Rotofix 32a

NCI-H69 cells (ATCC) were cultured in Rosewell Park Medium Institute 1638 medium (RPMI-1638) (Sigma-Aldrich) supplemented with penicillin (50 units/mL), streptomycin (50 µg/mL) (Pen/Strep) and 10% fetal calf serum (FCS). CA20948 14 (link) cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) supplemented with Pen/Strep and 10% FCS. Cells were cultured at 37 ^oC and 5% CO₂.
The molar activity of [¹⁷⁷Lu]Lu-DOTA-TATE used in vitro was 37 MBq/µg with a purity > 95%. Both CA20948 and NCI-H69 cells were incubated with 1 MBq/mL [¹⁷⁷Lu]Lu-DOTA-TATE for 4 h at 37 ^oC. NCI-H69 cells were spun down on a glass slide using a cytospin centrifuge (Rotofix 32A, Hettich) for 6 minutes at 347 g at room temperature (RT). CA20948 cells were washed and fixed on coverslips. Fixation was done using 2% paraformaldehyde (PFA) for 20 minutes at RT and samples were stained according to protocol (see below).

Rosehips were treated using different methods of preservation such as freezing, air-drying, and lyophilization. Freezing was performed in a commercial freezer at −20 °C, air-drying was performed in the dark at room temperature for 20 days, and lyophilization was performed with a vacuum freeze-dryer (Christ Freeze Dryer ALPHA 1-2, Milan, Italy) at 0.01 atm and −48 °C for 72 h.
The seeds were removed from differently preserved rosehips and their pulp was finely chopped; 10 g of pulp was put in a mixture of ethanol/water 50:50 v/v (150 mL) and sonicated (Transonic TP690 by Elma, Singen, Germany) for 40 min at room temperature. After extraction, the mixture was centrifuged at 4000 rpm for 20 min and the supernatants were filtered using a Buchner filter (Rotofix 32A by Hettich, Tuttlingen, Germany). The residue was further extracted by repeating the procedure mentioned above. The supernatants were collected and evaporated under a vacuum at 40 °C (Buchi Rotavapor Heating Bath B-490) to remove alcohol and most of the water. The concentrated extract was freeze-dried at 48 °C for 48 h and stored at +4.0 ± 1.0 °C in the dark until use.

Cells (2×10⁵ cells/mL) cultivated in different tumor environments for 72 h were treated with perifosine (20 µM) for 2 h. Then, 6×10⁵ cells in 100 µl of 1×PBS was cytocentrifuged (Rotofix 32A, HETTICH Zentrifugen, Tuttlingen, Germany, 1,000 rpm/5 min) on 4 positions of an indium tin oxide (ITO) conductive glass slide. The diameter of each spot was approximately 5 mm. The samples were covered by sublimated 2,5-dihydroxybenzoic acid (DHB, 0.24 mg/cm²) and dried in a desiccator for at least 30 min. Perifosine in spots of cytocentrifuged cells was measured using MALDI MSI (see the Supplementary Data Sheet 1, Methodology). However, since the imaging mode was not employed to obtain spatial information, but rather to automatically measure selected areas of all 4 spots at once, the laser beam diameter was set to a relatively large value of 70–80 µm and the pixel size was 200 µm. To obtain maximum information from the 200-µm pixel area, a total number of 1,000 laser shots were summed within the pixel, with random movement of the laser firing 200 shots/position. The number of pixels measured from each cytocentrifuged spot was approximately 40 to control the pixel-to-pixel reproducibility. The cytocentrifugation experiment was performed in duplicates the same day (2 glass slides) and in triplicates during a longer period.

The cytotoxicity analysis of the electrospun nanofibres membrane was performed using Human Dermal Fibroblasts adult. The cells were grown in DMEM (Dulbecco’s Modified Eagle Medium), supplemented with 10% FBS (Fetal Bovine Serum) and an antibiotic cocktail consisting of 1% (v/v) penicillin-streptomycin and 1% (v/v) non-essential amino acids. The medium was changed every day. The cell culture flasks (NuncTM EasYFlask TM, ThermoFisher Scientific, USA) were incubated at 37 °C in a 95% humidified atmosphere and 5% CO₂ (MCO-5AC CO₂ Incubator, Sanyo). Cells were allowed to grow in four culture flasks to reach 80% confluence and then were trypsinised with 0.25% trypsin solution at 37 °C for 3 min, followed by the addition of fresh medium to neutralize trypsin. The concentration of cells was 1 × 10⁴ cells/cm². After centrifugation (Roto-fix-32A, Hettich, Tuttlingen, Germany) and re-suspension in fresh medium, the viable cells were incubated in the presence of sterilised membrane samples on flat-bottom 96-well plates for 24 and 48 h. After the incubation time, the cell viability was determined using 3-(4,5-Dimethyl-2-thia zolyl)-2,5-diphenyl-2H-tetrazolium bromide (Merck, Darmstadt, Germany) following the MTT assay. Each type of nanofibre membrane was tested in triplicate.

The anti-oxidant properties of the fresh pasta were analysed for two weeks. For that, the phenolic compounds present in each formulation were extracted using ethanol. Briefly, 4 mL of ethanol were added to 2 g of fresh pasta and the extraction procedure (1 min of vortex followed by 5 min in an ultrasonic bath) was performed three times. The solutions were centrifuged for 20 min at 3000 rpm using a Rotofix 32 A centrifuge (Hettich, Tuttlingen, Germany). The supernatant was collected and, again, 4 mL of ethanol were added to the fresh pasta and the whole procedure was repeated once more. The supernatant was used to analyse the anti-oxidant capacity (ABTS and DPPH) of the different formulations according to the methods described in Section 3.3.