The use of fluorescent nanostructures can bring several benefits on the signal to background ratio for in vitro microscopy, in vivo small animal imaging, and image-guided surgery. Fluorescent quantum dots (QDs) display outstanding optical properties, with high brightness and low photobleaching rate. However, because of their toxic element core composition and their potential long term retention in reticulo-endothelial organs such as liver, their in vivo human applications seem compromised. The development of new dye-loaded (DiO, DiI, DiD, DiR, and Indocyanine Green (ICG)) lipid nanoparticles for fluorescence imaging (lipidots) is described here. Lipidot optical properties quantitatively compete with those of commercial QDs (QTracker®705). Multichannel in vivo imaging of lymph nodes in mice is demonstrated for doses as low as 2 pmols of particles. Along with their optical properties, fluorescent lipidots display very low cytotoxicity (IC50 > 75 nM), which make them suitable tools for in vitro, and especially in vivo, fluorescence imaging applications.
Fabrice Navarro, Denise Bechet, Thomas Delmas, Pierre Couleaud, Céline Frochot, Marc Verhille, Ezatul Kamarulzaman, Régis Vanderesse, Patrick Boisseau, Isabelle Texier, Julien Gravier, Françoise Vinet, Muriel Barberi-Heyob, Anne Claude Couffin
PhotoDynamic Therapy (PDT) has been established as a potent and less invasive treatment for different kinds of
cancer. Among various attempts to enhance the therapeutics efficacy of PDT, the specific delivery of the PhotoSensitizer
(PS) in the tumor is expected to increase its clinical applications, since unwanted accumulation, especially in the skin,
impairs the patients' quality of life (prolonged cutaneous photosensitivity). The aim of this study was to engineer Lipid
Nanoparticles (LNP) with different sizes and various PS contents, using simple, solvent-free and easily scale up
manufacturing processes. Meso-tetra (hydroxyphenyl) chlorin (mTHPC) is one of the most potent photoactive
compounds for clinical use and it has been successfully applied in the treatment of various indications, such as the head
and neck, prostate and pancreatic cancers. Here, a derivative of mTHPC was efficiently incorporated into the lipid core
of LNP, leading to a large range of stable and reproducible mTHPC-loaded LNP with narrow size distribution. The
photophysical and photochemical properties of mTHPC-loaded LNP were studied by measuring absorbance and
fluorescence spectra, colloidal stability, particle size and zeta potential, as well as singlet oxygen luminescence. The
photocytotoxicity of three selected mTHPC-loaded LNP (25 nm, 45 nm and 95 nm of diameter, respectively) was
evaluated on MCF-7 cells, in comparison to free mTHPC, under irradiation at 652 nm with a range of light fluence from
1 to 5 J/cm2. All the physico-chemical, photophysical and biological measurements allow us to conclude that LNP is a
promising nano-drug delivery system for PDT.
KEYWORDS: Fluorescence resonance energy transfer, Luminescence, Nanoparticles, In vivo imaging, Absorbance, Magnesium, Atmospheric particles, Particles, Energy transfer, Confocal microscopy
Different approaches have been reported in the recent years for the in vivo delivery and targeting of poorly
soluble contrast agents and active ingredients in diseased tissues. In this context, we developed new lipid nanoparticles
(Lipidots®) with size being easily varied from 25 to 120 nm. Lipidots® display numerous advantages: they are
composed of low-cost and biocompatible lipids; they can be stored in injection-ready formulations for long duration;
their manufacturing process is versatile and up-scalable. Several indocyanines have been efficiently encapsulated in the
particles while retaining their spectroscopic properties, with emission wavelengths ranging from 500 to 820 nm. Thus,
dye loaded-Lipidots® have been proved suitable for both in vitro and in vivo applications.
To better understand Lipidots®' behavior in biological systems, formulations based on Förster Resonance
Energy Transfer (FRET) have been studied. Different pairs of the selected indocyanines were co-encapsulated and
calculations proved that transfer efficiency within nanoparticles (i) behaves as in a continuous medium; (ii) depends on
local acceptor concentration. Thanks to the local dye concentration dependence of FRET, these formulations are used to
understand where and when the particles are assimilated in biological systems. FRET-based Lipidots® contrast agents
are also promising tools to enhance imaging contrast in vivo by making a clear difference between circulating and
uptaken particles.
Fluorescence imaging is a valuable tool for the study of living systems. It can be used with good resolution from
the micro- to the macroscopic range. However, for macroscopic use in living animals or humans, fluorescent probes must
overcome several obstacles such as aqueous solubility, suitable circulating lifetime and clearance. Fluorescent probes
should also display high molar extinction coefficient and fluorescence quantum yield. In this article, we report the
encapsulation of five hydrophobic or amphiphilic fluorophores (DiO, DiI, DiD, DiR and ICG) with emission wavelength
ranging from 500 to 800 nm, in long-circulating Lipid NanoParticles (LNP). Loading of these commercially available
indocyanines in LNP is highly efficient (from 77 to 97 %), and fluorescence quantum yields range from 7 to 53%,
depending on the dye, in the standard formulation (50 nm diameter nanoparticles). Given the wide range of wavelengths
covered and the stability of particle dispersion in aqueous buffer, dye-loaded LNP should be a valuable tool for both in
vivo and in vitro fluorescence imaging.
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