3.2.6.3. THP-1 Cell Culture

Human THP-1 monocytes (passage was maintained in RPMI-1640 medium with glutamate supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin (GIBCO, Winsford CW7 3GA, UK). Cells were cultured at a density of 5 × <sup>10</sup><sup>5</sup> cells/mL in 5% CO2 humidified atmosphere at 37 ◦C and subcultured twice a week. Macrophage differentiation was induced by incubation with 500 nM phorbol myristate acetate (PMA; Sigma-Aldrich, MO, USA) for 24 h. Culture medium was then replaced and cells cultured for an additional 24 h. For medium conditioning, 6 × <sup>10</sup><sup>6</sup> cell were seeded in 75 cm<sup>2</sup> flask, differentiate into macrophages as described before and treated with 1 ng/mL LPS for 6 h. At the end of the LPS treatment, medium was recovered and stored at −80 ◦C until use.

## 3.2.6.4. WPMY-1 Cell Culture

Human prostate stromal (myofibroblast) (WPMY-1) (passage 40 to 50) were cultured in DMEM, supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin-streptomycin mix. As for Caco-2, cells were seeded at 2000 cells/cm<sup>2</sup> and subcultivated by tryspinization every 7 d when 80–90% confluent. The medium was refreshed every other day. For cell contraction experiments, 1 × <sup>10</sup><sup>6</sup> cells were loaded into each collagen gel.

#### 3.2.7. Determination of Active Principle Absorption Rate

To evaluate the effectiveness of Lipomatrix technology in increasing the enteric absorption rate of lipophilic active principles, we compared the performance of a Lipomatrix-based formulation (LBF) as described in *3.2.1.1* against two commercial formulations, as indicated in Table 7.


#### **Table 7.** List and composition of analyzed formulations.

#### 3.2.7.1. Digestion Process

A single dose of each formulation listed in Table 7 was exposed to in vitro digestion process simulating the physiological human digestion in the oral, gastric and intestinal compartments. Briefly, the formulations were incubated for 5 min in saliva at 37 ± 1 ◦C, rotating head-over-heels at 55 rpm, simulating peristaltic movements. Subsequently, gastric juice (pH 1.3 ± 0.1) was added to the mixture and the pH of the sample was checked and, if necessary, adjusted to 2.5 ± 0.5 with NaOH (1 M) or HCl (37% *w*/*w*). The sample was further incubated rotating at 37 ◦C for 2 h. Subsequently, duodenal juice (pH 8.1 ± 0.1), bile (pH 8.2 ± 0.1) and sodium bicarbonate were added. The pH of this mixture was set at 6.5 ± 0.5 with NaOH (1 M) or HCl (37%) and it was rotated head-over-heels for another 2 h. For simulated digestive fluids composition refer to Walczak et al., 2013. Once completed the digestion process, SRO bioaccessibility was determined by measuring fatty acids with GC/MS, while proanthocyanidin (PAC) and lycopene loaded in Lipomatrix formulation were determined by high pressure liquid chromatography (HPLC).

#### 3.2.7.2. HPLC Analysis of LYC and PCA

Bioaccessible fractions of LYC and PAC, released during the digestive process and absorbed at the intestinal epithelium level, were determined by HPLC. Reversed phase HPLC with absorbance detection (at 475 nm) based on the modified method of Thadikamala et al. (2009) was used for analyzing LYC. HPLC separation was carried out using a Varian Prostar 210 pump system (Agilent) and a UV–VIS detector (Variant Prostar, Agilent), operated at 25 ◦C. A C18 column (4.6 × 150 mm, Agilent) was used with methanol-acetonitrile-methanol-tetrahydrofuran (THF) (70:25:5, *v*/*v*) as an isocratic eluent. Each sample was dissolved first in methanol-THF (50:50, %*v*/*v*) and diluted as needed in the same solvent. Quantification of the eluted LYC was accomplished by the peak area method using the calibration range of 15 to 150 ppm of LYC (Sigma-Aldrich, Milan, Italy) as external standards. For PACs, HPLC analysis was performed with Variant Prostar HPLC (Agilent) with the same pump system and UV–VIS detector described above. The phenolic compounds were detected at 280 nm with a flow rate of 1 mL/min. The column was operated at a temperature of 25 ◦C. Separations were carried out with a C18 column (Agilent) in a dual pumping system by varying the proportion of 2.5% (*v*/*v*) acetic acid in water (mobile phase A) and 70% methanol in water (mobile phase B). The

solvent gradient elution program was as follows: 10% to 26% B (*v*/*v*) in 10 min, to 70% B at 20 min and finally to 90% B at 25 to 31 min. The injection volume for all samples was 100 μL. The phenolic compounds were analyzed by matching the retention time and their spectral characteristics against those of standards PAC A, B1, or B2 (20 to 400 ppm).
