Abstract
Tumor extracellular matrix (ECM) is a high-capacity target for the precision delivery of affinity ligand-guided drugs and imaging agents. Recently, we developed a PL1 peptide (sequence: PPRRGLIKLKTS) for systemic targeting of malignant ECM. Here, we map the dynamics of PL1 binding to its receptors Fibronectin Extra Domain B (FN-EDB) and Tenascin C C-isoform (TNC-C) by computational modeling and cell-free binding studies on mutated receptor proteins, and study cellular binding and internalization of PL1 nanoparticles in cultured cells. Molecular dynamics simulation and docking analysis suggested that the engagement of PL1 peptide with both receptors is primarily driven by electrostatic interactions. Substituting acidic amino acid residues with neutral amino acids at predicted PL1 binding sites in FN-EDB (D52N-D49N-D12N) and TNC-C (D39N-D45N) resulted in the loss of binding of PL1 nanoparticles. Remarkably, PL1-functionalized nanoparticles (NPs) were not only deposited on the target ECM but bound the cells and initiated a robust cellular uptake via a pathway resembling macropinocytosis. Our studies establish the mode of engagement of the PL1 peptide with its receptors and suggest applications for intracellular delivery of nanoscale payloads. The outcomes of this work can be used for the development of PL1-derived peptides with improved stability, affinity, and specificity for precision targeting of the tumor ECM and malignant cells.
Highlights
Introduction iationsSystemic homing peptides are used for the precision delivery of drugs and nanoparticles (NPs)
The PL1 peptide (PPRRGLIKLKTS) with free carboxyl terminus and 5(6)-carboxyfluorescein (FAM) or biotin coupled via the 6-aminohexanoic acid spacer to the N-terminus of the peptide was ordered from TAG Copenhagen (Frederiksberg, Denmark)
We perturbed the peptide by moving it a few angstroms away from the docked position to see if the peptide returns to its original position, suggesting the stability of that conformation
Summary
Systemic homing peptides are used for the precision delivery of drugs and nanoparticles (NPs). In addition to affinity targeting of plasma membrane receptors expressed on the surface of malignant and tumor-associated cells, homing peptides with specificity towards tumor-associated ECM molecules have been developed [1,2,3,4,5,6]. ECM is an abundant high-capacity target that is primarily deposited by stromal cells and not by rapidly mutating malignant cells. Fibronectin has over 20 different alternatively spliced isoforms, and some of these variants are expressed only during embryonic development and in activated tissues, including solid tumors. The expression of extra domain B of fibronectin (FN-EDB) and tenascin-C C isoform (TNCC) is associated with angiogenesis and tissue remodeling, whereas resting normal adult
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