Vegf165

The anti‐VEGF ability and resulting inhibition efficiency of cell growth for the recombinant proteins and control were assessed using the HUVEC proliferation assay. In brief, a series of 4‐fold dilutions starting from 750 nM were prepared using 0.5% FBS MEM (Zhong Qiao Xin Zhou Biotech., Shanghai, China) for the test protein. Next, 50 μL of 210 ng/mL VEGF₁₆₅ (GenScript, Nanjing, China) in 0.5% FBS MEM was added to each dilution of the test protein in a 96‐well plate (Costar, Corning, Inc. USA), followed by incubation at 37°C for 1.5–2 h. Then, HUVEC cells (NSCTRB, Shanghai, China) were prepared at a concentration of 2.4 × 10⁵ cells/mL, and 50 μL of the cell suspension was added to each well. After incubation for 68–72 h, 16 μL of CCK‐8 (Solarbio, Beijing, China) was added to each well of the plate, followed by incubation at 37°C for 2.5 h. The absorbance at 450 nm was measured using a microplate reader (Multiskan™ Spectrophotometer Thermo Scientific, Singapore).

The binding affinity of nanoFc to VEGF was determined using surface plasmon resonance (SPR) on a Biacore S200 instrument (GE, USA). VEGF₁₆₅ (GenScript, Nanjing, China) was immobilized onto flow channels of an activated CM5 sensor‐chip by diluting it to 5 μg/mL in 10 mM sodium acetate (pH 5.5) and injecting into the SPR system to obtain a final immobilization level of 160 RU. A range of nanoFc analyte dilutions were subsequently injected, with HBS‐EP running buffer (GE‐Healthcare, USA) at a flow rate of 45 μL/min for 150 s, followed by a dissociation time of 1200 s. The sensor chip surface was regenerated with 100 mM HCl (Merck, China) between each injection. The binding sensorgrams were analyzed using the 1:1 Langmuir model to determine the binding kinetics and dissociation constant.

First, human pulmonary microvascular endothelial cells (HMVEC-L) (Lonza, Allendale, NJ) were lysed on ice with Laemmli buffer after 5 minutes of treatment with human VEGF₁₆₅ (GenScript, Piscataway, NJ) at 10 ng/mL and activation of VEGFR2 was confirmed with immunoblot for P-VEGFR2.
Once activation was confirmed, HMVEC-L were treated without (control) or with VEGF₁₆₅ for 8 hours. Protein content of conditioned media from control and VEGF-treated HMVEC-L were analyzed with a Proteome Profiler^™ Angiogenesis Array (R&D Systems, Minneapolis, MN) while cell lysate was characterized with a Protease Array (R&D Systems) according to the manufacturer’s protocol. Briefly, nitrocellulose membranes embedded with capture antibodies were blocked for 1 hour prior to the addition of conditioned medium or cell lysate samples. Samples were incubated with an antibody cocktail for 1 hour at room temperature before being applied on the membranes for overnight incubation at 4°C. After conjugation with streptavidin-HRP, signals were developed with a hydrogen peroxide and luminol-based reagent mix and quantified using ImageJ (NIH, Bethesda, MD).