Polyethylene glycol adsorbed in microemulsion (MLP) was formulated from an aqueous phase (distilled water), oily phase (isopropyl myristate-IPM^®-EHL 11.5 (Bianquimica^®, São Paulo, Brazil)), tensoactive (polysorbate 80-Tween 80^® (TW)-EHL = 15.0 (Dynamics^®, São Paulo, Brazil)) and polyethylene glycol 400-PEG 400@EHL = 8.5 (Dynamics^®, São Paulo, Brazil) modified according to the method of Torres et al. (2021) where each component ranged from 10% to 75%. Being used for formulation, 10% of the oily phase, 15% of the phase aqueous, and 75% of tensoactive [44] .

Caffeine was weighed on an analytical scale, diluted in Buffered Saline Solution (PBS), and adjusted at a 100 ng/mL concentration. Diluted caffeine was associated with the microemulsified system containing PEG 400 in the equivalent amount of the aqueous phase of the formulation, where diluted caffeine was homogenized with PEG 400, plus the oily phase, and then the tensoactive was added. Caffeine concentration was determined based on a previous study in which cellular modulation was observed [26] .

A 10 mL of the peripheral blood sample from 18 clinically healthy donors aged 18 to 35 was collected in Vacutainer tubes with EDTA (Beckton Dickinson, Franklin Lakes, NJ, USA®) to obtain MN cells. Then, the cell populations were separated by density gradient with Ficoll-Paque (Pharmacia, Upsala, Sweden) by centrifugation for 40 minutes at 160 ×g at laboratory temperature (25˚C). Next, the mononuclear phagocyte ring (MN) was removed with a Pasteur pipette and centrifuged twice at 160 ×g for 10 minutes with buffered saline solution (PBS) for cell washing. Finally, the supernatant was discarded, and the pellet was added 1 ml of PBS. The cells were then counted in the Neubauer chamber, and the cell concentration was adjusted to 2 × 10⁶ cells/mL according to Honorio-frança [45] .

ATCC (American Type Culture Collection, USA) cell lines of breast adenocarcinoma (MCF-7) were used for the biological assays with the immunomodulator. The cells were cultivated in RPMI medium (Sigma), plus 10% Bovine fetal serum (FBS, Cultilab), 1.0 ml pyruvate, 1.0 ml Hepes, 4.5 ml glucose, 100 μl gentamicin, the final volume of 100 ml of the medium. The cells were grown in bottles and maintained at 5% CO₂ and 37˚C until the formation of a cell monolayer. Every 96 hours, the cells were removed from the CO₂ oven, added in a 15 ml falcon tube, and 1 ml of trypsin was placed in each bottle for 5 minutes. Trypsin was removed from the bottles and added to the corresponding falcon tubes. The bottles with RPMI medium were washed and added to the corresponding falcon tubes to avoid cell loss. The cells in the falcon tube were centrifugated for 10 minutes at 160 g. The Avernum was discarded, and the pellet was suspended in a 2 ml RPMI medium. Next, 1 ml of MCF-7 cells were added in two new bottles; each bottle was completed with 4 ml of RPMI medium and taken to a CO₂ kiln repeating the cycle until the number of cells suitable for the experiment was obtained.

The MN and MCF-7 cells were incubated with caffeine associated or not with polyethylene glycol adsorbed in microemulsion to verify the release of superoxide anion, using the chromogen Ferricitochrome C, according to the Pick and Mizel method [46] and adapted by Honório-França et al. [47] . They were analyzed by spectrophotometer at 550 nm absorbance. For the assays, the samples were divided into groups containing 500 μL of MN, MCF-7 cells, and cocultures incubated with 50 μL of caffeine associated or not with polyethylene glycol adsorbed in microemulsion and without stimuli used as control.

The samples were incubated for 24 hours at 37˚C in a greenhouse with 5% CO₂.

At the and of this time, the samples were centrifuged at 1600 g for 10 min. The supernatant was collected and reserved for analysis of the SOD enzyme, while the pellets were resuspended in 150 μL of ferricytochrome C solution (2 mg/mL of glucose PBS). Subsequently, the samples were transferred to a 96-well plate and accommodated in an oven at 37˚C for 1 hour without light. Finally, the reading was performed in a spectrophotometer (Thermo Plate TP-Reader) with a 540 nm filter.

The concentration of the superoxide anion was calculated according to the method adapted from Pick and Mizel [46] by the equation:

Concentration O 2 − = (DO/6.3) × 100

where: DO: Optical density.

The spectrophotometer performed the enzymatic activity of superoxide dismutase (Cu-Zn-SOD) at 560 nm absorbance. For the dismutation assays, 500 μL of the cell suspension overeating was pipetted in different tubes, and the stimulus was removed from the O 2 − dosage. Then, in each tube, in the following order were added: 500 μL of the chloroform-ethanol mixture (1:1), 500 μL of the reactive mixture of Nitro Bluetetrazolium (NBT) and tetraacetic ethylenediamine acid (EDTA) (1:1.5) and 2 mL of carbonate and hydroxylamine buffer solution. For calibration of the device, from the reactive mixture of NBT and EDTA. The standard absorbance was measured from the following solutions: 500 μL of the hydroalcoholic mixture (1:4), 500 μL of the chloroform-ethanol mixture (1:1), 500 μL of NBT and EDTA reactive mixture, and 2 mL of carbonate buffer and hydroxylamine.

SOD values were expressed in SOD g⁻¹ units, i.e., in terms of enzyme activity, a SOD unit is defined as the amount of enzyme required to inhibit 50% of the NBT reduction. The equation gives the calculation of % inhibition:

% reduction of NBT = (Abs. standard − Abs. sample)/Abs. standard × 100

where:

Abs. standard: Absorbance of standard sample;

Abs. Sample: Absorbance of each sample at 560 nm.

Rheological parameters were determined in Modular Compact Rheometer—MCR 102 (Anton Paar®, GmbH, Ostfildern, Germany). For the hemorheological analyses, 600 μL of blood and 60 μL of MLP stimulus and caffeine associated or not with polyethylene glycol adsorbed in microemulsion were added to the surface of the reading plate, and the excess sample was removed. The readings were performed with permanent control of the measurement gap with TruGap^TM support at 0.099 mm, unit measuring cell Toolmaster^TM CP 50 (angle 1˚), and precise temperature control with T-Ready feature^TM, using Software Rheoplus V3.61. The rheological charts were all treated with Rheoplus Software. For the flow and viscosity curves, the established parameters were based on the control of shear stress (τ), and the last 5 points of the ascending curve and the first 5 points of the descending curve for statistical analysis were used, modified according to the method of France et al. [48] . The tests were carried out under isothermal conditions at 37˚C.

Statistical analyses were performed in the BioEstat 5.3 program through a variance analysis (ANOVA), followed by the Tukey test for the analyses that presented p < 0.05. Spearman’s test performed the correlation between the values of the viscosity and apoptosis assays.

The rheological evaluations of the blood with caffeine associated or not with polyethylene glycol adsorbed in microemulsion (MLP) were able to alter the rheological behavior, increasing viscosity (Figure 1) and altering the behavior of the blood (Figure 2). A recent study with melatonin associated with microemulsion and polyethylene glycol 400 (MLMP) also showed that there was a rheological change, where it was observed that the viscosity of the blood treated with MLMP increased [44] .

A similar result was observed in a study conducted with Dillenia, indicating a change in the viscosity of peripheral blood in the presence of Dillenia indica extract at the concentration of (100 ng/ml) [49] . The viscoelastic properties of cells are important biomarkers of disease status and progression. A simpler approach to defining cells’ viscoelastic properties examines two parameters: stiffness and viscosity [50] .

The rheological evaluation of MCF-7 cells with caffeine associated or not with polyethylene glycol adsorbed in a microemulsion is presented in Figure 3 and Figure 4. An increase in viscosity was obtained (Figure 4) in MCF-7 cells treated

with MLP and caffeine associated with polyethylene glycol adsorbed in microemulsion. It is observed that there was also a change in the rheological behavior of MCF-7 cells (Figure 3).

Research conducted with cancerous liver cells of the HepG2 line treated with dexamethasone also observed an increase in cellular viscosity, corroborating our study [51] .

A study using mononuclear phagocytes (MN), MCF-7 cells, and coculture treated with Parrychloride (BaCl2) adsorbed to PEG microspheres and a study using Bryophytes spp. and MCF-7 cells observed that blood flow and viscosity curves were not affected [41] [52] .

The release of O 2 − was higher in MCF-7 cells treated with caffeine adsorbed in MLP (Figure 6), suggesting the stimulating effect of MLP in these cells. A study previously carried out by the group also observed an increase in the release of superoxide in MN phagocytes co-cultivated with MCF-7 cells treated with mangaba fruit extract (Hancornia speciosa), adsorbed or not in PEG microspheres [26] .

A similar result was observed in the combined treatment of menadione (vitamin K) and calcitriol (vitamin D) in MCF-7 cells. In addition, it was observed that many trials performed with menadione and calcitriol increased O 2 − , but these values intensified when both drugs were administered simultaneously [53] .

In general, when compared to normal cells, tumor cells produce more ROS due to metabolic and signaling alterations [54] . This may explain why MN phagocytes treated with caffeine associated with MLP did not demonstrate a significant increase in the release of O 2 − (Figure 5) compared to MCF-7 cells (Figure 6).

Caffeine showed modulating activity in MCF-7 cells, decreasing O 2 − (Figure 7). The same was observed in MN and MCF-7 cells in coculture, probably due to caffeine’s antioxidant properties. Several studies have shown that coffee reduces oxidative stress [40] [42] [43] . A previous study with pomiferin will identify the significant regulation of two important genes, the MN-SOD and ACOX1, of antioxidant enzymes that help remove excess superoxide anions in MCF-7 cells [55] .

The regulation of oxidative stress is important in tumor development and responses to anticancer therapies. Cell survival and death can be influenced by oxidative stress. Although high levels of ROS cause cell death, low levels of free radicals can directly modulate transcription factors that regulate apoptosis [56] . Because of this, scientists are looking for potential modulators of oxidative stress as anticancer strategies [26] [44] .

In metabolic reactions, they also have antioxidant enzymes that control THE concentration. Superoxide dismutase (SOD) is a very important enzyme that functions in the dismutation of O 2 − in H₂O and O₂ [42] . The antioxidant action of caffeine can aid in eliminating ROS by avoiding cellular damage. Many studies have shown that caffeine can eliminate free radicals and increase the activity of superoxide dismutase (SOD) in vivo [57] [58] . SOD activity was increased in MN phagocytes treated with MLP and caffeine associated with MLP (Figure 8). The result suggests that the antioxidant system of MN phagocytes was maintained, where we observed increased SOD activity and O₂-reduction. A similar result performed with melon-são-Caetano (Momordica charantia) altered the SOD production of mononuclear cells, increasing their antioxidant effects [59] .

There was a reduction in the sod activity of MCF-7 cells when treated with caffeine (Figure 9) this is a good factor, as it can prevent the antioxidant escape mechanism used by the pre-established tumor. Tumor cells increase their antioxidant defense mechanisms to prevent the high concentration of ROS in the tumor microenvironment from inducing cell death [60] .

In the present work, modulation was observed MCF-7 cells treated with MLP and caffeine associated or not with polyethylene glycol adsorbed in microemulsion, in which the MLP and caffeine associated with MLP significantly increased the activity of sod of MCF-7 cells (Figure 9). A study conducted with Hancornia speciosa adsorbed in PEG microsphere in MCF-7 breast cancer cells and cocultured with blood cells also shows high levels of SOD in these cells when treated with H. speciosa ethanol extract (HSEE) [26] .

Caffeine can potentially increase the antioxidant defenses of breast tumor cells. The same was observed in MCF-7 and MDA-MB-231 cells treated with caffeine [38] .

Phagocytes MN and MCF-7 in coculture treated with MLP and caffeine associated with MLP had the reduction of SOD (Figure 10). A similar result was observed in a study conducted with MN phagocytes with enteropathogenic Escherichia coli (EPEC) bacteria treated with Psidium guajava leaf extract. It was observed that at concentrations of 200 ng/mL and 200 pg/mL, the extract significantly reduced the activity of the SOD enzyme [61] . A study conducted with cells treated with guarana and their combinations with caffeine and/or taurine showed an exponential decrease in SOD activity in human neuronal cells (SH-SY5Y) [62] .