**5. Characterization**

Loading of drugs by MOFs can be confirmed by various methods. Spectroscopic techniques such as UV–visible and fluorescence spectroscopy are useful to confirm encapsulation/loading of chromophoric and fluorescent drugs. Encapsulation of zinc phthalocyanine (ZnPc), which shows characteristic absorbance peaks at 605 and 670 nm was confirmed by the presence of these peaks in the absorbance spectrum of ZnPc@ZIF-8 but absence in the spectrum of only ZIF-8 [26]. This technique is also helpful to quantify the loaded drug. Determination of Brunauer–Emmett–Teller

(BET) surface area and pore volume can also help in verifying successful encapsulation of drug. Nitrogen adsorption analysis of ZIF-90, ZIF-90-DOX, and 5-FU@ ZIF-90-DOX showed BET surface areas 1045.7, 890.4, and 48.3 m<sup>2</sup> /g, respectively. A decrease in BET surface area validated drug loading [34]. Deviations in the TGA curve of drug incorporated MOF are also indicative of drug loading. TGA curve of ZIF-90 showed no significant loss in weight between 300 and 500° C, whereas ZIF-90-DOX showed much larger weight loss in the same temperature range. Zeta potential and hydrodynamic size measurement by dynamic light scattering (DLS) experiments can also confirm the nature of the association of MOF with the drug. Pure ZIF-8 nanospheres had a more positive zeta potential value of +31.4 mV, as compared to +22.9 mV for fluorescein adsorbed ZIF-8 nanospheres. This indicated surface adsorption of negatively charged fluorescein dye on the surface of positively charged nanospheres [35]. A very small change in negative zeta potential value for MIL-100 and DM NPs (DOX loaded MIL-100 NPs) confirmed encapsulation of DOX majorly inside the particle with some surface adsorption [36]. Other techniques such as FT-IR spectroscopy, PXRD, NMR, and electron microscopy techniques such as SEM and TEM are also frequently used for characterizing MOFs, with and without drugs [30, 37–39].
