title: Folic acid-capped PEGylated magnetic nanoparticles enter cancer cells mostly via clathrin-dependent endocytosis.
abstract: This work is focused on mechanisms of uptake in cancer cells of rationally designed, covalently assembled nanoparticles, made of superparamagnetic iron oxide nanoparticles (SPIONs), fluorophores (doxorubicin or Nile Blue), polyethylene glycol (PEG) and folic acid (FA), referred hereinafter as SFP-FA.
  
  
  
  SFP-FA were characterized by DLS, zetametry and fluorescence spectroscopy. The SFP-FA uptake in cancer cells was monitored using fluorescence-based methods like fluorescence-assisted cell sorting, CLSM with single-photon and two-photon excitation. The SFP-FA endocytosis was also analyzed with electron microscopy approaches: TEM, HAADF-STEM and EELS.
  
  
  
  The SFP-FA have zeta potential below -6mW and stable hydrodynamic diameter close to 100nm in aqueous suspensions of pH range from 5 to 8. They contain ca. 109 PEG-FA, 480 PEG-OCH3and 22-27 fluorophore molecules per SPION. The fluorophores protected under the PEG shell allows a reliable detection of intracellular NPs. SFP-FA readily enter into all the cancer cell lines studied and accumulate in lysosomes, mostly via clathrin-dependent endocytosis, whatever the FR status on the cells.
  
  
  
  The present study highlights the advantages of rational design of nanosystems as well as the possible involvement of direct molecular interactions of PEG and FA with cellular membranes, not limited to FA-FR recognition, in the mechanisms of their endocytosis.
  
  
  
  Composition, magnetic and optical properties of the SFP-FA as well their ability to enter cancer cells are promising for their applications in cancer theranosis. Combination of complementary analytical approaches is relevant to understand the nanoparticles behavior in suspension and in contact with cells.
authors.affiliation: Université François Rabelais de Tours, EA 6295 Nanomédicaments et Nanosondes, UFR des Sciences Pharmaceutiques, 31 avenue Monge, F-37200 Tours, France.
authors.forename: Emilie
authors.initials: E
authors.lastname: Allard-Vannier
authors.affiliation: Université François Rabelais de Tours, EA 6295 Nanomédicaments et Nanosondes, UFR des Sciences Pharmaceutiques, 31 avenue Monge, F-37200 Tours, France.
authors.forename: Katel
authors.initials: K
authors.lastname: Hervé-Aubert
authors.affiliation: Université François Rabelais de Tours, EA 6295 Nanomédicaments et Nanosondes, UFR des Sciences Pharmaceutiques, 31 avenue Monge, F-37200 Tours, France.
authors.forename: Karine
authors.initials: K
authors.lastname: Kaaki
authors.affiliation: Université François Rabelais de Tours, EA 6295 Nanomédicaments et Nanosondes, UFR des Sciences Pharmaceutiques, 31 avenue Monge, F-37200 Tours, France.
authors.forename: Thibaut
authors.initials: T
authors.lastname: Blondy
authors.affiliation: Biological Faculty, Lomonosov Moscow State University, Vorobyevi Gori 1, Moscow 119992, Russia.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Shebanova
authors.affiliation: Biological Faculty, Lomonosov Moscow State University, Vorobyevi Gori 1, Moscow 119992, Russia.
authors.forename: Konstantin V
authors.initials: KV
authors.lastname: Shaitan
authors.affiliation: Biological Faculty, Lomonosov Moscow State University, Vorobyevi Gori 1, Moscow 119992, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya, 16/10, 117997 Moscow, Russia.
authors.forename: Anastasia A
authors.initials: AA
authors.lastname: Ignatova
authors.affiliation: MPMC-Université Pierre et Marie Curie, Case courrier 115, 4 place Jussieu, F-75252 Paris - Cedex 5, France; Functional Nano & Soft Materials Laboratory (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Soochow University, Suzhou 215123, China.
authors.forename: Marie-Louise
authors.initials: ML
authors.lastname: Saboungi
authors.affiliation: Biological Faculty, Lomonosov Moscow State University, Vorobyevi Gori 1, Moscow 119992, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya, 16/10, 117997 Moscow, Russia.
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Feofanov
authors.affiliation: Université François Rabelais de Tours, EA 6295 Nanomédicaments et Nanosondes, UFR des Sciences Pharmaceutiques, 31 avenue Monge, F-37200 Tours, France. Electronic address: igor.chourpa@univ-tours.fr.
authors.forename: Igor
authors.initials: I
authors.lastname: Chourpa
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D021941:Caveolae / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D004317:Doxorubicin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D004337:Drug Carriers; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005260:Female; D005492:Folic Acid / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D061986:MCF-7 Cells; D008280:Magnetics / Q000379:methods*; D058185:Magnetite Nanoparticles / Q000737:chemistry; D018613:Microscopy, Confocal; D017348:Microscopy, Electron, Scanning Transmission; D036641:Microscopy, Fluorescence, Multiphoton; D050997:Nanomedicine / Q000379:methods*; D011092:Polyethylene Glycols / Q000737:chemistry*; D046590:Spectroscopy, Electron Energy-Loss; D002583:Uterine Cervical Neoplasms / Q000188:drug therapy / Q000378:metabolism*
chemical_list: D000903:Antibiotics, Antineoplastic; D002966:Clathrin; D004337:Drug Carriers; D058185:Magnetite Nanoparticles; D011092:Polyethylene Glycols; D004317:Doxorubicin; D005492:Folic Acid
keywords: Caveolae-dependent endocytosis (CvDE); Clathrin-dependent endocytosis (CDE); Confocal laser-scanning microscopy (CLSM); Electron energy loss spectroscopy (EELS); Folic acid (FA); High angle annular dark field scanning transmission electron microscopy (HAADF-STEM); Superparamagnetic iron oxide nanoparticles (SPIONs); Two-photon excited (TPE) fluorescence