A new contrast agent, LipImage™ 815, has been designed and compared to previously described indocyanine green (ICG)-loaded lipid nanoparticles (ICG-lipidots®). Both contrast agents display similar size (50-nm diameter), zeta potential, high IC50 in cellular studies, near-infrared absorption and emission wavelengths in the “imaging window,” long-term shelf colloidal and optical stabilities with high brightness (>10 6 L mol −1 cm −1 ) in ready-to-use storage conditions in aqueous buffer (4°C in dark), therefore being promising fluorescence contrast agents for in vivo imaging. However, while ICG-lipidots® display a relatively short plasma lifetime, LipImage™ 815 circulates in blood for longer times, allowing the efficient uptake of fluorescence signal in human prostate cancer cells implanted in mice. Prolonged tumor labeling is observed for more than 21 days.
Anne-Claude Couffin, Thomas Delmas, Jean-Sébastien Thomann, Ismail Cheibani, Eric Bayma, Emilie Heinrich, Marie Escudé, Thomas Courant, Antoine Hoang, Rachel Auzély, Isabelle Texier
The controlled delivery of drugs and biologicals (proteins, antibodies, DNA and derivatives) is a growing need to take the full benefit of new therapeutic strategies. However these new molecules or biomolecules display solubility issues, or high degradation rates once injected. Therefore, both suitable delivery materials for their encapsulation and protection from the surrounding environment, and smart delivery devices (such as micro-needles or implanted pumps) are necessary to achieve controlled delivery of these precious therapeutic agents. We have developed bio-inspired gel materials, based on lipid nanoparticles which act as reservoirs for lipophilic drugs. The lipid nanoparticles, termed lipidots™, are biocompatible, colloidally stable, non-immunogenic, and obtained from a cheap and simple solvent-free process. The particles can be assembled to form physical or chemical gels, with tunable rheological properties. Physico-chemical studies have been carried out to determine the limits of the stability domains for colloidal and gel formulations (choice of surfactants for nanoparticle surface, and composition ratios of lipids, surfactants and co-surfactants). In particular, it is demonstrated that lipid nanoparticles keep their integrity in the gels. Gels of lipidots™ could therefore constitute biocompatible materials for the efficient encapsulation and tuned delivery of lipophilic drugs and biomolecules.
Near-infrared fluorescence-enhanced diffuse optical tomography (fDOT) is used to localize tumors in mice using fluorescent nanoparticles as a blood pool contrast agent. The infrared dye DiR is loaded in the lipid core of nontargeted nanoparticles (DiR-lipidots) and injected systemically via the tail vein in mice bearing U87 tumors. Distribution and time-course of DiR-lipidots are followed using in vivo fluorescence reflectance imaging and reveal enhanced fluorescent signal within the subcutaneous tumors up to seven days due to the enhanced permeability and retention effect. Tumor growth into the brain is followed using bioluminescent imaging, and tumor localization is further determined by magnetic resonance imaging. The fDOT provides three-dimensional fluorescent maps that allow for consistent localization for both subcutaneous and brain tumors.
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.
A large number of different approaches for the aqueous phase transfer of quantum dots have been proposed. Surface
ligand exchange with small hydrophilic thiols, such as L-cysteine, yields the lowest particle hydrodynamic diameter.
However, cysteine is prone to dimer formation, which limits colloidal stability. We demonstrate that precise pH control
during aqueous phase transfer dramatically increases the colloidal stability of InP/ZnS quantum dots. Various
bifunctional thiols have been applied. The formation of disulfides, strongly diminishing the fluorescence QY has been
prevented through addition of appropriate reducing agents. Bright InP/ZnS quantum dots with a hydrodynamic diameter
<10 nm and long-term stability have been obtained. Finally we present in vitro studies of the quantum dots
functionalized with the cell-penetrating peptide maurocalcine.
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.
Fluorescence is a very promising radioactive-free technique for functional imaging in small animals and, in the future, in humans. However, most commercial near-infrared dyes display poor optical properties, such as low fluorescence quantum yields and short fluorescence lifetimes. In this paper, we explore whether the encapsulation of infrared cyanine dyes within the core of lipid nanoparticles (LNPs) could improve their optical properties. Lipophilic dialkylcarbocyanines DiD and DiR are loaded very efficiently in 30-35-nm-diam lipid droplets stabilized in water by surfactants. No significant fluorescence autoquenching is observed up to 53 dyes per particle. Encapsulated in LNP, which are stable for more than one year at room temperature in HBS buffer (HEPES 0.02 M, EDTA 0.01 M, pH 5.5), DiD and DiR display far improved fluorescence quantum yields (respectively, 0.38 and 0.25) and longer fluorescence lifetimes (respectively, 1.8 and 1.1 ns) in comparison to their hydrophilic counterparts Cy5 (=0.28, =1.0 ns) and Cy7 (=0.13, =0.57 ns). Moreover, dye-loaded LNPs are able to accumulate passively in various subcutaneous tumors in mice, thanks to the enhanced permeability and retention effect. These new fluorescent nanoparticles therefore appear as very promising labels for in vivo fluorescence imaging.
Fluorescence imaging (FLI) allows the in vivo monitoring of biological events associated with disease and
represents a new promising tool for drug discovery. In particular, it speeds up the development and assessment of new
therapies in oncology, helps in diagnosis, and improves surgery by fluorescence-guided tumor resection. This technique
is highly sensitive, non-ionizing, easy to use and relatively inexpensive.
Nevertheless, the main limitation of FLI lies in the optical properties of biological tissues. Mainly because of
haemoglobin and water absorption, only near-infrared (NIR) light is adapted to image tissues in depth. Using a
contrasting agent absorbing and emitting in the NIR region is therefore necessary to improve the background signal ratio,
and thus the image contrast.
Among many commercially available NIR optical contrast agents, only indocyanine green (ICG), has been
approved by the United State Food and Drug Administration (FDA) for various medical applications. However, its
instability (photo-degradation, thermal-degradation and low aqueous solubility) limits its applications as a fluorescent
probe for imaging purposes. In order to improve the effectiveness of ICG, we engineered ICG-doped lipid nanoparticles
(LNP).
In this communication, we will report the design of these novel fluorescent nanoparticle probes. These low cost
nanocarriers have numerous advantages, including their high chemical stability and biocompatibility. The
characterization of the optical properties of the nanoparticles entrapping ICG will also be discussed. Finally, the
biodistribution in mice of ICG when delivered through nanoparticles in comparison to free ICG in solution is presented.
It demonstrates the efficient accumulation of ICG-doped nanoparticles in the tumor site.
Non-invasive near infrared fluorescence imaging of mice models is a very attractive tool for fastening the
development of new therapeutics. Two classes of labels exist for the near infrared domain: organic dyes and quantum
dots (QDs). QDs are inorganic luminescent semi-conductor nano-crystals which display very attractive optical features.
They are now commercially available for in vivo mouse tests, and new compositions with less toxic elements are
currently being developed.
The concept of activatable probes, which fluorescence is activated specifically upon the biological process to be
visualized, has also been demonstrated to improve the fluorescence image contrast.
The construction of activatable probes based on quantum dot labels has therefore been undertaken. Commercial
PEGylated quantum dots bearing around 80-100 amino pending groups are used. Long PEG chains are demonstrated to
be essential in order to increase the blood circulation time of the particles and avoid their massive storage into the liver.
The amino groups coating the QD surface can be used for their further functionalization by either a tumor-targeting
ligand, a cleavable spacer bearing a fluorescence inhibitor I, or both. Functionalization of 80% of the amino groups by
the inhibitors I leads to more than 99% fluorescence quenching. Cleavable spacers X-L-S-S-L'-I in which S-S is a
disulfide bond cleavable by cell internalization, and X a chemical group for QD grafting have been synthesized. The
functionalization of the QD by 12 cleavable spacers leads to more than 85% fluorescence inhibition, which can be
recovered upon cleavage of the disulfide bonds.
The framework of fluorescent targeting probes for optical imaging is similar to that of contrast agents for other
modalities. They generally include a biological ligand, specific of the biological process to image, and a label, which
confers the probe its optical properties. Moreover, more sophisticated labeling functions, termed "activatable" can be
designed. An "activatable probe" will be initially non fluorescent. Only a specific molecular process, such as an
enzymatic reaction or cell internalization, is able to activate the probe fluorescence. Such probes are particularly easy to
design for the optical imaging modality, because of the easily triggered and well known fluorescence inhibition
processes. The optical properties of different commercially available organic dyes and their fluorescence inhibition are
therefore examined. Three classes of activatable probes have been listed: (i) activatable probes which rely on the selfquenching
of the label; (ii) activatable probes which use RET (Resonance Energy Transfer) between the label acting as a
donor, and an organic non-emissive acceptor; (iii) activatable probes which use an inorganic nanostructure as the
inhibitor, such as a gold nano-particle. Whereas activatable probes of class (i) can lead to higher fluorescence levels after
activation, their initial fluorescence inhibition can be hindered by structural constraints. Probes of class (ii) can therefore
be more interesting according to the probe design. The efficiency of probes of class (iii) using nanometer gold particles is
reduced because of their plasmon band lying in the visible and not near-infrared domain.
Luminescent nanoparticles are increasingly used as emitting labels in the design of fluorescence imaging probes, because
of their outstanding optical properties, such as in the case of quantum dots, or their role in nano-assembling different
functionalities, such as imaging, drug delivery, and therapy. In this work, the potentiality of up-converting nano-crystals
for non invasive fluorescence imaging of small animal is explored. These up-converting nano-crystals are lanthanide
doped oxide matrices, such as Y2O3:Er,Yb compositions. They are produced by a bottom-up process and a thermal
treatment. They are functionalized by the coating of a thin polysiloxane shell layer, which can be further derivatized in
order to graft biomolecules such as antibodies, peptides, or DNA. Contrary to classical luminescent particles for which
light emission occurs at a wavelength superior to that of excitation, these materials emit at 564 and 661 nm upon
excitation at 980 or 815 nm. These unique emission properties, due to a multi-photonic process, can allow imaging
without any auto-fluorescence from the tissues in the wavelength detection window. Experiments in phantoms
mimicking the optical absorption and diffusion of tissues show that these crystals can be detected 4 mm deep at a 10
mg/mL concentration. Luminescence measurements in mouse and the potentiality of these nano-crystals for in vivo
imaging are discussed.
Going along with instrumental development for small animal fluorescence in vivo imaging, we are developing molecular fluorescent probes, especially for tumor targeting. Several criteria have to be taken into account for the optimization of the luminescent label. It should be adapted to the in vivo imaging optical conditions : red-shifted absorption and emission, limited overlap between absorption and emission for a good signal filtering, optimized luminescence quantum yield, limited photo-bleaching. Moreover, the whole probe should fulfill the biological requirements for in vivo labeling : adapted blood-time circulation, biological conditions compatibility, low toxicity. We here demonstrate the ability of the imaging fluorescence set-up developed in LETI to image the bio-distribution of molecular probes on short times after injection. Targeting with Cy5 labeled holo-transferrin of subcutaneous TS/Apc (angiogenic murine breast carcinoma model) or IGROV1 (human ovarian cancer) tumors was achieved. Differences in the kinetics of the protein uptake by the tumors were evidenced. IGROV1 internal metastatic nodes implanted in the peritoneal cavity could be detected in nude mice. However, targeted metastatic nodes in lung cancer could only be imaged after dissection of the mouse. These results validate our fluorescence imaging set-up and the use of Cy5 as a luminescent label. New fluorescent probes based on this dye and a molecular delivery template (the RAFT molecule) can thus be envisioned.
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