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|a Engineering of a physiologically compatible, stable and targetable delivery vehicle superparamagnetic iron oxide nanoparticles-Bromelain-folic acid (SPIONs-Br- FA) was reported. Initially, the synthesized bare SPIONs were coated with citric acid (CA) in order to increase biocompatibility, stability and solubility of the SPIONs. Moreover, through CA coating, carboxyl functional groups for further reactions were produced. Br (as an anti-cancer agent) and FA (as a targeting agent to the folic acid receptor positive (FAR+) cancer cells) were conjugated to the synthesized nanocarrier through 1-ethyl-3-(3-dimethylaminpropyl)carbodiimide hydrochloride/ Nhydroxysuccinimide (EDC/NHS) click chemistry. Subsequently, characterization and physico-chemical analyses were carried out through methods such as Fourier transform infrared spectroscopy, atomic absorption spectroscopy (AAS), dynamic light scattering, vibrating sample magnetometer, x-ray diffraction, transmission electron microscopy (TEM) and field emission scanning electron microscopy. The in vitro tetrazolium dye (MTT) assay and blood compatibility tests were performed to confirm the biocompatibility of the engineered nano delivery system. High level of SPIONs-FA binding to FAR+ cell lines (HeLa, MDA-MB-231 and 4 T1) compared to folic acid receptor negative (FAR-) cell lines (HSF 1184 and MDA-MB-468) was assured via qualitative and quantitative in vitro binding studies (Prussian blue assay and AAS analysis). The reason may be higher transport of SPIONs-FA through the mechanism of receptor endocytosis pathway into FAR+ cells in comparison with the mechanism of passive diffusion of SPIONs into the FAR- cells. Cytotoxicity studies carried out in human cell lines (HSF 1184, MDA-MB-468, MDA-MB-231 and HeLa) and mouse breast cancer cells (4 T1) showed significant dose advantage with SPIONs-Br-FA in reducing the half maximal inhibitory concentration (IC50) values compared with neat Br. Through morphological observation studies by inverted microscope and acridine orange/ethidium bromide fluorescent staining method, it was disclosed that the cells had undergone apoptosis since the shrinkage as well as the apoptotic bodies were obviously seen. The results showed that SPIONs-Br-FA was a rewarding candidate to suppress the migration of the FAR+ cancer cells as well as to inhibit colony formation of the FAR+ cancer cells compared to neat Br. The percentage of apoptotic cells (apoptotic index) with more condensed and fragmented chromatin increased sharply in SPIONs-Br-FA treated cells compared to the neat Br. Overall, the SPIONs-Br-FA induced higher percentage of apoptotic cells than the neat Br. Moreover, after treatment protocol performance on 4 T1 tumor bearing mice, the qualitative and quantitative biodistribution study were carried out in vital organs and tumor using colorimetric method (AAS) and TEM method which indicate significant tumor targetability of SPIONs-FA. Finally, the tumor volume and inhibition growth rate were measured in 4 T1 tumor bearing mice treated with different SPIONs formulations to investigate the effectiveness of SPIONs- Br-FA. Administration of SPIONs-Br-FA through tail vein (three times a week) during the four-week treatment period reduced the tumor burden of tumor bearing mice and also increased their life-span when compared with SPIONs-Br and neat Br at same concentration of bromelain. In conclusion, the current results indicated the dualfunctional synthesized SPIONs-Br-FA is a promising tool in the field of biomedicine, chiefly cancer therapy.
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