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Modular Nanotransporters: A Multi-purpose Platform for Cell

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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
Modules:
endosomolytic
Рќ+
3. Escape from
endosomes
carrier
nuclear localizatuion
sequence
Nucleus
2.Endocytosis
ligand
1. Receptor
binding
4. Entry
into the
nucleus
Receptors
Endosome
Importins пЃЎ/пЃў
Nuclear
pore
complex
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
Carrier module
Pharmaceutical
Ligand modules: 1) пЃЎMSH, 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.
Expression and purification of HMP-NLS-пЃЎMSH
(A) and DTox-HMP-NLS-пЃЎMSH (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
All MNT modules are
functional.
They retain their activities within
the modular nanotransporters:
Displacement of [125I]-DTox-HMP-EGF by
DTox-HMP-NLS-EGF (�) and
HMP-NLS-DTox-EGF (п‚ї) from ErbB1 receptors
Gilyazova D.G. et al. Cancer Res. 2006; 66: 10534-10540.
Interaction of DTox-HMP-NLS-EGF and HMPNLS-DTox-EGF with пЃЎ/пЃў-importin heterodimer
k
Рђ+Р’
a1
k a2
РђР’
k d1
k d2
РђР’*
Modular
nanotransporter
ka1, Рњ-1В·s-1
kd1, s-1
ka2, s-1
kd2, s-1
Ka, M-1
DTox-HMP-NLS-EGF
(9.48п‚±0.11)В·103
(5.08п‚±0.15)В·10-3
(2.81п‚±0.12)В·10-3
(1.83п‚±0.30)В·10-4
3.06В·107
HMP-NLS-DTox-EGF
(1.75п‚±0.04)В·103
(6.08п‚±0.05)В·10-3
(2.54п‚±0.04)В·10-3
(4.76п‚±0.44)В·10-5
1.57В·107
Gilyazova D.G. et al. Cancer Res. 2006; 66: 10534-10540.
Atomic force microscopy of a supported lipid
bilayer at СЂРќ 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.
Subcellular MNT localization
A
B
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
hn
20-40 nm
PS
Subcellular localization of photosensitizers
chlorin Рµ6
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
C6 glioma
Photosensitizers delivered by MNT to the
target cell nuclei acquire significantly
higher efficacy…
A, (chlorin e6)-HMP-NLS-DTox-EGF (п‚ї) and free chlorin e6 (п‚Ї). B, (bacteriochlorin p)-HMP-NLSDTox-EGF (пЃ®) and free bacteriochlorin p (пЃЇ).
Gilyazova D.G. et al. Cancer Res. 2006; 66: 10534-10540.
… and cell specificity
C, (chlorin e6)-DTox-HMP-NLS-EGF acting on A431 target cells (в—Џ) and on non-target NIH 3T3
cells (в–І). D, free chlorin e6 acting on target A431 cells (п‚Ї) and on non-target NIH 3T3 cells (пЃІ).
Gilyazova D.G. et al. Cancer Res. 2006; 66: 10534-10540.
In vivo application of the MNT,
photosensitizers
Tumor/non-tumor ratios
3 h after i.v. injection of [125I]-DTox-HMP-NLS-MSH
MNT to C57Black mice with B16-F1 melanoma
14
12
Tumor/muscle
Tumor/skin
10
8
6
4
2
4.7
1.4
8.2 9.8
13.4 4.9
0
10.8 mg/mouse
213.5 mg/mouse
850 mg/mouse
Slastnikova, T.A. et al. (submitted)
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)
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 e6)-DTox-HMP-NLS-EGF
1000
Tumor volume, mm
3
Chl
800
600
control
400
200
N
MRT-Chl
0
0
10
20
30
40
50
Time after inoculation, days
Slastnikova, T.A. et al. (submitted)
PDT of human A431 epidermoid carcinoma
xenografts on nude Balb/c ByJIco-nu/nu mice
with (chlorin e6)-DTox-HMP-NLS-EGF
100
MNT-Chl
N
Survival, %
80
60
40
Chl
control
20
0
0
10
20
30
40
50
60
70
80
90
Time after inoculation, days
Slastnikova, T.A. et al. (submitted)
MNT is a versatile platform for cell specific
subcellular drug delivery:
пЃ®
MNT modules retain their functions within the MNT
пЃ®
MNT are highly expressed and easily purified
пЃ®
MNT modules are interchangeable, meaning that they can be
tailored for particular applications
пЃ®
Anti-cancer therapeutics (photosensitizers, alpha-emitters)
carried by MNT acquired 20-3000 times greater cytotoxicity
and cell specificity if compared with free therapeutics
пЃ®
The MNT demonstrated very low toxicity and immunogenicity
on mice
пЃ®
MNT accumulated in tumors with high tumor:non-tumor ratios
and displayed preferential nuclear accumulation.
пЃ®
The MNT are effective both in vitro and in vivo
MNT in action
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