Escolar Documentos
Profissional Documentos
Cultura Documentos
Alberto Bianco
Outline Integration of functionalized carbon nanotubes into living systems Capacity of functionalized carbon nanotubes to penetrate into the cells Delivery of therapeutic molecules by carbon nanotubes
Peptides Nucleic acids (DNA, RNA, siRNA) Antibodies Small drugs
Use of carbon nanotubes for imaging applications Biodistribution and pharmacokinetics Conclusions
H HO H HO HO H H OH OH H O
Glucose
Virus
Cancer cell
Tennis ball
Water
Antibody
Bacteria
10-1
10
102
103
104
105 106
107
108
Nanometers
Nanodevices: Nanoparticles
Dendrimers Quantum Dots Carbon nanotubes
- Carbon nanotubes for cancer therapy: hyperthermia (by NIR, RF activation); chemotherapy; radiotherapy; gene therapy
A549
C. neoformans
NH3+ClO OH O HO2C N HN S H N O O NH O O
Cytoplasm
NHCOCH3 O O N O H N O S HO O O
HeLa
Macrophages
CO2H NH
T Cells
O O NH3 Cl
+ -
B Cells
7
O HO O OH OH
3
OH OH OH OH O OH H N O O HO H2N OH O O N N O O H N HN S N N NH2 O OH NH2 HO2C O N O O N O N H Glu-NH N N O O O N OH O NH3+Cl-
MOD-K
4 6
S O NH N H O O
Jurkat
HO2C O
HN
N O OH O O
H N
HN S HO2C
Jurkat
B Lymphocytes
S O N O N H H N CO2H O OH N O O NH S N H HO2C O
T Lymphocytes
Macrophages
OH O O
O HO2C
OH
HN S
H N
n NH O
O NH O O n H N S NH CO2H HO O O
Functionalized carbon nanotubes penetrate into the cells mainly following two mechanisms:
i) via an energy-independent mechanism (passive insertion and diffusion through lipid bilayer of the cell membrane nanoneedle penetration)
Solution of
Cell culture on coverslips
TEM images
Thickness= 90 nm
NH3+ClO O N
CELL MEDIUM
NH3+Cl-
O O
nuclear membrane
CYTOPLASM
Carbon nanotubes as suitable scaffold for the presentation of antigens to the immune system
Development of vaccines based on synthetic peptides
- Conjugation to protein carriers (BSA) are necessary to improve the Abs production
- The carrier presents the peptide to the immune system in the correct conformation
- Inert carriers system are ideal Molecular model of a helical peptide-CNT conjugate
N O O
O O O O O N O N N O
1)
(4) in DMF, 3h
6
Ac-Cys-FMDV O HN N O O S
NH2 O O N
O N H N O
S Ac-Cys-FMDV
O N O
3)
Pantarotto et al. J. Am. Chem. Soc. 2003, 125, 6160 Pantarotto et al. Chem. Biol. 2003, 2003, 10, 961
The peptide antigen should adopt the correct conformation when bound to the carrier system
The conformation should be the same as in the case of the native structure present in the protein, which contains the peptide epitope
Molecular model of the complex between a helical peptide-CNT conjugate and an antibody fragment (Fab: Fragment antigen binding)
Antigenic properties
Immunogenic properties
FMDV(141-159)-BSA
Control-peptide-BSA
CNT
80 RU
Bis FMDV-NT 7
40
Potential of carbon nanotubes on gene delivery: delivery and expression of plasmid DNA by functionalized CNT
Plasmid DNA condenses on the surface of the cationic carbon nanotubes forming supercoiled and globular-like structures
1.2E+05
1.0E+05
8.0E+04
The charge ratio between NH3+ at the SWNT surface and the phosphates of the DNA backbone is a determinant factor of the resulting levels of gene expression SWNT:DNA +/charge ratios between 2:1 and 6:1 offer 5 to 10 times higher levels of gene expression compared to DNA alone This first generation of functionalized carbon nanotubes is less effective for transfection in vitro than lipid:DNA system
6.0E+04
4.0E+04
2.0E+04
0.0E+00 Nave DNA only SWNT:DNA SWNT:DNA SWNT:DNA SWNT:DNA SWNT:DNA SWNT:DNA 1:1 2:1 4:1 6:1 8:1 10:1
Pantarotto D et al. Angew. Chem. Int. Ed. 2004, 43, 5242; Singh R et al. J. Am. Chem. Soc. 2005, 127, 4388
Functionalized multi-walled carbon nanotube delivery of cytotoxic siRNA for in vivo cancer therapy
Carbon nanotubes functionalized with ammonium groups can be exploited to form stable supramolecular complexes with nucleic acids based on +/- charge interactions. The complexes have a potential use for the development of novel therapies based on gene transfer or siRNA silencing
System 1
System 2
Tumor growth and survival following intratumoral administration of CNT:siRNA and liposome:siRNA complexes
Tumor growth was blocked and survival of the animal prolonged after intratumoral administration of cationic MWNT:siTOX in comparison to the control liposome:siTOX complexes in Calu 6 xenografts.
Cationic MWNT:siTOX complexes induce tumor collapse and apoptosis of human lung carcinoma (Calu 6 cells) xenograft tumors provoking extended apoptosis of the tumor cells.
Efficient intracellular transport and delivery of siRNA are critical to RNA interference potency. Cleavable functionalized SWNT carriers transport, release, and deliver siRNA in mammalian cells achieving highly efficient lamin A/C gene silencing compared to existing transfection agents.
Kam NWS et al. JACS 2005, 127, 12492
Double functionalization approach to preparation of carbon nanotubes carrying different active molecule for:
i)
O S HN N H O O NH
AmB
HO2C O O OH
FITC
OH HO H N O O O N OH O O OH NH2 O
OH O OH OH OH OH O OH
Control
AmB
O S HN N H O O NH
HO2C O O OH
AmB-CNT rapidly enter the cells and accumulate into the cytoplasm The conjugation of AmB to carbon nanotubes clearly reduces the toxic affect of the antifungal agent on mammalian cells
Wu W et al. Angew. Chem. Int. Ed. 2005, 44, 6358
*The MIC corresponds to the lowest concentration of compound that inhibited visible growth of the organism. Results given are mean values of two independent determinations performed in duplicate. C.i.: clinical isolate. In this table, the MIC values for MWNT-AmB and SWNT-AmB refer to the concentration of AmB in the conjugates (approximately one third by weight).
AmB-CNT preserve a high antifungal activity When equal amount of free and conjugated AmB are administered the CNT conjugates are more potent against certain strains
The reduced mammalian cell cytotoxicity and the increased antimycotic activity can be explained with: i) ii) iii) iv) Rapid internalization of AmB into the cytoplasm by the CNT reduces the possibility of disruption of the cell membrane Prevention of aggregation Increased solubility of the drug Binding to CNT and the presence of multiple copies of AMB per CNT favor the interaction of the drug with the fungal membrane
N N N
N N
NH2
NH2
N O O
H HN N S HO2C
0.5g
0.05g
0.5g
5g
0.05g
0.5g
5g
CNT-FITC-NH3
Apoptosis induced after 48 h
CNT-FITC-MTX
Apoptosis induced after 72 h
, O O N O N H LinkerNH O Glu_NH O N
N N NH2 N N NH2
OH O
N N NH2 N N NH2
120 100 80 60 40 20 0
***
CONTROL
10 % DMSO
MTX
MWNT 2
MWNT 3
MWNT 4
Cancer cell targeting using biotin and paclitaxel functionalized carbon nanotubes
Intracellular glutathione in the leukemia cells should be able to cleave the disulfide linkage over longer incubation times, but the endogenous glutathione level in cancer cells varies due to the significant difference in physiological conditions between cultivated cancer cells and those in the actual leukemia or solid tumors in vivo. The extracellular addition of excess glutathione ethyl ester is beneficial for rapid visualization of the drug release inside the leukemia cells. The fluorescein-labeled taxoid released from the conjugate inside the leukemia cells binds to the tubulin/microtubule that is the target protein of the drug. The fluorescent taxoid does bind to the target protein to light up the large bundles of microtubule.
Targeted killing of cancer cells in vivo using EGF functionalized carbon nanotubes
Raman spectroscopy
Inhibition of pre-established HN12 HNSCC (head and neck squamous cell carcinoma) tumor growth by SWNT-cisplatin-EGF bioconjugates. The nanotube bioconjugates were injected intravenously through the tail vein and observed for the tumor progression.
Antibody functionalized carbon nanotube for thermal ablation of cancer cells (covalent approach)
Specific killing of target cells by MAb-CNTs following exposure to NIR light AFM images of CNTs functionalized with MAbs
Morphology of Daudi cells following treatment with MAbCNT and NIR irradiation
Viability of different folate positive or negative cell lines treated with the platinum-based prodrug linked to SWNTs or alone in comparison to cisplatin.
The nanotube-PTX conjugates suppress tumor growth of 4T1 breast cancer mice model.
Lipo-Pegylated nanotubes distribute in the different organs of mice after 24 hour post injection as detected by measuring the retained activity of 64Cu-labelled material.
Water-soluble carbon nanotubes (SWNTs) were functionalized with PEG, radio labels and targeting RGD peptide. The hydrophobic carbon chains of the phospholipids strongly bind to the sidewalls of the nantubes, and the PEG chains render the material water soluble. The DOTA molecules on the nanotubes were used to chelate 64Cu for radio labelling.
The nanotubes were detected in mice tissues using characteristic Raman signatures of SWNTs.
Fluorescence signal
Intrinsic Fluorescence signal (Cherukuri et al, PNAS 2006)
Raman signal
Intrinsic Raman signal (Keren et al, PNAS 2008)
Raman Spectroscopy
Photoacoustic signal
Intrinsic Photoacoustic signal (De la Zerda et al, Nature Nanotech 2008)
Ultrasound
Radiometal signal from filled nanotubes (Hong et al, Nature Mater 2010)
SPECT (single photon emission computer tomography; PET (positron emission tomography)
Gadolinium
Grafted Paramagnetic signal (Al Faraj et al, NanoLetters 2009)
MRI
Gold
Grafted Photoacoustic/NIR signals (Kim et al, Nature Nanotech 2009)
Fluorescence Microscopy
NN
Radiometals
R
O R
(e.g.
Quantum Dots
Chemically Conjugated Fluorescence signal (Shi et al, Adv.Mat. 2007)
MWNT
N HO2C HO2C HO2C N N HO2C O = O O O N O N O O In O N H N O O
MWNT
N NH O O
N O O
[111In]DTPA
25
i.v. administration
20
30 min
15
3 hrs
10 5 0
24 hrs
Organ localization of radiolabelled nanotubes following tail-vein injection from time zero to five minutes (0-5 min)
Lacerda L et al. Adv. Mater. 2008, 20, 225
DTPA-CNT (SWNT, A,B; MWNT, C-H) collected from the urine 18 h post injection
ox-MWCNTs 2
ox-MWCNTs 3
MWCNTs from NANOAMOR ox-MWCNTs ox-MWCNTs + HRP (60 days) ox-MWCNTs + PSF (60 days)
MWCNTs from NANOCYL ox-MWCNTs ox-MWCNTs + HRP (60 days) ox-MWCNTs + PSF (60 days)
Conclusions
Translation to clinical applications is still early Toxicity issues are under strong debate Carbon nanotubes are potentially biodegradable Innovative technology
Acknowledgements
Immunologie et Chimie Thrapeutiques Strasbourg (France) Ccilia Mnard-Moyon Davide Pantarotto Cdric Klumpp Giorgia Pastorin Wei Wu Shouping Li Claire Gaillard Raquel Sainz Prabhpreet Singh Cristian Samor Enrica Venturelli Alessia Battigelli Giuseppe Lamanna Department of Pharmaceutical Sciences, University of Trieste (Italy) Maurizio Prato Nanomedicine Laboratory, Centre for Drug Delivery Research, The School of Pharmacy, University of London (UK) Kostas Kostarelos Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste (Italy) Renato Gennaro University of Besanon (France) Hlne Dumortier Marion Dcossas Julie Russier Christophe Ramseyer
Financial Support