Yuri V. Khramtsov

Targeting Cancer Cells by Novel Engineered Modular Transporters

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.

Modular nanotransporters: a multipurpose in vivo working platform for targeted drug delivery

Background 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 immuno-fluorescence 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.

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.

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

BackgroundGene-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.MethodsWe 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.ResultsWe 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.ConclusionsWe 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.

Modular drug transporters with diphtheria toxin translocation domain form edged holes in lipid membranes

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 pHs 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.

Live imaging of transgene expression in Cloudman S91 melanoma cells after polyplex-mediated gene delivery

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.

Development and evaluation of a new modular nanotransporter for drug delivery into nuclei of pathological cells expressing folate receptors

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.

Use of intracellular transport processes for targeted drug delivery into a specified cellular compartment

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.

MNT Optimization for Intracellular Delivery of Antibody Fragments

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.