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

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

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

Properties of PEI-based Polyplex Nanoparticles That Correlate With Their Transfection Efficacy

We have evaluated the key properties of the polyethylenimine (PEI)-polyethylene glycol (PEG)-TAT peptide polyplex nanoparticles including their behavior in cells and compared them with the transfection efficacy (TE) using 11 different cell lines. We found statistically significant positive correlation between TE and the share of 50–75 nm fraction in the whole mixture of nanoparticles estimated with atomic force microscopy. Variations in PEG/PEI and N/P ratios (PEI nitrogen to DNA phosphate ratio) enabled us to find their optimal combinations, which resulted in up to 100% TE for several cell lines. Surfaces of the TE dependence of both PEG/PEI and N/P turned out to be similar in appearance for all investigated cell lines, while maximum TEs were different. We investigated subcellular transport kinetics and unpacking of the polyplex nanoparticles labeled with quantum dots (plasmid DNA) and AlexaFluor647 (block-copolymer part) using Förster Resonance Energy Transfer approach. The results demonstrated clear and statistically significant positive correlation of TE with the cellular uptake rate of the nanoparticles and negative correlation with the rate constant of their unpacking within endo/lysosomal compartments in the living cells