Controlling the Porosity and Biocidal Properties of the Chitosan-Hyaluronate Matrix Hydrogel Nanocomposites by the Addition of 2D Ti3C2Tx MXene

A recent discovery of the unique biological properties of two-dimensional transition metal carbides (MXenes) resulted in intensive research on their application in various biotechnological areas, including polymeric nanocomposite systems. However, the true potential of MXene as an additive to bioactive natural porous composite structures has yet to be fully explored. Here, we report that the addition of 2D Ti3C2Tx MXene by reducing the porosity of the chitosan-hyaluronate matrix nanocomposite structures, stabilized by vitamin C, maintains their desired antibacterial properties. This was confirmed by micro computed tomography (micro-CT) visualization which enables insight into the porous structure of nanocomposites. It was also found that given large porosity of the nanocomposite a small amount of MXene (1–5 wt.%) was effective against gram-negative Escherichia coli, gram-positive Staphylococcus aureus, and Bacillus sp. bacteria in a hydrogel system. Such an approach unequivocally advances the future design approaches of modern wound healing dressing materials with the addition of MXenes.


Preparation of Composites
The prepared samples were frozen at −45 °C, then dried using an ALPHA 2-4 LDplus freeze dryer, from CHRIST. The materials were dried for 24 hours at −20 °C under 1 mbar pressure. The best parameters were found in the composite composition in which the ratio of chitosan to sodium hyaluronate was 2:1 and the content of LAA was 3 wt.%. A smaller addition of sodium hyaluronate meant that the obtained composites were hard, and fragile, in the case of a larger addition, the composites were too delicate and gelified quickly. The addition of LAA was also optimized and the optimal choice was 3 wt.% because a larger addition of L-ascorbic acid caused an increase in the hardness and porosity of the tested material, and thus increased its fragility, in the case of a smaller addition, the composite obtained changed after one week from white to light brown.
The height of the liquid column for freeze-drying was also optimized concerning the thickness of the composites produced. Samples with previously selected optimal composition were made (ratio of CH to SHA 2:1 and addition of 3 wt.% LAA). Samples of thickness: 0.7 cm, 0.4 cm, and 0.2 cm were made. The optimal thickness was 0.2 cm because in the 0.7 cm and 0.4 cm thick the material was quite stiff and difficult to deform, which reduced its application properties.

Examination of Composite Morphology Using SEM
SEM analysis of chitosan composites with 1 and 5 wt.% of LAA content was performed at various magnifications. The results are presented in Figure S1.

Computed Tomography Results
Micro CT (Computed Tomography) analysis was performed, samples composed of chitosan without the addition of MXenes phase, and with 5 and 10 wt.% addition of 2D Ti3C2Tx MXene flakes. As a result of photo processing, 8-bit images were obtained, which allowed obtaining a threedimensional tomographic model of the tested samples. The drawings show examples of slices of samples in the XY, YZ, XZ planes, and 3D reconstruction of individual materials.   Table S2. Analyzing the above table, it can be concluded that the addition of the 2D Ti3C2Tx MXene flakes causes a significant decrease in the closed porosity in chitosan-hyaluronate sodium composites. As the addition of MXene phase increases, a decrease in open porosity can also be observed.

Swelling experiment
The swelling properties of hydrogels correspond to their good ability to release contained in them macromolecules, e.g. drugs, Xu et al. [1] in their work, investigated the swelling capacity of hydrogels with different weight ratios of chitosan to hyaluronic acid 1:1, 2:1 and 3:1. The results showed that hydrogels with a 2:1 ratio of chitosan to hyaluronic acid had the lowest swelling capacity compared to the 1:1 and 3:1 ratio composites, which may be due to the 2:1 hydrogel composition: 1 is close to the highest gel density which results in the smaller pore size of network structure preventing the water absorption. Kim et al. [2] on the other hand, proved in his work that the increase in the amount of chitosan in hydrogels in relation to hyaluronic acid increased the swelling capacity of the tested material.
In our work, we conducted swelling tests for composites with a CH/SHA composition in the ratio of 2:1 with different content of the MXenes phase. The swelling ratios (ESR) presented in Figure.S4, were obtained by weighing the initial and swollen samples. To measure the swelling ratio, previously weighted dry sampels (Wdry) were treated with deionized water. After 2 h the excess water was removed from the surface with filter paper and the swollen samples (Wwet) were weighted at room temperature. The swelling ratio was determined according to the equation. / Figure S4. The swelling of the reference CH/SHA matrix nanocomposite as well as modified with 1, 5 and 10 wt.% of 2D Ti3C2Tx MXene flakes.
As can be seen, the addition of the Ti3C2Tx MXenes phase to the CH/SHA composite increases its swelling capacity. The highest swelling ratio is observed for composites with the addition of 1% by weight of Ti3C2Tx MXenes phase. As the amount of MXenes phase additive increased, the swelling decreased.