Screening of natural compounds is critically important, because they are increasingly used to prevent morbidities without proper monitoring . In particular, curcumin has many beneficial effects; however, its low bioavailability and rapid metabolism suggest the need for nanoformulations [21, 36].
In the present study, two curcumin nanoformulations were prepared, NC-EDG and NC-PCL. We also prepared two nanoformulations without curcumin to serve as controls. The physio-chemical characteristics of both curcumin nanocapsules showed an efficiency of encapsulation and uniformity of nanometric size; these results were satisfactory according to the production protocol established by our research group [29, 30].
Curcumin has been considered a promising approach to the treatment of several brain disorders. We determined cytotoxicity parameters of free curcumin and curcumin-loaded nanocapsules measured as cell death, proliferation and oxidative profile. In vitro experimental models are useful tools because they provide preliminary profiles of cellular responses and possible damages that could occur, guiding subsequent studies [37, 38].
A review by Soleimani, Sahebkar, and Hosseinzadeh discussed the safety and toxicity of curcumin in in vitro and in vivo models, both in animal and human trials . According to the authors, curcumin was non-toxic up to specific concentrations, depending on the model studied. Nevertheless, the use of curcumin nanoformulations needs to be explored in more depth. The authors indicated that curcumin is non-mutagenic and safe for pregnant animals; however, this assertion should be tested carefully . In agreements, some authors defended the notion that nanoencapsulated medications should be tested for their safety. We evaluated NC-PCL and NC-EDG and free curcumin in microglial and neuronal-like cell lines at different incubation periods.
In BV-2 cells, at 24 h after exposure, we found that free curcumin at the two highest concentrations decreased ROS production and 0.5–2.5 µM of NC-EDG and 2.5 and 5 µM for NC-PCL were safe to use in vitro, in that they did not compromise cell homeostasis. However, at 48 h of exposure, specific concentrations of the nanoformulations unbalanced the oxidative profile. The amount of free DNA increased, suggesting that the non-toxic concentrations were 0.5–1.0 µM for NC-EDG and 2.5 µM for NC-PCL, in this situation. With longer exposure times, the amount of free dsDNA was highly significant for all nanoformulations, suggesting possible membrane damage; this finding can be explained by the interaction of the polymers that make up the nanocapsule with the cell membrane, increasing permeability [36, 39]. By contrast, curcumin alone did not cause membrane damage at 72 h.
Exposure of these agents to neuronal-like cells showed that curcumin nanocapsules were associated with increased ROS levels, suggesting oxidative stress. NC-EDG was associated with high dsDNA levels at all concentrations, suggesting membrane damage, possibly due to EDG interacting with the cell membrane. At 48 h, the amount of free dsDNA also increased. By contrast, from 1.0 µM of nanoformulations, ROS production decreased, suggesting that perhaps the nanocapsules positively modulated oxidative mechanism. Curcumin was safe up to 10 µM; however, it was associated with ROS levels from 2.5 µM. At 72 h, the changes were more significant, suggesting compromised cellular homeostasis.
The antioxidant properties of curcumin are among the essential features that raise its therapeutic value. Severe oxidative stress is a cause of neural loss in the context of brain disorders . The production of ROS and NO, under conditions of homeostasis, occurs as part of cellular physiological metabolism and is essential for the organism to identify and eliminate stressors agent. Decreased ROS levels can be harmful under normal conditions but not during diseases treatment. The mechanisms by which curcumin decrease ROS levels, unbalancing the compromising antioxidant profile, involve its antioxidant activity, which neutralizes ROS formed by cells [1, 2, 5, 6, 40]. Studies showed that cancer cells are more susceptible to curcumin than normal cells; curcumin’s mechanism of action involves the regulation of ROS production, depending on cell type and experimental conditions [40–43].
Here, we demonstrated that 10 and 20 µM of curcumin, depending on the exposure time, were harmful to cells, causing lower cell viability. In contrast, intermediate curcumin concentrations showed less neurotoxicity compared to the same concentrations of nanoformulations, as these obtained more significant statistical results when compared with negative control. This can be explained by the fact that nanoformulations have components that generate cytotoxicity, including the surfactant polysorbate 80. This fact also explains decreased or increased cell viability, membrane damage, and imbalance in levels of ROS and NO in nanoformulations that did not contain curcumin, used here as controls .
Comparing the two curcumin nanoformulations, NC-PCL showed better results than NC-EDG, indicated by the lower significance levels when compared to negative control; however, the optimal choice for use in subsequent experiments will depend on the investigation experimental design because NC-PCL is more suitable for parenteral administration and NC-EDG is better for the oral route; this is because the constituent polymers differ for each nanocapsule. It is also important to highlight that in vitro screening tests are very important; however, results obtained in vivo may present different results, due to the consideration of an organism and its metabolism as a whole [20, 36].
It is important to note that cellular behavior, oxidative status depended on the exposure time. Damage increased over time primarily due to the high levels of free DNA in the supernatants in BV-2 cells and high levels of NO for 20 µM of NC-EDG. These characteristics must be considered when formulating the experimental design of a study.
No studies focused on safe concentration-response curves of curcumin in healthy cells originating from the CNS,; few studies are the same in healthy cells from tissue sources [28, 44–47]. However, in animals, the ethanolic extract of the rhizomes of C. longa, when administered chronically, changed the weight of the heart and lung . Corroborating this, Balaji and Chempakam demonstrated the toxicity of several components of C. longa, among them curcumin, with dose-dependent hepatotoxicity .
Cancer cell lines have been used to determinate the ideal concentration of curcumin treatment and neural lines for brain injuries in vitro models.
In the PC12 cancer cells, curcumin decreased cell viability in a concentration dependent manner . Using microglial BV-2 cells, Zhang et al. found that curcumin inhibited lipopolysaccharide-induced neuroinflammation at 1, 5, 10, 20, and 40 µM . Curcumin also inhibited oxidative stress by decreasing ROS in a model of neurodegenerative disease with SH-SY5Y cells at 1, 2.5, and 5 µM . Mursaleen et al. used curcumin nanoformulation 5 and 10 µM to protect the cells against neurotoxicity induced by rotenone in SH-SY5Y cells .
These studies demonstrate that our results for safe concentrations of curcumin are in line with those used to treat diseases that affect the CNS. Therefore, concentrations between 1.0 to 5 µM are not harmful to healthy cells. Nevertheless, because of the disadvantages of the substance in free form, nanoformulations at the same concentrations or even lower than those mentioned may deliver more satisfactory results by controlling the release of curcumin and increasing its bioavailability, in addition to crossing the blood-brain barrier and reaching the sites of brain injuries [18, 21, 22].
According to our introductory innovative study, that determined the non-neurotoxic concentrations of curcumin nanoformulations and their free form, researchers can use our findings as a basis for choosing the optimal concentrations for their experimental models. It is essential to use safe concentrations to analyze the pharmacological potential of natural products. Because the present study was a proof of concept study, more specific investigations and in vivo experimental models must be carried out to test our findings.