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Therapeutic biomacromolecules are highly specific, which results in controlled therapeutic effect and less toxicity than small molecules. However, proteins and nucleic acids are large and have significant surface hydrophilicity and charge, thus cannot diffuse into cells. These chemical features render them poorly encapsulated by nanoparticles. Protein vesicles are self-assembling nanoparticles made by warming elastin-like polypeptide (ELP) fused to an arginine-rich leucine zipper and a globular protein fused to a glutamate-rich leucine zipper. To impart stimuli-responsive disassembly and small size, ELP was modified to include histidine and tyrosine residues. Additionally, hydrophobic ion pairing (HIP) was used to load and release protein and siRNA cargos requiring endosomal escape. HIP vesicles enabled delivery of cytochrome c, a cytosolically active protein, and significant reduction in viability in traditional two-dimensional (2D) human cancer cell line culture and a biomimetic three-dimensional (3D) organoid model of acute myeloid leukemia. They also delivered siRNA to knockdown protein expression in a murine fibroblast cell line. By examining uptake of positive and negatively charged fluorescent protein cargos loaded by HIP, this work revealed the necessity of HIP for cargo release and how HIP influences protein vesicle self-assembly using microscopy, small angle x-ray scattering, and nanoparticle tracking analysis. HIP protein vesicles have the potential to broaden the use of intracellular proteins for various diseases and extend protein vesicles to deliver other biomacromolecules. 

Protein vesicles made from bioactive proteins have potential value in drug delivery, biocatalysis, and as artificial cells. As the proteins are produced recombinantly, the ability to precisely tune the protein sequence provides control not possible with polymeric vesicles. The tunability and biocompatibility motivated this work to develop protein vesicles using rationally designed protein building blocks to investigate how protein sequence influences vesicle self-assembly and properties. We have reported an elastin-like polypeptide (ELP) fused to an arginine-rich leucine zipper (ZR) and functional, globular proteins fused to a glutamate-rich leucine zipper (ZE) that self-assemble into protein vesicles when warmed from 4 to 25 °C due to the hydrophobic transition of ELP. Previously, we demonstrated the ability to tune vesicle properties by changing protein and salt concentration, ZE:ZR ratio, and warming rate. However, there is a limit to the properties that can be achieved via assembly conditions. In order to access a wider range of vesicle diameter and stability profiles, this work investigated how modifiying the hydrophobicity and length of the ELP sequence influenced self-assembly and the final properties of protein vesicles using mCherry as a model globular protein. The results showed that both transition temperature and diameter of protein vesicles were inversely correlated to the ELP guest residue hydrophobicity and the number of ELP pentapeptide repeats. Additionally, sequence manipulation enabled assembly of vesicles with properties not accessible by changes to assembly conditions. For example, introduction of tyrosine at 5 guest residue positions in ELP enabled formation of nanoscale vesicles stable at physiological salt concentration. This work yields design guidelines for modifying the ELP sequence to manipulate protein vesicle transition temperature, size and stability to achieve desired properties for particular biofunctional applications. 

Abstract Recombinant proteins have emerged as promising building blocks for vesicle self‐assembly because of their versatility through genetic manipulation and biocompatibility. Vesicles composed of thermally responsive elastin‐like polypeptide (ELP) fusion proteins encapsulate cargo during assembly. However, vesicle stability in physiological environments remains a significant challenge for biofunctional applications. Here, incorporation of an unnatural amino acid, para‐azido phenylalanine, into the ELP domain is reported to enable photocrosslinking of protein vesicles and tuning of vesicle size and swelling. The size of the vesicles can be tuned by changing ELP hydrophobicity and ionic strength. Protein vesicles are assessed for their ability to encapsulate doxorubicin and dually deliver doxorubicin and fluorescent protein in vitro as a proof of concept. The resulting photocrosslinkable vesicles made from full‐sized, functional proteins show high potential in drug delivery applications, especially for small molecule/protein combination therapies or targeted therapies.