Controlling Polymer Morphologies by Intramolecular and Intermolecular Dynamic Covalent Iron(III)/Catechol Complexation – From Polypeptide Single Chain Nanoparticles to Hydrogels

Responsive biomaterials, tunable from the molecular to the macroscopic scale, are attractive for various applications in nanotechnology. Herein, a long polypeptide chain derived from the abundant serum protein human serum albumin was cross-linked by dynamic-coordinative iron(III)/catechol bonds. By tuning the binding stoichiometry and the pH, reversible intramolecular folding into polypeptide nanoparticles with controllable sizes was achieved. Moreover, upon varying the stoichiometry, intermolecular cross-links became predominant yielding smart and tunable macroscopic protein hydrogels. By adjusting the intra-and intermolecular interactions, biocompatible and biodegradable materials were formed with varying morphologies and dimensions covering several lengths scales featuring rapid gelation without toxic reagents, fast and autonomous self-healing, tunable mechanical properties and high adaptability to local environmental conditions. Such material characteristics could be particularly attractive for tissue engineering approaches to recreate soft tissues matrices with highly customizable features in a fast and simple fashion. This article is protected by copyright. All rights reserved.

The reaction was stirred for 2 h on an orbital shaker at room temperature.
The reaction mixture was stirred for 26 hours with 1150 rpm. After that, n-propylmaleimid  The solutions were measured in the fluorescence top reading mode with an excitation wavelength of 348 nm and an emission wavelength of 416 nm.

UV-Vis experiments
The backbone material dcHSA-PEG-C3 and the cross-linker Fe 3+ were dissolved as separate aquous solutions (MilliQ), mixed in the required composition ( Table 2)

Sample preparation for DLS and SEC
dcHSA-PEG-C3 solutions (0.1 mg/mL, 0.7 nM) were produced with MilliQ water/urea (8M) as a solvent. After that, the mixture was filtered by a syringe filter (pore size: 0.45 µm). An iron(III)chloride solution (1 mg/mL, 6.15 mM) was mixed by adding water-free iron(III)-chloride to MilliQ water and subsequent vortexing. The filtered iron(III)-solution (filter pore size: 0.45 µm) was dropped in the dcHSA-PEG-C3 solutions (amounts in volume and equivalents of Fe 3+ in Table 3) and immediately mixed with the used eppendorf pipette tip and by vortexing (1200 rpm). The pH values were adjusted to pH 4 (acetic acid), to pH 7.4 (phosphate buffer, 50 mM) and to pH 10 (NaOH solution). The solutions were mixed for 30 min at 1150 rpm. The resulting solutions were filtered through a syringe filter (pore size: 0.45 µm).

DLS and SEC measurement
The DLS measurements were conducted by a Malvern Pananalytical Zetasizer Nano ZS® device (Figure 4). The volumes of the samples were 1 mL, measured in disposable cuvettes.

Sample preparation for AFM
The initial sample (dcHSA-PEG-C3, 1 mM in 150 mM Phosphate buffer, pH 7.4 with 8 M urea) was diluted to 700 nM with phosphate buffer (50 mM, pH 7.4) and subsequently as a 30 μL sample applied onto the freshly cleaved mica substrate. The solution was left to incubate for 15 minutes in order to deposit the desired species on the mica substrate. After successful adsorption, the supernatant was removed and fresh phosphate buffer (250 μL) was added for the measurement.

AFM measurement
AFM measurements were conducted on a Dimension FastScan BioTM atomic force microscope from Bruker, which was operated in the PeakForce mode. AFM probes with a nominal spring constant of 0.25 Nm-1 were employed (FastScan-D, Bruker) for measurement in liquid. A circular mica disc (15 mm) was used as the substrate. Measurements were performed at scan rates between 0.8 and 2 Hz. Different areas of the mica substrate were scanned in order to ensure the integrity of the shown images. The images were finally processed by the software NanoScope Analysis 1.8.

Rheology
Rheological characterization was performed using a DHR3 rheometer (TA Instruments) which is provided with a temperature controller and a solvent reservoir to avoid dehydration of the hydrogel.
The experiments were conducted using a parallel-plate geometry (8 mm plate, hydrogels of 30 µL volume, gap size of 0.45 -0.60 mm). The experiments were performed at 25 °C.

Sample preparation for rheological measurements
The backbone material dcHSA-PEG-C3 and the cross-linker Fe 3+ were dissolved as separate aquous solutions (MilliQ), mixed in the required composition (Table S2) Table 5.

Rheological measurements
To investigate the hydrogels mechanical properties, several different experiments were performed: (i) Time sweep: The linear viscoelastic region was found to be in the range of 1% strain and 1 Hz frequency. Therefore, oscillatory time-sweep measurements were performed at a fixed strain of 1% and a fixed frequency of 1 Hz at 25 °C for 400 s.
(iv) Thixotropy measurement: Combined measurements of an oscillatory strain sweep (0.01-1000%) at 25 °C with a fixed frequency of 1 Hz followed by an oscillatory time sweep measurement with a fixed strain of 0.1% and frequency of 1 Hz for 800 s at 25 °C were conducted to analyze the self-healing capacity of the hydrogels. The data was normalized to the mean storage modulus of the respective gel after the first strain sweep.
The following figures (all figures: G' > G'') point out the storage moduli (G') and loss moduli (G'') of the different gels, listed in Table 5.