Synthesis and characterization of hyperthermia induced spions-peg-her in targeted breast cancer

Abstract

Hyperthermia has opened up key avenues in cancer therapy. Nevertheless, engineering a smart and efficient tumor targeting superparamagnetic fluid agent capable of elevating the temperature of targeted sites as well as exposing a safe level of biocompatibility remain remarkably demanding and challenging. In this study, a novel core-shell tumor-targeting superparamagnetic iron oxide nanoparticle-polyethylene glycol-Herceptin (SPIONs-PEG-HER) was developed and evaluated for an efficient hyperthermia treatment of HER2+ breast cancer using an alternating magnetic field (AMF). Both in vitro and in vivo treatment models using four different cell lines and 7, 12 dimethylbenz (a) anthracene (DMBA)-induced balb/c mice were developed, respectively. SPIONs modification was carried out by PEGylation to provide biocompatibility and conjugation of Herceptin to add the tumortargeting features to the SPIONs. The morphological characterization and physico-chemical analyses of SPIONs-PEG-HER, were carried out using transmission electron microscopy (TEM), field emission scanning electron microscopy, dynamic light scattering (DLS), x-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), vibrating sample magnetometer (VSM) and specific absorption rate (SAR). The in vitro biocompatibility confirmation of SPIONs-PEG-HER was performed using tetrazolium dye (MTT) assay, Trypan blue staining method and blood compatibility tests. The ability of SPIONs-PEG-HER binding to HER2+ cell line (SK-BR-3) was measured comparatively to HER2- cell lines (HSF 1184, MDA-MB-231, MDA-MB-468) via in vitro binding studies. The qualitative and quantitative data were obtained using Prussian blue staining, TEM assay and atomic absorption spectroscopy (AAS) analysis. The comparison on the changes in growth inhibition rates in HSF 1184, MDA-MB-231, MDA-MB-468 and SK-BR-3 cell lines at different temperatures (40 °C, 42 °C, and 45 °C) were studied using a post-hyperthermia MTT assay. Then, post-hyperthermia morphological investigations were performed via inverted microscope and acridine orange/ethidium bromide (AO/EB) staining method. The in vivo model using DMBA-induced balb/c mice were injected with SPIONs-PEG-HER via tail vein. The biodistribution of SPIONs-PEG-HER at tumor site as well as in vital organs were qualitatively and quantitatively measured using colorimetric methods (AAS and TEM). Finally, the survival rate of the DMBA-induced balb/c mice injected with SPIONs-PEG-HER was measured in the presence and absence of AMF and the daily monitoring of the weight of the treated DMBA-induced babl/c mice during the treatment period was carried out. The morphological characterization and physico-chemical analyses revealed that the synthesized SPIONs- PEG-HER had a size of almost 17 nm and possessed a nearly spherical appearance as well as greater hydrodynamic diameter (~84 nm) and a wider distribution compared to the bare SPIONs. Moreover, XRD and FT-IR analyses confirmed that the processes of PEGylation and conjugation were successfully accomplished while the VSM and SAR analyses showed that the SPIONs-PEG-HER possess efficient magnetic properties to be used as a hyperthermia fluid agent. SPIONs-PEG-HER showed relatively low levels of toxicity even at the extremely high concentration of 1000 µg/ml. The results of the binding studies indicated that the SPIONs-PEG-HER could selectively bind to the HER2+ cancer cells. The results obtained from the post-hyperthermia MTT assay indicated that exposing the HER2+ cells to the temperature of 45 °C for 20 minutes inhibited the growth of the cells by 90% where they did not regain their normal proliferation like the HER2- cell lines which corroborates the results obtained through post-hyperthermia morphological analyses where it was shown that the temperature of 45 °C induced significant apoptosis compared to the other temperatures. Through morphological alteration studies by inverted microscope and AO/EB staining method, it was disclosed that the SK-BR-3 cells had undergone apoptosis since apoptotic signs such as shrunk cells as well as apoptotic bodies were obviously seen. The results of biodistribution studies showed significantly higher accumulation of the SPIONs-PEG-HER in the tumor site compared to SPIONs- PEG. Based on the results obtained through hyperthermia treatment of DMBA-induced balb/c mice, it was revealed that the survival rate in the experimental group treated with SPIONs-PEG-HER in the presence of AMF was much higher than other experimental groups where 50% of the DMBA-induced balb/c mice survived and maintained their average body weight. Findings in this study illustrated that SPIONs-PEG-HER-mediated hyperthermia is a potent breast cancer treatment.