Presentation on theme: "Modular nanotransporters: a multi-purpose platform for cell-specific intranuclear drug delivery Alexander S. Sobolev Institute of Gene Biology RAS Moscow."— Presentation transcript:
Modular nanotransporters: a multi-purpose platform for cell-specific intranuclear drug delivery Alexander S. Sobolev Institute of Gene Biology RAS Moscow State University
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:-
Modular drug nanotransporters and their modules endosomolytic nuclear localizatuion sequence Modules: carrier ligand Nucleus Nuclear pore complex Receptors 1. Receptor binding Н+Н+ 3. Escape from endosomes Importins / 4. Entry into the nucleus 2.Endocytosis Endosome
Modular nanotransporters (MNT), a scheme Ligand module enabling target cell recognition/penetration Endosomolytic module enabling escape from endocytotic vesicles NLS-containing module enabling transport into the cell nucleus Carrier module Pharmaceutical Ligand modules: 1) MSH, 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.
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 Expression and purification of HMP-NLS-MSH (A) and DTox-HMP-NLS-MSH (B)
All MNT modules are functional. They retain their activities within the modular nanotransporters:
Displacement of [ 125 I]-DTox-HMP-EGF by DTox-HMP-NLS-EGF ( ) and HMP-NLS-DTox-EGF ( ) from ErbB1 receptors Gilyazova D.G. et al. Cancer Res. 2006; 66: 10534-10540.
Interaction of DTox-HMP-NLS-EGF and HMP- NLS-DTox-EGF with -importin heterodimer Modular nanotransporter k a1, М -1 ·s -1 k d1, s -1 k a2, s -1 k d2, s -1 K a, M -1 DTox-HMP-NLS-EGF (9.48 0.11)·10 3 (5.08 0.15)·10 -3 (2.81 0.12)·10 -3 (1.83 0.30)·10 -4 3.06·10 7 HMP-NLS-DTox-EGF (1.75 0.04)·10 3 (6.08 0.05)·10 -3 (2.54 0.04)·10 -3 (4.76 0.44)·10 -5 1.57·10 7 А+В АВ k a1 k d1 k a2 k d2 АВ* Gilyazova D.G. et al. Cancer Res. 2006; 66: 10534-10540.
DTox-HMP-NLS-EGF at pH 5.5; egg lecithin on mica Atomic force microscopy of a supported lipid bilayer at рН 5.5 after addition of the MNT Gilyazova D.G. et al. Cancer Res. 2006; 66: 10534-10540; Khramtsov Yu.V. et al. J. Contr. Release 2008; 128: 241-247.
Subcellular MNT localization BA 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.
In vitro application of the MNT, photosensitizers
Photoactivation of photosensitizer PS 20-40 nm h
Subcellular localization of photosensitizers C 6 glioma chlorin е 6 protoporphyrin IX 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
Photosensitizers delivered by MNT to the target cell nuclei acquire significantly higher efficacy… A, (chlorin e 6 )-HMP-NLS-DTox-EGF ( ) and free chlorin e 6 ( ). B, (bacteriochlorin p)-HMP-NLS- DTox-EGF ( ) and free bacteriochlorin p ( ). Gilyazova D.G. et al. Cancer Res. 2006; 66: 10534-10540.
C, (chlorin e 6 )-DTox-HMP-NLS-EGF acting on A431 target cells ( ● ) and on non-target NIH 3T3 cells ( ▲ ). D, free chlorin e 6 acting on target A431 cells ( ) and on non-target NIH 3T3 cells ( ). … and cell specificity
In vivo application of the MNT, photosensitizers
Tumor/non-tumor ratios 3 h after i.v. injection of [ 125 I]-DTox-HMP-NLS-MSH MNT to C 57 Black mice with B16-F1 melanoma 10.8 g/mouse 213.5 g/mouse850 g/mouse Tumor/muscle Tumor/skin Slastnikova, T.A. et al. (submitted)
MNT localize in mainly in 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. tumor cells non-tumor cells Slastnikova, T.A. et al. (submitted)
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)
PDT of human A431 epidermoid carcinoma xenografts on nude Balb/c ByJIco-nu/nu mice with (chlorin e 6 )-DTox-HMP-NLS-EGF N Slastnikova, T.A. et al. (submitted)
PDT of human A431 epidermoid carcinoma xenografts on nude Balb/c ByJIco-nu/nu mice with (chlorin e 6 )-DTox-HMP-NLS-EGF N 0 20 40 60 80 100 0102030405060708090 Time after inoculation, days Survival, % control Chl MNT-Chl Slastnikova, T.A. et al. (submitted)
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