2 g HSA (Beyotime Biotechnology, China) was dissolved in 30 mL 0.1 m NaHCO3 (pH 8.2). Then, 2 g diethylenetriaminepentaacetic acid dianhydride was dissolved in 10 mL dimethyl sulfoxide (DMSO) and added to the HSA solution. The pH was adjusted to 8.2 using 1 m NaOH. The solution was stirred for 2 h at room temperature and dialyzed against deionized water. Then, 1 g GdCl3 (J&K Scientific, China) was added at a pH of 6.5 to generate GdDTPA–HSA. Mass spectrometry was then conducted to verify the Gd linking efficiency. Then, 60 mg GdDTPA–HSA solution was mixed with free ICG (J&K Scientific, China) (10 mg dissolved in 2 mL DMSO). The solution was stirred for 12 h at room temperature. Finally, the mixture was purified using a Sephadex G50 column (GE 121 Healthcare, USA).
Next, 25 mg GdDTPA–HSA@ICG nanoparticles were linked to 2 mg Bev antibodies (Roche Pharma, Switzerland) using N‐hydroxy‐succinimide (NHS)—polyethylene glycol (PEG) 2000—COOH (Aladdin, China). The carboxylic group was activated by N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide hydrochloride (EDC•HCl) and NHS (Energy Chemical, Shanghai) to generate a fluorescence/magnetic resonance dual‐mode functionalized tumor‐targeting VEGF‐A multifunctional probe, NPs‐Bev. Also, NPs‐IgG was synthesized as a control probe.
Next, 25 mg GdDTPA–HSA@ICG nanoparticles were linked to 2 mg Bev antibodies (Roche Pharma, Switzerland) using N‐hydroxy‐succinimide (NHS)—polyethylene glycol (PEG) 2000—COOH (Aladdin, China). The carboxylic group was activated by N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide hydrochloride (EDC•HCl) and NHS (Energy Chemical, Shanghai) to generate a fluorescence/magnetic resonance dual‐mode functionalized tumor‐targeting VEGF‐A multifunctional probe, NPs‐Bev. Also, NPs‐IgG was synthesized as a control probe.