Sephadex g 75

The ratiometric pH-sensitive fluorophore pyranine (from Molecular Probes, Eugene, OR) was prepared at a concentration of 10 mM in milli-Q water. pyranine (final concentration of 300 μM) was mixed with the stocked liposomes (4 mg of lipid) and 100 mM KPi (pH 7.0) in a total volume of 1 mL. pyranine was encapsulated in the liposomes by three freeze-and-thaw cycles at 30°C (65°C for mixtures containing DPPE, DPPC, and DPPG lipids). The osmolality of the liposome lumen is ∼190 mosmol/kg. This value equals the osmolality of the assay buffer (100 mM KPi (pH 7.0)). After extrusion through a 200 nm polycarbonate filter at room temperature (65°C for mixtures containing DPPE, DPPC, and DPPG lipids) to homogenize the vesicles, the liposomes were eluted through a 22-cm-long Sephadex-G75 (Sigma-Aldrich) column pre-equilibrated with the assay buffer to remove the external pyranine. For blank correction, empty liposomes were prepared using the same procedure without the addition of pyranine. The collected 1 mL fractions containing the liposomes were identified using either an ultraviolet lamp (for liposomes filled with pyranine) or a NanoDrop spectrophotometer (for empty liposomes) and diluted in a total volume of 12 mL of the assay buffer.

E. coli Rosetta(DE3)pLysS cells were from Novagen (Rome, Italy); ECL plus, Hybond ECL membranes were from GE Healthcare; l-[³H]Arg was from Perkin Elmer (Waltham, MA, USA); conjugated anti-His6 antibody, TX-100, Amberlite XAD-4, egg yolk phospholipids (3-sn-phosphatidylcholine from egg yolk), Sephadex G-75, His-Select resin, l-Arg and all the other reagents were from Sigma-Aldrich (Saint Louis, MO, USA).

All the reagents used in enzyme purification were procured from HiMedia Laboratories except the matries for column chromatography viz Sephadex G-75 and DEAE-Sepharose which were obtained from Sigma-Aldrich (St. Louis, MO, USA). Flavonoid and isoflavonoid standards daidzein, daidzin, genistin, genistein, hesperidin, hesperetin, naringin, naringenin, rutin hydrate and quercetin used were also obtained from Sigma-Aldrich, while the solvents hexane, toluene, chloroform, ethyl acetate, methanol, acetone, formic acid and glacial acetic acid (AR grade), from HiMedia. The TLC plates (precoated with aluminum-backed silica gel 60 F₂₅₄) were procured from Merck (Darmstadt, Germany).

The following materials were used: BL21 pLysS (DE3) strain (Novagen, Cat. No.: 69451–3, Darmstadt, Germany), pAED4 (BV Tech, Sofia, Bulgaria), isopropyl β-d-1-thiogalactopyranoside or IPTG (Sigma-Aldrich Cat. No.: I6758, St. Louis, MO, USA), Luria Bertani broth or LB broth (Merck, Cat. No.: 1.10285.0500, Whitehouse Station, NJ, USA), diethylaminoethyl (DEAE)-Sepharose Fast Flow (Amersham, Cat. No.: 17-0709-01, Amersham, UK), Sephadex G-75 (Sigma-Aldrich, Cat. No.: G7550), l-asparagine (Sigma-Aldrich, Cat. No.: A0884), Nessler's reagent (Sigma-Aldrich, Cat. No.: 72190), and CS (low molecular weight (% deacetylation 75% to 85%, viscosity 20 to 300 cP, average MW ~ 50 kDa), Sigma-Aldrich; Cat. No.: 448869), sodium tripolyphosphate (Sigma-Aldrich, Cat. No.: 238503).

1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] ammonium salt (DSPE-m-PEG2000 or 18:0PEG2000PE), and cholesterol were bought from Avanti Polar Lipids LLC (Alabaster, AL, USA), L-α-Phosphatidylethanolamine (PE) from egg yolk was purchased as a lyophilized powder from Sigma Aldrich (St. Louis, MO, USA). All lipids purchased were used without further purification. SN-38 in powder was purchased from Fisher Scientific (Waltham, MA, USA) and its stock solution was prepared in dimethyl sulfoxide (DMSO; 10 mg/mL). Reagents such as methanol and chloroform were purchased from Fisher Scientific and used as provided. Sephadex G-75 and carboxyfluorescein were purchased from Sigma Aldrich (St. Louis, MO, USA). Indocyanine green (ICG) was purchased from Fisher Scientific (Waltham, MA, USA). Vivotrack 680 was purchased from PerkinElmer (Hopkinton, MA, USA).

HEPES salt (Sodium 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonate), cholesterol, dicetyl phosphate (DCP), Sephadex G75, pentamidine isethionate, pyrene, 1,6-diphenyl-1,3,5-hexatriene (DPH), mucin from porcine stomach type II powder, Tween 20 (polysorbate 20), chitosan medium molecular weight powder, and sodium hydroxide were purchased from Sigma-Aldrich (Milan, Italy). l-Glutamic acid was supplied by PanReac Applichem (Milan, Italy). All other products and reagents were of analytical grade.