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Modular nanotransporters: a multi-purpose platform for cell-specific

Modular nanotransporters: a multi-purpose platform for cell-specific intranuclear drug delivery. Alexander S. Sobolev Institute of Gene Biology RAS Moscow State University.

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Modular nanotransporters: a multi-purpose platform for cell-specific

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  1. Modular nanotransporters: a multi-purpose platform for cell-specific intranuclear drug delivery Alexander S. Sobolev Institute of Gene Biology RAS Moscow State University

  2. Every drug transporter, which ultimate goal is to deliver the drug not only into a target cell but also into its specific subcellular compartment, should achieve several sub-goals:-

  3. Modules: 3. Escape from endosomes endosomolytic Н+ carrier 2.Endocytosis nuclear localizatuion sequence Nucleus ligand 4. Entry into the nucleus Endosome 1. Receptor binding Receptors Importins / Modular drug nanotransporters and their modules Nuclear pore complex

  4. Modular nanotransporters (MNT), a scheme Ligand module enabling target cell recognition/penetration NLS-containing module enabling transport into the cell nucleus Endosomolytic module enabling escape from endocytotic vesicles Pharmaceutical Carrier module Ligand modules: 1) aMSH, or 2) EGF, or 3) IL-3, or 4) somatostatin, enable recognition of the following target cell types: 1) melanoma, or 2) head-and-neck cancer, glioblastoma multiforme, oesophagus cancer, epidermoid carcinoma, or 3) acute myeloid leukemia, or 4) neuroblastoma, respectively.

  5. Expression and purification ofHMP-NLS-aMSH (A) and DTox-HMP-NLS-aMSH (B) A B 1 2 3 1 2 3 1, total soluble protein of E. coli; 2, total soluble protein of E. coli expressing the MNT; 3, purified MNT Rosenkranz A.A. et al. FASEB J. 2003;17:1121-1123

  6. All MNT modules are functional.They retain their activities within the modular nanotransporters:

  7. Displacement of [125I]-DTox-HMP-EGF by DTox-HMP-NLS-EGF () and HMP-NLS-DTox-EGF ()fromErbB1 receptors Gilyazova D.G. et al. Cancer Res. 2006; 66: 10534-10540.

  8. k k a2 a1 АВ АВ* А+В k k d2 d1 Interaction of DTox-HMP-NLS-EGF andHMP-NLS-DTox-EGF with a/b-importin heterodimer Gilyazova D.G. et al. Cancer Res. 2006; 66: 10534-10540.

  9. Atomic force microscopy of a supported lipid bilayerat рН 5.5 after addition of the MNT DTox-HMP-NLS-EGF at pH 5.5; egg lecithin on mica Gilyazova D.G. et al. Cancer Res. 2006; 66: 10534-10540; Khramtsov Yu.V. et al. J. Contr. Release 2008;128: 241-247.

  10. A B Subcellular MNT localization A, DTox-HMP-NLS-EGF in A431 cells; B, the same A431 cells with DNA stained with ToPro-3 Gilyazova D.G. et al. Cancer Res. 2006; 66: 10534-10540.

  11. In vitro application of the MNT,photosensitizers

  12. 20-40 nm Photoactivation of photosensitizer Reactive oxygen species hn PS

  13. Subcellular localization of photosensitizers chlorin е6 protoporphyrinIX Photosensitizers do not localize into the cell nuclei, the subcellular compartments which are most sensitive to the action of reactive oxygen species produced by the photosensitizers C6 glioma

  14. Photosensitizers delivered by MNT to the target cell nuclei acquire significantly higher efficacy… A, (chlorin e6)-HMP-NLS-DTox-EGF () and free chlorin e6 (). B, (bacteriochlorin p)-HMP-NLS-DTox-EGF () and free bacteriochlorin p (). Gilyazova D.G. et al. Cancer Res. 2006; 66: 10534-10540.

  15. … and cell specificity C, (chlorin e6)-DTox-HMP-NLS-EGF acting on A431 target cells (●) and on non-target NIH 3T3 cells (▲). D, free chlorin e6 acting on target A431 cells () and on non-target NIH 3T3 cells (). Gilyazova D.G. et al. Cancer Res. 2006; 66: 10534-10540.

  16. In vivo application of the MNT,photosensitizers

  17. 10.8 mg/mouse 213.5 mg/mouse 850 mg/mouse Tumor/non-tumor ratios3 h afteri.v. injection of [125I]-DTox-HMP-NLS-MSH MNT to C57Black mice with B16-F1 melanoma Tumor/muscle Tumor/skin Slastnikova, T.A. et al. (submitted)

  18. MNT localize in mainly in tumor cells… non-tumor cells tumor cells MNT was i.v. injected in DBA/2 mice bearing Cloudman S91 (M3) melanoma transformed with EGFP. (a)immunofluorescent staining for MNT; (b)fluorescence of M3 melanoma cells expressing green fluorescent protein; (c)DAPI staining (cell nuclei); (d)an overlay of a, b, c. Slastnikova, T.A. et al. (submitted)

  19. Within tumor cells, MNT localize mainly in the nuclei MNT was i.v. injected in nude mice bearing human epidermoid carcinoma cells. (A) DAPI staining (cell nuclei); (B)immunofluorescent staining for MNT; (C)an overlay of A and B. (A’), (B’), and (C’) are controls, injected with saline. Slastnikova, T.A. et al. (submitted)

  20. N PDT of human A431 epidermoid carcinoma xenografts on nude Balb/c ByJIco-nu/nu mice with (chlorin e6)-DTox-HMP-NLS-EGF Slastnikova, T.A. et al. (submitted)

  21. 100 MNT-Chl 80 60 Survival, % 40 Chl control 20 0 0 10 20 30 40 50 60 70 80 90 Time after inoculation, days PDT of human A431 epidermoid carcinoma xenografts on nude Balb/c ByJIco-nu/nu mice with (chlorin e6)-DTox-HMP-NLS-EGF N Slastnikova, T.A. et al. (submitted)

  22. MNT is a versatile platform for cell specific subcellular drug delivery: • MNT modules retain their functions within the MNT • MNT are highly expressed and easily purified • MNT modules are interchangeable, meaning that they can be tailored for particular applications • Anti-cancer therapeutics (photosensitizers, alpha-emitters) carried by MNT acquired 20-3000 times greater cytotoxicity and cell specificity if compared with free therapeutics • The MNT demonstrated very low toxicity and immunogenicity on mice • MNT accumulated in tumors with high tumor:non-tumor ratios and displayed preferential nuclear accumulation. • The MNT are effective both in vitro and in vivo

  23. MNT in action

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