Alexander S. Sobolev

Targeted intracellular delivery of photosensitizers to enhance photodynamic efficiency

Photodynamic therapy (PDT) is a novel treatment, used mainly for anticancer therapy, that depends on the retention of photosensitizers (PS) in tumour cells and irradiation of the tumour with appropriate wavelength light. Photosensitizers are molecules such as porphyrins and chlorins that, on photoactivation, effect strongly localized oxidative damage within target cells. The PS used for PDT localize in various cytoplasmic membranous structures, but are not found in the most vulnerable intracellular sites for reactive oxygen species, such as the cell nucleus. The experimental approaches discussed in the present paper indicate that it is possible to design highly efficient molecular constructs, PS carriers, with specific modules conferring cell‐specific targeting, internalization, escape from intracellular vesicles and targeting to the most vulnerable intracellular compartments, such as the nucleus. Nuclear targeting of these PS‐carrying constructs results in enhanced photodynamic activity, maximally about 2500‐fold that of free PS. Future work is intended to optimize this approach to the point at which tumour cells can be killed rapidly and efficiently, while minimizing normal cell and tissue damage.

Receptor-mediated endocytosis and nuclear transport of a transfecting DNA construct

A soluble construct consisting of a plasmid carrying the gene of the SV40 large T-antigen and an insulin-poly-L-lysine conjugate is able to selectively transfect PLC/PRF/5 human hepatoma cells which possess insulin receptors. Transfection can be efficiently competed by excess free insulin. To examine intracellular transport of the construct, it was fluorescently labeled and its accumulation on and in cells visualized by video-enhanced microscopy and quantitative confocal laser scanning microscopy. After 2 h at 37 degrees C, the labeled construct was found predominantly in intracellular acidic compartments, with a substantial portion of fluorescence localized both near and in the cell nucleus. Binding, endocytosis, and nuclear localization of the labeled conjugate could all be competed by excess free insulin, thus indicating that entry of the conjugate into cells was specifically mediated by the insulin receptor.

Targeted intracellular delivery of photosensitizers

Targeted intracellular delivery of photosensitizers Alexander S. Sobolev*, David A. Jans, Andrey A. Rosenkranz Department of Biophysics, Biological Faculty, Moscow State University, 119899, Moscow, Russia Laboratory of Molecular Genetics of Intracellular Transport, Institute of Gene Biology, Russian Academy of Science, Vavilov St. 34/5, 117984, Moscow, Russia Nuclear Signaling Laboratory, Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra, ACT 2601, Australia

Current Approaches for Improving Intratumoral Accumulation and Distribution of Nanomedicines

The ability of nanoparticles and macromolecules to passively accumulate in solid tumors and enhance therapeutic effects in comparison with conventional anticancer agents has resulted in the development of various multifunctional nanomedicines including liposomes, polymeric micelles, and magnetic nanoparticles. Further modifications of these nanoparticles have improved their characteristics in terms of tumor selectivity, circulation time in blood, enhanced uptake by cancer cells, and sensitivity to tumor microenvironment. These “smart” systems have enabled highly effective delivery of drugs, genes, shRNA, radioisotopes, and other therapeutic molecules. However, the resulting therapeutically relevant local concentrations of anticancer agents are often insufficient to cause tumor regression and complete elimination. Poor perfusion of inner regions of solid tumors as well as vascular barrier, high interstitial fluid pressure, and dense intercellular matrix are the main intratumoral barriers that impair drug delivery and impede uniform distribution of nanomedicines throughout a tumor. Here we review existing methods and approaches for improving tumoral uptake and distribution of nano-scaled therapeutic particles and macromolecules (i.e. nanomedicines). Briefly, these strategies include tuning physicochemical characteristics of nanomedicines, modulating physiological state of tumors with physical impacts or physiologically active agents, and active delivery of nanomedicines using cellular hitchhiking.

Recombinant modular transporters for cell-specific nuclear delivery of locally acting drugs enhance photosensitizer activity

The search for new pharmaceuticals that are specific for diseased rather than normal cells in the case of cancer and viral disease has raised interest in locally acting drugs that act over short distances within the cell and for which different cell compartments have distinct sensitivities. Thus, photosensitizers (PSs) used in anti‐cancer therapy should ideally be transported to the most sensitive subcellular compartments in order for their action to be most pronounced. Here we describe the design, production, and characterization of the effects of bacterially expressed modular recombinant transporters for PSs comprising 1) α‐melanocyte‐stimulating hormone as an internalizable, cell‐specific ligand; 2) an optimized nuclear localization sequence of the SV40 large T‐antigen; 3) an Escherichia coli hemoglobin‐like protein as a carrier; and 4) an endosomolytic amphipathic polypeptide, the translocation domain of diphtheria toxin. These modular transporters delivered PSs into the nuclei, the most vulnerable sites for the action of PSs, of murine melanoma cells, but not non‐MSH receptor‐overexpressing cells, to result in cytotoxic effects several orders of magnitude greater than those of nonmodified PSs. The modular fusion proteins described here for the first time, capable of cell‐specific targeting to particular subcellular compartments to increase drug efficacy, represent new pharmaceuticals with general application.

Adenoviruses synergize with nuclear localization signals to enhance nuclear delivery and photodynamic action of internalizable conjugates containing chlorin e6.

Photosensitizers, molecules that produce active oxygen species upon activation by visible light, are currently being used in photodynamic therapy (PDT) to treat cancer and other conditions, where limitations include normal cells and tissue damage and associated side effects, and the fact that cytotoxic effects are largely restricted to the plasma and other peripheral membranes. In this study, we used insulin‐containing conjugates to which variants of the simian‐virus‐SV40 large‐tumor antigen (T‐ag) nuclear localization signal (NLS) were linked in order to target the photosensitizer chlorin e6 to the nucleus. NLSs were included either as peptides coupled co‐valently to the carrier bovine serum albumin, or within the coding sequence of β‐galactosidase fusion proteins. The most potent photosensitizing conjugate was the NLS‐containing T‐ag β‐galactosidase fusion protein (P10)‐(chlorin e6)‐insulin, exhibiting an EC50 more than 2400‐fold lower than the value for free chlorin e6, and more than 15‐fold lower than that of an NLS‐deficient β‐galactosidase‐(chlorin e6)‐insulin construct, thus demonstrating that NLSs can increase the photosensitizing activity of chlorin e6. Attenuated adenoviruses were used to increase the nuclear delivery of conjugates through its endosomal‐membrane‐disrupting activity. In the case of the NLS‐containing P10‐conjugate, co‐incubation with adenovirus increased the proportion of cells whose nuclear photosensitizing activity was higher than that in the cytoplasm by 2.5‐fold. This use of adenoviruses in conjunction with photosensitizers has clear implications for achieving efficient cell‐type‐specific PDT. Int. J. Cancer 81:734–740, 1999.

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.

Optimization of the phase-locked flux-flow oscillator for the submm integrated receiver

The Superconducting Integrated Receiver (SIR) comprises in one chip a planar antenna integrated with an SIS mixer, a superconducting Flux Flow Oscillator (FFO) acting as Local Oscillator (LO) and a second SIS harmonic mixer (HM) for FFO phase locking. Free-running FFO linewidth well below 10 MHz is required to ensure phase-locked operation of an SIR. Comprehensive experimental study of the Nb-AlOx-Nb FFO linewidth and other main parameters has been carried out in order to achieve this goal. Essential dependence of the FFO linewidth on its width and idle region dimension has been found. It makes possible an optimization of the FFO design and selection of the best FFO parameters for practical operation of the SIR.

Engineered Modular Recombinant Transporters: Application of New Platform for Targeted Radiotherapeutic Agents to α-Particle Emitting 211At

PURPOSE To generate and evaluate a modular recombinant transporter (MRT) for targeting 211 At to cancer cells overexpressing the epidermal growth factor receptor (EGFR). METHODS AND MATERIALS The MRT was produced with four functional modules: (1) human epidermal growth factor as the internalizable ligand, (2) the optimized nuclear localization sequence of simian vacuolating virus 40 (SV40) large T-antigen, (3) a translocation domain of diphtheria toxin as an endosomolytic module, and (4) the Escherichia coli hemoglobin-like protein (HMP) as a carrier module. MRT was labeled using N-succinimidyl 3-[211 At]astato-5-guanidinomethylbenzoate (SAGMB), its 125 I analogue SGMIB, or with 131 I using Iodogen. Binding, internalization, and clonogenic assays were performed with EGFR-expressing A431, D247 MG, and U87MG.wtEGFR human cancer cell lines. RESULTS The affinity of SGMIB-MRT binding to A431 cells, determined by Scatchard analysis, was 22 nM, comparable to that measured before labeling. The binding of SGMIB-MRT and its internalization by A431 cancer cells was 96% and 99% EGFR specific, respectively. Paired label assays demonstrated that compared with Iodogen-labeled MRT, SGMIB-MRT and SAGMB-MRT exhibited more than threefold greater peak levels and durations of intracellular retention of activity. SAGMB-MRT was 10-20 times more cytotoxic than [211 At]astatide for all three cell lines. CONCLUSION The results of this study have demonstrated the initial proof of principle for the MRT approach for designing targeted alpha-particle emitting radiotherapeutic agents. The high cytotoxicity of SAGMB-MRT for cancer cells overexpressing EGFR suggests that this 211 At-labeled conjugate has promise for the treatment of malignancies, such as glioma, which overexpress this receptor.

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.