V6 - Bioorthogonal Activation of Prodrugs using a Cancer Targeting Peptide Amphiphile

Introduction: : Chemotherapies often cause dose-limiting side effects and they are not site-specific.1 In this context, the interaction between nanomaterials such as peptide amphiphiles (PA) and biomolecules in vivo can be utilized for improved treatment outcomes. PAs are biodegradable, biocompatible with prolonged blood circulation that can deliver payloads to their intended target.2 Recent work in our group has demonstrated the ability of a self-assembled peptide amphiphile nanostructure, namely SA-E, to accumulate in a broad range of solid tumors by exploiting circulating lipoproteins.3 Furthermore, it was shown that the SA-E retained in tumors for up to 2 weeks, while it was mostly cleared from other tissues in a day. While this cancer specificity improves the therapeutic window of chemotherapies attached to them, side effects are still possible. In this scenario, bioorthogonal reactions have gained significant interest for their ability to selectively manipulate molecules. Among these reactions, the cleavable linkers are especially compelling for biological media use due to the possible selectivity of the linkers.4 This work aims to utilize the SA-E and the bioorthogonal reactions for site-specific activation of different prodrugs. The amphiphilic SA-E labeled with tetrazine molecule will serve as the vehicle into the tumor followed by a trans-ciclooctene (TCO)-caged prodrug and after a rapid bioorthogonal reaction occurring specifically at the tumor site, the active drug is released (Fig. 1A). This method ensures the targeted delivery of the prodrug to the designated location, reducing side effects.

Materials and

Methods: : Maleimide thiol chemistry was used to conjugate tetrazine to the SA-E peptide as well as to conjugate the prodrug to the TCO. All products were purified by HPLC and the successful reaction confirmed by LCMS. Click release capabilities were tested in PBS, where TCO-caged prodrug and free drug signals were monitored by HPLC over time. Then, anticancer activity was demonstrated in an in vitro setup utilizing glioblastoma (GL-261, RG2), colon cancer (HCT116) and breast cancer (4T1). Confocal imaging confirmed the bioorthogonal reaction, and the SN-38 release.

Results, Conclusions, and Discussions:: Successful bioorthogonal chemistry using the SA-E peptide and the SN-38 drug was achieved, as was seen after monitoring the reaction by HPLC over time. The TCO-conjugated prodrug was almost completely consumed after 2h and gone entirely overnight, converting into free SN-38. In addition, the successful reaction was visualized by confocal imaging. The distinct blue fluorescence was mainly visible in the presence of the free, bioorthogonally released SN-38, while no drug signal was present in the case of the TCO-caged counterpart (Fig. 1B). Anticancer activity studies were performed, and the results implies that the sequential addition of SAE-tetrazine followed by SN38-TCO, resulted in successful bioorthogonal reaction with the release, delivery and activation of SN-38 drug at the tumor site, as was tested on different cell lines (Fig. 1C). In undergoing work, an in vivo model of GL-261 is being established to monitor the tumor growth and test this novel treatment strategy.
Overall, these results show the advantageous use of the bioorthogonal chemistry to selectively deliver anti-cancer drugs to solid tumors and the rapid and efficient release of the drug. With its simple and modular design and high accumulation, SA-E represents a promising platform for broad applications in cancer imaging and therapy.

Acknowledgements and/or References (Optional)::

References:
[1] Watanabe T., J Med. Chem. 2024, 67:18124-18138
[2] Pitman M., Anal. Methods. 2020, 12:2068-2081
[3] Fischer J., bioRxiv. 2024
[4] Versteegen R., Angew Chem. 2013, 52: 14112-14116