title: Multifunctional mitoxantrone-conjugated magnetic nanosystem for targeted therapy of folate receptor-overexpressing malignant cells.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Research Center for Pharmaceutical Nanotechnology, Tabriz, Iran. jbarar@tbzmed.ac.ir.|Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. jbarar@tbzmed.ac.ir.
authors.forename: Jaleh
authors.initials: J
authors.lastname: Barar
authors.affiliation: Research Center for Pharmaceutical Nanotechnology, Tabriz, Iran. vkafil@yahoo.com.|Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. vkafil@yahoo.com.
authors.forename: Vala
authors.initials: V
authors.lastname: Kafil
authors.affiliation: Research Center for Pharmaceutical Nanotechnology, Tabriz, Iran. dr_mr_majd@yahoo.com.
authors.forename: Mostafa Heidari
authors.initials: MH
authors.lastname: Majd
authors.affiliation: Research Center for Pharmaceutical Nanotechnology, Tabriz, Iran. barzegari.abolfazl@gmail.com.
authors.forename: Abolfazl
authors.initials: A
authors.lastname: Barzegari
authors.affiliation: Research Center for Pharmaceutical Nanotechnology, Tabriz, Iran. khani.sajjad@gmail.com.
authors.forename: Sajjad
authors.initials: S
authors.lastname: Khani
authors.affiliation: Research Center for Pharmaceutical Nanotechnology, Tabriz, Iran. johari_ahar@yahoo.com.|Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. johari_ahar@yahoo.com.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Johari-Ahar
authors.affiliation: Research Center for Pharmaceutical Nanotechnology, Tabriz, Iran. d.asgari@yahoo.com.|Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. d.asgari@yahoo.com.
authors.forename: Davoud
authors.initials: D
authors.lastname: Asgari
authors.affiliation: Ludwig Centre for Cancer Research, University of Lausanne, Lausanne, Switzerland. George.Coukos@chuv.ch.
authors.forename: George
authors.initials: G
authors.lastname: Coukos
authors.forename: George
authors.initials: G
authors.lastname: Cokous
authors.affiliation: Research Center for Pharmaceutical Nanotechnology, Tabriz, Iran. yomidi@tbzmed.ac.ir.|Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. yomidi@tbzmed.ac.ir.
authors.forename: Yadollah
authors.initials: Y
authors.lastname: Omidi
mesh_terms: D000970:Antineoplastic Agents / Q000008:administration & dosage* / Q000737:chemistry; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D058973:Folate Receptors, GPI-Anchored / Q000378:metabolism*; D005492:Folic Acid / Q000008:administration & dosage / Q000737:chemistry; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D061986:MCF-7 Cells / Q000187:drug effects; D058185:Magnetite Nanoparticles / Q000008:administration & dosage* / Q000737:chemistry; D018625:Microscopy, Atomic Force; D008942:Mitoxantrone / Q000008:administration & dosage* / Q000737:chemistry; D058990:Molecular Targeted Therapy / Q000379:methods*; D010316:Particle Size; D011092:Polyethylene Glycols / Q000737:chemistry
chemical_list: D000970:Antineoplastic Agents; D058973:Folate Receptors, GPI-Anchored; D058185:Magnetite Nanoparticles; D011092:Polyethylene Glycols; D005492:Folic Acid; D008942:Mitoxantrone