title: Chitosan functionalisation of gold nanoparticles encourages particle uptake and induces cytotoxicity and pro-inflammatory conditions in phagocytic cells, as well as enhancing particle interactions with serum components.
abstract: BACKGROUND
  Gold nanoparticles (AuNPs) are a popular choice for use in medical and biomedical research applications. With suitable functionalisation AuNPs can be applied in drug delivery systems, or can aid in disease diagnosis. One such functionalisation is with chitosan, which enables efficient interaction and permeation of cellular membranes, providing an effective adjuvant. As both AuNPs and chitosan have been shown to have low toxicity and high biocompatibility their proposed use in nanomedicine, either individually or combined, is expanding. However, further toxicological and immunological assessments of AuNP-chitosan conjugates are still needed. Therefore, we have evaluated how AuNP functionalisation with chitosan can affect uptake, cytotoxicity, and immunological responses within mononuclear cells, and influence the interaction of AuNPs with biomolecules within a complex biofluid. The AuNPs used were negatively charged through citrate-coating, or presented either low or high positive charge through chitosan-functionalisation. Uptake by THP-1 cells was assessed via transmission electron microscopy and electron energy loss spectroscopy, pro-inflammatory responses by ELISA and qRT-PCR, and cell death and viability via lactate dehydrogenase release and mitochondrial activity, respectively. Interactions of AuNPs with protein components of a frequently used in vitro cell culture medium supplement, foetal calf serum, were investigated using mass spectrometry.
  
  
  RESULTS
  Although cells internalised all AuNPs, uptake rates and specific routes of intracellular trafficking were dependent upon chitosan-functionalisation. Accordingly, an enhanced immune response was found to be chitosan-functionalisation-dependent, in the form of CCL2, IL-1β, TNF-α and IL-6 secretion, and expression of IL-1β and NLRP3 mRNA. A corresponding increase in cytotoxicity was found in response to chitosan-coated AuNPs. Furthermore, chitosan-functionalisation was shown to induce an increase in unique proteins associating with these highly charged AuNPs.
  
  
  CONCLUSIONS
  It can be concluded that functionalisation of AuNPs with the perceived non-toxic biocompatible molecule chitosan at a high density can elicit functionalisation-dependent intracellular trafficking mechanisms and provoke strong pro-inflammatory conditions, and that a high affinity of these NP-conjugates for biomolecules may be implicit in these cellular responses.
authors.affiliation: Department of Molecular Biology, Division of Allergy and Immunology, Paris-Lodron University Salzburg, Hellbrunnerstr. 34, 5020, Salzburg, Austria. m.boyles@hw.ac.uk.
authors.forename: Matthew S P
authors.initials: MS
authors.lastname: Boyles
authors.affiliation: Department of Molecular Biology, Division of Chemistry and Bioanalytics, Paris-Lodron University Salzburg, Salzburg, Austria. theresa.kristl@med.lu.se.
authors.forename: Theresa
authors.initials: T
authors.lastname: Kristl
authors.affiliation: Department of Cell Biology, Paris-Lodron University Salzburg, Salzburg, Austria. ancuela.andosch@sbg.ac.at.
authors.forename: Ancuela
authors.initials: A
authors.lastname: Andosch
authors.affiliation: Department of Molecular Biology, Division of Allergy and Immunology, Paris-Lodron University Salzburg, Hellbrunnerstr. 34, 5020, Salzburg, Austria. mirjam_zimmermann@hotmail.com.
authors.forename: Mirjam
authors.initials: M
authors.lastname: Zimmermann
authors.affiliation: Institut Català de Nanotecnologia, Bellaterra, Barcelona, Spain. ngoc.tran1011@gmail.com.
authors.forename: Ngoc
authors.initials: N
authors.lastname: Tran
authors.affiliation: Institut Català de Nanotecnologia, Bellaterra, Barcelona, Spain. eudald.casals@icn.cat.
authors.forename: Eudald
authors.initials: E
authors.lastname: Casals
authors.affiliation: Department of Molecular Biology, Division of Allergy and Immunology, Paris-Lodron University Salzburg, Hellbrunnerstr. 34, 5020, Salzburg, Austria. martin.himly@sbg.ac.at.
authors.forename: Martin
authors.initials: M
authors.lastname: Himly
authors.affiliation: Institut Català de Nanotecnologia, Bellaterra, Barcelona, Spain. victor.puntes@icn.cat.|Institut Catala de Recerca i Estudis Avancats, Barcelona, Spain. victor.puntes@icn.cat.
authors.forename: Victor
authors.initials: V
authors.lastname: Puntes
authors.affiliation: Department of Molecular Biology, Division of Chemistry and Bioanalytics, Paris-Lodron University Salzburg, Salzburg, Austria. c.huber@sbg.ac.at.
authors.forename: Christian G
authors.initials: CG
authors.lastname: Huber
authors.affiliation: Department of Cell Biology, Paris-Lodron University Salzburg, Salzburg, Austria. ursula.luetz-meindl@sbg.ac.at.
authors.forename: Ursula
authors.initials: U
authors.lastname: Lütz-Meindl
authors.affiliation: Department of Molecular Biology, Division of Allergy and Immunology, Paris-Lodron University Salzburg, Hellbrunnerstr. 34, 5020, Salzburg, Austria. albert.duschl@sbg.ac.at.
authors.forename: Albert
authors.initials: A
authors.lastname: Duschl
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism; D016923:Cell Death; D002460:Cell Line; D048271:Chitosan / Q000737:chemistry*; D003470:Culture Media / Q000737:chemistry; D004705:Endocytosis; D006046:Gold / Q000737:chemistry*; D006801:Humans; D058847:Inflammasomes / Q000378:metabolism; D007249:Inflammation / Q000473:pathology; D053768:Metal Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D010586:Phagocytes / Q000378:metabolism* / Q000473:pathology
chemical_list: D002352:Carrier Proteins; D003470:Culture Media; D058847:Inflammasomes; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C453881:NLRP3 protein, human; D006046:Gold; D048271:Chitosan