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

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 nanotransporte

Integrity of the Mod(mdg4)-67.2 BTB Domain Is Critical to Insulator Function in Drosophila melanogaster

The Drosophila gypsy insulator contains binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein. Enhancer and silencer blocking require Su(Hw) recruitment of Mod(mdg4)-67.2, a BTB/POZ domain protein that interacts with Su(Hw) through a carboxyl-terminal acidic domain. Here we conducted mutational analyses of the Mod(mdg4)-67.2 BTB domain. We demonstrate that this domain is essential for insulator function, in part through direction of protein dimerization. Our studies revealed the presence of a second domain (DD) that contributes to Mod(mdg4)-67.2 dimerization when the function of the BTB domain is compromised. Additionally, we demonstrate that mutations in amino acids of the charged pocket in the BTB domain that retain dimerization of the mutated protein cause a loss of insulator function. In these cases, the mutant proteins failed to localize to chromosomes, suggesting a role for the BTB domain in chromosome association. Interestingly, replacement of the Mod(mdg4)-67.2 BTB domain with the GAF BTB domain produced a nonfunctional protein. Taken together, these data suggest that the Mod(mdg4)-67.2 BTB domain confers novel activities to gypsy insulator function