Double functionalisation of carbon nanotubes for multimodal drug delivery

Article abstract of DOI:10.1039/b516309a

Multi-walled carbon nanotubes have been covalently functionalised via 1,3-dipolar cycloaddition of azomethine ylides with orthogonally protected amino functions that can be selectively deprotected and subsequently modified with drugs and fluorescent probe

Full text of DOI:10.1039/b516309a

Double functionalisation of carbon nanotubes for multimodal
drug delivery
Giorgia Pastorin,a Wei Wu,a Sébastien Wieckowski,a Jean-Paul Briand,a Kostas
Kostarelos,b Maurizio Prato*c and Alberto Bianco*a
Supporting Information
Experimental Section
General. The reactions were monitored by thin-layer chromatography (TLC) on silica gel
(F254 Merck) and the products were visualised on aqueous potassium permanganate or
ninhydrine spray followed by heating. Infrared spectra (IR) were measured on a Perkin Elmer
1
13
Spectrum One ATR-FT-IR Spectrometer. H and C NMR spectra were recorded in CDCl3
or CD3OD using a Bruker DPX 300 spectrometer (300 MHz for proton and 75 MHz for
carbon), the peak values were given as ppm (δ), using the tetramethylsilane or the residual
deuterated solvent protons as reference. Chromatographic purifications were done with silica
gel Merck (Kiesegel 60, 40-60 µm, 230-400 mesh ASTM) in standard column. Organic
phases were dried with sodium sulphate. Transmission electron microscopy (TEM) analyses
were performed on a TEM Hitachi 600 HS.
Synthesis of {2-[2-(2-Amino-ethoxy)-ethoxy]-ethyl}-carbamic acid benzyl ester (8)
O
O
O
O
NH2
O
N
H
O
O
NH2
N
+
H2N
O
O
O
O
8
A solution of Z-OSu (1.68 g, 7 mmol) in 1,4-dioxane (30 ml), was added dropwise, over a
period of 3 hours, to a solution of 2,2’-(ethylene-dioxy)bis(ethylamine) (10 g, 68 mmol) in
1,4-dioxane (50 ml). The reaction mixture was stirred overnight at room temperature. The
solvent was removed under reduced pressure and the residue dissolved in DCM (40 ml),
1

washed with water (3×100 ml), dried (Na2SO4) and evaporated. The unreacted diamine was
removed by flash chromatography in DCM/ MeOH 7:2. Yield: 1.4 g (5 mmol, 70%). FT-IR:
3334, 1718 cm-1.1H NMR (300 MHz, CDCl3): δ 1.78 (s, 2 H), 2.8-3.6 (m, 12 H), 5 (s, 2 H),
13
5.6 (bs, 1 H), 7.25-7.33 (m, 5 H). C NMR (75 MHz, CDCl3): δ 40.85, 41.56, 66.59, 70.02,
70.28, 73.14, 128.05, 128.30, 128.48, 136.63, 156.50.
Synthesis of {2-[2-(2-Benzyloxycarbonylamino-ethoxy)-ethoxy]-ethylamino}-acetic acid
tert-butyl ester (9)
O
O
O
H
N
O
O
O
NH2
Br
O
N
H
O
O
N
H
O
O
+
O
8
9
To a solution of {2-[2-(2-amino-ethoxy)-ethoxy]-ethyl}-carbamic acid benzyl ester 8 (1.4 g, 5
mmol) in 1,4-dioxane (30 ml) cooled to 0 °C, was added a solution of tert-butyl-bromoacetate
(0.32 g, 1.7 mmol) in 1,4-dioxane (15 ml), dropwise over a period of 1.5 hours.
The reaction mixture was allowed to reach the room temperature and was stirred for 6 hours.
The solvent was removed under reduced pressure and the residue dissolved in DCM (40 ml),
washed with water (3×100 ml), dried (Na2SO4) and evaporated. The final product was
purified by flash chromatography in DCM/MeOH 9.5:0.5. Yield: 0.5 g (1.3 mmol, 76%). FT-
1
IR: 3334, 1718, cm-1. H NMR (300 MHz, CDCl3): δ 1.25 (s, 9 H) 2.1 (bs, 2 H), 2.5-3.5 (m,
14 H), 4.88 (s, 2 H), 5.93 (bs, 1H), 7.13 (m, 5H). 13C NMR (75 MHz, CDCl3): δ 27.96, 40.77,
48.46, 51.38, 66.36, 69.95, 70.01, 70.12, 70.44, 80.99, 127.87, 127.94, 128,33, 136.66,
156.58, 171.42.
Synthesis of {2-[2-(2-Benzyloxycarbonylamino-ethoxy)-ethoxy]-ethylamino}-acetic acid
(10)
O
O
O
O
O
NH
O
NH
O
N
H
O
OH
O
N
H
O
O
9
10
2

To a solution of {2-[2-(2-benzyloxycarbonylamino-ethoxy)-ethoxy]-ethylamino}-acetic acid
tert-butyl ester 9 (0.5 g, 1.3 mmol) in MeOH (10 ml), 2 ml of trifluoroacetic acid were added.
The reaction mixture was stirred for 5 hours at room temperature. The solvent was removed
under reduced pressure and the residue was precipitated in diethyl ether as trifluoroacetic acid
1
salt. Yield: 0.4 g (1.3 mmol, 93 %). FT-IR: 3378, 1730, 1703 cm-1. H NMR (300 MHz,
CDCl3): δ 2.1 (bs, 1 H), 2.5-3.5 (m, 14 H), 5.06 (s, 2 H), 7.3 (m, 5H), 7.79 (bs, 1 H), 10.56
13
(bs, 1 H). C NMR (75 MHz, CDCl3): δ 40.45, 47.16, 65.60, 66.52, 66.91, 69.70, 116.57,
127.05, 128.49, 128.81, 136.62, 157.16, 161.87, 169.29.
Synthesis of {2-[2-(2-Amino-ethoxy)-ethoxy]-ethyl}-carbamic acid tert-butyl ester1 (11)
O
O
O
O
NH2
O
NH2
+
O
N
H
O
H2N
O
O
O
O
11
To a solution of 2,2’-(ethylene-dioxy)bis(ethylamine) (10 g, 68 mmol) in 1,4-dioxane (50 ml)
was added a solution of Boc2O (1.47 g, 6.8 mmol) in 1,4-dioxane (30 ml), dropwise over a
period of 3 hours. The reaction mixture was stirred overnight at room temperature. The
solvent was removed under reduced pressure and the residue dissolved in DCM (40 ml),
washed with water (3×100 ml), dried (Na2SO4) and evaporated. The unreacted diamine was
removed by flash chromatography in DCM/MeOH 8:2. Yield: 1.4 g (5.6 mmol, 84 %). FT-IR:
3356, 1691 cm-1.1H NMR (300 MHz, CDCl3): δ 1.39 (s, 9 H) 1.69 (bs, 2 H), 2.8-3.7 (m, 12
13
H), 5.1 (bs, 1 H). C NMR (75 MHz, CDCl3): δ 28.27, 40.16, 41.51, 70.04, 73.14, 78.83,
155.92.
Synthesis of {2-[2-(2-Benzyloxycarbonylamino-ethoxy)-ethoxy]-ethyl}-carbamic acid
tert-butyl ester2 (12)
O
O
O
O
NH2
O
+
O
NH
O
N
H
O
Br
O
N
H
O
O
O
11
12
3

To a solution of {2-[2-(2-amino-ethoxy)-ethoxy]-ethyl}-carbamic acid tert-butyl ester 11 (1.4
g, 5.6 mmol) in 1,4-dioxane (30 ml) cooled to 0 °C, was added a solution of benzyl-
bromoacetate (0.3 ml, 1.9 mmol) in 1,4-dioxane (15 ml), dropwise over a period of 1.5 hours.
The reaction mixture was allowed to reach the room temperature and was stirred for 6 hours.
The solvent was removed under reduced pressure and the residue dissolved in DCM (40 ml),
washed with water (3×100 ml), dried (Na2SO4) and evaporated. The final product was
purified by flash chromatography in DCM/ MeOH 9:1. Yield: 0.58 g (1.5 mmol, 78%). FT-
IR: 3338, 1739, 1709 cm-1. 1H NMR (300 MHz, CDCl3): δ 1.36 (s, 9 H), 2.1 (bs, 1 H), 2.7-3.6
13
(mt, 14 H), 5.1 (s, 2 H), 5.5 (bs, 1 H); 7.28 (m, 5 H). C NMR (75 MHz, CDCl3): δ 28.42,
40.36, 48.67, 50.79, 66.55, 70.14, 70.23, 70.55, 79.10, 128.37, 128.59, 135.58, 156.03,
172.07.
Synthesis of {2-[2-(2-tert-Butoxycarbonylamino-ethoxy)-ethoxy]-ethylamino}-acetic acid2
(13)
O
O
O
O
O
NH
O
NH
O
N
H
O
O
O
N
H
O
OH
12
13
To a solution of {2-[2-(2-benzyloxycarbonylamino-ethoxy)-ethoxy]-ethyl}-carbamic acid
tert-butyl ester 12 (0.58 g, 1.5 mmol) in methanol (20 ml), were added 50 mg of Pd/C (10%),
in the presence of H2. The reaction mixture was stirred for 5 hours at room temperature. The
solution was filtered through a celite pad and the solvent was evaporated under reduced
1
pressure. Yield: 0.4 g (1.4 mmol, 93%). FT-IR: 3346, 1694, 1626 cm-1. H NMR (300 MHz,
CDCl3): δ 1.39 (s, 9 H) 1.69 (bs, 2 H), 3.2-3.78 (m, 14 H), 7.8 (bs, 1 H), 10.56 (bs, 1 H). 13C
NMR (75 MHz, CDCl3): δ 28.46, 40.35, 46.54, 50.02, 66.54, 70.04, 70.15, 70.29, 79.05,
156.14, 170.83.
Synthesis
of
(2-{2-[2-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-ethoxy]-ethoxy}-ethyl)-
carbamic acid tert-butyl ester3 (14)
O
O
O
O
OCH3
OH
N
O
+
H2N
O
O
N
H
O
O
N
H
O
O
O
14
13
4

A solution of {2-[2-(2-benzyloxycarbonylamino-ethoxy)-ethoxy]-ethyl}-carbamic acid tert-
butyl ester 13 (1.5 g, 6 mmol), mono-methylphthalate (1 g, 5.76 mmol), BOP (2.55 g, 5.76
mmol) and DIEA (3 µl, 17.28 mmol) in CH3CN (40 ml) was stirred at room temperature for 3
hours. A solution of Na2CO3 (1.2 g, 11.52 mmol) in water (20 ml) was subsequently added
and the reaction was stirred for 5 hours at room temperature. The solvent was removed under
reduced pressure. AcOEt was added and the solution was washed with saturated NaHCO3,
water, 1N KHSO4 and water. The organic phase was dried with Na2SO4 and the solvent was
evaporated. The product was obtained as an oil and used without further purifications. Yield:
1.66 g (4.4 mmol, 76%). FT-IR: 3388, 1774, 1707 cm-1. 1H NMR (300 MHz, CDCl3): δ 1.36
(s, 9 H), 3.2-3.6 (m, 12 H), 7.5 (bs, 1 H), 7.75 (dd, 2 H), 7.81 (dd, 2 H). 13C NMR (75 MHz,
CDCl3): δ 27.28, 36.08, 39.24, 59.04, 66.66, 68.80, 69.02, 77.41, 121.98, 130.88, 132.93,
154.85, 166.84.
Synthesis of 2-{2-[2-(2-Amino-ethoxy)-ethoxy]-ethyl}-isoindole-1,3-dione (15)
O
O
O
N
O
N
O
+
O
N
H
O
O
NH3 TFA-
O
O
15
14
To the derivative (2-{2-[2-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-ethoxy]-ethoxy}-ethyl)-
carbamic acid tert-butyl ester 14 (1.66 g, 4.4 mmol) was added a solution of TFA/CH2Cl2 (1:1
v/v). The reaction mixture was stirred at room temperature for 30 min. The solvent was
removed under reduced pressure and the residue was precipitated several times from
DCM/Et2O as trifluoroacetic acid salt. Yield: 1.7 g (6.9 mmol, 93 %). FT-IR: 3438, 3332,
1771 cm-1. 1H NMR (300 MHz, CDCl3): δ 3.2-3.87 (m, 12 H), 7.75 (dd, 2 H), 7.81 (dd, 2 H).
13C NMR (75 MHz, CDCl3): δ 37.31, 39.68, 68.51, 68.24, 69.64, 70.05, 118.60, 123.37,
131.87, 134.17, 161.89, 168.57.
Synthesis of (2-{2-[2-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-ethoxy]-ethoxy}-ethylamino)-
acetic acid benzyl ester (16)
O
O
O
+
Br
O
N
O
O
N
O
O
NH2
O
NH
O
O
O
16
15
5

To a solution of 2-{2-[2-(2-amino-ethoxy)-ethoxy]-ethyl}-isoindole-1,3-dione 15 (1.7 g, 6.9
mmol) and DIEA (2 µl, 6 mmol) in 1,4-dioxane (20 ml) cooled to 0 °C was added benzyl-
bromoacetate (0.4 ml, 2.3 mmol) in 1,4-dioxane (5 ml), dropwise over a period of 1.5 hours.
The reaction mixture was allowed to warm to room temperature and stirred for 6 hours. The
solvent was removed under reduced pressure. The final product was purified by flash
cromathography in AcOEt/cyclohexane 8:2. Yield: 0.5 g (1.3 mmol, 55%). FT-IR: 3438,
3430, 1770, 1700 cm-1. 1H NMR (300 MHz, CDCl3): δ 2.1 (bs, 1 H), 2.7-3.8 (m, 14 H), 4.95
13
(s, 2 H), 7.16 (s, 5 H), 7.49 (dd, 2 H), 7.62 (dd, 2H). C NMR (75 MHz, CDCl3): δ 37.09,
48.53, 50.70, 60.13, 67.72, 69.85, 70.09, 70.47, 122.99, 128.13, 128.40, 131.96, 133.76,
135.67, 167.94, 171.94.
Synthesis of (2-{2-[2-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-ethoxy]-ethoxy}-ethylamino)-
acetic acid (17)
O
O
OH
N
O
O
N
O
O
NH
O
NH
O
O
O
O
16
17
To
a
solution
of
(2-{2-[2-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-ethoxy]-ethoxy}-
ethylamino)-acetic acid benzyl ester 16 (0.5 g, 1.3 mmol) in methanol (20 ml), were added 50
mg of Pd/C (10%), in the presence of H2. The reaction mixture was stirred for 5 hours at room
temperature. The solution was filtered through a celite pad and the solvent was evaporate
under reduced pressure. Yield: 0.4 g (1.2 mmol, 93%). FT-IR: 3438, 3332, 1771, 1701 cm-1.
1H NMR (300 MHz, CD3OD): δ 3.18 (bs, 1 H), 3.24-3.86 (m, 14 H), 7.49 (dd, 2 H), 7.62 (dd,
2H), 10.56 (bs, 1 H). 13C NMR (75 MHz, CD3OD): δ 36.97, 46.87, 47.30, 65.59, 67.66,
69.77, 69.99, 122.75, 131.89, 134.04, 168.37.
6

Procedures for the double functionalisation of carbon nanotubes
Procedure 1: functionalisation with Boc- and Z-protected amino acids (2).
O
O
N
H
O
O
O
O
N
N
HN
HOOC
O
N
H
O
O
MWNT
H
N
O
HOOC
N
O
H
H
O
O
N
O
O
O
(CH2O)n in DMF 125 °C
1
2
CNT (100 mg) and paraformaldehyde (3 × 150 mg every 24 hours) were suspended in 100 ml
of DMF. 150 mg of Boc-NH-(CH2CH2O)2-CH2CH2-NHCH2COOH (13) and 167 mg Z-NH-
(CH2CH2O)2-CH2CH2-NHCH2COOH (10) (1:1 molar ratio) were added and the reaction was
heated at 125 °C for 72 hours. The heating was stopped. Unreacted CNT remained completely
insoluble in DMF and were separated from the soluble functionalised tubes by centrifugation
and filtration through a 0.22 µm PTFE filter. The filtrate was evaporated and the brown
residue was dissolved in DCM, washed once with water and dried over Na2SO4. The solvent
was evaporated and the product was reprecipitated several times from methanol/diethyl ether.
MWNT 2 were characterised by TEM (Fig. S1) and 1H-NMR (Fig. S2).
Figure S1. TEM image of MWNT 2.
7

1.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
Figure S2. 1H-NMR spectrum of MWNT 2.
Cleavage of the Boc protecting group (3)
O
O
N
H
O
O
O
O
O
O
NH3+Cl-
N
N
N
N
4 M HCl in dioxane
H
N
H
O
O
N
O
O
O
O
3
2
A solution of HCl 4 M in dioxane is added to MWNT 2 (5 mg) and the mixture was stirred for
5 hours at room temperature. The solvent was removed under reduced pressure and the
product was reprecipitated several times from methanol/diethyl ether. MWNT 3 were
characterised by TEM and 1H-NMR. Quantitative Kaiser test: 0.35 mmol/g of free NH2.
Acetylation of MWNT 3 and cleavage of Z protecting group4 (4)
O
NH3+Cl-
O
O
O
O
O
O
N
H
N
N
1) Ac2O
2) TFA/TMSOTf/p-cresol
N
N
H
NH3+TFA-
O
N
O
O
O
4
3
MWNT 3 (5 mg) were solubilised in 600 µl of DMF and 200 µl of acetic anhydride were
added. The solution was stirred for 1.5 hours and then precipitated several times in cold
8

diethyl ether. Qualitative Kaiser test was negative. A solution (400 µl) of TFA/TMSOTf/p-
cresol (325:87:42.5, µl/µl/mg) (TMSOTf, trimethylsilyltrifluoromethanesulfonate) was added
to the acetylated MWNT 3 (5 mg) and the mixture was stirred for 12 hours at room
temperature. The product was directly precipitated in diethyl ether and washed several times
by reprecipitation from methanol/diethyl ether. MWNT 4 were characterised by TEM and 1H-
NMR. Quantitative Kaiser test: 0.35 mmol/g of free NH2.
Procedure 2: functionalisation with Boc- and phthalimide-protected amino acids (5)
O
O
N
H
O
O
O
N
N
HOOC
HN
O
N
H
O
O
MWNT
O
O
O
N
O
HOOC
NH
O
O
N
O
O
(CH2O)n in DMF 125 °C
1
5
CNT (100 mg) and paraformaldehyde (3 × 150 mg every 24 hours) were suspended in 100 ml
of DMF. 150 mg of Boc-NH-(CH2CH2O)2-CH2CH2-NHCH2COOH (6) and 165 mg Pht-NH-
(CH2CH2O)2-CH2CH2-NHCH2COOH (10) (1:1 molar ratio) were added and the reaction was
heated at 125 °C for 72 hours. The heating was stopped. Unreacted CNT remained completely
insoluble in DMF and were separated from the soluble functionalised tubes by centrifugation
and filtration through a 0.22 µm PTFE filter. The filtrate was evaporated and the brown
residue was dissolved in DCM, washed once with water and dried over Na2SO4. The solvent
was evaporated and the product was reprecipitated several times from methanol/diethyl ether.
MWNT 5 were characterised by TEM (Fig. S3) and 1H-NMR (Fig. S4).
Figure S3. TEM image of MWNT 5.
9

8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
Figure S4. 1H-NMR spectrum of MWNT 5. The two multiplets at about 1.0 and 3.4 ppm
correspond to residual diethyl ether.
Cleavage of the phthalimide protecting group and functionalisation with fluorescein
isothiocyanate (6)
O
O
O
O
N
H
O
O
O
N
H
O
N
N
1) N2H4 in ethanol
2) FITC in DMF
OH
O
O
HN
S
N
H
N
N
O
O
N
O
O
HO2C
O
O
6
5
Hydrazine (36.5 µl, 752 µmol) in ethanol (10 ml) was added to double functionalised MWNT
5 (9.4 mg) and the mixture was stirred for 16 hours at room temperature under argon. The
solvent was removed, methanol was added and the solution was centrifuged to eliminate
phthalhydrazide. The product was reprecipitated several times from methanol/diethyl ether.
Amino-functionalised MWNT 5 (7 mg, 4.2 µmol corresponding to the amount of free NH2
measured by quantitative Kaiser test) were solubilised in 200 µl of DMF. A solution of
fluorescein isothiocyanate (FITC) (4.9 mg, 12.6 µmol) in 200 µl of DMF was added and the
solution was stirred overnight at room temperature. The solvent was removed and the product
was precipitated several times from methanol/diethyl ether. Qualitative Kaiser test was
negative. MWNT 6 were characterised by TEM (Fig. S5). Quantitative Kaiser test: 0.65
mmol/g of NH2.
10

Figure S5. TEM image of MWNT 5 after the cleavage of Pht protecting group.
Reaction of fluorescein functionalised carbon nanotubes with methotrexate5 (7)
N
N
N
NH2
α,γ
O
O
N
N
O
O
O
Glu-NH
NH2
O
O
N
H
O
O
N
H
1) 4M HCl in dioxane
N
N
N
N
O
2) Methotrexate, Bop/DIEAin DMF
OH
OH
O
O
O
HN
HN
S
H
H
N
N
O
O
HO2C
HO2C
S
O
6
7
A solution of HCl 4 M in dioxane is added to MWNT 6 (6 mg) and the mixture was stirred for
5 hours at room temperature. The solvent was removed under reduced pressure and the
product was reprecipitated several times from methanol/diethyl ether. The functionalised
1
carbon nanotubes were characterised by TEM and H-NMR. Quantitative Kaiser test: 0.30
mmol/g of free NH2. Methotrexate (650 µg, 1.44 µmol), DIEA (1 µl) and BOP (637 µg, 1.44
µmol) were mixed in 50 µl of DMSO. After stirring for 30 min, a solution of MWNT 6
without Boc (6 mg, 1.2 µmol corresponding to the amount of free NH2 measured by
quantitative Kaiser test) in DMSO and DIEA (1µl) was added. The mixture was stirred
overnight at room temperature. The amino-PEGA resin (36 mg, 14.4 µmol) was added and
the solution was stirred slowly for 2 hours. Then the resin was filtered and washed several
11

times (≥ 5) with MeOH. The solvent was removed and the product was precipitated in Et2O.
MWNT 7 were characterised by TEM and UV-Vis (Fig. S6).
1,4
1,2
1,0
0,8
MTX
0,6
FITC
0,4
0,2
0,0
250 300 350 400 450 500 550 600 650 700
Wavelength (nm)
Figure S6. UV-Vis spectrum of 7 in methanol.
Cells and cell cultures
Preparation of MWNT 7
MWNT 7 were dissolved in DMSO/water (0.09/1, v/v) at different concentrations (0.05, 0.5,
+
and 5 µg/ml). To have some controls, two other solutions of MWNT-FITC-NH3 , derived
from MWNT 6 after Boc cleavage, (0.05, 0.5 and 5 µg/ml) and MTX alone (0.005, 0.05 and
0.5 µg/ml) were prepared in DMSO/water (0.09/1, v/v).
Cell culture and treatment with MWNT 7
Jurkat human T leukemia cell line was cultured in RPMI 1640 (Cambrex Bioscience)
supplemented with gentamicin (10 µg/ml) and 10% of heat-inactivated fetal bovine serum.
Cells were grown in suspension at 37 °C in a humidified atmosphere with 5% CO2. For the
tests cell suspensions were prepared at a final concentration of 5×105 cells/ml in 1 ml of
medium containing 10 µl of indicated MWNT 7 solutions (final concentration: 0.05 to 5
µg/ml). As resulted from the dilution on cell medium the maximum final percentage of
DMSO in cell culture is lower than 0.01%. The cells were then incubated at 37 °C for 1 and
24 hours. After incubation, all of the cells were washed twice with 1 ml of phosphate buffered
12

saline (PBS) and then analysed for intracellular MWNT 7 content by flow cytometry and
fluorescence microscopy.
Flow cytometry measurements
After two washes in PBS, cells were resuspended in 300 µl of PBS and analysed with the flow
cytometer FACSCalibur® operating at 488 nm excitation wavelength and detecting emission
wavelengths with a 530/30 nm band-pass filter. At least 25,000 cells were acquired using the
CellQuest 3.3 software (Becton & Dickinson) and distribution of the FITC fluorescence was
analysed with the WinMDI 2.8 freeware (Joseph Trotter, Scripps Research Institute).
Epifluorescence and confocal microscopy
After two washes in PBS, cells were resuspended in ready to use Fluorescent Mounting
Medium (DakoCytomation) and mounted between glass slide and coverslip. The distribution
of fluorescence was analysed on an Olympus BX51 microscope with a FITC wide-band cube
(460-490 nm excitation filter and 515-550 nm emission transmission range) using the
AnalySIS 3.0 software. A Zeiss LSM 510 Meta confocal microscope was also used operating
at 488 nm excitation wavelength and detecting emission wavelengths with a 505-550 nm
band-pass filter. Data were analysed using the ImageJ 1.33u freeware (Wayne Rasband,
National Institutes of Health) and the LSM Reader 3.2d plugin (Patrick Pirrotte, Yannick
Krempp and Jerome Mutterer, Institute for Molecular Biology of Plants, Strasbourg, France).
Cell penetration of the precursor of MWNT 7
For comparison to the behaviour of MWNT 7, we have verified the capacity of MWNT 6
without the Boc protecting group to penetrate into the cells by epifluorescence microscopy.
Again human Jurkat cells were cultured using RPMI 1640 medium at 37 °C. Functionalised
MWNT were added to the cells at room temperature in a range of concentration between 0.05
and 5 µg/ml, and the cells were incubated at 37 °C for 1 h. After this period, the cells were
carefully rinsed, mounted on a microscope slide and observed under the microscope. Fig. S7
shows the bright light and epifluorescence images of the cells after being treated with 0.5
µg/ml of MWNT 6 without Boc. It is evident that CNT accumulated into the cell. In addition,
we have studied the dose dependence of the internalisation process. Fig. S8 shows the flow
cytometry analysis of the cells treated with three different amounts of MWNT 6 without Boc
for 24 hours. Finally, we have measured the effect of time on the internalisation of MWNT 6
without Boc in comparison to MWNT 7 at the different doses (Fig. S9). The process was
13

followed between 0 and 24 hours. We have observed a linear correlation with a rapid kinetics
of uptake for the highest dose used (5 µg/ml). The dose of 0.05 µg/ml was too low to detect a
clear shift in the fluorescence signal after 24 hours.
Figure S7. Bright field (left) and epifluorescence (right) images of Jurkat cells incubated for 1
h at 37 °C with 0.5 µg/ml of MWNT 6 after cleavage of Boc protecting group.
Figure S8. Dose-response of the internalisation after incubation of Jurkat cells for 24 h at 37
°C with increasing amount of MWNT 6 after cleavage of Boc protecting group [0.05 (red
line), 0.5 (blue line) and 5 (magenta line) µg/ml]. FL1-H corresponds to FITC intensity.
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A
B
C
D
Figure S9. Effect of the time on the internalisation of MWNT 6 without Boc (A, B) and
MWNT 7 (C, D) at 37 °C. A, C: 0.5 µg/ml. B, D: 5 µg/ml. Black line, 0 h; red line, 1 h; blue
line, 24 h. FL1-H corresponds to FITC intensity.
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