Identification of peptide coatings that enhance diffusive transport of nanoparticles through the tumor microenvironment

Rashmi Mohanty; Xinquan Liu; Jae You Kim; Xiujuan Peng; Sahil Bhandari; Jasmim Leal; Dhivya Arasappan; Dennis C. Wylie; Tony Dong; Debadyuti Ghosh

We wanted to fluorescently label the phage to image and determine the diffusivity through the tumor ECM. However, it was important to label the phage without affecting the coat protein, thus conserving the surface physicochemical properties of the phage clones. As a result, the encapsidated DNA of the selected peptide-presenting T7 phage was labeled with SYBR Gold dye with a slight modification of the protocol described by Mosier-Boss et al. (2003) 88 . Briefly, the SYBR Gold dye (ThermoFisher Scientific) was diluted to 100× from the 10,000× stock concentration using 1× PBS. Sixty microliters of each clone (10 8 pfu/µL) was incubated with 500 µL of diluted dye for 20 minutes on a rocker (ThermoFisher Scientific) at room temperature. One hundred microliters of sterile 50% PEG 8000 was added to the labeled clone, incubated overnight at 4 °C, and centrifuged at 17000g for 30 minutes. The supernatant containing unlabeled free dye was decanted away from the labeled phage pellet, which was subsequently resuspended with 1× PBS. The suspension was filtered using a syringe filter 0.2 µm PES membrane (VWR). To ensure complete removal of any trace amounts of free dye, the phage suspension underwent a second round of purification using ultrafiltration with Amicon Ultra 3 kDa molecular weight cutoff of 0.5mL centrifugal filters (MilliporeSigma) and five 1× PBS washes. After centrifugation, the resulting phage pellet was resuspended with 150 µL of 1× PBS and transferred to a fresh tube.