Human serum albumin (hsa)

The human tubular epithelial cell line HK-2 and the human myeloid leukemia mononuclear cell line THP-1 were obtained from the Cell Bank of the Type Culture Collection (Chinese Academy of Sciences, Shanghai, China) and maintained in Roswell Park Memorial Institute 1640 medium containing 10% fetal bovine serum (FBS) (Gibco, Australia) at 37 °C under a 5% CO₂ atmosphere.
For treatment with human serum albumin (HSA), HK-2 cells were cultured in RPMI 1640 medium containing 2% FBS for 24 h and then stimulated with 20 mg/ml HSA for 48 h. For lipopolysaccharide (LPS) stimulation, THP-1 cells were induced to macrophages using 100 nM phorbol myristate acetate (PMA) (S1819; Beyotime, China) for 48 h and treated with 100 ng/ml LPS (S1732; Beyotime) for another 24 h. For investigations with the prolyl hydroxylase inhibitor FG-4592, macrophages were treated with 5 mM FG-4592 (SC1135; Beyotime) for 24 h.
HK-2 cells and macrophages were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, USA). To knock down Rab27a, HK-2 cells were transfected with 50 nM Rab27a siRNA (RiboBio, Guangzhou, China). For HIF-1α knockdown, macrophages were transfected with 50 nM HIF-1α siRNA (RiboBio, Guangzhou, China). Transwell co-culture systems were applied for co-culture with HK-2 cells and macrophages (Corning, MA, USA).

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.