Targeted delivery of anticancer chemotherapeutics such as mitoxantrone (MTX) can significantly intensify their cytotoxic effects selectively in solid tumors such as breast cancer. In the current study, folic acid (FA)-armed and MTX-conjugated magnetic nanoparticles (MNPs) were engineered for targeted eradication of folate receptor (FR)-positive cancerous cells. Polyethylene glycol (PEG), FA and MTX were covalently conjugated onto the MNPs to engineer the PEGylated FA-MTX-MNPs. The internalization studies were performed using fluorescein isothiocyanate (FITC)-labeled FA-decorated MNPs (FA-FITC-MNPs) in both FR-positive MCF-7 cells and FR-negative A549 cells by means of fluorescence microscopy and flow cytometry. The cellular and molecular impacts of FA-MTX-MNPs were examined using trypan blue cell viability and FITC-labeled annexin V apoptosis assays and 4′,6-diamidino-2-phenylindole (DAPI) staining, DNA ladder and quantitative polymerase chain reaction (qPCR) assays. The FR-positive MCF-7 cells showed significant internalization of the FA-FITC-MNPs, but not the FR-negative A549 cells. The FR-positive cells treated with the PEGylated FA-MTX-MNPs exhibited the IC50 values of 3 μg/mL and 1.7 μg/mL, 24 h and 48 h post-treatment, respectively. DAPI staining and DNA ladder assays revealed significant condensation of nucleus and fragmentation of genomic DNA in the FR-positive MCF-7 cells treated with the PEGylated FA-MTX-MNPs as compared to the FR-negative A549 cells. The FITC-labeled annexin V assay confirmed emergence of late apoptosis (>80%) in the FR-positive MCF-7 cells treated with the PEGylated FA-MTX-MNPs, but not in the FR-negative A549 cells. The qPCR analysis confirmed profound cytotoxic impacts via alterations of apoptosis-related genes induced by MTX-FA-MNPs in MCF-7 cells, but not in the A549 cells. Our findings evince that the engineered PEGylated FA-MTX-MNPs can be specifically taken up by the FR-positive malignant cells and effectively demolish them through up-regulation of Bcl-2–associated X protein (Bax) and Caspase 9 and down-regulation of AKt. Hence, the engineered nanosystem is proposed for simultaneous targeted imaging and therapy of various cancers overexpressing FRs.

We report on the synthesis of bifunctional mitoxantrone (MTX)-grafted magnetic nanoparticles (MNPs) modified by dopamine-polyethylene glycol-folic acid (DPA-PEG-FA) for targeted imaging and therapy of cancer. MNPs (∼7–10 nm) were synthesized using the thermal decomposition reaction of Fe(acac)₃. Bromoacetyl (BrAc) terminal polyethylene glycol dopamine (DPA-PEG-BrAc) was synthesized and treated with ethylene diamine to form bifunctional PEG moiety containing dopamine at one end and amino group at the other end (i.e. DPA-PEG-NH₂). It was then reacted with Fe₃O₄ nanoparticles (NPs) to form Fe₃O₄-DPA-PEG-NH₂ NPs. The activated folic acid (FA) was chemically coupled to Fe₃O₄-DPA-PEG-NH₂, forming Fe₃O₄-DPA-PEG-FA. MTX was then conjugated to Fe₃O₄-DPA-PEG-FA, forming Fe₃O₄-DPA-PEG-FA-MTX. Physicochemical characteristics of the engineered MNPs were determined. The particle size analysis and electron microscopy showed an average size of ∼35 nm for Fe₃O₄-DPA-PEG-FA-MTX NPs with superparamagnetic behavior. FT-IR spectrophotometry analysis confirmed the conjugation of FA and MTX onto the MNPs. Fluorescence microscopy, cytotoxicity assay and flow cytometry analysis revealed that the engineered Fe₃O₄-DPA-PEG-FA-MTX NPs were able to specifically bind to and significantly inhibit the folate receptor (FR)-positive MCF-7 cells, but not the FR-negative A549 cells. Based upon these findings, we suggest the Fe₃O₄-DPA-PEG-FA-MTX NPs as an effective multifunctional-targeted nanomedicine toward simultaneous imaging and therapy of FR-positive cancers.