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

20The mechanism of cellular uptake by endocytosis and subsequent oxidative stress has been 21 identified as the paradigm for the toxic response of cationically surface charged nanoparticles. In 22 an attempt to circumvent the process, the effect of increased cellular membrane permeability on 23 the uptake mechanisms of poly (amidoamine) dendrimers generation 4 (G4) and 6 (G6) in vitro 24 was investigated. Immortalised, non-cancerous human keratinocyte (HaCaT) cells were treated 25 with DL-Buthionine-(S,R)-sulfoximine (BSO). Active uptake of the particles was monitored 26 using fluorescence microscopy to identify and quantify endosomal activity and resultant 27 oxidative stress, manifest as increased levels of reactive oxygen species, monitored using the 28 carboxy-H 2 DCFDA dye. Dose dependent cytotoxicity for G4 and G6 exposure was registered 29 using the cytotoxicity assays Alamar Blue and MTT, from 6 to 72 hours. 30Reduced uptake by endocytosis is observed for both dendrimer species. A dramatic change, 31 compared to untreated cells, is observed in the cytotoxic and oxidative stress response of the 32 BSO treated cells. The significantly increased mitochondrial activity, dose dependent anti-33 oxidant behaviour and reduced degree of endocytosis for both dendrimer generations, in BSO 34 treated cells, indicates enhanced permeability of the cell membrane, resulting in the passive, 35 diffusive uptake of dendrimers, replacing endocytosis as the primary uptake mechanism. The 36 complex MTT response reflects the importance of glutathione in maintaining redox balance 37 within the mitochondria. The study highlights the importance of regulation of this redox balance 38 for cell metabolism, but also points to the potential of controlling the nanoparticle uptake 39 mechanisms, and resultant cytotoxicity, with implications for nanomedicine. 40 41 3

PAMAM dendrimer - cell membrane interactions

“…the impact of membrane asymmetry, which is found in most cellular membranes, on interactions between bilayers and dendrimers. It also highlights the ability of dendrimers to translocate passively through cellular membranes, as observed by several others experimentally [14,57,85].…”

“…The size of these dendrimer-lipid aggregates has been measured using DLS and it was found that lipid bilayers wrap around higher generation dendrimers (>G5) forming 'dendrisomes'. Using NR, dendrimers have been found to translocate through SLBs, which indicates the possibility of passive transfer through cell membranes as suggested by in vitro studies discussed in Section 3 [57]. The energetics of these interactions has been explored using DSC and Raman spectroscopy, revealing changes in the fluidity of lipid acyl chains in the presence of dendrimers dependent upon dendrimer generation and charge.…”

“…Recently, the in vitro uptake mechanisms of PAMAM dendrimers were investigated for non-cancerous human keratinocyte (HaCaT) cells by Maher and Byrne [57]. Endocytosis [6] was the primary mechanism for the uptake of G4 and G6 PAMAM dendrimers, identified by ROS production and dendrimer localisation in the mitochondria after their escape from endosomes, using MTT and Alamar Blue dye-based assays.…”

“…Passive uptake of dendrimers decreased oxidative stress, with the dendrimers acting as antioxidants. [57] fluidity, e.g. via the lipid-substrate electrostatic interactions and mismatch between the membrane natural curvature and the flat substrate, thus affecting lateral molecular diffusion, lipid domain formation and the reactivity of the SLB.…”

Determination of spectral markers of cytotoxicity and genotoxicity using in vitro Raman microspectroscopy: cellular responses to polyamidoamine dendrimer exposure

“…[16][17][18] The toxic effects of the PAMAM dendrimers on different cell lines have also been widely studied using cytotoxicity assays such as Alamar Blue (AB), MTT, Neutral Red (NR) and Clonogenic assays. [19][20][21][22][23][24] Cellular exposure to PAMAM dendrimers has been shown to result in oxidative stress, activation of inflammatory cascades. As a general mechanism, dendrimers are found to cause a generation dependent, systematic cytotoxicity, oxidative stress and genotoxicity due to the proton sponge effect in endosomes and/or lysosomes, which causes lysosomal rupture and mitochondrial accumulation, resulting in cell death.…”