Targeted delivery of therapeutic proteins to triple negative breast cancer cells using modular hepatitis B virus-like particles
Date
2023
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Protein therapeutics offer enormous clinical impact in treating a variety of diseases, such as cancer, by offering high selectivity with limited off-target effects in vivo. However, delivery challenges, such as short circulation half-lives, protein instability, and proteolytic degradation, severely reduce functional proteins from reaching their target and require frequent administration. To address these problems, nanocarrier encapsulation can provide enhanced targeted transportation of functional proteins with protease protection to the tumor site. Non-viral vectors, such as polymer nanoparticles, offer high loading capacity and design flexibility by leveraging established chemical syntheses; however, large particle size and harsh synthesis conditions may cause rapid hepatic clearance and affect protein stability. In nature, viruses have developed an efficient delivery system because of their regular, multifunctional architecture and their ideal size, conducive to mononuclear phagocytic system evasion. Inspired by their viral analogues, virus-like particles (VLP) are non-infectious viral capsids, which have potential for drug delivery applications because of their shared structural characteristics. The well-characterized hepatitis B virus (HBV) VLP is an ideal protein delivery vehicle because it has regular solvent exposed features for incorporating surface modifications, a large interior cavity amenable for loading various cargos, and a diameter of 36 nm for favorable pharmacokinetics. This makes it an ideal candidate as the platform to develop a modular nanocarrier for simultaneous tunable interior loading of protein cargos and exterior modification of surface decoration to facilitate targeted, intracellular delivery. ☐ To engineer the HBV VLP as a nanocarrier for targeted protein delivery, genetic insertions within the solvent-exposed c/e1 immunodominant loop on the HBV core protein monomer have facilitated incorporation of targeting peptides or proteins and modifications at the C-terminus have demonstrated cargo encapsulation within the VLP. Nevertheless, these approaches are not tunable and cannot be generalized to larger proteins and multiple cargos, respectively, as they are limited by the physical dimensions of the capsid monomer and interior lumen, such that larger proteins would prevent proper protein folding and capsid assembly due to steric effects. To address these challenges, we focused on two strategies for tunable VLP modification: SpyCatcher/SpyTag bioconjugation and a VLP multi-expression system. With these techniques, we have demonstrated simultaneous and tunable loading of multiple protein cargos within the HBV VLP and tailored uptake and cytotoxicity to treat our triple negative breast cancer disease model. ☐ To target our modular VLP delivery platform for target delivery of therapeutic proteins, we first incorporated the SpyCatcher-SpyTag protein-peptide pair, which facilitates site- and orientation-specific nanocarrier modification onto the HBV capsid in a plug-and-play manner. SpyTag was genetically inserted into the solvent-exposed exterior loop, while SpyCatcher was fused to potential delivery moieties, such as elastin-like polypeptide (ELP) stealth moieties and epidermal growth factor receptor (EGFR)-targeting designed ankyrin repeat proteins (DARPin). For cargo encapsulation, we used our VLP multi-expression system for encapsulation of two model proteins, green fluorescent protein (GFP) and the prodrug converting enzyme yeast cytosine deaminase (yCD). Upon delivering the assembled and targeted VLP, we observed enhanced uptake to EGFR-overexpressing triple negative breast cancer (TNBC) cells in contrast to non-malignant breast epithelial cells that exhibit only basal levels of EGFR. Corresponding targeted cytotoxicity was also observed in the cancer cells upon prodrug activation by the internalized yCD cargos in the targeted VLPs. ☐ Cancer cell-specific recognition is one of the predominant challenges of targeted delivery and the lack of commonly overexpressed estrogen (ER), progesterone (PR) and human epidermal growth factor receptor 2 (HER2) in triple negative breast cancer cells make it more difficult to target. Nevertheless, cancer cells generally exhibit dysregulated expression of numerous cell surface markers, which result in their uncontrollable growth and invasiveness. To enhance the recognition capabilities of our modular VLP, we decided to employ the colocalization dependent protein switch (CoLOCKR) system previously developed by the Baker lab, which can use overexpressed receptors as Boolean inputs in logic-gated nanocarrier uptake. In addition to EGFR, epithelial cell adhesion molecule (EpCAM) is also commonly overexpressed in TNBC cells, so we used EGFR and EpCAM as receptor inputs to activate a CoLOCKR AND-gate architecture to control delivery of our previously developed GFP- and yCD-loaded VLPs. AND-gate-mediated uptake of CoLOCKR-compatible VLPs activated by EGFR and EpCAM cell marker inputs demonstrated significantly greater uptake in TNBC cells in contrast to non-cancerous cells that lack high levels of both cell receptors. Furthermore, trends in corresponding CoLOCR-mediated VLP cytotoxicity correlated with that of uptake in TNBC cells. These results demonstrate our ability to use logic-gating to control therapeutic protein delivery by integrating the CoLOCKR system to our VLP platform. ☐ Lastly, we investigated if we could broaden the therapeutic proteins that could be delivered with our modular VLP system to cargo proteins with subcellular targets through the incorporation of endosomolytic peptides. To facilitate endosomal escape of targeted VLPs after endocytosis, we added an endosomal disruption module, containing the INF7 peptide, on the exterior of the VLP. Previous work has also demonstrated that incorporation of intra-endosomal enzyme protease sites for furin and cathepsin L protease cleavage releases the peptide from their protein cargo and enhances endosomal escape. To investigate whether this could be applied on our VLP, we also developed a second module with the two proteolytic cleavage sites to trigger enzyme-responsive INF7 release. Our results demonstrated that incorporation of both cleavable and non-cleavable endosomal disruption modules enhanced the intracellular half-lives of targeted VLPs via disruption of the endosomal membrane, but the cleavable module had a greater effect. Lastly, to test that INF7 incorporation could facilitate delivery of cytosolically active cargos, we demonstrated targeted delivery in TNBCs and corresponding cytotoxicity when our targeted, endosomolytic VLPs were loaded with ribosomal inactivating protein, gelonin. These results confirm the importance of endosomolytic peptides to facilitate the targeted delivery of cytosolically active protein therapeutics and how enzyme-triggered release of endosomolytic peptides can further enhance endosomal disruption in nanocarriers. ☐ These findings demonstrate the benefits of a modular protein nanocarrier for targeted therapeutic protein delivery in treating TNBC cells by specifically highlighting how the exterior can be modified in a plug-and-play manner with SpyCatcher/SpyTag bioconjugation to achieve an elevated level of cancer cell specificity. Furthermore, the multi-expression system provides a facile strategy to tunably encapsulate drugs within the VLP interior and provides a platform to investigate simultaneous delivery of multiple drug cargos and potential therapeutic synergy.
Description
Keywords
Drug delivery, Protein engineering, Synthetic biology, Virus-like Particles