title: Hollow microspheres based on - Folic acid modified - Hydroxypropyl Cellulose and synthetic multi-responsive bio-copolymer for targeted cancer therapy: controlled release of daunorubicin, in vitro and in vivo studies.
abstract: OBJECTIVE
  Conventional chemotherapy drugs such as anthracyclines show no specific activity. They destroy cancer cells but also and the healthy ones, and for that reason exhibit high toxicity. In order to alleviate the toxic effects of chemotherapeutic drugs, the administration dose is being minimized, while their reactivity against tumor cells is lessened. This problem can be overcome or at least reduced by using nanoscale drug delivery systems to target the pathogenic area. The present work deals with the synthesis, characterization and biological evaluation of multi-responsive hollow microspheres coated with Hydroxypropyl Cellulose (HPC)-a biocompatible and thermosensitive polysaccharide-conjugated with folic acid as well promising drug vehicles for targeted cancer therapy.
  
  
  METHODS
  The synthetic route consists of two steps. In the first step, a single layer of sensitive copolymers is ((Methacrylic acid (MAA), N-(2-Hydroxypropyl) methacrylamide (HPMA) and N,N'-(disulfanediylbis(ethane-2,1-diyl))bis(2-methylacrylamide) (DSBMA)) fabricated on a sacrificial template of SiO2 and in the second step, an additional layer of the folic acid modified HPC coat the microspheres' surface. The layers fabrication is performed through a combination of distillation precipitation co-polymerization and chemical deposition method. The loading capacity (% LC) and encapsulation efficiency (% EE) percentages of the chemotherapeutic agent daunorubicin (DNR) in the fabricated microspheres were calculated through the standard curve methodology. In addition, the releasing properties of the resulting spheres are investigated, using the above mentioned methodology. It is worth mentioning that, spheres release the entrapped drug under combined conditions such acidic and reductive environment along with conventional hyperthermia. Cytotoxic activity of the synthesized spheres was investigated by using the well-established method of MTT assay in MCF-7 (breast cancer), HeLa (cervical cancer) and HEK 293 (Human Embryonic Kidney healthy cells) cell lines. Confocal and fluorescence microscopy were used to confirm the in vitro targeted ability of folic acid modified drug loaded microspheres in HeLa, to that overexpress folate receptors, MCF-7 and 3T3 cells, as negative folate cell substrate. Finally, radiolabelling of the spheres is performed, with a gamma emitting radionuclide ((99m)Tc), to assess their in vivo profile by means of scintigraphic imaging and biodistribution studies.
  
  
  RESULTS
  Hollow spheres release the encapsulated drug under acidic environment, conventional hyperthermia or in the presence of glutathione (reductive environment). The ability of modified drug carriers to target the HeLa cells, was confirmed by confocal and fluorescence microscopy. The resulting spheres are observed to be promising drug-carriers for cancer treatment due to their releasing properties under tumor's environment and high concentration in HeLa cells via endocytosis. In addition, the empty vehicles have no toxicity in healthy cells and present antimicrobial activity.
authors.affiliation: Laboratory for Sol-Gel, Institute of Nanoscience and Nanotechnology, NCSR "Demokritos", 153 10 Aghia Paraskevi Attikis, Greece.
authors.forename: Aikaterini-Foteini
authors.initials: AF
authors.lastname: Metaxa
authors.affiliation: Laboratory for Sol-Gel, Institute of Nanoscience and Nanotechnology, NCSR "Demokritos", 153 10 Aghia Paraskevi Attikis, Greece. Electronic address: elefth@chem.demokritos.gr.
authors.forename: Eleni K
authors.initials: EK
authors.lastname: Efthimiadou
authors.affiliation: Laboratory for Sol-Gel, Institute of Nanoscience and Nanotechnology, NCSR "Demokritos", 153 10 Aghia Paraskevi Attikis, Greece.
authors.forename: Nikos
authors.initials: N
authors.lastname: Boukos
authors.affiliation: Radiochemical/Radiopharmacological Quality Control Laboratory, Institute of Nuclear and Radiological Sciences and Technology, Energy & Safety, N.C.S.R. 'Demokritos', 15310 Aghia Paraskevi, Greece; Department of Biomedical Technology Engineering, Technological Educational Institute, GR 122 10 Athens, Greece.
authors.forename: Eirini A
authors.initials: EA
authors.lastname: Fragogeorgi
authors.affiliation: Department of Biomedical Technology Engineering, Technological Educational Institute, GR 122 10 Athens, Greece.
authors.forename: George
authors.initials: G
authors.lastname: Loudos
authors.affiliation: Laboratory for Sol-Gel, Institute of Nanoscience and Nanotechnology, NCSR "Demokritos", 153 10 Aghia Paraskevi Attikis, Greece. Electronic address: gkordas@ims.demokritos.gr.
authors.forename: George
authors.initials: G
authors.lastname: Kordas
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002482:Cellulose / Q000031:analogs & derivatives* / Q000737:chemistry; D003692:Delayed-Action Preparations; D004317:Doxorubicin / Q000008:administration & dosage / Q000494:pharmacology*; D016503:Drug Delivery Systems; D005260:Female; D005492:Folic Acid / Q000737:chemistry*; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D008863:Microspheres; D015394:Molecular Structure
chemical_list: D000970:Antineoplastic Agents; D003692:Delayed-Action Preparations; D004317:Doxorubicin; D002482:Cellulose; D005492:Folic Acid; C008079:hydroxypropylcellulose
keywords: Cytotoxicity; Daunorubicin; Drug delivery systems; Folic acid; HPC; IC(50); MTT assay; Microspheres; Multi responsive carriers; Polysaccharides