Enhanced anti-tumor activity of transferrin/folate dual-targeting magnetic nanoparticles using chemo-thermo therapy on retinoblastoma cancer cells Y79

Abstract

Malignant neoplasms are one of the main causes of death, especially in children, on a global scale, despite strenuous efforts made at advancing both diagnostic and therapeutic modalities. In this regard, a new nanocarrier Vincristine (VCR)-loaded Pluronic f127 polymer-coated magnetic nanoparticles conjugated with folic acid and transferrin (PMNP-VCR-FA-TF) were synthesized and characterized by various methods. The cytotoxicity of these nanoparticles was evaluated in vitro and ex vivo conditions. The in vitro anti-tumor effect of the nanoparticles was evaluated by colony formation assay (CFA) and reactive oxygen species (ROS) in Y79 cell line. The results showed that nanoparticles with two ligands conferred greater toxicity toward Y79 cancer cells than ARPE19 normal cells. Under an alternating magnetic field (AMF), these nanoparticles demonstrated a high specific absorption rate. The CFA and ROS results indicated that the AMF in combination with PMNP-VCR-FA-TF conferred the highest cytotoxicity toward Y79 cells compared with other groups (P < 0.05). PMNP-VCR-FA-TF could play an important role in converting externally applied radiofrequency energy into heat in cancer cells. The present study confirmed that dual targeting chemo-hyperthermia using PMNP-VCR-FA-TF was significantly more effective than hyperthermia or chemotherapy alone, providing a promising platform for precision drug delivery as an essential component in the chemotherapy of retinoblastoma.

Figure 1

Synthetic steps of SPIONs, FA-F127, and COOH-F127 (A) and schematic illustration of final nanoparticles preparation (B).

Figure 2

The 1HNMR spectra of (A) carboxyl functionalized Pluronic F127 polymer and (B) FA-conjugated Pluronic F-127 polymer in CDCl_3, FT-IR spectrum of Transferrin conjugated nanoparticles (PMNP-FA-TF) (C), nanoparticle size distribution of PMNP-FA-TF (D), PMNP-VCR-FA-TF (E).

Figure 3

FE-SEM images of final nanoparticles PMNP-FA-TF with two magnifications (A,B), EDX elemental mapping including Fe, O, C, and N (C–F, respectively), Elemental composition (G), and XRD pattern of final nanoparticles (H).

Figure 4

Properties of nanoparticles. TEM images of PMNP-Tf-FA (A) and PMNP-Tf-FA-VCR (B), In vitro vincristine release profile in 37 °C (C), Thermometry of different concentrations of PMNPs and control groups by IR camera during 12 min (D).

Figure 5

Hemolysis caused by different concentration of nanoparticles. (A) Image of samples after centrifugation at 1500 rpm for 5 min: Negative control (PBS), positive control (water), and nanoparticle suspension (4, 2, 1, 0.5, 0.25, 0.125, 0.0625 and 0.3125 mg/mL). (B) Evaluation of the percentage of hemolysis of RBCs induced by nanoparticles.

Figure 6

Cytotoxicity of different concentration of VCR, PMNP-VCR-FA-TF and PMNP-FA-TF after 48 h incubation with Y79 (A) and ARPE-19 (B) cell lines.

Figure 7

IR camera images of Y79 cells treated with and without nanoparticles under AMF (70 W, 13.56 MHz, 14 min) (A), Heating Profile of Y79 cells during AMF exposure with and without PMNP incubation (B).

Figure 8

Percentage of DCF fluorescence intensity that represents the intracellular ROS after different treatments (****P < 0.0001).

Figure 9

Colony formation assay after treatment whit two types of NPs (PMNP-VCR-FA-TF and PMNP-FA-TF) and vincristine with and without AMF hyperthermia (*P < 0.05, ***P < 0.001, ****P < 0.001) (A). Image of Y79 cell colonies under light microscope with 10 × magnification (B).