Functionalization of nano-emulsions with an amino-silica shell at the oil–water interface

Nano-emulsions are very promising nano-carriers with high potential for loading lipophilic drugs. However, the surface of oil nano-droplets is a dynamic oil/water interface stabilized by surfactants, and its chemical modification to graft ligands is highly challenging. In this study we developed a new protocol for modification of the nano-droplets surface through a silica shell terminated by amine functions. It enabled preparation of nanocapsules of 65, 85 and 120 nm diameters with a surface coverage of ca. 2 amino groups per nm2. The nanocapsule surface was then functionalized (41% efficiency) by a model fluorescent ligand (coumarin blue) with a carboxylic function. The evidence for the successful grafting was provided by spectrofluorometry, Forster resonance energy transfer, atomic force microscopy coupled with fluorescence imaging and fluorescence correlation spectroscopy. This simple protocol for surface functionalization of the liquid/liquid interface of lipid droplets may constitute a real advance regarding potential applications that need efficient decoration of droplets with ligands.


Introduction
Nanomedicines for therapeutics and diagnosis purposes always have a common aim: bringing the drugs to the specic target. Targets are, for instance, organs, lymph nodes, cancer tumors, and the accumulation mechanisms rely on the targets and can follow passive or active targeting. In cases of liver, spleen, or in some case tumor targeting, passive accumulation mechanisms can be sufficient to bring to the desired site a signicant amount of active molecules. These passive mechanisms are driven by the uptake and metabolization of the nanoparticle systems that are undertaken by the liver and/or the spleen, depending on the chemical nature of the carrier. 1 In case of tumors, the passive accumulation results from the enhanced permeation and retention effect (EPR) 2 that is related to the half-life of the nanomedicines in the bloodstream. On the other hand, the selective accumulation in targets can also be improved by an active mechanism, involving ligand/receptor interactions. 3 This approach also depends on the circulation time in blood of the nanoparticles (NPs), in order to ensure efficient ligand/receptor interactions. The higher the half-life, the better the chance of their interactions. Ligands graed onto the NPs surface recognize the receptors and, as a result, NPs concentrate their active principles at the targeted site. Many ligands have already been proven to be effective for cancer active targeting and they can be graed onto the surface at high concentrations. Typical examples are cyclic pentapeptides c(RGD) that, when graed on the surface of polymeric PLGA-PEG NPs (poly(D,L-lactic-co-glycolic acid)-block-polyethylene glycol), showed increasing in vivo accumulation in cancer cells. 4 The co-encapsulation of anticancer active molecules results in specic treatment of the tumor. Other peptides like bombesin graed onto gold NPs have shown an in vitro and in vivo cancer cell specicity allowing detection of tumors cells by X-ray imaging owing to the gold NPs accumulation. 5 E-selectin peptides graed on liposome surface, 6 or 2-deoxy-D-glucose onto gold NPs 7 have also shown interesting accumulation in cancer cells due to their enhanced glucose consumption. Specic targeting of pancreatic ductal adenocarcinoma can be performed through plectin-1 targeted peptides (PTP) at the surface of hybrid iron oxide/polymeric NPs. 8 Another approach relies on the use of specic antibodies targeting cancer cells receptors, like anti-Her2 (ref. 9) or anti-CD4. 10 Different other types of ligands could also be efficient for active targeting of tumors such as folic acid for which receptors are overexpressed in gastric cancer cells 11 or cationic polymers that target sialic acid overexpressed on colonic malignant tissues. 12 These examples clearly emphasized that specic targeting is efficient only if the surface concentration is high enough to provide a signicant response or contrast in vivo. Active targeting is generally performed with polymeric or inorganic (particularly gold) NPs, because the surface chemistry performed on these systems is facile and efficient, providing a high number of available functional groups on the NP surface, and a high stability aer ligand graing. In the case of polymeric NPs, the functional groups (like carboxylic acids 3c,4,13 ) are part of the polymer constituting the NP matrix. Therefore a large number of sites are available for ligands, which aer graing are strongly anchored onto the NP surface. Functionalization of gold NPs is generally performed through strong thiol/gold interactions in order to decorate the gold NP by thiolpolyethylene glycol-COOH molecules. 10,14 In this case also, the reactive groups are strongly anchored to the NP core, presenting a high number of available reactive groups at the interface. Generally, the simple and efficient surface functionalization is one of the main advantages of polymeric 15 and inorganic NPs for active targeting strategies. In contrast, one of their main drawbacks is their limited capability in encapsulating active molecules, drugs or contrast agents, into the polymeric matrix or within the shell of inorganic NPs.
Other important family of nanomedicines is lipid nanodroplets, so-called nano-emulsions or nanocapsules. They are kinetically stable class of emulsions, in contrast to microemulsions that are thermodynamically stable. 16 Nanoemulsions can be easily formulated and they enable high loading levels of guest molecules in their core. We recently obtained 17 lipid nano-emulsions encapsulating in their lipid core, uorescent probes with concentrations as high as 8 wt%, opening new possibilities for single-particle tracking in vivo. 17b In addition, we formulated nano-emulsions as lipid reservoirs containing more than 50 wt% of iodine, and thus highly efficient for X-ray imaging. 1,18 In addition to iodine, we cosolubilized in the same droplets signicant amounts of a uorescent dye, which allowed monitoring their cellular uptake. 1 Nano-emulsions are also prospective for co-encapsulating drugs and contrast agents, and following their actual dosage in real time by imaging. In contrast, the active targeting of nanoemulsions is not presently well developed, due the technical difficulties related to the structure of the nano-emulsions. Indeed, oil droplets are stabilized by a monolayer of nonionic surfactants, so that there is no reactive groups for conjugation with ligands, and no possibilities for covalent anchoring of reactive chemical species as the liquid/liquid interface is a dynamic medium. Strategies such as post-insertion at the oil/ water interface of reactive function-bearing surfactants, like DSPE-PEG 2000-maleimide (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000]) 2b, 19 were developed, but the obtained surface modication is still largely below that of polymeric or inorganic NPs. The DSPE-PEG 2000-maleimide molecules are inserted in the surfactant monolayer, by incubating the lipid nano-droplets with a micellar solution of these molecules. However, in this approach the interfacial concentration is not controlled and is signicantly lower compared to polymeric or inorganic NPs. In addition, the interfacial adsorption is denitively weaker than covalent bonds, and can even be reversible leading to ligand release into the biological medium.
In the present study, we propose a new and simple method to functionalize lipid nano-droplets. The idea is rst to formulate nano-emulsions, and second to build amino-silica shell at the droplet interface. As a result, covalent graing of ligands to the lipid nanocarriers becomes possible. The silica precursor, (3aminopropyl)triethoxysilane (APTES), beforehand solubilized in the oil core of the nano-emulsion droplets, reacted by ultrasounds with the aqueous phase at the droplets interface to generate the silica shell specically at the interface. A signicant part of the APTES 0 NH 2 groups nally appears decorating the nanocapsules, oriented towards the aqueous bulk phase. We quantied the reactive NH 2 groups by using the uorescamine-based method, and further characterized the physicochemical properties of the functional nanocapsules by dynamic light scattering (DLS), transmission electron microscopy (TEM), and atomic force microscopy coupled with uorescence microscopy (AFM-Fluo). Moreover, as a proof of concept, we graed a uorescent model ligand presenting activated COOH functions (coumarin blue dye) that react with the amino groups onto the capsules. Thus, we describe a robust approach to cover lipid droplets with a solid silica shell that enables their covalent functionalization.

Methods
Formulation of amino-decorated lipid nano-emulsions. First, the silica monomer (APTES) is dissolved in the oil at different concentrations from 0.05 M to 0.5 M. This {oil + APTES} phase is then homogenized (vortex) 3 minutes at room temperature to obtain a transparent sample. It is then mixed with the non-ionic surfactant at different surfactant-to-oil weight ratio, SOR ¼ 40%, 50% and 60%, i.e. respectively {w surf. ¼ 0.32 g/w oil. ¼ 0.48 g}, {w surf. ¼ 0.40 g/w oil. ¼ 0.40 g} and {w surf. ¼ 0.48 g/w oil. ¼ 0.32 g}. This mixture is homogenized (vortex) during one minute and heated at 60 C for only one minute to avoid premature reaction of APTES. The mixture is then homogenized for 10 seconds, and nally mixed with the water phase (1.2 mL, to have 60 wt% of water in the nal nanoemulsion), and homogenized (vortex) for 5 minutes. The suspension of nano-emulsion droplets is placed in a sonication bath (Thermo Scientic, T310/H) at 35 kHz during four minutes, and then homogenized (vortex) one minute, and this procedure is repeated 6 times. The result is a hydrolysis followed by a condensation of APTES, building the aminofunctionalized silica capsule at the oil/water interface.
Quantication of primary amines available on the nanocapsules. The uorescamine-based method is a common procedure used for the quantication of primary amines in proteins or peptides. We adapted it for the nano-emulsion system. Before reaction with primary amines, the uorescamine is not uorescent, but aer reaction with primary amines uorescamine appear yellow and show uorescence properties with absorption/emission around 288 nm/490 nm, respectively. Stock solution of uorescamine (10 mM in anhydrous dioxane) was mixed with nano-emulsions and different APTES concentrations. The condensation reaction between uorescamine and primary amines is very fast, being typically completed within seconds, so that the samples are mixed (vortex) for only one minute. Fluorescence intensities of the samples (diluted 30 times) were measured immediately aer reaction by uorescence spectroscopy. Each experiment was done in triplicate.
Graing of activated coumarin blue (7-(diethylamino) coumarin-3-carboxylic acid N-succinimidyl ester dye) on primary amines decorating the nanocapsules. In this step, our aim was to show that activated coumarin blue that reacted with the available NH 2 groups at the surface of the nanocapsules as well as to quantify the graing efficiency. To this end, we used the quantication data of available NH 2 groups given by the uorescamine method described above, and we added in the bulk phase the desired amount of activated coumarin blue able to react with NH 2 . Coumarin is a dye with absorption/emission at 394/473 nm, respectively. A stoichiometric ratio (1 : 1) was tested, corresponding to 25 mL of the stock solution of activated coumarin blue (32 mM in dry dimethyl formamide) diluted in 1 mL of nano-emulsions having a concentration of available primary amines at 0.8 mM. The solution was then incubated for 24 hours under gentle stirring. Then, the sample was dialyzed through a 12 kDa membrane (Spectra/Por®, Spectrum Europe B.V., Breda, the Netherlands) for another 48 h, to ensure complete removal of excess unreacted dye (water changed three times). Finally, samples were characterized via uorescence spectroscopy (see above), uorescence correlation spectroscopy (FCS), single-particle measurement by TIRF microscopy, and atomic force microscopy coupled with uorescence microscopy (AFM-Fluo).
Physicochemical characterization of nano-emulsions Dynamic light scattering, zeta potential measurements. Size distributions and polydispersity indices (PDI) were measured by DLS, along with zeta potentials, with a NanoZS Malvern apparatus (Malvern, Orsay, France). The helium/neon laser, 4 mW, was operated at 633 nm, with the scatter angle xed at 173 and the temperature maintained at 25 C. DLS data were analyzed using a cumulant-based method.
Transmission electron microscopy. The silica shell formed gives rise to a signicant contrast in TEM. Therefore, samples were used without any staining agent and were diluted (1/100) with Milli-Q water. A drop of the suspension was placed on a carbon grid (carbon type-A, 300 mesh, copper, Ted Pella Inc. Redding, PA) and dried at 40 C. Observations were carried out using a Philips Morgagni 268D electron microscope.
Fluorescence spectroscopy. Absorption and uorescence spectra were recorded on a Cary 4000 spectrophotometer (Varian) and a Fluorolog (Jobin Yvon, Horiba) spectrouorometer, respectively. Fluorescence emission spectra were recorded at room temperature with 365, 405 and 552 nm excitation wavelengths for uorescamine-, coumarin blue-, and Nile red-loaded nanocapsules respectively. All uorescence measurements were done using solutions with absorbance #0.1. Förster resonance energy transfer (FRET) experiments were carried on nanocapsules decorated with coumarin blue as a donor. The acceptor dye Nile red was inserted into the oil core of the nanocapsules. FRET experiments were performed using the following preparation protocol: to 1 mL of MilliQ water we added 20 mL of stock solution of Nile red (526 mM in DMSO), and 7.5 mL of stock solution of silica-covered nano-emulsion decorated with coumarin (aer dialysis, concentration was 600 mM). Control measurements were done with: (i) Nile red-loaded nanocapsules with silica shell but without coumarin graing, and (ii) nanocapsules decorated with coumarin that did not contain Nile red.
Fluorescence correlation spectroscopy (FCS). FCS measurements were done on a two-photon platform including an Olympus IX70 inverted microscope. 21 Two-photon excitation at 800 nm (15 mW laser output power) was provided by an Insight Deepsee femtosecond laser (Spectra-Physics). The measurements were performed in a 96 well plate, using a 200 mL volume per well. The focal spot was set about 20 mm above the bottom of the plate. Following the assumption that the NPs undergo a Gaussian diffusion in the two-photon excitation volume, the correlation function G(s), calculated from the uorescence uctuations was tted according to Thompson: 22 GðsÞ where N is the mean number of uorescent species within the two-photon excitation volume, s d is the diffusion time and s is the ratio between the axial and lateral radii of the excitation volume. The NPs were diluted 200-fold from the originally prepared nano-emulsion. Using 6-carboxytetramethylrhodamine (TMR from Sigma-Aldrich) in water as a reference (D TMR ¼ 421 mm 2 s À1 ), 23  Atomic force microscopy coupled with uorescence microscopy. The AFM/uorescence microscope is a home-made association of an AFM composed of a SMENA Head driven by NTEGRA electronics (NT-MDT, Ru) combined with a wide-eld uorescence inverted microscope (Olympus IX-71). The uorescence microscope is working in TIRF mode (Total Internal Reection Fluorescence) with an oil immersion objective (NA ¼ 1.49, 100Â). A DPPS (cobolt) CW laser emitting at 488 nm was used to excite the NPs with a power of 3 W cm À2 . Fluorescence imaging was performed using an EMCCD camera IXON 897 (ANDOR, UK). AFM images were acquired in the tapping mode, using a NSG03 (NT-MDT, RU) cantilever tip, with a spring constant of 0.9 N m À1 , in liquid environment.

Results and discussion
Nano-emulsions present many advantages such as high encapsulation efficiency, 17 stealth properties in the blood pool, 1,18 and also facile formulation. 16,24 Therefore, the challenge in the functionalization of oil droplets relies in the postfabrication of a functional shell without affecting all these points. Several examples in the literature have shown the fabrication of a silica shell at the oil/water interface of emulsion droplets using TEOS (tetraethyl orthosilicate) as a silica precursor. 25 Here, we replaced TEOS for its derivative APTES bearing a primary amine as reactive group with the aim of building a silica shell decorated with NH 2 functions. As schematically illustrated in Fig. 1, APTES is introduced in the oil phase during the spontaneous nano-emulsication process, to allow its spreading over all the population of forming droplets.
In general, the condensation of a silica precursor with TEOS uses a low pH in bulk as a catalyst. However, alternative methods using ultrasounds instead of chemical catalyst have been developed, 25a,26 giving rise to the same TEOS interfacial condensation reaction. The advantage of the ultrasound approach is the possibility to use physiological pH and salt concentrations.
The obtained silica-treated nano-droplets, as imaged by TEM ( Fig. 2(a)) appear as spherical structures with a diameter around 100-120 nm. In contrast, the control without silica (Fig. 2(b)) shows large and light oil puddles on the support, indicating that the droplets have merged during the drying process. Thus, while the sample drying on the carbon greed destroys the parent nano-droplets, the silica-treatment seems to strengthen the droplet structure. This corroborates the hypothesis that a silica framework or shell is built at the interface of the droplets. For the higher magnications, we can note the malleability of the interface when the nanocapsules are stuck together, indicating that the nanocapsules are still deformable.
Next, we studied the size distribution, polydispersity, and zeta potential of the obtained nano-emulsions, as well as the impact of the silica concentration (APTES in oil) on these parameters. Fig. 3(a) shows a representative size distribution for the highest APTES concentration studied (0.5 M in oil). This distribution is centered at 115 nm, in line with the size range observed on the TEM micrographs, and with a polydispersity index (PDI) of 0.16. In Fig. 3(b), we show the impact of the initial APTES concentration in oil on the nano-emulsion properties. Interestingly, the silica shell has no signicant inuence on the size distribution and PDI. This suggests that when solubilized in oil, APTES does not interfere with the spontaneous emulsi-cation process.
The surface charge clearly decreases with increasing APTES concentrations, from an almost neutral value without APTES (around À3 mV), up to around À20 mV above 0.25 M. This evolution of the surface properties likely reects the density of silica at the water/oil interface, thus showing the gradual formation of the shell. These data also suggest that the shell covers the entire surface when [APTES] oil $ 0.25 M. Considering the TEM pictures (Fig. 2) and the changes in the surface potential ( Fig. 3(b)), likely related to the modication of the surface composition, we can conclude that the silica shell has effectively formed at the oil-water interface. Complementary studies on stability over time of the nanocapsules in FBS were carried out and reported in ESI Fig. S1. † The results did not reveal signicant changes in the size over time, but showed a slight size increase for the higher dilution (1/100 and 1/1000), which could mean a weak aggregation. However, in the conditions of usual in vivo administration (e.g. 1/10 for X-ray imaging 1,18 ) no signicant changes were observed.
In this study, we used APTES instead of TEOS because it bears a primary amine, in order to decorate the nanocapsules formed aer the silica poly-condensation. However it is obvious Fig. 1 General scheme describing the formulation of amino-functionalized nano-emulsion droplets.
that a signicant part of the NH 2 of the shell will be also entrapped into the oil core. To quantify the amino groups available for reaction at the nanocapsule interface, we used a method based on uorescamine. This dye is routinely used in primary amine quantication of peptides or proteins, because it becomes uorescent only aer reaction with primary amines. To elaborate the protocol to the nanocapsules, we used a model water-soluble primary amine, ethanolamine. Different ethanolamine concentrations were mixed with increasing concentrations of uorescamine, and the uorescence intensity was measured (see scheme in Fig. 4, top). These experiments enabled us to calibrate the uorescamine assay with our instrumental settings (ESI section †). It was found that for a uorescamine concentration of 500 mM, the linearity region is obtained for ethanolamine concentrations in the range between 0 and 500 mM.
This calibration was used to determine the apparent NH 2 concentration in the suspension of NH 2 -decorated nanocapsules. In order to be in the linear region, the nano-emulsions were diluted to a maximum theoretical NH 2 concentration of 500 mM and were mixed with 500 mM uorescamine. The  uorescamine reaction with the silica shell is schematically reported in Fig. 4 (top), giving rise to uorescent nanocapsules. Nanoparticles with silica shell gave strong uorescence, whereas no uorescence was observed for blank nanoemulsions without silica shell (Fig. 4, bottom). This result conrmed the presence of reactive NH 2 groups at the surface of the nanocapsules, as well as their accessibility for reacting with uorescamine.
In addition, this method allowed evaluating the impact of the APTES (i.e. silica) concentration on the available NH 2 groups at the interface, shown in Fig. 5 for 3 different nanocapsule diameters, 120, 85 and 65 nm. The surface of the nanocapsules presents a very high coverage, up to 9 Â 10 4 available NH 2 groups per particle of 120 nm (i.e. 2 groups per nm 2 ), prepared in the presence of 0.5 M APTES in oil. The obtained value corresponds to around 50% of the amino groups introduced with APTES in the formulation (ESI section †), indicating that half of the amine functions are entrapped in the droplets while the remaining half is available. As expected, the higher the silica concentration, the higher the number of amine functions per drop. Moreover, the larger is droplets size, the higher is the number of reactive groups. Finally, the straight lines tting the experimental points indicate that the silica concentration at the interface is still growing and does not stabilize around [APTES] oil ¼ 0.25 M as it was shown with the zeta potential (in Fig. 3). This difference is probably because the zeta potential only reects the surface coverage by the silica and once the surface is fully covered, the zeta potential does not change. On the other hand, increasing the silica concentration could increase the thickness and/or compactness of the layer thus increasing the total amount of amino groups.
In a next step, our objective was to gra model ligands on the available NH 2 functions. As a proof of concept for the graing of ligands bearing COOH function, we selected coumarin blue that presents a N-hydroxysuccinimide activated acid. Aer reaction for 24 h, the samples are dialyzed for another 24 h to remove the free coumarin, and the emission of the samples is measured. The results reported in Fig. 6(a), show that coumarin ligands are graed on the silica shell and not simply adsorbed on the nanoemulsion.
Surprisingly, the emission spectrum of coumarin blue is strongly red-shied in comparison to the free dye, with a maximum emission wavelength at 554 nm vs. 473 nm for the free dye in water. A possible explanation is that the uorescence properties of the dye are modied by their high local concentration once they are graed onto the capsule surface. The light absorption measurements at the maximum of the peaks, before and aer dialysis allows us evaluating the proportions of dye graed onto the nanocapsule, giving 41.5% graed. That is to say around 3.7 Â 10 4 coumarin dyes are graed per particle. Such a huge local accumulation can modify the dye emission due to formation of ground or excited-state aggregates. To prove this hypothesis, we recorded the excitation spectra of the dyelabeled particles before dialysis (ESI †). When recorded at 554 and 465 nm, the excitation bands were both located around 400 nm, with some blue shied maximum for the former. These spectra were close to the corresponding absorption spectrum as well as to that of free coumarin dye in water. The small blue shi of the species emitting at 554 nm suggests a ground-state aggregation of the coumarin dyes at the particle surface. These  aggregates may present a strongly red-shied emission, as other dye aggregates. 27 In addition, quantum yields ratio between free coumarin and graed on nanocapsules (aer dialysis) has been calculated from the spectra, giving the graed dyes 5.3 times brighter than free dye in solution. This corroborates the drastic changes in the uorescence properties aer the surface conjugation.
Then, the dialyzed suspensions of nanocapsules decorated with coumarin, in which we have loaded the oil core with Nile red were studied with Förster Resonance Energy Transfer (FRET) experiments. Since the Nile red is homogeneously distributed in all the nanocapsules, our aim here was to study by FRET the co-localization of the two dyes in order to show that coumarin is graed to all the nanocapsules. The control experiment with only coumarin gives a signicant uorescence peak. In the presence of Nile red as FRET acceptor, the uorescence of coumarin is strongly inhibited giving rise to the uorescence of the acceptor. Thus, the distance from coumarin to the closest Nile red molecules in the oil phase appears to be well below the Forster radius ($5 nm).
Moreover, another physical method, combined AFM and uorescence microscopy, shows the co-localization of the particles and the uorescence signal of coumarin (Fig. 8). Indeed, the nanocapsules detected by AFM could be observed in the uorescence images as bright spots, indicating that these nanostructures were labeled with the dye. However, the uorescence spots appeared much larger due to diffraction limited resolution of uorescence microscopy. The AFM micrographs   This journal is © The Royal Society of Chemistry 2015 evidence the spherical shape of the particles, in line with the TEM image (Fig. 2). The uorescent dots correspond well to the particles seen by AFM. Thus, AFM-Fluo further conrms that dialyzed samples show signicant uorescence originated from the nanocapsules.
To further conrm the successful surface modication of the nanocapsules, we studied by uorescence correlation spectroscopy (FCS) the silica coated and non-coated nanocapsules aer treatment with coumarin blue. FCS is a powerful technique to study uorescent nanoparticles because it can provide simultaneously multiple parameters such as the correlation time, the number of particle per volume and the photon count rate, which provide access to the particle size, concentration and brightness, respectively. Corresponding values are reported in Table 1. Two powers of laser were adopted to compare nanocapsules with graed dyes and free dyes, since on the one hand free dye is not sensitive enough at 5 mW (does not correlate) and on the other hand, we observed a strong bleaching at the graed ones at 10 mW. It can be seen that the graed particles have a signicantly higher correlation time than free species, TMR of free dyes. This indicates a bigger size calculated at 100.1 nm, in agreement with DLS and TEM data. The same trend is observed with brightness, and to be comparable we consider the value of the brightness over the one of TMR as reference (Br/Br(TMR)), giving the graed ones around 633 times higher than free dye.

Conclusion
To summarize, the aim of this study was to propose a new method for functionalizing nano-emulsion droplets, aer their formulation. Lipophilic silica precursor (APTES) was added to the oil before the formulation, and turned into silica shell aer nano-droplet fabrication by sonochemistry that avoids addition of catalyst in the bulk phase. In comparison with existing methods to functionalize nano-droplets like post-insertion of functionalized lipids, this novel procedure brings real advantages: covalent graing of functional groups at the nano-droplet surface i.e. strongly anchored on the silica nanocapsules, as well as a large number of reactive sites per capsule (up to 9 Â 10 4 , $2 amino groups per nm 2 ). The functional silica shell fabrication is simple, quick, and affects neither the formulation process nor the droplets size. Amino-group quantication allowed understanding the impact of formulation parameters, like the droplet size or APTES concentration, on the number of reactive NH 2 groups decorating the resulting nanocapsules. Finally, the last part of the study was focused on the graing of a model ligand though the reaction of an activated carboxylic acid (coumarin blue) on the amine functions available onto the nanocapsule surface. We achieved a graing efficiency of around 41% and characterized the conjugate by various spectroscopic and microscopic methods, proving the concept of this new original protocol for functionalizing oil droplets. Nano-emulsions are a particularly interesting type of nano-carriers, able to solubilize a large range of active molecules or contrast agents, and their simple surface functionalization is a real step forward regarding potential applications like active targeting in vivo.