*3.1. Preparation and Characterization of Cu-Carrying Chitosan Powders*

Chitosan modified with metal NPs was prepared in two steps. Firstly, Cu NPs were prepared by interaction of metal vapors with acetone vapors according to the MVS protocol (see experimental part). During the second step, freshly prepared Cu-acetone organosol was deposited in situ onto chitosan powders. During the deposition procedure, the flask was stirred manually to obtain homogeneous material. Discoloration of organosol indicated the completeness of the nanoparticle's deposition on the biopolymer support and the color of powders changed from beige to dark-green. XRF analysis shows that the metal proportion of the Cu-carrying powders based on ChitLMW and ChitHMW is 0.5 and 0.83%, respectively. Previously, the same method for preparing Cu-carrying chitosan powders with a high copper concentration of 3–5% *w*/*w* was used [20].

The TEM images in the bright field and the selected area diffraction pattern (SAED) for the newly prepared Cu-acetone organosol have been seen in Figure 2A. As can be shown, SAED has diffuse reflexes suggesting the production of a significant number of very small particles. NPs have a mainly spherical shape and blurry boundaries. The NPs' sizes estimated from Figure 2B are in the range of 1 < d < 4 nm. The good solvating properties of acetone for preparing Cu NPs were shown in previously published works [38,39].

**Figure 2.** TEM image in bright field (**A**) and selected area diffraction pattern (SAED) (**B**) from a region **Figure 2.** TEM image in bright field (**A**) and selected area diffraction pattern (SAED) (**B**) from a region for Cu-acetone organosol.

Five rings correspond to the lattice planes of Cu and Cu2O. As a result of the proximity of some interplanar distances of Cu and Cu2O and relatively broad rings, their superposition was observed. The formation of a core-shell structure of copper NPs with metallic copper as a core and copper oxide

(I) as a shell can be assumed. A similar structure of Cu NPs in organosols prepared with different solvents via MVS was detected [40].

In Figure 3 TEM images in bright/dark field and SAED of powdered chitosan doped with Cu NPs using the impregnation step are shown. It was detected that Cu-carrying chitosan composite contains Cu and Cu2O phases as well as Cu-acetone organosol (Figure 3B). The crystallite sizes estimated from dark field image are in the range of 2–4 nm (Figure 3D).

**Figure 3.** TEM images in bright (**A**,**C**) and dark fields (**D**) of chitosan with a high molecular weight (ChitHMW) doped with copper nanoparticles (Cu NPs) as well as SAED (**B**) of highlighted field.

It was previously demonstrated that the surface of the composite prepared with Cu-acetone organosol contains two oxidized copper states Cu2<sup>+</sup> and Cu+, with concentrations (at. %) of 10.7 and 3.6%, respectively [20]. Experimental SAXS curves of Cu-carrying chitosan and pristine non-modified chitosan (ChitLMW) are described in Figure 4A.

Volume size distribution functions *DV(R)* of heterogeneities presented in pristine non-modified chitosan and that of Cu nanoparticles embedded in the chitosan are shown in Figure 4B. To obtain *DV(R)* only for the Cu nanoparticles, a difference SAXS curve was calculated by subtraction of the scattering of the pristine non-modified chitosan from the scattering of the Cu-carrying chitosan (Figure 4A insert). As we can see from Figure 4B, Cu nanoparticles in this system are practically monodisperse and more compact (average size is about 1.5–2 nm) to compare with the sizes of the pores of the pristine non-modified chitosan (about 3 nm), where the Cu nanoparticles are located. A small detectable

*χ*

number of larger particles (possible aggregates or clusters of Cu nanoparticles) is also present in the sample. Due to the practically monodisperse character of the *DV(R)* function for Cu nanoparticles, one can reconstruct an average shape of the Cu nanoparticles [21]. For the ab initio restoration by the program DAMMIN [24], distance distribution function *p*(r) was calculated. A shortened curve with no initial part at the range of momentum transfer < 0.7 nm−<sup>1</sup> was used for the calculation to minimize the influence of scattering from large aggregates on the result of the shape restoration. The distance distribution function *p(r)* is shown in Figure 5 (insert on the top right) along with a model scattering curve from a restored shape and with a smoothed curve after the of collimation corrections.

**Figure 4.** (**A**) Experimental (SAXS) curves: 1—Cu-carrying chitosan; 2—pristine non-modified chitosan (chitosan low molecular weight (ChitLMW)). Insert—difference SAXS curve for the embedded Cu nanoparticles. (**B**) Volume size distribution functions *DV(R)*: 1—Cu nanoparticles; 2—pristine non-modified chitosan (ChitLMW).

**Figure 5.** Reconstruction of the shape of the Cu nanoparticles in the Cu-carrying chitosan: 1—difference SAXS curve; 2—a model scattering curve calculated from the restored shape of the Cu nanoparticles; 3—extrapolated to zero angles smoothed scattering curve after the introduction of collimation corrections. Inserts: top right—distance distribution function *p(r)*; bottom left—restored shape of the Cu nanoparticles.

The restored shape of the Cu nanoparticles is a cluster consisting of 5–6 individual Cu nanoparticles with the average sizes of about 1.5–2.0 nm and with the length of the cluster of about 5 nm. Due to the presence of some amount of large aggregates it is impossible to restore the shape of the individual Cu nanoparticles. However, the shape of the cluster is restored with very good accuracy: χ *<sup>2</sup>* = 0.92, and the separate nanoparticles in the cluster are clearly visible.

−

Chitosan gels have been produced by ionic physical gelation of the oxalic acid-biopolymer [41,42]. Two types of Cu-carrying chitosan gels were obtained with the consecutive procedures of dissolution in oxalic acid at high temperature, gelation and thorough washing procedures (Figure 6). *χ*

**Figure 6.** Scheme of preparation of chitosan hydrogels from Cu-carrying chitosan powders.
