Concentration-Independent Multivalent Targeting of Cancer Cells by Genetically Encoded Core-Crosslinked Elastin/Resilin-like Polypeptide Micelles

Abstract

Valency is a fundamental principle to control macromolecular interactions and is used to target specific cell types by multivalent ligand-receptor interactions using self-assembled nanoparticle carriers. At the concentrations encountered in solid tumors upon systemic administration, these nanoparticles are, however, likely to show critical micelle concentration (CMC)-dependent disassembly and thus loss of function. To overcome this limitation, core-crosslinkable micelles of genetically encoded resilin-/elastin-like diblock polypeptides were recombinantly synthesized. The amphiphilic constructs were covalently photo-crosslinked through the genetically encoded unnatural amino acid para-azidophenylalanine in their hydrophobic block and they carried different anticancer ligands on their hydrophilic block: the wild-type tenth human fibronectin type III domain, a GRGDSPAS peptide-both targeting α_vβ₃ integrin-and an engineered variant of the third fibronectin type III domain of tenascin C that is a death receptor 5 agonist. Although uncrosslinked micelles lost most of their targeting ability below their CMC, the crosslinked analogues remained active at concentrations up to 1000-fold lower than the CMC, with binding affinities that are comparable to antibodies.

Figure 1 –. Construct overview and stability analysis of unfunctionalized RLP/ELP nanoparticles before and after photocrosslinking:

To enable photocrosslinking, the diblock polypeptide contained 5 evenly spaced pAzF residues in the RLP block. Successful crosslinking was shown by incubation of nanoparticles with 7.2 m guanidinium hydrochloride: Cryo-TEM micrographs show that crosslinked samples (particles indicated by arrows) retained their spherical morphology upon exposure to guanidinium hydrochloride, whereas the uncrosslinked nanoparticles disassembled into unimers that are not detectable by cryo-TEM. Scale bars represent 200 nm.

Figure 2 –. Architecture of the ligand functionalized, photocrosslinkable RLP/ELP constructs:

The anticancer ligands were genetically encoded to be presented on the C-terminal, hydrophilic end of the construct with a hydrophilic K₈D₄ linker between the ligand and the ELP block.

Figure 3 –. Cell viability experiments for DR5-targeting constructs:

Cell survival curves for Colo205 cells after coincubation with DR5-targeting constructs over 24 h in complete media showed that the EC₅₀ value for the uncrosslinked sample closely matched the CMC of the unfunctionalized RLP/ELP diblock micelles. Photocrosslinking significantly increased the potency of Tn3-functionalized nanoparticles to concentrations well below the CMC.

Figure 4 –. Quantification of cellular uptake for integrin-targeting constructs:

A: Representative images of K562 cells after coincubation with AF488-labeled (green), functionalized nanoparticles over 1.5 h in PBS. Scale bars represent 20 μm. B: Quantification of cell uptake levels using flow cytometry. Boxes indicate 3^rd and 7^th percentiles, bars represent 1^st and 9^th percentiles over >30000 events. Note that all constructs have a RLP_{40, 5pAzF}-ELP₈₀ base and carry the C-terminal functionalization denoted on the respective images or figure legend. See figures S8–10 for additional microscopy images and the flow cytometry histograms***: p < 0.001 (unpaired student’s t test).

Figure 5 –. SPR data for the integrin-targeting RLP/ELP diblock polypeptides both above and below their CMC:

A-D: SPR sensorgrams for ligand-functionalized RLP/ELP constructs at concentrations below the CMC showing that for both ligands, integrin-binding is only retained for photocrosslinked nanoparticles but not for their uncrosslinked analogs. E/F: SPR sensorgrams at concentrations above the CMC show moderate binding avidities for uncrosslinked, functionalized nanoparticles. Note that the dotted, vertical line represents the point at which the buffer was exchanged during the SPR measurement. See figure S12 for SPR sensorgrams for the untargeted control constructs.