## 3 Results and Discussion
### CO Release, Drug Release, and Chemical Images of Loaded CF's  
In order to further corroborate the presence of chemisorbed CORMs on CF and to evaluate if the same retained their CO-releasing ability, CF-1 and CF-12 were suspended in a newly prepared buffered solution of horse skeletal myoglobin (Mb) reduced under a nitrogen atmosphere with excess Na\({}_{2}\)S\({}_{2}\)O\({}_{4}\) at 25 \({}^{\circ}\)C and at pH 7.4. As mentioned in Section 2.3, this test (known as the myoglobin assay) is the standard technique to assess the ability of molecules to release CO. It is grounded on the spectroscopic evaluation of the conversion of Mb to carboxymyoglobin (Mb-CO) as evidenced by the change in the absorption spectrum of the Q band region (500-600 nm) of the protein [67]. Figure 4 (top left spectrum) shows the UV-visible spectrum of reduced Mb in the presence of CF and CF-12. As evident from the spectra, i.e., the split of the single Q band absorption of Mb at 555 nm to the two peaks of Mb-CO at 540 and 576 nm, only CF-12 was able to induce the spectroscopic change associated with CO-binding to the protein. This assay confirmed the presence of B\({}_{12}\)-ReCORM-2 in the material and that the same is still able to release CO. Similar results were obtained, under the same conditions, if CF-1 was assessed. Figure 3: MCT detector generated chemical images of single _Coscindiscus_ valves exposed to CH\({}_{3}\)OH solutions of the CO releasing drugs B\({}_{12}\)-ReCORM-2 (left) and ReCORM-2 (right). (**A**) and (**A**): Optical image of single _Coscindiscus_ valve. (**B**) and (**B**’): chemical mapping of the Si-O-Si stretching vibration at 1100 cm\({}^{-1}\). (**C**) and (**C**’): chemical mapping of the symmetric C\(\equiv\)O stretching mode of B\({}_{12}\)-ReCORM-2 at 2010 (left) and ReCORM-2 at 1996 (right) cm\({}^{-1}\) respectively. (**D**) and (**D’**): overlay of (**B**) and (**C**), and (**B’**) and (**C**’) respectively. (**E**) and (**E’**): 3D surface representations of chemical maps shown in (**B**) and (**B’**) respectively. (**F**) and (**F’**): 3D surface representations of chemical maps shown in (**C**) and (**C’**) respectively. Images were recorded with frustules allowed to settle in a thin film of paratone oil on round CaF\({}_{2}\) windows (2 cm in diameter; 2 mm thickness). After exposure to the Mb solution, CF-12 was recovered and analyzed once again by FTIR-SMI, in order to evaluate changes in its IR spectrum. A comparison of the average FTIR spectromicroscopy spectra of CF-12 before and after reaction with Mb is given in Figure 4 (bottom left spectrum). There are two fundamental changes in the spectrum. First, the relative intensity of the two weak absorptions in the 2000 and 1880 cm\({}^{-1}\) region decreased after the reaction of CF-12 with Mb. This is consistent with the release of the drug and/or loss of CO by B\({}_{12}\)-ReCORM-2, resulting in a lower relative concentration of its symmetric and asymmetric Re-C\(\equiv\)O \(\gamma\)(CO) stretching modes. Furthermore, there appears to be an increase in intensity of the 952 cm\({}^{-1}\) band (see red spectrum). This vibration has been associated to the stretching mode of vicinal silanols (Si-OH) resulting from an increase of surface concentration of the species following surface hydration [72]. This assignment is fully consistent with CF-12 being exposed to an aqueous solution of Mb. In the same region, however, the frequency of stretching Re = O mode is also reported [76]. As mentioned above, a Re = O species (in the form of perhrenate, ReO\({}_{4}^{-}\)) is the final product in the aerobic decomposition of the ReCORMs following CO release [27]. We doubt, however, that this band can be assigned to ReO\({}_{4}^{-}\) given the high solubility of the ion in water. To evaluate the relative distribution of the above-mentioned vibration, FTIR-SMI maps of Mb-exposed CF-12 were recorded. Figure 4 (right) shows optical and MCT detector generated chemical images acquired in this experiment.