Two-Step Targeted Drug Delivery via Proteinaceous Barnase-Barstar Interface and PLGA-Based Nano-Carrier

The conventional methods of treating cancer with chemo- and radiotherapy present plenty of serious problems, such as low therapeutic index and high systemic toxicity. The advanced cancer treatment strategies utilize nanoformulations of drugs that can enter a tumor due to the enhanced permeability and retention (EPR) effect. However, EPR fails in the treatment of several human diseases. Therefore, mainstream biomedical studies are focused on creating the drugs that would enter the tumor with higher effectiveness and require smaller doses for administration. A two-step targeted drug delivery system (DDS), involving the tumor pre-targeting with the first non-toxic module and subsequent targeting with the second complementary toxic module, is a solution for decreasing systemic toxicity. To meet the challenge, we have developed the two-step DDS mediated by the high-affinity binding of the barnase*barstar protein pair, one of the strongest known protein*protein complexes with Kaff = 1014 M−1. Ribonuclease barnase (12 kDa) from Bacillus amyloliquefaciens and its natural inhibitor barstar (10 kDa) act as lego bricks linking the first and the second modules on the surface of the cancer cell. Artificial scaffold polypeptide DARPin9_29 (14 kDa) recognizing HER2 oncomarker was genetically fused with barstar to serve the first module of the developed two-step DDS. A therapeutic PLGA-based nanocarrier coupled to barnase was used as a second module. The PLGA nanoparticles were loaded with diagnostic dye, Nile Blue, and chemotherapeutic drug, doxorubicin. We showed that the two-step DDS increases the performance of chemotherapy-loaded nanocarriers both in vitro and in vivo compared to the one-step DDS. Namely, we showed that IC50 of doxorubicin delivered via two-step DDS is more than 100 times lower than that for one-step DDS: IC50 = 43 ± 3 nM for two-step DDS vs. IC50 = 4972 ± 1965 nM for one-step DDS. Moreover, the developed two-step DDS based on barnase*barstar showed superior efficiency in image-guided cancer therapy: HER2-positive tumors’ treatment tests showed the tumor growth inhibition index TGI = 68.4% for one-step DDS and TGI = 94.9% for two-step DDS. The obtained results demonstrate the significant efficiency of two-step drug delivery over the classical one-step one. We believe that the obtained data will significantly change the direction of research in developing targeted anti-cancer drugs and promote the creation of new-generation cancer treatment strategies.