Amicon membrane

HUVEC cells were grown in plates using EGM2 with 1% (v/v) growth factors (hFGF, VEGF, R3-IGF-1), hydrocortisone, ascorbic acid, glutamine and heparin, keeping them incubated at 37°C in a 5% CO₂ atmosphere and collected in the first phase of the cell cycle (G1). The cells were harvested by centrifugation (600 g, 5 min), washed twice with PBS (NaH₂PO₄ 50 mM and NaCl 150 mM, pH 7.4) and resuspended in 450 μl of PBS at 0.1% of Igepal together with 50 μl of protease inhibitor cocktail (AESBF (4- (2-Aminophenylbenzenesulfonyl fluoride hydrochloride), aprotinin, bovine hydrogen chloride, E-64 [N- (trans-epoxy succinyl] -L-leucine, 4-guanidiobutylamide, EDTA, salt of leupeptine bisulfate) to avoid protein degradation. The resuspended pellet was subjected to homogenization by a manual “Dounce” homogenizator and centrifuged at 10,000 rpm for 5 min at 4°C (Centrifuge 5424-R, Eppendorf). Protein-rich supernatant was extracted and thus separated from insoluble pellets, consisting of membranes and cytoplasmic organelles. The concentration of protein lysate was estimated by Bradford assay and diluted to obtain a concentration of 1 mg/ml (Bradford, 1976 (link)).
The secretome was harvested from the cultured cells and concentrated using an Amicon membrane (Millipore); protein concentration was evaluated by Bradford's assay to obtain a final value of 6 mg/ml.

Supernatant of Nalm-6/CD63-nanoluc cells were concentrated on a 100-kDa Amicon membrane (Millipore, USA). Further, the supernatant was added to the 0.2 × 10⁶ CD19-CAR Jurkat and control Jurkat cells at various concentrations and incubated at 37 °C for 10 min. The cells were pelleted, washed before measurement of the nanoluciferase activity. A similar experiment was carried out with purified EVs.

H. polygyrus somatic antigen (referred to as H. polygyrus antigen) and H. polygyrus excretory/secretory products (HES) were prepared using established methods described by Hewitson et al. and Johnston et al. [43 (link),60 (link)]. In brief, male, 8-week-old, C57BL/6 mice were infected with 400 L3 H. polygyrus larvae by gavage, and adult worms were recovered 14 days post-infection. Adult worms were washed extensively before incubation in RPMI 1640 medium, supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 U/mL streptomycin, and 100 µg/mL gentamicin. Pooled culture supernatants were filtrated into phosphate buffered saline PBS over a 3000 MW Amicon membrane (Merck Millipore, Darmstadt, Germany). The resulting HES was a well characterized preparation containing 374 proteins, novel O-linked glycoproteins, and small RNAs [43 (link),45 (link),61 (link)]. H. polygyrus antigen was prepared by uniformly homogenizing adult worms in PBS on ice using a Polytron homogenizer (Kinematica AG, Lucerne, Switzerland; 3 × 30 s pulses at speed 3, with 30 s intervals). The resulting mixture of 446 proteins was centrifuged at 13,000 g for 30 min, from which the supernatant was collected [43 (link)]. The protein concentration of HES and H. polygyrus antigen was quantified using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, USA) before storing at −80 °C.

The plasmid-free C29 strain was grown in BHI broth without antibiotics and supplemented with ampicillin at 0.25×, 0.5×, and 0.75× MIC (Sigma‒Aldrich). The culture was incubated for 18 h at 37 °C under shaking at 200 rpm, followed by centrifugation at 10,000× g. The supernatant was filtered with a 0.22-µm membrane followed by ultrafiltration in an Amicon membrane^® (PM30 polyethersulfone, Merck Millipore, Burlington, MA, USA) and sterilization at 121 °C/20 min as previously reported [1 (link),3 (link)].

To prepare D-HFnX and
D-HCX, Doxo was loaded using the pH disassembly-reassembly method
already described by our group, with slight modifications.^{14 (link)} The protein was diluted down to 0.5 mg/mL into
a 150 mM NaCl solution, adjusted to pH 2 to disassemble protein nanocages,
and incubated at 180 rpm at room temperature (RT). After 15′,
Doxo (200 μM) was added, the pH was adjusted back to 7.5, and
the mixture was incubated for 2 h in agitation (180 rpm, RT). At the
end of incubation, the solution was centrifuged (3500g, 15′) through 4 mL of 100 kDa Amicon membranes (Millipore)
several times to simultaneously concentrate the nanodrug and remove
nonencapsulated Doxo.
Finally, the nanodrugs were centrifuged
through 7K MWCO Zeba Spin Desalting columns (Thermo Fisher) previously
equilibrated with PBS for buffer exchange and further purification.
Encapsulated Doxo was extracted by diluting the samples in a 1:1 isopropanol/chloroform
solution, with SDS 0.01% and K₂SO₄ 0.01%, and
incubated O/N at −20 °C. The following day, Doxo concentration
was measured by spectrofluorimetry and compared with a predetermined
calibration curve.

The selected recombinant E. coli clones were grown as previously described [57 (link),76 (link)]. Kanamycin 0.05 mg/mL was added into the medium for the cultivation of cells harboring plasmids. The target protein expression was induced by lactose added to the expressed culture at a density of A600 1.9 to a final concentration of 0.2%. The cells were grown for an additional 17–20 h and pelleted by centrifugation at 4000× g for 15 min.
All enzyme purification stages were performed at +4 °C. Three grams of biomass was suspended in buffer (20 mM sodium phosphate buffer pH 7.2, 1 mM glycine, 1 mM EDTA) and destroyed by ultrasound treatment [57 (link)]. Cell debris and unbroken cells were removed by centrifugation (35,000× g, 30 min). Supernatant, containing the enzyme, was applied to SP-Sepharose column. Protein was eluted with a linear gradient of 0–1.0 M NaCl. Column fractions, containing enzyme (0.46–0.7 M NaCl), were collected. Ultrafiltration, desalting and buffer exchange was performed using Amicon membranes (Millipore, Burlington, MA, USA) as described previously [77 ]. Samples were frozen and stored at −20 °C.
Protein concentration was determined by the method of Sedmak [78 (link)] with bovine serum albumin as the standard. SDS-PAGE was carried out to visualize and determine protein purity as previously described [79 (link)].