Butylated hydroxyanisole (BHA) is a synthetic phenolic food additive that is used to prevent oils, fats, and shortenings from oxidative deterioration and rancidity. It was tested for potential cytotoxicity and genotoxicity upon A549 lung cancer cells. MTT assay and flow cytometry analysis were used for cytotoxicity assessment, whereas genotoxicity was evaluated in vitro by DAPI staining, alkaline comet, and DNA fragmentation assays. BHA dose- and time-dependently decreased the growth of A549 cells with an IC50 of ∼0.55, 0.4, and 0.3 mM BHA at 24, 48, and 72 h, respectively. Primary and late apoptosis in the treated cells was determined using the flow cytometry analyses. In addition, single-strand DNA breakage has been observed through the comet assay technique. In addition, the morphology of DAPI-stained cells and DNA fragmentation assay using gel electrophoresis showed clear fragmentation in the chromatin and DNA rings within the nucleus of cells treated with BHA.
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.
BackgroundTargeted 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.ResultsThe 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.ConclusionOur 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.
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