## 3 Results and Discussion
### Assessment of Biological Activities of B\({}_{12}\)-ReCORM-2 and Re-CORM-2 Molecules
The tested Re-CORM-2 molecules were applied to 6- hpf embryos at a maximum dose of 100 \(\upmu\)M (corresponding to the highest safety dose of B\({}_{12}\)-ReCORM-2, Figure 7) and inspected at 48 hpf and 72 hpf for the intersegmental vessels (ISVs) and subintestinal vessels (SIVs) development (ISV angiogenesis precedes SIV angiogenesis), respectively. The anti-angiogenic phenotype was characterized by the decreased number/length of SIVs and/or ISVs along the whole body. While in untreated embryos, 28-30 ISVs and 6-9 arcades in the SIV basket-like structure were present, the embryos treated with both molecules at doses \(\geq\)25 \(\upmu\)M had markedly reduced the number of both ISVs and SIVs vessels. In the experiment, B\({}_{12}\)-ReCORM-2 demonstrated higher anti-angiogenic potential than ReCORM-2, particularly on the SIVs development (\(p>0.001\), Figure 7). We note that the majority of ISV vessels were affected at 100 \(\upmu\)M of B\({}_{12}\)-ReCORM-2 (see Supplementary Materials Figure S1), showing a markedly decreased EGFP expression as an indication of the cells viability loss, without affecting other inner organs' functioning and development. None of the tested molecules inhibited angiogenesis at the doses below 25 \(\upmu\)M, according to the fluorescence intensity of the vessels in hematopoietic tissues. Although vessel fluorescence in these areas was not measured, observably much higher fluorescence intensity indicates that both CO-releasing molecules increased vessel density in embryos exposed to the doses from 5 and 10 \(\upmu\)M,  
Figure 6: Toxicity evaluation of ReCORM-2, B\({}_{12}\)-ReCORM-2, NaReO\({}_{4}\) and vitamin B\({}_{12}\) addressed in the zebrafish (_Danio rerio_) embryos exposed to different doses in a period from 6 hpf to 120 hpf. contrary to their inhibitory effect at higher doses (\(\geq\)25 \(\upmu\)M). This observation is in line with previously reported studies [25]. Having established that the examined CO-releasing molecules show no toxic responses in zebrafish embryos at doses as high as 100 \(\upmu\)M, we finally loaded Re-CORM-2 and B\({}_{12}\)-ReCORM-2 into _Aulacoseira_ frustules (13 and 20 \(\upmu\)m in size) and evaluated the toxicity of the drug-free micro-frustules in vivo. In these experiments, we evaluated _Aulacoseira_ diatoms to address their effects on embryos' survival, development (teratogenicity), inner organs functioning, and particularly their inflammatory response. The zebrafish model has been proven particularly suitable for the toxicity assessment of nanoparticles and biomaterials [82; 83; 84; 85]. In our experiment, embryos were in direct contact with diatoms of 13 and 20 \(\upmu\)m in size for a period of 114 h (from 6 to 120 hpf). The results obtained after 5-days exposure showed no evidence of any detrimental effects to the organisms at concentrations up to 200 \(\upmu\)g/mL (Figures 7A and 8A), indicating that the tested silicate frustules are safe. Inflammation is the most common adverse tissue reaction towards drug carrier-materials, nanoparticles, and released drugs. Since neutrophils migration, infiltration and accumulation within inflamed tissue represent the hallmark of primary host response to inflammation [86; 87; 88], we have evaluated whether the tested micro-frustules provoked an inflammatory response in zebrafish embryos after 5-days exposure. Accordingly, we used embryos of the transgenic _Tg_(_mpx_:EGFP) zebrafish line with fluorescently labeled neutrophils, which enabled us to visualize the neutrophils' burden, recruitment, chemotaxis, and accumulation within damaged tissues after exposure to the diatoms, as previously shown [71; 89; 90]. The zebrafish embryonic epithelium is very thin and consists of only two layers of epidermal cells [91]. This characteristic makes the zebrafish embryos very sensitive to exposed materials and tractable for the inflammatory response.