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. 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-stage drug delivery system is an encouraging alternative solution. At first, the primary, non-toxic targeting module is delivered to the tumor cells, followed by injection of the second complementary targeting module at a considerably lower dose, thus decreasing systemic toxicity. To meet the challenge, we have developed a two-stage drug delivery system (DDS), mediated by the high-affinity binding of the Barnase*Barstar protein pair. Barnase and Barstar act as lego bricks linking the first and the second modules on the surface of the cancer cell. Barnase (12 kDa) is a natural ribonuclease from Bacillus amyloliquefaciens, while Barstar (10 kDa) is its natural inhibitor. The Barnase*Barstar is one of the strongest known protein*protein complexes with Kaff = 1014 M−1 exhibiting extraordinarily stability in severe conditions. Artificial scaffold polypeptide DARPin9_29 genetically fused with Barstar served is a first module of the developed two-step DDS. DARPin9_29 (14 kDa) specifically recognizes the tumor marker HER2 overexpressed on human breast cancer cells. As a second module, a therapeutic nano-cargo was developed based on fluorescent polymer PLGA nanoparticles loaded with diagnostic Nile Blue dye and the chemotherapeutic drug doxorubicin. This nano-PLGA structure was covalently coupled to Barnase. We showed two-stage efficient labeling of HER2-overexpressing cancer cells using the first non-toxic module DARPin9_29-Barstar and the second toxic nano-module PLGA-Barnase. We demonstrated the doxorubicin-induced cytotoxicity of this two-step DDS based on polymer nanoparticles and proteinaceous Barnase-Barstar interface and showed more than 10-fold therapeutic dose reduction versus free doxorubicin. We believe that the developed two-step DDS based on PLGA nano-cargo and protein interface will promote the creation of new-generation cancer treatment strategies.