Eliminating and preventing toxic Aβ aggregations, as one of the most momentous AD hallmarks, has been the focal point of recent researches.[33] With this respect, researchers have synthesized a wide range of NPs with different biological functions for drug delivery, among which is magnetic NPs. Considering the importance of functionalizing the NPs in enhancing the biocompatibility and efficiency of the NPs, as well as side effect reduction, in particular for a better brain/ Aβ targeting,[17, 34] wide range of biomaterials have been taken into consideration for the aforementioned purpose. In light of this, polyphenolic compounds, as natural polymers, have attracted much attention because not only do they possess high anti-amyloidogenic activities that can directly eradicate the aggregations, but they also are able to impede the amyloid formation by affecting the indirect mechanisms of AD (including the acetylcholine esterase reduction, neuroinflammation, and presence of reactive oxygen species (ROS) level formed as a result of mitochondria dysfunction), and therefore, are great options for treating AD. Fulvic acid is one of the polyphenolic compounds with strong anti-inflammation and anti-tau aggregation impacts.[35–38]
In the current experiment, we have assessed the impedimental impacts of Fe3O4-FA NPs with a mean size of 10.7 ± 2.7nm on the amyloid aggregations in a common Aβ in-vitro model, i.e., HEWL, via ThT fluorescence enhancement, FESEM, and CD spectroscopy. Our findings have demonstrated that Fe3O4-FA NPs suppressed β-sheet formation and aggregation proportional to their concentrations. Further than inhibition of the aggregation process of HEWL, these NPs could remodel toxic amyloid aggregates into off-pathway aggregates that turned out to be non-toxic, as was confirmed by MTT assay. Our results corroborate entirely with the data obtained from the four previous studies in terms of the anti-Alzheimer’s impact of fulvic acid [22–25]. Based on the FESEM, the aggregations were diminished completely upon addition of the Fe3O4-FA NPs, while in the earlier studies [24–26], either fulvic acid or Fe3O4 NPs alone could decrease the length of the aggregations, but could not remove the accumulations completely; therefore, it can be inferred that Fe3O4 NPs could enhance the fulvic acid’s ability such a way that enabled fulvic acid to entirely suppress the amyloid aggregations.
A possible interpretation for why the higher concentrations of the Fe3O4-FA NPs is more effective in inhibiting aggregation formation, can be made through looking at the interactions occur between the
NPs and the amyloid aggregation under the low pH condition (which was applied to drive the aggregation formation), because increasing the concentration of the NPs under low pH, increases the iron atom concentrations, which leads to decrement in oxidation by decreasing the ratio of Fe+ 2 (magnetic) to Fe + 3 (maghemite)[39, 40] (because maghemite is more stable against oxidation at high temperature and different pH ranges than magnetic),[40] thus, the high-concentration samples will not become oxidated and thus they will not gain oxygen and electrons, on the other hand, decreasing the concentrations under low pH, promotes oxidation of the surface of the Fe3O4-FA NPs by increasing the Fe+ 2: Fe+ 3 ratio and therefore, enhances the magnetic form, which is not stable against oxidation. Thus, the FA-Fe3O4 NPs gain more oxygen and electrons, and their surface charges become more negative as compared with their high-concentration counterparts; [39, 40] considering that all proteins (including amyloid beta protein) have more affinity to bind to more positive surfaces than negative ones ,[41] therefore, the low-concentration nano particles would have less interaction with amyloid beta proteins, and hence, would not be able to remove all of the amyloid aggregations, whereas the high- concentration samples can have more interactions with the amyloid beta aggregations, and hence, eliminate them efficiently.
However, looking at this phenomenon from another aspect, it can be inferred that, since the surface charges of the low-concentration NPs are more negative, the NPs would interact with neuroblastoma SHSY5Y ‘s membrane through electric repulsion (because the surface charge of the cell membrane is negative),[41, 42] and, therefore, the NPs cannot bind to the proteins on the cell membrane of neurons, this makes the lower concentrations gain an advantage in terms of developing anti-Alzheimer’s drugs that are devoid of side effects; since the lower concentrations will not interact with the neural membrane proteins, they will not activate other unwanted pathways that are activated as a result of interacting with membrane proteins; meanwhile, they can induce their anti-Alzheimer’s effect that although it is not effective compared to higher concentrations, their anti-Alzheimer’s impact could be enhanced by adding another material to them, thus, they can affect the cells and remove some portion of the amyloid aggregations in an indirect way and without binding to membrane proteins.
In general, there are numerous advantages of using FA- Fe3O4 NPs, which are as follows: the NPs subsume low cytotoxicity (no toxicity till 1000µg/mL), high level of cell protection, have the ability to pass the BBB, and inhibiting AD progression through overshadowing multifarious AD-related pathways simultaneously (inflammation, amyloid and tau aggregations, along with acidic pH), as the NPs can efficiently target the amyloid aggregations and hence prohibit the formation of tau aggregations (because that amyloid accumulation triggers tau deposition formation), furthermore, due to the alkaline nature of the FA- Fe3O4 NPs (because in the final step of synthesizing we raised the pH to 7.2), these NPs can also target the acidic pH of the brain, which is considered as one of the important factors that lead to amyloid aggregations[43–47]. Moreover, the magnetic nature of the iron oxide NPs facilitates their quick and easily evacuation through applying small magnetic field, which itself lowers the level of Aβ plaques.[18] Therefore, the anti-Alzheimer’s impact of Fe3O4 NPs would be reinforced while excreting from the body.
To sum up, in this study, we investigated the inhibitory effect of FA-Fe3O4 NPs in-vitro on neuroblastoma SHSY5Y cell line. According to the results, the concentrations used in this study were all non-toxic to the cells, but the highest concentration exhibited the most efficient anti-amyloidogenic activity and best cell viability percentage (100%), however, further future investigation through in-vivo model is required to better confirm the results and there are some question to be answered, which would require future investigations, such as how would the NPs behave if they are applied at concentrations higher than 1000µg/mL? what other mechanisms can the NPs trigger that are effective in AD treatment (assessing the nanoparticles’ impact on gut bacteria, beta secretase enzyme, etc.)?