Effects of multiple injections on the efficacy and cytotoxicity of folate-targeted magnetite nanoparticles as theranostic agents for MRI detection and magnetic hyperthermia therapy of tumor cells

Abstract

Folate-targeted iron oxide nanoparticles (FA@Fe₃O₄ NPs) were prepared by a one-pot hydrothermal method and then used as cancer theranostic agents by combining magnetic resonance imaging (MRI) and magnetic hyperthermia therapy (MHT). Crystal structure, morphology, magnetic properties, surface functional group, and heating efficacy of the synthesized nanoparticles were characterized by XRD, TEM, VSM, FTIR, and hyperthermia analyses. The results indicated that the crystal structure, magnetic properties, and heating efficacy of the magnetite nanoparticles were improved by hydrothermal treatment. Toxicity of the prepared NPs was assessed in vitro and in vivo on the mammary cells and BALB/c mice, respectively. The results of the in vitro toxicity analysis showed that the FA@Fe₃O₄ NPs are relatively safe even at high concentrations of the NPs up to 1000 µg mL^-1. Also, the targetability of the FA@Fe₃O₄ NPs for the detection of folate over-expressed cancer cells was evaluated in an animal model of breast tumor using MRI analysis. It was observed that T₂-weighted magnetic resonance signal intensity was decreased with the three-time injection of the FA@Fe₃O₄ NPs with 24 h interval at a safe dose (50 mg kg^-1), indicating the accumulation and retention of the NPs within the tumor tissues. Moreover, the therapeutic efficacy of the MHT using the FA@Fe₃O₄ NPs was evaluated in vivo in breast tumor-bearing mice. Hyperthermia treatment was carried out under a safe alternating magnetic field permissible for magnetic hyperthermia treatment (f = 150 kHz, H = 12.5 mT). The therapeutic effects of the MHT were evaluated by monitoring the tumor volume during the treatment period. The results showed that the mice in the control group experienced an almost 3.5-fold increase in the tumor volume during 15 days, while, the mice in the MHT group had a mild increase in the tumor volume (1.8-fold) within the same period (P < 0.05). These outcomes give promise that FA@Fe₃O₄ NPs can be used as theranostic agents for the MRI and MHT applications.

Figure 1

Synthesis process for the preparation of FA@Fe₃O₄ NPs.

Figure 2

(a) XRD spectra of (i) sample A, and (ii) sample B, (b) Magnetization curves of (i) sample A, and (ii) sample B, the inset shows the magnified hysteresis loop of both samples, (c) FTIR spectra of (i) folic acid, (ii) dextran-coated Fe₃O₄ NPs, and (iii) FA@Fe₃O₄ NPs.

Figure 3

(a) TEM image and (b) particle size distribution of the FA@Fe₃O₄ NPs, (c) temperature rise vs. time curves of the magnetic suspension containing FA@Fe₃O₄ NPs (4 mg mL⁻¹), and (d) cell viability of MC4-L2 cells after exposing to the FA@Fe₃O₄ NPs at different concentrations and times.

Figure 4

Chronic toxicity effects of FA@Fe₃O₄ NPs on the major hematological and blood biochemical parameters.

Figure 5

Magnetic resonance images of the mouse with a breast tumor, (a) mouse without injection dose (control), (b) mouse with one injection dose, (c) two injection doses, and d) three injection doses.

Figure 6

MRI signal intensity of the tumor tissue in the breast tumor-bearing mice with several receiving doses.

Figure 7

(a) magnetic hyperthermia unit used for in vivo MHT experiments, (b) Schematic of in vivo magnetic hyperthermia therapy on a mouse, and (c) The mean tumor volume vs days after onset of the treatment for all groups, (*) indicates P < 0.05, (**) indicates P < 0.005.

Figure 8

In vivo MRI experiments.