## 2 Functionalization of CDs
### Secondary Functionalization or Surface Modification  
As depicted in Figure 3, there exist various surface modification methods, including amidation, silylation, esterification, sulfonylation, copolymerization, Schiff base interactions, \(\pi\)-interactions, metal ion complexation, and electrostatic interactions [84; 85; 86; 87; 88]. The amidation is widely used to modify the CD surfaces with carboxyl or amine groups in aqueous media (Figure 4a) [89; 90]. This method involves the use of coupling reagents such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). After EDC reacts with carboxyl groups of CDs or molecules, an unstable O-acylisourea intermediate is produced and then stabilized by NHS to form a semi-stable NHS ester, which reacts with amine groups of molecules or CDs to form an amide bond. This modification allows for the conjugation of various molecules, including targeting ligands, photosensizers, and anticancer drugs [39; 47; 91; 43]. For example, [PERSON] et al. prepared CDs with carboxyl groups by oxidizing carbon nanopowder, and covalently conjugated the carboxyl groups of CDs with amine groups of transferrin and doxorubicin to form CD-Trans/Dox using EDC/NHS coupling reaction [91]. The surface modification of CDs via silylation involves the reaction between silane derivatives such as (3-aminopropyl)triethoxysilane (APTES) or tetraethyl orthosilicate (TEOS) and active hydrogens on the CD surfaces in ammonium solution, which results in the covalent O-Si bond (Figure 4b). Compared to TEOS, APTES has the advantage of having amine groups (-NH\({}_{2}\)), which can be used for further functionalization and bonding with carboxyl groups. This functionalization can enhance the biocompatibility and dispersion of CDs in aqueous solution [92]. For example, [PERSON] et al. prepared CDs with hydroxyl groups using alanine and histidine as carbon sources in a hydrothermal method. The as-prepared CDs were then conjugated with APTES through a silylation reaction [92]. The surface modification of CDs via esterification is achieved by reacting alcohol groups of CDs or molecules with carboxylic acids of molecules or CDs to form the ester bond in the presence of heat and an acid catalyst (Figure 4c). This surface modification provides a route for conjugating drugs with -COOH groups with -OH groups of CDs and vice versa. It is known that ester bonds are susceptible to hydrolysis at low pH values, which  
Figure 3: Various functionalizations of CD surfaces. could contribute to the enhanced drug release at pH 5, making esterification an effective strategy to use in drug delivery systems [93]. For example, [PERSON] et al. synthesized CDs with hydroxyl groups using lactose as the carbon source. The functionalization of CDs was achieved by adding mercaptosuccinic acid (MSA) through an esterification reaction, resulting in the formation of CDs-MSA [94]. Figure 4: Schematic illustration of surface modifications of CDs via (**a**) amidation, (**b**) silylation, (**c**) esterification, (**d**) sulfonylation, (**e**) copolymerization, (**f**) Schiff-base reaction, (**g**) \(\pi\)-interaction, (**h**) metal ion complexation, and (**i**) electrostatic interaction. The surface modification of CDs via sulfonylation involves the attachment of sulfonyl groups to the amine groups of CDs. The nucleophilic amine group attacks the sulfonyl chloride to displace the chloride ion to obtain the sulfonated CDs (Figure 4d). This modification greatly increases the water solubility, enabling better dispersion of CDs in aqueous environments. For example, [PERSON] et al. prepared CDs with amine groups using phenylenediamines as the carbon precursor and obtained the 2,4-dinitrobenzene-conjugated CDs (CD-DNS) by reacting 2,4-dinitrobenzenesulfonyl chloride (DNS-Cl) with CDs in the presence of triethylamine and dichloromethane [95]. The surface modification of CDs via copolymerization involves the attachment of cyclic monomers to CDs with hydroxyl groups through polymerization.