**4. Conclusions**

This paper reported the development via a physical method of three topical gel formulations loaded with CD-NHF, which were evaluated from the point of view of the modulatory activities of putative anticancer agents. The obtained results indicate that CD-NHF-loaded gels matrices present complex and interesting cell modulatory activities which are relevant for cancer control in potential clinical applications. The mechanical properties of gels were investigated, concluding that the presence of CD-NHF in the gels induced a slight decrease of the dynamic moduli, indicating a more flexible gel structure. The fluorescence analysis confirmed the presence of the embedded CD-NHF in all gel formulations. Also, worth noting is the potential antitumoral activity of the gel formulations loaded with CD-NHF. The most important finding is that the CD-NHF-loaded gel formulations are able to affect the number, size, and cellular organization of spheroids, while also having a significant impact on the individual tumor cell's ability to proliferate and aggregate in spheroids.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4923/11/7/303/s1, Figure S1. Experimental setup used to prepare carbon dots. Figure S2. FT-IR spectra for CD-NHF. Figure S3. Photos of gels without and with CD-NHF under (a) white and (b) UV light. Figure S4. Spheroid fluorescent staining using green/FITC (left column) for live cells, blue/NucBlue for nuclei (middle column), and merged signals (right column) from 3D human melanoma cell cultures. Figure S5. The analytical segmentation procedure for a typical spheroid image. Table S1. Fluorescence results obtained for CD-NHF-loaded gels at di fferent excitation wavelengths: 370–410 nm.

**Author Contributions:** Gel formulation experimental part, results interpretation, and writing and editing of the manuscript: C.-L.S. and C.A.P.; carbon dot preparation and fluorescence analysis: C.S.S.; rheological analysis and rheology results interpretation: C.A.I.; conceptualization and supervision: B.C.S.; in vitro experiments, biochemical tests, writing, and results interpretation: C.T. and E.C. All authors have given approval to the final version of the manuscript.

**Funding:** This work has been performed in the frame of the Complex Projects Partnership Program CDI (PCCDI) in priority areas—PN III under UEFISCDI authority, project code PN-III-P1-1.2-PCCDI-2017-0083 (project number 37PCCDI/2018).

**Acknowledgments:** We wish to thank Florin Zugun-Eloae for his scientific involvement in the in vitro experimental part and the critical review of the manuscript and Adrian Tiron for his involvement in performing the microscopy analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.
