title: Modification of the in vitro uptake mechanism and antioxidant levels in HaCaT cells and resultant changes to toxicity and oxidative stress of G4 and G6 poly(amidoamine) dendrimer nanoparticles.
abstract: The mechanism of cellular uptake by endocytosis and subsequent oxidative stress has been identified as the paradigm for the toxic response of cationically surface charged nanoparticles. In an attempt to circumvent the process, the effect of increased cellular membrane permeability on the uptake mechanisms of poly(amidoamine) dendrimers generations 4 (G4) and 6 (G6) in vitro was investigated. Immortalised, non-cancerous human keratinocyte (HaCaT) cells were treated with DL-buthionine-(S,R)-sulfoximine (BSO). Active uptake of the particles was monitored using fluorescence microscopy to identify and quantify endosomal activity and resultant oxidative stress, manifested as increased levels of reactive oxygen species, monitored using the carboxy-H2DCFDA dye. Dose-dependent cytotoxicity for G4 and G6 exposure was registered using the cytotoxicity assays Alamar Blue and MTT, from 6 to 72 h. Reduced uptake by endocytosis is observed for both dendrimer species. A dramatic change, compared to untreated cells, is observed in the cytotoxic and oxidative stress response of the BSO-treated cells. The significantly increased mitochondrial activity, dose-dependent antioxidant behaviour and reduced degree of endocytosis for both dendrimer generations, in BSO-treated cells, indicate enhanced permeability of the cell membrane, resulting in the passive, diffusive uptake of dendrimers, replacing endocytosis as the primary uptake mechanism. The complex MTT response reflects the importance of glutathione in maintaining redox balance within the mitochondria. The study highlights the importance of regulation of this redox balance for cell metabolism but also points to the potential of controlling the nanoparticle uptake mechanisms, and resultant cytotoxicity, with implications for nanomedicine.
authors.affiliation: FOCAS Research Institute, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland. marcus.maher@mydit.ie.|School of Physics, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland. marcus.maher@mydit.ie.
authors.forename: Marcus A
authors.initials: MA
authors.lastname: Maher
authors.affiliation: FOCAS Research Institute, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland.
authors.forename: Hugh J
authors.initials: HJ
authors.lastname: Byrne
mesh_terms: D000975:Antioxidants / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D050091:Dendrimers / Q000493:pharmacokinetics* / Q000633:toxicity*; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D053758:Nanoparticles / Q000737:chemistry / Q000633:toxicity* / Q000648:ultrastructure; D018384:Oxidative Stress / Q000187:drug effects* / Q000502:physiology; D010316:Particle Size; D011073:Polyamines / Q000493:pharmacokinetics* / Q000633:toxicity*; D013329:Structure-Activity Relationship
chemical_list: D000975:Antioxidants; D050091:Dendrimers; C531249:Poly(amidoamine); D011073:Polyamines
keywords: Dendrimer nanoparticles; Endocytosis; Membrane permeability; Oxidative stress; PAMAM nanoparticles; Passive diffusion