Freezone 1

PDOPAC was synthesized via controlled oxidatively induced polymerization under nitrogen atmosphere in a Schlenk flask. In this study, we used only one optimized product with high catechol content for an effective BZM binding. The synthesis of this selected PDOPAC was carried out under the following conditions: 5 mM DOPAC; 10 mM boronic acid; 1 M NaCl; 2.5 mM sodium periodate at pH 8.5 (adjusted with 1 M NaOH), and room temperature. After seven days, the reaction was terminated by filling the reaction mixture in a 50 kDa dialysis membrane (Carl Roth GmbH & Co. KG, Karlsruhe, Germany). Purging was performed for seven days with daily water exchange. Afterwards, the product was freeze dried in a lyophilizer (Free Zone 1, Labconco Corporation, Kansas City, MO, U.S.A.) to obtain a brownish solid powder. Investigations concerning the influence of the reaction conditions such as pH, temperature, and content of oxidizer as well as boronic acid and salt to the product will be published elsewhere.

The biomass collected from the different treatments was lyophilized using LABCONCO^® Freezone1^® equipment, with a vacuum of 0.017 mBar and a temperature of −35 °C for five days until the desired dryness was achieved. This allowed the obtaining of ~100 mg of total dry biomass per sample. Subsequently, the samples were macerated until a fine powder was obtained. To extract secondary metabolites, the dry biomass previously processed was used, and we analyzing the shoot (rosette) and the root separately. For this, the assisted extraction equipment Dionex ASE 350 (Accelerated Solvent Extractor) was used, using SST (stainless steel)-type cells, at a temperature of 60 °C for 5 min, a static time of 5 min, a washing volume of 30%, and a purge time of 90 s, using only HPLC (high-performance liquid chromatography)-grade methanol as the solvent, and performing two extraction cycles per sample. Once the above was done, the extracts were stored at −20 °C until later use. Subsequently, the previously obtained supernatants were concentrated with a rotary evaporator (Büchi, R100) to complete dryness, and later, by re-suspension, recovered in 300 µL of 50% methanol [20 (link),21 (link)].

pNP morphology was characterized by scanning electron microscopy (SEM). For this, 1 mL of the selected pNP samples were flash frozen on liquid nitrogen and then lyophilized at −50 °C under high vacuum (0.002 mbar) during 24 h in a Freezone 1 (Labconco, Kansas City, MO, USA). Trehalose 5% w/v in a 1:3 ratio was used as cryoprotectant. Then, lyophilized powders were coated with a thin layer (10–12 nm of thickness) of Au/Pt in a high vacuum evaporator (108 Auto, Cressington Scientific Instruments Ltd., Watford, United Kingdom) and were analyzed in a SEM Inspector F50 (FEI, Hillsboro, OR, USA) operated at 5 kV.

Leaves and stems of C. nocturnum were washed with distilled water, cut into small pieces and frozen for 24 h at −80 °C (Thermo Scientific, 900 series, Waltham, MA, USA). Subsequently, the material was lyophilized (Labconco, Freezone 1, Kansas City, MO, USA) and pulverized in a blade mill (Pulverissette 15, Fritsch GmbH, Idar-Oberstein, Germany). Then the material (946.2 g) was subjected to an extraction process by maceration with methanol (MeOH) in a 1:10 weight/volume ratio at room temperature under constant stirring for 24 h. This procedure was repeated three times with the resulting residual material. Finally, supernatants of the extractions were combined, filtered and the solvent was eliminated by rotary evaporation under reduced pressure (Buchi, RII, Flawil, Switzerland) to yield 64.7 g of ECn.

The hydrogels were analyzed using the FTIR equipment described in Section 3.7. The hydrogels were frozen with liquid nitrogen, and then dried by freeze-drying using a FreeZone 1 (Labconco Corp., Kansas City, MO, USA). The dry samples were measured in attenuated total reflectance (ATR) mode using 1 cm⁻¹ resolution and 200 scans. The data analysis was then performed using the OMNIC software 9.8.286.

For the oxidation reaction, 0.5 g of chitin powder (600–150 µm) were added in 50 mL of a 45 wt.% ammonium persulfate solution (APS) at 40 °C under stirring for diverse reaction times in a round bottom flask. For gel production, β-chitin was stirred for 24 h.
After that, the solution appears as an opalescent dispersion. The material in suspension was collected and washed with deionized water by consecutive centrifugation with an Eppendorf Centrifuge 5810 R at 8000 rpm for 5 min until it reached neutrality.
The powder was then resuspended in 30 mL of milliQ water and left under magnetic stirring overnight, to eliminate possible reaction residues. The next day, the powder was centrifuged, as was carried out previously, and resuspended in about 15 mL of milliQ water, frozen in the freezer, and desiccated by freeze-drying using a FreeZone 1 (Labconco Corp., Kansas City, MO, USA). The product was conserved dry at room temperature.

Tissue samples underwent thermogravimetric analysis (TGA) to assess the thermal stability and to find any matrix degradation due to the decellularization protocol. For TGA, both native and decellularized samples were freeze-dried at 3.6 Pa and −47 °C in a Freeze Dryer (FreeZone 1, Labconco, Kansas City, MO, USA). The experiment was carried out in a thermogravimetric analyzer (Q5000, TA Instruments, New Castle, DE, USA) with a heating rate of 10 °C/min under a nitrogen atmosphere with a temperature range from 32 °C to 500 °C (from near human body normal temperature to elevated temperatures to characterization purposes of the proteins components of the biomaterial).