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Carbon-based nanomaterials: biomedical applications

Alberto Bianco

CNRS, Laboratoire dImmunologie et Chimie Thrapeutiques, Strasbourg, France

Immunologie et Chimie Thrapeutiques

NOIS 2011, May 16-20, 2011, Anglet, France

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

Dimensions of the main classes of nanoparticles

Pharmaceutical Research 2006, 23, 1417

The right size of nano-objects


Nanoscale devices are of the same scale of biologically important molecules

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

Biomedical applications of carbon nanotubes


- Carbon nanotubes as substrates for neuronal and cell growth

- Carbon nanotubes for implants

- Carbon nanotubes as biosensors for sugars, antigens, proteins, antibodies

- Carbon nanotubes for the delivery of therapeutic agents

- Carbon nanotubes for cancer therapy: hyperthermia (by NIR, RF activation); chemotherapy; radiotherapy; gene therapy

Carbon nanotubes in medicine


Only a few types of CNTs used in medical applications

Kostarelos K, Bianco A, Prato M, Nature Nanotech. 2009, 4, 627

Do functionalized carbon nanotubes penetrate into the cells?


Culture medium

A549

S. cereE. coli visiae


O

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

Kostarelos K et al. Nature Nanotech. 2007, 2, 108

Cell uptake of functionalized CNT by immunocompetent cells

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

Dumortier H et al. Nano Lett. 2006, 6, 1522

Possible cell uptake mechanisms of functionalized carbon nanotubes

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)

ii) via an energy-dependent mechanism (endocytosis, phagocytosis)

Direct visualization of carbon nanotubes into the cells

Solution of
Cell culture on coverslips

f-MWNT or f-SWNT in PBS

Washing Fixation Staining

Embedding into a resin Sectioning


DIAMOND KNIFE

TEM images

Thickness= 90 nm

Bianco A et al. Chem. Commun. 2005, 571

Direct visualization of carbon nanotubes into the cells

NH3+ClO O N

Pantarotto D et al. Angew. Chem. Int. Ed. 2004, 43, 5242

Nanoneedle cell penetration of carbon nanotubes


Functionalized carbon nanotubes penetrate like nanoneedles piercing the cell membrane without inducing cell death

CELL MEDIUM
NH3+Cl-

Golgis complex chromatin internalised MWNT


N

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

- Peptide antigens are poorly immunogenic

- 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

- Protein carrier is intrinsically immunogenic

- Generated Abs present low specificity

- Inert carriers system are ideal Molecular model of a helical peptide-CNT conjugate

Peptide antigens conjugated to carbon nanotubes for synthetic vaccine delivery


Ac-Cys-FMDV S O
H N

N O O

O O O O O N O N N O

1)

(4) in DMF, 3h

2) Ac-Cys-GSGVRGDFGSLAPRVARQL (5) (Ac-Cys-FMDV) in H2O, 6h

6
Ac-Cys-FMDV O HN N O O S

NH2 O O N

H N 1) Boc-Lys(Boc)-OH, DIC/HOBt in DMF, 3h N 2) TFA, 2h O O N O in DMF, 3h O O O

O N H N O

S Ac-Cys-FMDV

O N O

3)

4) Ac-Cys-GSGVRGDFGSLAPRVARQL (Ac-Cys-FMDV) in H2O, 9h

Pantarotto et al. J. Am. Chem. Soc. 2003, 125, 6160 Pantarotto et al. Chem. Biol. 2003, 2003, 10, 961

Foot-and-Mouth Disease Virus (FMDV)

Immunological characterization of peptide-carbon nanotubes

Antigenicity is the capacity of a peptide antigen to be specifically recognized by an antibody

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

Immunogenicity is the capacity of a peptide antigen to elicit an immune response

Molecular model of the complex between a helical peptide-CNT conjugate and an antibody fragment (Fab: Fragment antigen binding)

Antigenic and immunogenic characterization of peptide-carbon nanotubes

Antigenic properties

Immunogenic properties

FMDV(141-159)-BSA

Control-peptide-BSA

CNT

Surface plasmon resonance (SPR) analysis


120

6 Log10 antibody titer 5 4 3 2 1 0


Free peptide Mono conjugate 6 Bis conjugate 7

80 RU

Bis FMDV-NT 7

40

Mono FMDV-NT 6 FMDV-peptide

0 Ac-NT 0 200 Time (s) 400 600

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

B-gal Expression (RLU/well)

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

Podesta JE et al. Small 2009, 5, 1176

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.

Collapse and apoptosis of human xenograft tumor by CNT:siRNA complexes

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.

Functionalized carbon nanotubes with a cleavable linker for siRNA delivery

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

Antibiotic carbon nanotube conjugates


Amphotericin B (AmB) is a potent antifungal agent for the treatment of chronic fungal infections AmB is highly toxic for mammalian cells (likely because of the formation of aggregates which reduce the solubility in water)

Double functionalization approach to preparation of carbon nanotubes carrying different active molecule for:

i)

Optical signal for imaging


OH HO H N O O O N OH O O OH NH2 O OH O OH OH OH OH O OH

ii) Specific targeting iii) Recognition capacity

O S HN N H O O NH

AmB

HO2C O O OH

FITC

Wu W et al. Angew. Chem. Int. Ed. 2005, 44, 6358

Functionalized carbon nanotubes with antibiotics


Activity of AmB conjugated to carbon nanotubes

OH HO H N O O O N OH O O OH NH2 O

OH O OH OH OH OH O OH

60 % Apoptotic and/or dead cells 50 40 30 20 10 0

Control

MWNT 4 (40 g/ml)

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

Antifungal activity of AmB conjugated to carbon nanotubes


Minimum inhibitory concentration (MIC)* in g/ml
C. parapsilosis ATCC90118 C. famata (c.i.) C. albicans (c.i.) C. neoformans ATCC90112 S. cerevisiae (c.i)

AmB + SWNT-NH3 MWNT-AmB SWNT-AmB

20 > 80 1.6 1.6

20 > 80 0.8 1.6

> 80 > 80 6.4 13.8

5 > 80 0.8 0.8

2.5 > 80 0.8 1.6

*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

Carbon nanotubes functionalized with anticancer molecules: Methotrexate


, O O N O N Glu-NH H O OH
D

N N N

N N

NH2

NH2

N O O

H HN N S HO2C

100 % of specific apoptosis 80 60 40 20 0 5ng 0.05g MTX


Apoptosis induced after 24 h Pastorin G et al. Chem. Commun. 2006, 1182

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

Enhanced anticancer activity


, O O N O N H Glu_NH O OH O N N N NH2 N N NH2

, O O N O N H LinkerNH O Glu_NH O N

N N NH2 N N NH2

OH O

O O N H N O O N H LinkerO O Glu_NH O O O O S N H O SO3O N N+ N

Percentage Cell Survival

N N NH2 N N NH2

120 100 80 60 40 20 0

***

Samor C et al. Chem. Commun. 2010, 46, 1494

CONTROL

10 % DMSO

MTX

MWNT 2

MWNT 3

MWNT 4

Functionalization of carbon nanotubes with anticancer molecules: paclitaxel

Chen J et al. JACS 2008, 130, 16778

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

Transmission electron microscopy

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

Marches et R al. Int. J. Cancer 2009, 125, 2970

Non covalent functionalization with therapeutic molecules

Targeting carbon nanotubes as delivery systems for platinum(IV) anticancer based-drugs


Folate receptor (FR)-mediated targeting of SWNT-platinum based-drugs by endocytosis Fluorescent SWNTs uptake by folate overexpressing cells

Viability of different folate positive or negative cell lines treated with the platinum-based prodrug linked to SWNTs or alone in comparison to cisplatin.

Dhar S et al. JACS 2008, 130, 11467

Drug delivery with carbon nanotubes for cancer treatment


Carbon nanotube were functionalized with paclitaxel (PTX) using phospholipids with branched PEG chains. The PTX molecules are bound via a cleavable ester bonds. This allows for releasing of PTX from nanotubes by ester cleavage in vivo. The SWNT-PTX conjugates are stably suspended in normal physiological buffer and serum without aggregation.

The nanotube-PTX conjugates suppress tumor growth of 4T1 breast cancer mice model.

Liu Z et al. Cancer Res. 2008, 68, 6652

Drug delivery with carbon nanotubes for cancer treatment

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.

Liu Z et al. Nature Nanotech. 2007, 2, 47

Drug delivery with carbon nanotubes for cancer treatment


Integrin positive tumour (U87MG) in mice was targeted by RGD-functionalized SWNTs. Micro positron emission tomography (PET) images clearly show accumulation of the nanotube conjugates into the tumour. High tumour uptake of SWNTPEG5400RGD is observed in the U87MG tumour in contrast to the low tumour uptake of SWNTPEG2000RGD.

The nanotubes were detected in mice tissues using characteristic Raman signatures of SWNTs.

Different Carbon Nanotubes in Biomedical Imaging

Carbon nanotubes in biomedical imaging Intrinsic CNT signals


NIR Fluorescence Microscopy

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

Carbon nanotubes in biomedical imaging

Filled CNT signals


Iron Selenium Copper

Magnetic Resonance Imaging (MRI) Raman Spectroscopy

Radiometals (e.g. Iodine)

SPECT PET MRI

Radiometal signal from filled nanotubes (Hong et al, Nature Mater 2010)

SPECT (single photon emission computer tomography; PET (positron emission tomography)

Carbon nanotubes in biomedical imaging Coated/Grafted (non-chemically conjugated) signals


Raman Spectroscopy Mass Spectroscopy MRI Synchrotron X-ray Fluorescence microscopy

Metal Impurities (e.g. Iron) Carbon Isotopes (13C)

Gadolinium
Grafted Paramagnetic signal (Al Faraj et al, NanoLetters 2009)

MRI

Gold
Grafted Photoacoustic/NIR signals (Kim et al, Nature Nanotech 2009)

NIR spectroscopy and Ultrasound

Carbon nanotubes in biomedical imaging Chemically conjugated signals to CNT

Fluorescent molecules (e.g. FITC, Alexa, Rhodamine)


O OO
O

Fluorescence Microscopy

NN

Radiometals
R
O R

(e.g.

111In, 64Cu, 90Y, 131I)

Chemically Conjugated Radiation signal (McDevitt et al, PLoS One 2007)

SPECT PET MRI

Quantum Dots
Chemically Conjugated Fluorescence signal (Shi et al, Adv.Mat. 2007)

Fluorescence Microscopy Luminescence

Biodistribution and elimination of functionalized carbon nanotubes

Tissue biodistribution of functionalized carbon nanotubes


O O N O N H N HO2C CO2H N N CO2H CO2H N O O N H

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

% injected Dose/g tissue

20

30 min
15

3 hrs
10 5 0

24 hrs

Tissues and organs


Singh R et al. PNAS 2006, 103, 3357

Real-time distribution of functionalized carbon nanotube

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

Tissue biodistribution of radiolabelled carbon nanotubes

Lacerda L et al. Adv. Mater. 2008, 20, 225

Carbon nanotube excretion via the urine

Scale bar 100 nm

DTPA-CNT (SWNT, A,B; MWNT, C-H) collected from the urine 18 h post injection

Mechanism of carbon nanotube renal excretion


The blood purification process is guaranteed by the typical leaky tissue of the glomerular capillary bed, rich of thousands of small orifices that act like a filter. Since the dimensions of the CNTs are smaller than the pore of the glomerulus, they are able to pass through and be excreted by the urine.

Lacerda L et al. Small 2008, 4, 1130

Biodegradation of functionalized carbon nanotubes


Day 0
ox-SWCNTs 1

PSF Day 7 Day 30 Day 60 Day 7

HRP Day 30 Day 60

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)

Russier J. et al. Nanoscale 2011, 3, 893

Conclusions

Organic functionalization improves dispersibility/solubility of CNTs


in physiological conditions

Functionalized CNTs are particularly promising for treating cancer


diseases

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

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