Supplementary material. Appendix 1.
Determination of LNC concentration after the separation process
To determine LNC concentration before and after centrifugation, derived count rate (scattered light intensity, DCR, in kcps) of aqueous LNC suspensions, a parameter obtained during size measurements, was used (see 2.4. Hydrodynamic diameter, PdI and Derived Count Rate (DCR) measurements). There was a linear correlation (r2 > 0.99) for DCR and LNC concentration for a given LNC hydrodynamic diameter (27, 65 and 110-nm) (Supplementary information figure S1).
Figure S1. Derived Count Rate (DCR) (kcps) vs. LNC concentration (mg.g-1) for LNC Z-Ave of 27 ( ), 65 ( ) and 110 nm ( ). Dot straight lines corresponded to linear fit of the experimental values.
This was in agreement with light scattering theory. Debye’s equation showed the linearity of scattered light intensity vs. the scattering particle concentration in solution, with constant scattering angle (P. Debye, E.W. Anacker, Micelle shape for dissymmetry measurements, J.
Phys. Colloid Chem. 55 (1951) 644-655). In addition, the slope of the linear function was different when Z-Ave was modified. The proportionality DCR = f(LNC concentration) was checked with a constant Z-Ave and a constant PdI of the scattering particles. So it was possible to evaluate the LNC concentration using DCR measurements before and after centrifugation, with constant nanocarrier Z-Ave and PdI.
Variations of DCR values for aqueous LNC suspensions before and after centrifugation were less than 3% in all the experiments described in the paper. Some LNC suspensions were described in table S1. Therefore LNC concentrations in aqueous suspension before and after centrifugation were considered similar, meaning that the totality of LNC was recovered in the aqueous phase and no LNC was present in the oil compartment after separation. With
centrifugation, it was possible to separate the aqueous LNC suspension from the LC after mixture of these 2 media.
Table S1. Z-Ave, PdI, DCR and LNC concentration of LNC suspension before and after separation process (mixture with LC and centrifugation) for non-loaded LNC and NR, 6-Cou, DiI, DiD or DiO-loaded LNCs. Constant parameters: florescent dye concentration was 1 mg.g-1 (dye/Labrafac in LNC w/w), LNC Z-Ave = 65 nm, R = 1/1 (Labrafac in LNC/Labrafac as LC w/w), LC was Labrafac. (n = 3; mean ± SD).
a) DCR value after a 1/90 dilution in pure water b) No dilution
Z-Ave
(nm) PdI DCRa
(104 kcps)
LNC concentrationb
(mg.g-1) Non-loaded
LNC
Before separation process 60 ± 2 0.07 ± 0.01 49.7 ± 0.6 385 ± 5 After separation process 57 ± 3 0.08 ± 0.02 49.6 ± 4.3 384 ± 33 NR-loaded
LNC
Before separation process 66 ± 2 0.07 ± 0.02 49.9 ± 1.5 385 ± 12 After separation process 61 ± 1 0.08 ± 0.01 50.0 ± 2.0 385 ± 15 6-Cou-
loaded LNC
Before separation process 69 ± 1 0.09 ± 0.01 54.8 ± 1.3 385 ± 9 After separation process 70 ± 0 0.08 ± 0.02 52.5 ± 1.3 369 ± 9 DiI-loaded
LNC
Before separation process 61 ± 2 0.07 ± 0.01 48.5 ± 2.0 385 ± 16 After separation process 61 ± 1 0.06 ± 0.01 46.8 ± 3.0 372 ± 24 DiD-loaded
LNC
Before separation process 67 ± 1 0.08 ± 0.01 51.2 ± 1.1 385 ± 8 After separation process 69 ± 0 0.06 ± 0.02 51.6 ± 3.5 388 ± 26 DiO-loaded
LNC
Before separation process 65 ± 2 0.08 ± 0.02 49.0 ± 1.7 385 ± 13 After separation process 64 ± 1 0.07 ± 0.01 51.5 ± 1.1 405 ± 9
Supplementary material. Appendix 2.
Formula of the fluorescent dyes.
N
H3C H3C
N
O O
N O
H3C
H3C O
N S
!
O N CH2 H2C H3C
16
O N H2C H2C
CH3
16 + ClO4-
N CH2 H2C H3C
16
N H2C
CH2 CH3
16 ClO4-
+
N CH2 H2C H3C
16
N H2C
CH2 CH3
16 +
-O3S Cl
" # # $ ! $ $
! % # & ' $
# & ( $ ! !
#
Supplementary material. Appendix 3.
Calculation of the number of surfactant and dye molecules per LNC
Calculation of the number of surfactant and dye molecules per LNC was based on the one reported by Texier et al. [31]. First, the number of particles N after the formulation step can be evaluated as follows:
LNC Lip Kol Lab
V
V V
N V + +
=
with VLab, VKol and VLip the volume of Lab, Kol and Lip, respectively, used for formulation.
VLNC is the volume of one LNC.
(
H)
3Lip Lip Kol
Kol Lab
Lab
' k 3 R
4
d m d
k m d
m N
×
× π
×
× + +
=
mLab, mKol and mLip are the masses of Lab, Kol and Lip, respectively, used for formulation.
The densities were obtained from suppliers: dLab = 0.93 g.mL-1, dKol = 0.96 g.mL-1. dLip was fixed at 1 g.mL-1. k = 0.7 because 70% of Kol are polyethylene glycol 660 hydroxystearate whereas 30% correspond to free polyethylene glycol 660 (supplier information), not implied in the LNC structure (A. Vonarbourg, P. Saulnier, C. Passirani, J.-P. Benoit, JP.
Electrokinetic properties of noncharged lipid nanocapsules: influence of the dipolar distribution at the interface, Electrophoresis 26(11) (2005) 2066-2075). k’ = 0.774 and corresponds to the ratio RH / RG (hydrodynamic radius / gyration radius, respectively) for an homogeneous sphere and RG corresponds to the physical radius of the particle measured by light scattering (S. K. Schmitz, An introduction to dynamic scattering by macromolecules, New-York: Academic Press, 1990).
The number of Kol molecules (NKol), Lip molecules (NLip), and dye molecules (Ndye) per LNC can be calculated as follows:
N M N
k m N
a Kol
Kol
Kol
× ×
= N
M N m N
a Lip Lip
Lip
×
= N
M N m N
a dye dye
dye
×
=
with Na the Avogadro number, MKol = 961 g.mol-1, MLip = 790 g.mol-1, MDiO = 882 g.mol-1, MDiI = 934 g.mol-1 and MDiD = 1052 g.mol-1.
Supplementary material. Figure S2.
Figure S2. Proportions of Nile Red (NR) in LNCs (grey part bar) and in LC (white part bar) after the direct method (solid line bar) and the reverse method (dot line bar) transfers (see figure 1) vs. LNC Z-Ave: 25, 65 and 110 nm; and LNC concentration: 110, 193 and 385 mg.g-1 in suspension. Constant parameters: NR concentration was 1 mg.g-1 (dye/Labrafac in LNC w/w), R = 1/1 (Labrafac in LNC/Labrafac as LC w/w), LC was Labrafac. (n = 3; mean ± SD; ***: p<0.005).
LNC Z-Ave 27 65 110 65 65 (nm) LNC conc. 385 385 385 193 110 (mg.g-1)
*** *** *** ***
Supplementary material. Figure S3.
Figure S3. Proportions of Nile Red (NR) in LNCs (grey part bar) and in LC (white part bar) after the direct method transfer (see figure 1) vs. LC nature: Labrafac® WL 1349 (Lab), ethyl oleate (EO), Captex® 200 (Captex200), Captex® 300 (Captex300) and perfluoro-15-crown-5- ether (PFCE). Constant parameters: LNC Z-Ave = 65 nm, LNC concentration = 385 mg.g-1 NR concentration was 1 mg.g-1 (dye/Labrafac in LNC w/w), R = 1/1 (Labrafac in LNC/Labrafac as LC w/w). (n = 3; mean ± SD; ****: p<0.001).
LC Lab EO Captex300 Captex200 PFCE
****
Supplementary material. Figure S4.
Figure S4. Proportions of Coumarine-6 (6-Cou) in LNCs (grey part bar) and in LC (white part bar) after the direct method (solid line bar) and the reverse method (dot line bar) transfers (see figure 1) vs. 6-Cou concentration (dye/Labrafac in LNC w/w): 1, 0.5 and 0.1 mg.g-1 and R (Labrafac in LNC/Labrafac as LC w/w): 1/1, 1/2 and 2/1. Constant parameters: LNC Z-Ave = 65 nm, LNC concentration = 385 mg.g-1, LC was Labrafac. (n = 3; mean ± SD; ***: p<0.005;
****: p<0.001).
R 1/1 2/1 1/2 1/1 1/1
6-Cou conc. 1 1 1 0.5 0.1 (mg.g-1)
**** ***
