Sample preparation

ELP samples were dialyzed into PBS and filtered through a 0.2-μm filter to trap larger aggregates. Spectrophotometry was used to determine the concentration after correction for light scattering (22). Samples were prepared gravimetrically. The Dox-ELP labeling reaction was achieved by covalently linking Dox-maleimide, purchased from MedKoo Biosciences (Morrisville, NC) (Dox-succinimidyl-4-(N-maleimidethyl)cyclohexane-1-carboxylate), to ELP via the reaction of the thiol group available in the N-terminus domain of ELP (Table 1). The reaction was performed overnight at 5°C on a rotary plate after cysteine reduction with 1 mM tris(2-carboxyethyl)phosphine hydrochloride at room temperature for 20 min. The excess dye was removed using three 5-mL Zeba columns (CO MW 1000 Da; ThermoFisher Scientific) and equilibrated into PBS. The concentration of Dox-ELP was determined using absorbance spectroscopy by correcting the ELP concentration for Dox absorbance at 280 nm according to the following formula:

The covalent Dox concentration [Dox] is estimated by the Abs_496nm/9250 M⁻¹cm⁻¹, and the ratio [Dox]/[Dox−ELP] is used to determine the labeling efficiency.