Aptamer-Conjugated Superparamagnetic Ferroarabinogalactan Nanoparticles for Targeted Magnetodynamic Therapy of Cancer

Abstract

Nanotechnologies involving physical methods of tumor destruction using functional oligonucleotides are promising for targeted cancer therapy. Our study presents magnetodynamic therapy for selective elimination of tumor cells in vivo using DNA aptamer-functionalized magnetic nanoparticles exposed to a low frequency alternating magnetic field. We developed an enhanced targeting approach of cancer cells with aptamers and arabinogalactan. Aptamers to fibronectin (AS-14) and heat shock cognate 71 kDa protein (AS-42) facilitated the delivery of the nanoparticles to Ehrlich carcinoma cells, and arabinogalactan (AG) promoted internalization through asialoglycoprotein receptors. Specific delivery of the aptamer-modified FeAG nanoparticles to the tumor site was confirmed by magnetic resonance imaging (MRI). After the following treatment with a low frequency alternating magnetic field, AS-FeAG caused cancer cell death in vitro and tumor reduction in vivo. Histological analyses showed mechanical disruption of tumor tissues, total necrosis, cell lysis, and disruption of the extracellular matrix. The enhanced targeted magnetic theranostics with the aptamer conjugated superparamagnetic ferroarabinogalactans opens up a new venue for making biocompatible contrasting agents for MRI imaging and performing non-invasive anti-cancer therapies with a deep penetrated magnetic field.

Keywords: aptamers; arabinogalactan; drug delivery; magnetic resonance imaging; magnetically induced cell disruption; magnetodynamic therapy; superparamagnetic ferroarabinogalactans.

Figure 1

Synthesis of aptamer-conjugated ferroarabinogalactan nanoparticles (AS-FeAGs). (a) Schematic representation of the synthesis of ferroarabinogalactan nanoparticles and their conjugation with aptamers. (b) A part of the aptamer-arabinogalactan complex. C—Cytosine, G—Guanine, A—Adenine, and T—Thymine. Non-covalent hydrogen bonds between atoms are shown as blue dashed lines. Hydrogen—gray, oxygen—red, carbon—blue, nitrogen—purple, phosphorus—yellow, sodium—pink.

Figure 2

Magnetic properties of ferroarabinogalactan. (a) Pure arabinogalactan does not have a magnetic moment. (b) Subtraction of a diamagnetic curve of solvent from a curve of ferroarabinogalactan solution. (c) The magnetization of superparamagnetic nanodispersed ferroarabinogalactan.

Figure 3

The molecular structure and results of the fragment molecular orbital method (FMO) applied to the aptamer-arabinogalactan complex. (a) Fragments of arabinogalactan and aptamer AS-14 are shown in different colours. (b) Total pair interactions between arabinogalactan (Ar9–Ar13) and aptamer fragments (A1, T1, C1, G1, A2, T2, C2, G2,). All values are in kcal/mol. (c) Main interactions between arabinogalactan (Ar9–Ar13) and aptamer fragments (T1, A1, C1). The total values are shown below each bar. All values are in kcal/mol. Each pair interaction is divided into electrostatic (ES), quantum-mechanical (QM), and van-der-Waals (vdW) components.

Figure 4

Schematic representation of asialoglycoprotein receptor-mediated delivery and cell damage by AS-FrFeAG nanoparticles at a low alternating magnetic field. Due to a magnetic moment (M) of nanoparticles, they oscillate in an alternating magnetic field. AS-FrFeAG binds to fibronectin via aptamer AS-14 and disrupts extracellular matrix; AS-FrFeAG is internalized via asialoglycoprotein receptors and binds to Hsc70 via aptamer AS-42 causing lysosome damage and leakage of proteolytic enzymes resulting in cell lysis.

Figure 5

Effects of AS-FeAG and low frequency alternating magnetic field (LFAMF) in vitro. (a) Fluorescence microscopy of Ehrlich cells with FAM-labeled As-FeAG before and after LFAMF treatment for 10 min represented in reflected light with fluorescence (a1,4), white light with fluorescence (a2,5) and only fluorescence (a3,6). Scale bar: 5 µm. (b1) Flow cytometry analysis of apoptosis 3 h after the treatment using the caspase 3/7 activity; (b2) the level of necrosis determined by propidium iodide accumulation in dead cells. The red curve corresponds to intact cells without staining; the green curve represents the basic level of non-treated cells; purple—to cells treated with FrFeAG (6%); blue—to FrFeAG (4.5%), dark blue—to FeAG (6%), orange—to FeAG (4.7%).

Figure 6

Application of AS-FrFeAG as a contrast agent for tumors in magnetic resonance imaging (MRI). A mice with solid Ehrlich carcinoma transplanted into the right leg (a,b) and brain (c) without contrasting (1); with omniscan (2), and AS-FrFeAG (3) as a contrast agent.

Figure 7

Distribution of AS-FrFeAG in different organs of mice analyzed by MRI before (a) and 15 min (b), 1.5 h (c) after the intravenous administration.

Figure 8

Histological features of the treated tumors. (a) Non-treated Ehrlich carcinoma. The invasive tumor has a solid structure composed of atypical cells with pleomorphic, hyperchromatic nuclei of different shapes and volume and grows into the muscle tissue. No immune response. (b) Treated with an aptamer mix. Carcinoma cells are with cytoplasm vacuolization; lymphocytic infiltration is moderate. (c,d) FrFeAG and LFAMF treated carcinoma has scattered tumor necrosis among the remaining carcinoma tissue with inflammatory infiltration around them. (e,f) AS-FrFeAG in LFAMF caused irreversible damaging effects on the treated tumor, such as karyo and plasmolysis, and karyo and plasmorrhexis. Large tumor necrosis area, on the periphery of which remains small amounts of dead carcinoma cells with destructive changes of the cancer tissue microenvironment. Visible inflammatory infiltration of segmented leukocytes and swellings observed. Magnification × 100.