Samplecase

1D ¹H NMR spectroscopy was used to analyze the degree of substitution as well as the uronic acid composition of the alginates. The samples were subjected to mild acid hydrolysis as previously described [53 ] to reduce the viscosity prior to the NMR analysis. 6–10 mg of the samples were then dissolved in 600 µL D₂O (99.9%) and added 5 µL 3-(Trimethylsilyl)propionic 2,2,3,3-d₄ acid (TSP, Sigma-Aldrich, Oslo, Norway) as an internal standard and 15 µL Triethylenetetraamine-hexaacetic acid (TTHA, Sigma Aldrich) as a chelating agent for residual divalent ions. The latter was not added to samples already added EDTA (analysis of leaked alginate). 1D ¹H spectra were recorded at 90 °C on a Bruker Ascend 400 MHz Avance III HD spectrometer, equipped with a 5-mm SmartProbe z-gradient probe and SampleCase (Bruker BioSpin AG, Fällanden, Switzerland). The spectra were recorded using TopSpin 3.2 software (Bruker BioSpin, Fällanden, Switzerland) and processed and analyzed with TopSpin 3.5 software (Bruker BioSpin).

NMR spectra were recorded on a Bruker Avance II 600 MHz Spectrometer equipped with a TCI cryo probe and a “SampleCase”, using DMSO-d₆ as solvent. The sample of the DHP was dissolved in DMSO-d₆ at 55–100 mg/mL depending on sample amount and solubility. NMR spectra were acquired and processed using Topspin 3.2 pl6 and ¹³C NMR spectra were recorded using a z-restored spin-echo pulse program to achieve a straight baseline free of hump, dip or roll37 (link). An acquisition time of 0.9 s, relaxation delay of 10 s, a dead time of 70 μs, 5120 scans and a spectral width of 239 ppm at 300 K was used for all acquisitions. Assignment of main lignin-like features of DHPs was performed according to Ralph et al38 (link).

The PEGylation of NPs was quantified by ¹H nuclear magnetic resonance (NMR), as previously described [18 (link)]. Briefly, a Bruker 400 MHz Avance III HD equipped with a 5-mm SmartProbe z-gradient probe and SampleCase (parameters: zg30 pulse sequence, 30° pulse, 1 s delay time, 65,536 points spectral width, 3.96 min acquisition time) was used. Preceding NMR, the dialyzed NPs were centrifuged, the supernatant was removed, and the particles were resuspended with deionized water before a second centrifugation step followed by drying of the pellet at 50 °C for 12−18 h. The sample was dissolved in acetone-d6. The spectra were processed using Spectres (v. 2019.1.0) with automatic baseline and phase correction. Integration of the corresponding protons from PEG (3.6 ppm) and methylene protons at (1.75 ppm) was used to quantify the number of ethylene units on the nanoparticles. Due to two different PEGs being used and no way of distinguishing the two by NMR, it was not possible to calculate the number of PEG chains on the nanoparticles.