title: Poly-lactic acid nanoparticles (PLA-NP) promote physiological modifications in lung epithelial cells and are internalized by clathrin-coated pits and lipid rafts.
abstract: BACKGROUND
  Poly-lactic acid nanoparticles (PLA-NP) are a type of polymeric NP, frequently used as nanomedicines, which have advantages over metallic NP such as the ability to maintain therapeutic drug levels for sustained periods of time. Despite PLA-NP being considered biocompatible, data concerning alterations in cellular physiology are scarce.
  
  
  METHODS
  We conducted an extensive evaluation of PLA-NP biocompatibility in human lung epithelial A549 cells using high throughput screening and more complex methodologies. These included measurements of cytotoxicity, cell viability, immunomodulatory potential, and effects upon the cells' proteome. We used non- and green-fluorescent PLA-NP with 63 and 66 nm diameters, respectively. Cells were exposed with concentrations of 2, 20, 100 and 200 µg/mL, for 24, 48 and 72 h, in most experiments. Moreover, possible endocytic mechanisms of internalization of PLA-NP were investigated, such as those involving caveolae, lipid rafts, macropinocytosis and clathrin-coated pits.
  
  
  RESULTS
  Cell viability and proliferation were not altered in response to PLA-NP. Multiplex analysis of secreted mediators revealed a low-level reduction of IL-12p70 and vascular epidermal growth factor (VEGF) in response to PLA-NP, while all other mediators assessed were unaffected. However, changes to the cells' proteome were observed in response to PLA-NP, and, additionally, the cellular stress marker miR155 was found to reduce. In dual exposures of staurosporine (STS) with PLA-NP, PLA-NP enhanced susceptibility to STS-induced cell death. Finally, PLA-NP were rapidly internalized in association with clathrin-coated pits, and, to a lesser extent, with lipid rafts.
  
  
  CONCLUSIONS
  These data demonstrate that PLA-NP are internalized and, in general, tolerated by A549 cells, with no cytotoxicity and no secretion of pro-inflammatory mediators. However, PLA-NP exposure may induce modification of biological functions of A549 cells, which should be considered when designing drug delivery systems. Moreover, the pathways of PLA-NP internalization we detected could contribute to the improvement of selective uptake strategies.
authors.affiliation: Laboratory of Bioengineering and in Vitro Toxicology, Directory of Metrology Applied to Life Sciences (Dimav), National Institute of Metrology Quality and Technology (INMETRO), Duque De Caxias, RJ, Brazil.
authors.forename: Camila Macedo
authors.initials: CM
authors.lastname: da Luz
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.|Heriot-Watt University, Edinburg, UK.
authors.forename: Matthew Samuel Powys
authors.initials: MS
authors.lastname: Boyles
authors.affiliation: Laboratory of Bioengineering and in Vitro Toxicology, Directory of Metrology Applied to Life Sciences (Dimav), National Institute of Metrology Quality and Technology (INMETRO), Duque De Caxias, RJ, Brazil.
authors.forename: Priscila
authors.initials: P
authors.lastname: Falagan-Lotsch
authors.affiliation: Laboratory of Chemical Signaling in Nervous System, Biology Institute, Fluminense Federal University, Niteroi, RJ, Brazil.
authors.forename: Mariana Rodrigues
authors.initials: MR
authors.lastname: Pereira
authors.affiliation: Laboratory of Bioengineering and in Vitro Toxicology, Directory of Metrology Applied to Life Sciences (Dimav), National Institute of Metrology Quality and Technology (INMETRO), Duque De Caxias, RJ, Brazil.
authors.forename: Henrique Rudolf
authors.initials: HR
authors.lastname: Tutumi
authors.affiliation: Laboratory of Bioengineering and in Vitro Toxicology, Directory of Metrology Applied to Life Sciences (Dimav), National Institute of Metrology Quality and Technology (INMETRO), Duque De Caxias, RJ, Brazil.|Laboratory of Biochemistry, State University Center of West Zone (UEZO), Rio de Janeiro, RJ, Brazil.
authors.forename: Eidy
authors.initials: E
authors.lastname: de Oliveira Santos
authors.affiliation: Laboratory of Bioengineering and in Vitro Toxicology, Directory of Metrology Applied to Life Sciences (Dimav), National Institute of Metrology Quality and Technology (INMETRO), Duque De Caxias, RJ, Brazil.
authors.forename: Nathalia Balthazar
authors.initials: NB
authors.lastname: Martins
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Martin
authors.identifier: http://orcid.org/0000-0001-5416-085X
authors.initials: M
authors.lastname: Himly
authors.affiliation: Department of Cell Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Aniela
authors.initials: A
authors.lastname: Sommer
authors.affiliation: Department of Cell Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Ilse
authors.initials: I
authors.lastname: Foissner
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Albert
authors.initials: A
authors.lastname: Duschl
authors.affiliation: Laboratory of Bioengineering and in Vitro Toxicology, Directory of Metrology Applied to Life Sciences (Dimav), National Institute of Metrology Quality and Technology (INMETRO), Duque De Caxias, RJ, Brazil.|Dental School, Fluminense Federal University, Niteroi, RJ, Brazil.
authors.forename: José Mauro
authors.initials: JM
authors.lastname: Granjeiro
authors.affiliation: Laboratory of Bioengineering and in Vitro Toxicology, Directory of Metrology Applied to Life Sciences (Dimav), National Institute of Metrology Quality and Technology (INMETRO), Duque De Caxias, RJ, Brazil. leitepec@gmail.com.|, Av. Nossa Senhora das Gracas 50, LABET - Dimav, Predio 27, Duque de Caxias, Xerem, Rio de Janeiro, 25250-020, Brazil. leitepec@gmail.com.
authors.forename: Paulo Emílio Corrêa
authors.initials: PE
authors.lastname: Leite
mesh_terms: D000072283:A549 Cells; D001672:Biocompatible Materials / Q000737:chemistry*; D021941:Caveolae / Q000378:metabolism*; D002470:Cell Survival; D002966:Clathrin / Q000737:chemistry; D016503:Drug Delivery Systems; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D018664:Interleukin-12 / Q000378:metabolism; D021962:Membrane Microdomains; D035683:MicroRNAs / Q000378:metabolism; D053758:Nanoparticles / Q000737:chemistry*; D010316:Particle Size; D010873:Pinocytosis; D011091:Polyesters / Q000737:chemistry*; D020543:Proteome; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D001672:Biocompatible Materials; D002966:Clathrin; C524942:MIRN155 microRNA, human; D035683:MicroRNAs; D011091:Polyesters; D020543:Proteome; D042461:Vascular Endothelial Growth Factor A; D018664:Interleukin-12; C033616:poly(lactide)
keywords: Clathrin-coated pits; Drug delivery; Endocytosis; Lipid rafts; Nanoparticles