Self-Assembling VHH-Elastin-Like Peptides for Photodynamic Nanomedicine

Abstract

Recombinant llama heavy-chain antibody fragments (VHHs) are promising tools in the field of targeted nanomedicine. 7D12, a VHH against the epidermal growth factor receptor (EGFR) that is overexpressed in various cancers, has been evaluated as an effective cancer-targeting VHH in multiple studies. The small size of VHHs (15–20 kDa) results in a low circulation half-life, which can be disadvantageous for certain applications. A solution to this problem is to attach VHHs to the surface of nanoparticles to increase the hydrodynamic radius of the conjugate. This approach simultaneously allows the incorporation of different VHHs and other targeting moieties and therapeutic components into one structure, creating multispecificity and versatility for therapy and diagnosis. Here, we present the construction of highly defined 7D12-containing nanoparticles by utilizing thermoresponsive diblock elastin-like peptides that reversibly self-assemble into micellar structures. The resulting particles have a hydrodynamic radius of 24.3 ± 0.9 nm and retain full EGFR-binding capacity. We present proof of concept of the usability of such particles by controlled incorporation of a photosensitizer and show that the resulting nanoparticles induce EGFR-specific light-induced cell killing. This approach is easily extended to the controlled incorporation of various functional modules, improving therapy and diagnosis with targeted nanomedicine.

Pathway‐Dependent Co‐Assembly of Elastin‐Like Polypeptides

“…We subsequently proceeded to add a single‐domain antibody fragment‐ELP conjugate, 7D12‐[A 3 G 2 ‐60]‐[I‐60], to the co‐assembly formulation to stimulate uptake of particles containing both fluorescent proteins by cells expressing the epidermal growth factor receptor (EGFR) (Figure 3c). [ 44 ] A431, a cell line known to highly express EGFR, was selected as the model cell line, since 7D12 efficiently targets A431 cells and induces uptake of attached cargo as shown in our previous work. [ 44 ] Live cell confocal imaging showed that functionalized particles were taken up after an incubation time of 1 hour as expected.…”

“…[ 44 ] A431, a cell line known to highly express EGFR, was selected as the model cell line, since 7D12 efficiently targets A431 cells and induces uptake of attached cargo as shown in our previous work. [ 44 ] Live cell confocal imaging showed that functionalized particles were taken up after an incubation time of 1 hour as expected. The punctuated fluorescence within cells indicates endosomal or lysosomal localization, as would be expected given the size of co‐assembled particles and the targeting moiety.…”

Multivalent display of chemical signals on self‐assembled peptide scaffolds

“…[182] The Peptide amphiphiles, made from elastin-like peptides (ELPs), have also been used as scaffolds for multivalent display of bioactive motifs. In an example from Pille et al, [183] recombinant llama heavy-chain antibody fragments (VHHs) that target epidermal growth factor receptor (EGFR), which is overexpressed in cancer cells, were multivalently displayed on a diblock ELP composed of 60 pentameric units of GXCVP (where X is either A or G in a 3:2 ratio) and 60 units of GICVP. Tumor cells that overexpressed EGFR had higher uptake of the ELP, indicating their potential as vehicles for targeted drug delivery.…”

Nanoparticles based on natural, engineered or synthetic proteins and polypeptides for drug delivery applications

“…Modifications are possible by recombinant fusion and/or chemical modification. Di-blocks with varying modifications can be mixed, creating diverse ELP micelles displaying and/or encapsulating drugs and targeting moieties (Pille et al, 2017). Adapted from Biomacromolecules, Vol 18 (4), 2017, J. Pille et al, Self-Assembling VHH-Elastin-Like Peptides for Photodynamic Nanomedicine, 1302-1310.…”