Structural and Hydrodynamic Analysis of a Novel Drug Delivery Vector: ELP[V5G3A2-150]

Abstract

The therapeutic potential of elastin-like polypeptide (ELP) conjugated to therapeutic compounds is currently being investigated as an approach to target drugs to solid tumors. ELPs are hydrophobic polymers that are soluble at low temperatures and cooperatively aggregate above a transition temperature (TT), allowing for thermal targeting of covalently attached drugs. They have been shown to cooperatively transition from a disordered structure to a repeating type II β-turn structure, forming a β-spiral above the TT. Here we present biophysical measurements of the structural, thermodynamic, and hydrodynamic properties of a specific ELP being investigated for drug delivery, ELP[V5G3A2-150]. We examine the biophysical properties below and above the TT to understand and predict the therapeutic potential of ELP-drug conjugates. We observed that below the TT, ELP[V5G3A2-150] is soluble, with an extended conformation consisting of both random coil and heterogeneous β structures. Sedimentation velocity experiments indicate that ELP[V5G3A2-150] undergoes weak self-association with increasing temperature, and above the TT the hydrophobic effect drives aggregation entropically. These experiments also reveal a previously unreported temperature-dependent critical concentration (Cc) that resembles a solubility constant. Labeling ELP[V5G3A2-150] with fluorescein lowers the TT by 3.5°C at 20 μM, whereas ELP[V5G3A2-150] dissolution in physiological media (fetal bovine serum) increases the TT by ∼2.2°C.

Effect of Basic Cell-Penetrating Peptides on the Structural, Thermodynamic, and Hydrodynamic Properties of a Novel Drug Delivery Vector, ELP[V5G3A2-150]

“…All data sets exhibited the J–O or Johnston–Ogston effect, 13,36−38 a classic boundary sharpening anomally caused by high concentrations of other components. All three constructs demonstrate a decrease in the weight average sedimentation coefficient with an increase in the level of FBS (Figure S1 of the Supporting Information).…”

“…4,6,8,13 Therefore, there are two major questions. (1) Do the modified constructs retain the ability to thermally aggregate in a reversible manner?…”

Effects of Doxorubicin on the Liquid-Liquid Phase Change Properties of Elastin-Like Polypeptides

“…The drug carrier SynB1-Cys-ELP1 used in this study is made of a 150-pentapeptide repeat of [VPG(V 5 G 3 A 2 )G] 150 , where V, G, and A are the guest residues in a 5:3:2 ratio (Table 1). (ELP1 refers to a construct with a 5:3:2 V/G/A ratio used in many of these early studies (6,8,(10)(11)(12)(13)(14)). A cysteine was engineered into the sequence for covalent drug attachment, and a SynB1 cell-penetrating peptide (CPP) was included at the N-terminus for ELP drug targeting.…”

“…A cysteine was engineered into the sequence for covalent drug attachment, and a SynB1 cell-penetrating peptide (CPP) was included at the N-terminus for ELP drug targeting. The presence of a SynB1 CPP is designed to enhance ELP uptake to the cytoplasm of cells and tumors via endocytosis (10,11). Cleavable hydrazone linkers are typically used in these studies to take advantage of the low pH environment of the lysosome to release the drug for targeting to the site of action (12)(13)(14).…”

“…Previously, we used complementary physical methods (circular dichroism (CD), turbidity, dynamic light scattering (DLS), sedimentation velocity analytical ultracentrifugation (SV-AUC), and differential scanning calorimetry (DSC)) to investigate the properties of various ELP1 constructs, comparing a Lys-ELP1, a Cys-ELP1, and two basic CPPcontaining constructs, Bac-Cys-ELP1 and SynB1-Cys-ELP1 (see Table 1 for abbreviated sequence information) (10,11). CD consistently revealed the temperature-dependent loss of random coil and the formation of b-turns with increasing temperature.…”