Modular DNA nanopore (NP) for targeted cell killing

Abstract

Cancer treatment strategies often rely on combination therapies that can be aggressive, time-consuming, and present severe side effects. To overcome these challenges, we have developed a modular therapeutic platform for targeted cell killing using self-assembling DNA nanopores (NPs). This study focuses on designing 6-helix bundle NP, featuring hydrophobic moieties that enable membrane insertion, leading to uncontrolled ion flow and ultimately to cell death. These NPs are conjugated site-specifically to rituximab, a CD20-targeting monoclonal antibody, using a dibromopyridazinedione-linker which rebridges interchain disulphides. This conjugation results in a precise 4:1 NP-to-antibody ratio, ensuring targeted delivery and cytotoxicity in B lymphocyte cells.
The NPs were synthesised in a one-step assembly process and verified using electromobility shift assays (EMSA). Successful conjugation was demonstrated through SDS-PAGE, and flow cytometry confirmed the preservation of the antibody's specificity to the CD20 receptor. Furthermore, the stability of the construct was verified in serum over a three-week period at 37°C. In co-culture with a malignant B-cell line (Ramos), the cholesterol-modified NPs exhibited a 20% increase in cell death compared to untreated controls after 48 hours.
This work describes the development of a stable and specific antibody-NP conjugate with defined antibody-drug ratios. The findings elucidate the potential of membrane-spanning NPs as a novel therapeutic platform for cancer treatment, with implications for future applications in various types of cancer.

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Identifiers

ORCID for Eugen Stulz: orcid.org/0000-0002-5302-2276

ORCID for Sean Lim: orcid.org/0000-0002-2768-4858

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