Versatile Platform for Nanoparticle Surface Bioengineering Based on SiO₂-Binding Peptide and Proteinaceous Barnase*Barstar Interface

Abstract

Nanoparticle surface engineering can change its chemical identity to enable surface coupling with functional biomolecules. However, common surface coupling methods such as physical adsorption or chemical conjugation often suffer from the low coupling yield, poorly controllable orientation of biomolecules, and steric hindrance during target binding. These issues limit the application scope of nanostructures for theranostics and personalized medicine. To address these shortfalls, we developed a rapid and versatile method of nanoparticle biomodification. The method is based on a SiO-binding peptide that binds to the nanoparticle surface and a protein adaptor system, Barnase*Barstar protein pair, serving as a "molecular glue" between the peptide and the attached biomolecule. The biomodification procedure shortens to several minutes, preserves the orientation and functions of biomolecules, and enables control over the number and ratio of attached molecules. The capabilities of the proposed biomodification platform were demonstrated by coupling different types of nanoparticles with DARPin9.29 and 4D5scFv-molecules that recognize the human epidermal growth factor receptor 2 (HER2/neu) oncomarker-and by subsequent highly selective immunotargeting of the modified nanoparticles to different HER2/neu-overexpressing cancer cells in one-step or two-step (by pretargeting with HER2/neu-recognizing molecule) modes. The method preserved the biological activity of the DARPin9.29 molecules attached to a nanoparticle, whereas the state-of-the-art carbodiimide 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/ N-hydroxysulfosuccinimide method of conjugation led to a complete loss of the functional activity of the DARPin9.29 nanoparticle-protein complex. Moreover, the method allowed surface design of nanoparticles that selectively interacted with antigens in complex biological fluids, such as whole blood. The demonstrated capabilities show this method to be a promising alternative to commonly used chemical conjugation techniques in nanobiotechnology, theranostics, and clinical applications.

Advanced in developmental organic and inorganic nanomaterial: a review

“…This approach allows the robust self-assembly of multifunctional superstructures consisting of different singlefunction particles such as labels, carriers, recognition, and targeting agents, including antibodies. This bioengineering method employs two unique proteins from Bacillus amyloliquefaciens, barnase and barstar, to rapidly bring together the structural components directly in solution [137]. The properties of the superstructures can be designed on demand by linking different agents of various sizes and chemical nature, as a function of the specific purpose.…”

Elucidating the Binding Mechanism of a Novel Silica-Binding Peptide

“…In this platform technology, LPG acts as an anchor for the rapid and oriented immobilization of antibodies onto silica surfaces, thereby ensuring the functional display of conjugated antibodies without the need for complex conjugation chemistry. Although this linker technology has been widely used in several biotechnology and biomedical applications [14][15][16][17], there is still a lack of understanding about the interaction mechanism which facilitates the binding of this SBP to the silica surface.…”

A Novel Approach to Anticancer Therapy: Molecular Modules Based on the Barnase:Barstar Pair for Targeted Delivery of HSP70 to Tumor Cells

“…The expression level of the tumor-associated HER- 2/neu antigen on the surface of the cultured cell lines was tested using fluorescence microscopy and the previously designed recombinant constructs for visualizing cancer cells expressing the HER-2/neu antigen (anti-HER2/neu mini-antibody-barstar●GFP-barnase) [36, 37]. It has been demonstrated that the cell culture samples being used are characterized by a sufficiently high level of expression of the HER-2/neu surface antigen (the data are not shown).…”