Integrin αvβ3-targeting human serum albumin for improved blood circulation and targeting efficiency

Objectives: The RGD specifically recognizes the integrin αvβ3 which is overexpressed in various malignant tumors. One major drawback of small peptide such as RGD, however, is short half-life in the blood, which greatly compromises their targeting efficacy. To improve blood circulation time and targeting efficacy, we developed cRGDyK conjugated human serum albumin (HSA) using click chemistry reaction.

Methods: HSA was conjugated with DBCO-NHS (dibenzyl cyclooctyne) under physiologically favorable reaction condition for the preparation of strain promoted azide-alkyne reaction. Using this reaction group, DBCO-HSA was conjugated with N₃-cyclic RGDyK (Azido cyclic RGDyK) for integrin αvβ3 targeting and ⁶⁴Cu labeled N₃-NOTA (3-azidopropyl-NOTA) for radiolabeling. Cellular uptake of ⁶⁴Cu-cRGDyK-HSA were tested for integrin αvβ3 specific binding at cell level. PET images were acquired after tail vein injection of ⁶⁴Cu-HSA, ⁶⁴Cu-cRGDyK-HSA in integrin αvβ3 positive tumor (SK-OV3, ovarian cancer cell line) bearing BALB/c nude mice. PET signals were quantitatively analyzed with PET image analysis program, AMIDE.

Results: The number of cRGDyK on DBCO-HSA conjugates was confirmed using MALDI-TOF MS. cRGDyK-HSA was successfully conjugated with ⁶⁴Cu labeled N₃-NOTA. Integrin αvβ3 mRNA and protein was highly expressed in SK-OV3 cell line. When compared to cellular uptake level, ⁶⁴Cu-cRGDyK-HSA were more accumulated at integrin αvβ3 positive cells (SK-OV3) than integrin αvβ3 negative cells (P < 0.05) and there were no difference in ⁶⁴Cu-HSA. Serial PET images were acquired 0, 4, 24, 48 hours after tail vein injection of ⁶⁴Cu-cRGDyK-HSA. Radioactivity of ⁶⁴Cu-cRGDyK-HSA in SKOV3 tumor were higher than that of ⁶⁴Cu-HSA. ⁶⁴Cu-labeled-cRGDyK-HSA can be observed at 48 hours after injection, which shows longer circulation time in mice.

Conclusion: We successfully conjugated cyclic RGDyK to HSA using click chemistry approach. We demonstrated that cRGDyK-HSA specifically bind to integrin αvβ3 in vitro and in vivo level. In animal model, ⁶⁴Cu-labeled-cRGDyK-HSA can be observed at 48 hours after injection, which shows longer circulation time. Our results indicated that cRGDyK-HSA have a potential to tumor diagnosis and tumor therapy response monitoring. Research Support: .