Inter-nanocarrier and nanocarrier-to-cell transfer assays demon- strate the risk of an immediate unloading of dye from labeled lipid
nanocapsules.
Carl Simonssona,b,‡, Guillaume Bastiata,b,‡,*, Marion Pitorrea,b, Andrey S. Klymchenkoc, Jérôme Béjauda,b, Yves Mélyc, Jean-Pierre Benoita,b
a INSERM, U 1066, Micro et Nanomédecines biomimétiques – MINT, Angers, F-49933 France.
b LUNAM Université, UMR-S1066 Angers, F-49933 France.
c Laboratoire de biophotonique et de Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch, France.
‡ Equal contribution to the work.
* Corresponding author: Guillaume Bastiat. Tel.: +33 244 688531. Fax: +33 244 688546. E-mail guillaume.
bastiat@univ-angers.fr. Complete postal address: MINT – UMR_S1066, Institut de Biologie en Santé – IRIS, 4 rue Larrey CHU, 49933 Angers Cedex 9, France.
Supporting Information.
Table S1. Size characterization (Z-Ave and PdI) of fluorescent dye-labeled LNCs, for FRET, cell culture, di- alysis and size-exclusion chromatography assays.
Z-Ave (nm) PdI
Methoda Dye [Dye]b mean SD Mean SD nc
F+C+D
LNC60
--- 0 63 3 0.1 0.02 14
C + D NR 1 65 4 0.12 0.05 13
C DiO 1 67 2 0.09 < 0.01 2
C NR / DiO 1 / 1 64 1 0.07 < 0.01 3
F NR 5 58 1 0.15 0.01 3
F F888 5 54 1 0.03 0.01 3
F NR / F888 5 / 5 59 1 0.16 0.01 3
F DiI 3.75 53 1 0.06 0.01 3
F + S
LNC30
--- 0 30 < 1 0.1 < 0.01 2
S NR 1 30 2 0.1 0.01 3
S DiO 1 31 na 0.11 na 1
F NR 5 27 < 1 0.08 0.04 3
F F888 5 28 1 0.05 0.01 3
F NR / F888 5 / 5 27 < 1 0.11 0.02 3
F DiI 3.75 28 1 0.17 0.04 3
F + S
LNC120
--- 0 131 4 0.1 < 0.01 3
S NR 1 150 7 0.13 0.02 3
S DiO 1 118 na 0.09 na 1
F NR 5 126 1 0.1 0.02 3
F F888 5 102 2 0.04 0.01 3
F NR / F888 5 / 5 123 2 0.1 0.02 3
F DiI 3.75 91 1 0.04 0.01 3
a) F: FRET; C: cell culture; D: dialysis; S: size-exclusion chromatography;
b) Dye concentration in Labrafac (mg/mL);
c) Experiment number;
0 5000 10000 15000
400 500 600 700
Wavelength (nm)
Fluorescence (a.u.)
F888 NR−LNC F888−LNC+LNC F888−LNC+NR−LNC F888 NR−LNC+LNC NR−LNC+LNC
A
0 4000 8000 12000
600 650 700 750 800
Wavelength (nm)
Fluorescence (a.u.)
DiI DiD−LNC DiI−LNC+LNC DiI−LNC+DiD−LNC DiI DiD−LNC+LNC DiD−LNC+LNC
B
0 5000 10000 15000
400 500 600 700
Wavelength (nm)
Fluorescence (a.u.)
F888 NR−LNC F888−LNC+LNC F888−LNC+NR−LNC F888 NR−LNC+LNC NR−LNC+LNC
A
0 5000 10000 15000
600 650 700 750 800
Wavelength (nm)
Fluorescence (a.u.)
DiI DiD−LNC DiI−LNC+LNC DiI−LNC+DiD−LNC DiI DiD−LNC+LNC DiD−LNC+LNC
B
Figure S1. Fluorescence emission spectra for (A) F888/NR-LNC and mixtures of F888-LNC and LNC, F888-LNC and NR-LNC, F888/NR-LNC and LNC, NR-LNC and LNC; with an excitation wavelength of 390 nm; and (B) DiI/DiD-LNC and mixtures of DiI-LNC and LNC, DiI-LNC and DiD-LNC, DiI/
DiD-LNC and LNC, DiD-LNC and LNC; with an excitation wavelength of 549 nm. LNC size was 30 nm and the Labrafac weight ratio was 1:1 for all mixtures. The experiments were repeated in triplicate.
Figure S2. Fluorescence emission spectra for (A) F888/NR-LNC and mixtures of F888-LNC and LNC, F888-LNC and NR-LNC, F888/NR-LNC and LNC, NR-LNC and LNC; with an excitation wavelength of 390 nm; and (B) DiI/DiD-LNC and mixtures of DiI-LNC and LNC, DiI-LNC and DiD-LNC, DiI/DiD-LNC and LNC, DiD-LNC and LNC; with an excitation wavelength of 549 nm. LNC size was 120 nm and the Labrafac weight ratio was 1:1 for all mixtures. The experiments were repeated in triplicate.
0 25 50 75 100
0.5 1 5 10
Number of THP-1 Cells (x106)
% NR or DiO in LNC after Incubation
DiO-LNC (DiO) NR-LNC (NR) NR/DiO-LNC (DiO) NR/DiO-LNC (NR)
0 100 200
0.5 min 1 min 15 min 30 min Control Incubation Time
Fluorescence Intensity in THP-1 Cells (Geo-Mean, a.u.)
Figure S3. Percentage of NR and DiO in LNCs after incubation for 30 sec. at 25°C of non-labeled THP-1 cells (0.5, 1, 5 and 10 × 106 cells) with DiO-LNC, NR-LNC, or NR/DiO-LNC suspension (1 mg/mL, 60 nm). (n = 3, mean ± standard deviation).
Figure S4. Fluorescence intensity in THP-1 cells after incubation for 30 sec., 1min, 15 min and 30 min at 25°C of non-labeled THP-1 cells (0.5 × 106 cells) with NR-LNC suspension (1 mg/mL, 60 nm), using flow cytometry with NR (FL2 channel) detection. (n = 3, mean ± standard deviation).
0 100 200
40 20 10 5 1 Control
LNC Concentration (mg/ml)
Fluorescence Intensity in THP-1 Cells (Geo-Mean, a.u.)
0 1 2 3 4 5
DiO NR
Dye Label
% NR or DiO in LNC after Incubation
LNC Concentration:
1 mg/ml 5 mg/ml 10 mg/ml Control
Figure S5. Fluorescence intensity in THP-1 cells after incubation for 30 sec. at 25°C of non-labeled THP-1 cells (0.5 × 106 cells) with NR-LNC suspension (1, 5, 10, 20 and 40 mg/mL, 60 nm), using flow cytometry with NR (FL2 channel) detection. (n = 3, mean ± standard deviation).
Figure S6. Percentage of NR and DiO in LNCs after incubation for 30 sec. at 25°C of NR-THP-1 cells (10
× 106 cells) or DiO-THP-1 cells (2.5 × 106 cells) with non-labeled LNC (1, 5 or 10 mg/mL, 60 nm). Propor- tions were calculated considering NR or DiO-LNC original formulation as 100%. (n = 3, mean ± standard deviation).
