In Vitro Evaluation of Hyperthermia Magnetic Technique Indicating the Best Strategy for Internalization of Magnetic Nanoparticles Applied in Glioblastoma Tumor Cells

Abstract

This in vitro study aims to evaluate the magnetic hyperthermia (MHT) technique and the best strategy for internalization of magnetic nanoparticles coated with aminosilane (SPION_Amine) in glioblastoma tumor cells. SPION_Amine of 50 and 100 nm were used for specific absorption rate (SAR) analysis, performing the MHT with intensities of 50, 150, and 300 Gauss and frequencies varying between 305 and 557 kHz. The internalization strategy was performed using 100, 200, and 300 µgFe/mL of SPION_Amine, with or without Poly-L-Lysine (PLL) and filter, and with or without static or dynamic magnet field. The cell viability was evaluated after determination of MHT best condition of SPION_Amine internalization. The maximum SAR values of SPION_Amine (50 nm) and SPION_Amine (100 nm) identified were 184.41 W/g and 337.83 W/g, respectively, using a frequency of 557 kHz and intensity of 300 Gauss (≈23.93 kA/m). The best internalization strategy was 100 µgFe/mL of SPION_Amine (100 nm) using PLL with filter and dynamic magnet field, submitted to MHT for 40 min at 44 °C. This condition displayed 70.0% decreased in cell viability by flow cytometry and 68.1% by BLI. We can conclude that our study is promising as an antitumor treatment, based on intra- and extracellular MHT effects. The optimization of the nanoparticles internalization process associated with their magnetic characteristics potentiates the extracellular acute and late intracellular effect of MHT achieving greater efficiency in the therapeutic process.

Keywords: AMF; C6 cells; PLL; dynamic magnetic field; extracellular hyperthermia; glioblastoma; intracellular hyperthermia; magnetic nanoparticles; magneto hyperthermia; static magnetic field; superparamagnetic iron oxide nanoparticles.

Figure 1

Experimental design. The first stage (A–C): (A) Aminosilane-coated SPION; (B) Calculation of the SPION_Amine SAR according to magnetic field intensity, frequency, and SPION_Amine diameter; (C) C6 cells after transfection of luciferase and (¥) Selection of the SPION_Amine with the best SAR value. The second stage: (D) Internalization strategies (I–VI) of the SPION_Amine into the C6 cells in (#) three different concentrations (100, 200, and 300 µgFe/mL), with and without filtration, and also with and without magnetic field (static or dynamic) and combined or not with poly-L-lysine (PLL) transfection agent; (£) Selection the C6 cell labeled with SPION_Amine resulting from the best strategy of internalization. The third stage: (E) MHT assay in vitro. The fourth stage: (F) evaluation of MHT therapy efficiency.

Figure 2

Evaluation of the SPION_Amine 50 and 100 nm features for MHT: (A) SPION_Amine size distribution characterization by DLS, SPION_Amine of 50 nm (blue) and 100 nm (red); (B,C) Heating curves acquired using the following values of magnetic field: 50 Gauss (black), 150 Gauss (blue), and 300 Gauss (red) curves combined with the frequencies 305 kHz (dashed line) and 557 kHz (continuous line); (D) Heating curves of SPION_Amine of 50 nm (red dashed line) and 100 nm (back continuous line) in the best condition of magnetic field and frequency; (E,F) Graphic in bars of mean SAR values of the SPION_Amine of 50 nm and 100 nm, respectively, analyzing each one of the three parameters of magnetic field with each frequency.

Figure 3

Bioluminescent Kinetics of the C6Luc according to cell concentrations (represented by lines with solid symbols) and the respective controls (represented by lines with empty symbols). The inside figure shows the BLI signal inferior of 0.20 × 109 photons/s. The BLI signal intensity was represented by color score bar (inferior right corner) and the correspondent BLI intensity of the C6Luc cells concentrations and control were represented using a ROI at 180 min.

Figure 4

Microscopy optical image of 6_Luc labeled with SPION_Amine of 100 nm with following strategies of internalization: 100 (#), 200 (§), or 300 (@) μgFe/mL of SPION_Amine concentration; without PLL transfection agent and without filter (A,F,K); without PLL and with filter (B,G,L); with PLL and without filter (C,H,M); with PLL and with filter (D,I,N); control conditions (E,J,O); without magnet field (A–E), with static magnet field (F–J), and with dynamic magnet field (K–O). Abbreviations: PLL: Polylysine; WM: without magnet; SM: static magnet; DM: dynamic magnet. The images are shown in 10× magnification.

Figure 5

Evaluation of the SPION_Amine polydispersity resuspended in different colloidal solutions used in cellular labeling, in the presence of the transecting agent and the filtering process by the technique of dynamic light scattering. * RPMI culture medium supplemented with 10% fetal bovine serum.

Figure 6

MTT assay for cellular viability evaluation after process of C6_Luc labeling with SPION_Amine for concentrations: of 100 (A), 200 (B) and 300 μgFe/mL (C). Control conditions are demonstrated by the white bars, without PLL condition in light grey bars and with PLL condition in dark grey bars. The conditions that combined the use of filter were represented by bars with fill of vertical lines. Abbreviations: WM: without magnet; SM: static magnet; DM: dynamic magnet.

Figure 7

MHT technique efficiency evaluation. (I) C6_Luc submitted to MHT; (I*) C6_Luc did not submit to MHT; (II) C6_Luc labeled with 100 μgFe/mL SPION_Amine submitted to MHT; (II*) C6_Luc labeled with 100 μgFe/mL SPION_Amine did not submit to MHT; (III) the cellular viability quantification using the BLI intensity signal for each condition. MHT applications in C6_Luc and in C6_Luc labeled with SPION_Amine, where (A,B) heating planning curve, (C,D) C6_Luc heating curve, (E,F) Magnetic field curve, (G,H) frequency of field during MHT. ** p < 0.001 compared with (II) group. Abbreviations: MHT: Magnetic hyperthermia therapy, Luc: Luciferase, min: minutes, SPION: superparamagnetic iron oxide nanoparticles, MF: magnetic field.

Figure 8

C6_Luc cells viability analysis by flow cytometry. (A) C6_Luc without MHT application; (B) C6_Luc submitted to MHT; (C) C6_Luc labeled with 100 μgFe/mL SPION_Amine without MHT application; (D) C6_Luc labeled with 100 μgFe/mL SPION_Amine submitted to MHT at 44 °C; and (E) cell viability in each experimental group. ** p < 0.001 compared with (D) group. Abbreviations: MHT: Magnetic hyperthermia therapy, Luc: Luciferase, min: minutes, SPION: superparamagnetic iron oxide nanoparticles, FITC: Fluorescein isothiocyanate. Q1: early necrotic cells (AnxV+/PI-); Q2: necrotic or late apoptotic cells (AnxV+/PI+); Q3: necrotic cells (AnxV-/PI+); Q4: viable cells (AnxV-/PI-).