Papers by Alexander Sobolev
Theranostics, 2015
The ability of nanoparticles and macromolecules to passively accumulate in solid tumors and enhan... more 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.
The search for new pharmaceuticals has raised interest in locally-acting drugs which act over sho... more The search for new pharmaceuticals has raised interest in locally-acting drugs which act over short distances within the cell, and for which different cell compartments have different sensitivities. Thus, photosensitizers used in anti-cancer therapy should be transported to the most sensitive subcellular compartments where their action is most pronounced. Earlier, we described the effects of bacterially expressed modular recombinant transporters
Biulleten' eksperimental'noĭ biologii i meditsiny, 1978
Early alterations in the enzyme activities controlling cyclic adenosine-3',5'-monophospha... more Early alterations in the enzyme activities controlling cyclic adenosine-3',5'-monophosphate (cAMP) metabolism after the irradiation (800 rad) of mice were found in the lymphoid cells of the spleen and thymus. Postradiation disturbances in these activities' ration induced alterations in cAMP steady-state concentrations in the cell. It was also demonstrated that irradiation reduced lymphoid cell ability to accumulate cAMP in response to isoproterenol administration.
Doklady Akademii nauk SSSR, 1977
Doklady Akademii nauk SSSR, Jan 21, 1975
Biochemistry. Biokhimii͡a, 2014
Targeted drug delivery into the cell compartment that is the most vulnerable to effects of the co... more 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.
Current pharmaceutical design, 2015
The review is devoted to a subcellular drug delivery system, modular nanotransporters (MNT) that ... more The review is devoted to a subcellular drug delivery system, modular nanotransporters (MNT) that can penetrate into target cells and deliver a therapeutic into their subcellular compartments, particularly into the nucleus. The therapeutics which need such type of delivery belong to two groups: (i) those that exert their effect only when delivered into a certain cell compartment (like DNA delivered into the nucleus); and (ii) those drugs that are capable of exerting their effect in different parts of the cells, however there can be found a cell compartment that is the most sensitive to their effect. A particular interest attract such cytotoxic agents as Auger electron emitters which are known to be ineffective outside the cell nucleus, whereas they possess high cytotoxicity in the vicinity of nuclear DNA through the induction of non-reparable double-strand DNA breaks. The review discusses main approaches permitting to choose internalizable receptors permitting both recognition of tar...
Nauchnye doklady vyssheĭ shkoly. Biologicheskie nauki, 1975
Journal of Translational Medicine, 2015
Background: Gene-directed enzyme prodrug therapy (GDEPT) represents a technology to improve drug ... more 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.
Doklady Biochemistry and Biophysics, 2012
Photodynamic therapy (PDT) is a promising treatment method, which was shown to be effective for v... more Photodynamic therapy (PDT) is a promising treatment method, which was shown to be effective for various types of cancer . This method is based on selective accumulation of photosensitizers in cancer cells, with subsequent irradiation of the tumor at one of the absorption peaks of photosensitizers . The active cytotoxic principle of photosensitizers are reactive oxygen species (ROS), primarily singlet oxygen and hydroxyl radical, whose radius of action is limited to 20 nm . The most sensitive cellular compartment to the damaging effect of ROS, and consequently, to the photocytotoxic effect of photosensitizers is the cell nucleus . However, it was shown that free photosensitizers do not accumulate in the nucleus in detectable amount . Thus in order to reach the maximum effect of PDT with the minimum dose of a photosensitizer (and, thus, to minimize the side effects of therapy), it seems reasonable to ensure not only selective accumulation of photosensitizers in tumor cells but also their targeted delivery into the cell nucleus. Applying this strategy there were developed modular nanotransporters (MNTs) utilizing natural intracellular transport and macromolecule sorting machinery for selective targeted delivery of locally acting drugs to the nucleus of melanoma cells. MNTs are recombinant polypeptides whose functional modules ensure (1) selective recognition of the target cell with its subsequent endocytosis, due to the ligand module, α-melanocyte stimulating hormone (αMSH), a ligand for internalized melanocortin receptors-1 overexpressed on the surface of melanoma cells [4]; (2) release from the endosome into the cytoplasm, due to the translocation domain of the diphtheria toxin (DTox); (3) transport into the nucleus, due to the optimized nuclear localization sequence (NLS) of the SV40 large T antigen; and (4) effective attachment of the delivered drug, ensured by the carrier module, hemoglobin like protein (HMP) of E. coli. Earlier, it was shown that the photosensitizers conjugated to MNTs were two orders of magnitude more efficient than the free photosensitizers on the melanoma cells in vitro .
International Journal of Nanomedicine, 2012
Modular nanotransporters (MNT) are recombinant multifunctional polypeptides created to exploit a ... more 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. In vivo targeting of the modular nanotransporter was determined by immuno-fluorescence confocal laser scanning microscopy and by accumulation of (125)I-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. 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 (125)I-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 e(6)- MNT{EGF} resulted in 94% tumor growth inhibition compared with free chlorin e(6), with 75% of animals surviving at 3 months compared with 0% and 20% for untreated and free chlorin e(6)-treated groups, respectively. 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.
EJNMMI Research, 2012
Background: This study evaluates the potential utility of a modular nanotransporter (MNT) for enh... more Background: This study evaluates the potential utility of a modular nanotransporter (MNT) for enhancing the nuclear delivery and cytotoxicity of the Auger electron emitter 125 I in cancer cells that overexpress the epidermal growth factor receptor (EGFR). Methods: MNTs are recombinant multifunctional polypeptides that we have developed for achieving selective delivery of short-range therapeutics into cancer cells. MNTs contain functional modules for receptor binding, internalization, endosomal escape and nuclear translocation, thereby facilitating the transport of drugs from the cell surface to the nucleus. The MNT described herein utilized EGF as the targeting ligand and was labeled with 125 I using N-succinimidyl-4-guanidinomethyl-3-[ 125 I]iodobenzoate (SGMIB). Membrane binding, intracellular and nuclear accumulation kinetics, and clonogenic survival assays were performed using the EGFR-expressing A431 epidermoid carcinoma and D247 MG glioma cell lines.
Nuclear medicine and biology, 2014
Modular nanotransporters (MNTs) are vehicles designed to transport drugs from the cell surface vi... more Modular nanotransporters (MNTs) are vehicles designed to transport drugs from the cell surface via receptor-mediated endocytosis and endosomal escape to nucleus. Hence their conjugation to Auger electron emitters, can cause severe cell killing, by nuclear localization. Herein we evaluate the use of MNT as a platform for targeted radiotherapy with (67)Ga. EGF was the targeting ligand on the MNT, and NOTA was selected for its radiolabeling with (67)Ga. In the radiolabeling study we dealt with the precipitation of MNT (pI 5.7) at the labeling pH (4.5-5.5) of (67)Ga. Cellular and nuclei uptake of (67)Ga-NOTA-MNT by the A431 cell line was determined. Its specific cytotoxicity was compared to that of (67)Ga-EDTA, (67)Ga-NOTA-BSA and (67)Ga-NOTA-hEGF, in A431 and U87MGWTT, cell lines, by clonogenic assay. Dosimetry studies were also performed. (67)Ga-NOTA-MNT was produced with 90% yield and specific activity of 25.6mCi/mg. The in vitro kinetics revealed an increased uptake over 24h. 55% of...
The FASEB Journal, 2003
The search for new pharmaceuticals that are specific for diseased rather than normal cells in the... more 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. M any macromolecular medicines acting within the cell (e.g., foreign DNA, antisense oligonucleotides, polypeptide toxins) must be transported into specific intracellular compartments in order to exert their effects. By themselves, many of these locally acting compounds are unable to reach the site of their action, needing additional macromolecular systems for targeted intracellular delivery. The list of locally acting drugs includes not only macromolecules but also low-molecular weight drugs that act over very short distances comparable with the dimensions of the cell compartments, where additionally, the sensitivities of distinct cell compartments to the action of these drugs are different. Such drugs accordingly need to be transported efficiently to the most sensitive/vulnerable site(s) of the target cell. Photosensitizers (PSs), used for anti-cancer photodynamic therapy, are an example of this type of locally acting drugs, with cytotoxic action not exceeding 40 nm from the site of subcellular localization since it is effected via reactive oxygen species (mainly, singlet oxygen and free radicals úOH and HOú 2 ), which have a very short mean range within the cell (1). Keeping in mind that the efficacy of cell killing induced by reactive oxygen species depends on the site of their action, and that the cell nucleus is the most vulnerable/sensitive site, in contrast to cell membranes and other cytoplasmic organelles (1), there is little doubt that PS anti-tumor efficiency will depend on the subcellular distribution of PSs. Consistent with this, many laboratories have demonstrated that PSs of medical interest localize in various cytoplasmic structures, but not in the cell nucleus, and that the ultrastructural damage caused by PSs coincides with their localization (1, 2). Earlier (3, 4), we tried to change the subcellular distribution of PSs in favor of the cell nucleus, by exploiting targeting signals recognized by the cellular nuclear transport machinery. PSs can be successfully redirected within cells by using cross-linked modular polypeptide transporters possessing 1) an internalizable ligand providing for cell-specific delivery; 2) a nuclear localization sequence (NLS) conferring interaction with importins, the cellular proteins mediating active translocation into the nucleus; and modules enabling 3) escape from endosomes and 4) attachment of the PSs. The PSs transported to the cell nucleus by the modular carriers proved to be several orders of magnitude more efficient than nonmodified, free PSs, the best results being obtained using model modular transporters composed of 1) insulin as the internalizable ligand, 2) an optimized NLS from the SV40 large tumor antigen (T-ag), 3) attenuated human adenovirus Ad5 dl312 as an endosomolytic component, and 4) bacterial β-galactosidase as a cross-linked carrier (3, 4). Analogous results have been recently obtained by others (5). Notably, internalized PSs are more efficient in cell killing than those localized at the cell surface (6-8), whereas PSs transported to the nuclei are more efficient than those internalized (3, 4) and, as just mentioned, substantially more efficient than free, nonmodified PSs. The nucleus is thus a hypersensitive site for photodynamic damage. Although insulin was used as a model ligand conferring both cell-type specificity and receptormediated endocytosis of a photosensitizing construct, the approach is clearly applicable to a wide variety of cancer cell types, if ligands are used that are recognized by receptors overexpressed on these cells, e.g., α-melanocyte-stimulating hormone (MSH) specific for a number of melanomas (9-12), epidermal growth factor receptor family binding ligands , and somatostatin . There are options to simplify the transporting constructs, e.g., by replacing adenoviruses
Molecular Reproduction and Development, 1999
Mouse and rabbit preimplantation embryos with intact zona pellucida were incubated for 3 hr with ... more Mouse and rabbit preimplantation embryos with intact zona pellucida were incubated for 3 hr with DNA-carrying constructs containing insulin as an internalizable ligand: (insulin-polylysine)-DNA and (insulinpolylysine)-DNA-(streptavidin-polylysine)-(biotinylated adenovirus). Video-intensified microscopy demonstrated that the constructs penetrated the zona pellucida and accumulated in the blastomere perinuclear space. The percentage of blastocysts formed was about 70% after incubation of zygotes and two-cell embryos with the constructs. Foreign DNA was detected after 51 hr in 80% of rabbit embryos and after 96 hr in 73% of mouse embryos. Inclusion of various adenoviruses into the construct improved foreign DNA preservation in early embryos. Blot hybridization revealed genome-integrated foreign DNA in 12-and 15-day mouse embryos and in a newborn. Thus, the ligand-mediated mechanism can be employed for introducing foreign genetic material into early mammalian embryos; insulin provides for delivery inside the cell and to the nucleus, while adenoviruses ensure release from endosomes. Mol. Reprod. Dev. 54:112-120, 1999. 1999 Wiley-Liss, Inc.
Molecular and Cellular Biochemistry, 1988
Page 1. Molecular and Cellular Biochemistry 81:19-28 (1988) @ Kluwer Academic Publishers - Printe... more Page 1. Molecular and Cellular Biochemistry 81:19-28 (1988) @ Kluwer Academic Publishers - Printed in the Netherlands 19 Original Article Application of percolation theory principles to the analysis of interaction of adenylate cyclase complex proteins in cell membranes ...
International Journal of Radiation Oncology*Biology*Physics, 2008
Purpose: To generate and evaluate a modular recombinant transporter (MRT) for targeting 211 At to... more 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 a-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. Ó
Uploads
Papers by Alexander Sobolev