*2.3. Proteins*

G. J. Bernardes et al. disclosed that CORMs was compatible with the protein complexes transportation (Figure 14) [121]. They presented that the RuII(CO)2-protein complexes using the reaction between CORM-3 and histidine fragment at the protein surface, the spontaneous CO is released to deliver in cells and mice. They also discussed that plasma protein acts as a CO carrier for in vivo by the CORM-3 formulation. Therapeutically, the controlled CO release favors in downregulation of the cytokines interleukin, i.e., IL-6 and IL-10.

**Figure 14.** The reactivity of the *fac*-[RuL3(CO)3]<sup>2</sup>+ complex (**A**) and CORM-3 react with single-His protein (**B**).

Another Ruthenium (II) carbonyl reagen<sup>t</sup> *cis*-[Ru(CO)2(H2O)4]<sup>2</sup>+ has been reported for the spontaneous CO release in live cells using histidine (His) metalloprotein and retained at IL-8 (Figure 15a) [148]. The *cis*-[Ru(CO)2]<sup>2</sup>+ carbonyl segmen<sup>t</sup> could be produced by aqua dirutheniumcarbonyl *cis*-[Ru(CO)2(H2O)4]<sup>2</sup>+ (Figure 15b). It was also explained that metalloproteins can be modified as organometallic pro-drugs rather than catalysis. Such artificial metallohydrolase performance can be compared with the human carbonic anhydrase (CA)-II.

**Figure 15.** The spontaneous CO release by metalloprotein: (**A**) The carbonyl reagen<sup>t</sup> *cis*-[Ru(CO)2(H2O)4]<sup>2</sup>+ spontaneous CO release in live cells using histidine (His) metalloprotein and retained at IL-8; (**B**) the *cis*-[Ru(CO)2]<sup>2</sup>+ carbonyl segmen<sup>t</sup> can be produced by the aqua carbonyl *cis*-[RuII(CO)2(H2O)4]2<sup>+</sup>.

In spite of the CORMs fragment incorporation with protein, Takafumi Ueno et al. explored the cages of protein for CO releasing [149]. They administrated the ferritin (Fr) cage of protein for capturing the CORM-3 moiety (Figure 16). Furthermore, it was observed that the half-life of the CO release could be enhanced; which indicates a good sign for ideal drug development. When they interrogated their performance at the biological sites, they described that the nuclear factor kappa B (NF-κB) becomes 10-times higher than the parent CORM-3. The CORM-3 protein cage is quite a unique way of CORM's engineering.

**Figure 16.** The recombinant L-chain apoferritin (apo-rHLFr) of Ru carbonyl complexes.

Additionally, Takafumi Ueno et al. explored the immobilization of the crosslinked hen egg white lysozyme CL-HEWL crystal deposit on MCCs for therapeutic purposes [146]. The scientist disclosed that NF-κB is remarkably high in order to respond to the pathological signals. The extra scaffold Ruthenium carbonyl moiety (Ru·CL-HEWL), was used to induce the NF-κB activation and immobilized Ruthenium carbonyl [*cis*-Ru(CO)2X4]2- moieties inside the protein cage (Ru·CL-HEWL). Optimist approaches of this transport service bear the potential of the artificial extracellular scaffold.

NF-κB can be regulated by the protein fragment. Susumu Kitagawa et al. disclosed the crystalline assembly of protein with CORM-2 in polyhedra crystals (PhC) [147]. They introduced the ruthenium carbonyls immobilized on hexahistidine. The activation of NF-κB was significantly improved up to six folds. This therapeutic research will lead to further investigation on the extracellular scaffold.
