[Construction and in vitro Testing of Genipin Cross-linked Hemerythrin Nanoparticles]

To explore the feasibility of modifying hemerythrin molecules with natural cross-linker genipin, and evaluate its efficacy and safety.

Hemerythrin was isolated and purified from sipunculid worms using tangential flow ultrafiltration. Subsequently, genipin cross-linked hemerythrin nanoparticles (GHrNPs) were constructed by adding 20% w/w genipin under mildly acidic conditions, and glutaraldehyde cross-linked hemerythrin nanoparticles (GAHrNPs) were constructed by adding 10% w/w glutaraldehyde under mildly alkaline conditions. The diameter, dispersity index, zeta potential, functional group structure, P₅₀, and Hill coefficient of the two nanoparticle groups were measured. The two nanoparticle groups at different concentrations were co-cultured with vascular endothelial cells for 24 hours, then the cell viability and NO concentration in the culture medium were measured.

After glutaraldehyde/genipin molecular cross-linking, infrared spectra showed the continuous presence of amide bands I and II. The hydrated particle sizes of hemerythrin, GHrNP and GAHrNP were (93.14±2.11), (109.53±3.54), and (115.65±2.65) nm, dispersity indexes were 0.30±0.06, 0.27±0.05, and 0.25±0.03, zeta potentials were (-24.00±1.54), (-19.52±1.31), and (-18.90±1.25)mV, P₅₀ values were (9.28±0.22), (8.50±0.54), and (5.75±0.90) mmHg, and Hill coefficients were 1.61±0.14, 1.58±0.17, and 1.41±0.22, respectively. The average hydrated particle size increased after cross-linking with hemerythrin, the negative value of the zeta potential decreased (both P < 0.05). The P₅₀ value of GAHrNP was significantly decreased than that of hemerythrin and GHrNP (P < 0.05). The viability of vascular endothelial cells in the GHrNP group was higher than that in the GAHrNP group at different mass concentrations (P < 0.05). The NO concentration in the culture medium of vascular endothelial cells in the GHrNP group was higher than that in the GAHrNP group only at 2.0 mg/ml (P < 0.05).

Hemerythrin molecules cross-linked by genipin can form stable nanoparticles with good oxygen-carrying activity and lower cytotoxicity compared to glutaraldehyde.