Michel Bessodes

Nanoprobes with near-infrared persistent luminescence for in vivo imaging

Fluorescence is increasingly used for in vivo imaging and has provided remarkable results. Yet this technique presents several limitations, especially due to tissue autofluorescence under external illumination and weak tissue penetration of low wavelength excitation light. We have developed an alternative optical imaging technique by using persistent luminescent nanoparticles suitable for small animal imaging. These nanoparticles can be excited before injection, and their in vivo distribution can be followed in real-time for more than 1 h without the need for any external illumination source. Chemical modification of the nanoparticles surface led to lung or liver targeting or to long-lasting blood circulation. Tumor mass could also be identified on a mouse model.

Controlling Electron Trap Depth To Enhance Optical Properties of Persistent Luminescence Nanoparticles for In Vivo Imaging

Focusing on the use of nanophosphors for in vivo imaging and diagnosis applications, we used thermally stimulated luminescence (TSL) measurements to study the influence of trivalent lanthanide Ln(3+) (Ln = Dy, Pr, Ce, Nd) electron traps on the optical properties of Mn(2+)-doped diopside-based persistent luminescence nanoparticles. This work reveals that Pr(3+) is the most suitable Ln(3+) electron trap in the diopside lattice, providing optimal trap depth for room temperature afterglow and resulting in the most intense luminescence decay curve after X-ray irradiation. This luminescence dependency toward the electron trap is maintained through additional doping with Eu(2+), allowing UV-light excitation, critical for bioimaging applications in living animals. We finally identify a novel composition (CaMgSi(2)O(6):Eu(2+),Mn(2+),Pr(3+)) for in vivo imaging, displaying a strong near-infrared afterglow centered on 685 nm, and present evidence that intravenous injection of such persistent luminescence nanoparticles in mice allows not only improved but highly sensitive detection through living tissues.

EFFECT OF CORE DIAMETER, SURFACE COATING, AND PEG CHAIN LENGTH ON THE BIODISTRIBUTION OF PERSISTENT LUMINESCENCE NANOPARTICLES IN MICE

A growing insight toward optical sensors has led to several major improvements in the development of convenient probes for in vivo imaging. Efficient optical detection using quantum dots (QDs) as well as near-infrared organic dyes relies on several key driving principles: the ability to lower background absorption or autofluorescence from tissue, a good photostability of the probe, and a high quantum yield. In this article, we report the real-time biodistribution monitoring of lanthanide-doped persistent luminescence nanoparticles (PLNP), emitting in the near-infrared window, in healthy and tumor-bearing mice. We focused on the influence of hydrodynamic diameter, ranging from 80 to 180 nm, and polyethylene glycol (PEG) surface coating on the behavior of our probes. Tissue distribution was found to be highly dependent on surface coverage as well as core diameter. The amount of PLNP in the blood was highly increased for small (d < 80 nm) and stealth particles. On the opposite, PEG shield molecular weight, ranging from 5 to 20 kDa, had only negligible influence on the in vivo biodistribution of our silicate-based material.

Growth Factor Delivery Approaches in Hydrogels

The controlled delivery of growth factors is a very challenging task because many different issues have to be addressed to develop the best suited system. A wide range of approaches have been employed for the controlled delivery of growth factors by hydrogels. Direct loading, electrostatic interaction, covalent binding, and the use of carriers are the main strategies presented in the literature. They are all detailed in the first part of this review. Recent work emphasizing biologically inspired strategies is also included. Also, both natural and synthetic materials are discussed. The second part comprises the methods to evaluate such delivery approaches. Both in vivo and in vitro techniques are presented. Improvements based on the discussed approaches may illustrate future paths toward the development of an ideal growth factor delivery system.

In Vitro Targeting of Avidin-Expressing Glioma Cells with Biotinylated Persistent Luminescence Nanoparticles

Far red emitting persistent luminescence nanoparticles (PLNP) were synthesized and functionalized with biotin to study their targeting ability toward biotin-binding proteins. First, the interaction of biotin-decorated PLNP with streptavidin, immobilized on a plate, was shown to be highly dependent on the presence of a PEG spacer between the surface of the nanoparticles and the biotin ligand. Second, interaction between biotin-PEG-PLNP and free neutravidin in solution was confirmed by fluorescence microscopy. Finally, in vitro binding study on BT4C cells expressing lodavin fusion protein, bearing the extracellular avidin moiety, showed that such biotin-covered PLNP could successfully be targeted to malignant glioma cells through a specific biotin-avidin interaction. The influence of nanoparticle core diameter, incubation time, and PLNP concentration on the efficiency of targeting is discussed.

Cell microcarriers and microcapsules of stimuli-responsive polymers.

Cell microcarriers and microcapsules have presented a wide range of potential applications. This article overviews their role in biotechnology with focus on the progress accomplished using stimuli-responsive polymers. Key properties of cell microcarriers and microcapsules are identified, followed by a description of the chemistry and gel formation mechanism of some of the stimuli-responsive polymers used to design them. Production methods are introduced and characterization techniques for evaluating such microsystems are equally presented.

Anionic polyethyleneglycol lipids added to cationic lipoplexes increase their plasmatic circulation time

Cationic liposomes have been widely sensed as good DNA compacting delivery agents. Although their use generally met with encouraging results in vitro, the results in vivo were rather disappointing, as they strongly interact with the blood components before they can reach the therapeutic target. Polyethyleneglycol (PEG) shielding has been proposed as a way to alleviate this effect, but was still found unsatisfactory in most instances for systemic administration. We demonstrate here that the insertion of anionic functions between the lipid part and the PEG, at a correct distance to favor electrostatic interactions with the outer cationic layer of the lipoplexes, provides not only a decrease in the mean peripheral charge of the lipoplex (ζ potential), but also a greater colloidal stability of the particles in the presence of serum. Transfection in the lung is also decreased with negatively charged PEG shielding, although no significant changes are observed in the tumor. This encouraging new approach should consequently be combined with active extra-cellular receptor targeting to achieve the desired delivery of the therapeutic DNA to tumor tissues.

Anionic polymers for decreased toxicity and enhanced in vivo delivery of siRNA complexed with cationic liposomes

We recently reported a cationic lipid-based vector of siRNA, termed siRNA lipoplex that was very efficient in specific gene silencing, both in cell culture and in mouse disease models. To be more efficient, this vector included the addition of a plasmid DNA as an anionic cargo. Although this plasmid DNA was devoid of any eukaryotic expression cassette, we decided to replace it by an anionic polymer that would be more acceptable for clinical applications. We identified seven anionic polymers, regarded as non-toxic, biodegradable, of various characteristics and nature. The addition of polymers to siRNA lipoplexes led to the formation of particles with similar characteristics to crude siRNA lipoplexes, decreased cellular toxicity and variable in vitro gene silencing efficiency depending on the type of polymer used. Upon i.v. injection in mice, siRNA lipoplexes prepared with the best polymer, polyglutamate, led to significantly increased recovery of siRNA in liver and lung compared with lipoplexes without polymer.

Cationic Lipids for Transfection

Among other strategies, the use of cationic lipids as autoassembling vehicles for non viral DNA transfection has received considerable attention. An exponentially growing litterature has been published on this topic (over 700 hits for the past decade, including 400 in the last two years). The present review focuses on the main present strategies aiming at improving cationic lipids induced transfection, and on some of the frequently encountered problems that should be solved to apply these non-viral vectors for human health. The review contains several sections dealing with the chemistry, physico-chemistry, cell biology, in vivo biology, and targeting of cationic-lipid DNA complexes.

Liposomal encapsulation of the natural flavonoid fisetin improves bioavailability and antitumor efficacy.

The natural flavonoid fisetin (3,3,4,7-tetrahydroxyflavone) has shown antiangiogenic and anticancer properties. Because of fisetin limited water solubility, we designed a liposomal formulation and evaluated its biological properties in vitro and in Lewis lung carcinoma (LLC) bearing mice. A liposomal formulation was developed with DOPC and DODA-PEG2000, possessing a diameter in the nanometer range (173.5±2.4nm), a high homogeneity (polydispersity index 0.181±0.016) and high fisetin encapsulation (58%). Liposomal fisetin incubated with LLC cells were internalized, induced a typical fisetin morphological effect and increased the sub-G1 cell distribution. In vivo, liposomal fisetin allowed a 47-fold increase in relative bioavailability compared to free fisetin. The effect of liposomal fisetin on LLC tumor growth in mice at low dose (21mg/kg) allowed a higher tumor growth delay (3.3 days) compared to free fisetin at the same dose (1.6 day). Optimization of liposomal fisetin therapy was attempted by co-treatment with cyclophosphamide which led to a significant improvement in tumor growth delay (7.2 days) compared to cyclophosphamide with control liposomes (4.2 days). In conclusion, fisetin liposomes markedly improved fisetin bioavailability and anticancer efficacy in mice and this formulation could facilitate the administration of this flavonoid in the clinical setting.