3.2.5.2. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis on Gastric-Resistance Test Samples

To proceed with GC-MS assay, 5 mL of liquid coming from gastric resistance test chamber were added of 100 μL of metyl pentadecanoate solution (115 μg/mL) as internal standard. Extraction with ethyl acetate was performed three times. The obtained organic phases were pooled together, anhydrified with sodium sulfate and led to dryness. The extracts were added of 3 mL of methanol and 1 mL of dichloromethane and were acidified with concentrated sulfuric acid. Samples were heated under reflux for 40 min, then the solution were cooled down and diluted with water and diethyl ether. The etheric phase was investigated.

The standard was prepared mixing 100 mg of SRO fatty acids GC with 1 mL of metyl pentadecanoate solution (115 μg/mL) as internal standard, and adding 2 mL of dichloromethane, 10 mL of methanol and 0.2 mL of concentrated sulfuric acid. Standard sample was heated under reflux, then cooled down and diluted with water and diethyl ether. The etheric phase was investigated.

The GC-MS system was comprised of a Varian 3800 equipped with autosampler and coupled with a Varian Saturn 2100 MS/MS ion trap mass spectrometer. A HP-88 column (60 m × 0.25 mm) was used for separation (J and W Scientific, Agilent technologies Inc., Santa Clara, CA 95051, USA)

#### 3.2.5.3. Dynamic Light Scattering (DLS) Analysis of Lipomatrix Dispersion in FaSSIF-V2

DLS was performed on emulsified dispersion of Lipomatrix in *FaSSIF-V2* as described in Section 3.2.1.2, corresponding to 640 mg of SRO resulting from in vitro emulsification test described in Section 3.2.3. The size distribution of the particles was evaluated as a signal intensity function only. The conversion of the signal intensity distribution into particles volume or number distribution can cause error propagation since it requires some unavailable parameters (e.g., the particles refractive index). The liquid samples (FaSSIF-V2 and Lipomatrix dispersion in FaSSIF-V2) were analyzed by DLS (Zetasizer Nano S, Malvern).

#### 3.2.6. Cell Cultures

#### 3.2.6.1. Caco-2 Cell Culture

The Caco-2 human colon adenocarcinoma cells (passage 32 to 42) were seeded in adhesion flask at a density of 2 × 103 cell/cm2 in Caco-2 Complete Medium (CCM) (high glucose DMEM, 10% heat inactivated FBS (FBS), 1% non-essential amino acids, 4 mM L-glutamine, and 1% penicillin-streptomycin mix) and cultured at 37 ◦C and 5% CO2 in a humidified incubator. Cell 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.

#### 3.2.6.2. LNCaP Cell Culture

The human prostate cancer cell line LNCaP cells (passage 25 to 40) were maintained in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin mix. The cells were grown at 37 ◦C in a humidified atmosphere with 5% CO2. Cell were seeded at 10,000 cell/cm2 and medium changes every other day. As for Caco-2, cells were subcultivated by tryspinization every 7 d when 80–90% confluent. For vitality and anti-inflammatory experiments, LNCaP were seeded 96-well plates and six-well plates, respectively, at a density of 1 × <sup>10</sup><sup>5</sup> cell/cm2 and allowed to adhere for two days prior to experiments, while for prostate-specific antigen (PSA) experiment cells were seeded at a density of 50,000 cells/cm<sup>2</sup> in 24-well plates.
