## 3 Results and Discussion
### Assessment of Biological Activities of B\({}_{12}\)-ReCORM-2 and Re-CORM-2 Molecules  
The toxicity profile of B\({}_{12}\)-ReCORM-2 and Re-CORM-2 molecules was addressed in vivo using zebrafish embryos, a creature model generally utilized in the current preclinical medication improvement pipeline. Due to their remarkable genetic, physiological, and immunological resemblance to human beings, and high response correspondence to drugs, zebrafish has risen as a flexible biotechnological stage for the toxicity and bioactivity evaluation of chemically synthesized molecules, streamlining the way to preclinical trials and lessening the failure at advanced phases of experimentation and testing [77; 78]. To date, the toxicological profile of the rhenium-based CO-releasing molecules has remained unexplored in vivo. Herein, the toxicity assays were initiated from the 6 h post-fertilization (hpf) stage, when embryos are highly sensitive to applied xenobitiocs. The results obtained after 120-h treatment have revealed that both types of molecules were safe at the doses \(<\)150 uM, while at higher doses, B\({}_{12}\)-ReCORM-2 was more toxic than Re-CORM-2. On the other side, NaReO\({}_{4}\), the CO-free water metabolite of the CORMs, did not provoke any adverse toxic effect in the treated animals by 120 hpf. Interestingly, see Figure 6 and Table S1 in Supplementary Materials, none of the CORMs provoked the appearance of teratogenic malformations at any applied dose, nor had adverse effects on embryos hatching. This suggests that mortality caused at higher COMs doses (\(\geq\)150 uM) could be due to the liberated CO, which could diffuse into embryos via the chorion pores and might interfere with the oxygen exchange within the  
Figure 5: Cumulative [%] of Re metal released in PBS buffer, pH 7.4, by CF-1 (black line) and CF-12. The cumulative [%] of Re metal in solution is taken as a measure of drug release. embryo tissues [79]. In a previous study, [PERSON] et al., investigating CORM-3, have found dose-dependent mortality, cardiotoxicity, and inhibitory effect on the embryos hatching, suggesting that CO-mediated inhibition of embryos hatching might play a critical role in the embryo's survival [79]. To address whether the chorion acted as a protective barrier limiting Re-CORM-2 and B\({}_{12}\)-ReCORM-2 passage to embryos, we mechanically removed the chorion at 20 hpf (earlier removal is not safe). B\({}_{12}\)-ReCORM-2 and Re-CORM-2 were then applied onwards at a doses ranging from 50 to 250 \(\upmu\)M. Data recorded by 120 hpf showed no difference in the survival rates between the groups of embryos with and without chorion, nor any adverse effect of the CO-releasing molecules at the highest applied dose of 250 \(\upmu\)M (mortality rate of 10% compared to that in the control group). Taken together, the data suggest that the chorion had no effect on embryos survival and that embryos' death (Figure 6) is likely due to embryos' sensitivity at the early developmental stage to CO-releasing molecules. In line with this assumption is the fact that treated embryos died by 48 hpf (data not shown). Stimulated by the observation of the reduced circulation in some embryos treated with B\({}_{12}\)-ReCORM-2 at doses \(\geq\)50 \(\upmu\)M, we explored both B\({}_{12}\)-ReCORM-2 and ReCORM-2 for the possibility of inhibiting angiogenesis. Neo-angiogenesis (new vessels formation) represents a crucial process for cancer development, intrusion, and metastasis [80], therefore, inhibiting tumor vascularization is a recognized clinical approach for solid malignancies therapy, which, in combination with cytotoxic drugs, could greatly increase the anticancer effectiveness of medications [81]. Suppression of angiogenesis was studied using transgenic zebrafish _Tg(fli1:EGFP)_ embryos. In this transgenic line, endothelial cells expressing EGFP can be directly observed by fluorescence microscopy, enabling the detection of the anti-angiogenic drugs without adverse toxic effects.