*2.2. Methods*

#### 2.2.1. Preparation of Gel Formulations Based on CARB, AS, and CMC

Antitumoral gels based on CARB, AS, and CMC, respectively, and loaded with CD-NHF were prepared according to the method reported by Hellerbach et al. [19] with slight modification. CD-NHF were prepared according to a protocol reported by Stan et al. [4]. Prior to gel embedment, the carbon dots were prepared through pyrolytic processing of an NHF precursor using an experimental setup presented in Figure S1. Briefly, in a typical synthesis procedure, 0.1 g of NHF is thermally processed in carefully controlled conditions (temperature of the main sequence of the pyrolytic process is 253 ◦C within 30 min), leading to a typical configuration of the carbon dots consisting of a carbonaceous core with a surface highly decorated by various functional groups. The slightly di fferent processing parameters compared with the previously reported preparation path are due to the di fferent experimental setup, which in this work, was reconfigured and extended in order to increase both the reproducibility of the carbon dots' preparation path and the resultant per batch quantities. The resultant reaction mass is primarily dispersed in high-purity water kept cooled at 4–5 ◦C. In the next stages, the suspension is centrifuged at 10,000 rpm for 15 min and the supernatant is collected and freeze-dried, with a fine powder of carbon dots being obtained. A series of gel formulations were prepared by mixing di fferent quantities of CARB, AS, or CMC (Table 1) in a solution mixture consisting of 10 mL distilled water and 3 mL ethanol in glass vessels at room temperature. In order to achieve a uniform gel formulation, a T 18 digital ULTRA-TURRAX disperser by IKA was used (10 min, 10,000 rpm). The gelling agent's concentration ranged from 3.8 to 5.8% (Table 1). In order to prepare the gel formulations containing CD-NHF (CARB-F4, AS-F6, or CMC-F6, respectively), the CD-NHF (3.84 mg/mL) was added into the water–ethanol solution and dispersed by sonication for 1 min at room temperature using a sonication bath (Bandelin Sonorex, Berlin, Germany), followed by adding CARB, AS, or CMC. The obtained gel formulations and CD-NHF gel (Figure S3) were stored at 4◦ C in a refrigerator. The basic molecular formulas for the polymers and CD-NHF used in order to obtain the gel formulations with and without CD-NHF are: sodium alginate (AS)—(C6H8O6)*<sup>n</sup>*, carboxymethyl cellulose (CMC)—[C6H7O2(OH)2OCH2COONa]*<sup>n</sup>*, Ultrez 10 carbomer (CARB)—(C3H4O2)*<sup>n</sup>*, and *N*-hydroxyphthalimide (NHF)—C8H5NO3.


**Table 1.** Polymeric composition of various CARB, AS, and CMC gel formulations. CARB: carbomer Ultrez 10; AS: sodium alginate; CMC: carboxymethyl cellulose; GLY: glycerin.

2.2.2. Characterization of the Prepared NHF Carbon Dots

The morpho-structural characteristics of the prepared NHF carbon dots were essentially similar to those previously reported [4]. In brief, the XPS survey and high-resolution O1s, N1s, and C1s spectra revealed the presence of the defect-rich graphitic core highly decorated with various terminal functional groups, while the recorded FT-IR spectra revealed the modifications/reconfigurations occurring through thermal processing of the NHF precursor and confirmed by XPS results which were revealing the presence of various functional groups attached to the graphitic core, which are essentially responsible for both optical and antitumoral properties of this type of carbon dots. In Figure S2 are presented the recorded spectra of the NHF precursor and resultant carbon dots, and several specific vibrations of various groups are detailed. Interesting details regarding the morphology of the prepared carbon dots were highlighted by the HR-TEM investigation. Figure 1 presents the recorded micrographs of two samples obtained by depositing a highly diluted chloroform-dispersed carbon dot solution on 300-mesh carbon-plated copper grids. Both images sugges<sup>t</sup> a cluster organization of smaller entities, which presumably are individual carbon dots. While their aspect could sugges<sup>t</sup> individual carbon dots, it is also possible to be attributed to even smaller clusters, as can be seen in Figure 1b.

**Figure 1.** Recorded HR-TEM images of the carbon dots prepared from *N*-hydroxyphthalimide (CD-NHF): (**a**) CD-NHF with an average size of the clusters in 100–300 nm range and (**b**) CD-NHF with an average size of the smaller entities in 2–4 nm range
