 48 hours. Post-transfection cells were stained with calcein-AM/PI staining solution and images were captured using a fluorescence microscope. Scale bar: 100 μm. Cells stained in green indicate live cells and cells stained in red indicate dead cells, probably undergoing apoptosis after transfection. In contrast, cells treated with PEI 25K and PolyMag showed higher cytotoxicity, which caused surface detachment during the staining process after washing with phosphate-buffered saline (PBS), leading to cell loss.

Figure 6

Flow cytometry analysis to study cell apoptosis. After transfection with miRNA34a/PEI@IONPs at a weight ratio of 5 in (a) MDA-MB-231 and (b) A549 cells, flow cytometry analysis showed that the maximum population of cells was located in late apoptosis quadrant Q2 (the upper right corner), indicating the capability of miRNA34a to induce apoptosis in cancer cells. The quantification of flow cytometry percentages in early apoptosis and late apoptosis was performed after transfection of miRNA34a for 48 hours in (c) MDA-MB-231 and (d) A549 cell lines and compared to PolyMag as a positive control. The results are represented as mean value ± standard deviation (n = 3, * p < 0.05, ** p < 0.01).