Snakeskintm dialysis tubing

All recPrP proteins were expressed in E. coli BL21 (DE3) and purified according to a previously reported protocol53 (link) with minor modifications. Briefly, inclusion bodies were collected (15,000 × g, 30 min, 4 °C), resuspended in 6 M Guanidine hydrochloride (GuHCl), 10 mM Tris-HCl, 100 mM Na-PO₄ buffer, 10 mM β-mercaptoethanol (βME), pH 8.0, sonicated on ice until completely solubilized, and applied to a Ni-NTA column (Qiagen). The recPrP was refolded on the column by running a GuHCl gradient (from 6 M GuHCl, 10 mM Tris-HCl, 10 mM βME, pH 8.0 to 10 mM Tris-HCl, 100 mM Na-PO₄ buffer, pH 8.0) at 1 mL/min. After wash, recPrP was eluted with 10 mM Tris-HCl, 100 mM Na-PO₄ buffer, and 500 mM imidazole, pH 5.8. All eluted fractions were combined and dialyzed using 7,000 MWCO, SnakeSkinTM Dialysis Tubing (Thermo Scientific) for 1.5 h against 10 mM Na-PO₄ buffer, pH 5.8, and then dialyzed overnight against deionized water. Protein samples were lyophilized (FreeZone 4.5, LABCONCO) and stored at −80 °C for further use.

Protein A affinity chromatography (ProSep-vA Ultra Chromatography Media, Millipore, Schwalbach, Germany) was used for the purification of L19 from the culture supernatant. For the purification, 500 mL of the collected culture supernatant containing the secreted L19 was mixed 2:1 with the protein A binding buffer (4 M NaCl, 2 M glycine, pH 8.5) and 750 mL were loaded onto the protein A column overnight at 4 °C. The column was washed with a washing buffer (protein A binding buffer diluted 1:3 in ddH2O). The elution of the recombinant heavy chain antibody was performed according to [50 (link)]. The purified L19 was dialyzed (Snake Skin^TM Dialysis Tubing, cut-off 30 kDa, Thermo Fisher Scientific, Waltham, MA, USA) against 1x PBS overnight at 4 °C. The concentration of dialyzed L19 was determined by using the BCA protein assay method according to the manufacturer’s instructions (Pierce^TM BCA Protein Assay Kit, Thermo Fisher Scientific, Waltham, MA, USA). L19 was stored at 4 °C.

The functionalization with Lf was obtained using a common method with 2-iminothiolane hydrochloride as the sulfhydrylization reagent [49 (link)]. For the preparation of CS/PEI-SA-Lf, Lf (10 mg) was dissolved in 1.0 mL aqueous solution of water and mixed with 1.0 mL aqueous solution of 2-iminothiolane hydrochloride (2-IOT, 0.7 mg), and the reaction proceeded for 1 h at room temperature, with moderate shaking. The excess of 2-IOT was removed by gel filtration chromatography using Sephadex G 100 (Hitrap desalting column) and with PBS 1× (pH 7.0) as the mobile phase. Fractions were collected according to the chromatogram obtained by analysis of the absorbance (a peak contains about 90% of thiolated Lf) [49 (link)]. Subsequently, CS/PEI-SA was dissolved in sodium acetate buffer (pH 4.5) and then added to the PBS solution of Lf, drop by drop, followed by incubation for 20 h at room temperature, in the dark. To remove unreacted Lf, the resulting mixtures were purified by dialysis (SnakeSkin^TM Dialysis Tubing, MWCO 3.5 kDa, 22 mm dry diameter, ThermoScientific) during 4 days against double deionized water and freeze-dried, for further usage. NMR spectroscopy and SDS-PAGE were used to confirm further the surface capping of the terminal amines of the CS/PEI-SA with Lf.

The release property of PF and P–S was conducted using Dexamethasone (Dex, Sigma-Aldrich, St. Louis, MO, USA), a type of corticosteroid drug. First, 20% (w/v) PF solution was fabricated, and SF solution was blended with PF solution with the final volumes of 0%, 0.5%, 1%, and 2% (w/v) and stirred at 4 °C. Then, Dex was dissolved in an amount of 1 mg/mL. The manufactured hydrogel solution was transferred into a Snakeskin^TM Dialysis Tubing (Thermo Fisher Scientific, Waltham, MA, USA) in an amount of 3 mL. The samples were incubated in 15 mL of PBS at 37 °C. At specific time points (0.5, 1, 3, 5, and 8 h), all of the extracted solutions were transferred to a new conical tube and fresh PBS was added to the samples. The released Dex was analyzed by a microplate reader (Synergy MX, BioTek, Vernusky, VT, USA) at an absorbance of 241 nm.

Forty grams of gelatin (Sigma-Aldrich, St. Louis, MO, USA) was gradually added to a 360 mL phosphate buffer solution (PBS, Invitrogen, Grand Island, NY, USA) and was stirred and heated to 50 °C in distillation-distillation water (ddH₂O) until fully dissolved. Next, 23 mL Methacrylic anhydride (MA) (Sigma-Aldrich) was slowly added to the mixed solution and left for 3 h until the reaction was complete. Next, the solution was diluted with 800 mL of warm ddH₂O in the oven at 37 °C. Subsequently, the diluted solution was transferred to 50 mL centrifuge tubes and centrifuged at 8000 rpm for 5 min. Before the supernatant was poured into 10 K MWCO SnakeSkinTM dialysis tubing (Thermo Fisher Scientific, Waltham, MA, USA) and kept at 37 °C, care was taken that there were no suspended substances present. The ddH₂O was changed, and the pH value of all samples was measured twice daily until dialysis was completed at a pH value of 6.1. Subsequently, the solution in the dialysis tubing was poured into a 1000 mL beaker and stirred evenly. Finally, the solution was transferred to plastic plates and kept in the −80 °C refrigerator for 1 d. All samples were freeze-dried until they became spongy solid specimens and stored at −20 °C for further use.

The conjugation of CS or PEI to SA (designated as CS-SA and PEI-SA, respectively) was performed as previously reported by Xie and co-workers [44 ], with modifications. In brief, first, SA (2.5 mg) and EDC/NHS (25 mg, at a ratio 1:10) were dissolved in 1.0 mL anhydrous DMSO and stirred at 60 °C for 1 h, until EDC and SA were well-dissolved and mixed. The resulting mixture (SA:EDC) was then added slowly to 1% (w/v) of CS and PEI in sodium acetate buffer (0.1 M sodium acetate/0.1 M acetic acid, pH 4.5), and the reaction solution was kept at 25 °C in the dark for 24 h, with stirring in a water bath. In the presence of EDC, the amino groups on the surface of polymers specifically reacted with the carboxyl groups of SA. Posteriorly, the resulting conjugate, CS/PEI-SA, was dialyzed first against phosphate-buffered saline (PBS, pH 7.4) for 1 day and then against double deionized water for 2 days, using a dialysis membrane (SnakeSkin^TM Dialysis Tubing, MWCO 3.5 kDa, 22 mm dry diameter, ThermoScientific). The conjugated CS/PEI-SA was isolated as a “sponge” by lyophilization and was further characterized by nuclear magnetic resonance (NMR) spectroscopy.

The proteins were isolated from the wet microalgal paste following an ultrasound-assisted solubilisation/precipitation methodology described elsewhere [16 (link)]. The only difference with that study is that in the present work, the precipitated proteins were dialysed against distilled water using 3.5 kDa molecular weight-cut off SnakeSkin^TM Dialysis Tubing (Thermo Scientific, Waltham, MA, USA). The isoelectric point at which the majority of the proteins were recovered was 3.9 and the pH was adjusted using either 1 M NaOH or 1 M HCl (0.1 M for fine adjustment). The centrifugation was carried out using a Sigma 3-18 KS centrifuge (Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany). The isolated proteins were freeze-dried and stored vacuum-sealed at −20 °C until further analysis.