Azide-functionalized nanoparticles as quantized building block
for the design of soft-soft fluorescent polystyrene core – PAMAM
shell nanostructures
Krystyna Baczko,a Hélène Fensterbank,a Bruno Berini,b Nadège Bordage,c Gilles Clavier,c
Rachel Méallet-Renault,c,d Chantal Larpenta and Emmanuel Allard*a
a
Institut Lavoisier de Versailles UMR-CNRS 8180, Université de Versailles-Saint-Quentinen-Yvelines. 45 avenue des Etats-Unis, 78035 Versailles cedex, France.
E-mail: emmanuel.allard@uvsq.fr; Tel: (+33) 139-25-44-12
Groupe d’Etude de la Matière Condensée UMR-CNRS 8635, Université de Versailles-SaintQuentin-en-Yvelines. 45 avenue des Etats-Unis, 78035 Versailles cedex, France.
b
c
PPSM, ENS Cachan, CNRS. 61 avenue du Président Wilson, 94230 Cachan Cedex, France.
Present address : Institut des Sciences Moléculaires d’Orsay, CNRS-Université Paris-Sud
(UMR 8214), Bât 350, 91405 Orsay Cedex, France
d
S-1
Tables of Contents
Pages
Figure S1: Structures and numbering of Bodipy 5 and 6 used
for 1H and 13C NMR signals assignments.
S4
Figure S2: Structures and numbering of PAMAM_[CO2CH3]16 (3-G2.5)
and PAMAM_[NH2]16 (3-G3) used for 1H and 13C NMR signals assignments.
S4
Synthesis of functionalized nanoparticles NP_Cl and NP_N3
S5
Table S1: Characteristics of chlorobenzyl- and azide-functionalized nanoparticles
S6
Synthesis of amino-terminated PAMAM[NH2]2 G0
S6
Synthesis of amino-terminated PAMAM[NH2]4 (1-G1)
S7
Synthesis of amino-terminated PAMAM[NH2]8 (2-G2)
S8
Synthesis of functionalized nanoparticles NP_PAMAM[NH2]4
S9
Synthesis of functionalized nanoparticles NP_PAMAM[NH2]8
S10
Synthesis of functionalized nanoparticles NP_PAMAM[NH2]16
S11
Synthesis of functionalized nanoparticles NP_Bodipy_PAMAM[NH2]8 (two steps)
S11
Synthesis of functionalized nanoparticles NP_Bodipy_PAMAM[NH2]16 (two steps)
S12
Synthesis of functionalized nanoparticles NP_Bodipy_PAMAM[NH2]4 (one pot)
S13
Synthesis of functionalized nanoparticles NP_Bodipy_PAMAM[NH2]8 (one pot)
S14
Figure S3: AFM Topographic images obtained at pH = 7
of NP_Bodipy_PAMAM[NH2]8 (A) and NP_Bodipy_PAMAM[NH2]16 (B)
S14
Figure S4. Absorption (lines) and Fluorescence emission (dotted lines)
spectra (exc = 495nm) of 6 diluted in DTAB 5 wt%.
S15
Figure S5. Absorption (A) and Fluorescence emission (B) spectra (exc = 495nm) of
NP_Bodipy_PAMAM[NH2]4 diluted in DTAB 1 wt% for pH values from 1.1 to 9.3
S16
Figure S6. Absorption (A) and Fluorescence emission (B) spectra (exc = 495nm) of
NP_Bodipy_PAMAM[NH2]8 diluted in water for pH values from 1.1 to 9.3
S17
Figure S7. Absorption (A) and Fluorescence emission (B) spectra (exc = 495nm) of
NP_Bodipy_PAMAM[NH2]16 diluted in water for pH values from 1.1 to 9.3
S18
Figure S8. Estimation of the radius of benzyl triazole functionalized
amino-terminated PAMAM dendron (1-G1) and (2-G2)
S19
S-2
Figure S9 and S10: Estimation of the radius of benzyl triazole functionalized
amino-terminated PAMAM dendron (3-G3)
S20
Table S2: Estimation of the maximum number (Nmax) of amino-terminated PAMAM
dendrons that could be grafted on the NP surface
S21
Table S3: Estimation of the number of Bodipy grafted on the NP surface
S22
S-3
Figure S1. Structures and numbering of Bodipy derivatives 5 and 6 used for 1H and 13C NMR signals
assignments.
PAMAM_[NH2]16 (3-G3)
PAMAM_[CO2CH3]16 (3-G2.5)
Figure S2. Structures and numbering of esters terminated PAMAM_[CO2CH3]16 (3-G2.5) and amino
terminated PAMAM_[NH2]16 (3-G3) used for 1H and 13C NMR signals assignments.
S-4
Synthesis of functionalized nanoparticles NP_Cl and NP_N3
Cl
Synthesis
of
chlorobenzyl-functionalized
nanoparticles
NP_Cl
A
microemulsion was prepared into a reactor by progressive addition, upon gentle magnetic stirring, of a
mixture of monomers (styrene/divinylbenzene/vinylbenzylchloride : 3.50 g (33.6 mmol) / 4.50 g (34.8
mmol) / 1.85 g (12.2 mmol), respectively) and DMPA (341 mg, 1.3 mmol) to 187 g of a 17.5-wt%
aqueous solution of DTAB. The resulting microemulsion was degassed with nitrogen for 30 min and
the polymerization was then carried out under white light irradiation using two 60 W lamps at room
temperature under nitrogen for 24 h. A stable translucent suspension of nanoparticles was obtained.
Chemical composition of the polymer: C, 84.30; H, 7.47; Cl, 3.85 % corresponding to a chloride
content of 1.08 mmol/g of polymer. Mean diameter: 17 nm (PDI: 0.007). IR (KBr, ν, cm-1): 3056, 3023
(C-H arom), 2922 (a C-H), 2853 (s C-H), 1600, 1498, 1448 (C=C), 834, 796, 759, 702 (-C-H et C=C).
NP_Cl bis Chemical composition of the polymer: C, 78.36; H, 8.26; Cl, 3.08 % corresponding to a
chloride content of 0.87 mmol/g of polymer. Mean diameter: 16 nm (PDI: 0.014).
N3
Synthesis
of
azide-functionalized
nanoparticles
NP_N3
Azide-coated
nanoparticles NP_N3 were prepared by adding 27 mL an aqueous solution of sodium azide (8.04 g,
123.7 mmol) to 218 g of the freshly prepared crude suspension of NP_Cl. The resulting suspension
was stirred at room temperature for one week. The excess of sodium azide was then removed by
dialysis through a porous cellulose membrane (MWCO 12000-14000D) toward an aqueous solution of
DTAB (5 wt %) leading to a aqueous suspension of azide-functionalized nanoparticles NP_N3. The
suspension was kept in the presence of DTAB (15 wt%). Polymer particle content in suspension was
1.5 wt%. Chemical composition: C, 85.65; H, 7.26; N, 1.92 % corresponding to an azide content of
0.46 mmol/g of polymer. Mean diameter (QELS): 16 nm (PDI: 0.141). IR (KBr, ν, cm-1): 3083, 3058,
3025 (C-H arom), 2923 (a C-H), 2852 (s C-H), 2097 (N3), 1602, 1493, 1452 (C=C), 837, 797, 761, 700
(-C-H et -C=C).
NP_N3 bis Polymer content in suspension was 4.4 wt%. Chemical composition: C, 82.96; H, 7.17;
N, 2.18 % corresponding to an azide content of 0.53 mmol/g of polymer. Mean diameter (QELS): 15
nm (PDI: 0.033).
S-5
Table S1. Characteristics of chlorobenzyl- and azide-functionalized nanoparticles.
Entries elemental analysis loada (mmol/g of polymer)
NP_Cl
NP_N3
a.
b.
%C: 84.03
%H: 7.47
%Cl: 3.85
%C: 85.65
%H: 7.26
%N: 1.92
D (nm) b, c
[PDI]
Chloride
1.08
17
[0.007]
Azide content
0.46d
16
[0.141]
Deduced from elemental analysis.
The suspension was diluted one hundred times so that the content of DTAB in the suspension was close
to 0.15 wt%.
Particle diameter (D) and its polydispersity index (PDI) determined from QELS.
That corresponds to about 620 N3 per NP.
c.
d.
Procedure for the preparation PAMAM[NH2]n (denoted as G0, G1, and G3). Amino-terminated
PAMAM dendrons PAMAM[NH2]n were prepared using procedures described in the literature.
Synthesis of amino-terminated PAMAM[NH2]2 (G0)
CO2CH3
3
1
N
2
5
4
CO2CH3
6
7
Propargylamine (2.3 mL, 360 mmol) was mixed with 8 mL of dry methanol
and cooled to 0°C. To this cooled solution was added dropwise a 50% methanol (v/v) solution of
methyl acrylate (MA, 13 mL, 140 mmol) in methanol dry (15 mL) over a period of 30 min. The
reaction mixture was stirred 48h at room temperature. The excess of methyl acrylate and methanol
were then removed under vacuum at 60-70°C to afford the ester terminated PAMAM dendron (G-0.5)
as a light yellow oil (99 % yield). 1H NMR (CDCl3, δ, ppm): 2.17 (t, J 2.3, 1H, H1), 2.42 (t, J 7.1, 4H,
H5), 2.79 (t, J 7.0, 4H, H4), 3.37 (d, J 2.3, 2H, H3), 3.62 (s, 6H, H7); 13C NMR (CDCl3, δ, ppm): 32.7
(C5), 41.7 (C3), 48.8 (C4), 51.4 (C7), 73.2 (C1), 77.9 (C2); 172.5 (C6); ESI-HRMS: m/z calcd. for
C11H18NO4: 228.1236[M+ H]+; found 228.1236.
NH2
O
NH
3
1
2
N
5
4
6 H
N
O
8
7
NH2
Ethylenediamine (EDA, 35 mL, 525 mmol) was mixed with 35 mL of
methanol dry and cooled to 0°C. To this cooled solution was added dropwise a methanol solution (15
mL) of ester terminated PAMAM dendron (G-0.5) (8 g, 35 mmol). The reaction mixture was stirred
under nitrogen at 0°C for 30 min, then at room temperature for three days. Most of the excess of
ethylenediamine and methanol was removed under vacuum. The remaining ethylenediamine was
removed by azeotroping mixture of toluene and methanol 3/1 (v/v) then methanol to afford 10.4 g of
S-6
amino-terminated PAMAM dendron (G0) as a light yellow viscous oil (100% yield). 1H NMR (D2O, δ,
ppm): 2.43 (t, J 7.2, 4H, H5), 2.69 (t, J 6.2, 4H, H8), 2.83 (t, J 7.2, 4H, H4), 3.22 (t, J 6.2, 4H, H7), 3.33
(s, 1H, H1), 3.40 (s, 2H, H3); 13C NMR (D2O/CD3CN, δ, ppm): 33.2 (C5), 39.7 (C7), 41.2 (C3), 41.7
(C8), 48.9 (C4), 77.6 (C1), 174.8 (C6); IR (neat, ν, cm-1): 3275, 3066 (N-H), 2927 (as C-H), 2837 (s
C-H), 1639 (C=O amide), 1542 ( NH); ESI-HRMS: m/z calcd. for C13H26N5O2: 284.2087[M+ H]+;
found 284.2090.
Synthesis of amino-terminated PAMAM[NH2]4 (1-G1)
Starting from amino-terminated PAMAM[NH2]2 (G0) (7 g, 24.7
mmol) and MA (13.4 mL, 148 mmol), ester-terminated PAMAM[CO2CH3]4 (G0.5) was isolated after
purification by chromatography (SiO2, eluent: CHCl3/MeOH 9.5/0.5, then 9/1) as a pale yellowish oil
(11.3 g, 73%). 1H NMR (CDCl3, δ, ppm): 2.17 (t, J 2.3, 1H, H1), 2.35 (t, J 6.5, 4H, H5), 2.41 (t, J 6.6,
8H, H10), 2.52 (t, J 5.8, 4H, H8), 2.73 (t, J 6.6, 8H, H9), 2.82 (t, J 6.5, 4H, H4), 3.26 (m, 4H, H7), 3.43
(d, J 2.3, 2H, H3), 3.64 (s, 12H, H12), 7.08 (m, 2H, CONH) ; 13C NMR (CDCl3, δ, ppm): 32.5 (C10),
33.7 (C5), 36.9 (C7), 41.0 (C3), 49.2 (C9), 49.3 (C4), 51.5 (C12), 52.9 (C8), 73.3 (C1), 77.8 (C2), 171.7
(C6), 172.8 (C11); IR (neat, ν, cm-1): 3276 (N-H), 2951 (as C-H), 2830 (s C-H), 1729 (C=O ester), 1639
(C=O amide), 1528 ( NH); ESI-HRMS: m/z calcd. for C29H50N5O10: 628.3558[M+ H]+; found
628.3554 .
Starting from ester-terminated PAMAM[CO2CH3]4 (G0.5) (8.03 g, 12.7
mmol) and EDA (25 mL, 374 mmol), amino-terminated PAMAM[NH2]4 (1-G1) was isolated without
further purification as a pale yellowish oil (9.13 g, 97 %). 1H NMR (D2O, δ, ppm): 2.46 (t, J 7.0, 12H,
H5, H10), 2.67 (t, J 6.6, 4H, H8), 2.83 (m, 20H, H4, H9, H13), 3.32 (m, 12H, H7, H12), 3.35 (s, 1H, H1),
S-7
3.46 (s, 2H, H3); 13C NMR (D2O/CD3CN, δ, ppm): 33.4 (C10), 33.8 (C5), 37.4 (C7), 40.3 (C13), 41.8,
42.1 (C3, C12), 49.4, 49.5 (C4, C9), 51.8 (C8), 175.0, 175.7 (C6, C11); IR (neat, ν, cm-1): 3283, 3085 (NH), 2934 (as C-H), 2822 (s C-H), 1629 (C=O amide), 1549 ( NH); ESI-HRMS: m/z calcd. for
C33H66N13O6: 740.5259[M+ H]+; found 740.5262.
Synthesis of amino-terminated PAMAM[NH2]8 (2-G2)
Starting from amino-terminated PAMAM[NH2]4 (1-G1) (6.0 g,
8.1 mmol) and MA (8.8 mL, 97.3 mmol), ester-terminated PAMAM[CO2CH3]8 (G1.5) was isolated
after purification by chromatography (SiO2, eluent: AcOEt/MeOH 6/1, then 1/1) as a pale yellowish oil
(7.0 g, 60 %). 1H NMR (CDCl3, δ, ppm): 2.20 (t, J 2, 1H, H1), 2.40 (m, 28H, H5, H10, H15), 2.53 (t, J
5.9, 8H, H13), 2.60 (t, J 5.9, 4H, H8), 2.75 (t, J 6.7 24H, H9, H14), 2.82 (t, J 6.0, 4H, H4), 3.28 (m, 12H,
H7, H12), 3.45 (d, J 2, 2H, H3), 3.66 (s, 24H, H17), 7.08 (t, J 5.1, 4H, CONH); 7.74 (m, 2H, CONH); 13C
NMR (CDCl3, δ, ppm): 32.6, 33.7, 33.8 (C5, C10, C15), 37.2, 37.3 (C7, C12), 41.0 (C3), 49.2, 49.4, 49.9
(C4, C9, C14), 51.6 (C17), 52.5, 52.9 (C8, C13), 73.4 (C1), 78.0 (C2), 172.2, 172.3 and 173.0 (C6, C11, C16);
IR (neat, ν, cm-1): 3286. 3079 (N-H), 2951 (as C-H), 2824 (s C-H), 1732 (C=O ester), 1642 (C=O
amide), 1536 ( NH); ESI-HRMS: m/z calcd. for C65H114N13O22: 1428.8202[M+ H]+; found 1428.8690.
S-8
Starting from ester-terminated PAMAM[CO2CH3]8
(G1.5) (6.2 g, 4.3 mmol) and EDA (17 mL, 254 mmol), amino-terminated PAMAM[NH2]8 (2-G2) was
obtained without further purification as a pale yellowish oil (6.92 g, 100 %). 1H NMR (D2O, δ, ppm):
2.42 (m, 28H, H5, H10, H15), 2.61 (t, J 6.6, 12H, H8, H13), 2.78 (m, 44H, H4, H9, H14, H18), 3.11 (s, 1H,
H1), 3.25 (m, 30H, H7, H12, H17, H3); 13C NMR (D2O/CD3CN, δ, ppm): 33.3 (C5, C10, C15), 37.3 (C7,
C12), 40.3 (C3, C18), 41.6 (C17), 49.6 (C4, C9, C14), 51.8 (C8, C13), 175.3, 175.7 (C6, C11, C16); IR (neat,
ν, cm-1): 3276,3069 (N-H), 29327 (as C-H), 2832 (s C-H), 1634 (C=O amide), 1544 ( NH); ESIHRMS: m/z calcd. for C73H146N29O14: 1653.1604 [M+H]+; found 1653.1614.
Synthesis of functionalized nanoparticles NP_Bodipy_PAMAM[NH2]n using a two steps
process
Synthesis of functionalized nanoparticles NP_PAMAM[NH2]4
O
HN
N
O
NH2
HN
O
NH
NH2
N
N N
N
NH
O
N
O
HN
O
NH2
HN
NH2
Amino-terminated PAMAM dendron (1-G1)
(207 mg, 0.28 mmol) was added to 8.05 g of the aqueous suspension of azide-functionalized
nanoparticles NP_N3 bis (4.4 wt% in 15% DTAB). Then, 2 mL of a freshly prepared aqueous solution
of CuSO4 (74 mg, 0.46 mmol) and sodium ascorbate (167 mg, 0.84 mmol) was added. The resulting
yellow suspension was stirred at room temperature for 5 days. Then, the mixture was dialyzed with an
S-9
aqueous solution of DTAB (5 wt%) and EDTA (0.05 M), then with a solution of DTAB (1 wt%, ten
times) using a porous cellulose membrane (MWCO 12000-14000 D) leading to a stable translucent
aqueous suspension of PAMAM functionalized nanoparticles NP_PAMAM[NH2]4. Polymer particle
content in suspension was 1.8 wt%. IR (KBr, ν, cm-1): 3416 (N-H), 3081, 3052, 3027 (C-H arom),
2921 (as C-H), 2852 (s C-H), 2092 (N3), 1654 (C=O amide), 1597 (C=C), 1541 ( NH), 836, 796, 763,
702 (-C-H et -C=C).
Amino-terminated functionalized nanoparticles NP_PAMAM[NH2]8 and NP_PAMAM[NH2]16
were prepared using the same procedure as above.
NP_PAMAM[NH2]8
NH2
O
O
NH
H
N
N
HN
O
O
N
O
NH
HN
O
NH
NH2
NH2
N
NH
N
O
N N
N
NH2
NH
O
N
O
O
HN
O
NH2
NH
N
HN
N
O
NH
O
HN
O
NH
NH2
NH2
H2N
Starting from (2-G2) (315 mg, 0.19 mmol),
8.0 g of aqueous suspension of NP_N3 bis, 2 mL of aqueous solution of CuSO4 (74 mg, 0.46 mmol)
and sodium ascorbate (167 mg, 0.84 mmol). Polymer particle content in suspension was 1.2 wt%. IR
(KBr, ν, cm-1): 3428 (N-H), 3078, 3056, 3019 (C-H arom), 2921 (as C-H), 2847 (s C-H), 2092 (N3),
1658 (C=O amide), 1605 (C=C), 1544 ( NH), 833, 792, 764, 698 (-C-H et -C=C).
S-10
NP_PAMAM[NH2]16
H 2N
NH
O
NH2
HN
N
O
O
NH2
NH
NH
O
O
N
HN
O
O
O H
N
O
N N
N
N
NH
O
N
O NH2
O H
N
HN
N
N
H2 N
N
H
O
NH
O
O
N
H 2N
NH
O
N
O
NH2
HN
O
NH2
NH
O
NH
NH2
NH
HN
N
HN
O
NH2
N
H
N
O
O
NH2
NH2
NH
O
O
O
HN
H
N
O
O
N
H
NH
N
NH
N
N
O
NH
NH2
NH2
O
HN
N
O
H
N
N
HN
HN
H2N
Starting from (3-G3) (661 mg, 0.19
NH2
mmol), 8.0 g of aqueous suspension of NP_N3 bis, 2 mL of aqueous solution of CuSO4 (75 mg, 0.46
mmol) and sodium ascorbate (167 mg, 0.84 mmol). Polymer particle content in suspension was 1.1
wt%. IR (KBr, ν, cm-1): 3432 (N-H), 3077, 3052, 3019 (C-H arom), 2917 (as C-H), 2848 (s C-H), 2092
(N3), 1654 (C=O amide), 1544 ( NH), 837, 788, 760, 694 (-C-H et -C=C).
Synthesis of functionalized nanoparticles NP_Bodipy_PAMAM[NH2]8
NH2
O
O
NH
H
N
N
HN
O
O
NH
HN
N
O
O
NH
N
NH
N
F N
B
F N
O
N
NN
NH2
NH2
O
N N
N
NH2
NH
O
N
O
O
HN
O
NH2
NH
N
HN
N
O
HN
O
NH
H 2N
NH
O
NH2
NH2
Alkyne-Bodipy 5 (38 mg,
0.087 mmol) was added to 12 g of the aqueous suspension of NP_PAMAM[NH2]8 (1.2 wt% in 5 %
DTAB), the resulting mixture was stirred at room temperature for 2 hours. Then, 1 mL of a freshly
prepared aqueous solution of CuSO4 (17 mg, 0.104 mmol) and sodium ascorbate (40 mg, 0.20 mmol)
was added. The resulting suspension was stirred at room temperature for 6 days. Then, the mixture was
dialyzed against an aqueous solution of DTAB (5 wt%) and EDTA (0.05 M) and finally with DTAB (5
S-11
wt%) using a porous cellulose membrane (MWCO 12000-14000 D). The suspension was filtered and
purified by dialysis against an aqueous solution of DTAB (5 wt%) using an ultrafiltration device
(Vivascience, Vivaspin concentrator 20, 50 000 MWCO PES), leading to a stable translucent aqueous
suspension of Bodipy-functionalized nanoparticles NP_Bodipy_PAMAM[NH2]8. Polymer particle
content in suspension was 1.6 wt%. IR (KBr, ν, cm-1): 3432 (N-H), 3081, 3052, 3023 (C-H arom),
2921 (as C-H), 2852 (s C-H), 1650 (C=O amide), 1601 (C=C), 1540 ( NH), 833, 792, 760, 702 (-CH et -C=C).
Synthesis of functionalized nanoparticles NP_Bodipy_PAMAM[NH2]16
H 2N
NH
O
NH2
HN
N
O
O
O
NH
N
N
H
O
O
N N
N
N
NN
N
NH
O
N
O
O
O
HN
O H
N
HN
N
O
N
H
O
NH
N
HN
NH
O
O
O
N
N
O
HN
O
NH
NH2
N
H
NH2
NH
NH2
NH2
O
HN
H2N
O
N
NH
NH2
O
HN
NH2
NH2
NH
N
O
H 2N
H
N
O
O
N
H
NH
O
NH
N
N
N
F N
B
F N
NH2
NH2
O
O H
N
O
NH
H
N
O
H
N
N
O
NH
N
HN
O
NH2
H2N
NH2
Aqueous suspension of NP_Bodipy_PAMAM[NH2]16 was prepared using the same procedure
as above. Starting from alkyne-Bodipy 5 (29 mg 0.066 mmol), 5 g of aqueous suspension of
NP_PAMAM[NH2]16, 1 mL of aqueous solution of CuSO4 (13 mg, 0.079 mmol) and sodium ascorbate
(30 mg, 0.152 mmol). Polymer particle content in suspension was 1.1 wt%. IR (KBr, ν, cm-1): 3440 (NH), 3080, 3055, 3017 (C-H arom), 2920 (a C-H), 2849 (s C-H), 1650 (C=O amide), 1538 ( NH), 832,
800, 758, 702 (-C-H et -C=C).

S-12
Synthesis of functionalized nanoparticles NP_Bodipy_PAMAM[NH2]n using a one-pot process
Synthesis of functionalized nanoparticles NP_Bodipy_PAMAM[NH2]4
O
HN
N
O
NH2
HN
O
NH
NH2
N
N N
N
F N
B
F N
O
NH
O
N
NN
N
O
HN
O
NH2
HN
NH2
Amino-terminated
PAMAM
dendron (1-G1) (230 mg, 0.31 mmol) was added to 30 g of the aqueous suspension of azidefunctionalized nanoparticles NP_N3 (1.5 wt% in 15% DTAB). Then, 2 mL of a freshly prepared
aqueous solution of CuSO4 (80 mg, 0.50 mmol) and sodium ascorbate (167 mg, 0.84 mmol) was
added. The resulting suspension was stirred at room temperature for 4 days. Alkyne-Bodipy 5 (80 mg,
0.183 mmol) was then added. The resulting mixture was stirred at room temperature for 2h before
addition of 2 mL of a freshly prepared aqueous solution of CuSO4 (70 mg, 0.44 mmol) and sodium
ascorbate (167 mg, 0.84 mmol). The resulting red suspension was stirred at room temperature for 3
days. Aqueous suspension was then purified using the same procedure as for the two-steps process and
led
to
a
stable
translucent
aqueous
suspension
of
Bodipy-functionalized
nanoparticles
NP_Bodipy_PAMAM[NH2]4. Polymer particle content in suspension was 1.1 wt%. Chemical
composition: C, 77.21; H, 7.46; N, 3.71; B, 0.21 %; Mean diameter (QELS): 20 nm (PDI: 0.063); IR
(KBr, ν, cm-1): 3080, 3055, 3024 (C-H arom), 2922 (as C-H), 2851 (s C-H), 1650 (C=O amide), 1601
(C=C), 1541 ( NH), 834, 796, 760, 700 (-C-H et -C=C).
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Synthesis of functionalized nanoparticles NP_Bodipy_PAMAM[NH2]8
NH2
O
O
NH
H
N
N
HN
O
O
N
O
NH
HN
O
NH
N
NH
N
F N
B
F N
O
N
NN
NH2
NH2
O
N N
N
NH2
NH
O
N
O
O
NH
HN
O
NH2
N
HN
N
NH
O
O
O
HN
NH
H 2N
NH2
NH2
Aqueous suspension of
NP_Bodipy_PAMAM[NH2]8 was prepared using the same procedure as above. Starting from 2-G2
(512 mg, 0.31 mmol), 30 g of aqueous suspension of NP_N3, 2 mL of aqueous solution of CuSO4 (80
mg, 0.50 mmol) and sodium ascorbate (188 mg, 0.95 mmol), followed by addition of alkyne-Bodipy 5
(81 mg 0.19 mmol) and 2 mL of aqueous solution of CuSO4 (70 mg, 0.44 mmol) and sodium ascorbate
(167 mg, 0.84 mmol). Polymer particle content in suspension was 1.15 wt%. Chemical composition: C,
70.21; H, 7.59; N, 7.17; B, 0.12 %; Mean diameter (QELS): 24 nm (PDI: 0.172); IR (KBr, ν, cm-1):
3080, 3054, 3028 (C-H arom), 2921 (as C-H), 2846 (s C-H), 1649 (C=O amide), 1541 ( NH), 833,
795, 760, 700 (-C-H et -C=C).

Figure S3. AFM Topographic images obtained at pH = 7 of NP_Bodipy_PAMAM[NH2]8 (A) and
NP_Bodipy_PAMAM[NH2]16 (B) using the tapping mode onto a mica substrate.
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Figure S4. Absorption (full lines) and Fluorescence emission (dotted lines) spectra (exc = 495nm) of 6
diluted in DTAB 5 wt%.
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Figure S5. Absorption (A) and Fluorescence emission (B) spectra corrected from absorption (exc =
495nm, slits width 2.2 nm) of aqueous suspension of NP_Bodipy_PAMAM[NH2]4 (diluted 1000
times in 1 wt% DTAB aqueous solutions) for pH values ranging from 1.1 to 9.3. The pH has been
adjusted for pH = 1.1 with HCl (10-1 M); pH = 3.0 with HCl (10-3 M); pH = 5.0, 6.9 and 9.3 with
phosphate buffers.
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Figure S6. Absorption (A) and Fluorescence emission (B) spectra corrected from absorption (exc =
495nm, slits width 3.0 nm) of aqueous suspension of NP_Bodipy_PAMAM[NH2]8 (diluted 400 times
in water) for pH values ranging from 1.1 to 9.3. The pH has been adjusted for pH = 1.1 with HCl (10-1
M); pH = 2.9 with HCl (10-3 M); pH = 5.0, 7.0 and 9.3 with phosphate buffers.
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Figure S7. Absorption (A) and Fluorescence emission (B) spectra corrected from absorption (exc =
495nm, slits width 3.25 nm) of aqueous suspension of NP_Bodipy_PAMAM[NH2]16 (diluted 400
times in water) for pH values ranging from 1.1 to 9.3. The pH has been adjusted for pH = 1.1 with HCl
(10-1 M); pH = 2.9 with HCl (10-3 M); pH = 5.0, 7.0 and 9.3 with phosphate buffers.
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Figure S8. Estimation of the radius of benzyl triazole functionalized amino-terminated PAMAM
dendron (1-G1) using Chem Bio3D Ultra 11.0 (MMFF94 calculation) software.
Figure S9. Estimation of the radius of benzyl triazole functionalized amino-terminated PAMAM
dendron (2-G2) using Chem Bio3D Ultra 11.0 (MMFF94 calculation) software.
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Figure S10. Estimation of the radius of benzyl triazole functionalized amino-terminated PAMAM
dendron (3-G3) using Chem Bio3D Ultra 11.0 (MMFF94 calculation) software.
Using the Mansfield-Tomalia-Rakesh equation for parking spheres on a sphere, the maximum number
of amino-terminated PAMAM dendrons that could be arranged around each nanoparticle was
determined to be about 170, 105 and 55 for 1-G1, 2-G2 and 3-G3, respectively.
Equation 1:

r1/r2 > 1.20
 r1 = 8nm, is the nanoparticle radius of starting azide-functionalized nanoparticles (determined
from QELS).
Even if the dendrons do not adopt a spherical geometry, we have estimated the radii of benzyl-triazole
functionalized amino-terminated PAMAM dendrons considering that the dendrons have an extended
conformation without correction for charge, solvation, etc using Chem Bio3D Ultra 11.0 (MMFF94
calculation) software.

1-G1 (r2 = 1.375 nm),

2-G2 (r2 = 1.825 nm),

3-(G3) (r2 = 2.76 nm)
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Table S2. Estimation of the maximum number (Nmax) of amino-terminated PAMAM dendrons grafted
on the NP surface from the Equation 1 and number of charges per NP along with the estimation of the
diameter of dendronized NPs.
NP_PAMAM[NH2]4
NP_PAMAM[NH2]8
NP_PAMAM[NH2]16
Nmax
170
105
55
Number of primary
amines per NP
Number of tertiary
amines per NP
Number of (NH2 + N)
per NP
Number of (NH2 + N
+ CO-NH) per NP
Number of positive
charges per NP at
neutral pHa
Number of positive
charges per NP at acidic
pHb
Calculated diameter
(nm) of dendronized
NPsc
Measured diameter
(nm) of dendronized
NPsd
680
840
880
510
735
825
1190
1575
1705
2210
3045
3355
680
840
880
1190
1575
1705
21,5
23,3
27,05
20
24
27
a. Assuming complete protonation of the primary amine groups (pKa=7-9)
b. Assuming complete protonation of the primary and tertiary amine groups (pKa=7-9 and 3-6
respectively) at pH ≤ 3.
c. Calculated from the measured of the starting NP-N3 (16 nm) : D = DNP-N3 + 4*r2
d. Hydrodynamic diameter deduced from QELS measurements.
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Table S3. Estimation of the number of Bodipy grafted on the NP surface.
NP_Bodipy_PAMAM[NH2]4a NP_Bodipy_PAMAM[NH2]8b NP_Bodipy_PAMAM[NH2]16c
Number of NPsd
4,9 • 1018
5,1 • 1018
4,5 • 1018
Absorbance
0,30 (diluted 500 times)
0,26 (diluted 200 times)
0,25 (diluted 250 times)
[Bodipy]e
1,88
0,66
0,79
Number of
Bodipyf
1,1 • 1021
4,0 • 1020
4,8 • 1020
Number of
Bodipyg
230
80
105
a. Polymer content 1,1 wt%
b. Polymer content 1,15 wt%
c. Polymer content 1,0 wt%
d. Contained in 1L of the suspension
e. Concentration of the Bodipy (mmol.L-1) in the suspension assuming an  = 80 000 L.mol-1.cm-1
f. Contained in 1L of the suspension
g. Grafted on the NP surface
The number of nanoparticles (NPs) contained in 1L of the suspension was estimated assuming a size of
16 nm for the nanoparticle and a polymer density of 1.05. We have considered that all the mass of the
dendronized nano-object is concentrated in the polystyrene nanoparticle core. The volume of the NP
was calculated considering a spherical nanostructure and was estimated to be about 2,2 • 1018 cm3.
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