title: Co-association of methotrexate and SPIONs into anti-CD64 antibody-conjugated PLGA nanoparticles for theranostic application.
abstract: BACKGROUND
  Rheumatoid arthritis (RA) is an autoimmune disease with severe consequences for the quality of life of sufferers. Regrettably, the inflammatory process involved remains unclear, and finding successful therapies as well as new means for its early diagnosis have proved to be daunting tasks. As macrophages are strongly associated with RA inflammation, effective diagnosis and therapy may encompass the ability to target these cells. In this work, a new approach for targeted therapy and imaging of RA was developed based on the use of multifunctional polymeric nanoparticles.
  
  
  METHODS
  Poly(lactic-co-glycolic acid) nanoparticles were prepared using a single emulsion-evaporation method and comprisaed the co-association of superparamagnetic iron oxide nanoparticles (SPIONs) and methotrexate. The nanoparticles were further functionalized with an antibody against the macrophage-specific receptor, CD64, which is overexpressed at sites of RA. The devised nanoparticles were characterized for mean particle size, polydispersity index, zeta potential, and morphology, as well as the association of SPIONs, methotrexate, and the anti-CD64 antibody. Lastly, the cytotoxicity of the developed nanoparticles was assessed in RAW 264.7 cells using standard MTT and LDH assays.
  
  
  RESULTS
  The nanoparticles had a mean diameter in the range of 130-200 nm and zeta potential values ranging from -32 mV to -16 mV. Association with either methotrexate or SPIONs did not significantly affect the properties of the nanoparticles. Conjugation with the anti-CD64 antibody, in turn, caused a slight increase in size and surface charge. Transmission electron microscopy confirmed the association of SPIONs within the poly(lactic-co-glycolic acid) matrix. Both anti-CD64 and methotrexate association were confirmed by Fourier transform infrared spectroscopy, and quantified yielding values as high as 36% and 79%, respectively. In vitro toxicity studies confirmed the methotrexate-loaded nanosystem to be more effective than the free drug.
  
  
  CONCLUSIONS
  Multifunctional anti-CD64-conjugated poly(lactic-co-glycolic acid) nanoparticles for the combined delivery of methotrexate and SPIONs were successfully prepared and characterized. This nanosystem has the potential to provide a new theranostic approach for the management of RA.
authors.affiliation: REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal ; Faculty of Engineering, University of Porto, Porto, Portugal.
authors.forename: Catarina Costa
authors.initials: CC
authors.lastname: Moura
authors.affiliation: REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal.
authors.forename: Marcela A
authors.initials: MA
authors.lastname: Segundo
authors.affiliation: CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, Instituto de Ciências da Saúde-Norte, Gandra PRD, Portugal ; INEB - Instituto de Engenharia Biomédica, University of Porto, Porto, Portugal.
authors.forename: José das
authors.initials: Jd
authors.lastname: Neves
authors.affiliation: REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal.
authors.forename: Salette
authors.initials: S
authors.lastname: Reis
authors.affiliation: CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, Instituto de Ciências da Saúde-Norte, Gandra PRD, Portugal ; INEB - Instituto de Engenharia Biomédica, University of Porto, Porto, Portugal.
authors.forename: Bruno
authors.initials: B
authors.lastname: Sarmento
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D016503:Drug Delivery Systems / Q000379:methods*; D019344:Lactic Acid / Q000737:chemistry*; D058185:Magnetite Nanoparticles / Q000737:chemistry* / Q000633:toxicity; D008727:Methotrexate / Q000737:chemistry* / Q000633:toxicity; D051379:Mice; D010316:Particle Size; D011100:Polyglycolic Acid / Q000737:chemistry*; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D017452:Receptors, IgG / Q000037:antagonists & inhibitors*
chemical_list: D000906:Antibodies; D058185:Magnetite Nanoparticles; D017452:Receptors, IgG; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; D008727:Methotrexate
keywords: FcγRI; methotrexate; poly(lactic-co-glycolic acid); superparamagnetic iron oxide nanoparticles; targeted drug delivery