*3.2. Rheology Studies*

The viscoelastic nature of polymer gels plays an important role in their adhesion properties; a very important characteristic considering different biomedical applications. Gels based on natural or synthetic polymers exhibit an excellent adhesion property due to both the elastic and viscoelastic properties.

Using the rheological measurements, the mechanical properties of the best formulations were analyzed. All analyzed samples presented rheological characteristics of a gel. The obtained results showed that viscosity was directly dependent on the polymeric content of the formulations. During the rheological measurements, both dynamic moduli were constant. As can be observed from Figure 2, the storage modulus (G') exhibited higher values than the loss modulus (G")) over the entire strain range of 0.01 to 100%, indicating the gel-like behavior (Figure 2). The results obtained by rheological analysis confirmed that CARB-F2, AS-F5, and CMC-F3 samples were optimized and further considered for loading with CD-NHF, due to the gel equilibrium modulus. The presence of CD-NHF (for samples CARB-F4, AS-F6, and CMC-F6) in the gel structure has a significant influence over both dynamic moduli. In the case of the AS-F6 sample, the CD-NHF addition induced a slight increase of the dynamic moduli, indicating stiffness of the gel structure, whereas in CARB-F4 and CMC-F6 gels, the effect of CD-NHF addition was exactly the opposite, leading to a slight decrease of the dynamic moduli, indicating a flexible gel structure. Also, the amplitude sweep allows determination of the limits of the linear viscoelastic range for the prepared gels. These values are not affected by the presence of CD-NHF (Table 2).

> **Table 2.** The limits of the linear viscoelastic range (γLVE) for the tested gel formulations.


**Figure 2.** Amplitude sweep for simple gels (CARB-F2, AS-F5, and CMC-F3) and gels with CD-NHF (CARB-F4, AS-F6, and CMC-F6). G': storage modulus; G": loss modulus.

All tested gels showed a solid-like behavior, as the storage modulus (G') was always larger than the loss modulus (G"). As one can observe from Figure 2, the CARB and CMC gel formulations have a more flexible structure than the AS gel, confirmed by storage modulus (G') values (G'CARB (620 Pa) > G'CMC (540 Pa) > G'AS (437 Pa)). Frequency sweep tests allow observation of the elastic response of gels. The presence of the hydrogen bonding is evidenced by the frequency dependence of the moduli over the entire range of 0.05–500 1/s (γLVE(CARB) > γLVE(CMC) > γLVE(AS)) (Figure 3).

**Figure 3.** Frequency sweep for simple gels (CARB-F2, AS-F5, and CMC-F3) and gels with CD-NHF (CARB-F4, AS-F6, and CMC-F6).

## *3.3. Fluorescence Analysis*

In order to confirm the presence of CD-NHF in the gel matrices, the formulations were further evaluated through fluorescence analysis. Fluorescence spectra exhibit the maximum emission (λem) at 424 nm when using an excitation wavelength (λex) of 370 nm for CD-NHF (Figure 4 and Table S1). Also, Figure 4 illustrates the emission profile of CD-NHF-loaded gels (CARB-F4, AS-F6, and CMC-F6) at three different excitation wavelengths ranging from 370 to 410 nm. Moreover, the recorded results (Table S1) revealed that at 370 nm, in the case of CARB, the blue-light emission was not significantly affected by the CARB matrix. The observed difference between CD-NHF blue-light emission and CARB matrix was only 1.5% (Table S1). When AS and CMC were used as a matrix for the entrapment of CD-NHF, an 11% decrease of the blue-light emission was observed, compared to the case of the CARB matrix and CD-NHF. Herein, we can conclude that in this case, the polymer matrix clearly plays an important role for the modification of CD-NHF optical properties.

**Figure 4.** Emission spectra of CD-NHF suspended in H2O: CARB-F4, AS-F6, and CMC-F6 samples.

### *3.4. In Vitro Studies*

Analysis of the cytostatic or cytolytic or growing pattern modulatory activities may be done with end point assays or live cell imaging techniques. 3D cultures create a better similarity between the cultured cells and the living organism. The basic methods to evaluate modulatory activities of putative anticancer agents include reference cancer cell line cultures assays in both classic 2D and 3D cultures.

Viability of HDMVECn cells (primary dermal microvascular endothelial cells) in 2D culture treated with simple gel formulations (CARB-F2, AS-F5, and CMC-F3) and gel formulations containing CD-NHF (conc. 5%; CARB-F4, AS-F6, and CMC-F6) was not affected by CARB-F4 and CMC-F6, whereas, interestingly, AS-F6 had a significant effect (Figure 5).

**Figure 5.** Viability of normal dermal cells (Primary Dermal Endothelial Cells (PDMC)) for gel formulations without and with CD-NHF (*N* = 15 wells/column from two independent experiments). Ctr (Control).

Proliferation activity of melanoma cell lines (2D system) was not affected the by the presence of CARB-F2, CMC-F3, or AS-F5 formulations (Figure 6a), respectively, while cell viability was affected in a gradual manner in cells treated with CARB-F4, CMC-F6, and AS-F6 formulations (Figure 6b,c).

**Figure 6.** Murine and human melanoma cell viability for gel formulations without (**a**) and with (**b**,**<sup>c</sup>**) CD-NHF (conc. 5%). *N* = 15 wells/column from two independent experiments; viability of treated groups is expressed relative to the control group. (**a**) Skin cancer; (**b**) murine melanoma (B16F10); (**c**) human melanoma (A375); (**d**) scoring of significance effect, where +++ (\*\*\*) *p* = 0.0003, ++ (\*\*) *p* = 0.01, + (\*) *p* = 0.04, and — = no effect.

In the 3D Matrigel assay, the human melanoma cells form visible and consistently larger colonies compared with the same cells treated with CD-NHF-loaded gel formulations (conc. 5%) (Figure 7 and Figure S4).

By comparing the left column (Matrigel) with the median (simple gel formulations), it can be observed that spheroids are less compact, with decreased cellular uniformity and a tendency to dissociate. In the median column, we find that CMC and AS gel formulations are more unfavorable for maintaining a homogeneity of metabolic activity (the green shade in the L/D kit reflecting live cell population) and induce (or amplify) the dissociation tendency of cells from differentiated proliferation aggregates. The differences found in the analysis of 3D spheroids can be caused by the fact that the different types of gel formulations used either provide different stability conditions for extracellular molecules that favor maintaining a differentiated cellular phenotype, or employ different receptors for adhesion to cell membranes favorable to maintaining both differentiation and inhibition of the cellular dissemination tendency [20,21]. Comparing the median column (single gel formulations) with that on the right (gel formulations supplemented with 5% CD-NHF), we find that exposure to CD was unfavorable to cell proliferation, with global cell counts being significantly impaired and the quantitative weight of cells in invasion reduced. We specify that the shade of the green marker is proportional (L/D kit) to the level of cell metabolic activity. Smaller color intensities can be determined by both a decreased number of cells and more restricted metabolic activity in CD-NHF exposure. Also, the morphological aspect of spheroids under the influence of gel formulations containing CD-NHF is presented in Figure 8.

**Figure 7.** (**a**) 3D Matrigel assay for human melanoma cell cultures in standard conditions (left) and in the presence of added CD-NHF-free gel (middle) versus 5% CD-NHF-loaded gel (right). *N* = 3D Matrigel cultures per type of gel with or without CD-NHF; 20× microscope objective. (**b**) Sum intensity of fluorescence signal; (**c**) area of spheroids expressed in μm2.

**Figure 8.** The morphological aspect of spheroids under the influence of gel formulations. *N* = 3D Matrigel cultures per type of gel with or without CD-NHF; 20× magnification.

Also tested was the viability potential of two different breast cancer cell lines, namely 4T1 (mouse breast cancer) and MDA-MB-231 (human breast adenocarcinoma), in 2D and 3D culture models. The viability of the breast cancer cell lines 4T1 (mouse breast cancer) and MDA-MB-231 (human breast adenocarcinoma) in the 2D culture system treated with CARB-F4 was a ffected (Figure 9a). Mitochondrial activity was a ffected upon CARB-F4 treatment (Figure 9b). In the 3D Matrigel assay, the malignant cells form visible and consistently larger colonies compared with same cells treated with CARB-F4 (Figure 9c). Moreover, the apoptotic foci in 3D cultures treated with gels containing CD-NHF were significantly higher (Figure 9c; white squares in boxes 2 and 4). The apoptotic pattern in the whole spheroid body is also distorted: while in native culturing, inner cells (typically associated with hypoxic conditions inside large spheroids) in spheroids began the apoptotic program, at 5% CD-NHF in exposed spheroids, this form of cell death is initiated in all spheroid compartments. 3D cell cultures displayed visibly larger structure of cancer cells with reduced active phenotype appearance. This suggests that treatment with the CARB-F4 formulation could also either induce cell senescence or cell dormancy—a topic that deserves further clarification. Together, these data sugges<sup>t</sup> that CD-NHF-loaded gels a ffect the number, size, and cellular organization of spheroids and impacts individual cancer cell ability to proliferate and aggregate in spheroids.

**Figure 9.** The e ffects of gels with 5% CD-NHF in 2D and 3D culture. (**a**) Viability of MDA-MB-231 (human breast adenocarcinoma 33.3%) and 4T1 (mouse mammary breast cancer 44.87%) cell lines treated with gels containing 5% CD-NHF ( *N* = 8 wells/column from two independent experiments); (**b**) mitochondrial activity in MDA-MB-231 (human breast adenocarcinoma 2%) and 4T1 (mouse mammary breast cancer 49.2%) treated with gels containing 5% CD-NHF ( *N* = 8 wells/column from two independent experiments); (**c**) apoptosis evidence in MDA-MB-231 by immunofluorescence microscopy and 3D Matrigel culture treated with gels containing 5% CD-NHF ( *N* = 6; 3 Matrigel culture controls and 3 Matrigel cultures treated with 5% NHF). (c1,c3) represent NucBlue nuclei staining; (c2,c4) reflect apoptosis. Picture magnification 5×; where \*\*\* *p* < 0.0005, \*\*\*\*\* *p* < 0.000005, \*\*\*\*\*\* *p* < 0.0000005.
