Combined magnetic nanoparticle-based microRNA and hyperthermia therapy to enhance apoptosis in brain cancer cells

Abstract

A novel therapy is demonstrated utilizing magnetic nanoparticles for the dual purpose of delivering microRNA and inducing magnetic hyperthermia. In particular, the combination of lethal-7a microRNA (let-7a), which targets a number of the survival pathways that typically limit the effectiveness of hyperthermia, with magnetic hyperthermia greatly enhances apoptosis in brain cancer cells.

Keywords: drug delivery; hyperthermia; magnetic nanoparticles; microRNA; nanotechnology.

Figure 1

Magnetic nanoparticle-based microRNA and hyperthermia therapy to enhance the treatment brain cancer. (a) MNP complexes are first delivered to GBM cells, which is enhanced by magnetofection. Once inside the cell, let-7a miRNA is released thereby targeting downstream effectors of HSPs. This sensitizes the cancer cells to subsequent magnetic hyperthermia enhancing apoptosis. (b) MNPs will be complexed with let-7a miRNA using 10 kDa branched PEI via a layer-by-layer approach. (c) TEM micrograph of the MNPs (scale bar = 20 nm). Inset: High resolution TEM micrograph of the MNPs showing the lattice fringes (scale bar = 10 nm). (d) U87-EGFRvIII GBM cells readily uptake MNPs complexed with Cy3-labeled scrambled miRNA following magnetofection (scale bar = 50 μm). Blue = hoescht stained nuclei, red = cy3-labeled scrambled miRNA.

Figure 2

Magnetic nanoparticle-based let-7a delivery. (a) The delivery of let-7a can inhibit targets such as IGF1R, RAS, HMGA2, and c-MYC, which typically promote proliferation and cell survival while inhibiting apoptosis. (b) The delivery of let-7a to U87-EGFRvIII GBM cells significantly down-regulates expected targets of let-7a compared to scrambled miRNA controls as determined by qPCR (*p < 0.05, **p < 0.01). (c) Cell viability as quantified by MTS assay 48 hours after initial transfection with let-7a. Samples were normalized to untreated controls. (d) FACS analysis of Annexin-V and propidium iodide stained cells. (e) qPCR of downstream targets of let-7a compared to scrambled miRNA controls (*p < 0.05).

Figure 3

Magnetic nanoparticle-based magnetic hyperthermia. (a) MTS assay following the induction of magnetic hyperthermia (10 μg/mL MNP) in U87-EGFRvIII GBM cells. Conditions were assayed 48 hours after transfection and normalized to MNP controls (without exposure to AMF). (b) FACS analysis of Annexin-V and propidium iodide stained cells with and without treatment. (c) qPCR illustrates that, following magnetic hyperthermia, caspase-3 is significantly up regulated as are HSPs. Results were normalized to MNP controls without magnetic hyperthermia (*p < 0.05, **p < 0.01, N.S. = no significance). (d) The temperature of the solution was monitored using a fiber optic temperature probe (Lumasense) over the course of magnetic hyperthermia. Control consisted of the same conditions but without MNPs. (e) qPCR shows up regulation of let-7a targets following magnetic hyperthermia. Again, results were normalized to MNP controls in the absence of magnetic hyperthermia (*p < 0.01, **p < 0.001).

Figure 4

Combined magnetic nanoparticle-based let-7a delivery and magnetic hyperthermia therapy. (a) Timeline of combined treatment. (b) Cell viability following combined let-7a delivery and magnetic hyperthermia as quantified by MTS assay (*p < 0.05, **p < 0.01). (c) FACS analysis of combination treated cells compared to controls. (d) Combined let-7a delivery and magnetic hyperthermia results in up regulation of caspase-3 and a decrease in PI3K as well as HSPs as determined by qPCR (*p < 0.05, **p < 0.01). (e) qPCR analysis of let-7a targets following combined therapy (*p < 0.05, **p < 0.01). qPCR results were normalized to MNP-PEI/miRNA/PEI complex controls delivering scrambled miRNA without exposure to magnetic hyperthermia.