Avance 3 high resolution spectrometer

¹H NMR spectra of the samples were recorded with a 400 MHz Bruker^®Avance III High Resolution spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) using D₂O with acetic acid-d₄ (Sigma-Aldrich) as a solvent. The solutions (1% w/v) of CS, PNIPAAm and synthesized copolymer were prepared. Chemical shifts were reported in ppm using the signal of D₂O (4.79 ppm) as internal standard. To estimate the molar percentages of CS and NIPAAm in the copolymers, the ratio of the integration of the signal at 1.96 ppm corresponding to CS to that of the signal at 1.01 ppm corresponding to PNIPAAm side chains was used. The molar percentages of NIPAAm and CS were calculated as follows: $$\mathrm{NIPAAm}\%=\frac{\left( \frac{\mathrm{H}}{\mathrm{n}}·100\right)}{ \left( \frac{\mathrm{H}}{\mathrm{n}}\right)+\left( \frac{\mathrm{A}}{3}:0.24\right)}$$
$$\mathrm{CS}\%=100\%-\mathrm{NIPAAm}\%$$
where H is the peak area of the isopropyl group of NIPAAm in the grafted chain, n is the number of isopropyl group protons in NIPAAm, A is the peak area of the acetyl group signal, 3 is the number of acetyl group protons, and 0.24 is the degree of acetylation of CS.

CT (GC9009, batch 769FGD, molecular weight of ~50,000 g/mol, degree of deacetylation of ~94% and viscosity of ≤100 mPa.s) was purchased from Glentham Life Sciences (Corsham, UK). The degree of deacetylation measured in our laboratory by proton nuclear magnetic resonance (1H-NMR, 400-MHz Bruker^® Avance III High Resolution spectrometer, Bruker BioSpin GmbH, Rheinstetten, Germany with SpinWorks 4.0 software, University of Manitoba, Winnipeg, MB, Canada) and using 5% w/v deuterium oxide (D₂O, Sigma-Aldrich, St. Louis, MO, USA) solution and trifluoroAcetic acid (5% v/v in D₂O, Sigma-Aldrich) was 94.5% [16 (link),17 (link)]. The number- and weight-molecular weight of CT were 53,000 and 79,000 g/mol, respectively, as determined by gel permeation chromatography [17 (link)]. Sodium tripolyphosphate pentabasic (TPP) was supplied by Sigma-Aldrich (USA). Acetic acid was purchased from Merck Chemicals GmbH (Darmstadt, Germany) and IFNα-2b (BIOFERON^®, lyophilized powder) from BioSidus (Buenos Aires, Argentina). PEG-carboxymethyl (mPEG5000-COOH, molecular weight of 5000 g/mol) was supplied by Laysan Bio, Inc. (Arab, AL, USA).