This study evaluates the potential utility of a modular nanotransporter (MNT) for enhancing the nuclear delivery and cytotoxicity of the Auger electron emitter 125I in cancer cells that overexpress the epidermal growth factor receptor (EGFR).

Purpose: Modular nanotransporters (MNTs) are a polyfunctional platform designed to achieve receptor-specific delivery of short-range therapeutics into the cell nucleus by receptor-mediated endocytosis, endosome escape, and targeted nuclear transport. This study evaluated the potential utility of the MNT platform in tandem with Auger electron emitting ¹¹¹In for cancer therapy. Methods: Three MNTs developed to target either melanocortin receptor-1 (MC1R), folate receptor (FR), or epidermal growth factor receptor (EGFR) that are overexpressed on cancer cells were modified with p-SCN-Bn-NOTA and then labeled with ¹¹¹In in high specific activity. Cytotoxicity of the ¹¹¹In-labeled MNTs was evaluated on cancer cell lines bearing the appropriate receptor target (FR: HeLa, SK-OV-3; EGFR: A431, U87MG.wtEGFR; and MC1R: B16-F1). In vivo micro-single-photon emission computed tomography/computed tomography imaging and antitumor efficacy studies were performed with intratumoral injection of MC1R-targeted ¹¹¹In-labeled MNT in B16-F1 melanoma tumor-bearing mice. Results: The three NOTA-MNT conjugates were labeled with a specific activity of 2.7 GBq/mg with nearly 100% yield, allowing use without subsequent purification. The cytotoxicity of ¹¹¹In delivered by these MNTs was greatly enhanced on receptor-expressing cancer cells compared with ¹¹¹In nontargeted control. In mice with B16-F1 tumors, prolonged retention of ¹¹¹In by serial imaging and significant tumor growth delay (82% growth inhibition) were found. Conclusion: The specific in vitro cytotoxicity, prolonged tumor retention, and therapeutic efficacy of MC1R-targeted ¹¹¹In-NOTA–MNT suggest that this Auger electron emitting conjugate warrants further evaluation as a locally delivered radiotherapeutic, such as for ocular melanoma brachytherapy. Moreover, the high cytotoxicity observed with FR- and EGFR-targeted ¹¹¹In-NOTA–MNT suggests further applications of the MNT delivery strategy should be explored.

To compare the effectiveness of various bioactive agents reversibly acting within a cell on a target intracellular macromolecule, it is necessary to assess effective cytoplasmic concentrations of the delivered bioactive agents. In this work, based on a simple equilibrium model and the cellular thermal shift assay (CETSA), an approach is proposed to assess effective concentrations of both a delivered bioactive agent and a target protein. This approach was tested by evaluating the average concentrations of nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated-protein 1 (Keap1) proteins in the cytoplasm for five different cell lines (Hepa1, MEF, RAW264.7, 3LL, and AML12) and comparing the results with known literature data. The proposed approach makes it possible to analyze both binary interactions and ternary competition systems; thus, it can have a wide application for the analysis of protein–protein or molecule–protein interactions in the cell. The concentrations of Nrf2 and Keap1 in the cell can be useful not only in analyzing the conditions for the activation of the Nrf2 system, but also for comparing the effectiveness of various drug delivery systems, where the delivered molecule is able to interact with Keap1.

Treatment of various diseases, in particular cancer, usually requires the targeting of biologically active molecules at a selected subcellular compartment. We modified our previously developed modular nanotransporters (MNTs) for targeting mitochondria. The new MNTs are capable of binding to the protein predominantly localized on the outer mitochondrial membrane, Keap1. These MNTs possessing antiKeap1 monobody co-localize with mitochondria upon addition to the cells. They efficiently interact with Keap1 both in solution and within living cells. A conjugate of the MNT with a photosensitizer, chlorin e₆, demonstrated significantly higher photocytotoxicity than chlorin e₆ alone. We assume that MNTs of this kind can improve efficiency of therapeutic photosensitizers and radionuclides emitting short-range particles.

The proper viral assembly relies on both nucleic acids and structural viral proteins. Thus a biologically active agent that provides the degradation of one of these key proteins and/or destroys the viral factory could suppress viral replication efficiently. The nucleocapsid protein (N-protein) is a key protein for the SARS-CoV-2 virus. As a bioactive agent, we offer a modular nanotransporter (MNT) developed by us, which, in addition to an antibody mimetic to the N-protein, contains an amino acid sequence for the attraction of the Keap1 E3 ubiquitin ligase. This should lead to the subsequent degradation of the N-protein. We have shown that the functional properties of modules within the MNT permit its internalization into target cells, endosome escape into the cytosol, and binding to the N-protein. Using flow cytometry and western blotting, we demonstrated significant degradation of N-protein when A549 and A431 cells transfected with a plasmid coding for N-protein were incubated with the developed MNTs. The proposed MNTs open up a new approach for the treatment of viral diseases.