Lipid-core/polymer-shell hybrid nanoparticles: synthesis and characterization by fluorescence labeling and electrophoresis.

Among the lipid nanoparticles, lipid polymer hybrid nanoparticles (HNPs) composed of an oily core and a polymeric shell display interesting features as efficient drug carriers due to the high loading capability of the oil phase and the stability and surface functionalization of the polymer shell. Herein, we formulated lipid-core/polymer-shell hybrid nanoparticles (HNPs) using a simple nanoprecipitation method involving Vitamin E Acetate (VEA) as the oily core and a tailor-made amphiphilic polymer as a wrapping shell. The fluorescence labeling of the oil, using a newly developed green fluorogenic BODIPY tracker, and of the polymer using a covalent attachment of a red emitting rhodamine was done to assess the formation, the composition and the stability of these new hybrid nanoparticles using dual color electrophoresis gel analysis. This technique, combined to conventional DLS and electronic microscopy analysis, allowed us to quickly determine that 20 wt% of the polymer was an optimal ratio for obtaining stable HNPs by nanoprecipiation. Finally, we showed that using different polymeric shells, various HNPs can be obtained and finely discriminated using a combined approach of electrophoresis and two-color labeling.

Stealth and Bright Monomolecular Fluorescent Organic Nanoparticles Based on Folded Amphiphilic Polymer.

Fluorescent nanoparticles (NPs), owing to their superior brightness, are an attractive alternative to organic dyes. However, their cellular applications remain limited because of their large size, poor homogeneity, and nonspecific interactions in biological media. Herein, we propose a concept of monomolecular fluorescent organic nanoparticles of high brightness and very small size (10-14 nm) built of a single amphiphilic polymer bearing specially designed fluorescent dyes. We found that high PEGylation of poly(maleic anhydride-alt-1-octadecene (PMAO) favors a single-chain polymer folding into monomolecular stealth NPs with highly reduced nonspecific interactions with proteins and live cells. To ensure high stability of our NPs, the fluorophores (BODIPYs) are covalently linked to the polymer through an optimized linker. Among tested linkers of different lengths and polarity, a short medium-polar linker favoring location of the dyes at NPs interface ensures good fluorescence quantum yield and small particle size. The fluorescence brightness of these NPs has been dramatically enhanced by increasing the bulkiness of the BODIPY dyes that prevents their H-aggregation, reaching 2500000 M-1 cm-1 (extinction coefficient × quantum yield). Fluorescence microscopy revealed that the single-particle brightness of these NPs is ∼5-fold higher than that of QDot-585 using the same excitation wavelength (532 nm). Finally, when microinjected inside cells, these small and stealth NPs (10 nm diameter) distribute more evenly than 20 nm QDots inside the cytosol, showing similar spreading as a fluorescent protein. Thus, the developed monomolecular NPs, owing to their small size and stealth properties, are artificial analogues of fluorescent proteins, surpassing the latter >50-fold in terms of brightness.

Quantifying Release from Lipid Nanocarriers by Fluorescence Correlation Spectroscopy.

Understanding the release of drugs and contrast agents from nanocarriers is fundamental in the development of new effective nanomedicines. However, the commonly used method based on dialysis frequently fails to quantify the release of molecules poorly soluble in water, and it is not well-suited for in situ measurements in biological media. Here, we have developed a new methodology for quantifying the release of fluorescent molecules from lipid nanocarriers (LNCs) using fluorescence correlation spectroscopy (FCS). LNCs based on nanoemulsion droplets, encapsulating the hydrophobic Nile red derivative NR668 as a model cargo, were used. Our studies revealed that the standard deviation of fluorescence fluctuations in FCS measurements depends linearly on the dye loading in the nanocarriers, and it is insensitive to the presence of less-bright molecular emissive species in solution. In sharp contrast, classical FCS parameters, such as the number and the brightness of emissive species, are strongly influenced by the fluorescence of molecular species in solution. Therefore, we propose to use the standard deviation of fluorescence fluctuations for the quantitative analysis of dye release from nanocarriers, which is unaffected by the "parasite" fluorescence of the released dyes or the auto-fluorescence of the medium. Using this method, we found that LNCs remain intact in water, whereas in serum medium, they release their content in a temperature-dependent manner. At 37 °C, the release was relatively slow reaching 50% only after 6 h of incubation. The results are corroborated by qualitative observations based on Förster resonance energy transfer between two different encapsulated dyes. The developed method is simple because it is only based on the standard deviation of fluorescence fluctuations and, in principle, can be applied to nanocarriers of different types.

Light-triggered release from dye-loaded fluorescent lipid nanocarriers in vitro and in vivo.

Light is an attractive trigger for release of active molecules from nanocarriers in biological systems. Here, we describe a phenomenon of light-induced release of a fluorescent dye from lipid nano-droplets under visible light conditions. Using auto-emulsification process we prepared nanoemulsion droplets of 32nm size encapsulating the hydrophobic analogue of Nile Red, NR668. While these nano-droplets cannot spontaneously enter the cells on the time scale of hours, after illumination for 30s under the microscope at the wavelength of NR668 absorption (535nm), the dye showed fast accumulation inside the cells. The same phenomenon was observed in zebrafish, where nano-droplets initially staining the blood circulation were released into endothelial cells and tissues after illumination. Fluorescence correlation spectroscopy revealed that laser illumination at relatively low power (60mW/cm2) could trigger the release of the dye into recipient media, such as 10% serum or blank lipid nanocarriers. The photo-release can be inhibited by deoxygenation with sodium sulfite, suggesting that at least in part the release could be related to a photochemical process involving oxygen, though a photo-thermal effect could also take place. Finally, we showed that illumination of NR668 can provoke the release into the cells of another highly hydrophobic dye co-encapsulated into the lipid nanocarriers. These results suggest dye-loaded lipid nano-droplets as a prospective platform for preparation of light-triggered nanocarriers of active molecules.

Integrity of lipid nanocarriers in bloodstream and tumor quantified by near-infrared ratiometric FRET imaging in living mice.

Lipid nanocarriers are considered as promising candidates for drug delivery and cancer targeting because of their low toxicity, biodegradability and capacity to encapsulate drugs and/or contrasting agents. However, their biomedical applications are currently limited because of a poor understanding of their integrity in vivo. To address this problem, we report on fluorescent nano-emulsion droplets of 100nm size encapsulating lipophilic near-infrared cyanine 5.5 and 7.5 dyes with a help of bulky hydrophobic counterion tetraphenylborate. Excellent brightness and efficient Förster Resonance Energy Transfer (FRET) inside lipid NCs enabled for the first time quantitative fluorescence ratiometric imaging of NCs integrity directly in the blood circulation, liver and tumor xenografts of living mice using a whole-animal imaging set-up. This unique methodology revealed that the integrity of our FRET NCs in the blood circulation of healthy mice is preserved at 93% at 6h of post-administration, while it drops to 66% in the liver (half-life is 8.2h). Moreover, these NCs show fast and efficient accumulation in tumors, where they enter in nearly intact form (77% integrity at 2h) before losing their integrity to 40% at 6h (half-life is 4.4h). Thus, we propose a simple and robust methodology based on ratiometric FRET imaging in vivo to evaluate quantitatively nanocarrier integrity in small animals. We also demonstrate that nano-emulsion droplets are remarkably stable nano-objects that remain nearly intact in the blood circulation and release their content mainly after entering tumors.