Modular Nanotransporters Delivering Biologically Active Molecules to the Surface of Mitochondria.

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.

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.

Intracellular Degradation of SARS-CoV-2 N-Protein Caused by Modular Nanotransporters Containing Anti-N-Protein Monobody and a Sequence That Recruits the Keap1 E3 Ligase.

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.

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.

Nrf2/Keap1/ARE signaling: Towards specific regulation.

The Nrf2 transcription factor governs the expression of hundreds genes involved in cell defense against oxidative stress, the hallmark of numerous diseases such as neurodegenerative, cardiovascular, some viral pathologies, diabetes and others. The main route for Nrf2 activity regulation is via interactions with the Keap1 protein. Under the normoxia the Keap1 binds the Nrf2 and targets it to the proteasomal degradation, while the Keap1 is regenerated. Upon oxidative stress the interactions between Nrf2 and Keap1 are interrupted and the Nrf2 activates the transcription of the protective genes. Currently, the Nrf2 system activation is considered as a powerful cytoprotective strategy for treatment of different pathologies, which pathogenesis relies on oxidative stress including viral diseases of pivotal importance such as COVID-19. The implementation of this strategy is accomplished mainly through the inactivation of the Keap1 "guardian" function. Two approaches are now developing: the Keap1 modification via electrophilic agents, which leads to the Nrf2 release, and direct interruption of the Nrf2:Keap1 protein-protein interactions (PPI). Because of theirs chemical structure, the Nrf2 electrophilic inducers could non-specifically interact with others cellular proteins leading to undesired effects. Whereas the non-electrophilic inhibitors of the Nrf2:Keap1 PPI could be more specific, thereby widening the therapeutic window.

Low-resolution structures of modular nanotransporters shed light on their functional activity.

Modular nanotransporters (MNTs) are multifunctional chimeric polypeptides for the multistep transport of locally acting cytotoxic agents into the nuclei of cancer target cells. MNTs consist of several polypeptide domains (functional modules) for the recognition of a cell-surface internalizable receptor, pH-dependent endosomal escape and subsequent transport into the nucleus through the nuclear pores. MNTs are a promising means for cancer treatment. As has been shown previously, all of the modules of MNTs retain their functionalities. Despite their importance, there is no structural information available about these chimeric polypeptides, which hampers the creation of new MNT variants. Here, a low-resolution 3D structure of an MNT is presented which was obtained by atomic force microscopy, transmission electron microscopy and small-angle X-ray scattering coupled to size-exclusion chromatography. The data suggest that the MNT can adopt two main conformations, but in both conformations the protein N- and C-termini are distanced and do not influence each other. The change in the MNT conformation during acidification of the medium was also studied. It was shown that the fraction of the elongated conformation increases upon acidification. The results of this work will be useful for the development of MNTs that are suitable for clinical trials and possible therapeutic applications.

Targeted Delivery of 111In Into the Nuclei of EGFR Overexpressing Cells via Modular Nanotransporters With Anti-EGFR Affibody.

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 (MNTN-affibody) effectively delivered the Auger electron emitter 111In 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), MNTN-affibody and MNT with EGF on the C-terminus did not stimulate cancer cell proliferation.

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.

Transcription factors: Time to deliver.

Transcription factors (TFs) are at the center of the broad regulatory network orchestrating gene expression programs that elicit different biological responses. For a long time, TFs have been considered as potent drug targets due to their implications in the pathogenesis of a variety of diseases. At the same time, TFs, located at convergence points of cellular regulatory pathways, are powerful tools providing opportunities both for cell type change and for managing the state of cells. This task formulation requires the TF modulation problem to come to the fore. We review several ways to manage TF activity (small molecules, transfection, nanocarriers, protein-based approaches), analyzing their limitations and the possibilities to overcome them. Delivery of TFs could revolutionize the biomedical field. Whether this forecast comes true will depend on the ability to develop convenient technologies for targeted delivery of TFs.

Preparation, cytotoxicity, and in vivo antitumor efficacy of 111In-labeled modular nanotransporters.

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.

Therapeutic properties of a vector carrying the HSV thymidine kinase and GM-CSF genes and delivered as a complex with a cationic copolymer.

BACKGROUND: Gene-directed enzyme prodrug therapy (GDEPT) represents a technology to improve drug selectivity for cancer cells. It consists of delivery into tumor cells of a suicide gene responsible for in situ conversion of a prodrug into cytotoxic metabolites. Major limitations of GDEPT that hinder its clinical application include inefficient delivery into cancer cells and poor prodrug activation by suicide enzymes. We tried to overcome these constraints through a combination of suicide gene therapy with immunomodulating therapy. Viral vectors dominate in present-day GDEPT clinical trials due to efficient transfection and production of therapeutic genes. However, safety concerns associated with severe immune and inflammatory responses as well as high cost of the production of therapeutic viruses can limit therapeutic use of virus-based therapeutics. We tried to overcome this problem by using a simple nonviral delivery system. METHODS: We studied the antitumor efficacy of a PEI (polyethylenimine)-PEG (polyethylene glycol) copolymer carrying the HSVtk gene combined in one vector with granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA. The system HSVtk-GM-CSF/PEI-PEG was tested in vitro in various mouse and human cell lines, ex vivo and in vivo using mouse models. RESULTS: We showed that the HSVtk-GM-CSF/PEI-PEG system effectively inhibited the growth of transplanted human and mouse tumors, suppressed metastasis and increased animal lifespan. CONCLUSIONS: We demonstrated that appreciable tumor shrinkage and metastasis inhibition could be achieved with a simple and low toxic chemical carrier - a PEI-PEG copolymer. Our data indicate that combined suicide and cytokine gene therapy may provide a powerful approach for the treatment of solid tumors and their metastases.

Microdistribution of MC1R-targeted polyplexes in murine melanoma tumor tissue.

Targeted sodium-iodide symporter (NIS) gene transfer can be considered as a promising approach for diagnostics of specific types of cancer. For this purpose we used targeted polyplexes based on PEI-PEG-MC1SP block-copolymer containing MC1SP-peptide, a ligand specific for melanocortin receptor-1 (MC1R) overexpressed on melanoma cells. Targeted polyplexes demonstrated enhanced NIS gene transfer compared to non-targeted (lacking MC1SP) ones in vitro. Using dorsal skinfold chamber and intravital microscopy we evaluated accumulation and microdistribution of quantum dot-labeled polyplexes in tumor and normal subcutaneous tissues up to 4 h after intravenous injection. Polyplexes demonstrated significantly higher total accumulation in tumor tissue in comparison with subcutaneous ones (control). Targeted and non-targeted polyplexes extravasated and penetrated into the tumor tissue up to 20 µm from the vessel walls. In contrast, in normal subcutaneous tissue polyplexes penetrated not more than 3 µm from the vessel walls with the level of extravasated polyplexes 400-fold less than in tumor. Accumulated polyplexes in tumor tissue caused NIS gene expression. Subsequent (123)I(-) intravenous injection resulted in 6.8 ± 1.1 and 4.5 ± 0.8% ID/g (p < 0.001) iodide accumulation in tumors in the case of targeted and non-targeted polyplexes, respectively, as was shown using SPECT/CT.

Subcellular trafficking and transfection efficacy of polyethylenimine-polyethylene glycol polyplex nanoparticles with a ligand to melanocortin receptor-1.

We have synthesized and investigated properties of new PEI-PEG-based polyplexes containing MC1SP-peptide, a ligand specific for melanocortin receptor-1 (targeted polyplexes), and control polyplexes without this ligand peptide (non-targeted polyplexes). The targeted polyplexes demonstrated receptor-mediated transfection of Cloudman S91 (clone M-3) murine melanoma cells that was more efficient than with the non-targeted ones. Transfection with the targeted polyplexes was inhibited by chlorpromazine, an inhibitor of the clathrin-mediated endocytosis pathway, and, to a lesser extent, by filipin III or nystatin, inhibitors of the lipid-raft endocytosis pathway, whereas transfection with the non-targeted polyplexes was inhibited mainly by nystatin or filipin III. The targeted polyplexes caused significantly higher in vivo transfection of melanoma tumor cells after intratumoral administration compared to the non-targeted control. The targeted polyplexes carrying the HSVtk gene, after ganciclovir administration, more efficiently inhibited melanoma tumor growth and prolonged the lifespan of DBA/2 tumor-bearing mice compared to the non-targeted ones. Packed targeted polyplexes appeared and accumulated in the melanoma cells 6h earlier than the non-targeted ones. The targeted polyplexes enter into the nuclei of the melanoma cells more rapidly than the non-targeted control, and this difference may also be attributed to processes of receptor-mediated endocytosis. We believe that these data may be useful for the optimization of polyplex systems.

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.