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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

Tatiana A Slastnikova1,2, Andrey A Rosenkranz1,2, Pavel V Gulak1, Raymond M Schiffelers3, Tatiana N Lupanova1,4, Yuri V Khramtsov1, Michael R Zalutsky5, Alexander S Sobolev1,21Laboratory of Molecular Genetics of Intracellular Transport, Institute of Gene Biology, Moscow, Russia; 2Department of Biophysics, Biological Faculty, Moscow State University, Vorobyevy Gory, Moscow, Russia; 3Laboratory for Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, the Netherlands; 4Department of Bioengineering, Biological Faculty, Moscow State University, Vorobyevy Gory, Moscow, Russia; 5Department of Radiology, Duke University Medical Center, Durham, NC, USABackground: Modular nanotransporters (MNT) are recombinant multifunctional polypeptides created to exploit a cascade of cellular processes, initiated with membrane receptor recognition to deliver selective short-range and highly cytotoxic therapeutics to the cell nucleus. This research was designed for in vivo concept testing for this drug delivery platform using two modular nanotransporters, one targeted to the α-melanocyte-stimulating hormone (αMSH) receptor overexpressed on melanoma cells and the other to the epidermal growth factor (EGF) receptor overexpressed on several cancers, including glioblastoma, and head-and-neck and breast carcinoma cells.Methods: In vivo targeting of the modular nanotransporter was determined by immunofluorescence confocal laser scanning microscopy and by accumulation of 125I-labeled modular nanotransporters. The in vivo therapeutic effects of the modular nanotransporters were assessed by photodynamic therapy studies, given that the cytotoxicity of photosensitizers is critically dependent on their delivery to the cell nucleus.Results: Immunohistochemical analyses of tumor and neighboring normal tissues of mice injected with multifunctional nanotransporters demonstrated preferential uptake in tumor tissue, particularly in cell nuclei. With 125I-labeled MNT{αMSH}, optimal tumor:muscle and tumor:skin ratios of 8:1 and 9.8:1, respectively, were observed 3 hours after injection in B16-F1 melanoma-bearing mice. Treatment with bacteriochlorin p-MNT{αMSH} yielded 89%–98% tumor growth inhibition and a two-fold increase in survival for mice with B16-F1 and Cloudman S91 melanomas. Likewise, treatment of A431 human epidermoid carcinoma-bearing mice with chlorin e6- MNT{EGF} resulted in 94% tumor growth inhibition compared with free chlorin e6, with 75% of animals surviving at 3 months compared with 0% and 20% for untreated and free chlorin e6-treated groups, respectively.Conclusion: The multifunctional nanotransporter approach provides a new in vivo functional platform for drug development that could, in principle, be applicable to any combination of cell surface receptor and agent (photosensitizers, oligonucleotides, radionuclides) requiring nuclear delivery to achieve maximum effectiveness.Keywords: drug delivery, nanobiotechnology, nanomedicine, cancer therapy, photosensitizers, multifunctional nanotransporter

In the previously created modular nanotransporter (MNT) capable of delivering a monobody to Keap1 into the cytosol, the endosomolytic module, translocation domain of diphtheria toxin (DTox), was replaced by the endosomolytic peptide GALA3. It was found that this substitution more than doubles the lifetime of MNT in the blood. Using confocal microscopy, it was shown that MNT with GALA3 was internalized into AML12 cells mainly due to binding to the epidermal growth factor receptor, and is also able to exit from endosomes into the cytosol. Using cellular thermal shift assay, it was shown that MNT with GALA3 and MNT with DTox are equally effective in disrupting the formation of the Nrf2 complex with Keap1, which led to similar protection of AML12 cells from the action of hydrogen peroxide. The obtained results allow not only optimizing the systemic use of MNT, but can also serve as a basis for creating agents aimed at treating diseases associated with oxidative stress., Competing Interests: ETHICS APPROVAL AND CONSENT TO PARTICIPATE: Animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals ( http://oacu.od.nih.gov/regs/ index.htm ). Animal protocols were approved by the Bioethics Committee of the Institute of Gene Biology of the Russian Academy of Sciences, Moscow, Russia (Approval ID: no. 11, March 15, 2021). CONFLICT OF INTEREST: The authors of this work declare that they have no conflicts of interest., (© 2025. Pleiades Publishing, Ltd.)

Background/Objectives: The study of oxidative stress in cells and ways to prevent it attract increasing attention. Antioxidant defense of cells can be activated by releasing the transcription factor Nrf2 from a complex with Keap1, its inhibitor protein. The aim of the work was to study the effect of the modular nanotransporter (MNT) carrying an R1 anti-Keap1 monobody (MNT R1 ) on cell homeostasis. Methods: The murine hepatocyte AML12 cells were used for the study. The interaction of fluorescently labeled MNT R1 with Keap1 fused to hrGFP was studied using the Fluorescence-Lifetime Imaging Microscopy-Förster Resonance Energy Transfer (FLIM-FRET) technique on living AML12 cells transfected with the Keap1-hrGFP gene. The release of Nrf2 from the complex with Keap1 and its levels in the cytoplasm and nuclei of the AML12 cells were examined using a cellular thermal shift assay (CETSA) and confocal laser scanning microscopy, respectively. The effect of MNT on the formation of reactive oxygen species was studied by flow cytometry using 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate. Results: MNT R1 is able to interact with Keap1 in the cytoplasm, leading to the release of Nrf2 from the complex with Keap1 and a rapid rise in Nrf2 levels both in the cytoplasm and nuclei, ultimately causing protection of cells from the action of hydrogen peroxide. The possibility of cleavage of the monobody in endosomes leads to an increase in the observed effects. Conclusions: These findings open up a new approach to specifically modulating the interaction of intracellular proteins, as demonstrated by the example of the Keap1-Nrf2 system.

Modular nanotransporters (MNTs) are drug delivery systems for targeted cancer treatment. As MNTs are composed of several modules, they offer the advantage of high specificity and biocompatibility in delivering drugs to the target compartment of cancer cells. The large carrier module brings together functioning MNT modules and serves as a platform for drug attachment. The development of smaller-sized MNTs via truncation of the carrier module appears advantageous in facilitating tissue penetration. In this study, two new MNTs with a truncated carrier module containing either an N-terminal (MNT N ) or a C-terminal (MNT C ) part were developed by genetic engineering. Both new MNTs demonstrated a high affinity for target receptors, as revealed by fluorescent-labeled ligand-competitive binding. The liposome leakage assay proved the endosomolytic activity of MNTs. Binding to the importin heterodimer of each truncated MNT was revealed by a thermophoresis assay, while only MNT N possessed binding to Keap1. Finally, the photodynamic efficacy of the photosensitizer attached to MNT N was significantly higher than when attached to either MNT C or the original MNTs. Thus, this work reveals that MNT's carrier module can be truncated without losing MNT functionality, favoring the N-terminal part of the carrier module due to its ability to bind Keap1.

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.

Two eukaryotic cell lines, A549 and A431, with stable expression of the nucleocapsid protein (N-protein) of the SARS-CoV-2 virus fused with the red fluorescent protein mRuby3 were obtained. Using microscopy, the volumes of the cytoplasm and nucleus were determined for these cells. Using quantitative immunoblotting techniques, the concentrations of the N-mRuby3 fusion protein in their cytoplasm were assessed. They were 19 and 9 μM for A549 and A431 cells, respectively. Using these concentrations, the initial rate of N-protein degradation in the studied cells was estimated from the decrease in cell fluorescence. In A549 and A431 cells, it was the same (84 nM per hour). The approach of quantitatively describing the degradation process can be applied to analyze the effectiveness of a wide class of antiviral drugs that cause degradation of viral proteins., (© 2023. Pleiades Publishing, Ltd.)

Modular nanotransporters (MNTs) containing an antibody-like molecule, monobody, to the N‑protein of the SARS-CoV-2 virus, as well as an amino acid sequence that recruits the Keap1 E3 ligase (E3BP) were created. This MNT also included a site for cleavage of the E3BP monobody from the MNT in acidic endocytic compartments. It was shown that this cleavage by the endosomal protease cathepsin B leads to a 2.7-fold increase in the affinity of the E3BP monobody for the N-protein. Using A549 cells with transient expression of the N-protein fused with the fluorescent protein mRuby3, it was shown that incubation with MNT leads to a significant decrease in mRuby3 fluorescence. It is assumed that the developed MNTs can serve as a basis for the creation of new antiviral drugs against the SARS-CoV-2 virus., (© 2023. Pleiades Publishing, Ltd.)

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 e 6 , demonstrated significantly higher photocytotoxicity than chlorin e 6 alone. We assume that MNTs of this kind can improve efficiency of therapeutic photosensitizers and radionuclides emitting short-range particles.

On the basis of literature data, an antibody-like molecule, monobody, was selected that is capable of interacting with the nucleocapsid protein (N protein) of the SARS-CoV-2 virus with a high affinity (dissociation constant 6.7 nM). We have previously developed modular nanotransporters (MNTs) to deliver various molecules to a selected compartment of target cells. In this work, a monobody to the N protein of the SARS-CoV-2 virus was inserted in the MNT using genetic engineering methods. In this MNT, a site for the cleavage of the monobody from the MNT in endosomes was also inserted. It was shown by thermophoresis that the cleavage of this monobody from the MNT by the endosomal protease cathepsin B leads to a 12-fold increase in the affinity of the monobody for the N protein. Cellular thermal shift assay showed the ability of the obtained MNT to interact with the N protein in A431 cells transfected with the SARS-CoV-2 N protein fused to the mRuby3 fluorescent protein., (© 2023. Pleiades Publishing, Ltd.)

On the basis of known published data, six peptide sequences were selected that are potentially capable of being rapidly cleaved by the endosomal protease cathepsin B. For comparison, the cleavage of common linker sequences, polyglycine and polyglycine-serine, by cathepsin B was also studied. Different ends of these peptides were labeled with sulfoCyanine3 and sulfoCyanine5 fluorescent dyes, between which Förster resonant energy transfer (FRET) is possible. The kinetics of cleavage of peptides by cathepsin B was studied on a multimodal plate reader by FRET signal reduction. FKFL and FRRG cleavage sites have been shown to be the most suitable for potential use in various drug delivery systems. These sites are much more efficiently cleaved under slightly acidic conditions of endosomes than at neutral extracellular pH., (© 2023. Pleiades Publishing, Ltd.)

A modular nanotransporter (MNT) carrying the sequence of an antibody-like molecule, anti-c-Myc nanobody, was synthesized and characterized. It was demonstrated that the created MNT is able to interact with the target protein, c-Myc oncogene, with a dissociation constant of 46 ± 14 nM, internalize into target cells, change Myc-dependent expression, and exert an antiproliferative effect., (© 2023. Pleiades Publishing, Ltd.)

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.

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.

Based on previous studies, two antibody-like molecules, monobodies, capable of high-affinity interaction with the SARS-CoV-2 nucleocapsid protein (dissociation constant of tens of nM) were selected. For delivery to target cells, genetically engineered constructs containing monobody and TAT peptide, placed either at the N- or C-terminus of the resulting polypeptide, were produced and expressed in E. coli. The construct with the highest affinity to the SARS-CoV-2 nucleocapsid protein was revealed with the use of thermophoresis technique. Cellular thermal shift assay demonstrated the ability of this construct to interact with the nucleocapsid protein within HEK293T cells transfected with the SARS-CoV-2 nucleocapsid protein fused to the mRuby3 fluorescent protein. Replacement of TAT peptide to S10 shuttle peptide, containing endosomolytic peptide, significantly improved the penetration of the construct into the target cells., (© 2022. Pleiades Publishing, Ltd.)

Seven amino acid sequences of antibody mimetics molecules, monobodies, capable of interacting with the nucleocapsid protein of the SARS-CoV virus, were taken from the literature. Nucleotide sequences of monobody genes were obtained by gene synthesis, which were expressed in E. coli and isolated using Ni-NTA chromatography. It was shown by thermophoresis that three of the seven selected antibody-like molecules can interact with high affinity (dissociation constant of tens of nM) with the nucleocapsid protein of the SARS-CoV-2 virus. For the remaining four monobodies, only low affinity binding with a dissociation constant of several μM was found., (© 2022. Pleiades Publishing, Ltd.)

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.

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.)

It was found that the use of a new strain-producer Escherichia coli, expressing the heme receptor ChuA, enables obtaining a hemin-containing modular nanotransporter (MNT) for drug delivery into the nuclei of target cells. The hemin-containing MNT becomes stabilized, which leads to an increase in its thermal stability and prevents aggregation of this protein.

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.

Gamma-ray emitting 111 In, 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 111 In attached MNTs to kill human bladder cancer cells overexpressing epidermal growth factor receptor (EGFR). The cytotoxicity of 111 In delivered by the EGFR-targeted MNT ( 111 In-MNT) was greatly enhanced on EJ-, HT-1376-, and 5637-expressing EGFR bladder cancer cell lines compared with 111 In non-targeted control. In vivo microSPECT/CT imaging and antitumor efficacy studies revealed prolonged intratumoral retention of 111 In-MNT with t ½ = 4.1 ± 0.5 days as well as significant dose-dependent tumor growth delay (up to 90% growth inhibition) after local infusion of 111 In-MNT in EJ xenograft-bearing mice.

We studied the possibility of optimizing modular nanotransporters (MNTs) for the intracellular delivery of antibody fragments into the nuclei of cells of a specified type. Basic MNT with a reduced size retaining the desired functions was obtained, and the principal possibility of obtaining an MNT carrying an antibody fragment by microbiological synthesis was shown.

Modular nanotransporter (MNT) with C-terminal fragment of the p21 protein was synthesized and characterized, and its effect on DNA lesions was studied. This p21 fragment in MNT can significantly inhibit DNA repair in A431 human carcinoma cells after bleomycin treatment.

The distribution of modular nanotransporters (MNTs) that are used to deliver drugs into melanoma cell nuclei after their intravenous administration into mice with Cloudman S91 melanoma was studied. The modification of MNTs with polyethylene glycol (PEG) of different length and their administration during the treatment with docetaxel, nitroglycerin, and excess of nonspecific MNTs leads to an improved accumulation of MNTs in the tummor. Among the variants studied, the MNT with attached PEG with M r 40 kDa exhibited the best properties.

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.

The albumin-binding domain (ABD) with a site for its cleavage by tumor proteases was inserted in the structure of modular nanotransporters (MNTs), chimeric proteins for the delivery of anticancer drugs into the nuclei of cancer cells. The effectiveness of this cleavage was tested in both variants of created construct: "pure" ABD-MNT and the complex with albumin. The introduction of the ABD module into MNTs had no effect on the binding of MNT with receptors on the surface of the target cancer cells and on the preferential accumulation of MNTs in the nuclei of these cells. The use of thermophoresis allowed us to determine the equilibrium dissociation constants of the ABD-MNT complex with bovine and human serum albumins.

Utilizing nanoparticles made of cationic polymers as gene carriers is a promising approach in cancer gene therapy. One of the major requirements for successful gene delivery is DNA translocation into the nuclei of cancer cells. Nuclear envelope breakdown during mitosis has been considered as the most favorable opportunity for DNA translocation to the nucleus. Here, we aimed to study the influence of mitosis on polyplex-mediated gene delivery using time-lapse microscopy as a safe and accurate tool. Studying of reporter gene expression on a single cell level enabled to confirm the significance of cell division for gene delivery to Cloudman S91 melanoma cells, in spite of minor mitosis-independent transfection, and to discover some important details of polyplex delivery process. We have found that cell division can result in only one post-mitotic transfected cell of the two that could indicate non-uniform distribution of a very small number of intact plasmid DNA between daughter cells. According to our data, the shorter the time interval from polyplex addition to cell division, the longer time is required for the start of reporter gene expression after completed cytokinesis that presumably is a result of gradual polyplex dissociation in cell. Most probably, the development of new gene delivery carriers which would combine the strong ability to protect DNA and ability to release it during mitosis can provide an increase in intact DNA molecule number per cell, uniform DNA distribution between two post-mitotic cells, and fast reporter gene expression resulting in superior transfection of proliferating cells., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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.

Targeted drug delivery into the cell compartment that is the most vulnerable to effects of the corresponding drug is a challenging problem, and its successful solution can significantly increase the efficiency and reduce side effects of the delivered therapeutic agents. To accomplish this one can utilize natural mechanisms of cellular specific uptake of macromolecules by receptor-mediated endocytosis and intracellular transport between cellular compartments. A transporting construction combining the components responsible for different steps of intracellular transport is promising for creating multifunctional modular constructions capable of delivering the necessary therapeutic agent into a given compartment of type-specified cells. This review focuses on intracellular transport peculiarities along with approaches for designing such transporting constructions for new, more effective, and safer strategies for treatment of various diseases.

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

Modular/chimeric recombinant drugs or drug transporters usually contain a special translocation domain from bacterial toxins, e.g. diphtheria toxin, as a module enabling escape of the chimeric molecules from acidifying endosomes. This approach is limited by the shortage in the knowledge about the precise molecular mechanisms of translocation of diphtheria toxin and the toxin-based chimera across the endosomal membrane and its release into the cytosol. We present experimental data with the modular recombinant drug transporters (MRTs), containing the translocation domain of diphtheria toxin, developed by us earlier, demonstrating that the MRTs interact with lipid membranes (liposomes, free-standing and supported bilayer lipid membranes) and produce defects in them at endosomal pH's which are sufficient for escape of macromolecules: - MRTs induced leakage of calcein-loaded liposomes pH 3-6.5. Large fluctuating conductance states of 2-5 nS are formed in membranes at pH 5.5. Atomic force microscopy revealed two different types of defects in the supported lipid bilayers at pH 5.5: 1) giant pores (50-200 nm diameters) and 2) small depressions/holes (30-50 nm) produced by the MRTs which are inserted into the membranes thus forming the structures described above. The last was shown with the use of cantilever tips modified with anti-MRT antibodies.

A major problem in the treatment of cancer is the specific targeting of drugs to these abnormal cells. Ideally, such a drug should act over short distances to minimize damage to healthy cells and target subcellular compartments that have the highest sensitivity to the drug. We describe the novel approach of using modular recombinant transporters to target photosensitizers to the nucleus, where their action is most pronounced, of cancer cells overexpressing ErbB1 receptors. We have produced a new generation of the transporters consisting of (a) epidermal growth factor as the internalizable ligand module to ErbB1 receptors, (b) the optimized nuclear localization sequence of SV40 large T-antigen, (c) a translocation domain of diphtheria toxin as an endosomolytic module, and (d) the Escherichia coli hemoglobin-like protein HMP as a carrier module. The modules retained their functions within the transporter chimera: they showed high-affinity interactions with ErbB1 receptors and alpha/beta-importin dimers and formed holes in lipid bilayers at endosomal pH. A photosensitizer conjugated with the transporter produced singlet oxygen and (*)OH radicals similar to the free photosensitizer. Photosensitizers-transporter conjugates have >3,000 times greater efficacy than free photosensitizers for target cells and were not photocytotoxic at these concentrations for cells expressing a few ErbB1 receptors per cell, in contrast to free photosensitizers. The different modules of the transporters, which are highly expressed and easily purified to retain full activity of each of the modules, are interchangeable, meaning that they can be tailored for particular applications.