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Tatiana A Slastnikova,1 Andrey A Rosenkranz,1,2 Yuri V Khramtsov,1 Tatiana S Karyagina,1 Sergey A Ovechko,2 Alexander S Sobolev1,2 1Laboratory of Molecular Genetics of Intracellular Transport, Institute of Gene Biology, Russian Academy of Sciences, 2Department of Biophysics, Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia Purpose: Modular nanotransporters (MNTs) are artificial multifunctional systems designed to facilitate receptor-specific transport from the cell surface into the cell nucleus through inclusion of polypeptide domains for accomplishing receptor binding and internalization, as well as sequential endosomal escape and nuclear translocation. The objective of this study was to develop a new MNT targeted at folate receptors (FRs) for precise delivery of therapeutic cargo to the nuclei of FR-positive cells and to evaluate its potential, particularly for delivery of therapeutic agents (eg, the Auger electron emitter 111In) into the nuclei of target cancer cells.Methods: A FR-targeted MNT was developed by site-specific derivatization of ligand-free MNT with maleimide-polyethylene glycol-folic acid. The ability of FR-targeted MNT to accumulate in target FR-expressing cells was evaluated using flow cytometry, and intracellular localization of this MNT was assessed using confocal laser scanning microscopy of cells. The cytotoxicity of the 111In-labeled FR-targeted MNT was evaluated on HeLa and U87MG cancer cell lines expressing FR. In vivo micro-single-photon emission computed tomography/CT imaging and antitumor efficacy studies were performed with intratumoral injection of 111In-labeled FR-targeted MNT in HeLa xenograft-bearing mice.Results: The resulting FR-targeted MNT accumulated in FR-positive HeLa cancer cell lines specifically and demonstrated the ability to reach its target destination – the cell nuclei. 111In-labeled FR-targeted MNT demonstrated efficient and specific FR-positive cancer cell eradication. A HeLa xenograft in vivo model revealed prolonged retention of 111In delivered by FR-targeted MNT and significant tumor growth delay (up to 80% growth inhibition).Conclusion: The FR-targeted MNT met expectations of its ability to deliver active cargo into the nuclei of target FR-positive cells efficiently and specifically. As a result of this finding the new FR-targeted MNT approach warrants broad evaluation. Keywords: nuclear delivery, folic acid, cancer, radionuclide therapy, indium-111

Since cell nucleus is one of the most vulnerable compartments, the maximum therapeutic effect from a variety of locally acting agents, such as photosensitizers, alfa-emitters, Auger electron emitters, will be expected when they get there. Therefore, the targeted delivery of these agents into the nuclei of target tumor cells is necessary for their anticancer effects and minimization of side effects. Modular nanotransporters (MNT) are artificial polypeptides comprising several predefined modules that recognize target cell, launching their subsequent internalization, escape from endosomes, and transport the drug load to the nucleus. This technology significantly enhances the cytotoxicity of locally acting drugs in vitro and in vivo . Epidermal growth factor receptors (EGFR) are useful molecular targets as they are overexpressed in glioblastoma, head-and-neck cancer, bladder cancer, and other malignancies. Here, we examined the possibility of using internalizable anti-EGFR affibody as an EGFR-targeting MNT module for drug transport into the cancer cell nuclei. It binds to both murine and human EGFR facilitating preclinical studies. We showed that MNT with affibody on the N-terminus (MNT N-affibody ) effectively delivered the Auger electron emitter 111 In to target cell nuclei and had pronounced cytotoxic efficacy against EGFR-overexpressing human A431 epidermoid carcinoma cells. Using EGFR-expressing human adenocarcinoma MCF-7 cells, we demonstrated that in contrast to MNT with N-terminal epidermal growth factor (EGF), MNT N-affibody and MNT with EGF on the C-terminus did not stimulate cancer cell proliferation., (Copyright © 2020 Karyagina, Ulasov, Slastnikova, Rosenkranz, Lupanova, Khramtsov, Georgiev and Sobolev.)

New recombinant carriers-modular nanotransporters (MNTs)-with N-terminal ligand module to the epidermal growth factor receptor (EGFR) were developed and characterized. Human epidermal growth factor (hEGF) and antibody-like protein Z 1907 were used as a ligand module. We demonstrated that MNTs are able to internalize in a receptor-specific manner into the target cancer cells and to accumulate in the target cell nuclei. Conjugation of MNTs with the Auger electron emitter 111 In significantly enhanced the cytotoxic effect of 111 In on the target cells. It was found that the transfer of EGF from the C-terminus to the N-terminus of the MNT enhanced the proliferation of target cells, whereas the use of Z 1907 did not have a similar effect.

A new modular nanotransporter (MNT) for the delivery of anticancer agents into the nuclei of cells with folate receptor overexpression was created. An effective method for acceding labeling of this MNT with Auger electron emitter 111 In has been developed. A significant therapeutic effect was observed after a single intratumoral injection of the new 111 In-labeled MNT to mice grafted with human cervical carcinoma characterized by folate receptor overexpression.