2.3.1. Cell Viability by MTT and SRB Tests

NCUR-2, NMTX-1, and NCUR/MTX-2 were used for cell culture-based assays due to their known drug release profiles. In order to explore whether or not these formulations had a cytotoxic effect on the Calu-3 cell line, MTT and SRB assays were first performed. The cell viability results for NCUR-2 at high drug concentrations and NMTX-1 at low drug concentrations are shown in Table 2.


**Table 2.** Cell viability of Calu-3 by MTT and SRB assays after its treatment with NCUR-2 and NMTX-1 at different concentrations for 72 h.

Results are expressed as mean ± standard error of the mean from 4 independent experiments. Asterisks denote significance levels compared to control: significantly different, \*\*\* *p* < 0.0001.

NCUR-2 significantly reduced Calu-3 viability at 200 and 100 <sup>μ</sup>mol·L−<sup>1</sup> for both MTT and SRB assays. This formulation also provided a significant reduction of cell viability at 50 <sup>μ</sup>mol·L−<sup>1</sup> for the SRB test. No statistically significant decrease was observed for other concentrations tested of NCUR-2 and for all concentrations investigated of NMTX-1.

In this study, CUR demonstrated cytotoxicity only at high concentrations. This effect may be related to the nanoencapsulation procedure since PCL/PEG NCs may have provided a controlled drug release during the 72 h assays, resulting in low concentrations of released CUR in contact within Calu-3 cells. Different results were observed when free CUR was used. Dhanasekaran et al. [44] investigated the effect of CUR on KG-1 cells using MTT reduction assay, where CUR achieved a dose-dependent reduction in cell viability at different exposure times. Cell viability was reduced to 57% at 50 <sup>μ</sup>mol·L−<sup>1</sup> for 24 h and to 20% at 100 μmol.L−<sup>1</sup> for 48 h.

Although these data for pure CUR are more attractive, its free form is not a suitable option for its clinical use. CUR presents several problems related to its in vivo use due to its low aqueous solubility, low bioavailability, rapid hepatic metabolism, high decomposition rate at neutral or basic pH, and susceptibility to photochemical degradation. CUR generates inactive metabolites when administered orally, intraperitoneally, or intravenously, thus making it unfeasible to use in its free form [8].

In that sense, using CUR-loaded NCs is a viable and efficient therapeutic strategy in minimizing the negative features of CUR as well as to retain its antitumoral effect. Similar results have been reported for different tumor cell lines. Wang et al. [45] prepared solid lipid nanoparticles containing CUR and tested them on NCL-41299 and A549 cells. Yallapu et al. [46] obtained CUR-loaded polymeric nanoparticles and investigated their use against A2780CP and MDA-MB-231 lines.

Also, better results were obtained from the SRB assay instead of the MTT test. These findings were concerning to the high sensibility of SRB when cytotoxicity is investigated for cells growing adhered to dish bottom [47]. SRB evaluates cell viability through the ability of the dye to bind to protein components [47]. On the other hand, MTT examines the activity of mitochondrial dehydrogenase enzymes and their respective redox potential [48].

When cells were treated with NMTX-1, no significant reduction in Calu-3 viability was observed. This result is related to the very slow dissolution rate of MTX and the resistance phenomenon, in which MTX is a well-known substrate of P-gp [18,19]. Calu-3 is a tumor cell line that can express P-gp [49]. Therefore, lower intracellular accumulation of MTX was obtained since P-gp efflux pump was activated and led to the removal of the antitumor drug from within the cell.

To overcome this resistance mechanism, a co-loaded formulation (NCUR/MTX-2) was proposed since CUR even at low doses could block P-gp and could ensure the antitumor effect expected for MTX. The cell viability by MTT and SRB techniques of Calu-3, when treated with NCUR/MTX-2, is demonstrated in Figure 5.

NCUR/MTX-2 showed a significant reduction in Calu-3 cell viability by MTT procedure when 9.88:4 and 4.94:2 ratios of CUR:MTX were tested. For SRB assay, all concentrations (9.88:4; 4.94:2; and 2.47:1 ratio) provided a statistically significant decrease in tumor cell viability.

NMTX-1 at different drug concentrations of 1, 2, and 4 <sup>μ</sup>mol·L−<sup>1</sup> did not reduce Calu-3 viability when tested alone. However, co-loaded formulations containing the same MTX concentrations and low CUR concentrations achieved a statistical reduction of cell viability. This positive result may be due to the suitable dissolution profiles and the P-gp inhibition. NCUR/MTX-2 released both CUR and MTX in almost 72 h that was indeed the time interval during the in vitro cell culture-based assay was performed. Also, it is possible to infer that low CUR concentration in NCUR/MTX-2 plays a remarkable role as P-gp inhibitor, which allows that MTX remains within the cell and performs its antimetabolic/antitumor function. The effect of CUR was attributed only to its capacity of blocking P-gp because this drug demonstrated cytotoxicity against Calu-3 at concentrations higher than 50 μmol.L−<sup>1</sup> as represented in Table 2. Dey et al. [20] studied alginate-stabilized gold nanoparticles containing CUR and MTX and investigated their effect on C6 glioma and MCF-7 cancer cells. Cell viability was

reduced by the use of CUR:MTX ratios of 42:41 and 21:20.5 <sup>μ</sup>mol·L−1. In this study, a statistically significant decrease in tumor cell viability was observed by the use of NCs containing CUR even at lower concentrations than those reported in the literature [13].

**Figure 5.** Cell viability of Calu-3 by MTT and SRB assays after its treatment with NCUR/MTX-2 at different (low) concentrations for 72 h. Results are expressed as mean ± standard error of the mean from 4 independent experiments. Asterisks denote significance levels compared to control: significantly different, \*\* *p* < 0.001 and \*\*\* *p* < 0.0001.

Another mechanism that may be responsible for the improved cytotoxicity of MTX by CUR is the increase of folate receptors (FRs) expression. FRs are transport systems that show a high affinity for folate and are expressed in large numbers of cancer cells. They are important for cell uptake of folic acid for its metabolism and cell division. However, MTX use can lead to cell resistance since this drug reduces FRs activity [50]. Furthermore, polysorbate 80, a pharmaceutical excipient used as a surfactant during NCs preparation, is a well-known P-gp inhibitor. Its molecules insert themselves between lipid tails of the lipid bilayer and fluidize the cell membrane. It may also interact with the bilayer's polar heads and change the hydrogen bond or ionic bond forces which may contribute to its inhibitory action against P-gp [51]. In that sense, NC composition may enhance the effect of efflux pump inhibition provided by CUR and may lead to improved cytotoxicity against non-small-cell lung cancer cell growth even at low doses of CUR and MTX.

Taking all these into account, the combination of low doses of CUR and MTX by the use of colloidal suspensions of NCs may be a promising strategy for the treatment of lung cancer, reducing the toxic effects associated with the use of high doses of MTX [44,50].

#### 2.3.2. Combination Index

The next question addressed was whether NCUR/MTX-2 could provide a synergistic effect on Calu-3 lung cancer cells using MTT cytotoxicity assay. To reveal the synergistic activity, a combination index (CI) was performed considering the half-maximal inhibitory concentration (IC50) data of separate and combined cytotoxic effects of NCs containing CUR and/or MTX. IC50 (mean value ± SD) for CUR from NCUR-2 was 100.18 <sup>±</sup> 3.74 <sup>μ</sup>mol.L<sup>−</sup>1. NMTX-1 presented an IC50 of 23.06 <sup>±</sup> 1.13 <sup>μ</sup>mol·L<sup>−</sup>1. IC50 for CUR and MTX from NCUR/MTX-2 were 69.81 <sup>±</sup> 3.07 and 1.89 <sup>±</sup> 0.06 <sup>μ</sup>mol·L<sup>−</sup>1, respectively. Thereby, the CI value achieved was 0.78. In brief, the CI equal to 1 indicates that the two drugs have additive effects, the CI lower than 1 suggests a synergism and the CI higher than 1 indicates antagonism [52]. Consequently, a synergistic activity was demonstrated for NCUR/MTX-2 due to the combination of these drugs induced greater cytotoxicity against Calu-3. This finding shows that cytotoxic chemotherapy using NCUR/MTX-2 appears to be a promising strategy for the treatment of non-small-cell lung cancer which merits further preclinical and clinical investigation since it has allowed a suitable effect even at low chemotherapeutic doses into this nanocarrier.
