Antitumor Activity of Auger Electron Emitter 111In Delivered by Modular Nanotransporter for Treatment of Bladder Cancer With EGFR Overexpression

Gamma-ray emitting 111In, which is extensively used for imaging, is also a source of short-range Auger electrons (AE). While exhibiting negligible effect outside cells, these AE become highly toxic near DNA within the cell nucleus. Therefore, these radionuclides can be used as a therapeutic anticancer agent if delivered precisely into the nuclei of tumor target cells. Modular nanotransporters (MNTs) designed to provide receptor-targeted delivery of short-range therapeutic cargoes into the nuclei of target cells are perspective candidates for specific intracellular delivery of AE emitters. The objective of this study was to evaluate the in vitro and in vivo efficacy of 111In attached MNTs to kill human bladder cancer cells overexpressing epidermal growth factor receptor (EGFR). The cytotoxicity of 111In delivered by the EGFR-targeted MNT (111In-MNT) was greatly enhanced on EJ-, HT-1376-, and 5637-expressing EGFR bladder cancer cell lines compared with 111In non-targeted control. In vivo microSPECT/CT imaging and antitumor efficacy studies revealed prolonged intratumoral retention of 111In-MNT with t½ = 4.1 ± 0.5 days as well as significant dose-dependent tumor growth delay (up to 90% growth inhibition) after local infusion of 111In-MNT in EJ xenograft-bearing mice

Studies on Chromatin. Free DNA in Sheared Chromatin

Chromatin which has been hydrodynamically sheared in a low‐ionic‐strength buffer lacking divalent cations (I = 0.005 M) contains a heterogeneous set of deoxyribonucleoprotein particles but no molecules of free DNA. The main finding is that a transference of sheared chromatin to 1–2 mM MgCl₂ or to 0.1–0.2 M NaCl results in appearance of completely free DNA molecules. A salt‐induced rearrangement of DNA‐bound histories but not a partial loss of them is responsible for the observed phenomenon. Formation of free DNA molecules is accompanied by aggregation of the majority of remaining deoxyribonucleoprotein particles. The percentage of free DNA molecules in the chromatin which was sheared to an average DNA length of about 400 base pairs is increased from zero in the initial sample to 8–9% in 1 mM MgCl₂ and further to ∼ 25% of the total DNA in 0.15 M NaCl, 2 mM MgCl₂. Free DNA molecules in the sheared chromatin are observed not only upon isopycnic banding of formaldehyde‐fixed deoxyribonucleoproteins in CsCl gradients but also in non‐ionic metrizamide gradients with either fixed or unfixed deoxyribonucleoprotein samples. The process of free DNA formation is a reversible one; its direction and the equilibrium state depend in particular on the ionic conditions of the medium

An Approach to Evaluate the Effective Cytoplasmic Concentration of Bioactive Agents Interacting with a Selected Intracellular Target Protein

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

Mouse Syngeneic Melanoma Model with Human Epidermal Growth Factor Receptor Expression

The development of epidermal growth factor receptor (EGFR)-targeting agents for the treatment of malignant melanoma requires cheap and easy animal tumor models for high-throughput in vivo screening. Thus, the aim of this study was to develop mouse syngeneic melanoma model that expresses human EGFR. Cloudman S91 clone M3 mouse melanoma cells were transduced with lentiviral particles carrying the human EGFR gene followed by a multistep selection process. The resulting M3-EGFR has been tested for EGFR expression and functionality in vitro and in vivo. Radioligand assay confirmed the presence of 13,900 ± 1500 EGF binding sites per cell at a dissociation constant of 5.3 ± 1.4 nM. M3-EGFR demonstrated the ability to bind and internalize specifically and provide the anticipated intracellular nuclear import of three different EGFR-targeted modular nanotransporters designed for specific anti-cancer drug delivery. Introduction of the human EGFR gene did not alter the tumorigenicity of the offspring M3-EGFR cells in host immunocompetent DBA/2J mice. Preservation of the expression of EGFR in vivo was confirmed by immunohistochemistry. To sum up, we successfully developed the first mouse syngeneic melanoma model with preserved in vivo expression of human EGFR

An Approach to Evaluate the Effective Cytoplasmic Concentration of Bioactive Agents Interacting with a Selected Intracellular Target Protein

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