These are the first studies examining fullerene trafficking within human MC. It was observed that the C70-TR conjugated fullerenes were taken up through an endocytosis-dependent mechanism and persisted in the MC for up to one week. The intracellular localization was found predominately in the ER and to a lesser degree in mitochondria and lysosomes. No fullerenes were detected in the tryptase-containing secretory granules or in the nucleus at any time-points measured. Taken together, it is clear that MC actively take up fullerenes through an endocytotic pathway where they remain mostly localized to ER and to a lesser extent in lysosomes and mitochondria.
The ER is the site for synthesizing and ensuring proper folding of proteins within a cell and shuttles misfolded proteins through a degradative pathway. Recent evidence suggests this process involves the formation of disulfide bonds that stabilize the folding of nascent proteins resulting in an oxidizing environment and ROS generation (13
). In addition to the ER, mitochondria also produce ROS due to the production of ATP following oxidative phosphorylation. Our results using C70
-based derivatives are consistent with several publications examining fullerene localization in different cell types in which deposition in different intracellular organelles depends on the moieties added to the carbon cage (2
). For example, C63
appear to localize to mitochondria (14
) while C60
mixtures dispersed in tetrahydrofuran (and not purified from this solvent) localize to lysosomes and nuclei in macrophages (17
). Our results are also consistent with previously studies suggesting fullerenes can easily penetrate into a lipid membrane where they induce non-toxic changes in the structural and elastic properties of the lipid bilayer at very high concentrations (18
). Taken together, fullerenes as a general class have a varied localization profile within cells; how the fullerene cage is modified determines where the cage localizes in situ. Given that no fullerene formulation has yet been identified for treating a particular disease it is difficult to extrapolate in situ localization results from different fullerene preparations.
The results demonstrating that C70
-based fullerenes are endocytosed and localize to ER differentiate from previously published results showing that endocytosed C60
-based fullerenes localize to the mitochondria and lysosomes (19
). The ER accumulation of C70
-based fullerenes explains the data shown that pre-incubation of MC with the same fullerenes caused a reduction of FCεRI-mediated calcium release and ROS generation. These findings may help explain fullerenes mechanism of inhibition of FcεRI activated mediator release from MC. In a separate publication (J. Immunology.) we demonstrate that the C70
-based derivatives not unlike the one used for the TR conjugation blunts histamine degranulation and cytokine release when MC are pre-challenged with equivalent concentrations and times as described herein. The release of calcium stores and production of ROS occur in these organelles (21
); while release of calcium stores is absolutely required for FcεRI-mediator release it is still no clear if increased ROS levels parallel mediator release or is a consequence of FcεRI crosslinking. Calcium stores release from the ER in response to FcεRI crosslinking is controlled by inositol 1,4,5-trisphosphate. Thus, the observation that fullerenes localize to this organelle fits well with our data showing that calcium stores release and ROS production are inhibited when FcεRI-challenged MC are pre-challenged with fullerenes. Current efforts are aimed at determining what ER-associated signaling molecules (if any) are bound to fullerenes which would help explain how these molecules exert their inhibitory activities.
Fullerenes were also consistently found (but to a lesser extent as compared to ER accumulation) in lysosomes. Endocytosis from the plasma membrane can occur by a variety of mechanisms and once uptake occurs the molecules are then routed through lysosomes to various places within cells. We observed lysosomal accumulation of C70-TR predominately at four hour, 96 hour, and one week after washout perhaps indicating the cells shuffling the conjugates from the membrane early and to the membrane for possible excretion at the later times. Indeed, there appears to be a localization of fullerenes in the outer edges of the cell at later times () possibly indicated that their imminent transport out of the cells.
As mentioned above, the mitochondria is another organelle that is best known for its role in ROS production which is a result of the electrochemical membrane potential (ΔΨm) across the inner mitochondrial membrane (21
). We found sporadic localization of the C70
-TR in the mitochondria at some of the times examined. Mitochodrial localization has been demonstrated previously either directly or indirectly using other fullerene preparations (14
). Given that fullerenes are potent anti-oxidants that can react with ROS it has been speculated that much of their inhibitory capabilities are linked to this radical scavenging ability. However, we show here that fullerenes that are effective anti-inflammatory agents mainly target ER which is also a ROS-producing organelle. It is possible the C70
-TR may not reflect the same targeting specificity as the C70
derivative without TR. We are currently engineering fullerenes without bulky side chains that are structurally the same as those previously described so that results obtained on inhibitory capabilities can be extrapolated to organelle targeting more specifically.
In conclusion, we have identified the ER as a primary organelle for 70-carbon based fullerene derivatives localization in human MC as opposed to previous publications which show 60-carbon based fullerenes localize to the mitochondria This localization may help explain how the fullerenes exhibit their inhibitory activity through blunting of calcium and ROS spikes leading to subsequent reduction in histamine deganulation and cytokine production. Fullerenes are potent antioxidants and are being investigated as therapies for a wide range of diseases (24
). This may have clinical implications for developing future fullerene-based compounds, our efforts are focused on developing these lead candidates into novel ways to treat those diseases associated with MC activation including asthma, arthritis, and anaphylaxis.